Biology subjects

Whiting, P. J.

Publications and source records attributed to Whiting, P. J..

3 recordsLinked to original sources

bioRxiv2023

The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function: insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation.

Phospholipase C-gamma 2 (PLC{gamma}2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLC{gamma}2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLC{gamma}2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLC{gamma}2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLC{gamma}2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLC{gamma}2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLC{gamma}2 GOF on human microglia. PLC{gamma}2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLC{gamma}2 wild type. Despite the increase in PLC{gamma}2 enzymatic activity, the PLC{gamma}2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLC{gamma}2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLC{gamma}2 in the CNS. Drug candidates targeting PLC{gamma}2 will need to precisely mimic the effects of the PLC{gamma}2 P522R variant on microglial function, but not those of the PLC{gamma}2 S707Y variant. HighlightsO_LIThe impact of strongly hypermorphic variants of PLC{gamma}2 have not been studied in brain and yet PLC{gamma}2 is implicated in modifying risk of CNS disorders including Alzheimers disease C_LIO_LITo address this, we explored the role of the strongly hypermorphic PLC{gamma}2 S707Y variant in hiPSC-derived microglia C_LIO_LIS707Y increases PLC{gamma}2 enzymatic activity and intracellular calcium flux C_LIO_LIPhagocytosis and cytokine production are diminished in PLC{gamma}2 S707Y microglia C_LIO_LIPLC{gamma}2 S707Y downregulates expression of genes related to innate immunity and response C_LIO_LIModulation of PLC{gamma}2 for therapy must recapitulate the positive effects of moderate hypermorphic variants on microglial functions whilst avoiding detrimental effects of strongly hypermorphic variants like PLC{gamma}2 S707Y C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/564579v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9f0903org.highwire.dtl.DTLVardef@108d26forg.highwire.dtl.DTLVardef@2fb691org.highwire.dtl.DTLVardef@7af80c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Microglial expression of the Wnt signalling modulator DKK2 differs between human Alzheimer's disease brains and mouse neurodegeneration models

Wnt signalling is crucial for synapse and cognitive function. Indeed, deficient Wnt signalling is causally related to increased expression of DKK1, an endogenous negative Wnt regulator, and synapse loss, both of which likely contribute to cognitive decline in Alzheimers disease (AD). Increasingly, AD research efforts have probed the neuroinflammatory role of microglia, the resident immune cells of the central nervous system (CNS), which have furthermore been shown to be modulated by Wnt signalling. The DKK1 homologue DKK2 has been previously identified as an activated response and/or disease-associated microglia (DAM/ARM) gene in a mouse model of AD. Here we performed a detailed analysis of DKK2 in mouse models of neurodegeneration, and in human AD brain. In APP/PS1 and APPNL-G-F AD mouse model brains as well as in SOD1G93A ALS mouse model spinal cords, but not in control littermates, we demonstrated significant microgliosis and microglial Dkk2 mRNA upregulation in a disease-stage dependent manner. In the AD models, these DAM/ARM Dkk2+ microglia preferentially accumulated close to {beta}Amyloid plaques. Furthermore, recombinant DKK2 treatment of rat hippocampal primary neurons blocked WNT7a-induced dendritic spine and synapse formation, indicative of an anti-synaptic effect similar to that of DKK1. In stark contrast, no such microglial DKK2 upregulation was detected in the post-mortem human frontal cortex from individuals diagnosed with AD or pathological ageing. In summary, the difference in microglial expression of the DAM/ARM gene DKK2 between mouse models and human AD brain highlights the increasingly recognised limitations of using mouse models to recapitulate facets of human neurodegenerative disease. Significance statementThe endogenous negative Wnt regulator Dkk2 is significantly upregulated at the mRNA level in microglia of AD mouse models, implying that microglia derived Dkk2 protein may detrimentally contribute to a reduced Wnt signalling tone in the AD brain, a known pathophysiological manifestation. Indeed, recombinant DKK2 prevented Wnt- dependent synapse formation in cultured neurons. However, DKK2 upregulation was not recapitulated in post-mortem human AD brains. The success of neurodegeneration animal models has relied on pathophysiology that for the most part correctly modelled human disease. Increasingly however, limitations to the validity of mouse models to recapitulate human neurodegenerative disease have become apparent, as evidenced by the present study by the difference in microglial DKK2 expression between AD mouse models and human AD brain.

neuroscience↗

bioRxiv2017

Molecular and functional variation in iPSC-derived sensory neurons

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools to model biological processes and disease mechanisms, particularly in cell types such as neurons that are difficult to access from living donors. Here, we present the first map of regulatory variants in an iPSC-derived cell type. To investigate genetic contributions to human sensory function, we performed 123 differentiations of iPSCs from 103 unique donors to a sensory neuronal fate, and measured gene expression, chromatin accessibility, and neuronal excitability. Compared with primary dorsal root ganglion, where sensory nerves collect near the spinal cord, gene expression was more variable across iPSC-derived neuronal cultures, particularly in genes related to differentiation and nervous system development. Single cell RNA-sequencing revealed that although the majority of cells are neuronal and express the expected marker genes, a substantial fraction have a fibroblast-like expression profile. By applying an allele-specific method we identify 3,778 quantitative trait loci influencing gene expression, 6,318 for chromatin accessibility, and 2,097 for RNA splicing at FDR 10%. A number of these overlap with common disease associations, and suggest candidate causal variants and target genes. These include known causal variants at SNCA for Parkinsons disease and TNFRSF1A for multiple sclerosis, as well as new candidates for migraine, Parkinsons disease, and schizophrenia.

genomics↗