Biology subjects

Adhya, D.

Publications and source records attributed to Adhya, D..

5 recordsLinked to original sources

bioRxiv2026

Assessing the impact of mono- and bi-allelic deletions in NRXN1 on synaptic function

Neurexin 1 (NRXN1) is an adhesion protein involved in synapse development and function. Mutations in NRXN1 are strongly linked with neurodevelopmental and psychiatric conditions. Mono-allelic NRXN1 mutations are associated with autistic traits, with increased likelihood of co-occurring intellectual disability. However, mono-allelic mutations have variable penetrance and occur in individuals without neurodevelopmental phenotypes. Conversely, bi-allelic mutations, though rarer, are associated with more stable penetrance and severe neurodevelopmental phenotypes. Human induced pluripotent stem cells (iPSC) have been used to study how mutations in NRXN1 impacts its function, with most studies focusing on monoallelic mutations. In this study, we systematically compared monoallelic and biallelic mutations in NRXN1, characterising their effects on molecular, synaptic, and functional phenotypes. Using CRISPR-Cas9, we introduced indels in NRXN1 exon 19, in an iPSC line containing inducible NGN2. These edits caused either mono-allelic or compound bi-allelic frameshift mutations. iPSCs containing either mutation robustly generated glutamatergic neurons, but these neurons displayed reduced expression of major NRXN1 isoforms. Transcriptomic profiling revealed modest gene expression changes in mono-allelic mutant neurons, whereas bi-allelic mutants exhibited extensive dysregulation of gene networks associated with neuronal maturation and synaptic function. Furthermore, synaptic phenotypes were mild in mono-allelic mutants but pronounced in bi-allelic mutant neurons. Both mono-allelic and bi-allelic mutant neurons displayed alterations in neuronal network activity and reduced peak depolarisation responses to KCl stimulation. Together, these data demonstrate that NRXN1 exhibits gene-dosage sensitivity, with bi-allelic disruption of exon 19 unmasking molecular, synaptic, and functional phenotypes that are only modest in mono-allelic mutant neurons.

neuroscience↗

bioRxiv2025

A non-catalytic role for MLL2 in controlling chromatin organisation and mobility during the priming of pluripotent cells for differentiation

How the chromatin regulator MLL2 (KMT2B) influences cell differentiation remains poorly understood. MLL2 is the main histone 3 lysine 4 (H3K4) trimethyltransferase acting at bivalent promoters in embryonic stem cells (ESCs) and is required for ESCs to differentiate into neuroectoderm. We show here that this requirement occurs during exit from naive pluripotency, days before neuroectoderm differentiation is impaired. Although MLL2 knockout has only a subtle effect on transcription during exit, reducing the expression of a few important neuroectodermal transcription factors, it substantially remodels chromatin architecture, disrupting 3D chromatin loops associated with bivalent promoters. The enzymatic activity of MLL2 is not needed for stabilising these loops or for neuroectoderm differentiation. This non-catalytic function of MLL2 in stabilising 3D chromatin loops has implications for lineage specification. Because MLL2 shares features with all four MLLs, chromatin tethering, rather than H3K4 methylation, may represent the primary function of MLL proteins during lineage commitment.

cell biology↗

bioRxiv2024

Impact of intragenic NRXN1 deletions on early cortical development

BackgroundDeletions in NRXN1 are strongly associated with neurodevelopmental and psychiatric conditions. While exonic deletions are well-studied, intragenic deletions, particularly in intron 5, are less understood and generally consider benign. Recent studies show exonic deletions impact isoform diversity during neurodevelopment, affecting neurogenesis and neuronal function. However, whether intragenic deletions impact isoform expression and neurodevelopment remains underexplored. MethodsWe used hiPSCs from typically developing individuals (control) and those with NRXN1 intron 5 deletions to study neurodevelopment. HiPSCs were differentiated towards a cortical fate, with NRXN1 isoform expression, molecular differences, and neuronal morphology examined. ResultsWe observed distinct NRXN1 isoform expression dynamics during early neurodevelopment, with two expression peaks post-neuronal induction and NRXN1{beta} being most highly expressed. Both NRXN1 deletion and control lines showed similar acquisition of regional and cell fate identity, but significant differences in NRXN1 isoform expression were observed between deletion and control lines, and between deletion lines. RNA sequencing revealed genotype-dependent alterations, particularly in pathways related to synaptic function and neuronal morphology. Consistent with these findings, NRXN1 deletion lines exhibited altered dendrite outgrowth, with variations between deletion lines. ConclusionsOur results indicate a potential role for intron 5 in controlling NRXN1 isoform expression during neurodevelopment. Alterations in gene expression profiles, correlated with morphological changes, suggest a role for NRXN1 isoforms in shaping dendritic morphology. Molecular and cellular differences observed between lines with identical intronic deletions suggest that additional factors, such as genetic background or biological sex, may also play an important role in these phenotypes. Collectively, these findings indicate that NRXN1 intronic deletions are not benign, influencing isoform expression, cellular phenotypes, and neurodevelopment.

neuroscience↗

bioRxiv2020

Application of Airy beam Light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

BackgroundThe inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human Cortical Spheroids (hCSs) offer a pragmatic solution to this issue. In particular, hCSs are an accessible method of generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes. These neurogeneic niches give rise to neural progenitors that subsequently differentiate into neurons. Atypical formation of these structures has been associated with neurodevelopmental disorders such as autism spectrum conditions, from studies of patient-specific human induced pluripotent stem cells grown as 2D cultures. Thus far however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hSC or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. ResultsConventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution as compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. ConclusionWe present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. Furthermore, this process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes.

neuroscience↗

bioRxiv2018

Shared gene co-expression networks in autism from induced pluripotent stem cell (iPSC) neurons

BackgroundAutism is a heterogenous collection of disorders with a complex molecular underpinning. Evidence from post-mortem brain studies using adult brains have indicated that early prenatal development may be altered in autism. Induced pluripotent stem cells (iPSCs) generated from autistic individuals with macrocephaly also indicate prenatal development as a critical period for this condition. But little is known about early altered cellular events during prenatal stages in autism. MethodsIPSCs were generated from 9 unrelated autistic individuals without macrocephaly and with heterogeneous genetic backgrounds, and 6 typically developing, control, individuals. IPSCs were differentiated towards either cortical or midbrain fates. Gene expression and high throughput cellular phenotyping was used to characterise iPSCs at different stage of differentiation. ResultsA subset of autism-iPSC cortical neurons were RNA-sequenced to reveal autism-specific signatures similar to post-mortem brain studies, indicating a potential common biological mechanism. Autism-iPSCs differentiated towards a cortical fate displayed impairments in the ability to self-form into neural rosettes. In addition, autism-iPSCs demonstrated significant differences in rate of cell type assignment of cortical precursors, and dorsal and ventral forebrain precursors. These cellular phenotypes occurred in the absence of alterations in cell proliferation during cortical differentiation, differing from previous studies. Acquisition of cell fate during midbrain differentiation was not different between control- and autism-iPSCs. ConclusionsTaken together, our data indicate that autism-iPSCs diverge from control-iPSCs at a cellular level during early stage of neurodevelopment. This suggests that unique developmental differences associated with autism may be established at early prenatal stages.

neuroscience↗