Biology subjects

Oudakker, A.

Publications and source records attributed to Oudakker, A..

12 recordsLinked to original sources

bioRxiv2025

Whole-brain clearing reveals region- and cell type-specific imbalances in inhibitory neurons in a mouse model for Kleefstra Syndrome

GABAergic inhibition is essential for balanced brain function and is frequently disrupted in neurodevelopmental disorders such as autism spectrum disorder (ASD). The inhibition is generated by a diverse population of GABAergic interneurons, whose composition and density differs throughout the brain. Here, we applied an unbiased whole-brain clearing and light-sheet imaging approach to systematically map the distribution of the three major GABAergic neuron classes - Parvalbumin-positive (PV), Somatostatin-positive (SST), and Vasoactive Intestinal Polypeptide-positive (VIP) cells - across the mouse brain in a model of Kleefstra Syndrome (Ehmt1+/-), a monogenic intellectual disability disorder with a strong ASD component. Analyzing 895 brain regions we identified widespread, cell type- and region-specific alterations in GABAergic classes. Notably, we observed increased VIP neuron density in the Ehmt1+/- cortex and decreased SST neuron density in sensory cortical and subcortical regions. In the basolateral amygdala (BLA), PV neurons exhibited precocious maturation already at the juvenile stage, which persisted into adulthood and was associated with enhanced inhibitory input onto BLA principal neurons. We here demonstrate that Ehmt1 haploinsufficiency results in region- and cell-type specific changes throughout the brain. These results underscore the value of whole-brain, high-resolution mapping approaches in uncovering previously unrecognized patterns of neural vulnerability in neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

The Neuronal Primary Cilium is a Key Regulator of Homeostatic Plasticity

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 highlightsO_LIPrimary cilia adapt bidirectionally to neuronal activity by dynamically changing volume, expanding during suppressed activity and contracting with increased neuronal activity. C_LIO_LIDisruptions in ciliopathy-associated genes perturb homeostatic plasticity, impairing structural, functional, and transcriptional responses to activity changes. C_LIO_LIPrimary cilia are essential for neuronal network development. C_LI

neuroscience↗

bioRxiv2025

Targeting AASS improves neurotoxicity and mitochondrial function in astrocyte models for pyridoxine dependent epilepsy

Pyridoxine-dependent epilepsy (PDE) is a rare neurometabolic disorder of lysine catabolism caused by bi-allelic variants in ALDH7A1. This enzyme deficiency leads to the 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 lines (hiPSC) 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 and lipid peroxidation. In addition, dysregulated oxygen consumption rates suggested mitochondrial dysfunction in PDE astrocytes. Notably, these pathological phenotypes were alleviated by downregulating AASS, the first enzyme of the lysine catabolism, by using CRISPR/Cas9 editing or antisense oligonucleotides (AON). 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.

cell biology↗

bioRxiv2025

CHD2 Dosage Ties Autolysosomal Pathway to Cortical Maturation in Disease and Evolution

The mechanisms linking evolutionary changes in gene regulation to brain development and neurodevelopmental disease susceptibility remain poorly understood. Here, we identify a human-specific variant in an enhancer region that reduces expression of the chromatin remodeler CHD2. We investigate the variant's functional consequences using genome editing, cross-primate induced pluripotent stem cell models, cortical organoids, single-cell transcriptomics, patient-derived cells, and neuronal network analyses. We demonstrate that CHD2 dosage bidirectionally regulates lysosomal function and autophagosome flux to set the tempo of neuronal maturation. Higher CHD2 expression, as found in ancestralized and non-human primate models, enhances lysosomal degradative capacity and accelerates dendritic and synaptic maturation. Conversely, CHD2 haploinsufficiency yields reciprocal defects and disrupts broader neurodevelopmental transcriptional programs. Restoring lysosomal function genetically or pharmacologically rescues neuronal maturation in CHD2-haploinsufficient neurons, establishing lysosomal dysfunction as a causal and therapeutically tractable mechanism. These findings reveal that CHD2 and lysosomal homeostasis constitute a critical molecular axis regulating the pace of cortical development across evolution and disease.

neuroscience↗

bioRxiv2024

Integrative transcriptomics and electrophysiological profiling of hiPSC-derived neurons identifies novel druggable pathways in Koolen-de Vries Syndrome

Koolen-de Vries Syndrome (KdVS) is a neurodevelopmental disorder (NDD) with no treatment options due to a lack of understanding of its underlying pathophysiology. To investigate neuronal activity in KdVS, human induced pluripotent stem cell (hiPSC)-derived neurons from KdVS and control subjects were cultured on microelectrode arrays (MEAs). Our study identified reduced network burst rates, indicating disorganized network activity in KdVS neurons. To bridge molecular and functional aspects of the syndrome, we developed an experimental framework, MEA-seq, that integrates network activity measurements with high-throughput transcriptome profiling. This approach identified a negative correlation between the expression of the NDD-associated gene CLCN4 and the network burst rate. Consequently, knockdown of CLCN4 in KdVS neurons restored the activity to control level, confirming a causal relationship between increased CLCN4 expression and reduced network burst rate. Additionally, we identified a positive correlation between mitochondrial gene expression and the network burst rate, and identified impaired mitochondrial function in KdVS hiPSC-derived neurons. The transcriptomic signature of KdVS neurons was then used for computational screening against drug perturbation signatures of the LINCS Consortium database, predicting other drug targets and compounds capable of reversing the expression of affected genes in KdVS neurons. We selected 10 compounds for experimental validation, identifying the antioxidant phloretin and the Rho-kinase inhibitor fasudil as potential candidates for restoring the network activity dysfunction in KdVS. We conclude that the integrative molecular and electrophysiological of hiPSC-derived neurons with MEA-seq has excellent potential for identifying novel drugs and druggable pathways for KdVS and other NDDs.

neuroscience↗

bioRxiv2024

Navigating Human Astrocyte Differentiation: Direct and Rapid one-step Differentiation of Induced Pluripotent Stem Cells to Functional Astrocytes Supporting Neuronal Network development

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 human induced pluripotent stem cell (hiPSC)-derived astrocytes in neuronal networks remains limited. Here, we present a streamlined one-step protocol for the differentiation of hiPSCs directly into functional astrocytes without the need for ectopic gene expression or neural progenitor cell generation. We found that culturing hiPSCs directly in commercial astrocyte medium, was sufficient to differentiate hiPSCs into functional astrocytes within five weeks. Validation to varying extents across thirty hiPSC-lines demonstrated consistent astrocyte differentiation with minimal batch-to-batch variability. We confirmed astrocyte identity and functionality of the hiPSC-astrocyte monocultures by immunofluorescence, flowcytometry, RNA sequencing, glutamate uptake assays and calcium signaling recordings. Optimization of the protocol enabled co-culture of hiPSC-astrocytes with Ngn2 hiPSC-derived neurons (iNeurons), promoting neuronal differentiation and synapse formation. Lastly, we used single-cell electrophysiology and multi-electrode arrays to confirm robust neuronal network development in 5-week-old hiPSC-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. While validated across multiple hiPSC lines, we actively encourage researchers to test and provide feedback on this protocol to enhance its validation for future iterations.

neuroscience↗

bioRxiv2024

CACNA1A haploinsufficiency leads to reduced synaptic function and increased intrinsic excitability

Haploinsufficiency of the CACNA1A gene, encoding the pore-forming 1 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 modeling. We observed an altered network synchronization in CACNA1A+/- networks alongside synaptic deficits, notably marked by an augmented contribution of GluA2-lacking AMPA receptors. Intriguingly, these synaptic perturbations coexisted with increased non-synaptically driven activity, as characterized by inhibition of NMDA 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. In summary, our study pioneers the characterization of a human induced pluripotent stem cell-derived neuronal model for CACNA1A haploinsufficiency, and has unveiled novel 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.

neuroscience↗

bioRxiv2023

A human in vitro neuronal model for studying homeostatic plasticity at the network level

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell level; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.

neuroscience↗

bioRxiv2020

KANSL1 Deficiency Causes Neuronal Dysfunction by Oxidative Stress-Induced Autophagy

Autophagy is a finely tuned process of programmed degradation and recycling of proteins and cellular components, which is crucial in neuronal function and synaptic integrity. Mounting evidence implicates chromatin remodelling in fine-tuning autophagy pathways. However, this epigenetic regulation is poorly understood in neurons. Here, we investigate the role in autophagy of KANSL1, a member of the nonspecific lethal complex, which acetylates histone H4 on lysine 16 (H4K16ac) to facilitate transcriptional activation. Loss-of-function of KANSL1 is strongly associated with the neurodevelopmental disorder Koolen-de Vries Syndrome (KdVS). Starting from KANSL1-deficient human induced-pluripotent stem cells, both from KdVS patients and genome-edited lines, we identified superoxide dismutase 1, an antioxidant enzyme, to be significantly decreased, leading to a subsequent increase in oxidative stress and autophagosome accumulation. In KANSL1-deficient neurons, autophagosome accumulation at excitatory synapses resulted in reduced synaptic density, reduced AMPA receptor-mediated transmission and impaired neuronal network activity. Furthermore, we found that increased oxidative stress-mediated autophagosome accumulation leads to increased mTOR activation and decreased lysosome function, further preventing the clearing of autophagosomes. Finally, by pharmacologically reducing oxidative stress, we could rescue the aberrant autophagosome formation as well as synaptic and neuronal network activity in KANSL1-deficient neurons. Our findings thus point towards an important relation between oxidative stress-induced autophagy and synapse function, and demonstrate the importance of H4K16ac-mediated changes in chromatin structure to balance reactive oxygen species- and mTOR-dependent autophagy.

neuroscience↗

bioRxiv2020

Cadherin-13 is a critical regulator of GABAergic modulation in human stem cell derived neuronal networks

Activity in the healthy brain relies on concerted interplay of excitation (E) and inhibition (I) via balanced synaptic communication between glutamatergic and GABAergic neurons. A growing number of studies imply that disruption of this E/I balance is a commonality in many brain disorders, however, obtaining mechanistic insight into these disruptions, with translational value for the human patient, has typically been hampered by methodological limitations. Cadherin-13 (CDH13) has strongly been associated to attention-deficit/hyperactivity disorder and comorbid disorders such as autism and schizophrenia. CDH13 localises at inhibitory presynapses, specifically of parvalbumin (PV) and somatostatin (SST) expressing GABAergic neurons. However, the mechanism by which CDH13 regulates the function of inhibitory synapses in human neurons remains unknown. Starting from human induced pluripotent stem cells, we established a robust method to generate a homogenous population of SST and PV expressing GABAergic neurons (iGABA) in vitro, and co-cultured these with glutamatergic neurons at defined E/I ratios on micro-electrode arrays. We identified functional network parameters that are most reliably affected by GABAergic modulation as such, and through alterations of E/I balance by reduced expression of CDH13 in iGABAs. We found that CDH13-deficiency in iGABAs decreased E/I balance by means of increased inhibition. Moreover, CDH13 interacts with Integrin-{beta}1 and Integrin-{beta}3, which play opposite roles in the regulation of inhibitory synaptic strength via this interaction. Taken together, this model allows for standardized investigation of the E/I balance in a human neuronal background and can be deployed to dissect the cell-type specific contribution of disease genes to the E/I balance.

neuroscience↗

bioRxiv2019

Neuronal network dysfunction in a human model for Kleefstra syndrome mediated by enhanced NMDAR signaling

Epigenetic regulation of gene transcription plays a critical role in neural network development and in the etiology of Intellectual Disability (ID) and Autism Spectrum Disorder (ASD). However, little is known about the mechanisms by which epigenetic dysregulation leads to neural network defects. Kleefstra syndrome (KS), caused by mutation in the histone methyltransferase EHMT1, is a neurodevelopmental disorder with the clinical features of both ID and ASD. To study the impact of decreased EHMT1 function in human cells, we generated excitatory cortical neurons from induced pluripotent stem (iPS) cells derived from KS patients. In addition, we created an isogenic set by genetically editing healthy iPS cells. Characterization of the neurons at the single-cell and neuronal network level revealed consistent discriminative properties that distinguished EHMT1-mutant from wildtype neurons. Mutant neuronal networks exhibited network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes were mediated by the upregulation of the NMDA receptor (NMDAR) subunit 1 and correlate with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. Furthermore, we show that EHMT1 deficiency in mice leads to similar neuronal network impairments and increased NMDAR function. Finally, we could rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Together, our results demonstrate a direct link between EHMT1 deficiency in human neurons and NMDAR hyperfunction, providing the basis for a more targeted therapeutic approach to treating KS.

neuroscience↗

bioRxiv2018

Distinct pathogenic genes causing intellectual disability and autism exhibit overlapping effects on neuronal network development

An intriguing question in medical biology is how mutations in functionally distinct genes can lead to similar clinical phenotypes. For example, patients with mutations in distinct epigenetic regulators EHMT1, MBD5, MLL3 or SMARCB1 share the core clinical features of intellectual disability (ID), autism spectrum disorder (ASD) and facial dysmorphisms. To elucidate how these phenotypic similarities are reflected by convergence at the molecular, cellular and neuronal network level, we directly compared the effects of their loss of function in neurons. Interestingly, knockdown of each gene resulted in hyperactive neuronal networks with altered patterns of synchronized activity. At the single-cell level, we found genotype-specific changes in intrinsic excitability and excitatory-inhibitory balance, but in all cases leading to increased excitability. Congruent with our physiological findings, we identified dysregulated genes that converge on biological and cellular pathways related to neuronal excitability and synaptic function, including genes previously implicated in ID/ASD. Yet, our data suggests that the common cellular phenotypes depend on the ensemble of dysregulated genes engaged in neuronal excitability rather than the direction of transcriptional changes of individual genes. The demonstration of increasing convergence from molecular pathways to neuronal networks may be a paradigm for other types of ID/ASD.

neuroscience↗