Biology subjects

Srivastava, D. P.

Publications and source records attributed to Srivastava, D. P..

At least 19 recordsLinked to original sources

bioRxiv2026

A human neuron-microglia tri-culture platform to study the influence of microglia on developing neuronal networks in vitro

Human brain function is dependent on synaptic architecture and function between a range of different cell types. Glutamatergic and GABAergic neurons provide the basis by which the excitatory and inhibitory balance is achieved in cortical networks, and microglia interact with them to shape synaptic architecture and neural networks. Understanding the interactions between these cell types is crucial to elucidating mechanisms relevant to brain physiology and, potentially, to neurodevelopmental and neurological disorders. Here, we establish a rapid and reproducible human tri-culture platform comprising deterministically-programmed glutamatergic neurons, GABAergic neurons, and microglia to facilitate cell-cell interaction studies during human cortical development. Using these deterministically-programmed ioCells, we systematically optimised neuronal ratios, culture conditions, and the timing of microglial integration to generate a stable neuronal network prior to microglia incorporation. Multi-electrode arrays (MEAs) recordings identified an 80:20 glutamatergic-to-GABAergic ratio as the most robust configuration for sustained and reproducible network activity in this context. Structural characterisation using automated high-content imaging confirmed the formation of both excitatory and inhibitory synapses, while longitudinal MEA recordings demonstrated stable network maturation following microglial incorporation. Microglia incorporation influenced neuronal firing dynamics, increasing burst activity without disrupting early synapse formation. As a proof of concept for disease modelling, we incorporated microglia carrying the Alzheimers disease-associated TREM2 R47H mutation and detected subtle but reproducible alterations in neuronal burst dynamics. Together, this work establishes a defined human neuron-microglia triculture platform that enables scalable investigation of neuroimmune interactions and genetic variants, laying the foundations for more complex future models.

neuroscience↗

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.

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bioRxiv2025

Exploring the functions of JAKMIP1 in neuronal IL-6/STAT3 signaling and its relevance to chromosome 15q-duplication syndrome

Growing evidence supports neuroinflammation as a risk factor for neurodevelopmental and psychiatric disorders. Interleukin 6 (IL-6), a classical pro-inflammatory cytokine, has been associated with autism spectrum disorder (ASD)-related phenotypes. To better understand molecular factors that modify neuronal cytokine responses in ASD, we investigated potential roles for JAKMIP1, a gene linked to chromosome 15q-duplication syndrome (Dup15q; a form of syndromic ASD), in regulating IL-6/STAT3 signaling. We observe that JAKMIP1 deficiency impairs IL-6/STAT3 signaling and IL-6-induced neuritogenesis in SH-SY5Y cells; and discover that JAKMIP1 may regulate STAT3 expression via its C-terminus, which exhibits nucleoplasmic localization. Additionally, we find that IL-6/STAT3 signaling is altered in Dup15q hiPSCs-derived cortical neurons, which display heightened responsiveness to IL-6; though it is unclear whether and how JAKMIP1 contributes to this. Overall, our findings identify JAKMIP1 as a modulator of neuronal IL-6/STAT3 signaling and support that ASD-linked genetic variants can alter the inflammatory landscape of ASD.

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bioRxiv2025

Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

Parkinsons Disease (PD) is characterised by accumulation of -synuclein (-Syn), but how elevated -Syn alters presynaptic architecture during prodromal phases of the disease remains unclear. We investigated presynaptic morphology and two key presynaptic proteins: MYCBP2; and the Active Zone (AZ) protein ELKS. Primary rat cortical neurons were transfected with wildtype -Syn, or the familial A30P mutant, and nanoscopic changes to bouton morphology and protein localisation were investigated using super-resolution microscopy. Variant specific accumulation patterns for overexpressed -Syn were observed without changes to bouton structure. Additionally, increases of both -Syn variants affected presynaptic proteins, decreasing MYCBP2 puncta count and intensity, and reducing ELKS protein density. Since MYCBP2 potentially regulates ELKS, these findings suggest that elevated -Syn perturbs AZ components whilst presynaptic structure is preserved. Our study supports growing evidence that increased -Syn disturbs presynaptic function potentially through a MYCBP2-ELKS axis, a mechanism which may provide a valuable target for PD therapeutics.

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bioRxiv2025

Electrophysiological development and functional plasticity in dissociated human cerebral organoids across multiple cell lines

Microelectrode arrays (MEAs) are increasingly used to profile the development of synchronised activity in neural organoids, yet no organoid study has investigated the consistency of electrophysiological development across cell lines. Here, we used dissociated neural organoids derived from four cell lines on MEAs to characterise functional synapse development using multiple parameters across time. The dissociated organoids had increasing functional connectivity and network activity over time across all cell lines and plasticity in response to synaptic-like stimulation. Like the organoids they were derived from, dissociated organoid cultures contained a diverse mixture of cell types. Variability in activity parameters was associated with differences in cell type composition and regional identity, which in turn were affected by donor cell line and batch effects. These results demonstrate that dissociated cerebral organoids can generate functional neurons, akin to primary neuronal cultures from brain tissue, providing a scalable model for studies of neurodevelopment and synaptic function.

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bioRxiv2025

Proteomic Analysis Links Truncated Tau to Lysosome Motility, Autophagy and Endo-lysosomal Dysfunction

Tauopathies are characterized by the progressive accumulation of abnormal tau species, which disrupt the autophagy-lysosomal pathway (ALP), a critical system for degrading intracellular macromolecules and aggregated proteins, causing toxicity and cell death. This study investigates the impact of the N-terminally truncated Tau35 protein overexpression on proteolytic pathways, including effects on autophagy and endo-lysosomal processes. Using a Tau35 mouse model and SH-SY5Y cell lines stably expressing either the Tau35 fragment or full-length tau, we employed western blotting, proteomic analysis of lysosome-enriched brain fractions, proteolysis/endocytosis assays, and live-cell imaging with the lysotracker reporter to assess protein degradation and lysosomal function. Our findings identify early pathological changes in endo-lysosomal processes, including increased endocytosis, proteolytic dysfunction and lysosomal motility abnormalities, associated with Tau35-induced toxicity. This work extends previous research by providing new insights into the mechanisms of Tau35-induced neurotoxicity, offering a foundation for developing targeted therapeutic strategies to address tauopathies.

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bioRxiv2024

Properties of Alzheimers disease brain-derived tau aggregates define tau processing by human astrocytes.

The templated misfolding of tau proteins accounts for tau pathology spread in Alzheimers disease (AD). Post-translational modifications, including phosphorylation at specific residues, are closely linked with tau seeding ability and clinical disease progression. Increasing evidence supports a contributing role for astrocytes in tau spread. This study demonstrates that tau aggregates from postmortem AD brain are internalized and processed by human astrocytes. Differences in the efficiency of tau internalization, clearance and/or seeding were noted, which may reflect molecular properties of tau. Notably, we observed a direct relationship between alterations in tau handling by astrocytes and astrocyte responses, which were evident in transcriptomic data. Dysregulated genes include several previously identified as upregulated in reactive astrocytes in AD brain, as well as being implicated in pathological tau clearance by autophagy and other pathways. The study provides new insights into the complex interplay between tau molecular diversity and astrocyte responses in AD.

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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.

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bioRxiv2024

Schizophrenia risk gene ZNF804A controls ribosome localization and synaptogenesis in developing human neurons

ZNF804A was amongst the first genes robustly associated with schizophrenia based on findings from large-scale genomic studies. Previous research has implicated ZNF804A in the regulation of gene expression and synaptic function, but the role of this gene in neurodevelopment and in schizophrenia pathogenesis remains unclear. To study its function during neurodevelopment, we generated isogenic human induced pluripotent stem cells (hiPSCs) with reduced ZNF804A expression, differentiated them into developing cortical glutamatergic neurons and studied their transcriptomic, synaptic and protein signatures. Mutant neurons showed modest evidence changes in gene expression. However, high-content confocal imaging revealed increased excitatory synapse density in mutant neurons. Cell-compartment specific proteomic analysis further revealed that mutant neurons had higher levels of ribosomal and translational proteins within neurites, and high-content imaging confirmed increased local protein synthesis efficiency. Overall, these results demonstrate that in human developing cortical glutamatergic neurons, ZNF804A regulates excitatory synapse formation potential via increased local protein translation.

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bioRxiv2023

Transcriptional and Cellular Response of hiPSC-derived Microglia-Neural Progenitor Co-Cultures Exposed to IL-6

Elevated interleukin (IL-)6 levels during prenatal development have been linked to increased risk for neurodevelopmental disorders (NDD) in the offspring, but the mechanism remains unclear. Human-induced pluripotent stem cell (hiPSC) models offer a valuable tool to study the effects of IL-6 on features relevant for human neurodevelopment in vitro. We previously reported that hiPSC-derived microglia-like cells (MGLs) respond to IL-6, but neural progenitor cells (NPCs) in monoculture do not. Therefore, we investigated whether co-culturing hiPSC-derived MGLs with NPCs would trigger a cellular response to IL-6 stimulation via secreted factors from the MGLs. Using N=4 donor lines without psychiatric diagnosis, we first confirmed that NPCs can respond to IL-6 through trans-signalling when recombinant IL-6Ra is present, and that this response is dose-dependent. MGLs secreted soluble IL-6R, but at lower levels than found in vivo and below that needed to activate trans-signalling in NPCs. Whilst transcriptomic and secretome analysis confirmed that MGLs undergo substantial transcriptomic changes after IL-6 exposure and subsequently secrete a cytokine milieu, NPCs in co-culture with MGLs exhibited a minimal transcriptional response. Furthermore, there were no significant cell fate-acquisition changes when differentiated into post-mitotic cultures, nor alterations in synaptic densities in mature neurons. These findings highlight the need to investigate if trans-IL-6 signalling to NPCs is a relevant disease mechanism linking prenatal IL-6 exposure to increased risk for psychiatric disorders. Moreover, our findings underscore the importance of establishing more complex in vitro human models with diverse cell types, which may show cell-specific responses to microglia-released cytokines to fully understand how IL-6 exposure may influence human neurodevelopment.

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bioRxiv2023

Estradiol regulates local synthesis of synaptic proteome via sex-specific mechanisms.

Estrogens, specifically 17{beta}-estradiol (estradiol), can modulate synaptic function by regulating the expression and localisation of synaptic proteins. However, the mechanisms underlying estradiols regulation of synaptic protein expression, and whether if they occur in a sex specific manner, is not well understood. In this study, using sex-specific hippocampal slice cultures and mixed-sex primary hippocampal neurons, we investigated whether local protein synthesis is required for estradiol- induced synaptic protein expression. Estradiol rapidly increased the rate of protein synthesis and the number of actively translating ribosomes along dendrites and near synapses in both male and female hippocampal neurons. Importantly, these effects occurred independently of gene transcription. Moreover, estradiol also increased the abundance of nascent proteins localised to synapses, independently of gene transcription. Specifically, estradiol increased the synaptic expression of GluN2B- containing N-methyl-D-aspartate receptors and PSD-95 in male and female hippocampus. Mechanistically, mTOR signalling was required for estradiol-induced increases in overall local protein synthesis only in male but not female hippocampus. Consistent with this, mTOR signalling mediated estradiol increases in GluN2B in male, but not female, hippocampus. Conversely, mTOR inhibition, blocked estradiol-induced increased PSD-95 expression in both male and female hippocampus. Collectively, these data suggest that the rapid modulation of local protein synthesis by estradiol is required for changes in the synaptic proteome in male and female hippocampus, and that the requirement of the mTOR signalling pathway in these effects occur in both a sex-specific and protein-dependent manner, with this signalling pathway have a greater role in male compared to female hippocampus.

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bioRxiv2022

Social memory in female mice is rapidly modulated by 17β-estradiol through ERK and Akt modulation of synapse formation

BackgroundSocial memory is essential to the functioning of a social animal within a group. Estrogens can affect social memory too quickly for classical genomic mechanisms. Previously, 17{beta}-estradiol (E2) rapidly facilitated short-term social memory and increased nascent synapse formation, these synapses being potentiated following neuronal activity. However, what mechanisms underlie and co-ordinate the rapid facilitation of social memory and synaptogenesis are unclear. Here, the necessity of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling for rapid facilitation of short-term social memory and synaptogenesis was tested. MethodsMice performed a short-term social memory task or were used as task-naive controls. ERK and PI3K pathway inhibitors were infused intra-dorsal hippocampally 5 minutes before E2 infusion. Forty minutes following intrahippocampal E2 or vehicle administration, tissues were collected for quantification of glutamatergic synapse number in the CA1. ResultsDorsal hippocampal E2 rapid facilitation of short-term social memory depended upon ERK and PI3K pathways. E2 increased glutamatergic synapse number (GluA1/bassoon colocalization) in task-performing mice but decreased synapse number in task-naive mice. Critically, ERK signaling was required for synapse formation/elimination in task-performing and task-naive mice, whereas PI3K inhibition blocked synapse formation only in task-performing mice. ConclusionsWhilst ERK and PI3K are both required for E2 facilitation of short-term social memory and synapse formation, only ERK is required for synapse elimination. This demonstrates previously unknown, bidirectional, rapid actions of E2 on brain and behaviour and underscores the importance of estrogen signaling in the brain to social behaviour.

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bioRxiv2022

Acute IL-6 exposure triggers canonical IL-6R signalling in hiPSC microglia, but not neural progenitor cells.

BackgroundExposure to elevated interleukin (IL)-6 levels in utero is consistently associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ) and autism spectrum condition (ASC). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterised the response of hiPSC-derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture. ResultsWe observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both a protein and transcript level due to the absence of IL-6Ra expression and sIL-6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL-6, JMJD3 and IL-10 expression in MGL, confirming activation of canonical IL-6R signalling. Bulk RNAseq identified 156 upregulated genes (FDR <0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR, which significantly overlapped with an upregulated gene set from post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility suggestive of a gain of surveillance function, consistent with gene ontology pathways highlighted from the RNAseq data. Finally, MGLs displayed elevated CCL1, CXCL1, MIP-1A/B, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 secretion post 3h and 24h IL-6 exposure. ConclusionOur data provide evidence for cell specific effects of acute IL-6 exposure in a human model system and strongly suggest microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro.

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bioRxiv2021

Attenuated transcriptional response to pro-inflammatory cytokines in schizophrenia hiPSC-derived neural progenitor cells

Maternal immune activation (MIA) during prenatal development is an environmental risk factor for psychiatric disorders including schizophrenia (SZ). This link is particularly well established in the case of schizophrenia. Converging lines of evidence from human and animal model studies have suggested that elevated cytokine levels in the maternal and fetal compartments are an important indication of the mechanisms driving this association. However, there is variability in susceptibility to the psychiatric risk conferred by MIA, likely influenced by genetic factors. How MIA interacts with a genetic profile susceptible to SZ is challenging to test in animal models. To address this gap, we examined whether differential gene expression responses occur in forebrain-lineage neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSC) generated from three individuals with a diagnosis of schizophrenia and three healthy controls. Following acute (24 hour) treatment with either interferon-gamma (IFN{gamma}; 25 ng/l) or interleukin (IL)-1{beta} (10 ng/l), we identified, by RNA sequencing, 3380 differentially expressed genes (DEGs) in the IFN{gamma}-treated control lines (compared to untreated controls), and 1980 DEGs in IFN{gamma}-treated SZ lines (compared to untreated SZ lines). Of these, 359 genes responded differently to IFN{gamma} treatment in SZ relative to control lines, which were enriched for genes differentially expressed in adult SZ cases and in pathways related to the regulation of antigen processing, neuronal post-synaptic density, and the presynapse. There were no differentially expressed genes in the IL-1{beta}-treatment conditions after Benjamini-Hochberg correction. Gene set enrichment analysis however showed that IL-1{beta} impacts immune function and neuronal differentiation. Collectively, our data suggest that a) SZ NPCs show an attenuated transcriptional response to IFN{gamma} treatment compared to control NPCs; b) IL-1{beta} may be less relevant to NPC cultures than IFN{gamma} and c) the genes differentially regulated in schizophrenia lines are primarily mitochondrial, loss-of-function, pre- and post-synaptic genes. Our findings particularly highlight early synaptic development as a key target for future research into the mechanisms of association between maternal immune activation and schizophrenia risk. Highlights* There is a significant transcriptional response to IFN{gamma} treatment in human induced cortical neural progenitor cells (NPCs) * Differential expression implicates mitochondrial complex genes in this IFN{gamma}-dependent response, which are underexpressed in response to treatment in schizophrenia (SZ) NPCs. * SZ NPCs do not upregulate pre- and post-synaptic genes in response to IFN{gamma} as much as control NPCs * IL-1{beta} may be less relevant to NPC cultures than IFN{gamma} due to low IL1 receptor expression in NPCs.

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bioRxiv2021

Interferon-γ Exposure of Human iPSC-derived Neurons Alters Major Histocompatibility Complex I and Synapsin I Protein Expression

Human epidemiological data links maternal immune activation during gestation with increased risk for neurodevelopmental disorders including schizophrenia. Animal models of maternal immune activation (MIA) provide causal evidence for this association and strongly suggest that inflammatory cytokines act is a critical link between maternal infection and aberrant offspring brain and behavior development. This includes evidence for reduced synapse formation, consistent with post-mortem and in vivo evidence of reduced synaptic density in schizophrenia. However, to what extent specific cytokines are necessary and sufficient for these effects remains unclear. Using a human cellular model, we recently demonstrated that acute exposure to interferon-{square} (IFN{square}) recapitulates molecular and cellular phenotypes associated with neurodevelopmental disorders. Here, we extend this work to test whether IFN{square} affects synapse formation in an induced neuron model that generates forebrain glutamatergic neurons. Using immunocytochemistry and quantitative PCR, we demonstrate that acute IFN{square} exposure results in significantly increased MHCI expression at the mRNA and protein level. Furthermore, acute IFN{square} exposure decreases synapsin I protein in neurons but does not affect synaptic gene mRNA levels. Interestingly, complement component 4A (C4A) mRNA is also significantly increased following acute IFN{square} exposure. This study builds on our previous work by showing that IFN{square}-mediated disruption of relevant synaptic proteins can occur at early stages of synapse formation, potentially contributing to neurodevelopmental disorder phenotypes such as schizophrenia.

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bioRxiv2021

Schizophrenia Risk Proteins ZNF804A and NT5C2 Interact at Synapses

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes have been identified as robust susceptibility genes in large-scale genome-wide association studies of schizophrenia. The ZNF804A and NT5C2 proteins are highly expressed in developing and mature cortical neurons. ZNF804A has been implicated in regulating the development of neuronal morphology; it localises to synapses and is required for activity-dependent modifications of dendritic spines. NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and implicated in influencing protein synthesis in neural progenitor cells. But despite these findings, a better understanding of the role these proteins play in regulating neuronal function is needed. A recent yeast two-hybrid screen has identified ZNF804A and NT5C2 as potential interacting proteins, but whether this occurs in situ; and moreover, in cortical neurons, is unknown. Here we show that ZNF804A and Nt5C2 colocalise and interact in hEK293T cells. Furthermore, their rodent homolouges, ZFP804A and NT5C2, specifically colocalise at synapses and form a protein complex in cortical neurons. Knockdown of Zfp804A or Nt5c2 resulted in a significant decrease in synaptic expression of both proteins, suggesting that both proteins are required for the synaptic targeting of each other. Taken together, these data indicate that ZNF804A/ZFP804A and NT5C2 interact together in cortical neurons and indicate that these GWAS risk factors may function as a complex to regulate neuronal function.

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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.

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bioRxiv2019

Schizophrenia risk from locus-specific human endogenous retroviruses

Schizophrenia genome-wide association studies highlight the substantial contribution of risk attributed to the non-coding genome where human endogenous retroviruses (HERVs) are encoded. These ancient viral elements have previously been overlooked in genetic and transcriptomic studies due to their poor annotation and repetitive nature. Using a new, comprehensive HERV annotation, we found that the fraction of the genome where HERVs are located (the retrogenome) is enriched for schizophrenia risk variants, and that there are 148 disparate HERVs involved in susceptibility. Analysis of RNA-sequencing data from the dorsolateral prefrontal cortex of 259 schizophrenia cases and 279 controls from the CommonMind Consortium showed that HERVs are actively expressed in the brain (n = 3,979), regulated in cis by common genetic variants (n = 1,759), and differentially expressed in patients (n = 81). Convergent analyses implicate LTR25_6q21 and ERVLE_8q24.3h as HERVs of etiological relevance to schizophrenia, which are co-regulated with genes involved in neuronal and mitochondrial function, respectively. Our findings provide a strong rationale for exploring the retrogenome and the expression of these locus-specific HERVs as novel risk factors for schizophrenia and potential diagnostic biomarkers and treatment targets.

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