Biology subjects

Pak, C.

Publications and source records attributed to Pak, C..

9 recordsLinked to original sources

bioRxiv2025

Distinct Synaptic Mechanisms Drive NRXN1 Variant-Mediated Pathogenesis in iPSC-Derived Neuronal Models of Autism and Schizophrenia

Copy number deletions in the 2p16.3/NRXN1 locus confer genome wide risk for autism spectrum disorder (ASD) and schizophrenia (SCZ). Prior work demonstrated that heterozygous NRXN1 deletions decreases synaptic strength and neurotransmitter release probability in human-iPSC derived cortical glutamatergic induced neurons and this synaptic phenotype is replicated in SCZ patient iPSCs with varying NRXN1 genomic deletions. What is unknown, however, is whether similar synaptic impairment exists in ASD patients carrying NRXN1 deletions. Answering this question is important to determine whether all NRXN1 deletion carriers should be treated similarly or individually, based on their genetic backgrounds and deletion breakpoints. Here, using previously uncharacterized ASD patient iPSC lines, we show that ASD-NRXN1 deletions impact cortical synaptic function and plasticity in unique ways compared to SCZ-NRXN1 deletions. Specifically, at a single neuronal level, ASD-NRXN1 deletions alter basal spontaneous synaptic transmission by selectively enhancing excitatory synaptic signaling with no changes at inhibitory synapses while SCZ-NRXN1 deletions reduce both excitatory and inhibitory synaptic transmission. At the neuronal network level, there exists enhanced transmission probability and irregular firing patterns in ASD-NRXN1 deletions. Such changes at the synaptic and network level connectivity patterns influence a critical form of developmental cortical plasticity, synaptic scaling, as ASD-NRXN1 deletions uniquely fail to upscale their synaptic strength in response to chronic neuronal silencing. Together, these findings highlight the disorder-specific consequences of NRXN1 deletions on synaptic function and connectivity, offering mechanistic insights with implications for therapeutic targeting and refinement strategies for NRXN1-associated synaptopathies. HighlightsO_LINovel ASD patient-iPSC-derived E-I culture model to investigate pathogenic NRXN1 deletions on cortical synaptic function and plasticity C_LIO_LIASD-NRXN1 deletions enhance basal synaptic transmission by specifically increasing excitatory neurotransmitter release, opposite of SCZ-NRXN1 deletions C_LIO_LISynaptic scaling homeostasis is impaired in ASD-NRXN1 deletions C_LIO_LISpike train cross-correlation analysis reveals increased transmission probability and irregular firing patterns in ASD-NRXN1 deletions C_LI

neuroscience↗

bioRxiv2023

Patterning ganglionic eminences in developing human brain organoids using morphogen gradient inducing device

In early neurodevelopment, the central nervous system is established through the coordination of various neural organizers directing tissue patterning and cell differentiation. Better recapitulation of morphogen gradient production and signaling will be crucial for establishing improved developmental models of the brain in vitro. Here, we developed a method by assembling polydimethylsiloxane (PDMS) devices capable of generating a sustained chemical gradient to produce patterned brain organoids, which we termed Morphogen-gradient Induced Brain Organoids (MIBOs). At 3.5 weeks, MIBOs replicated Dorsal-Ventral patterning observed in the Ganglionic Eminence (GE). Analysis of matured MIBOs through single-cell RNA sequencing revealed distinct Dorsal GE derived CALB2+ interneurons (INs), Medium Spiny Neurons (MSNs), and MGE derived cell types. Finally, we demonstrate long term culturing capabilities with MIBOs maintaining stable neural activity in cultures grown up to 5.5 months. MIBOs demonstrate a versatile approach for generating spatially patterned brain organoids for embryonic development and disease modeling. MotivationThe use of small molecules for the guided differentiation of brain organoids has proven to be a useful tool for modeling various aspects of early neurodevelopment. Still, the embryonic brain is patterned through a combination of morphogen gradients stemming from organizer regions. To address this inconsistency, we designed a device capable of mimicking neural organizers by maintaining a steady morphogen gradient. We used this device to expose forebrain organoids to multiple gradient conditions and cataloged the diversity of cell types produced.

neuroscience↗

bioRxiv2022

Single cell transcriptomic profiling of human brain organoids reveals developmental timing- and cell-type-specific vulnerabilities induced by NRXN1 CNVs in schizophrenia

De novo mutations and copy number deletions in NRXN1 (2p16.3) pose a significant risk for schizophrenia (SCZ). It is unclear how NRXN1 deletions impact cortical development in a cell type-specific manner and disease background modulates these phenotypes. Here, we leveraged human pluripotent stem cell-derived forebrain organoid models carrying NRXN1 heterozygous deletions in isogenic and SCZ patient genetic backgrounds and conducted single-cell transcriptomic analysis over the course of brain organoid development from 3 weeks to 3.5 months. Intriguingly, while both deletions similarly impacted molecular pathways associated with ubiquitin-proteasome system, alternative splicing, and synaptic signaling in maturing glutamatergic and GABAergic neurons, SCZ-NRXN1 deletions specifically perturbed developmental trajectories of early neural progenitors and accumulated disease-specific transcriptomic signatures. Using calcium imaging, we found that both deletions led to long-lasting changes in spontaneous and synchronous neuronal networks, implicating synaptic dysfunction. Our study reveals developmental-timing- and cell-type-dependent actions of NRXN1 deletions in unique genetic contexts.

neuroscience↗

bioRxiv2022

Self-organized anteroposterior regionalization of early midbrain and hindbrain using micropatterned human embryonic stem cells

Anterior-posterior (AP) spatial regionalization is crucial for central nervous system development. Previous studies suggest that morphogen gradients can induce AP patterning in animal and organoid models. While self-organization in early embryogenesis and neurogenesis has been found using geometrically confined microtissues, spontaneously induced AP patterning has not been reported. Here, we show that circularly micropatterned human pluripotent stem cells self-organize into spatially distinct FOXG1-FOXA1+OTX2+ midbrain-like and HOXB4+ hindbrain/spinal cord-like regions. Notably, the tissue then folds inwardly to form a 3D annular structure, maintaining a distinct boundary between OTX2+ and HOXB4+ zones. The reaction-diffusion of BMP/Noggin plays a key role in the mechanism of AP patterning. Our model is validated by its capability to distinguish the teratogenic effects of valproic acid and isotretinoin. Our work suggests a novel regulatory mechanism for AP patterning and provides a tool for fast screening of teratogens.

bioengineering↗

bioRxiv2022

Loss of Neurodevelopmental Gene CASK Disrupts Neural Connectivity in Human Cortical Excitatory Neurons

Loss-of-function (LOF) mutations in CASK cause severe developmental phenotypes, including microcephaly with pontine and cerebellar hypoplasia, X-linked intellectual disability, and autism. Unraveling the pathogenesis of CASK-related disorders has been challenging due to limited human cellular models to study the dynamic roles of this molecule during neuronal and synapse development. Here, we generated CASK knockout (KO) isogenic cell lines from human embryonic stem cells (hESCs) using CRISPR/Cas9 and examined gene expression, morphometrics, and synaptic function of induced neuronal cells during development. While young (immature) CASK KO neurons show robust neuronal outgrowth, mature CASK KO neurons displayed severe defects in synaptic transmission and synchronized burst activity without compromising neuronal morphology and synapse numbers. In developing human cortical neurons, CASK functioned to promote both structural integrity and establishment of cortical excitatory neuronal networks. These results lay the foundation for future studies to identify suppressors of such phenotypes relevant to human patients. HighlightsO_LICASK LOF mutations increase neuronal complexity in immature developing neurons C_LIO_LICASK LOF does not alter synapse formation and neurite complexity in mature neurons C_LIO_LISynaptic transmission and network synchronicity are compromised in CASK KO neurons C_LIO_LIDifferential gene expression analysis reveals enrichment of synaptic gene networks in mature CASK KO neurons C_LI

neuroscience↗

bioRxiv2021

Analyses of the Autism-associated Neuroligin-3 R451C Mutation in Human Neurons Reveals a Gain-of-Function Synaptic Mechanism

Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C-mutation on human neurons has not been investigated. Here, we generated human knock-in neurons with the NLGN3 R451C-mutation. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C-mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses. No significant cell death and endoplasmic reticulum stress were detected. Importantly, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons that corresponded to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C-mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASDs.

neuroscience↗

bioRxiv2020

Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-Derived NRXN1-Mutant Neurons

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation predisposing to schizophrenia. Previous studies showed that engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multi-center effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. We show that in neurons that were trans-differentiated from induced pluripotent stem cells derived from three NRXN1-deletion patients, the same impairment in neurotransmitter release was observed as in engineered NRXN1-deficient neurons. This impairment manifested as a decrease in spontaneous synaptic events and in evoked synaptic responses, and an alteration in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

neuroscience↗

bioRxiv2019

Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel

Bioengineers designed numerous instructive brain extracellular matrix (ECM) environments that have tailored and tunable protein composition and biomechanics in vitro to study astrocyte reactivity during trauma and inflammation. However, a major limitation of both protein-based and model microenvironments is that astrocytes within fail to retain their characteristic stellate morphology and quiescent state without becoming activated under \"normal\" culture conditions. Here we introduce a synthetic hydrogel, that for the first time demonstrates maintenance of astrocyte quiescence, and control over activation on demand. With this synthetic brain hydrogel, we show the brain-specific integrin-binding and matrix metalloprotease (MMP)-degradable domains of proteins control astrocyte star-shaped morphologies, and we can achieve an ECM condition that maintains astrocyte quiescence with minimal activation. In addition, we can induce activation in a dose-dependent manner via both defined cytokine cocktails and low molecular weight hyaluronic acid. We envision this synthetic brain hydrogel as a new tool to study the physiological role of astrocytes in health and disease.

bioengineering↗

bioRxiv2017

DNA copy number gains of TCF4 (E2-2) are associated with poor outcome and the activated B-cell-like subtype of diffuse large B-cell lymphoma

The activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by the chronic activation of signaling initiated by immunoglobulin- (IgM). By analyzing DNA copy profiles of 1,000 DLBCLs, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. We show that these alterations target the TCF4 (E2-2) transcription factor, and that over-expression of TCF4 leads to its occupancy on immunoglobulin gene enhancers and increased expression of IgM at the transcript and protein level. The TCF4 gene is one of the top BRD4-regulated genes in DLBCL. Using a BET proteolysis-targeting chimera (PROTAC) we show that TCF4 and IgM expression can be extinguished, and ABC-like DLBCL cells can be killed in vitro and in vivo. This highlights a novel genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

genomics↗