Biology subjects

Linda, K.

Publications and source records attributed to Linda, K..

10 recordsLinked to original sources

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

Neuronal autophagosomes are transported to astrocytes for degradation

Autophagy is a vital catabolic process responsible for the degradation of cytosolic components, playing a key role in cellular homeostasis and survival. At synapses, autophagy is crucial for regulating neuronal activity and utilizes a specialized machinery. While considerable progress has been made in understanding the initiation of autophagy and autophagosome formation, the mechanisms governing the clearance of autophagosomes from synaptic sites remain poorly understood. Here, we identify a novel pathway in which astrocytes actively participate in the clearance of pre-synaptic autophagosomes. Using neurons derived from human induced pluripotent stem cell (hiPSC) lines expressing fluorescent autophagy markers and chimeric mouse models, we demonstrate that neuronal autophagosomal vesicles are physically transferred to astrocytes, a process that is enhanced when synaptic activity is suppressed. Autophagosome transfer does not require direct physical cellular contact, but it does require Dynamin and cholesterol-dependent endocytosis for the internalized neuronal autophagosomes to ultimately fuse with astrocytic lysosomes. Our findings reveal a previously unrecognized mechanism of neuronal autophagosome clearance that does not require slow axonal retrograde transport but their transfer to nearby astrocytes.

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↗

bioRxiv2021

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro

Micro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem-cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution and data analysis. Therefore, we benchmarked the robustness and sensitivity of MEA-derived neuronal activity patterns derived from ten healthy individual control lines. We provide recommendations on experimental design and analysis to achieve standardization. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field towards the use of MEAs for disease-phenotyping and drug discovery.

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

Mitochondrial dysfunction impairs human neuronal development and reduces neuronal network activity and synchronicity

Epilepsy, intellectual and cortical sensory deficits and psychiatric manifestations are among the most frequent manifestations of mitochondrial diseases. Yet, how mitochondrial dysfunction affects neural structure and function remains largely elusive. This is mostly due to the lack of a proper in vitro translational neuronal model system(s) with impaired energy metabolism. Leveraging the induced pluripotent stem cell technology, from a cohort of patients with the common pathogenic m.3243A>G variant of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), we differentiated excitatory cortical neurons (iNeurons) with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function on an isogenic nuclear DNA background. iNeurons with high levels of heteroplasmy exhibited mitochondrial dysfunction, delayed neural maturation, reduced dendritic complexity and fewer functional excitatory synapses. Micro-electrode array recordings of neuronal networks with high heteroplasmy displayed reduced network activity and decreased synchronous network bursting. The impaired neural energy metabolism of iNeurons compromising the structural and functional integrity of neurons and neural networks, could be the primary driver of increased susceptibility to neuropsychiatric manifestations of mitochondrial disease.

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↗