Biology subjects

Harwood, A. J.

Publications and source records attributed to Harwood, A. J..

7 recordsLinked to original sources

bioRxiv2025

Gain and loss of function changes in CACNA1C affect neuronal networks through divergent pathways

BackgroundCACNA1C encodes the pore-forming subunit of the L-type calcium channel Cav1.2. Common variants in CACNA1C are associated with psychiatric disorders, while rare single-nucleotide variants cause CACNA1C-related disorder (CRD), a multi-system disorder with symptoms that include autism spectrum disorder (ASD), intellectual disability and seizures. However, the cellular mechanisms linking CACNA1C dysfunction to neurodevelopmental phenotypes remain poorly understood. MethodsWe generated isogenic CACNA1C loss-of-function (LoF) induced pluripotent stem cell (iPSC) lines and reprogrammed a line from an individual carrying a novel gain-of-function (GoF) variant in CACNA1C. Neuronal activity was assessed using multi-electrode arrays (MEA), pharmacological manipulation, and gene expression analysis. Early developmental phenotypes were examined using qRT-PCR, immunocytochemistry, and RNA sequencing. ResultsLoF and GoF patient-derived neurons displayed opposing alterations in network dynamics. Pharmacological and molecular assays indicated that these network differences were associated with dysregulated GABAergic signalling. Early developmental analysis revealed that loss of CACNA1C altered rosette morphology, CREB phosphorylation, and transcriptional programs related to axonogenesis and synaptic signalling, indicating effects on neuronal differentiation. The patient line exhibited opposing phenotypes, reflecting variant-specific effects. ConclusionsThese findings demonstrate that Cav1.2 regulates excitatory-inhibitory balance, network organization, and aspects of neurodevelopment. Divergent effects of LoF and GoF variants highlight how altered Cav1.2 signalling contributes to variable neurodevelopmental phenotypes, including ASD and epilepsy, and establish a framework for defining CACNA1C variant effects in human neurons.

neuroscience↗

bioRxiv2023

The interplay between miRNAs and chromatin regulators underpins premature differentiation within neurodevelopmental disorders (NDDs)

Development of neural cells follows a unidirectional path, progressing from pluripotent stem cells into a variety of highly differentiated neuronal and glial cell types. Although the intermediate progenitor cells along this path are well characterised, the mechanisms that determine their directional flow remain to be established. Here, we identify a gene regulatory network that directs progression of neuronal differentiation. Previously, it was shown that loss of the epigenetic repressor, EHMT1, leads to premature neuronal differentiation, and this correlates with elevated miRNA expression and inhibition of a neuronal gene suppressor, REST. Here, we use a combination of in silico-analysis and targeted suppression of multiple miRNAs to reveal an unexpected degree of complexity to this gene regulatory pathway. We identity four miRNA, miR26a, miR140, miR142, miR153, that cooperatively block REST at the mRNA level. In contrast to most miRNA suppressors of REST, such as miR9a and miR124, which are in turn transcriptionally repressed by REST in a negative feedback loop (FBL), miR26a, miR140 and miR142 are not REST-targets. This creates a feedforward loop (FFL) that drives developing cells to switch from a high REST, non-neuronal state to a low REST, neuronal state, but not in the reverse direction. We show that together miR26a, miR140, miR142 and miR153 are necessary for neuronal differentiation and introduction of synthetic miRNAs mimics is sufficient to increase downstream miR-9 and miR-124 expression. Finally, we note that the Neurodevelopmental Disorder (NDD) Kleefstra Syndrome (KS) is caused by the loss of EHMT1, suggesting that the molecular mechanism described above may play a significant role in human brain development.

neuroscience↗

bioRxiv2023

Upregulated NF-κB pathway proteins may underlie APOE4 associated astrocyte phenotypes in sporadic Alzheimer's disease

The Apolipoprotein-E4 allele (APOE) is the strongest genetic risk factor for sporadic Alzheimers disease but its role in disease pathogenesis is incompletely understood. The APOE gene encodes Apolipoprotein E (ApoE). Astrocytes are the main source of ApoE in the central nervous system (CNS) and are essential for homeostasis in health and disease. In response to CNS insult, a coordinated multicellular inflammatory response is triggered causing reactive astrogliosis with changes in astrocytic gene expression, cellular structure and function. Human embryonic stem-cells with the neutral APOE33 genotype were edited using CRISPR Cas-9 gene-editing to create isogenic APOE lines with an APOE44 genotype. Quiescent astrocytes were differentiated then stimulated with TNF-, IL1 and C1q inducing an astrogliotic A1 phenotype. Several potentially pathological APOE44-related phenotypes were identified in both quiescent cells and reactive A1 astrocytes including significantly decreased phagocytosis, impaired glutamate and a defective immunomodulatory response. In quiescent APOE44 astrocytes there was significantly decreased secretion of IL6, IL8 and several oxylipins. In A1 astrocytes there was a pro-inflammatory phenotype in APOE44 astrocytes with increases in GRO, ENA78, IL6 and IL8, a decrease in IL10 as well as significant differences in oxylipin expression. As TNF- induced signaling in astrocytes is driven by Nuclear factor kappa B (NF-{kappa}B) proteins of this pathway were measured. Significantly higher levels of the p50, p65 and I{kappa}B sub-units were found in both quiescent and A1 APOE44 astrocytes. This suggests that perturbation of NF-{kappa}B signaling may contribute to the damaging APOE44 cell phenotypes observed providing a new direction for targeted disease therapeutics.

cell biology↗

bioRxiv2021

Dysregulation of NRSF/REST via EHMT1 is association with psychiatric disorders

Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association are undetermined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a neurodevelopmental disorder (NDD) leading to ID, and is associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA leading to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Importantly, the EHMT1-regulated miRNA gene set with elevated expression is enriched for NRSF/REST regulators with an association for ID and schizophrenia. This reveals a molecular interaction between H3K9 dimethylation and NSRF/REST contributing to the aetiology of psychiatric disorders.

neuroscience↗

bioRxiv2020

Synaptic protein DLG2 controls neurogenic transcriptional programs disrupted in schizophrenia and related disorders

Brain development requires a complex choreography of cell proliferation, specialisation, migration and network formation, guided by the activation and repression of gene expression programs. It remains unclear how this process is disrupted in neuropsychiatric disorders. Here we integrate human genetics with transcriptomic data from the differentiation of human embryonic stem cells into cortical excitatory neurons. This reveals a cascade of transcriptional programs, activated during early corticoneurogenesis in vitro and in vivo, in which genetic variation is robustly associated with neuropsychiatric disorders and cognitive function. Within these early neurogenic programs, genetic risk is concentrated in loss-of-function intolerant (LoFi) genes, capturing virtually all LoFi disease association. Down-regulation of these programs in DLG2 knockout lines delays expression of cell-type identity alongside marked deficits in neuronal migration, morphology and action potential generation, validating computational predictions. These data implicate specific cellular pathways and neurodevelopmental processes in the aetiology of multiple neuropsychiatric disorders and cognition.

genetics↗

bioRxiv2018

L-type voltage-gated calcium channel regulation of in vitro human cortical neuronal networks

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here, we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days, inhibitory GABergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that, although basal neuronal firing rate is unaffected, there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening.

neuroscience↗

bioRxiv2018

Nucleosome dynamics of human iPSC during the early stages of neurodevelopment

Regulation of nucleosome positioning is important for neurodevelopment, and mutation of genes mediating chromatin remodelling are strongly associated with human neurodevelopmental disorders. Unicellular organisms possess arrays of highly positioned nucleosomes within their chromatin, occupying up to 80% of their genomes. These span gene-coding and regulatory regions, and can be associated with local changes of gene transcription. In the much larger genome of human cells, the roles of nucleosome positioning are less clear, and this raises questions of how nucleosome dynamics interfaces with human neurodevelopment.\n\nWe have generated genome-wide nucleosome maps from an undifferentiated human induced pluripotent stem cell (hiPSC) line and after its differentiation to the neuronal progenitor cell (NPC) stage. We found that approximately 3% of nucleosomes are highly positioned in NPC. In contrast, there are 8-fold less positioned nucleosomes in pluripotent cells, with the majority arising de novo or relocating during cell differentiation. Positioned nucleosomes do not directly correlate with active chromatin or gene transcription, such as marking Transcriptional Start Sites (TSS). Unexpectedly, we find a small population of nucleosomes that remain positioned after differentiation, occupying similar positions in pluripotent and NPC cells. They flank the binding sites of the key gene regulators NRSF/REST and CTCF, but remain in place whether or not their regulatory complexes are present. Together, these results present an alternative view in human cells, where positioned nucleosomes are sparse and dynamic, but act to alter gene expression at a distance via structural conformation at sites of chromatin regulation, not local changes in gene organisation.

genomics↗