Astrocytes play a pivotal role in neuronal network development. Despite the well-known role of astrocytes in the pathophysiology of neurologic disorders, the utilization of induced pluripotent stem cell (iPSC)-derived astrocytes in neuronal networks remains limited. Here, we present a streamlined one-step protocol for the differentiation of iPSCs directly into functional astrocytes without the need for ectopic gene expression or neural progenitor cell generation. We found that culturing iPSCs directly in commercial astrocyte medium, was sufficient to differentiate iPSCs into functional astrocytes within 5 weeks. More than 60 iPSC lines were successfully differentiated into astrocytes by independent researchers across 10 independent laboratories. Validation of the iPSC-astrocyte cultures demonstrated consistent astrocyte differentiation with minimal batch-to-batch variability. In dept. characterization of a subset of iPSC lines confirmed astrocyte identity and functionality of the iPSC-astrocyte monocultures by immunofluorescence, flow cytometry, RNA sequencing, glutamate uptake assays and calcium signaling recordings. Optimization of the protocol enabled co-culture of iPSC-astrocytes with Ngn2 iPSC-derived neurons (iNeurons), promoting neuronal differentiation and synapse formation. Lastly, we used single-cell electrophysiology and multi-electrode arrays, by four independent researchers, to confirm robust neuronal network development in 5-week-old iPSC-astrocyte and iNeuron co-cultures. This protocol offers a rapid and efficient method to establish all-human astrocyte-neuron co-cultures, facilitating the investigation of cell-type-specific contributions to disease pathogenesis. Its validation across numerous iPSC lines in 10 independent laboratories highlights the reproducibility of the protocol and positions it as a platform for advancing disease modeling in human neural networks.

CACNA1A-related disorders constitute a diverse group of neurological conditions, including ataxia, migraine, and epilepsy. Despite extensive genetic studies, clear genotype-phenotype correlations remain elusive. Moreover, next-generation sequencing has identified many variants of uncertain significance (VUS). Here, we leveraged patient-derived and CRISPR-Cas9-engineered human neuronal networks to explore relationships between CACNA1A variants and neurophysiological activity. CACNA1A haploinsufficiency induced subtle alterations in glutamatergic network activity, whereas missense variants had a more pronounced effect on overall network function. Network fingerprints were most affected from patients where ataxia co-occurred with migraine or epilepsy. Furthermore, we analyzed the impact of CRISPR-Cas9-induced VUS on network developmental trajectories. Although functional changes could not be directly linked to clinical phenotypes, all tested variants induced measurable alterations in neuronal network function, supporting their classification as likely pathogenic. These findings highlight the potential of human neuronal networks as a translational model for evaluating CACNA1A variant effects and improving clinical variant interpretation.

Summary Induced pluripotent stem cells (iPSCs) are widely used as patient-specific disease models, yet substantial unexplained variability in molecular and functional readouts limits their reliability. Here, we systematically investigated the sources of variation in iPSC-derived neurons for three rare genetic disorders: Myotonic Dystrophy Type 1, chromodomain-DNA-helicase-binding protein 2-related disorder and N -acetylneuraminic acid synthase deficiency. This was performed by profiling multi-omics layers: genomics, epigenomics, transcriptomics, proteomics, metabolomics and lipidomics. Our study found that clonal variability was comparable to inter-patient differences and that neuronal differentiation state and nutrient-driven metabolic activity emerged as dominant contributors to variability observed across omics layers. Clonal differences could partly be attributed to stochastic differences in DNA methylation established during reprogramming. By modeling and correcting the observed variation, we improved the detection of disease-associated molecular signatures. Our study provides guidelines for improved study design and data analysis to minimize variability, enabling robust biomarker discovery and reliable iPSC-based disease modeling.

Microglia, the resident immune cells of the brain, act along a spectrum to maintain CNS homeostasis, respond to perturbations, and control neuronal activity. Disentangling the molecular mechanisms of human microglia-neuron crosstalk remains challenging due to the context-dependent, dynamic nature of their interaction. We introduce MEA-LINK, a systems-approach leveraging natural variation to screen for immune modulators of neuronal activity. This multi-modal platform integrates human induced pluripotent stem cell (hiPSC) technology with micro-electrode array (MEA) recordings and proteomic analyses of secreted immune factors, allowing for longitudinal samples and correlations across modalities. We applied MEA-LINK to explore microglia-neuron interactions during development and hyperactivity challenges. We show that human microglia accelerate neuronal network development and rescue hyperactive network phenotypes. Linking the secretome adaptations to neuronal network activity variations, we identified CCL4 as a top candidate in microglia-mediated hyperactivity control. Then, we functionally validated the context-dependent role of microglial CCL4 to neuronal CCR5 signaling in human neuronal networks. Our findings support a neuron-specific function of chemokines and their receptors in the brain and provide a new perspective for immune signaling in neuronal hyperactivity control. The MEA-LINK platform thus offers a foundation for comprehensive, systematic studies of human microglia-neuron interactions. ### Competing Interest Statement The authors have declared no competing interest. Simons Foundation, https://ror.org/01cmst727, #00010410 Simons Foundation SFARI, #890042 Stichting de Drie Lichten Dutch Research Council, #09120012110034, #OCENW.XS24.2.134

Kleefstra syndrome (KLEFS1) results from EHMT1 haploinsufficiency and is characterized by variable neurodevelopmental delays and psychopathology. Developmental regression, marked by the sudden loss of previously acquired daily life skills during late puberty or early adulthood, has emerged as a severe complication in individuals with KLEFS1. To investigate the clinical and molecular mechanisms underlying developmental regression and assess the therapeutic potential of olanzapine, we conducted a sequential study in an international cohort of 54 individuals with KLEFS1. Among 16 individuals treated with olanzapine, 10 exhibited a beneficial response based upon improvement of their adaptive functioning, and 4 showed temporary improvement. These clinical findings informed preclinical studies using human induced pluripotent stem cell-derived and ex-vivo cortical slices from a mouse model of KLEFS1. We identified hyperactivity in EHMT1+/- neuronal networks cocultured with EHMT1+/- astrocytes, a dysfunction reversible by olanzapine. Mechanistically, EHMT1+/- astrocytes displayed elevated levels of S100B, a neuroinflammatory marker contributing to neuronal network hyperactivity. Notably, olanzapine treatment reduced S100B levels, and pharmacological inhibition or genetic knockdown of S100B in EHMT1+/- astrocytes was sufficient to rescue the neuronal hyperactivity phenotype. These findings underscore a critical role for astrocytes in KLEFS1 pathophysiology and identify a potential cellular target for olanzapine in mitigating developmental regression.

Therapeutic options for neurodevelopmental disorders (NDD) are expanding. Targeting alternative splicing (AS) events linked to nonsense-mediated decay (NMD) offers a promising way to boost gene expression in haploinsufficiency disorders. However, naturally occurring NMD-coupled AS (NMD AS) events in brain cells remain poorly characterized. Here, we integrate long- and short-read RNA sequencing of NMD-inhibited induced pluripotent stem cell-derived excitatory neurons, astrocytes, and microglia to map and prioritize NMD AS events most suitable for therapeutic intervention. We developed an optimized prediction framework and provide an open access, queryable, database cataloging the existence and abundance of NMD AS events across these cell types. Querying this resource, we identified 936 NMD-sensitive AS events in 250 autosomal-dominant NDD genes and nominate 60 NMD AS events in haploinsufficient genes underlying 42 NDDs that are abundant in at least one cell type and thus represent promising therapeutic targets. These included a previously targeted NMD AS event in SCN1A, and known NMD AS events in CHD2, EZH2, and NR4A2, for which we confirm high cell type-specific abundance, as well as an newly identified abundant event in PHIP. Beyond NDD genes, we identify 1,817 differentially spliced AS events including NMD AS events, highlighting the potential functional role of (NMD) AS within brain cell-specific regulatory programs. This framework and resource enables systematic discovery and prioritization of therapeutically targetable NMD AS events and establish a cell-type resolved atlas to guide splice-modulating strategies in NDDs. ### Competing Interest Statement The authors have declared no competing interest. ZonMw, The Dutch Organisation for knowledge and innovation in health, healthcare and well-being, https://ror.org/01yaj9a77, 015.014.066, 10250022110002 Simons Foundation, https://ror.org/01cmst727, 890042

Microglia-neuron interactions play a key role in a variety of central nervous system disorders. Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation. Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing hiPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging, and neuronal activity readouts.In this protocol, astrocytes were required for long-term survival of microglia and for their integration into neuronal networks. Our co-maturation approach induced the typical ramified microglia morphology and characteristic microglia-neuron interactions. Homeostatic markers such as P2RY12 and TMEM119 and neuronal remodeling-associated genes were upregulated compared to microglia monocultures, highlighting the necessity of the environment to generate and maintain the context-dependent microglia signature in vitro. In this manuscript, we include the full optimization process of our co-maturation approach, a comprehensive description of the protocol, practical guidelines, and troubleshooting tips. Our co-maturation model provides a powerful tool to assess the role of human microglia in modulating neuronal function and development in health and disease.

While most protein–coding regions in our genome are highly conserved, phenotypic variation largely arises from differences in gene transcription, splicing, and translation. Intra–gene splicing variations are a known source of inter–individual differences. However, inter–gene splicing events, which generate fusion transcripts—RNA molecules combining exons from multiple distinct genes—are less explored. Fusion transcripts are well–studied in cancer, but their expression and inter–individual variability in normal human tissues has not been thoroughly investigated. We conducted a genome–wide fusion transcript analysis in postmortem human brain tissues from 276 individuals, identifying 717 distinct fusion transcripts. Many had protein coding potential, further supported in some cases by ribosome profiling data. Fusion transcripts that were present in only part of the human population were predominantly located within segmental duplication (SD) "hotspots"; highly copy number–variable genomic regions linked to neurodevelopmental and neurodegenerative diseases. None of these variable fusion transcripts were present in chimpanzee or rhesus macaque, suggesting they are human–specific. We confirmed that SD–derived fusion transcripts arise from structural genomic rearrangements in the human population, previously associated with neurodevelopmental and neurodegenerative disease risk. Inter–individual variation in fusion transcript expression represents an overlooked source of genetic diversity, with potential to contribute to differences in disease susceptibility. ### Competing Interest Statement The authors have declared no competing interest.

Microglia-neuron interactions play a central role in a variety of central nervous system disorders. Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation. Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing human iPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging and neuronal activity readouts. We show that astrocytes were required for long-term survival of microglia and for their integration into neuronal networks. Our co-maturation approach induced the typical ramified microglia morphology and characteristic microglia-neuron interactions. Homeostatic markers like P2RY12 and TMEM119 and neuronal remodeling associated genes were upregulated compared to microglia monocultures, highlighting the necessity of the environment to generate and maintain the context-dependent microglia signature in vitro. In this manuscript, we include the full optimization process of our co-maturation approach, a comprehensive description of the protocol, practical guidelines and troubleshooting tips. Our co-maturation model provides a powerful tool to assess the role of human microglia in modulating neuronal function and development in health and disease. Highlights ### Competing Interest Statement The authors have declared no competing interest. Simons Foundation, https://ror.org/01cmst727, 00010410 ZonMw, The Dutch Organisation for knowledge and innovation in health, healthcare and well-being, https://ror.org/01yaj9a77, 09120012110034 Radboud University Medical Center, https://ror.org/05wg1m734

Neuronal networks rely on a balance between the activity of excitatory and inhibitory neurons, each having distinct roles in regulating the flow of activity across brain circuits and signal processing. Recent work by Selten et al. uncovers how parvalbumin (PV)-expressing interneurons adjust their inhibitory inputs in response to activity changes, revealing a neuropeptide-based mechanism.

The capacity of neurons to maintain stable activity levels through homeostatic plasticity is essential for proper brain function. Primary cilia, which are non-motile, antenna-like organelles projecting from the surface of most vertebrate cells, serve as key hubs for signal transduction, playing crucial roles in tissue development and cellular homeostasis. In this study, we identify a previously unrecognised role for primary cilia in mediating neuronal homeostatic plasticity using human induced pluripotent stem cell-derived neurons. We show that neuronal cilia exhibit dynamic, bidirectional changes in volume in response to alterations in network activity: elongating during chronic activity suppression and shortening after increased activity. To assess the functional relevance of this ciliary plasticity, we modelled ciliary dysfunction in neurons carrying homozygous loss-of-function mutations in genes associated with neuronal ciliopathies, including NPHP1 and CEP290 . Mutations affecting ciliary function either increased ciliary length or led to ciliary loss, and these mutant neurons exhibited severe impairments in homeostatic regulation across multiple domains—morphological, functional, and transcriptional. Specifically, NPHP1 and CEP290 deficient neurons failed to adapt synaptic strength, intrinsic excitability, and ciliary morphology in response to prolonged activity suppression. They also displayed dysregulated baseline network activity, and exhibited blunted gene expression changes. Together, these findings establish the primary cilium as a critical regulator of homeostatic plasticity in human neurons and provide a new framework through which to examine neurodevelopmental and neuropsychiatric disorders linked to ciliary dysfunction. Key highlights ### Competing Interest Statement The authors have declared no competing interest. European Research Council, 861329 Dutch Research Council, NWO ENW-M2, OCENW.M20.216 Simons Foundation, 890042

Alternative splicing is a well-known contributor to transcriptomic diversity between individuals. Less explored are inter-gene splicing events, which generate fusion transcripts-RNA molecules combining exons from two or more distinct genes. In cancer, fusion transcripts frequently result from somatic structural rearrangements, yet it remains unclear to what extent germline structural variants in non-cancer tissues contribute to fusion transcript formation and inter-individual variability. We conducted a genome-wide fusion transcript analysis in post-mortem human brain tissues from 312 individuals, identifying 1458 distinct fusion transcripts in temporal cortex and cerebellum. We showed that many fusion transcripts have protein coding potential, further supported by ribosome profiling data. Fusion transcripts that were present in only part of the human population were predominantly located within segmental duplication (SD) 'hotspots,' highly copy number-variable genomic regions that are frequently linked to neurodevelopmental and neurodegenerative disorders. None of these variable fusion transcripts were present in chimpanzees or rhesus macaques, suggesting they are human-specific. Our study reveals that inter-individual variation in fusion transcript expression is a largely underappreciated source of genetic and transcriptomic diversity. Variable fusion transcripts originating from disease-associated SD hotspots in our genome may have yet unexplored functional consequences for physiological and pathophysiological processes in each individual.

Neuropsychiatric disorders impact over 3 billion individuals globally, posing significant challenges due to their molecular complexity, phenotypic diversity, and limited clinical translation of genetic insights. Advances in induced pluripotent stem cell (iPSC) technology offer unprecedented opportunities to model these disorders in human-relevant contexts. Human iPSC-derived two-dimensional neurons and glia, and three-dimensional organoids recapitulate key aspects of brain development and cellular functions, enabling the study of disease mechanisms and therapeutic responses on the relevant genetic background. Pioneering studies have begun to demonstrate the potential of iPSC models for precision medicine. However, translating these findings to clinical applications at scale requires robust validity assessments. Building on established frameworks of construct, face, and predictive validity derived from animal models, this perspective examines their application within an iPSC context. These approaches offer valuable insights to refine iPSC-based modeling systems and enhance their translational relevance as well as address the complexities of modeling neuropsychiatric disorders.

Pyridoxine-dependent epilepsy (PDE) is a rare neurometabolic disorder of lysine catabolism caused by bi-allelic variants in ALDH7A1. This enzyme deficiency leads to accumulation of neurotoxic metabolites, pyridoxal-phosphate inactivation, and consequently severe neurological symptoms. Current treatments, including vitamin B6 supplementation and lysine-restricted diets, partially alleviate seizures and intellectual disability but are not curative. To explore underlying mechanisms and potential therapies, we generated patient-derived human induced pluripotent stem cell (hiPSC) lines that were subsequently differentiated into astrocytes, the primary source of ALDH7A1 in the brain and key regulators of metabolic homeostasis. Metabolomic analyses confirmed elevated PDE biomarkers, and RNA sequencing revealed gene expression changes consistent with increased oxidative stress. Oxidative damage was validated by markers of DNA oxidation, increased reactive oxygen species (ROS) levels, and lipid peroxidation. In addition, dysregulated oxygen consumption rates suggested mitochondrial dysfunction in PDE astrocytes. Notably, these pathological phenotypes were alleviated by downregulating alpha-aminoadipic semialdehyde synthase (AASS), the first enzyme of the lysine catabolism, by using CRISPR-Cas9 editing or antisense oligonucleotides (AONs). This demonstrates that lysine catabolism underlies these phenotypes and highlights the therapeutic potential of AON therapy targeting AASS to reduce neurotoxic metabolite accumulation. These findings provide a promising strategy for developing targeted treatments for PDE and other rare neurometabolic disorders.

Haploinsufficiency of CACNA1A, encoding the pore-forming α1 subunit of P/Q-type voltage-gated calcium channels, is associated with a clinically variable phenotype ranging from cerebellar ataxia to neurodevelopmental syndromes with epilepsy and intellectual disability. To understand the pathological mechanisms of CACNA1A loss-of-function variants, we characterized a human neuronal model for CACNA1A haploinsufficiency by differentiating isogenic induced pluripotent stem cell lines into glutamatergic neurons and investigated the effect of CACNA1A haploinsufficiency on mature neuronal networks through a combination of electrophysiology, gene expression analysis and in silico modelling. We observed an altered network synchronization in CACNA1A+/- networks alongside synaptic deficits, notably marked by an augmented contribution of GluA2 subunit-lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Intriguingly, these synaptic perturbations coexisted with increased non-synaptically driven activity, as characterized by inhibition of N-methyl-D-aspartate and AMPA receptors on micro-electrode arrays. Single-cell electrophysiology and gene expression analysis corroborated this increased intrinsic excitability through reduced potassium channel function and expression. Moreover, we observed partial mitigation of the CACNA1A+/- network phenotype by 4-aminopyridine, a therapeutic intervention for episodic ataxia type 2. Positive modulation of small conductance calcium-activated potassium channels could reverse the CACNA1A+/- network electrophysiological phenotype. In summary, our study pioneers the characterization of a human induced pluripotent stem cell-derived neuronal model for CACNA1A haploinsufficiency and has unveiled new mechanistic insights. Beyond showcasing synaptic deficits, this neuronal model exhibited increased intrinsic excitability mediated by diminished potassium channel function, underscoring its potential as a therapeutic discovery platform with predictive validity.

The relatively slow pace of cortical development in humans has long been a topic of investigation. Studies seeking to understand the underlying mechanisms have mostly focused on neurogenetic comparisons with extant species. Here we ask if developmental tempo differences may have also existed between us and our extinct relatives for whom genomes are available. To do so, we nominate a sapiens-specific derived allele, virtually fixed in contemporary populations, which resides in an enhancer region active during early cortical development. The single nucleotide variant is predicted to significantly affect CHD2 expression, a chromatin remodeler known to play an important role in neural development and for which haploinsufficiency is associated with epilepsy and autism. We leverage patient induced pluripotent stem lines (iPSC) and engineered iPSCs in which we reintroduced the ancestral allele and generated heterozygous loss-of-function mutations. We reveal that CHD2 deficiency impairs lysosomal acidification and autophagosome flux. In contrast, ancestralized lines, which we find express higher levels of CHD2, exhibit enhanced lysosomal function and consequently accelerated autophagosome flux, consistent with our observations in chimpanzee and bonobo lines. This set of findings demonstrates that CHD2 dosage critically regulates the autolysosomal pathway. Through deep phenotyping of cortical organoid and neuron cultures, we show that the CHD2-modulated autolysosomal pathway impacts the timing of developmental programs, acquisition of neuronal functional properties and circuit maturation. Finally, we validate an estrogen-dependent rewiring of CHD2 regulation in the evolution of our lineage, providing a mechanistic understanding of how a single nucleotide variant in a regulatory region contributed to the modern pace of neuronal development and maturation. Together, our findings establish CHD2 as a regulator in setting neurodevelopmental tempo via the autolysosomal pathway. ### Competing Interest Statement The authors have declared no competing interest.

Early neuronal development relies on the proliferation of neural progenitor cells, making this developmental stage particularly vulnerable to DNA replication impediments. Here, we identify a non-canonical role for the non-specific lethal (NSL) complex in safeguarding DNA replication during neurodevelopment. The NSL complex, which acetylates histone 4 at gene promoters and is mutated in Koolen-de Vries syndrome (KdVS), prevents unscheduled R-loop accumulation at weakly transcribed promoters that are typically devoid of R-loops. Single-cell sequencing reveals that loss of NSL causes replisome stalling and delayed S-phase progression. Neural organoids derived from KdVS patients exhibit impaired DNA replication, developmental abnormalities, and reduced synapse formation, driven entirely by unscheduled R-loop accumulation. These findings reveal that faithful DNA replication is critical for early neurodevelopment. ### Competing Interest Statement The authors have declared no competing interest. Dutch Research Council, https://ror.org/04jsz6e67, NWO ENW-M2, OCENW.M20.216, NWO-VICI, 182.052 European Research Council, 101053581-scTranslatomics, 101043815-STOP-FIX-GO Novo Nordisk Fonden Synergy Programme, 0091873 Swiss National Science Foundation, https://ror.org/00yjd3n13, 310030-197003 Simons Foundation Autism Research Initiative, 890042 Koolen de Vries Foundation

BACKGROUND:Neurodevelopmental disorders (NDDs) are a challenging group of disorders to treat, but promising therapeutic interventions in the form of antisense oligonucleotides (AONs) have emerged in recent years. However, the applicability of AON therapy for NDDs varies based on genetic and phenotypic traits. In this study we systematically evaluated key characteristics for AON therapy suitability in NDDs, to estimate overall therapy potential and identify, both well- and less-studied, targetable NDDs. METHODS:An NDD dataset was created and evaluated to identify potentially targetable NDDs for seven AON strategies. This involved examining the presence of a combination of critical factors including disease-gene properties, such as regulatory elements, effects of pathogenic variants, and disease-associated phenotypic features. RESULTS:Through the systematic evaluation of the presence of targetable characteristic for each NDD and AON strategy, we identified 711 NDDs (38% of the total) with characteristics favorable for at least one AON strategy and predicted that 18% of affected individuals could benefit from AON therapy. CONCLUSIONS:The results from our analysis demonstrate that there might be a more extensive potential for the use of AON therapy in NDDs than was anticipated thus far, underscoring AON therapy as a promising treatment option for NDDs while simultaneously contributing to informed therapy selection.