Neurogenomics Conference

19 May 2025, 09:00 → 21 May 2025, 19:00 Europe/Rome

Human Technopole, Milan (Italy)

Overview

This groundbreaking conference aims to engage the leading communities from the main fields of research our institute is pursuing in Neurogenomics, focusing on the neurodiverse human condition and its neurodevelopmental and neurodegenerative vulnerabilities. By leveraging the interdisciplinarity of HT Neurogenomics themes, the goal is to foster international synergies across diverse academic communities through a vibrant mix of keynote lectures, talks, and discussions to celebrate the diversity and integrative challenges of human neuroscience. Whether you are an accomplished group leader or an emerging talent, whether neuroscience is already your home, or you are considering moving in, look no further to keep abreast and engage with the latest developments. 

Target Audience

Researchers, early-career scientists, postdoctoral fellows, and graduate students who wish to share and advance knowledge in neuropsychiatric and neurological diseases.

Registration Fee

Academic	Industry
500€	1500€

 

Important Deadlines

 

Abstract submission deadline [POSTPONED!]	Abstract notification deadline	Registration deadline	Payment deadline
March 16 April 14, 2025 h. 23:59 CEST	March 31, 2025 April 14, 2025 h. 23:59 CEST	March 31, 2025 h. 23:59 CEST April 14, 2025 h. 23:59 CEST	March 31, 2025 h. 23:59 CEST April 14, 2025 h. 23:59 CEST

Date and Place

May 19, May 20, May 21, 2025 @ Human Technopole (Milan, Italy). In presence only.

Monday, 19 May

09:00 → 09:30 Arrival and registration

09:30 → 10:00 Welcome coffee

10:00 → 10:15 Opening remarks

10:15 → 11:45 Neurodevelopment I

Convener: Nereo Kalebic (HT)

10:15 What made our brain so big and us smarter than Neandertals?

Speaker: Wieland Huttner (MPI-CGB, DE)

10:40 Importance of stereotypical migration routes for precise cortical interneuron positioning and circuit formation

Speaker: Philipp Abe (Technische Universität Dresden)

10:50 Temporal control of Neurogenesis

Speaker: Bassem Hassan (ICM, FR)

11:15 Selected speaker from abstractsHuman-specific morphoregulatory signatures in basal radial glia characterize neocortex evolution

Speaker: Mareike Albert (TUD Dresden University of Technology)

11:25 Single-cell and spatial transcriptomics reveal developmental conservation and adult divergence across tetrapod spinal cord evolution

Speaker: Yuri Ignatyev (ISTA Austria)

11:35 Single-cell multimodal history tracing reveals neuronal identity specification

Speaker: Taro Kitazawa (DANDRITE Nordic EMBL, Aarhus University)

11:45 → 13:55 Lunch

13:55 → 15:05 Neurodevelopment II

Convener: Elena Taverna (HT)

13:55 Exploring the role of the primary cilium in neuronal plasticity and development: How the quiet organelle claims center stage!

Speaker: Nael Nadif Kasri (Radboud University Medical Centre, Human genetics department,)

14:05 Gene regulation in response to neuronal activity

Speaker: Yukiko Gotoh (University of Tokyo)

14:30 Ankycorbin – a strong membrane shaper and its role in human neuron morphogenesis.

Speaker: Maria Schörnig (University hospital Jena, Institute for Biochemistry I)

14:40 Coordinated remodeling of neural stem cell epigenome and cell fate bias during mouse cortical development

Speaker: Boyan Bonev (Helmholtz Pioneer Campus, DE)

15:05 → 15:35 Coffee break

15:35 → 18:10 Brain evolution

Convener: Carmen Falcone (SISSA, IT)

15:35 Mapping development of the human brain using high-throughput genomics

Speaker: Tom Nowakowski (UCSF, US)

16:00 Investigating Evolutionary Expansion of the Human Cerebellum Using Cross-Species Cerebellar Organoids

Speaker: Luca Guglielmi (MRC Laboratory of Molecular Biology)

16:10 Timing mechanisms linking development and evolution of the human brain

Speaker: Pierre Vanderhaeghen (VIB, BE)

16:35 3D epigenome evolution underlies divergent gene regulatory programs in primate neural development

Speaker: Silvia Vangelisti (Helmholtz Pioneer Campus)

16:45 The interaction of genomes and mechanics in brain development and evolution

Speaker: Roberto Toro (Institut Pasteur, FR)

17:10 [Keynote Lecture] Constructing and deconstructing the human nervous system to study development and disease

Speaker: Sergiu Pasca (Stanford University, US)

18:10 → 18:30 Walking time to Triulza Academy

18:30 → 21:30 Poster Session: Drinks & Poster Session

18:30 A Combined Dynamical and Causal Framework for Identifying Gene Regulatory Targets in NeuroCOVID

Understanding causal mechanisms in biological systems is essential for decoding complex physiological and pathological processes. Causal learning, a branch of machine learning, establishes quantitative relationships between molecular variables, revealing regulatory dynamics. Identifying interactions between master regulators, their target transcripts, and external interventions is crucial for uncovering actionable targets in gene regulation.
Using single-cell RNA sequencing (scRNA-seq), we infer gene regulatory networks (GRNs) through classical statistical and causal learning approaches. While traditional correlation-based techniques capture co-expression patterns, they fail to distinguish direct regulatory interactions and causal directionality. In contrast, causal learning methods, such as probabilistic graphical models, directed acyclic graphs (DAGs), and Bayesian networks, enable a mechanistic understanding of transcriptional regulation by differentiating direct from indirect effects and identifying key causal regulators.
Once a GRN is established, we investigate the interplay between transcription factors (TFs), exogenous signalling, and endogenous gene products. Causal inference methodologies help deconvolute complex regulatory hierarchies and predict perturbation effects on cellular states. To further enhance causal discovery, we integrate causal kinetic models, which use dynamical systems approaches to infer gene regulatory interactions from pseudo-time-resolved scRNA-seq data. These models reconstruct transcriptional dynamics by capturing temporal dependencies and quantitative relationships in gene expression patterns, improving the identification of causal drivers of cellular state transitions and actionable targets.
A critical application of this approach is in understanding the molecular mechanisms underlying NeuroCOVID, and the neurological complications associated with SARS-CoV-2 infection. By leveraging causal learning on sc-multi-omic data from affected neural and microglial cells, we can unravel the acute from long-lasting dysregulated programs, identify key transcription factors driving chronic neuroinflammation, and map gene regulatory changes linked to neurological dysfunction.
This approach improves the interpretability of inferred GRNs and lays the foundation for novel therapeutic strategies in precision medicine. Such insights might facilitate the identification of therapeutic targets for mitigating LongCOVID-related pathologies.

Speaker: Vittorio Aiello (Fondazione Human Technopole)

18:30 A miRNA-based therapeutic approach for medulloblastoma in xenotransplanted wild-type mouse embryos

Medulloblastoma (MB) is the most common pediatric brain tumor, and current therapies fail in about 30% of cases due to fatal recurrences. To address effective therapeutic strategies against MB, a better modeling of the tumor microenvironment is needed.
Here, we firstly developed an orthotopic xenotransplantation model of human MB in wild-type mouse embryos, which recapitulates the MB microenvironment, including angiogenesis, cancer stem cell (CSC) niche and neuro-immune crosstalk, within a developmental context.
We then exploited the embryonic xenotransplantation model of MB to test the therapeutic potential of a combination of eleven microRNAs (miRNAs), which we previously showed to rescue impaired neurogenesis of miRNA-depleted neural stem cells. We demonstrate that the eleven miRNAs are downregulated across various human MB subtypes. Upon rescue of expression of the eleven miRNAs in human MB cells in vitro and in vivo, we show by multi-omics analyses that the pool of eleven miRNA exerts an anticancer activity by modulating key genes and pathways involved in proliferation, differentiation and migration.
This study demonstrates that our model recapitulates human MB development and microenvironment and is suitable for testing RNA-based therapies. Finally, our results emphasize the therapeutic potential of the synergistic action of multiple miRNAs as anticancer strategy for MB, and possibly other tumors.

Speaker: Letizia La Rosa (Istituto Italiano Tecnologia)

18:30 A Scalable and Reproducible Workflow for High-Throughput Analysis of Cortical Brain Organoids in Neurodevelopmental Disorders Research

"Neurodevelopmental Disorders (NDDs) include a wide range of conditions characterised by impairments affecting the development of the central nervous system, with implications for cognitive, motor, behavioral, and social functioning.
To tackle the complexity of the mechanisms underlying these dysfunctions suitable experimental models are needed. While animal models and 2D in vitro cultures are limited in recapitulating the complex, human-specific features of neurodevelopment, increasingly sophisticated 3D brain organoids have enabled researchers to study the dynamics of these alterations with unprecedented accuracy, opening new avenues for understanding the regulatory mechanisms of these diseases and testing potential drug therapies.

However, time-consuming procedures and technical challenges still hinder organoids modeling from being scaled-up in a high-reproducible and high-throughput fashion.

We developed a simple and reproducible workflow for the generation and profiling of patient-derived cortical brain organoids (CBOs) at scale. To strengthen the reliability of this pipeline we included over 40 stem cell lines from NDD case-control matched cohorts, along with iPSCs and embryonic stem cell isogenic lines harboring highly penetrant mutations in NDD-related genes.

First, we generated CBOs from iPSC and ESC lines, seeding and culturing them under identical conditions to ensure uniform confluence across all cell lines. Multiple time points were selected to profile the CBOs. At the gene expression level, single-cell data analysis was performed, and immunofluorescence analysis was integrated using an innovative tissue microarray platform and a slide scanner microscope. This approach significantly reduced sectioning, staining, and acquisition time while lowering antibody and reagent costs and minimizing procedural variability. Finally, a dedicated image- analysis pipeline was developed to characterise CBOs by assessing structure and expression using a panel of 16 selected NDD-related biomarkers.

In summary, we present a simplified and cost-effective approach that enhances efficiency, reproducibility, and scalability while minimizing technical variability in CBO characterization. This offers a promising new tool for studying NDD biomarkers.

With applications in disease modeling and drug screening, this study advances our understanding of neurodevelopmental disorders and paves the way for improved therapeutic strategies."

Speaker: claudio Maderna (Human Technopole)

18:30 A single cell atlas to unveil the diversity of mouse cerebellar astrocytes: insights into their molecular identities, development, and functions

Understanding astrocyte heterogeneity is crucial for gaining insights into the development and function of the central nervous system. While the main types of cerebellar astrocytes can be identified based on their morphology and location, it remains unclear whether this classification reflects the full extent of cerebellar astrocyte heterogeneity. Furthermore, our overall
knowledge on their molecular profile, development, and functions is very limited. We recently provided the first evidence that an ontogenetic program, tightly regulated in space and time, generates the main types of cerebellar astrocytes from embryonic and postnatal progenitors with distinct fate potencies. However, the molecular profiles of these progenitor pools and the mechanisms governing the ontogenesis of different astrocyte types remain to be elucidated.
Through single-cell RNA sequencing and spatial transcriptomics in both postnatal and adult mouse cerebella, we unveiled the transcriptome of established astrocyte types and identified novel subtypes. Moreover, by developing a multimodal computational approach, we were able to reconstruct their maturation trajectories from postnatal progenitors. This analysis highlighted previously unrecognized progenitor sources for some astrocyte types and uncovered the gene expression cascades along distinct trajectories. Intriguingly, we shed new light on the ontogenesis and physiology of cerebellar nuclei astrocytes (CNA), which had previously received limited attention. Our transcriptomics data, complemented by clonal analyses, suggest that CNA arise from a distinct embryonic lineage compared to other astrocytes, and share a common origin with oligodendrocytes. Hence, the developing cerebellum appears to include two astrocyte lineages, clearly segregated at birth. Yet, they eventually converge into mature astrocyte profiles that despite spatial segregation show in some cases a high degree of transcriptional overlap, particularly in genes associated with homeostatic functions. This suggests that environmental cues play a crucial role in shaping cerebellar astrocyte heterogeneity, driving astrocytes of different embryonic origins toward shared homeostatic roles.

Speaker: Valentina Cerrato (University of Turin and NICO (Neuroscience Institute Cavalieri Ottolenghi))

18:30 A stem cell platform for modelling triplet repeats somatic instability in Huntington’s Disease

"Huntington disease (HD) is a late-onset and progressive neurodegenerative condition caused by an expanded CAG repeat residing in exon1 of the Huntington gene (HTT). In recent years, there has been a shift in prospective about the pathogenic mechanisms underlying HD, moving beyond traditional protein-centric vision to embrace new DNA-based driving hypothesis. Accordingly, inherited CAG repeats can expand in somatic tissues, especially in post-mitotic neurons, giving rise to a HTT mosaicism that results in longer than inherited CAG tracts in affected tissues, such as the striatum and cortex. This expansion may continue during the lifetime of the individual contributing to exacerbate (or initiate) neuronal toxicity and selective degeneration. More recently, trans- and cis- modifiers of age of onset (AOO) have been identified. However, if and how they cause the progressive accumulation of CAG instability is not fully understood.
Here, we show a human stem cell-based platform (CAGinSTEM) capable of modelling HD repeat instability over time in mitotic cells and post-mitotic neurons. Thanks to the design based on targeted engineering of a Recombination Mediated Cassette Exchange at the HTT exon1, this platform allows for easy and flexible manipulation of the HTT CAG repeat with change in size and composition, providing a unique biological model system to study genotype-phenotype relationship in different cell types in vitro. Moreover, the platform is amenable to pooled screening approach that coupled to single nuclei RNA sequencing (snRNAseq) and CAG sizing, allows for high-throughput analysis of gene expression and repeat instability in individual cells. This powerful combination can offer unprecedented insights into the molecular mechanisms driving CAG instability and its downstream effects, paving the way for new therapeutic targets in HD research."

Speaker: Dario Besusso Besusso (Fondazione Telethon ETS)

18:30 Activity-dependent chromatin reorganization in cortical neurons depends on SATB2

When neurons are stimulated, a cascade of intracellular signaling events alters the chromatin landscape, leading to changes in gene expression. This process is essential for activity-dependent plasticity, learning, and memory. Activity-regulated genes often engage in chromatin looping, bringing enhancers and promoters into proximity.

However, the precise mechanisms governing 3D epigenome remodeling, chromatin accessibility modifications, and the radial repositioning of activity-induced loci relative to the nuclear periphery remain unclear. While CTCF and Cohesin contribute to chromatin reorganization in all cell types, specific chromatin scaffolding proteins are hypothesized to regulate activity-dependent transcriptional programs in neurons.

To investigate these questions, we analyzed transcriptomic and 3D epigenomic changes in cortical neurons from floxed versus Satb2 cKO mice following increased neuronal activity. We have previously shown that SATB2, a DNA-binding protein, plays a crucial role in 3D genome organization in pyramidal neurons. Here, we assessed the genome-wide effects of neuronal activity on chromatin architecture at multiple hierarchical levels in the presence and absence of SATB2.

Our multi-omics integration revealed a profound deficiency in the activity-dependent regulome at both early (1h) and late (6h) time points after bicuculline treatment in Satb2 cKO neurons. This was accompanied by a loss of newly gained open chromatin regions and reduced enhancer-promoter interactions. In floxed neurons, neuronal activity triggered highly specific alterations in FIREs and superFIREs, local interaction hotspots enriched for synaptic function-related genes, which were severely impaired or absent in cKO neurons.

At larger hierarchical scales, bicuculline stimulation in floxed neurons led to increased chromatin compaction, stronger compartmentalization, and enhanced inter- and intra-chromosomal interactions, as well as intra-TAD interactions. These changes were strongly diminished in cKO neurons.

Our findings indicate that SATB2-dependent chromatin architecture remodeling is a key component of the molecular mechanisms underlying activity-regulated transcription in neurons.

Speaker: Nico Wahl (Medical University of Innsbruck)

18:30 Application of 2D and 3D models on Neurodevelopmental disorders

Neurological disorders are highly prevalent (3 billion people around the globe are suffering from it), and due to the challenges associated with studying these pathologies in humans, the importance of in vitro human models is growing. The use of in vitro human models provides a more accurate representation of human biology, thereby enhancing our understanding of neurological disorders and the development of therapeutic interventions. Our group is focused on the generation of 2D and 3D human model.
2D human brain models. Building on a recently optimized protocol for differentiating iPSCs into glutamatergic neurons (iGluNeurons; Servetti et al., 2025), we generated iGluNeurons from iPSCs derived from patients affected by paroxysmal disorders caused by mutations in PRRT2 and SYN1. At the molecular level, PRRT2 influences sodium channel activity, playing a key role in regulating neuronal excitability, while SYN1 is involved in synaptic vesicle trafficking and neurotransmitter release at the presynaptic terminal. Mutations in these genes are associated with paroxysmal kinesigenic dyskinesia and infantile epilepsy (PRRT2) and focal seizures triggered by water contact (SYN1). iGluNeurons carrying PRRT2 mutations exhibited hyperexcitability, which we successfully rescued by applying subunit-specific sodium channel blockers. Meanwhile, iGluNeurons carrying SYN1 mutations are currently being characterized, with a focus on maturation and developmental differences.
3D human brain models. We have developed a novel Long-Term Air-Liquid Interface Culture protocol for generating hippocampal organoids and establishing recordings on HD-MEA, starting from DIV60 up to DIV180. Our results demonstrate that hippocampal organoids lack a necrotic core and exhibit functional, highly organized neural networks. Furthermore, we successfully integrated microglia into the hippocampal organoids. Notably, we observed a decrease in neuronal activity, which aligns with findings in mouse models of microglial deficiency, where an increase in activity has been reported.
These models could provide insights into pathophysiological mechanisms in human context and potential treatments.

Speaker: Martina Servetti (Institute Italian of Technology)

18:30 ATM-Knockout human neural progenitor cells: a powerful platform for identifying therapeutic targets in Ataxia-Telangiectasiac

Ataxia-telangiectasia (A-T) is a rare, autosomal recessive disorder affecting multiple systems, characterized by progressive cerebellar atrophy, neurodegeneration, and cognitive decline. A key neuropathological feature is the loss of Purkinje and granule cells in the cerebellum. Mutations in the ATM gene, located on chromosome 11q22-23, underlie A-T. This gene encodes ATM, a serine/threonine kinase involved in DNA repair and cell cycle regulation, impacting numerous substrates.

Our prior work has shown that human induced pluripotent stem cell-derived neural progenitor cells (hNPCs) possess self-renewal and multipotency, differentiating into both functional neurons and glial cells. Using CRISPR/Cas9 gene editing, we generated ATM-deficient hNPCs. Initial findings indicate that these ATM knockout hNPCs exhibit reduced proliferation and/or survival compared to wild-type hNPCs. Current investigations are focused on characterizing the ATM-deficient hNPCs, specifically examining established signaling pathways linked to ATM. We are also exploring the functional impact of ATM loss on hNPCs and their differentiated neuronal and non-neuronal progeny.

While the role of ATM in DNA damage repair and cell cycle control has been extensively studied in A-T patients and animal models, the precise molecular mechanisms driving neuronal and glial dysfunction in this disease remain to be fully elucidated. Given the absence of a targeted therapy for A-T, our research aims to identify novel cellular pathways disrupted by ATM deficiency in neural cells. This work seeks to uncover potential therapeutic targets for pharmacological intervention. Ultimately, ATM-deficient hNPCs offer a promising in vitro model to advance our understanding of A-T pathophysiology and facilitate drug discovery efforts.

Speaker: Emanuela Pessolano (University of Piemonte Orientale)

18:30 B7-H3/CD276 in Brain Tumors: A Paradoxical Role in Tumor Initiation Over Progression

B7-H3, also known as CD276, is an immune checkpoint molecule and has emerged as a significant player in cancer tumorigenesis across multiple malignancies, including the pediatric brain tumors medulloblastoma, gliomas and ependymoma. Its exact role in tumor formation, however, is unclear. Our investigations demonstrated high expression levels of B7-H3 in brain tumors, irrespective of tumor type or subgroup. To assess its functional relevance, we generated B7-H3 knockout medulloblastoma cells; however, loss of B7-H3 did not impact proliferation in vitro or tumor formation in vivo. This finding suggests that B7-H3 may not be critical for maintaining established tumors in this context, but may play a role in tumor initiation. We observed B7-H3 expression in early developmental stages of the mouse brain and in human neuroepithelial stem cells (NESC), indicating a possible role in normal brain development. In a syngeneic induced mouse model of ependymoma, B7-H3 knockout combined with ZFTA-RELA fusion overexpression failed to generate tumors, suggesting indeed a role for B7-H3 in tumor initiation.
Our findings collectively suggest a nuanced function of B7-H3 in cancer development. While its high expression in established brain tumors across various types implies a common malignancy feature, the lack of effect on tumor growth upon knockout in medulloblastoma indicates that its role may be context-dependent or compensated in established tumors. The inhibition of tumor formation in B7-H3 abscence during oncogene-induced transformation highlights its potential importance in the early stages of tumorigenesis, either by a supportive role of B7-H3 in tumorigenesis or by depleting tumor initiating cells.
In conclusion, our studies reveal a complex picture of B7-H3's role in brain tumor biology, emphasizing its potential involvement in tumor initiation and underscoring the need for further investigation, which we aim to use NESCs to module tumor initiation and progression to discover B7-H3 role in brain tumor initiation.

Speaker: Patricia Benites Goncalves da Silva (German Cancer Research Center)

18:30 Beyond Cerebrum and Neurosurgery: A Systematic Review on Chiari Malformation Type I integrating molecular and behavioral methodologies

Chiari Malformation Type I (CM1), characterized by cerebellar tonsillar herniation exceeding 5 mm through the foramen magnum, represents the most common subtype of Chiari Malformations, a group of developmental disorders involving posterior fossa deformities. Despite its prevalence characterized by a strong female predominance, its described neuroanatomical features, neurological symptoms, and neurosurgical treatments, CM1’s is often underdiagnosed because of interpersonal and sociodemographic factors, leading to disparities in treatment access. Additionally, classical manifestations of CM1 are often accompanied by the occurrence of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD), which are currently poorly considered as well as the genetic and molecular underpinnings which remain largely unknown.
This systematic review integrates findings from 50 original research articles, sourced from PubMed and Medline databases, and explores CM1 through neuropsychological, neuropsychiatric, genetic, and genomic research methodologies. We aim to bridge traditionally siloed research domains, proposing a discipline-overarching understandings of CM1, highlighting the cerebellum’s non-motor functions by deconstructing the genetic, familial and environmental factors through “CM1 Equifinality” in the occurrence of this disorder, investigating its frequent presence in both neurodevelopmental and psychiatric conditions, syndromes and its presentation along cognitive impairment and emotion dysregulation. Thereby, we address key research gaps including CM1’s multifactorial pathogenesis and population-level outcomes.
With this work we want to emphasize the critical need for psychiatric monitoring, neuropsychological interventions, and psychotraumatological implications in high-risk pediatric populations for CM1 within the general population. We want also to highlight how CM1 represents a unique model of study integrating developmental biology, cognition, and psychiatry through a cerebellar lens. Lastly, in light of the recent advances in patient-derived models foreseeing the use of 3D in vitro models to recapitulate cellular and molecular features of ASD and ADHD (both CM1 comorbidities), we propose cerebellar organoids as potential tool to investigate the neurogenomic bases of this disease.

Speaker: Irmak Oezdil (TU Dresden (Technische Universität Dresden))

18:30 Causal gene regulatory network reconstruction highlights cell type-specific interactions in neurodevelopment

Brain organoids can provide major insights into neurodevelopment and its disruption in neuropsychiatric disorders. To capture how individual chromatin remodellers and their dosage shape developmental trajectories, we profiled by single-cell RNA-seq brain organoids derived from a group of paradigmatic neurodevelopmental disorders (NDDs) caused by mutations in chromatin regulators. Currently existing tools for gene regulatory network reconstruction largely rely on co-expression. Advanced methods eventually constrain co-expression inference by considering only interactions backed by TF-gene interactions derived from single-cell ATAC or motif-enrichments at known regulatory regions. These approaches are limited since they consider only TFs as regulators. We repurposed a new algorithm, based on hierarchical Bayesian inference, to single-cell RNA-seq data, to allow for the reconstruction of causal relationships in dynamic systems. This approach is agnostic to ATAC- or motif-based prediction, thereby overcoming the TF-centric view of canonical tools. Our cell state-specific causal regulatory networks highlight known regulators of neurodevelopment as key hubs, as well as new candidate regulators of neuronal differentiation and cell identity maintenance. We benchmarked our new approach with existing tools and linked the activity of newly defined hubs to human cognitive-behavioural phenotypes. Collectively, by overcoming co-expression-based biases and identifying regulators beyond direct transcriptional rewiring, we provide a new approach for hypothesis generation and identify novel candidate mediators of human neurodevelopment.

Speaker: Lorenzo Basile (Fondazione Human Technopole)

18:30 Cell-based modulation of the brain’s immune response

Alzheimer's disease (AD) is the most common neurodegenerative disorder, yet current therapies have limited efficacy. Immune responses, primarily from microglia and regulatory adaptive immune cells, are closely linked to the pathological changes in amyloid-beta and tau observed in AD. We previously demonstrated that microglia in adult mice can be replaced through bone marrow transplantation (BMT), leading to improved cognitive and behavioral features in an AD model. However, the conditioning regimen was highly toxic, and single-cell analysis capabilities were limited at the time. Recent advances in reducing BMT toxicity have led us to explore a safer protocol, which has shown strong tolerability in older patients with and without cancer, potentially enabling cell-based modulation of the brain’s immune landscape in AD.

Methods: Wild-type recipient mice (C3B6F1) were conditioned with anti-thymocyte serum (ATS), low-dose total lymphoid irradiation (TLI), total body irradiation (TBI), and the CSF1R inhibitor PLX3397 before transplantation with whole bone marrow (BM) from sex- and age-matched Balb/c donors. CD45+ cells from the brain were sorted into donor and recipient populations based on haplotype and analyzed at the single-cell level.

Results: We observed sustained multilineage donor mixed chimerism in peripheral and hematopoietic tissues three months post-transplant, with similar chimerism levels detected in the brain. Single-cell RNA sequencing revealed a comprehensive immune map, capturing diverse microglial populations, including border-associated microglia (BAM) clusters, as well as T cells (CD4+ and CD8+), NK cells, and B cells. BM-derived microglia displayed distinct transcriptional profiles from resident microglia, and ongoing work aims to elucidate their phenotypic and functional differences.

Conclusions: Our results prompt further testing of this approach to modulate the immune environment in neurodegenerative mouse models and assess whether this therapy can delay or prevent neurodegeneration in AD.

Speaker: Amalia Perna (Stanford University)

18:30 Cell-type-specific proteome dysregulation in Fragile X Syndrome brain organoids

Fragile X Syndrome (FXS) is a rare neurodevelopmental disorder, representing a leading cause of inherited intellectual disability and monogenic cause of autism spectrum disorders. It results from abnormal CGG repeat expansion in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene, leading to its silencing and loss of FMRP, an RNA-binding protein crucial for synaptic plasticity and dendritic spine architecture. While FMR1 methylation occurs in the first trimester, the precise timing of FMRP loss and affected cell types remain unclear. Using a 3D human cortical development model derived from naïve induced pluripotent stem cells in a hypomethylated pluripotency state, we successfully recapitulated the FMR1 epigenetic dynamics observed during the early development of FXS patients’ brains. Proteomic analysis revealed that the number of dysregulated proteins in FXS increases along differentiation, half of them are direct targets of FMRP, and 1/5 are associated with autism. Most dysregulated proteins are involved in synaptic physiology and localized postsynaptically. Deconvolution of bulk proteomic with single cell RNAseq data showed that protein alterations are mainly in neuron subtypes, astrocytes and microglia. To dissect the cell type-specific contributions to FXS pathogenesis, we combined our model with a metabolic non-canonical amino acid (ncAA) tagging strategy. We generated transgenic FXS-derived naïve hiPSCs expressing the mutant tRNAse MetRSL274G under specific neuronal or glial promoters. The ncAA is incorporated into proteins which can be further coupled with different tags via click chemistry for direct visualization, quantification, or enrichment of the nascent proteome. We successfully validated the cell lines’ ability to differentiate into human brain organoids and to label proteins in specific cell-types. This system provides access to a previously inaccessible time window of FXS disease, allowing us to discover when and where FMR1 silencing occurs in a human model and new early-stage markers for potential pharmacological targets.

Speaker: Carmela Ribecco (Università degli studi di Padova)

18:30 Characterization of human healthy i3 lower motor neurons exposed to CSF from ALS patients stratified by UNC13A and C9ORF72 genotype

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motor neurons. Neurodegeneration in ALS might be driven by proteotoxicity or neuroinflammation, which have also been proposed to be promoted by toxic components of the cerebrospinal fluid (CSF).
We investigated the possible toxicity of ALS CSF on healthy induced pluripotent stem cells (iPSC)-derived integrated, inducible, and isogenic lower motor neurons (i3LMNs). CSFs were obtained from ALS patients homozygous for the risk UNC13A rs12608932 single nucleotide polymorphism (CC) and for the corresponding major allele (AA), ALS patients with C9ORF72 hexanucleotide repeat expansion, and individuals affected by normal pressure hydrocephalus, a non-degenerative condition, as controls (ND). Chronic sodium arsenite (ARS) treatment was used as positive control of oxidative stress.
We found that 10 % ALS CSF treatment for 48 h was not sufficient to induce significant alterations in viability, autophagic flux, axonal degeneration, DNA damage, and Golgi apparatus integrity in healthy i3LMNs, in contrast to ARS treatment. Only UNC13A CC CSF significantly increased protein aggregation and Golgi apparatus fragments dimension. RNA-sequencing revealed that all ALS and ND CSFs induced expression changes of few genes, while chronic ARS deregulated the expression of thousands of genes, mostly involved in inflammation and synapse biology.
In this work, we demonstrated that in our experimental settings only UNC13A CC CSF induced some ALS-associated pathological features in healthy i3LMNs. Further studies will be required to elucidate the mechanistic link between the risk UNC13A genotype and CSF composition and toxicity.

Speaker: Valeria Casiraghi (Università degli Studi di Milano)

18:30 CHD2 Dosage Links Autolysosomal Pathway to Cortical Maturation in Disease and Evolution

Cortical development is relatively slower in humans compared to other species. Here, we investigated whether our neurodevelopmental tempo also differs from that of our closest extinct relatives, the Neanderthals and Denisovans. To tackle this, we selected, through comparative paleogenomics analyses and machine learning, a sapiens-specific single nucleotide variant virtually fixed in contemporary populations and located within an enhancer region active during early cortical development. The single nucleotide variant was predicted to affect the expression of CHD2, a chromatin remodeler critical for neural development, for which both haploinsufficiency and overexpression are associated to severe neurodevelopmental conditions, thereby establishing the importance of optimal CHD2 dosage for physiological neurodevelopment. Using patient-derived and genome-edited models, we uncovered an evolutionary gradient in autolysosomal dynamics, enhanced by the ancestral allele and impaired by CHD2 deficiency, as well as mirroring differences in somatodendritic complexity and spontaneous neuronal activity. Through deep phenotyping of cortical organoids and neuronal cultures, we uncovered the CHD2-dosage dependent gene regulatory networks modulating the autolysosomal pathway and ultimately impacting the timing of developmental programs, acquisition of neuronal functional properties and circuit maturation. Finally, we traced the mechanistic impact of the allele to an estrogen-dependent rewiring of CHD2 regulation, establishing the significance of non-coding variants for the evolution of modern humans.

Speaker: Oliviero Leonardi (HUMAN TECHNOPOLE)

18:30 Cholesterol as a synaptogenic factor involved in the development of proper synaptic structure in an hiPSC-derived neurons model of Rett syndrome.

Rett syndrome is a neurodevelopmental disorder in which the involvement of astrocytes is recognized. Previous work from our laboratory underlined how synaptotoxic factors, such as IL-6, aberrantly expressed by MEPC2 knock-out astrocytes lead to detrimental effects on the synapses in development. Moreover, astrocyte role during development is to provide synaptotrophic factors which could be lacking in diseased MECP2 null or heterozygous environment. With cholesterol being one of the most important synaptogenic factors produced in central nervous system only by astrocytes we focused our attention on its production and distribution among the cell types involved. In our study we were unable to found differences in secreted cholesterol in the medium of co-cultured astrocytes and neurons thus indicating that, if impaired, a different cholesterol delivery mechanism is involved. Nonetheless, we found deregulated gene expression of cholesterol biosynthetic and trafficking pathway in MECP2-null astrocytes. Starting from the hypothesis that cholesterol flow from astrocytes to developing neurons is impaired in Rett syndrome, we are investigating its effect on synaptic compartment in terms of structure and synaptic maturity taking advantage of hiPSC-derived neurons. If proven to be the case that synaptic abnormalities are due to misregulated cholesterol synthesis or delivery our aim would be to evaluate the same results in an in vivo model as a preclinical validation for a feasible mechanism for therapeutic intervention.

Speaker: Fabio Biella (University of Milan)

18:30 Combining Human Stem Cells and Cortical Organoid Technologies to Explore the roles of the Transcription Factor FOXP1 in Neuroimmune Development

FOXP1 is a transcription factor critical for human brain function from early development. Indeed, heterozygous mutations in this gene cause FOXP1 syndrome, a rare neurodevelopmental disorder characterized by language impairment, intellectual disabilities, and autistic traits [1]. FOXP1 levels are affected also in Huntington disease and cancer. However, mouse models of FOXP1 haploinsufficiency or deficiency do not reveal major cortical phenotypes, suggesting human-specific pathogenic mechanisms.
To start to gain insight into relevant cell types, we analyzed its expression in human brain using human pluripotent stem cell (hPSC)-derived forebrain organoids (hCOs) [2]. IF analysis and gene expression data revealed FOXP1 expression in neural progenitor cells, excitatory cortical neurons and microglia, the brain's resident immune cells, which play essential roles in neurodevelopment and pathology. These results suggest the involvement of multiple cell types.
To start to gain insight into possible cell type specific targets, we have performed a FOXP1 CUT&RUN experiment [3] in hCOs enriched with neural progenitors. Additionally, we will then present several strategies that we are developing to functionally explore FOXP1 functions across cell types. We are employing the CRISPR-Cas9 system to knock out (KO) FOXP1 in hPSC lines to generate isoform-specific knock-out lines. Finally, we are developing an in vitro neuroimmune assembloid (NIA) model by engrafting hPSC-derived microglia progenitors into hCOs, allowing us to explore how FOXP1 function in microglia development and function.
In conclusion, our study underscores the utility of hPSCs and hCOs technologies for functional studies of human brain development and disease, particularly these approaches will provide valuable insights into the role of FOXP1 in brain development and its link to human brain dysfunctions.

Speaker: Sara Elisabetta Barilà (Università degli Studi Milano-Bicocca)

18:30 COMPASS: Comparative Organoid Mapping Platform for Assessment by Single-cell Similarity

"In the last decade, the widespread adoption of hiPSC technologies and their differentiation into brain organoids (BOs), together with the massive use of single-cell (SC) transcriptional profiling, has provided a significant boost to the study of human brain pathophysiology. This progress has enabled time-resolved characterization of cellular players and their interactions, which is particularly relevant to developmental neurobiology - a major field of application for BOs. Despite the expansion of protocols leading to increasingly accurate and multifaceted BO models, the dissemination of streamlined methods to assess their fidelity to primary tissues at SC level and with temporal attention is still lagging behind, ultimately slowing BOs optimization and translational potential.
Although recent efforts significantly contributed to the field (Fleck et al. 2021, He et al. 2024), releasing pre-trained models and methods focused on out-of-sample label transfer, the landscape remains sparse compared to the pace of experimental advances. Furthermore, these models do not guarantee sub-regional and temporal mapping capabilities directly onto fetal brain transcriptomes, leaving space for additional complementary tools.
Here, we present a lightweight resource designed to enable assessment of BO cultures profiled at SC resolution, whether generated using novel or established protocols. The data foundation of this project is based on primary samples collected from 14-previous studies and profiled at SC-level, from four major brain areas: cortex, cerebellum, thalamus and subpallium, six cortical regions: prefrontal, temporal, somatosensory, motor, parietal and visual, and 26 timepoints (spanning from post-conceptional week 5 to 36). Alongside the data core, the framework we propose encompasses a substantial set of functions and methods to carry-out in vivo – in vitro comparative analysis with tunable resolution focus, ranging from cell type to sub-region (cortical areas), region and developmental stage. The adoption of metacell aggregation and objective-aware reductionism in both building and querying methods allowed us to distill information to score similarities between BOs and primary samples, while minimizing computational burden, limiting confounding effects and keeping a low entry barrier for its utilization."

Speaker: Davide Castaldi (human technopole)

18:30 CRISPR-CAS9-BASED FUNCTIONAL INVESTIGATION OF THE “DARK GENOME” IN SEARCH OF PUTATIVE DOWNSTREAM EFFECTORS OF SOX2 IN NEURODEVELOPMENTAL DISEASE

Mutations in the SOX2 gene lead to defects in the development of multiple brain regions, causing blindness, intellectual disabilities, and seizures. SOX2 is a transcription factor that regulates many genes, some of which, when mutated, are involved in other neurodevelopmental disorders (NDDs).
Using RNA sequencing, we identified over a thousand genes that are downregulated following Sox2 deletion in neural stem cells derived from the developing mouse brain. Many of them have human homologs, and many belong to the ""T-Dark"" gene category, a group of genes of mostly unknown function.
We focused on 122 human T-Dark genes, whose expression is most reduced in Sox2-deleted mouse brain-derived neural stem cells. To explore their role in neurodevelopment, we applied a screening strategy based on growing in vitro human brain organoids derived from a previously established human embryonic stem cell line. This screening uses the CRISPR Lineage Tracing at Cellular Resolution in Heterogeneous Tissue (LICHT) technique, combining CRISPR-Cas9 mutagenesis and a dual barcode method. This approach allows us to identify mutated genes in underrepresented cell clones within brain organoids through genomic sequencing.
We designed and cloned a T-Dark guideRNAs library, and a control library that includes SOX2 and some of its known NDD-related targets. The libraries were then used to transduce the stem cells, from which brain organoids were differentiated. Afterwards, the organoids were dissociated to single cells, and DNA was extracted and prepared for sequencing, presently ongoing. Initial results will be discussed.
In parallel, we worked on optimizing this technique to make it more accessible and efficient for other laboratories.
We hypothesize that these T-Dark genes may act as functional effectors of SOX2. Identifying their neurodevelopmental roles will provide new insights into the SOX2-dependent regulatory network, not only in SOX2-related pathologies but also in other NDDs, with potential therapeutic perspective.

Speakers: Giorgia Pozzolini (University of Milano-Bicocca), Silvia Kirsten Nicolis (Università degli Studi di Milano Bicocca, Dipartimento di Biotecnologie e Bioscienze)

18:30 CROPseq-multi: A versatile solution for scRNA-seq based pooled multiplexed CRISPR screening in stem cell-derived neural models

Pooled CRISPR screenings are powerful functional genomics tools to assess how perturbing gene expression alters cellular fitness, differentiation, and transcriptomic landscapes in complex biological models. When studying systems that recapitulate brain development, it is crucial to resolve changes in developmental trajectories and cellular dynamics at single-cell resolution, as diverse neural cell types may respond differently to genetic perturbations. While tools like CROP-seq and Perturb-seq have advanced our understanding of gene effects on complex neurobiological traits, they primarily focus on single-gene perturbations. However, multiplexed CRISPR screening is essential for studying combinatorial interactions, yet current methodologies largely remain incompatible with pooled approaches requiring mRNA-embedded barcodes, thus limiting the possibility of pairing multiple genetic perturbations with morphological and transcriptome shifts at single-cell resolution.
Here, we present CROPseq-multi, a new construct enabling multiplexed Cas9-based perturbations while preserving mRNA barcoding of sgRNAs, allowing scRNA-seq and spatial in situ sequencing of perturbed cells. To validate this approach, we generated a lentiviral library delivering two sgRNAs per cell targeting 42 genes involved in neural progenitor differentiation. Additionally, we performed single and double knockout of GSK3A/B to investigate both individual and combinatorial perturbations of this key regulator of neural progenitor proliferation. Clonal barcodes were incorporated into the sgRNA sequence to track lineage dynamics throughout differentiation. We then transduced the library into stem cells and induced Cas9 expression during differentiation, challenging neural patterning with pooled gene knockouts.
In a two-phase pilot study, we benchmark the compatibility of CROPseq-multi with different sequencing strategies using both 3’ short- and long-read methods and 5’ targeted capture, ensuring compatibility with commercially available platforms without requiring modifications to sgRNA scaffolds.
This versatile solution provides the field with a new all-in-one vector that enhances functional genomics in neuroscience and beyond, unlocking new avenues for G × E studies and disease modelling at single-cell resolution.

Speaker: Manuel Lessi (Fondazione Human Technopole)

18:30 Deciphering the pathological mechanisms of Cohen Syndrome during cortical development

Cohen syndrome (CS) is a rare autosomal recessive disorder caused by a biallelic mutation in the VPS13B gene. It is a heterogeneous genetic disorder characterized by microcephaly, hypotonia, intellectual disability, neutropenia, and retinal degeneration. VPS13b is localized at the Golgi apparatus and is essential for the maintenance of organelle architecture. To understand how VPS13b mutation affects cortical development, we use cortical organoids as an in vitro model. We are electroporating a Sh construct against VPS13B at D39 and analyzing after 4 div or 7 div. By analyzing the samples, we were able to see defects in migration and an increase in TBR2+ intermediate progenitors. Further, we generate cortical organoids from iPSCs reprogrammed from CS patient’s fibroblast. Initial results show defects in Golgi integrity and endosomal vesicles. We are following up on these observations to understand the effects of these defects on the secretory pathway, cortical development, and neuronal maturation. Moreover, we are using in utero electroporation, electroporating Sh Vps13b in the cortex of mouse embryos at E15.5 and analyzing postnatally at P5 to investigate the role Vps13b has on neurogenesis in vivo. We intend to combine different omics approaches, live imaging on organoids and clearing to investigate the underlying molecular mechanism VPS13b exerts during cortical development.

Speaker: salma amin (Fondazione human technopole, milan, italy)

18:30 Deciphering the Role of WWOX in Oligodendrocyte Maturation and Remyelination

Myelination is essential for neuronal conductivity and brain function, yet the molecular mechanisms regulating this process remain unclear, limiting therapeutic advancements for demyelinating disorders. Our study aims to investigate the role of the WW domain-containing oxidoreductase (WWOX) gene in CNS myelination. We previously showed that murine Wwox deletion leads to hypomyelination and decreased mature oligodendrocyte numbers. Here, we examine how WWOX functions in oligodendrocyte progenitor cells (OPCs) as a key regulator of their differentiation and myelination. Using human embryonic stem cells (hESCs), we previously demonstrated that WWOX depletion disrupts the generation of mature oligodendrocytes (mOLs) in an oligocortical spheroid model, underscoring its crucial role in OPC differentiation. To further explore this, we generated an Olig2-Cre mouse model to delete Wwox specifically in OPCs. Our findings reveal that WWOX deficiency impairs OPC differentiation, leading to defective myelination and hindered remyelination following cuprizone-induced demyelination. Furthermore, single-nucleus RNA sequencing of the corpus callosum from wild-type and OPC-specific Wwox knockout mice identified novel WWOX-regulated effectors involved in OPC differentiation and proliferation during remyelination. Extending our findings to human pathology, we analyzed single-cell RNA sequencing data from chronic demyelinated lesions in multiple sclerosis (MS) patients and detected reduced WWOX expression in both oligodendrocytes and neurons. By integrating in vivo and in vitro models with transcriptomic analysis, our study provides new insights into WWOX’s pivotal role in CNS myelination and remyelination. These findings bridge a critical knowledge gap and highlight potential molecular targets for therapeutic strategies aimed at enhancing neural repair and promoting functional recovery in demyelinating disorders.

Speaker: Baraa Abudiab (Hebrew University of Jerusalem)

18:30 Decoding the interplay between morphology and stemness in glioblastoma: a neurodevelopmental perspective

Glioblastoma (GBM) is an aggressive and highly heterogeneous brain tumor, making treatment development particularly challenging. A key contributor to GBM's heterogeneity is glioblastoma stem cells (GSCs), which possess critical properties such as self-renewal, plasticity, and resistance to therapy. These characteristics are pivotal in tumor progression and recurrence, highlighting the need for a deeper understanding of GSC stemness mechanisms to improve therapeutic strategies. GBM cells utilize neurodevelopmental pathways, and GSCs share transcriptomic similarities with neural stem cells, particularly basal or outer radial glia (bRG). These bRG-like cells exhibit morphological diversity, which is thought to regulate cellular processes like proliferation and migration. Our recent research suggests that GSC morphology is strongly associated with their functional properties, leading us to investigate this further.
Using patient-derived 2D and 3D in vitro models, we identified GSC morphology as a novel and significant aspect of GSC heterogeneity. While bRG cells are categorized by number of cellular protrusions, GSCs displayed a much broader range of morphological variation both between and within individual patients. Specifically, GSCs exhibited differences in protrusion length, thickness, cell-cell network formation, and overall cell flatness. Based on these morphological features, we classified GSCs into distinct morpho-classes, which displayed different self-renewal potential in vitro. Transcriptomic profiling revealed that morpho-classes with higher clonogenic potential were enriched in molecular pathways related to tumor growth and aggressiveness. These pathways are also involved in neurodevelopmental processes, such as maintaining bRG cell properties, promoting neuronal process growth, and morphogenesis.
In conclusion, our study demonstrates that GSC morphology and stemness are intricately linked, likely through neurodevelopmental morphoregulatory pathways. These findings open the door to targeting morphoregulators as a potential therapeutic strategy to limit GBM aggressiveness.

Speaker: Stefania Faletti (Fondazione Human Technopole)

18:30 Dissecting the pathomolecular mechanisms of Prr12 gene loss leading to neurodevelopmental and eye abnormalities

PRR12 (Proline rich 12) gene is encoding for a nuclear protein but its function in chromatin regulation remains elusive. We collected preliminary evidence that PRR12 can interact with the transcription factor SOX2, a pivotal regulator of neural stem cell identity and self-renewal. In humans, heterozygous loss-of-function mutations cause the Neurocular syndrome. The aim of our project is to uncover the role of PRR12 and delve into the molecular consequences of its disruption in an in vitro and in vivo setting. We started by generating human embryonic stem cell (hESC) lines carrying targeted mutations through the CRISPR/Cas9 system. Targeted nucleotide insertions and/or deletions were introduced into the sequence leading to premature stop codons with consequent nonsense-mediated mRNA decay. As such, we have established a set of isogenic hESC lines carrying heterozygous or homozygous mutations in the PRR12 gene. Next, we differentiated these cells into neural progenitors and cortical neurons, two cell types highly affected in the disease. Interestingly, Prr12 mutant neural progenitor cells (NPCs) showed a higher cell proliferation rate accompanied by an impairment in neuronal differentiation and maturation. Prr12 mutant neurons displayed abnormal morphology with impaired neurite growth. Molecular and genomics studies are in progress to trace the underlying mechanisms of these significant deficits. In a parallel effort, we generated a transgenic mouse line carrying a Prr12 floxed allele, by flanking exon 6 with loxP sequences through homologous recombination in ES cells. Prr12 floxed mice have been crossed with mice expressing the Cre ubiquitously or only in the CNS. Full Prr12 knock-out mice are lethal perinatally suggesting that Prr12 plays a fundamental role in tissue functions. We are currently analyzing the cellular defects caused by Prr12 gene loss in both mouse models.

Speaker: Irina Cutei (San Raffaele Scientific Institute)

18:30 Distinct transcription factor networks specify neocortical versus hippocampal regionalization during early telencephalic development

The mammalian cerebral cortex arises from neocortical and hippocampal primordia (Ncp and Hcp respectively), which initially display cellular composition similarities and several molecular markers. Their trajectories diverge significantly during development, resulting in functionally distinct structures. The molecular mechanisms orchestrating the progressive regionalization of the Ncp and Hcp remain poorly understood. Using single-cell multiomic analysis, we demonstrate significant differences in the Ncp and Hcp as early as E12.5, regarding chromatin accessibility and transcriptional profiles. In particular, we identified differentially accessible enhancer regions in apical progenitors from Ncp and Hcp harboring both known and previously undescribed binding motifs for key transcriptional regulators. Ongoing work is focused on functionally validating some of these enhancer regions that modulate gene expression and may drive the gradual diversification of the Ncp and Hcp. Our findings provide critical mechanistic insights into early chromatin regulation underlying region-specific transcriptional programs that result in the functional divergence of the neocortex and hippocampus.

Speaker: Faye Chong (Helmholtz Zentrum München)

18:30 Elucidating SYNGAP1 Isoform Functions in Human Neurodevelopment Using Cerebral Organoids

SYNGAP1, encoding Ras/Rap GTPase-activating protein, is a critical gene involved in synaptic signaling and neurodevelopment. Mutations in SYNGAP1 are associated with intellectual disability and autism spectrum disorder (ASD). However, the specific functions of its multiple isoforms, generated by alternative splicing and transcription start sites, remain poorly understood. Current methods for studying SYNGAP1 functions rely on animal models, which do not fully recapitulate human neurodevelopment. This project aims to elucidate the roles of different SYNGAP1 isoforms using human cerebral organoids as an alternative to animal models. Cerebral organoids, derived from pluripotent stem cells, offer a promising 3R approach by mimicking the human brain architecture and functionality. We will employ cutting-edge techniques including single-cell RNA sequencing, CRISPR-based gene perturbation, and BaseScope in situ hybridization to comprehensively profile SYNGAP1 isoform expression, manipulate their levels, and visualize their spatiotemporal distribution across different developmental timepoints: 15, 30, 60 days, roughly equivalent to 4, 10, 18 post-conceptional weeks of human development in vivo. By developing this innovative 3D model system, we aim to uncover isoform-specific roles of SYNGAP1 in human brain development. Our approach will not only advance mechanistic understanding of SYNGAP1 biology, but also exemplify how cerebral organoids can serve as a powerful alternative to animal use.

Speaker: Ivanna Kupryianchyk-Schultz (MDC BIMSB)

18:30 EPM1 Epilepsy: Intrinsic and Extrinsic Mechanisms of Defective Neural Cell Fate

Progressive Myoclonus Epilepsy Type I (EPM1) is a rare form of epilepsy, caused primarily by mutations in the CSTB gene.
Our previous studies using cerebral organoids derived from somatic cells of EPM1 patients (EPM1-CO), have shown that CSTB is implicated in human cortical development and plays a role in extracellular signaling, cell proliferation, interneuron recruitment and synapse physiology.
Here we demonstrate that EPM1 COs display altered electrophysiological activity compared to control COs suggesting a disrupted excitatory/inhibitory balance. In order to dissect interneuron trajectory and maturation, we focused on ventrally patterned COs (vCO), which give rise to interneurons. EPM1-vCOs reveal abnormal neural cell fate specification, characterized by a shift toward dorsal neuron identities.
CSTB is secreted in extracellular vesicles during neurodevelopment and the levels of functional CSTB impact surrounding cells in a cell-non-autonomous way.
In EPM1-vCOs, we identify pathological alterations in extracellular vesicle biogenesis and cargo, including aberrant Sonic Hedgehog (SHH) signaling. The altered SHH signaling via EVs is likely to be a critical factor in influencing neural differentiation and fate, thereby playing a significant role in the pathogenesis of EPM1.
Our findings suggest that both intrinsic and extrinsic factors contribute to EPM1 pathology and highlights potential therapeutic strategies mediated by extracellular vesicles.

Speaker: Rossella Di Giaimo (Department of Biology, University of Napoli Federico II, Italy)

18:30 Exome sequencing identifies human knockouts for 34 candidate genes plausibly linking with severe neurodevelopmental manifestations.

Background:
Consanguineous unions enhance the risk of homozygous loss-of-function (LoF) variants in the subsequent generations. Hence, highly endogamous populations like Pakistan provides an opportunity to identify naturally occurring human knockouts (hKOs) to further understand the complexity of human genome. We designed this study to identify novel recessive candidate genes underlying neurodevelopmental disorders (NDDs) and to better understand the involved pathobiology.
Methods:
We recruited cases with NDDs, performed clinical evaluation, exome sequencing (ES), reverse phenotyping, and disease modelling to understand disease pathomechanisms.
Results:
We performed ES in 271 unrelated consanguineous Pakistani families for genetic characterization. Our cohort predominantly showed Delayed developmental milestones (90%), speech anomalies (75%), cognitive impairment (70%) with the history of seizures (65%). Less common features included peripheral neuropathy, ataxia, muscle atrophy, and skeletal curvature anomalies. Concordant with the published reports, we achieved a genetic diagnosis (pathogenic/ likely pathogenic variants in a phenotype-matched OMIM gene) in 76 (28%) families. We identified hKOs for 34 genes from 34 families (12%), of which, we are currently pursuing DDIAS, RELCH, and YAF2 for detailed investigations to understand the underlying pathobiology. Further, we observed that 37 (13%) families have VUS in a phenotypically relevant known gene. Fifteen families (5%) carry missense VUS in a gene wherein we have preliminary evidence of candidacy based on relevant biological pathways, functions, and matching cases in the GeneMatcher. Cases from 100 (37%) families harboured VUSs in one or more uncharacterized genes, six remained inconclusive, while three are under process.
Conclusion:
Our cohort of hKOs will serve as a resource to understand the role of involved genes in health and disease. Further, our study will potentially aid in establishing a genotype to phenotype correlation and add to the list of genes critical for neurodevelopment and improve the rate of disease diagnosis.

Speaker: Ambrim Fatima (Aga Khan University)

18:30 Exploiting hiPSC-derived neural stem cells with a radial glia-like signature and favorable long-term in vivo safety profile for CRISPRa-engineering in demyelinating disorders

Human somatic neural stem cells (NSCs) are emerging as promising advanced therapy medicinal products for treating neurodegenerative and demyelinating disorders. However, challenges remain, including the large-scale production of donor cells under GMP conditions and the necessity of immunosuppressive regimens in allogeneic transplant settings. Human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NSCs) offer a potential alternative but comprehensive studies investigating their identity and safety are still limited. Significantly, the hostile environment of demyelinating conditions, such as Multiple Sclerosis, could negatively impact transplanted NSC, highlighting the need for innovative approaches to enhance their therapeutic potential.
We performed genome-wide omics analyses, including RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and single-cell RNA sequencing (scRNA-seq), to compare hiPSC-NSCs with hiPSCs, human fetal NSCs (hfNSCs), and glioblastoma stem cells (GSCs) to examine their transcriptional and epigenetic signatures and assess their NSC identity and safety profile. Our analysis revealed that hiPSC-NSCs acquired a transcriptomic profile that represents an intermediate middle-radial glia (MRG) and late-radial glia (LRG) state, resembling primitive ventral RG (vRG) isolated from the human fetal brain. Notably, hiPSC-NSCs share key transcriptional and epigenetic characteristics with hfNSCs while significantly diverging from GSCs. Long-term transplantation in immunodeficient mice showed robust and stable engraftment of hiPSC-NSCs, with predominant differentiation into glial cells and no tumor formation. We are investigating whether hiPSC-NSCs engineered through CRISPR activation (CRISPRa) to overexpress therapeutic genes can promote oligodendrocyte maturation, immunomodulation, and neuroprotection. This approach aims to counteract the hostile environment of Multiple Sclerosis and address the remyelination failure.
These findings offer valuable transcriptional and epigenetic reference datasets that aid in defining the maturation stage of NSCs derived from different hiPSC sources. Additionally, the results demonstrate the long-term safety of hiPSC-NSCs. The potential to enhance their therapeutic effects through CRISPRa further underscores their viability as an alternative to hfNSCs for clinical applications.

Speaker: Marco Luciani (OSPEDALE SAN RAFFAELE SRL)

18:30 Exploiting advanced human induced pluripotent stem cell (hiPSC)-based 3D models to study Globoid Cell Leukodystrophy.

Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disorder due to mutations in the galactosylceramidase (GALC) gene, which is essential for sphingolipid metabolism. GALC deficiency leads to the accumulation of toxic psychosine, causing demyelination, neurodegeneration, and neuroinflammation in the central and peripheral nervous systems. While dysfunctional oligodendrocytes (OLs) were traditionally seen as the leading cause of GLD, new evidence suggests that neurons and astrocytes may play a significant role in white matter damage.
In previous studies, we used patient-specific human induced pluripotent stem cells (hiPSCs) to model GLD and explore gene therapy strategies. Using hiPSC-derived neural stem/progenitor cells and 2D neuronal/ glial cultures, We identify previously unrecognized early pathogenic events, uncovering mutation-dependent defects in the differentiation of neurons and OLs. Nevertheless, the 2D culture conditions do not support the full maturation of neurons and glial cells and lack the complex architecture needed to study cell-to-cell interactions in this disease.
To address these issues, we generated OL-enriched 3D spheroids (hOLS) from hiPSCs of healthy donors (HD), GLD patients, and isogenic GALC knock-out (KO) and knock-in (KI) lines generated via CRISPR-Cas9 editing. This research investigates the early neurodevelopmental defects associated with GLD pathology and the impact of GALC deficiency on glial and neuronal maturation. We demonstrated that hOLS were efficiently generated from all hiPSC lines and were composed by multiple populations of glial and neuronal progenitor cells and their differentiated progeny. Immunofluorescence data reflected the maturity of the evolving cell types, which included MBP-positive oligodendrocytes. Additionally, through single-cell RNA sequencing, we identified gene expression alterations and changes in cellular composition that were explicitly linked to GLD. The integration of transcriptomics with the phenotypic analysis of hOLS provides new insights into the molecular signature associated to GLD, offering mechanistic insight into the early pathogenesis of this disorder.

Speaker: Elisabeth Mangiameli (San Raffaele Telethon Institute for Gene Therapy (SR-Tiget); IRCCS San Ra&aele Scientific Institute)

18:30 Exploring the ontogenesis of adult neural stem cells across species

"Initial evidence for adult neurogenesis in the mammalian brain was already obtained in the 60s of the last century, when newly generated neurons were first described in the rat brain (Altman, 1962). Since then, numerous studies have provided experimental evidence of adult neurogenesis in several mammalian species, from rodents to non-human primates and humans. Despite the extensive characterization of neural stem cells (NSCs) in the adult brain, still relatively little is known about their origin.
Here, we investigate the regulatory mechanisms underlying adult NSCs (aNSCs) specification in the mouse Ventricular Sub-Ventricular Zone (V-SVZ) and explore the ontogeny of aNSCs across different mammalian species.
The majority of the aNSCs in the mouse V-SVZ is set aside during a specific window of the embryonic development (E13.5-E16.5) from slowly dividing progenitor cells (Furutachi et al., 2015; Fuentalba et al., 2015). To characterize the embryonic ancestors of aNSCs, we isolated slowly and fast dividing LGE progenitors from the iCOUNT mouse line (Denoth-Lippurner et al., 2021) and profiled their transcriptome via single-cell RNA sequencing. We identified distinct clusters of stem/progenitor cells. Via differential gene expression analysis, we detected a set of transcripts enriched in slowly versus fast dividing progenitors. We selected several candidates and explored their function in aNSCs specification via loss of function analysis, which will be presented at the meeting.
Finally, to address whether the establishment of aNSCs occurs similarly in other mammals, we performed cross-species comparison of single-cell datasets from the mouse, ferret and human embryonic brain, with the aim to identify a common molecular signature of the embryonic ancestors of aNSCs.
Taken together, our work aims to identify the molecular mechanisms specifying aNSCs with the long-term aim to possibly elicit their specification also in other brain regions lacking adult neurogenesis."

Speaker: Daniela Cimino (Helmholtz Center Munich - Institute of Stem Cell Research)

18:30 From pluripotency to germ cells and back: modeling the molecular impact of endocrine disruptors on intergenerational inheritance and human neurodevelopment in a dish

Whether and how the impact of environmental exposures can be inherited, with parental environments shaping phenotypes across generations independent of DNA sequence carried in gametes, is a fundamental question in biology, with far-reaching implications for human health. Despite evidence of such epigenetic inheritance in animals, the mechanisms and relevance to humans remain elusive due to lack of (i) multi-generational human data on environmental exposures with quantified health outcomes, and (ii) access to germ cells and tissues from exposed individuals to deduce and validate molecular mechanisms.
Considering the paradigmatic case of the heritable impact of the exposure to endocrine disruptors (EDC), a widespread and hazardous class of chemicals that interfere with hormonal signaling causing a wide range of adverse health effects, including adverse neurodevelopmental outcomes, we are overcoming these challenging by (i) leveraging a unique multigenerational human cohort (Carl-Gustaf Bornehag et al., 2021; Caporale et al., 2022), where we are measuring the parental prenatal (F1) exposure to EDC and relating it to quantified neurodevelopmental outcomes in their children (F2), (ii) developing an innovative in vitro system to make epigenetic inheritance tractable in humans. Specifically, in collaboration with Harry Leitch's lab, for the first time, we converted human primordial germ cell-like cells (hPGCLC, differentiated from hiPSC and model of F1 generation developing germline) back to the pluripotent state, namely human embryonic germ cell-like cells (hEGCLC, proxy model of F2 generation post-implantation epiblast). By scRNAseq and DNA methylation profiling, we confirmed hEGCLCs pluripotency and showed how the initial demethylation occurring in hPGCLCs is largely reversible, as hEGCLCs exhibit methylation levels comparable to hiPSCs. This new in vitro model represents a highly tractable system to study epigenetics transitions and dynamics in humans. Therefore, we exposed differentiating hPGCLCs to EDCs at epidemiologically relevant concentrations, converted them into hEGCLCs, and further differentiated these into cortical brain organoids (CBOs, model of F2 fetal cortex). We performed transcriptome and epigenome profiling (focusing on DNA methylation, key player in epiallele inheritance) of hPGCLCs, hEGCLCs, and CBOs to assess which EDC-induced epigenetic changes are inherited and maintained in the next generation and with what phenotypic consequences.
Overall, by integrating experimental and epidemiological evidence, this project will contribute to shed light on the impact of environmentally induced, epigenetically inherited changes on human development, providing scientific evidence to guide the reduction of adverse effects on present and future generations resulting from widespread environmental exposures to EDCs.

Speaker: SARAH STUCCHI (Human Technopole)

18:30 Functional characterization of putative non-coding regulatory elements in Autism Spectrum Disorder risk

Autism spectrum disorder (ASD) is highly inherited and arises from a complex interplay between three major categories of genetic risk: common polygenic variation, rare inherited and de novo mutations. The efforts to identify rare variants of large effect have mainly focused on the common variants in coding regions of the genome. However, rare genetic variants, especially in non-coding regions, remain elusive. Most identified DNA variants associated with ASD indeed map to non-coding regions of the genome, which often carry epigenetic features of enhancers. Often located far away on the linear chromosome map, enhancers are associated with the regulated promoter of the gene by long-range interactions (LRI). We previously performed RNApolIII-ChIA-PET and ChIP-seq profiling of mouse brain-derived neural stem cells (mNSCs), and identified over 10,000 enhancers linked to LRI, many of which are syntenic to human enhancers with neurodevelopmental relevance. Several such LRIs connect “epigenetic enhancers” to high-confidence ASD genes. The most promising enhancers hosting prioritized variants are presently being selected, to identify de novo variants that are specific to patients, that map onto enhancers, and that may contribute to the disease, looking for variants that are predicted to interfere with transcription factor binding sites. An interesting case is an enhancer connected to the FOXG1 gene (whose mutation causes an autism-related disorder), which is the object of microdeletions in various ASD patients, that leave the FOXG1 gene intact. We decided to analyse and evaluate potential mechanisms for the contribution of regulatory elements likely corresponding to neural enhancers to the regulation of the gene(s) connected to them by LRI, by silencing variant-containing regulatory regions using a CRISPR interference (CRISPRi) system in vitro and analysing the effect on the expression of the gene connected by LRI. To ask whether the identified enhancers do regulate the connected gene, we introduced vectors expressing guide RNAs (gRNAs) targeting a specific enhancer region, and the gene transcription start site (TSS) as a positive control in mNSC stably expressing dCas9/KRAB protein from nuclei. qRT-PCR analysis showed strong (>90%) downregulation of the connected FOXG1 gene following anti-FOXG1-TSS gRNA expression in our preliminary experiments. We are presently comparatively testing how repression of the enhancer connected with FOXG1 affects FOXG1 mRNA levels.

Speaker: Elvira Zakirova (University of Milano-Bicocca)

18:30 GENERATION AND CHARACTERISATION OF CORTICAL BRAIN ORGANOIDS FROM HUMAN EMBRYONIC GERM CELL-LIKE CELLS

Studying the epigenetic landscape during the transition between pluripotency and the germline at multi-omics level enables to study the molecular basis of germ cell tumorigenesis and epigenetic inheritance. We recently developed and characterised a protocol to differentiate human pluripotent stem cells (hPSC) into Primordial Germ Cell-Like Cells (hPGCLC), and to go back again to pluripotency, deriving human Embryonic Germ Cell-Like Cells (hEGCLC) (Stucchi et al., biorXiv). This demonstrated the overlapping identities in terms of pluripotency and transcriptome between hPSC and hEGCLC and represents the first fully-defined and reproducible protocol to obtain hEGCLC.

To further characterise this cell type and its potency, we differentiated them into human cortical brain organoids (hCBO) and we are now investigating to which extent hEGCLC-derived CBO recapitulate human fetal cortex development, comparing them to hPSC-derived CBO. To this aim, we are characterizing this model at three different levels:
- At the transcriptomic level, by scRNAseq performed longitudinally at different timepoints (25, 50, 100, 200 and 300 days of differentiation);
- At the protein level, by 2D immunostaining for key neural markers;
- At the electrophysiological level, by Multi-Electrode Array (MEA).

The epigenetic dynamics that characterise hiPSC-to-hPGCLC-to-hEGCLC transition, recapitulate some of the key epigenetics changes occurring during the passage from one generation to the next. Combining this with the generation of CBOs from both hiPSC and hEGCLC represents a unique in vitro modelling strategy to study the inter-generational impact of environmental exposure on neurodevelopment.

By combining specific exposures, during hPGCLC differentiation, hiPSC-derived and hEGCLC-derived CBO differentiation, we are isolating the differential impact of environmental factors on parental and offspring neurodevelopmental outcomes, dissecting windows of vulnerability across parental prenatal and offspring prenatal periods.

Overall, this work will contribute to shed light on the impact of environmentally induced, epigenetically inherited changes on human neurodevelopment, providing scientific evidence to guide the reduction of adverse effects on present and future generations resulting from widespread environmental exposures.

Speaker: Riccardo Nagni (Fondazione Human Technopole)

18:30 Generation of seven human induced pluripotent stem cell lines with CRISPR/Cas9-mediated deletions in the DMD gene

"Duchenne muscular dystrophy (DMD) is an X-linked progressive neuromuscular disorder caused by mutations in the DMD gene, resulting in the absence of dystrophin protein. In healthy muscle, only the full-length dystrophin isoform Dp427m is expressed, while the brain expresses the full-length isoforms Dp427c and Dp427p, and the shorter isoforms Dp140 and Dp71/40. Depending on the position of the mutation within the DMD gene, one or multiple dystrophin isoforms are lacking in the brain, which is associated with cognitive and behavioral problems. However, knowledge on the role of the dystrophin isoforms in brain cell development and the consequences of their absence is limited.
To improve our understanding, we utilized the CRISPR/Cas9 genome editing technology to generate isogenic human iPSC lines lacking one or multiple dystrophin isoforms from two healthy male control hiPSC lines. The expression of single isoforms (either Dp427c, Dp427p, Dp140 or Dp71) was abolished by targeting their unique promoter. The expression of multiple isoforms was abolished by the deletion of specific exons. Exon 11, 52 or 66 was deleted to respectively disrupt expression of either Dp427, Dp427+Dp140 or all isoforms. After gene editing, clones were picked and expanded, and mutated clones were selected by Sanger sequencing. The quality of the hiPSCs and neuronal progenitor cells (NPCs) is currently being assessed by a panel of standard quality control assays for morphological appearance, expression of pluripotency markers, differentiation potential, and microbiological contamination. NPCs will be used to generate control and DMD 2D neuronal cultures.
These isogenic DMD hiPSC cultures will be a valuable resource for the characterization of the role of dystrophin isoforms in brain cell development by studying the morphological, molecular and functional consequences of their absence."

Speaker: Kayleigh Putker (Leiden University Medical Center (LUMC))

18:30 Golgi traffic and brain development in health and disease

"During neocortex development neural stem progenitor cells display polarity features that are fundamental in determining their identity and fate. Interestingly, Golgi apparatus (GA) is differentially positioned in the two main neural stem cell types, apical and basal progenitors (APs and BPs, respectively). GA is known to be the main hub for glycosylation and defects in GA and glycosylation can affect brain development. Previous results from our lab suggested that the GA fragmentation in mouse embryonic brain and in human brain organoids favors the APs to BPs cell fate transition. To investigate the influence of GA on cell identity and lineage progression during brain development, we leverage the genetics of Congenital Disorders of Glycosylation (CDGs), a group of multi organ rare and ultra-rare diseases caused by mutations of structural proteins or enzymes in the secretory pathway. We focus our attention on COG5-CDG, caused by mutations in COG5, a GA-resident proteins involved in Golgi homeostasis and function. COG5-CDG patients suffer from neurodevelopmental defects, including seizures and microcephaly. The knock-down of COG5 in human brain organoids through electroporation causes the fragmentation of Golgi apparatus, an increase in Tbr2-positive intermediate progenitors (IP) and an increase in the Ctip2-positive neuronal population. No significant changes were found at the level of apical progenitors. These results suggest an effect of Golgi apparatus on NPCs identity and fate transition.
To gain further insight in the contribution of COG5, and more in general of GA, on brain development, we generated human brain organoids from iPSCs obtained from two siblings harboring a mutation in COG5. We are now combining different phenotyping strategies including staining for fate markers, scRNA seq to follow lineage progression and glycomics to investigate the effect of COG5 mutation on the glycosylated proteins and lipids and correlate the glyco-makeup of progenitors with their fate choice.
In conclusion, by leveraging the genetics of a human glycosylation disease we aim at filling the gap between the cell biological role of GA in progenitor cells and the pathophysiological manifestations associated with defective GA glycosylation."

Speaker: Martina Polenghi (Fondazione Human Technopole)

18:30 Hi-C and RNA-seq analyses on hiPSC-derived astrocytes allow to further decipher pathomechanisms in Autosomal Dominant adult-onset demyelinating LeukoDystrophy (ADLD)

Background/Objectives. ADLD is a rare neurodegenerative disorder caused by LMNB1 overexpression due to gene duplications (classical ADLD) or noncoding deletions in the locus (atypical ADLD). Our group collected > 20 families worldwide carrying structural variants (SVs) in the LMNB1 locus and reported strong clinical variability, even among patients carrying duplications of the entire LMNB1 gene, ranging from classical and atypical ADLD to asymptomatic carriers. We performed Hi-C and RNA sequencing on ADLD fibroblasts, providing preliminary insights into ADLD pathomechanisms and the genotype/phenotype correlation of SVs at the LMNB1 locus. Here, we aim to validate these findings in disease-relevant cellular models, namely hiPSC-derived astrocytes.
Methods. We generated hiPSC-derived astrocytes from four subjects carrying a SV in the LMNB1 locus (a classical ADLD and two atypical ADLD patients, and a asymptomatic carrier) and an healthy control. Hi-C were performed on 1 million astrocytes per sample following standard protocols. Statistically significant contacts have been highlighted using Fit-Hi-C v.2.0.8 (FitHiC2).
Results. We validate that: classical ADLD is due to an intra-TAD duplication, resulting in a simple gene dose gain; atypical ADLD is caused by LMNB1 forebrain-specific misexpression due to SVs encompassing a TAD boundary. Finally, in the asymptomatic subject we identified an inter-TAD duplication, which extends centromerically and crosses two boundaries. FitHiC2 has detected a significant physiological regulatory contact between the LMNB1 promoter and the LMNB1 regulatory element at genomic coordinates chr5:125995687-125996125 (hg19), previously reported by our group in the control cell line.
Conclusion. Our results highlight the importance of considering the effects of SVs at the LMNB1 locus on the TAD structure before assuming their pathogenicity and corroborate that a duplication encompassing the LMNB1 gene is not sufficient per se to diagnose ADLD, with implications for ADLD diagnosis and genetic counseling.

Speaker: Anna Basile (Università degli Studi di Pavia)

18:30 hiPSC-derived models to dissect CAPRIN1-linked neurodevelopmental disorder: the role of CAPRIN1 loss on neuronal differentiation, neurogenesis, and proliferation.

"CAPRIN1 is a ubiquitous protein involved in cell proliferation and migration. In neurons, it regulates the transport and translation of mRNAs involved in synaptic plasticity. We have previously associated CAPRIN1 loss-of-function variants with a rare autosomal dominant neurodevelopmental disorder (NEDLAAD, OMIM #620782), showing several defects on hiPSCs-derived neurons, including decreased process length, overall disruption of the neuronal structure, enhanced neuronal death, impaired calcium signaling, increase protein translation and oxidative stress.
We performed bulk transcriptomics analysis in early and late differentiated neurons to further define the mechanisms behind those neuronal defects. The sequencing data explained the previously observed defects on CAPRIN1 deficient neurons, highlighting impaired oxidative phosphorylation, neuronal outgrowth, and translation. We also found a significant increase in genes involved in cell cycle control, DNA replication, proliferation, and dopaminergic neurogenesis, as well as a decrease in GABA pathway-related genes. Preliminary data on CAPRIN1+/- neuronal progenitor cells showed an increased proliferative rate and impaired expression of neuronal progenitors’ markers, suggesting a possible loss of balance in neuronal proliferation/differentiation. To explore these features, we generated forebrain cortical organoids (fBOs) from CAPRIN1 patient-derived hiPSCs lines and controls to analyze their cellular architecture, neuronal activity, and cellular composition, combining immunofluorescence analysis and a multicolor immunophenotyping panel. We observed an essential impact of CAPRIN1 loss on the architecture development of fBOs and an impaired proportion of GFAP+ and MAP2B+ cells. Metabolic analysis on fBO-derived cells showed a switch toward aerobic glycolysis, further supporting defects in the cellular organization of the fBO. To further dissect fBOs composition, single-cell RNA-seq was performed at 40, 60 and 90 days of differentiation; data analysis and pseudotime trajectory study are ongoing.
In conclusion, our study will unravel the impact of CAPRIN1 loss on neurodevelopment, shedding light on its role in neurogenesis, neuronal differentiation, and proliferation."

Speaker: Lisa Pavinato (Institute of Oncology Research (IOR), Bellinzona Institute of Science)

18:30 Human neurodiversity in a dish: the regulatory logic of cortical neurodevelopment unmasked by chromatinopathies

The polygenic underpinnings of human neurodiversity require new maps to trace its neurodevelopmental physiopathology. Brain organoids can provide major insights into neuropsychiatric disorders, yet the focus is still on one or a few specific disorders at a time, typically caused by highly penetrant genetic mutations studied in isolation. CRISPR-based screenings of heterogenous collections of genes involved in separate functions or pathways has been the main alternative. To capture how individual genomes and chromatin remodellers dosage shape developmental trajectories, we generated single-cell resolved models of paradigmatic neurodevelopmental disorders (NDDs) caused by mutations in chromatin regulators. We collected 50+ cell lines from case-control matched cohorts and isogenic series. This massive design allowed us to uncover the gene regulatory mechanisms defining the specificity of differentiation trajectories underlying NDDs. Through machine learning we reconstructed cell state-specific causal regulatory networks and characterized the impact of chromatin regulators dosage on salient cognitive/behavioral phenotypes. Our integration between gene regulatory networks with the differential expressed genes in a cell type- and disease-specific manner identified key mediators orchestrating biological processes and signalling pathways, inferring intricate gene interactions. By comparing disease gene virtual knockouts with effective cell type abundance changes in organoids, we assessed each gene impact on cellular dynamics, offering insights into cell differentiation fates and their physiopathological mechanisms. Together with this systematic reconstruction of gene regulation along neurogenesis, we provide an NDD atlas and easy to query resources, that can be leveraged to generate new hypotheses and integrate larger datasets accruing from national and international cohorts.

Speaker: Filippo Prazzoli (Fondazione Human Technopole)

18:30 Identification and Functional Study of Non-Coding DNA Variants in Microphthalmia, Anophthalmia, and Coloboma Patients

Microphthalmia, Anophthalmia, and Coloboma (MAC) are severe ocular malformations resulting
from mutations in both protein-coding genes, such as the transcription factor SOX2, and non-coding
regulatory elements. Approximately 50% of affected individuals lack a definitive molecular diagnosis based
on exome sequencing aimed at detecting protein-coding genes mutations, suggesting that mutations in noncoding regions may play a critical role in disease pathogenesis.
We previously performed RNApolII-ChIA-PET analysis on neural stem cells derived from the mouse brain,
mapping long-range chromatin interactions between enhancers and gene promoters. These interactions
were subsequently mapped to the human genome, identifying 7.698 syntenic regions, termed human-mouse
syntenic long-range interactions (hmsLRI). DNA sequence variants associated with MAC (identified through
whole-genome sequencing of affected individuals) were then overlapped with this enhancer dataset.
This comparison identified significant copy number variants (CNVs) associated with genes involved in eye
development and inherited ocular diseases, though not affecting protein-coding regions; our study focuses
on four CNVs. We started from this selection to plan further functional studies, including transgenesis
experiments in zebrafish and the generation of brain and eye organoids from human pluripotent stem cells.
For in vivo analysis, CNVs were cloned into a Gateway vector, then transferred to a ZED vector optimized for
transgenesis in Danio rerio. This vector enables precise expression of transgenes and real-time monitoring
of gene activity using fluorescent reporters. These tools will provide valuable insights into how the identified
CNVs influence eye development and contribute to inherited ocular diseases

Speakers: Delia Morciano (Università Milano-Bicocca), Gabriele Antoniazzi (Università Milano-Bicocca)

18:30 Immune activation during pregnancy drives TREM2 dysregulation and synaptic defects via maternal Type-I Interferon responses

Maternal infections during gestation cause perturbed neuroimmune interactions and increased risk of neurodevelopmental disorders in the offspring. Microglia, the immune surveillant of the brain, express Triggering Receptor Expressed on Myeloid Cell 2 (TREM2), which regulates synapse elimination and neuronal cell metabolism during development. Using a maternal immune activation model with polyriboinosinic-polyribocytidilic acid [Poly(I:C)], Trem2-/- mice, and anti-Type I Interferon receptor (αIFN-IR) treatment, we investigated how maternal infections reshape the hippocampal landscape of the newborn. Poly(I:C) exposure elevated Type I Interferon (IFN-I) in pregnant dams, disrupting neuronal transcriptional trajectories and synapse homeostasis in juvenile offspring. Poly(I:C) offspring microglia exhibited reduced TREM2 expression, suppressed IFN-I-responsive substate, and impaired phagocytosis. Downregulation of synaptic genes and microglial IFN-I-signature was Trem2-dependent, highlighting its regulatory role in neurodevelopmental pathologies. Blocking maternal IFN-I signaling restored synaptic defects and TREM2 function, suggesting that monitoring IFN-I responses during pregnancy may represent viable therapeutic approaches to mitigate neurodevelopmental dysfunction in newborn.

Speaker: Matteo Bizzotto (IRCCS Humanitas Research Hospital)

18:30 IMPACT OF EBV INFECTION ON CORTICAL NEURODEVELOPMENT

The Epstein-Barr Virus (EBV) is a DNA virus belonging to the Herpesvirus family. EBV infections are extremely common, and linked to various diseases. Recent studies have highlighted the role of herpesviruses in the central nervous system (CNS) and some reports suggest a possible link between EBV infection and Rasmussen’s encephalitis. This is a progressive neurological disease that primarily affects children between the ages of 6 and 10 and it suggests a potential involvement of early-stage EBV infection. Due to EBV's inability to infect rodent cells lacking the necessary receptors, iPSC-derived cortical cultures provide a unique opportunity to study the interactions between EBV and human brain cells across neurodevelopmental stages. To this end, 2D cell cultures were infected at a neurodevelopmental stage in which mature neurons and neural progenitors coexist. Real-time PCR analysis confirmed that EBV successfully infected cortical cultures, detecting latent and lytic transcripts, including BZLF1 LMP1 and GP220. Strikingly, infected cultures displayed higher levels of the neural progenitor marker PAX6, suggesting an alteration in neuronal differentiation.
Focusing on an earlier neurodevelopmental stage, we observed an increase in LAMP1 and CLEAVED CASPASE 3, associated with an abnormal structuring of neural rosettes after EBV infection. Typically, neural rosettes recapitulate neural tube closure with a well-defined organization, however, EBV infection profoundly disrupted the morphology of neural rosettes in 2D cultures leading to chaotic formations. To further investigate this phenomenon, we employed a 3D micropatterned system (RosetteArray technology) that forces cell aggregation into 3D neural rosettes. Across three different control cell lines, we observed that EBV infection impaired neural rosette formation, leading to a reduced number of rosettes with significantly smaller areas supporting the idea that EBV infection impacts neurodevelopment. To further explore its effects on neuronal networks, we will analyze morphological, molecular, and functional changes using multielectrode array recordings and calcium imaging.

Speaker: Anna Dell'Armi (Sapienza Università di Roma)

18:30 Induced Pluripotent Stem Cell (iPSC)-derived Microglia and Organoids for neuronal disease modeling

Microglia are the principal resident immune cells of the central nervous system (CNS) representing 5-12% of the total cell population in the brain. They are of myeloid origin and their survival and maintenance depend on several cytokines. Microglia play a role in development of the CNS and are constantly engaged in detecting changes in their environment, maintaining homeostasis and protecting against endogenous or environmental injurious agents. Microglia has emerged as an interesting target in many neurodegenerative diseases which deserve to be studied in more detail. Indeed, the mechanisms by which aberrant microglia activation causes neurodegeneration remains unclear. In its activated state, microglia play a role in neurodegeneration of most CNS disorders such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease and Parkinson’s disease. Microglia is implicated in the initiation and in the progress of these diseases, switching from a neuroprotective activated state to a neurotoxic one. Phagocytosis, which involves engulfment and elimination of large cellular structures, is carried out by microglia and can be regulated by an autophagic process which impairment is associated with many neurodegenerative disorders, in part related to an increased release of inducers of the inflammatory response. We preliminary optimized a protocol to differentiate microglia from iPSCs generated from both healthy donor and patients affected by neurodegenerative diseases such as ALS and FTD. Microglia cells were characterized for the expression of specific markers (CD11b, Vimentin, TREM2, TMEM119, IBA1) by immunofluorescence, while their functionality was evaluated by assessing fluorescent latex-beads internalization which confirmed their phagocytic activity. We also evaluated the presence of lipid droplets, consisting in organelles that contain neutral lipids, increasingly being accepted as structural markers of inflammation. In addition, both neural and spinal cord organoids were obtained from iPSCs. Given the important modulatory role in neural death in neurodegenerative diseases, we will establish co-cultures of microglia and iPSC derived organoids combining microglia obtained from patients with organoids derived from controls and vice-versa in order to analyze engraftment of microglia on organoids and to study the interactions between microglial and neural cells.

Speaker: Patrizia Bossolasco (IRCCS Istituto Auxologico Italiano)

18:30 Inference of Possible Novel Autism Risk Genes by Comparative Socio-Genomics and Molecular Network Analysis

Comparative socio-genomics is an emerging field that integrates behavioral science, molecular, and evolutionary biology, genomics, neuroscience, and bioinformatics to explore the genetic basis of social behavior across species.
Our aim is to uncover the genetic roots of social behavior to infer novel risk genes for autism, a neurodevelopmental disorder characterized by social and communication deficits, and with a strong sex bias (males: females=4.5: 1). To this purpose, we conducted molecular network analysis on 659 sociality-related genes across different animal species, including humans.
We found that 240 sociality-related genes are in network proximity in the gene-centric human interactome. Interestingly, the network is highly enriched in genes associated with autism (p < 10⁻¹⁵). It shows a modular structure composed of 23 communities and it involves genes primarly regulating cell-cell communication, junction organization, and adherens junction interactions. Enrichment analysis also revealed nine chromosomal bands and five genes (MED12, GJB1, SRGAP2, CFTR, AVPR2) in linkage with key autism genes, while 24 are targets of Human Accelerated Regions (HARs). The network contains genes that could have a role in autism, e.g.: MED12, a regulator of gene transcription acting in neurodevelopment, is a differentially expressed gene (DEG) in the brain of autistic individuals, belongs to chXq13.1, is in linkage with the autism associated gene NLGN3X; FZD9, a receptor for WNT signaling proteins, is a DEG in the brain of autistic individuals, and maps within the WBS locus, whose dosage imbalance leads to behavioral disorders; DMD, is a DEG in the brain of autistic individuals, maps on chX and is a HAR target.
Multiple animal species may serve as a more complete model to define the genomic pillars of sociability. If validated, the inferred genes could serve for advancing the understanding of human social behavior and as novel biomarkers of autism. Funding: GEMMA project (H2020, grant number:00002771-13001252).

Speaker: Alessandra Mezzalani (Institute for Biomedical Technologies, National Research Council (CNR-ITB))

18:30 Insights from cell villages: imbalance in multi-donor iPSC differentiation models

Induced pluripotent stem cell (iPSC) models combined with single-cell transcriptomics enable the study of cellular consequences resulting from human disease alleles, particularly in early-onset developmental disorders. A significant bottleneck lies in the scalability of iPSC-based experiments. Pooled cell culture or ‘cell villages’ has emerged as a key strategy to enable large-scale studies. However, achieving a balanced representation of cell lines within villages remains challenging, likely due to differences in growth dynamics, non-cell autonomous effects and intrinsic variations in developmental signaling pathways during early embryonic lineages.

To better understand the factors that cause donor imbalance in 2D iPSC villages, we systematically evaluated the effects of different experimental and cell-line related factors on the composition of eight villages (5-18 donors each) differentiated to cortical neurons. We also assessed donor imbalance in the endoderm lineage by differentiating two of the villages to a pancreatic fate, and maintained them in pluripotency as a baseline. All villages included wild type, patient-derived and engineered mutant lines, and their donor composition was deconvoluted using natural genetic variation from either single-cell RNA-seq reads or low-coverage whole-genome sequencing.

We observed that specific cell lines consistently become dominant in villages at neuronal precursor (NPC) stage (50.4% representation), regardless of village size, and were more likely to be female-derived lines (p=0.018). Comparing the transcriptomic profiles of lines differentiated individually and in pools, we confirmed donor identity was preserved at NPC stage. We also found significant shifts in donor representation between neuronal and pancreatic fates, indicating pool imbalance was not constant across cellular lineages. This suggests that pool imbalance arises immediately upon induction due to donor-intrinsic variation in developmental cues that affect fate commitment programs differently. Our future aim is to mitigate donor imbalance in cell villages to create more accurate and accessible disease models for neurodevelopment disorders.

Speaker: Pau Puigdevall Costa (University of Helsinki)

18:30 Insights into Mitochondrial Homeostasis and mTOR/AKT Signaling in Peripheral Neuropathies: A Focus on CMT2A2, ADOA, and CMT4B3

Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and dominant optic atrophy (ADOA), are frequently associated with mitochondrial dysfunction. Through transcriptomic analysis, we identified the mTOR/AKT axis as a key regulator of mitochondrial homeostasis and quality control. We investigated fibroblast lines derived from three patients: two carrying mutations in mitochondrial fusion proteins MFN2 (CMT2A2) and OPA1 (ADOA), and one with a mutation in the non-mitochondrial protein MTMR5/SBF1 (CMT4B3). Our study assessed mitochondrial function, autophagy/mitophagy processes, and their impact on cellular proliferation and senescence. All patient-derived fibroblasts exhibited mitochondrial dysfunction, with MFN2 and OPA1 mutations impairing autophagosome formation but exerting divergent effects on proliferation: CMT2A2 cells displayed enhanced growth, while ADOA cells underwent premature senescence. Crucially, inhibition of the mTOR/AKT pathway restored autophagy and normalized proliferation and senescence in both conditions. These findings underscore the significance of mTOR/AKT dysregulation as a common pathological mechanism in mitochondrial-related neuropathies. Further insights into the role of CMT4B3 in mitochondrial quality control will be discussed. Our results pave the way for targeted therapeutic strategies aimed at restoring cellular homeostasis by modulating mTOR/AKT signaling in peripheral neuropathies.

Speaker: Paola Zanfardino (University of Bari Aldo Moro)

18:30 Investigating gene-environment interactions in neurodevelopmental disorders through single-cell profiling of cortical brain organoids

Mental health outcomes are shaped by a complex interplay between genetics and environmental factors throughout life. Early-life exposure to endocrine-disrupting chemicals (EDCs), widespread environmental compounds that interfere with the endocrine system, has been associated with neurodevelopmental disorders (NDDs). Our research aims to identify risk and protective factors for mental health by investigating how prenatal EDC exposure interacts with genetic predisposition.

To explore these interactions, we leverage the Swedish SELMA cohort, which includes extensive data on environmental exposures, genetic profiles, and cognitive-behavioral phenotypes from over 600 children. From this cohort, we are selecting a subset for human-induced pluripotent stem cell (hiPSC) reprogramming and cortical brain organoid (CBO) differentiation. Selection is based on polygenic risk scores for NDDs and psychiatric disorders, prenatal EDC exposure levels, and behavioral phenotypes.

CBOs are 3D in vitro models that recapitulate key features of the developing human cortex, housing essential cell types such as progenitors and neurons. This controlled experimental setting enables precise manipulation of EDC exposure levels and allows us to model susceptibility and resilience to mental health outcomes at single-cell resolution.

We will perform single-cell RNA sequencing (scRNA-seq) to investigate the molecular mechanisms underlying these interactions. Furthermore, by integrating data from large scRNA-seq atlases of human fetal brain tissue, NDD-related genes identified through genome-wide association studies (GWAS), and our lab's ongoing work on a transcriptomic atlas of EDC and hormonal impacts on CBOs, we aim to pinpoint genes involved in NDDs that are altered by EDC exposure. We will develop cell-type-specific gene-environment interaction scores, offering insights into how EDCs modify gene expression in cortical cells depending on an individual’s genetic background.

Ultimately, this work will unravel how environmental exposures shape neurodevelopment at the molecular level, contributing to advancements in precision medicine for NDDs and supporting EU regulatory efforts concerning EDC exposure.

Speaker: Gaja Matassa (Fondazione Human Technopole)

18:30 Investigating the transcriptional landscape of cerebellar primary cilia across development

Primary cilia (PCs) are single-copy organelles functioning as important signaling hubs for multimodal stimuli. Dysregulation of PCs leads to syndromic phenotypes (including hindbrain malformations) and developmental disorders referred to as primary ciliopathies. Aberrant activation of ciliary pathways is a major driver in the formation of brainstem and cerebellar tumors. It is well known that the structure, function, and composition of PCs is tightly regulated by cell cycle, but it is becoming increasingly clear that other factors also regulate ciliary dynamics. However, little is known about the transcriptional profiles associated with such ciliary dynamics at a single-cell resolution in intact tissue environments. We use the chicken cerebellum and brainstem in combination with scRNA-seq and spatial transcriptomics to investigate the ciliary transcriptional landscape across cerebellar development. Classical in-situ hybridization of candidate primary cilia genes across cerebellar and spinal cord development revealed differential expression patterns in both space and time. Using scRNA-seq, we were able to identify 827 chicken homologs of the 956 genes listed in the CiliaCarta (a validated compendium of ciliary genes) across different cerebellar cell types and states of differentiation. Unsupervised clustering based on the CiliaCarta homologs identifies distinct ciliary transcriptional clusters. We found that ciliary transcriptional clusters map closely to general cell type clusters. To investigate the spatial distribution and relationships between these ciliary transcriptional clusters, we have established a BARseq2 pipeline. BARseq2 allows for highly-multiplexed spatial transcriptomic readouts by combining nucleotide barcodes with Illumina in-situ sequencing. Initial small-scale pilot experiments have shown the BARseq2 pipeline to be compatible with chicken embryonic cerebella. We are scaling up the BARseq2 panel to > 100 genes to investigate spatially-resolved co-expression at single-cell resolution in an intact cerebellar environment. By ultimately collecting data across different stages of cerebellar development, we will be able to map ciliary transcriptional clusters across space and time.

Speaker: Reto Cola (University of Zurich)

18:30 Leveraging Brain Organoids to Explore Neurodevelopmental Mechanisms in Congenital Central Hypoventilation Syndrome (CCHS)

The human brain is a complex organ, and its features of neurodevelopment and disease remain largely unexplored due to limited accessibility of living tissue. Recently, the human induced pluripotent stem cells (hiPSC) technology allowed to model the human brain in vitro, generating 3D organoids.
We developed brain and region-specific brainstem organoids, characterized them by fluorescent IHC to evaluate neuronal differentiation and brain regionalization, and bulk RNA sequencing followed by deconvolution to infer cell compositions and gene expressions. In parallel, label-free Raman images were collected on organoid slices for their non-invasive biomolecular characterization.
Since energy metabolism is a crucial element of neurodevelopment and can constitute a common pathway through different diseases, we performed untargeted metabolic signature analysis of developing brain organoids over time.
Comprehensively, the analyses performed on control brain organoids paved the way to model and characterize neurodevelopmental disease-derived organoids. They constitute an unvaluable asset in the study of neurodevelopmental disorders for which viable animal models are not available: in this context, we focused on the study of Congenital Central Hypoventilation Syndrome (CCHS). CCHS is a rare genetic disorder affecting the autonomic nervous system (ANS) and central chemosensitivity caused by the loss of retrotrapezoid nucleus (RTN) neurons in the brainstem. Mutations of the PHOX2B master gene affect the development of the ANS and central structures (RTN) that participate in breathing control. Given its fundamental role, CCHS animal models die in utero: to overcome this issue and have an insight in PHOX2B’s function during the first stages of human neurodevelopment, we used CCHS patient-derived iPSCs carrying different PHOX2B mutations to generate brain and brainstem organoids that contain cytoarchitectures resembling central chemoreceptors.
This new personalized disease-in-a-dish model of CCHS will allow to identify molecular and cellular defects induced by PHOX2B mutations as well as modelling for drug discovery/screening for therapeutic perspectives.

Speaker: Eleonora Piscitelli (ITB-CNR)

18:30 Modeling activity-dependent transcription in iPSC-derived human cortical organoids at single-cell resolution

Neurons are capable of adapting their gene expression in response to electrical signals, converting synaptic activity into dynamic and lasting functional changes. This activity-dependent transcription is tightly regulated by neuronal firing and specialized pathways, shaping neural circuits and synaptic plasticity (Greenberg et al., 2018). Sustained hyperexcitability can lead to several pathologies, both during developmental stages as well as in the mature brain, including epilepsy. However, the precise impact of prolonged hyperexcitability on cellular composition and gene regulation is not well understood. Here, we use iPSC-derived cortical organoids - an in vitro model mirroring key aspects of human cortical development (Velasco et al., 2019) - to investigate how hyperexcitability alters cell populations and gene networks at single-cell resolution. KCl-induced hyperexcitability is a commonly used approach to study aberrant neuronal activity in vitro, while TTX/5AP provides a baseline for comparison. Our computational analyses relies on a metacell-based approach (SEACells) to compare TTX/5AP and KCl conditions in human cortical organoids, minimizing technical noise and enhancing lineage-specific resolution. Pseudotime analysis allows us to capture gene expression shifts across progenitor stages, and by applying variational autoencoders (scPoli, scArches), we mapped organoid cell states to developing human cortex datasets to reveal parallel trajectories in progenitor cell differentiation and neuronal maturation.Our preliminary analyses highlighted selective susceptibility of specific lineages under sustained activity, suggesting dynamic changes in early cell fate. Pseudotime analysis revealed key gene expression shifts in progenitor and immature neuron populations, while functional pseudo-bulk analysis identified putative immediate-response regulons. Overall, these findings reveal that KCl-induced activity modulates transcription factors and pathways crucial for cortical development and synaptogenesis. Integrating metacells differential expression and VAE comparative mapping, we uncover potential mechanisms linking hyperexcitability to neurodevelopmental disorders like infantile epilepsy. This framework integrates multi-omics data, enabling a deeper exploration of hyperexcitability’s regulatory architecture in organoids at single-cell resolution.

Speaker: Illia Simutin (Humanitas University)

18:30 Modeling Autosomal Dominant LeukoDystrophy pathology with human iPSC-derived glial cells: altered phenotypes and rescue strategies

"Autosomal Dominant Leukodystrophy (ADLD) is a rare genetic disease associated with white matter loss in the CNS and characterized by autonomic dysfunction and motor impairment. The genetic cause is the presence of three copies of the lamin B1 (LMNB1) gene, which encodes for a structural protein located in the nuclear lamina. Pathogenic mechanisms in ADLD have only initially been explored and a therapy to treat this disease is currently not available.
Based on evidence showing glial pathology in ADLD patients, we generated human glial cells from both ADLD patient- and healthy donor (CTRL)- derived human-induced pluripotent stem cells (hiPSCs), and specifically investigated ADLD astrocytes. Compared to CTRL cells, ADLD astrocytes displayed increased LMNB1 expression, at both RNA and protein level, and morphological and neurochemical cell alterations. Transcriptional profiling of the disease astrocytes pointed to functional defects in a number of key astrocytic functions comprising extracellular matrix composition, calcium signaling and mitochondrial metabolism. The analysis further revealed the acquisition of signs of cellular senescence and abnormalities in RNA processing. Importantly, ADLD Astrocyte Contidioned Medium affects murine and human oligodendrocytes survival and maturation, suggesting a detrimental effect of ADLD astrocytes on oligodendroglia. Moreover, ADLD astrocytes seeded on murine demyelinated organotypic cerebellar cultures have a detrimental effect on the re-myelination process and on myelin maintenance.
Finally, these detrimental effects on oligodendroglia and myelination were successfully reduced by using a specific RNA interference technique called Allele SPecific (ASP) RNAi, which selectively silenced the non-duplicated LMNB1 allele.
Our “disease-in-a-dish” platform reveals previously unknown ADLD astroglial dysfunctions, shedding light on their potential contribution to the white matter loss observed in the disease. Moreover, our results provide direct evidence that ASP RNAi can effectively target and alleviate ADLD pathology in human glial cells."

Speaker: Martina Lorenzati (Dipartimento di Neuroscienze "Rita Levi Montalcini", Neuroscience Instititute Cavalieri Ottolenghi)

18:30 Modeling patient-specific long-term neurological vulnerabilities to post-acute SARS-CoV-2 infection using patient-derived models

Long COVID refers to persistent symptoms lasting over a year after a SARS-CoV-2 infection, with no other underlying cause. These include neurological symptoms (NeuroCOVID) such as memory disorders and concentration impairment, which disrupt daily life and can persist even after other viral symptoms resolve. The NeuroCOV Consortium was established to address the emerging clinical need to investigate the long-term neurological impacts of COVID-19. As part of this consortium, our aim is to decipher the cellular and molecular mechanisms underlying the diverse NeuroCOVID trajectories using patient-derived immunocompetent brain organoids. As a first step to achieve this goal, we are coupling clinical metadata with transcriptomic profiles at single cell resolution from peripheral blood mononuclear cells (PBMCs) of patients belonging to two European cohorts (from Italy and Germany) to perform a deep phenotyping of the patients to identify key molecular signatures associated with their neurological profiles. Using that information, we are generating a NeuroCOVID specific human induced pluripotent stem cells (hiPSCs) cell bank that will allow us to ensure representation across the spectrum of neurological manifestations when differentiated to brain organoids (BOs). Additionally, we are using a combination of small molecules to fine-tune a previously described protocol in our lab (Caporale et al. 2024) that will allow us to obtain organoids with a more matured phenotype to produce a scalable model applicable to cohorts’ studies. Immunostainings to assess neuronal population markers (SATB2, TBR1, TBR2, among others) were performed on cortical BOs at different timepoints (D25, 38 and 50) and calcium dynamics were assessed by Fluo-4 probe based imaging. Finally, to dissect neuronal and immune factors contributing to NeuroCOVID in a post SARS-CoV-2 infection setting, we are comparing two hiPSC derived microglia protocols to establish their reproducibility and robustness in different cell lines to generate both patient-derived microglia and cortical BOs in order to subsequently perform an integrated omics approach that will allow us to investigate the cellular and molecular mechanisms underlying neuroinflammation and neurodegeneration in Long COVID, with the ultimate goal of identifying potential drivers of NeuroCOVID vulnerability and resilience.

Speaker: Luciana Isaja (Fondazione Human Technopole)

18:30 Modelling dopamine/α-synuclein interplay in Parkinson’s disease using differentiated SH-SY5Y cells

PD is characterized by the degeneration of dopaminergic neurons of the Substantia Nigra pars compacta (SNpc), disruption of dopamine (DA) homeostasis, and the formation of Lewy bodies (LB), intracellular protein inclusions primarily composed of α-synuclein (α-syn). Alpha-syn plays a key role in synaptic function regulating vesicle trafficking, membrane dynamics, and neurotransmitter release. The mechanisms underlying α-syn aggregation are not fully understood, but it is believed that α-syn undergoes a multistep aggregation process, forming toxic oligomers that disrupt DA homeostasis by damaging DA-containing vesicles. The SH-SY5Y cell line, derived from human neuroblastoma cells, is a commonly used model for studying neurodegenerative diseases like Parkinson’s disease (PD). However, differentiating SH-SY5Y cells into mature dopaminergic neurons remains a challenge due to the lack of a standardized protocol. Although several differentiation methods exist, their efficacy and reproducibility are often limited. To address this, we modified a previously proposed protocol to develop a more reliable and effective method for differentiating SH-SY5Y cells into dopaminergic neurons. This new protocol provides a robust model to investigate the molecular mechanisms underlying PD pathology. More specifically, we aimed at developing a cellular model to investigate how the interplay between DA and α-syn may guide the aggregation and spreading pathways. First, in-vitro α-syn aggregation studies were undertaken to explore the effects of DA-induced modifications on oligomers. Then, using our differentiated dopaminergic SH-SY5Y neurons, we investigated α-syn uptake, release, and cell-to-cell spreading in trans-well co-cultures exposed to fluorescently labelled recombinant α-syn protein monomers and oligomers. Eventually, we started the setup of methods to explore the effects of DA-induced modifications on α-syn seeding and spreading in three-dimensional cellular models (midbrain organoids). Collectively, our findings offer new insights into the formation and stabilization of α-syn oligomers in the presence of DA, contributing to a deeper understanding of the molecular mechanisms driving PD.

Speaker: Francesca Martorella (University of Insubria)

18:30 Modelling Sex Bias and Tumor Initiation of Group 3 and Group 4 Medulloblastoma in Human Cerebellar Organoids

Medulloblastomas are malignant cerebellar tumors that represent approximately 20% of all pediatric brain tumors. Molecular profiling categorizes these tumors into four subgroups: WNT, SHH, Group 3 (G3), and Group 4 (G4). G3 and G4 tumors account for more than 65% of pediatric cases and exhibit the highest rates of metastasis, relapse, and mortality. Current models predominantly reflect G3 tumors, inadequately capturing the biological complexity and diversity of G3 and G4 medulloblastomas. These tumors likely originate from excitatory progenitors of the rhombic lip, a structure with developmental differences between mice and humans, potentially explaining the limited efficacy of murine models. To develop accurate human-specific models of medulloblastoma tumorigenesis, we differentiated iPSCs into cerebellar organoids and validated their cerebellar identity and cellular diversity. Remarkably, G3 and G4 medulloblastoma tumors predominantly affect boys over girls, indicating a potential role of sex hormones in tumor initiation. Supporting this, recent findings by Kelava et al. (2022) demonstrated that male sex steroids (androgens) enhance the proliferation of excitatory neurogenic progenitors in cerebral organoids. Consequently, increased proliferation of rhombic lip progenitors in males could elevate their susceptibility to oncogenic events and malignant transformation. To investigate this hypothesis, we engineered male and female iPSC lines with an mNeonGreen reporter downstream of transcription factors specific to G3 (LMX1A) and G4 (EOMES) progenitor populations. Differentiation of these lines into cerebellar organoids followed by androgen treatment significantly increased the proportion of LMX1A+ and EOMES+ progenitors, confirming androgen-driven expansion of these rhombic lip progenitors. Currently, we are evaluating the tumorigenic potential of these progenitors through lineage-specific, conditional activation of oncogene combinations identified in G3/G4 medulloblastoma patients. These novel organoid models offer valuable tools for investigating human-specific mechanisms of G3/G4 medulloblastoma initiation, facilitating therapeutic discovery and improving outcomes for pediatric patients.

Speaker: Frederik Arnskötter (German Cancer Research Center (DKFZ))

18:30 Molecular diversity of adult mouse cerebellar astrocytes

In the mature cerebellum astrocytes with different morphologies occupy distinct cerebellar territories. This enables their classification into 4 distinct types based on their morphology and topographical localization. Such a well-defined classification offers an excellent platform to challenge the concept of astrocyte heterogeneity as well as to attempt cross-correlating the multilevel expression of such diversity.
Through single-cell RNA sequencing of the adult mouse cerebellum, we shed light to an unprecedented degree of transcriptional heterogeneity within cerebellar astrocytes and uncovered the transcriptional profiles of nine distinct astrocyte subtypes, whose regional segregation was unveiled by spatial transcriptomics.
Many of these subtypes showed strong enrichment in one cerebellar layer, enabling us to confirm the correspondence between morphologically and topographically cell classes and transcriptional heterogeneity. Of note, among the four identified BG subtypes, two showed complementary distributions along the anteroposterior axis. Some subtypes instead showed enrichment in multiple layers. Among them, a subtype localized in the Purkinje cell layer and in the white matter showed transcriptional features similar to progenitors. Another subgroup of cells, spanning both the cerebellar cortex and white matter, showed an enrichment in terms suggesting their involvement in vesicular gliotransmission and synaptic communication. While these clusters highlighted specialized functional traits, the overall transcriptional heterogeneity among all nine profiles underscored a predominant broad functional divergence between BG and non-BG astrocytes. BG transcriptional signature confirmed their specialization in interacting with Purkinje cells and modulating their synaptic inputs, while the other non-BG astrocytes displayed enrichment for genes that pointed to their novel potential roles in energy homeostasis via regulation of blood supply,glucose and glycogen metabolism and in neuroprotection.These findings set the stage for investigating cerebellar astrocyte heterogeneity in diseased conditions and for comparative studies between mouse and human cerebellum.

Speaker: Giacomo Turrini (University of Turin)

18:30 Molecular mechanisms of neurodevelopmental SPTBN1 syndrome

βII-spectrin, encoded by SPTBN1, binds F-actin to form a submembrane cytoskeletal lattice that organizes transmembrane proteins in neurons. We discovered that heterozygous SPTBN1 variants cause a neurodevelopmental disorder marked by developmental delay, intellectual disability, ASD, ADHD, epilepsy, and cortical deficits –SPTBN1 syndrome. Brain βII-spectrin haploinsuficient mice display developmental delays as well as cortical development and behavioral deficits in line with patients’ presentations, supporting a loss-of-function pathogenic mechanism. Notably, most probands carry missense variants within the N-terminal calponin homology domains (CHDs), which bind F-actin. However, the unique and/or shared mechanisms through which CHD variants cause these morphological alterations and impact downstream neuronal processes are unknown.
Here, we combine studies in stem cell-derived human neurons, mouse models and heterologous systems to investigate the molecular and cellular effects of SPTBN1 CHD syndrome variants. We find across disease models that endogenous heterozygous expression of a subset of these variants cause protein aggregates, which sequester wild-type and mutant βII-spectrin and other key protein partners. This alters the neuronal proteome and affect the integrity of the axon initial segment. Knock-in mice expressing aggregation-prone disease variants exhibit disruptions in cortical and cerebellar development and behavioral deficits that recapitulate patients’ clinical presentations. We demonstrate that βII-spectrin aggregates recruit the proteostasis machinery and are susceptible to targeted degradation by disaggregases and pharmacological chaperone activators. These findings offer potential therapeutic strategies to restore βII-spectrin function.

Speaker: Damaris Lorenzo (University of Pennsylvania School of Medicine)

18:30 Multiomic Analysis Reveals a Regulatory Network of miRNAs, circRNAs, and genes in Proneural Glioblastoma

Glioblastoma (GBM) is an aggressive malignant tumor arising from neuroglial progenitor cells, characterized by its remarkable ability to infiltrate healthy brain parenchyma. Multiomic studies have revealed a complex heterogeneity, identifying three distinct GBM subtypes: proneural, mesenchymal, and classical. In this study, we focused exclusively on the proneural subtype, using multiomics patients data from Wang et al. 2021 and LSD1 ChIP-Seq data (Faletti et al. 2021) in GBM#22 TICs, a tumor stem cell (TIC) model derived from a GBM proneural patient.
LSD1 is a significant epigenetic regulator in GBM TICs and its possible molecular mediators are miRNAs, a class of short non-coding RNAs with great importance and regulatory roles in tumors. Recently, also circRNA roles in cancer are acquiring more importance.
We performed an integrative multiomics analysis to identify a regulatory network composed by miRNAs, circRNAs and genes, also defining miRNAs and genes characterized by LSD1 peaks in their promoter regions, in order to understand their involvement in the proneural subtype.
MiRNA-Seq, RNA-Seq, and circRNA-Seq data from Wang et al. 2021 were analyzed, identifying differentially expressed miRNAs (DEMs) and genes (DEGs) between GBM proneural samples and normal samples. A Spearman anticorrelation analysis (p-value <0.05, rs < -0.6) between DEMs and DEGs was performed considering only miRNA-gene Targetscan interactions, identifying 42 significant interactions corresponding to 19 DEMs and 40 DEGs. We identified 225 circRNAs targeting these 19 DEMs using the CSCD2 database. Lastly, we verified the LSD1 presence in the DEM and DEG promoter regions and we found that 15 out of 19 DEMs and 26 out of 40 DEGs showed a LSD1 peak around their promoters.
In conclusion, we provide for the first time a regulatory network involving circRNAs in patients with proneural GBM, providing a set of regulatory interactions with miRNAs and protein coding genes that could be experimentally explored.

Speaker: Teresa Gravina (University of Piemonte Orientale)

18:30 Multiomic features associated with risk genes for epilepsy

Genomic studies have identified an ever-expanding set of de novo gene mutations in Epilepsy Risk Genes (ERGs) conferring high risk for neurodevelopmental epilepsy. ERGs are known to converge on synaptic and ion channel pathways in neurons, but we currently lack a systematic annotation of their functional genomic features.

To address this, we collated twelve diverse multiomic data sets spanning diverse spatiotemporal scales of the human brain. Ten were whole-neurogenomic transcriptomic studies, capturing expression patterns across brain regions, embryonic to adult development, cell types, and subcellular structures; one capturing reported direct protein-protein interactions between proteins coded for by the genes; one with metrics of intolerance to loss-of-function mutations. We included 18,450 protein-coding genes which were present in at least 6 of the 12 datasets. 557 (3%) are identified as ERGs because single-nucleotide variants have been linked to epilepsy risk.

Comparison of ERGs with null gene sets recovered their relative intolerance to loss of function mutations and their enrichment in gene sets marking inhibitory neurons, but also revealed several other novel associations. For example, regional expression is highest in the frontal and temporal cortices, especially in neonatal samples. At a cellular level alongside GABA-ergic interneurons, expression was high in intratelencephalic excitatory projection neurons in cortical layers 2 to 4. Subcellularly, ERG proteins mostly localised to the cellular membrane and mitochondria, consistent with previous protein function studies. At the scale of protein-protein interactions, ERGs have significantly higher degree and are more commonly connected with other ERGs, particularly SCN1A, SCN2A, CACNA1A, but with notable neuro-related non-ERGs (e.g. CREB1, CAMK2A, TH).

This work provides an integrative view of the genomic features that distinguish ERGs - pointing towards a multiscale signature threading through cortical regions, layers, cell-types compartments and biological pathways, informing mechanistic models and setting the stage for deeper predictive modelling.

Speaker: Jack Highton (King's College London)

18:30 Multiscale Insights into BBSOAS: Linking NR2F1 Mutations to Brain Development and Mitochondrial Dysfunction

Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS; OMIM 615722) is a rare neurodevelopmental disorder caused by mutations in NR2F1, a transcriptional regulator essential for brain and visual system development. Patients display a broad clinical spectrum—including intellectual disability, optic atrophy, autistic traits, and hypotonia—reflecting NR2F1 pleiotropic roles and suggesting genotype-phenotype correlations. Most mutations lead to haploinsufficiency or dominant-negative effects that impair NR2F1 transcriptional activity.

To uncover the molecular underpinnings of BBSOAS, we applied a multidisciplinary approach combining structural bioinformatics, genetic manipulation, patient-derived 3D cerebral organoids, animal models, imaging, and -omics analyses. We first characterized how specific NR2F1 point mutations alter protein structure, DNA binding, and localization, linking these defects to cortical malformations in mouse models and clinical neuroimaging. These studies highlight region-specific roles for NR2F1 in regulating neural stem cell maintenance and cortical folding.

In parallel, we identified a novel role for NR2F1 in mitochondrial regulation. Transcriptomic and proteomic profiling revealed that NR2F1 controls a network of nuclear-encoded mitochondrial genes, affecting mitochondrial mass and morphology. In Nr2f1-heterozygous mice, we observed significant disruption of mitochondrial pathways and reduced levels of key mitochondrial proteins in the brain. We are now extending these findings to human iPSC-derived telencephalic neurons. Preliminary imaging data suggest increased mitochondrial fragmentation in NR2F1-deficient human neurons, pointing to mitochondrial dysfunction as a key pathological mechanism in BBSOAS.

Altogether, our work shows that NR2F1 orchestrates both developmental and metabolic programs, with its dysfunction leading to impaired cortical architecture and mitochondrial homeostasis. This integrative framework advances our understanding of BBSOAS pathophysiology and supports the development of genotype-driven therapeutic strategies.

Speaker: MICHELE STUDER (Institut de Biologie Valrose - University Côte d'Azur)

18:30 Neural organoids as a model to track the formation and maturation of dendritic spines

Neural organoids provide a great tool to decipher human brain diseases at the molecular and physiological levels. The organoids are of great value in understanding neurodevelopmental diseases, as they can reflect changes occurring even in the prenatal period. Those diseases are often accompanied by aberrant changes in the formation of dendritic spines harboring excitatory synapses. Yet, none of the studies focused on detailed characterization of dendritic spines in organoids. Herein, we present the novel protocol to visualize and characterize single dendritic spines in matured organoids.
Human induced pluripotent stem cells were differentiated to cortical spheroids. On subsequent stages of development organoids were evaluated by whole organoids’ imaging and western blot analysis. Till day 200 the organoids were evaluated for proper differentiation: rosettes formation, cortex layering and glial/neuronal differentiation. After day 200 organoids were evaluated for their maturation properties. Live calcium imaging was performed to analyze the spontaneous activity of cells. Next, organoids older than one year were evaluated for dendritic spines formation. Spines were characterized using biolistic delivery of lipophilic dye combined with subsequent immunolabeling of pre- and postsynaptic markers.
We show that organoids’ maturation can be manifested by spontaneous activity of neurons with visible synchronization. This maturation is accompanied by changes in expression of repertoire of synaptic-related proteins (glutamate receptors, postsynaptic scaffolding proteins). Importantly, we were able to optimize protocol to successfully visualize dendritic spines in neurons within organoids. Furthermore, we were able to immunolabel dendritic spines with antibodies directed to proteins forming either pre- or postsynaptic compartments. This method enables a more detailed characterization of complex dendritic spine structure and function in human neurons in both health and disease.

Speaker: Bogna Badyra (Nencki Institute of Experimental Biology PAS)

18:30 Next-Generation Electrophysiology for Functional Characterization of Human Neural Organoids and Assembloids

"Three-dimensional neural models derived from human-induced pluripotent stem cells (hiPSCs), such as organoids and assembloids, are essential tools for replicating key features of human brain development. They play a vital role in advancing research on neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Real-time, label-free measurement of electrical activity is needed for unraveling the intricate dynamics of the neuronal networks formed within these self-organizing in-vitro systems.
High-density microelectrode arrays (HD-MEAs) provide a powerful, non-invasive platform for high-resolution electrical imaging, enabling real-time recordings of electrical signals from a wide range of electrogenic samples, including neural organoids, assembloids, and brain or retinal tissue slices. In this study, we employed the MaxOne and MaxTwo HD-MEA platforms, featuring 26.400 electrodes per well, to record extracellular action potentials from 3D neural models across multiple levels of resolution, spanning entire networks to individual cells and subcellular structures. We demonstrated the systems’ flexible electrode selection, leading to enhanced reproducibility and statistical power of the collected data. Extracted and analyzed parameters included firing rate, spike amplitude, and network burst profile.
To further explore subcellular dynamics, the AxonTracking Assay was used to trace the propagation of action potentials along axonal branches, enabling precise investigation of axonal properties, such as conduction velocity, latency, axonal length and branching patterns. This revolutionary assay offers a high-resolution approach for studying disease models focusing on axon initial segment, axonal development, branching and conduction.
The capacity of these HD-MEA platforms to selectively target specific electrodes enhances the quality of the collected data while ensuring higher reproducibility. Together with the integrated tools for automated data visualization and metric extraction, the here presented systems offer a user-friendly and robust platform for disease modelling and drug testing, supporting both acute and long-term electrophysiological studies."

Speaker: Silvia Oldani (MaxWell Biosystems)

18:30 Non-coding structural variants identify a commonly affected regulatory region steering FOXG1 transcription in early neurodevelopment

The FOXG1 transcription factor is a crucial regulator of embryonic brain development. Pathogenic FOXG1 variants cause FOXG1 syndrome. Although structural variants (SVs) in the non-coding region downstream of FOXG1 have been reported in 38 individuals with similar characteristics, the regulatory pathomechanisms remain unknown.

We identified a de novo non-coding deletion in an individual with FOXG1 syndrome-like features, allowing us to delineate a ~124 kb commonly affected regulatory region (CARR). By integrating epigenomics data, 3D chromatin interaction profiles (Hi-C, UMI-4C), and in vivo enhancer assays in zebrafish, we uncovered multiple regulatory elements within this CARR, including a neuronal enhancer cluster and a conserved boundary of the FOXG1-containing topologically associating domain (TAD). Hi-C analysis on case lymphoblastoid cells revealed increased interactions with the adjacent TAD. Moreover, sequential UMI-4C and CUT&RUN assays during neural progenitor cell (NPC) differentiation demonstrated dynamic activation of, and interaction with the enhancer cluster. Finally, CRISPR-Cas9 deletion of the enhancer cluster and TAD boundary in NPCs resulted in decreased FOXG1 transcription.

We identified and characterized enhancer and architectural elements essential for proper FOXG1 transcription. Our findings provide new insights into chromatin architecture and gene regulation at the FOXG1 locus, improving interpretation of SVs in this region.

Speaker: Eva D'haene (Ghent University)

18:30 OPEN-array: a simple and powerful tool to increase the throughput of imaging-based organoid characterization

The advent of three-dimensional (3D) biological models, particularly organoids, has revolutionized biomedical research by providing physiologically relevant in vitro models that mimic the complexity of human tissue and organs, samples that may not be routinely available to study. Amongst the fields more impacted by this revolution is neuroscience, thanks to the development of several protocols of brain organoids resembling cell types from different regions of the brain. These models can be produced in large quantities by individual researchers from multiple patients’ hiPSC lines or to test several conditions in parallel. Given the peculiarities of organoid models, such as the heterogeneity of cell types and their spatial patterning, their characterization often necessitates imaging-based immunophenotyping or transcriptional characterization via RNA fluorescence in situ hybridization (RNA-FISH). However, researchers lack access to automated systems, and high-content screening equipment requires accessible, off-the-shelf, open methodologies to facilitate and accelerate sample acquisition and analysis. In this study, we introduce OPEN-array, a user-friendly and cost-effective pipeline designed to enhance the throughput of array-wise histological characterisation, from the automatic identification of organoids’ position on the array up to nuclei segmentation and the measurements of morphological and intensities properties.
We demonstrate its use by analysing microarrays of cortical brain organoids (CBO) exposed to GSK343 and XAV939 at two different concentrations and their combinations, with the goal of identifying markers of an enhanced maturation in the exposed organoids with respect to a previously established protocol for CBO generation.

Speaker: Alessia Valenti (Human Technopole)

18:30 Optimized hESC-based approaches for restoring striatal function in Huntington’s Disease: insights into in-vivo cellular identity and functionality

"Huntington’s disease (HD) is a devastating neurodegenerative disorder characterized by the progressive loss of medium spiny neurons (MSNs) in the striatum, leading to severe motor, cognitive, and psychiatric impairments. As no disease-modifying treatments are currently available, cell replacement therapy has emerged as a potential promising approach to restore striatal function.
From this perspective, developing an optimized differentiation protocol is key to generating the most effective cell product from human embryonic stem cells (hESCs), capable of functionally replacing degenerated striatal neurons upon transplantation. Equally crucial is ensuring their integration into host circuits to restore lost connections and regain function.
In this study, we employed multiple approaches to explore the characteristics and behavior of second generation hESC-derived striatal neurons transplanted into the striatum of an HD rat model.
Through snRNA-seq analysis, we identified the specific cellular composition of our transplants, uncovering a diverse mix of neuronal and non-neuronal populations typically found in the striatum, including MSNs, interneurons, and astrocytes.
To study the graft activity and its integration into host circuitries, we further engineered the cells for chemogenetic modulation to induce detectable phenotypic changes in behavioral tests. The obtained Bi-DREADD cell lines responded properly to different ligands in vitro. We also demonstrated that by 6 months post transplantation, grafted cells are functionally active and capable of responding to external stimuli.
Collectively, these findings highlight the robustness of our transplant, offering a wealth of new insights into the therapeutic potential of stem cell-derived cell replacement approaches for HD."

Speaker: linda scaramuzza (Università degli studi di Milano)

18:30 Patient-derived cortical organoids to decode HCN1 mutations in Developmental and Epileptic Encephalopathy 24

Epileptic disorders represent high burden diseases characterized by recurrent and unprovoked seizures, which are due to the excessive and hypersynchronous discharge of neuronal networks. Strikingly, 75% of epilepsies arise during childhood, such as developmental epileptic encephalopathies (DEEs), which are genetic conditions characterized by recurrent and drug-resistant seizures and by cognitive and developmental delays. Recently, de novo mutations in the gene encoding for hyperpolarization-activated cyclic nucleotide gated channel 1 (HCN1) have been associated with DEE24, representing a rare and severe DEE form. Noteworthy, HCN1 is present in cortical neurons, where it conducts an inward depolarizing current that contributes to the maintenance of their resting membrane potential. Even though mouse models for Hcn1 mutations have been generated, patient-specific in vitro models able to recapitulate the genetic landscape and human brain development are still missing. To this aim, we managed to reprogram somatic cells from three patients bearing different HCN1 mutations into human induced pluripotent stem cells (hiPSCs) and to differentiate them into cortical organoid models. Aiming at uncoupling the effects of HCN1 mutations on the phenotype from the patient-specific genetic background, we also leveraged isogenic hiPSCs lines genetically edited to insert the mutations under investigation. Interestingly, we observed that patient-derived cortical organoids present significantly decreased size compared to their wild-type (WT) counterpart, thus recapitulating the microcephalic phenotype observed in DEE24 patients. Preliminary analysis revealed profound alterations in neural progenitors of both patient-derived and genetically edited cortical organoids compared to WT samples. Strikingly, HCN1 mutant organoids displayed epileptic-like high-frequency calcium waves compared to their WT counterpart by calcium imaging. We are going to identify the molecular signature of DEE24-derived cortical organoids through transcriptomic analyses. Ultimately, our work will contribute to unveil the underlying pathogenic mechanisms and the genotype-phenotype relationship in DEE24, unprecedently providing personalized models to test potential therapies for this incurable disease.

Speaker: Elena Florio (Humanitas University)

18:30 Patient-specific mechanisms in SYNGAP1 syndrome: toward targeted therapeutic strategies

"SYNGAP1 syndrome is a rare neurodevelopmental disorder caused by heterozygous de novo mutations in the SYNGAP1 gene. Affected individuals present with a complex clinical phenotype, including intellectual disability, developmental epileptic encephalopathy, autism spectrum disorder, and other comorbidities. SYNGAP1 encodes SynGAP, a Ras GTPase-activating protein critical for excitatory synapse formation, maturation, and plasticity. Beyond its well-established synaptic role, emerging evidence suggests a broader function in early brain development. However, the mechanisms linking SYNGAP1 dysfunction to the clinical heterogeneity remain poorly understood.
We hypothesize that SYNGAP1 variants of different nature may underlie the diverse spectrum of neurodevelopmental impairments observed in patients. Investigating patient-specific mutations may provide insights into genotype–phenotype correlations and support the development of personalized therapeutic strategies.
To address this, we reprogrammed fibroblasts from patients carrying two distinct SYNGAP1 mutations into induced pluripotent stem cells. From these lines, we generated both 2D and 3D in vitro models to recapitulate early stages of brain development and study neural circuit formation. Our analyses revealed that the two mutations differentially affect SYNGAP1 mRNA stability. Using early brain organoids, we observed variant-dependent alterations in neural rosette organization and progenitor proliferation. In parallel, we derived 2D cortical neurons and tracked network maturation over time, detecting an accelerated development of neuronal circuits in SYNGAP1-mutant lines. Additionally, we identified changes in mitochondrial morphology and dynamics during early neurodevelopment, suggesting a possible role for SYNGAP1 in regulating mitochondrial function.
Together, our findings point to both synaptic and metabolic contributions to SYNGAP1-related pathophysiology and support the use of patient-specific stem cell models to unravel mutation-specific mechanisms. This approach offers a promising avenue toward the development of targeted, stratified therapeutic strategies."

Speaker: Bernadette Basilico (Università Sapienza di Roma)

18:30 Placenta-brain axis and neurovascular communication during mammalian brain development

The bidirectional communication between placenta and embryonic brain during pregnancy, known as placenta-brain axis, is an important aspect of embryonic development in placental mammals, including humans. Recently we have shown that the maternal environment of pregnancy dominantly regulates the length of neurogenesis in the embryonic brain. The brain vascular system is a key intermediate to relay this dominant regulation. Additionally, the molecular signaling from the cells forming the brain vasculature – endothelial cells and perivascular cells – include several other key factors that mediate neurovascular interactions to ensure proper nutrient delivery, neuronal differentiation, and blood-brain barrier formation. Disruption in placenta-brain axis and/or neurovascular communication can therefore lead to neurodevelopmental disorders, thus underscoring their fundamental role in brain morphogenesis. A deeper understanding of these bidirectional communications can offer valuable insights into congenital brain anomalies. We are using our previously developed inter-strain embryo transfer system as a tool to understand how placenta-brain axis and neurovascular communication regulate embryonic brain development.

Speaker: Samir Vaid (Neurologischen Klinik, Universitätsklinikum Carl Gustav Carus Dresden, Technische Universität Dresden)

18:30 PRC2 gatekeeps the balance between direct and indirect neurogenesis in human corticogenesis

"Weaver syndrome (WVS) is a multisystem disorder, characterized by pre- and post-natal overgrowth and intellectual disability. WVS genetic cause was identified in heterozygous mutations in Polycomb repressive complex 2 (PRC2). This complex catalyzes the tri-methylation of Lysine 27 on histone 3 (H3K27me3), promoting transcriptional repression during corticogenesis. Our knowledge about the impact of PRC2 misfunction in corticogenesis and the impact of the WVS heterozygous mutations on the landscape of H3K27me3 and the resulting transcriptomic changes are incomplete and based on mouse data. To better understand the molecular mechanisms underlying WVS, we conducted transcriptomic and epigenomic analyses of patient-derived cortical brain organoids (CBOs) cultured for up to 250 days in vitro.
Our multi-omic approach integrated bulk and single-cell RNA sequencing, EZH2 and H3K27me3 genomic distribution mapping and DNA methylation profiling. These analyses revealed disruptions in neuronal maturation and migration from day 25 to 250 of differentiation. We observed temporally regulated gene expression defects and asynchronous cell type emergence in WVS, particularly affecting indirect neurogenesis. By identifying specific transcription factors responsible for cell-type dysregulation, we highlighted defects in cell fate commitment and migration. This finding is consistent with clinical data from WVS patients, whose MRI profiles show altered cortical development associated with defects in later stages of neuronal migration. Moreover, we identified AJAP1 as upregulated in WVS CBOs at day 25. AJAP1 cooperates with multiple proteins to organize the adherens junctions belt, potentially determining radial glial cell fate. We developed a CRISPRa system to upregulate AJAP1 in control CBOs highlighting its relationship with EZH2 in neuronal migration. This study provides the first comprehensive molecular characterization of PRC2's role in human cortical development and its involvement in neuronal migration, addressing a significant knowledge gap. Our findings advance the understanding of WVS pathogenesis and establish new resources for investigating neurodevelopmental disorders."

Speaker: Martina Pezzali (Fondazione Human Technopole)

18:30 Presenting the proposed project: The gut microbiota in the first 1000 days of life and its relationship to a healthy neurodevelopment in child

Among the myriad captivating relationships forged by the gut microbiota (GM) within the human body, one of the most intriguing and intricate is the connection between the gut and the brain. This pathway has garnered increasing interest in recent years as a potential mechanism involved in the complex interplay of processes related to neurodevelopment, especially in critical periods as the ‘first 1000 days of life’, when neuronal plasticity is heightened (rendering being exceptionally susceptible to any form of stimulation) and blood-brain barrier and intestinal tight junctions are still developing and more permeable. Neurodevelopmental models are primarily animal-based or epidemiological studies, but the recent advancement of organoid technology has opened up incredible possibilities for research in this field.
The aim of this PhD project is to deepen the study of the role of metabolites derived from the gut microbiota on neurodevelopment. The study consists of two main parts: i) a prospective observational study will be conducted using faecal samples from children at risk for neurodevelopmental disorders (preterm) and control children (full-term), from birth up to 2 years of age. The goal is to correlate microbiota composition with neurodevelopmental outcomes (assessed over a 2-year period) and inflammatory markers; ii) brain organoids will be generated and co-cultured with metabolites derived from in vitro fermentation inoculated with the infant faecal samples, to investigate the effect of the microbiota metabolites on the neurodevelopment, using the organoid model.
The project has started with contacting hospital institutions and preparing the necessary documentation for ethics committee approval for sample collection. The immediate activity will be to proceed with in vitro fermentation of fecal samples and treatment of cell cultures (2D and 3D neural cultures derived from hPSCs) with fermentation fluids and selected bacterial metabolites.

Speaker: Lucia Occhigrossi (Università degli Studi di Camerino)

18:30 Reconstruction of cortical development through multi-modal epigenetic perturbation in organoids

"The development of the human cortex entails a complex series of cell fate decisions that are both genetically encoded and epigenetically orchestrated. Understanding how these mechanisms work at the single-cell level is crucial for deciphering the molecular basis of neuronal development and disease. In this context, we investigate the role of chromatin modulators in controlling neural cell identity while capturing their impact on the complex regulatory landscape characterizing human cortical maturation. We explore the potential of pooled CRISPRi-based perturbation screening coupled with single-cell transcriptomics and epigenomics to identify key regulators of neural fate decisions during cortical organoid development.
We engineered and validated an iPSC line that harbors an endogenously inducible CRISPRi cassette, along with a dual-sgRNA lentiviral library to establish time-dependent and targeted perturbations of chromatin remodellers. Subsequently, we will harness this system to generate cortical organoids and interrogate the effects of chromatin remodellers disruption on developmental trajectories by defining gene expression, accessibility and perturbation profiles at single-cell level.
The objective of our study is to outline and functionally validate the fundamental role of chromatin modifiers acting in the main patterning stages of the developing human cortex, and particularly how transcription factors interact and ultimately shape the gene-regulatory network required for neurodevelopment in health and disease states."

Speaker: Ludovico Rizzuti (Helmholtz Munich)

18:30 Reduced H2AK119ub levels during early neurodevelopment sensitise the genome to ectopic transcription factor-mediated gene activation

During brain development, Polycomb group proteins regulate the spatiotemporal expression of neurodevelopmental genes to control cell fate decisions. Polycomb repressive complex 1 (PRC1) is a key regulator of neural development and is frequently deregulated in neurodevelopmental disorders. PRC1 mediates gene repression through both its catalytic activity (H2AK119ub deposition) and non-catalytic functions (chromatin architecture modulation) in neural stem and progenitor cells. Here, we investigate the molecular and developmental consequences of catalytically hypomorphic RING1B (Ring1bI53A/I53A), the core PRC1 subunit. Impaired PRC1 ubiquitin ligase activity leads to a global reduction in H2AK119ub, resulting in the ectopic expression of lineage-inappropriate PcG target genes. This effect is observed during neuroectodermal specification in both in vitro neural progenitor differentiation and ex vivo primary neural stem cells (E10.5), with the emergence of mesendodermal gene expression. Hypomorphic RING1B also causes morphological defects in E12.5 mouse embryos, including a reduction in the size of the telencephalon and a corresponding reduction in the population of SOX2-positive stem cells. Thus, PRC1 ubiquitin ligase activity is essential for maintaining neural progenitor cell identity, and its impairment leads to forebrain developmental abnormalities, including a loss of apical progenitors in the ventricular zone. Next, we aimed to determine whether global loss of H2AK119ub sensitizes cells to mis-activation by lineage-inappropriate transcription factors. Using a TALE-based approach with the transcriptional activator VP64, we have activated master developmental transcription factors in wild-type cells, partially recapitulating the transcriptional and developmental consequences of H2AK119ub loss. These findings suggest that PRC1-mediated H2AK119ub is required to safeguard neural lineage integrity by preventing inappropriate gene activation. Disruptions in this mechanism may contribute to the aetiology of neurodevelopmental disorders, highlighting the importance of epigenetic regulation in early brain development.

Speaker: Lucy Doyle (University of Edinburgh)

18:30 Regulatory logic of human cortex evolution by combinatorial perturbations

Comparative genomic studies between contemporary and extinct hominins revealed key evolutionary modifications, but their number has hampered a system level investigation of their combined roles in scaffolding modern traits. Through multi-layered integration we selected 15 genes carrying nearly fixed sapiens-specific protein-coding mutations and developed a scalable design of combinatorial CRISPR-Cas9 bidirectional perturbations to uncover their regulatory hierarchy in cortical brain organoids. Interrogating the effects of overexpression and downregulation for all gene pairs in all possible combinations, we defined their impact on transcription and differentiation and reconstructed their regulatory architecture. We uncovered marked cell type-specific effects, including the promotion of alternative fates and the emergence of interneuron populations, alongside a core subnetwork comprising KIF15, NOVA1, RB1CC1 and SPAG5 acting as central regulator across cortical cell types.

Speaker: Alessandro Vitriolo (Fondazione Human Technopole)

18:30 RNA ligase 1 contributes to neuronal resilience and sensory function in zebrafish

RNA ligases play critical roles in RNA repair, splicing, and editing, but their significance in vertebrate neural development remains poorly understood. Here, we investigated RNA ligase 1 (Rlig1), a recently characterized 5’-3’ RNA ligase, using zebrafish (Danio rerio) as an in vivo model. We discovered that rlig1 mRNA is maternally deposited and later becomes enriched in the developing brain and eyes. While CRISPR/Cas9-mediated knockout of rlig1 did not lead to obvious morphological defects, mutant zebrafish exhibited impaired behavioral responses to visual stimuli. Consistent with these behavioral changes, in vivo calcium imaging revealed reduced neuronal activity in the pretectum and anterior hindbrain – key regions involved in visual processing. Notably, the number of motion-responsive neurons was significantly reduced in the anterior hindbrain of rlig1 KO larvae, while no reduction was observed in the pretectum. Despite the reduction in the anterior hindbrain, the direction selectivity of the responsive neurons was preserved. These findings suggest that Rlig1 may contribute to the establishment or maintenance of neuronal function during development. To further explore the molecular basis of this phenotype, we performed transcriptome analysis of larval heads. This revealed distinct genotype-specific expression changes in rlig1 KO larvae, including genes related to nucleotide metabolism, redox regulation, and regulated cell death pathways such as ferroptosis and necroptosis. These results suggest that Rlig1 may contribute to neuronal homeostasis during development, potentially by influencing transcript stability under metabolic or oxidative stress. By linking RNA metabolism to changes in both neural activity and gene expression, our findings open new perspectives on the molecular mechanisms that shape neural circuit functionality and sensory perception.

Speaker: Fiona Klusmann (Universität Konstanz)

18:30 RNA Sequencing Reveals a Strong Predominance of THRA Splicing Isoform 2 in the Developing and Adult Human Brain

Thyroid hormone receptor alpha (THRα) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development, metabolism, and reproduction. In mammals, THRα has two major splicing isoforms, THRα1 and THRα2. The better-characterized isoform, THRα1, is a transcriptional stimulator of genes involved in cell metabolism and growth. The less-well-characterized isoform, THRα2, lacks the ligand-binding domain (LBD) and is thought to act as an inhibitor of THRα1 activity. The ratio of THRα1 to THRα2 splicing isoforms is therefore critical for transcriptional regulation in different tissues and during development. However, the expression patterns of both isoforms have not been studied in healthy human tissues or in the developing brain. Given the lack of commercially available isoform-specific antibodies, we addressed this question by analyzing four bulk RNA-sequencing datasets and two scRNA-sequencing datasets to determine the RNA expression levels of human THRA1 and THRA2 transcripts in healthy adult tissues and in the developing brain. We demonstrate how 10X Chromium scRNA-seq datasets can be used to perform splicing-sensitive analyses of isoforms that differ at the 3′-end. In all datasets, we found a strong predominance of THRA2 transcripts at all examined stages of human brain development and in the central nervous system of healthy human adults.

Speaker: Eugenio Graceffo (Charité – Universitätsmedizin Berlin)

18:30 Shifting perspectives: the Impact of the Immune System on Neurodegeneration in Parkinson’s Disease

Parkinson’s Disease (PD) is a neurodegenerative pathology characterized by loss of dopaminergic neurons (DANs) of the substantia nigra, accumulation and aggregation of 𝜶-synuclein (αSYN), and neuroinflammation. Neuroinflammation is a risk factor to PD development, moreover the pathology is characterized by a correlation between levels of proinflammatory cytokines and disease severity, a consistent activation of microglia and recruitment of CD4+ and CD8+ T cells from the perifery since early stages of the disease. Studying the role of neuroinflammation in PD requires establishing its reproducibility across various mouse models, a fundamental step in the research process. Therefore, our group conducted an extensive comparative analysis of the levels of neurodegeneration, microglial activation and T lymphocytes infiltration levels in the substantia nigra pars compacta (SNpc) of different mouse models. These parameters were assessed by tyrosine hydroxylase (TH) staining for DANs counting, immunodecoration for microglial Iba1 activation marker and CD3, CD4 and CD8 to detect lymphocytic infiltrations. Specifically, we studied mice exhibiting pathology through αSYN-induced neurodegeneration. The overexpression of αSYN was obtained by local SNpc administration of αSYN preformed fibrils (PFFs) or delivery of the human wild-type SNCA gene by either lentiviral or adeno-associated viral vectors. Additionally, we utilized Cre-inducible models with DAN or microglial specificity for the expression of αSYN limited to specific cell types. We demonstrated a tight correlation between αSYN accumulation, neurodegeneration and neuroinflammation in most of the models. We also showed that accumulation of αSYN in glial cells rather than in DANs alone determined higher levels of neuroinflammation and neurodegeneration, highlighting the role of these cells in the pathogenic process. Furthermore, microglial activation and T lymphocytes infiltration precedes the loss of DANs in our LV-SNCA mouse model, showing how the immune response and neuroinflammation are key aspects to determine neurodegeneration and the development of the pathology.

Speaker: Alice Calderoni (San Raffaele Scientific Institute)

18:30 Study of the function of the natural antisense lncRNA PHOX2B-AS1 in 2D and 3D iPSc derived neuronal models of Congenital Central Hypoventilation Syndrome

Congenital Central Hypoventilation Syndrome (CCHS) is a rare life-long threatening genetic disorder caused by mutations in the PHOX2B gene, a master transcription factor of the autonomic nervous system (ANS). It is characterized by ANS dysfunctions, most importantly a deficient control of autonomic ventilation that leads to hypoventilation during sleep. In vivo and in vitro studies suggest that a loss of function mechanism, combined with a dominant-negative effect and/or toxic gain of function of the mutated proteins, is responsible for the entire disease spectrum. Since no pharmacological treatment has been identified, CCHS patients rely on mechanical ventilation for life support.
We have recently identified a natural antisense lncRNA (PHOX2B-AS1), transcribed in the opposite direction and partially complementary to the PHOX2B gene transcript, favoring PHOX2B translation.
Due to the unavailability of viable CCHS mice models to study the function of PHOX2B-AS1, we used reprogramming technology to generate induced pluripotent stem cells (iPSC) from patient’s fibroblasts carrying different PHOX2B mutations and we generated 2D autonomic neurons and 3D cerebral organoids with cytoarchitectures resembling central chemoreceptors from CCHS patients and controls to give insight into possible developmental defects at both peripheral and central level.
We found that PHOX2B-AS1 and PHOX2B gene expression is dysregulated at earlier developmental stages and that PHOX2B-AS1 splicing defects may be involved in CCHS pathogenesis. Overall our data support the possibility to modulate PHOX2B-AS1 expression as a new therapeutic strategy aimed at reducing the expression of mutant PHOX2B proteins.

Speaker: Simona Di Lascio (University of Milan)

18:30 SUPERNUMERARY X CHROMOSOMES SHAPE BRAIN ORGANOID ARCHITECTURE AND FUNCTIONS IN A DOSE-DEPENDENT FASHION

Klinefelter syndrome (KS, 47,XXY) is the most prevalent aneuploidy in males (1:400-1:600). High-grade sex chromosome aneuploidies (HGA-SCAs), such as 48,XXXY, and 49,XXXXY are rarer conditions occurring in 1:40.000-1:80.000 males. KS and HGA-SCA patients exhibit a broad spectrum of neuronal impairment, including cognitive deficits, seizures, autistic traits, and motor, speech, and language delays. While KS patients typically display a milder phenotype, HGA-SCAs are associated with profound cognitive defects. Despite the prevalence of X chromosome aneuploidies, there is a critical need for cellular models to define the transcriptional, epigenetic, and functional consequences of X chromosome overdosage during neurodevelopment. To this end, we derived cortical organoids from 47,XXY, 48,XXXY, and 49,XXXXY iPSCs. Allele-specific expression (ASE) analysis on X aneuploid organoids demonstrated a preserved epigenetic X inactivation status at different time points, from one to 12 months of differentiation in vitro. Through a multi-layered analysis integrating morphological, functional, bulk, and single-cell transcriptomics, we found that the additional X chromosomes lead to impaired neural patterning, disrupted cortical architecture, and altered electrophysiological properties of cortical organoids in a dose-dependent manner. While 47,XXY organoids are phenotypically and functionally similar to 46,XY controls, HGA-SCAs display severe functional defects and aberrant transcriptomes. Through single-cell RNA analysis, we profiled the genes that escape X inactivation in neuronal and non-neuronal cell populations and revealed a dysregulated proliferation of neural progenitor in organoids carrying supernumerary X chromosomes. Additionally, severe astrocyte differentiation defects were observed in HGA-SCAs organoids, potentially contributing to synaptic dysfunction. Moreover, high-density microelectrode arrays (MEA) analysis revealed a higher mean spike firing rate and amplitude of HGA-SCAs compared to 46,XY organoids. Finally, patch-clamp studies demonstrated significant hyperexcitability of HGA-SCA organoids and X dosage-sensitive deficits in long-term potentiation (LTP). Our work leveraged the inaugural cohort of X aneuploid cortical organoids to unravel the functional consequences of X-linked gene overdosage during neurodevelopment.

Speaker: Antonio Adamo (King Abdullah University of Science and Technology)

18:30 The continuum of human neurodiversity: from in vitro to in silico and back for elucidating the molecular mechanisms of cortical neurodevelopment.

The polygenic architecture of human neurodiversity requires new maps to model the developmental continuum it underpins. While pluripotent stem cell and brain organoid modelling has begun to provide major insights into the pathogenesis of neuropsychiatric disorders, the focus is still on one or, at best, a few specific disorders at a time, mostly caused by highly penetrant genetic mutations and studied in isolation. The combination of single cell analysis of developmental trajectories through brain organoids modelling and advanced mathematical formalism has the potential to overcome the limitations of such classical “disease vs control” dichotomy and shift the paradigm towards the study of the continuous distributions of clinical and molecular phenotypes. For that, however, a significant scaling up is needed in the numbers of timepoints, cells and individuals profiled. Starting from anchor points from our single cell resolved reference of paradigmatic neurodevelopmental disorders (including Kabuki, Gabriele De Vries, Weaver, Williams Beuren, 7 dup and several others) at multiple time points and multiple individual per conditions, we developed a sophisticated mathematical approach to enable the continuous and probabilistic definition of trajectories, by extending nonlinear filtering and statistical learning methods. This unique combination of generative properties, computational and experimental alike, allows us to uncover hidden cellular microstates from noisy single-cell omics data.Through iterative refinement and experimental validation, this approach enables mapping, with unprecedented resolution, the molecular underpinnings of cellular dynamics onto clinical and neurobehavioral traits.

Speaker: Carlo Emanuele Villa (Human Technopole Foundation)

18:30 The role of lipid droplets during mouse and human brain development

Lipid droplets (LDs) are intracellular lipid storage organelles. Recently, we showed that adult mouse neural stem/progenitor cells (NSPCs) contain a large number of LDs, which directly influence NSPC proliferation and metabolism. To further study LDs in the brain, we have developed a novel endogenous fluorescent LD reporter mouse (tdTom-Plin2 mouse) to allow staining-free visualization of LDs. We have demonstrated that LDs are highly abundant in various cell types in the healthy adult brain, and we also found numerous LDs in the developing brain. Furthermore, adding lipids to the medium of ex vivo embryonic brain sections resulted in increased LDs. This suggests that the build-up, breakdown, and storage of lipids in LDs might play an important role in NSPC regulation during brain development. However, very little is known about how LDs influence mouse brain development and their role in human brain development is even less explored.
We here use the novel tdTom-Plin2 mouse to characterize the distribution and dynamics of LDs over a developmental time-course. Using genetic and pharmacological means, we will further perturb LD usage and numbers and assess the consequences for brain development. We also utilize human induced pluripotent stem cell (hiPSCs) derived NSPCs and cerebral organoids to characterize and dissect the role of LDs in human brain development. These different model systems allow us to better understand the importance of LDs during early mouse and human brain development.

Speaker: Carla Marie Igelbüscher (University of Lausanne)

18:30 The role of PHF3 during neuronal differentiation and in neuronal disorders

During neural development, neuronal stem cells differentiate into functional neurons that make up the human brain. Transcription regulators ensure the timely expression of genes required for neuronal differentiation. Perturbed transcription can cause neurological and neurodevelopmental disorders as well as lead to the development of cancer. It is therefore crucial to identify and characterize transcriptional regulators involved in these processes to understand disease origin and to develop new treatment strategies. We identified PHD finger protein 3 (PHF3) as a key player in the regulation of neuronal gene expression and neuronal differentiation. PHF3 directly binds RNA polymerase II and regulates transcription and mRNA stability. The molecular mechanism of how PHF3 chooses its targets and regulates neuronal differentiation remains unresolved. Our preliminary data indicate that loss of PHF3 in human induced pluripotent stem cells (hiPSCs) due to gene knock-out (KO) or acute degradation impairs differentiation into functional neurons. Single-cell RNA-sequencing (scRNA-seq) revealed accelerated neuronal differentiation in PHF3 KO cells with a higher proportion of neurons and a lower proportion of radial glial and intermediate progenitor cells. This is coupled with deregulation of transcription factors known to be essential for neurogenesis. As a result, neuronal functionality is compromised in PHF3 KO, which show reduced neurite length and impaired spontaneous excitatory input measured by patch-clamp recordings. Insights gained from this project will provide a comprehensive understanding of PHF3 function in neuronal differentiation, paving the way for new therapeutic approaches tackling neurodevelopmental diseases.

Speaker: Magdalena Engl (Max Perutz Labs)

18:30 The synaptic transcriptome of human iPSC-derived motoneurons

Neurons mediate the reception and transmission of nervous stimuli with their polarized shape and processes. Each nervous cell makes thousands of synaptic contacts with other cells. Synapses are well characterized in their protein content but there is less concordance about the resident RNA species that contribute to synapse function and maintenance. mRNAs that localize at synaptic boutons can undergo local translation and produce synaptic proteins, and also non-coding RNA species like lncRNAs, miRNAs and circRNAs could play a fundamental but still uncharacterized role in synapse biology. Furthermore, many studies focused on mouse models, while the human synaptic transcriptome remains still unexplored. Recent evidence shows that synaptic aberrations might represent a shared pathological feature across some neurodegenerative diseases like Amyotrophic Lateral Sclerosis, an illness specifically targeting motoneurons. In this study we characterize the human synaptic transcriptome of in vitro iPSC-derived motoneurons. We exploit an adaptation of the mouse brain synaptosome isolation protocol, to isolate the motoneuron synaptosome-associated RNAs. With these techniques, we aim at identifying the molecular mechanisms that underlie the synaptic localization of coding and non-coding RNAs and their local function. We aim to analyze the differences between wild type ad ALS motoneuron synapses with mutations in the FUS protein, that are associated with a juvenile and aggressive form of ALS. In mice, FUS also localizes at the pre-synaptic compartment and a recent report outlined that its synaptic accumulation upon NLS deletion triggers early misregulation of synaptic RNAs (Sahadevan et al., 2021). With this work we aim at the characterization of the human synaptome to highlight the RNA contribution to synapse function and ultimately degeneration.

Speaker: Vittorio Padovano (Sapienza Università di Roma)

18:30 Tissue-Specific 3D Genome and Splicing Signatures of Clinically Accessible Tissues Inform Sample Selection for Assessing the impact of Genomic Aberrations in Neurodevelopmental Disorders

The recent expansion of multi-omic molecular methods, including transcriptomics, proteomics, epigenomics and whole-genome sequencing, has led to huge improvements in the diagnosis and research of rare diseases, such as neurodevelopmental disorders (NDDs). The accuracy of these methods in pathology profiling is in part determined by the closeness of the tested biological sample to the diseased tissue of interest. The norm to avoid the need for invasive biopsy is the sequencing of clinically accessible tissues (CATs), commonly peripheral blood mononuclear cells (PBMCs) or lymphoblastoid cell lines (LCLs) from blood, fibroblasts from skin, or now with increasing frequency Urine-Derived Renal Epithelial Cells (UREC). Previous studies have revealed that only about 60% of topologically associating domains (chromatin features) are conserved between tissues. Similarly, a study in non-CATs reported that 40.2% of genes have splicing that is inadequately represented by at least one CAT with 6.3% of genes having splicing inadequately represented by all assessed CATs.
By combining previous research mapping RNA splicing variations in CATs and 56 different nonaccessible tissues, including foetal and adult cortex (Aicher et al., 2020), with new RNA-sequencing and chromatin confirmation mapping (Hi-C) of neuronal tissues (adult neurons and glia, foetal cortex at different developmental timepoints) and CATs (PBMC, LCLs, and fibroblasts), we have outlined the expression levels and splicing events of 1182 NDD related genes, and 3D chromatin features absent/inadequately represented from commonly used CATs. Initial analysis revealed only 3.53% of NDD genes within adult cortex neurons display adequate expression, 3D structure, and splicing within tested CATs, demonstrating the consideration required for CAT choice in the majority of diagnostic settings. With this list, we can now begin feasibility testing of novel CATs for use in NDD research and diagnostics, based on their ability to adequately represent neurogenomic features lacking in established CAT systems.

Speaker: Michael B Vaughan (UGent)

18:30 TMEM151A, a new causative gene for Paroxysmal Kinesigenic Dyskinesia

TMEM151A, an almost unknown gene, has been recently associated to Paroxysmal Kinesigenic Dyskinesia (PKD), an autosomal dominant movement disorder. Our project aims to identify the role of TMEM151A in the brain from both a physiological and pathological point of view through a multi-level approach. We studied the topology of TMEM151A and showed that it is a membrane protein with two membrane-spanning helices and a large cytosolic domain with alpha-helix and beta-sheet structures. We explored TMEM151A expression from macro-areas of the brain down to the cellular level by real-time PCR experiments and showed that it is enriched in the cerebral cortex, hippocampus and spinal cord. In primary neuronal cultures TMEM51A is developmentally regulated with a peak of expression between 14 and 21 days in vitro, a time of intense synaptogenesis. Overexpression experiments showed an enrichment in endoplasmic reticulum and Golgi apparatus. To go in depth about the role of TMEM151A in the disease, we investigated the effects of some pathological mutations by in vitro assays. We show that some of them cause alteration in the protein expression, suggesting a possible loss of function mechanism. Therefore, to mimic the pathology, we used induced pluripotent stem cells (iPSCs) carrying a null mutation in TMEM151A. KO-TMEM151A iPSCs and isogenic control have been differentiated to glutamatergic excitatory neurons and functional characterization is ongoing. To define the molecular interactome of TMEM151A in mouse brain, we used a pull down-based proteomic approach and identified several potential interacting proteins involved in membrane trafficking processes. We are validating these potential candidates by biochemical approaches. Our results may help to understand the physio-pathological mechanisms at the basis of these paroxysmal disorders and to get a step forward for the identification of new targeted therapeutic strategies.

Speaker: Lisastella Morinelli (Università di Genova)

18:30 Transcriptional signatures of hippocampal tau pathology in primary age-related tauopathy and Alzheimer’s disease

Spatial multi-omic technology has the potential to reveal the molecular foundations of neurodegeneration, uncovering both shared pathological responses and discrete signatures of vulnerability across clinically distinct neurodegenerative diseases. Using GeoMx spatial profiling, we quantified mRNA in CA1 pyramidal neurons with and without tau pathology in 6 primary age-related tauopathy (PART cases), 6 Alzheimer's disease (AD) cases, and 4 control cases. We found that tau pathology was the primary factor influencing transcriptional variation rather than disease classification. Differential gene expression analysis revealed an unexpected pattern: while synaptic genes were downregulated in disease neurons overall, tau-positive neurons showed upregulation of synaptic genes compared to tau-negative neurons in the same disease. Using unsupervised machine learning, we identified two distinct expression patterns associated with tau pathology that spanned both conditions. These transcriptional patterns were validated using transfer learning in an independent dataset of cortical neurons with tau pathology in AD, demonstrating that these changes are consistent across brain regions. Hybridization chain reaction studies in an expanded cohort confirmed the upregulation of select genes in tau-positive neurons.
Our findings highlight the power of molecular analysis stratified by pathology and suggest that the tau pathology in PART and AD may induce a shared molecular program despite occurring in different contexts. This has potential implications for therapeutic approaches targeting tau pathology in both conditions. We are currently developing an affordable, FFPE-compatible platform to spatially interrogate mRNA concurrently with a panel of known neuropathological markers. This expanded capability will allow us to correlate gene expression patterns with multiple protein markers simultaneously at subcellular resolution, providing a more comprehensive understanding of the cellular microenvironment in tauopathies and other neurodegenerative conditions.

Speaker: Ryan Palaganas (Johns Hopkins SOM)

18:30 UBE2I mutations unveil the critical role of SUMOylation in neurodevelopmental disorders

SUMOylation pathway is a post-translational modification process that regulates the function of hundreds of proteins by SUMO protein attachment. This three-step pathway requires activation of SUMO proteins by the E1 enzyme, transfer to the E2 conjugating enzyme and SUMO-protein ligation directly or through E3 ligase. Defects in SUMOylation are being linked to abnormal neuronal development and synaptic function. This study focuses on UBE2I, which encodes the sole human E2 enzyme (UBC9); the goal is to evaluate the role of UBE2I in neurodevelopmental disorders (NDDs). Trio-exome sequencing of a cohort of >600 NDD families led to the identification of a de novo (dn) missense variant in UBE2I [NM_194260.3:p.(Asn124Ile)] in a pediatric patient with a syndromic phenotype. In GeneMatcher, we found three further NDD patients harboring UBE2I variants (p.Val25Met dn; p.Trp53Leu dn; p.Glu143Gly). All have shared clinical features, such as neurodevelopmental delay, hypertelorism/telecanthus, almond-shaped eyes, thick eyebrows, and hand anomalies. All variants are absent in GnomAD ver 4.0. The substituted amino acids are evolutionarily conserved from C.elegans to humans and the changes are predicted to be highly damaging by multiple bioinformatic tools (CADD ≥26). All variants destabilize protein structure according to 3D modelling predictions (ΔΔG ≤ 0.5 kcal/mol). When stable cell lines expressing either WT or variant UBC9-mCherry fusion proteins were analyzed by western blot and flow cytometry, expression of the UBC9 variants was found to be reduced by ⁓50% with respect to WT. Proteasome inhibition significantly restored expression of the UBC9 variant proteins, indicating they likely destabilize protein structure, leading to proteasomal degradation. Moreover, reduced UBC9 expression was observed in one patient’s lymphoblastic line, corroborating the results obtained before.
In conclusion, we propose that UBE2I be considered as a candidate gene for a novel NDD, a finding that will provide insights into the role of SUMOylation in brain function and development.

Speaker: Verdiana Pullano (University of Turin)

18:30 Uncovering the pivotal role of lncRNAs in cortical neuron differentiation from human embryonic stem cells

This study presents a comprehensive computational and experimental analysis of long non-coding RNAs (lncRNAs) in cortical neuron differentiation from human embryonic stem cells (hESCs). Using a systematic workflow, we identified lncRNAs overexpressed at late stages of neural differentiation and characterized their expression, subcellular localization, sequence features, and regulatory interactions. K-mer profiling revealed distinct lncRNA clusters with unique sequence signatures, suggesting diverse functional roles in neural differentiation and brain tissue specificity. Notably, certain clusters were linked to ribosomal function, epigenetic regulation, and chromatin remodeling, highlighting the multifaceted contributions of lncRNAs to neural development. By illuminating the landscape of lncRNA expression during cortical neuron differentiation, this work advances our understanding of lncRNA functions in neurodevelopment and underscores their potential as biomarkers or therapeutic targets in neurological disorders.

Speaker: ANNALAURA TAMBURRINI (University of Turin)

18:30 Understanding the physiological and pathological roles of the tRNA deaminase complex ADAT2/ADAT3 during cortical development

With the identification of ~150 modifications found in RNA, epitranscriptomic modifications emerged as critical posttranscriptional modulators of brain development and function. tRNAs, the adaptor molecules that deliver specific amino acids to the polypeptide chain during translation, are the most modified RNA species, with an average of 13 modifications per tRNA. These modifications, catalyzed by over 70 different tRNA modifying enzymes, influence tRNA structure, stability and function depending on their chemical nature and position on the tRNA. Strikingly, while more than half of these modifications is linked to human diseases, most of the diseases linked to mutations in genes encoding for tRNA-modifying enzymes are NDDs suggesting a clear vulnerability of the developing human brain to disturbances of tRNA modifications. One such tRNA modification introduced by the ADAT2/3 complex, the conversion of Adenine (A) to Inosine (I) at position 34 (I34) in ANN-tRNAs, extends the base-pairing capacity of tRNA’s as -I can base pair with -U, - A and -C and is essential for decoding the C-ending codons encoding the 8 amino acids, as GNN-tRNAs do not exist in eukaryote genomes. While mutations in ADAT3, the catalytically inactive subunit of the ADAT2/ADAT3 complex, have been identified in patients presenting with severe neurodevelopmental disorders (NDDs) the biological role of the ADAT complex during cortical development and the effect of NDD related ADAT3 mutations remains uncharacterized.

Here we show that maintaining a proper level of ADAT2/ADAT3 catalytic activity is required for correct radial migration of projection neurons in the developing mouse cortex. Through structural, biochemical and molecular analysis, we demonstrated that all the identified variants alter both the abundance and the activity of the complex leading to a significant decrease of I 34 with direct consequence on the steady-state levels of ADAT target tRNAs. Using in vivo complementation assays, we correlated the severity of the migration phenotype with the degree of the loss of function caused by the variants. Altogether, our results indicate a critical role of ADAT2/ADAT3 during cortical development and provide cellular and molecular insights into the pathogenicity of ADAT3-related neurodevelopmental disorder.

Speaker: Efil Bayam (CERBM GIE)

18:30 Understanding the subcellular architecture of mRNAs networks in neural stem cells during brain development

In mammals, neural stem cells proliferation and differentiation contribute to organize sophisticated brain architecture during neocortical development.

Among different classes of progenitors, apical progenitors (APs) possess unique apicobasal polarity that has driven increasing interest in understanding how subcellular regulation of gene expression occurs within these cells. Similarly to other highly polarized cells such as neurons, transcripts distribution and local processing in APs apical and basal compartments might be differentially regulated in response to external stimuli.

Taking advantage of embryonic mouse brain as a model system and single cell labeling techniques, we designed a strategy to separate and enrich the apical and basal domains of APs from tissue. Downstream bulk-RNAseq provided us with a list of differentially distributed mRNAs. GO analysis revealed the enrichment, in the basal compartment of APs, of terms related to mRNA translation and processing, suggesting that the basal process might function as a dynamic compartment in which transcripts are transported and translated differentially from the apical cell body to match cellular needs.

In this scenario, the dichotomy between intracellular trafficking and transcripts localization takes on a fascinating role. Peculiar asymmetric distribution of traffic organelles within APs, suggests indeed that intracellular traffic could be the key mediator of local regulation of gene expression, influencing both transcripts localization and levels. Moreover, alterations in the distribution of traffic organelles have been associated to severe neurodevelopmental disorders, thus uncovering mRNA subcellular dynamics would contribute to the dissection of the molecular mechanisms underlying such pathological conditions.

Our work is intended to deepen the knowledge in the promising field of mRNA subcellular metabolism, providing novel insights on APs regulation during neocortical development.

Speaker: Giulia Visani (Human Technopole)

18:30 Unlocking the Guardian of the Brain: Choroid Plexus and Neurodevelopmental Insights

The choroid plexus (ChP), a vascularized structure, plays a pivotal role in cerebrospinal fluid (CSF) production and brain homeostasis by regulating ion transport, nutrient delivery, and waste clearance. Beyond these functions, the ChP acts as a sensor and modulator of environmental signals, influencing brain development and contributing to the pathophysiology of neurodevelopmental disorders. Growing evidence implicates inflammation and immune dysregulation in childhood neuropsychiatric conditions such as schizophrenia and autism, highlighting the ChP as a key amplifier of these pathological processes. Given its anatomical proximity to the subventricular zone, deciphering the mechanisms of ChP function is crucial for understanding brain maturation and exploring its potential as a therapeutic target. Animal models incompletely model human physiology, while in vitro systems lack key components to replicate ChP complexity. To address this, we developed VIChOs (Vascular Immune ChP Organoids) as a novel 3D human ChP model capturing the cellular heterogeneity of the ChP, extending beyond its well-known epithelial barrier function. VIChOs faithfully replicate the histological and ultrastructural features of native ChP tissue. Using single-cell RNA sequencing, we longitudinally characterized the diverse cell-type composition of VIChOs, revealing a dynamic cellular landscape regulated by subtype-specific signaling pathways. The development of VIChOs into tissue with neural, endothelial, and immune cell populations is accompanied by the dynamic secretion of a CSF-like fluid, which contains actively synthesized neurotrophic and signaling molecules. By leveraging the inherent heterogeneity of VIChOs, we conducted hypothesis-driven perturbations to dissect how microenvironmental cues influence ChP cytoarchitecture and secretory profiles. These experiments unveiled robust, context-dependent shifts in cellular organization and secretome composition, underscoring the utility of VIChOs as a platform for mechanistic studies.
Our work establishes VIChOs as a novel human ChP model that captures the tissue’s full complexity. It provides a scalable, physiologically relevant system for exploring ChP biology in health, disease, and therapeutic discovery.

Speaker: Vanessa Aragona (Humanitas University)

18:30 Unravelling Convergence and Divergence Mechanisms in Neurodevelopmental Disorders Through Single-Cell Transcriptomics

Neurodevelopmental disorders (NDDs) encompass a heterogeneous spectrum of conditions that impact the development of the nervous system, leading to impairments in cognitive function, behaviour, and social abilities. The genetic basis of these disorders ranges from highly penetrant monogenic diseases to complex polygenic influences, in addition to a wide array of environmental factors. Despite the heterogeneity of genetic and environmental factors, many NDDs converge at distinct functional impairments. In vitro modelling has emerged as a crucial approach to studying neurodevelopmental physiology, providing a controlled system to investigate disease mechanisms. Among these models, cortical brain organoids have demonstrated transcriptional similarities to fetal tissues, making them a valuable tool for understanding early brain development.
We generated cortical brain organoids from 52 cell lines of 10 NDDs representing anchor points in neurodevelopmental pathology. We profiled with single-cell transcriptomics, whole genome sequencing and Imaged-based phenotyping at three-time points recapitulating transcription changes in fetal cortex at 8 and 12 post-conceptional weeks. Differential expression analysis revealed that transcriptional changes are specific for each disease at an early stage and converge at one particular shared set of genes at later stages. Diseases like Kabuki-Syndrome, GADEVS, and CHD8 Haploinsufficiency converged at all stages in functions related to synaptic activity and excitatory/inhibitory imbalance.
A comprehensive analysis of the mechanisms underlying NDDs facilitates the identification of shared and unique pathways, providing a foundation for future drug discovery and therapeutic interventions.

Speaker: Mazen Khaddour (Human Technopole)

18:30 Unravelling the molecular basis of DLG4-related Synaptopathy

DLG4-related Synaptopathy, also known as SHINE syndrome, is a newly identified neurological disorder associated with variants in the Discs Large Homolog 4 (DLG4) gene, encoding for the post-synaptic density 95 (PSD-95) protein, the most abundant scaffolding MAGUK protein in excitatory post synaptic densities (PSDs). Owing to the diversity of signalling pathways associated with PSD-95, 225 DLG4 variants, of which 83 pathogenic, have been identified, with disease manifestations consistent with those of its numerous interacting partners. De novo heterozygous pathogenic mutations in 53 individuals have been reported to be associated with moderate to severe intellectual disability, developmental delay, autism spectrum disorder, epilepsy, ataxia, hypotonia, ADHD, language delay and vision problems. No genotype-phenotype correlations have been established thus far, with treatment being restricted to management of symptoms and supportive care. We employ patient-derived induced pluripotent stem cell lines (hiPSCs) for in vitro characterization of glutamatergic NGN2-induced neurons and 3D cerebral organoids for disease modelling. To this end, we aim to establish the mutational effects of three different variants on the gene expression levels while elucidating the primary affected pathways by studying the domain-specific protein interactions. We also strive to restore neuronal function in haploinsufficient patients through AAV9-hSynI-hDLG4 gene therapy. Data thus far uncovered a loss-of function phenotype in all three patients, with patient-specific electrophysiological deficits, as revealed by patient cerebral organoids recorded using a high-density microelectrode array system (HD-MEA). Additionally, preliminary results from transcriptomic analysis at single cell resolution of patient-derived cerebral organoids revealed convergence with ASD and ADHD pathways. Intriguingly, AAV9-mediated DLG4 delivery reveals promising phenotype restoration. On the whole, our research holds promise for establishing the groundwork for future studies on other DLG4 variants and insights for gene therapy approaches for other monogenic brain disorders.

Speaker: Dania Abdellatif (Hebrew University of Jerusalem)

18:30 Unveiling myeloid-mediated enzymatic correction of ARSA-deficient neural cells in hematopoietic stem cell gene therapy for Metachromatic Leukodystrophy

Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficiency of Arylsulfatase A (ARSA), a critical enzyme that breaks down sulfatides. Accumulation of sulfatides results in neurological manifestations related to white matter loss in the central and peripheral nervous systems (CNS, PNS), accompanied by neuroinflammation and neurodegeneration.
Ex vivo hematopoietic stem cell gene therapy (HSC-GT) using autologous HSCs engineered by lentiviral vectors (LV) to express supraphysiological ARSA levels is the only approved treatment for MLD. While it is acknowledged that engraftment of metabolically proficient HSC myeloid progeny plays an indispensable role in immunomodulation and neuroprotection, the precise mechanism of myeloid-to-neural enzymatic cross-correction is debated. Therefore, we have investigated myeloid-mediated cross-correction mechanisms using relevant in vitro human models and ARSA-/- mice.
Our research unveiled that ARSA overexpression in human monocyte-derived MLD macrophages did not affect the M1/M2 macrophage polarization, which did not influence ARSA secretion from myeloid cells. Transgenic ARSA released by macrophages purified from HSC-GT-treated MLD patients efficiently cross-correct MLD hiPSC-derived neurons and oligodendrocytes. Metabolic cross-correction reduces sulfatide storage at levels similar to healthy donor-derived neural cultures. Of note, ARSA released in the cerebrospinal fluid of HSC-GT-treated MLD patients retains similar cross-correction properties. Delving deeper into the mechanism of cross-correction, we showed that the mannose-6-phosphate receptor partly mediates the uptake of transgenic ARSA enzyme.
Transplantation of murine HSC transduced with LV.ARSA resulted in engraftment of myeloid progeny in the brain and restoration of physiological enzyme levels in ARSA-deficient neural cells. We are performing sn-RNAseq analysis in HSC-GT-treated MLD mice to uncover the effects of cross-correction in reverting neurodegenerative/inflammatory pathways.
Our findings underscore the consistent occurrence of myeloid-mediated enzymatic cross-correction of ARSA-deficient neurons and glial cells within a clinically relevant HSC-GT framework, offering profound insights into the therapeutic potential of this approach for genetic neurological diseases.

Funding: GR-2019-12368930

Speaker: Vasco Meneghini (Ospedale San Raffaele SRL)

18:30 Wireless Integration: Temporal Interference Stimulation to Enhance Stem Cell Therapies in Parkinson’s Disease

Parkinson’s disease (PD) is a pervasive neurodegenerative disorder characterized by a loss of dopaminergic neurons starting from the substantia nigra, which triggers motor and cognitive symptoms. Existing treatments such as dopamine replacement therapies and deep brain stimulation (DBS) provide symptomatic relief, but they are limited by side effects and transient efficacy. Cell replacement therapies (CRTs) are emerging as a promising alternative in ongoing clinical trials, where mesencephalic dopaminergic neurons (mesDAs) derived from human pluripotent stem cells are transplanted into the striatum to restore dopamine functionality.
Cell survival, healthy neural maturation and functional integration of mesDAs into host brain circuits represent major challenges in CRTs. Behavioral improvements can require months in animal studies, and up to two years in humans, underscoring the clinical need for enhancing cell maturation and functionality. Temporal interference (TI) stimulation is a revolutionary technique for non-invasive DBS in the clinic (Grossman et al., 2017), which has shown potential to increase neural differentiation and functionality in vitro and in vivo (Peressotti et al., in progress). However, the optimal TI stimulation parameters for improving CRT outcomes remain unknown, due to the wide range of potential protocols.
Understanding the functionality and maturation dynamics of transplanted cells is a complex endeavor requiring diverse and longitudinal readouts, which are challenging to obtain in vivo. Thus, a high-throughput in vitro platform will be developed to systematically evaluate the influence of TI on an in vitro CRT model. The construct will consist in 3D spheroids from primary striatal cells, mixed with optogenetically-tagged mesDA progenitors. The impact of different stimulation protocols on differentiation, maturation, and functionality will be evaluated using longitudinal optogenetics-based electrophysiological studies, dopamine release quantifications, immunofluorescence and mRNA sequencing at multiple time points. The results will be validated in vivo, towards a highly translatable approach to improve the efficacy of CRTs for PD.

Speaker: Sofia Peressotti (University of Geneva)

Tuesday, 20 May

09:00 → 11:00 Neurodegeneration and regeneration

Convener: José Davila Velderrain (Human Technopole, IT)

09:00 Regeneration of a tetrapod nervous system

Speaker: Elly Tanaka (IMBA, IMP, AT)

09:25 Single-Cell Multiomic Atlas of Human Cortical Development in Down Syndrome

Speaker: Vincenzo De Paola (Duke-NUS Medical School)

09:35 Body-brain communication at the choroid plexus

Speaker: Aleksandra Deczkowska (Institut Pasteur, FR)

10:00 Decoding Cerebellar Cellular and Molecular Dynamics in Sudden Infant Death Syndrome (SIDS) Across Developmental and Corrected Ages

Speaker: Javid Ghaemmaghami (University of Michigan)

10:10 Single-Cell and Spatial Transcriptomics and Epigenomics of Oligodendroglia in Development and in Multiple Sclerosis

Speaker: Gonçalo Castelo-Branco (Karolinska Institutet, SE)

10:35 [The EMBO Keynote Lecture] From correlation to mechanisms: how genetics determine your risk of Alzheimer’s disease

Speaker: Bart De Strooper (VIB-KU Leuven Center for Brain & Disease Research, BE)

11:00 → 11:30 Coffee

11:30 → 13:05 Neuroimmunology

Convener: Oliver Harschnitz (HT)

11:30 Leveraging single cell technologies to engineer the immune system

Speaker: Ido Amit (Weizmann Institute, IL)

11:55 Exploring the role of the piRNA pathway in microglia and neuroinflammation

Speaker: Silvia Beatini (Istituto Italiano di Tecnologia)

12:05 Tau uptake, processing and secretion by human iPSC-microglia

Speaker: Sally Cowley (University of Oxford, UK)

12:30 Village-based neural progenitor cell proliferation and viability assays reveal genetic and environmental determinants of cellular fitness

Speaker: Michael Wells (University of California Los Angeles)

12:40 Mapping and modeling brain macrophages

Speaker: Florent Ginhoux (A-STAR, SG)

13:05 → 14:15 Lunch

14:15 → 19:30 Stem cell and organoid disease modelling

Convener: Veronica Krenn (Milano-Bicocca, IT)

14:15 From Spikes to Structure: Spontaneous Activity in Cortical Development

Speaker: Simona Lodato (Humanitas RC, IT)

14:40 Benchmarking cerebellar organoids to model autism spectrum disorder and human brain evolution

Speaker: Davide Aprile (Human Technopole)

14:50 Cellular Crosstalk in Brain Development

Speaker: Silvia Cappello (Ludwig Maximilian University of Munich – LMU, DE)

15:15 Passage of time in brain organoids: the journey to understand human brain development and maturation

Speaker: Irene Faravelli (Harvard University)

15:25 Engineering brain organoids for modeling neural development and diseases

Speaker: Guo-li Ming (University of Pennsylvania, US)

15:50 Coffee break

16:20 Meet the speaker

17:20 Transfer

18:30 [Keynote Lecture] The idea of the brain and the future of neuroscience

Speaker: Matthew Cobb (The University of Manchester, UK)

19:30 → 21:30 Light refreshment

Wednesday, 21 May

08:45 → 10:20 Cancer neuroscience

Convener: PhD/postdoc Rep Human Technopole, IT

08:45 A brain-wide neuronal circuit connectome of human glioblastoma

Speaker: Hongjun Song (University of Pennsylvania, US)

09:10 Glioblastoma stem cell morphotypes convey distinct cell states and clinically relevant functions

Speaker: Carlotta Barelli (Human Technopole)

09:20 The neural environment of cancer and beyond

Speaker: Claire Magnon (INSERM, FR)

09:45 Mapping tumour heterogeneity in glioblastoma with integrated single cell and spatial genomics

Speaker: Fani Memi (Wellcome Sanger Institute)

09:55 Targeting the neural blueprint of brain tumors

Speaker: Varun Venkataramani (DKFZ, DE)

10:20 → 10:50 Coffee break

10:50 → 12:40 Neurodiversity in language and cognition

Convener: Giuseppe Testa (Human Technopole, IT)

10:50 The Human Condition through Human Conditions

Speaker: Cedric Boeckx (ICREA, ES)

11:15 The genetic architecture of autism: from medicine to neurodiversity

Speaker: Thomas Bourgeron (Institut Pasteur, FR)

11:40 Neurodiversity in a dish: integrating epidemiology and cortical brain organoid modelling through a hormonal signalling atlas

Speaker: Nicolò Caporale (Human Technopole)

11:50 Autism, ability and the human brain on a continuum

Speaker: Daniel Geschwind (UCLA, US)

12:15 Tracing Neurogenomic Disorders Across Human Phenotypes

Speaker: Armin Raznahan (NIH, US)

12:40 → 13:45 Lunch

13:45 → 14:00 Best poster award

14:00 → 15:00 Neurodiversity in language and cognition

Convener: Giuseppe Testa (Human Technopole, IT)

14:00 [Keynote Lecture] (Epi)Genomics Of Stress - Implications For Psychiatry

Speaker: Elisabeth Binder (Max Planck Institute of Psychiatry, DE)

15:00 → 15:30 Closing remarks & departure