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Biology subjects

Doble, B. W.

Publications and source records attributed to Doble, B. W..

5 recordsLinked to original sources

CK2 variant function and disease modelling in Drosophila reveal allelic heterogeneity and Wnt/β-catenin-mediated phenotypes

Heterozygous pathogenic variants in CSNK2A1 or CSNK2B encoding the Casein Kinase 2 (CK2) protein complex, lead to pediatric neurodevelopmental disorders, Okur-Chung Neurodevelopmental Syndrome (OCNDS) and Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS). OCNDS and POBINDS are characterized by a range of symptoms, including developmental delay, intellectual disability, facial dysmorphism, and seizures. Despite over 250 reported cases of OCNDS and POBINDS, we do not fully understand how specific alterations in CK2 relate to the heterogeneity observed in patients. To investigate this, we used the fruit fly, Drosophila melanogaster, as a model system. To assess variant impact, we co-expressed human CSNK2A1 and CSNK2B reference or disease-causing variants in flies. In parallel, we determined the role of Drosophila CkII in the developing and mature nervous system, specifically in neurons and glia. We found that 12/13 variants tested act as full or partial loss-of-function with one CSNK2A1 variant showing gain-of-function. Phospho-proteomic studies in neurons revealed separate signatures for loss- and gain-of-function variants. We found that neuronal and glial CkII is critical for organismal development. Reduction of neuronal CkII in the adult nervous system causes motor and seizure-like phenotypes. Finally, given the known role of CK2 in potentiating Wnt/{beta}-catenin signalling, we show that Wnt agonists partially rescue phenotypes associated with adult-specific neuronal reduction of CkII. This work generates Drosophila models of CSNK2A1 and CSNK2B expression to functionally assess variant impact, as well as an adult-specific neuronal loss-of-function model for drug screening and mechanistic studies.

genetics↗

Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease relevant pathologies

Manuscript summaryThere are hundreds of risk genes associated with autism spectrum disorder (ASD), but signaling networks at the protein level remain unexplored. We use neuron-specific proximity-labeling proteomics (BioID) to identify protein-protein interaction (PPI) networks for 41 ASD-risk genes. Neuron-specific PPI networks, including synaptic transmission proteins, are disrupted by de novo missense variants. The PPI network map reveals convergent pathways, including mitochondrial/metabolic processes, Wnt signaling, and MAPK signaling. CRISPR knockout reveal an association between mitochondrial activity and ASD-risk genes. The PPI network shows an enrichment of 112 additional ASD-risk genes and differentially expressed genes from post-mortem ASD patients. Clustering of risk genes based on PPI networks identifies gene groups corresponding to clinical behavior score severity. Our data reveal that cell type-specific PPI networks can identify individual and convergent ASD signaling networks, provide a method to assess patient variants, and reveal biological insight into disease mechanisms and sub-cohorts of ASD.

neuroscience↗

Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome

Copy number variations (CNV) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase (DUB) function. The OTUD7A protein-protein interaction (PPI) network revealed interactions with synaptic, axonal, and cytoskeletal proteins and was enriched for known ASD and epilepsy risk genes. The interactions between OTUD7A and the NDD risk genes Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment (AIS), while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Further, our study highlights the utility of targeting CNV genes using cell-type specific proteomics to identify shared and unexplored disease mechanisms across NDDs.

neuroscience↗

Lfc/Arhgef2 regulates mitotic spindle orientation in hematopoietic stem and progenitor cells and is essential for productive hematopoiesis

How hematopoietic stem cells (HSCs) coordinate their divisional axis relative to supportive niche cells and whether or not their divisional orientation is important for stem cell-driven hematopoiesis is poorly understood. Single cell RNA sequencing data from patients with the inherited bone marrow failure Shwachman-Diamond syndrome (SDS) show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor (GEF) and determinant of mitotic spindle orientation, is one of a restricted group of genes specifically downregulated in SDS HSCs and multipotent progenitors. Here, we describe Lfc/Arhgef2 as an important regulator of hematopoiesis in vivo. Transplanted Lfc/Arhgef2-/- bone marrow shows impaired hematopoietic recovery and a production deficit of long-term HSCs. These phenotypes cannot be explained by differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output or homing ability. Using live imaging of dividing hematopoietic stem and progenitor cells (HSPCs), we show an increased frequency of misoriented divisions in the absence of Lfc/Arhgef2. Functional ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller hematopoietic xenografts. Together, these data provide evidence demonstrating a conserved role for Lfc/Arhgef2 in orienting HSPC division and suggest that HSCs divide in certain orientations to establish hematopoiesis, the loss of which leads to their exhaustion in a mechanism that may underlie certain bone marrow failure syndromes.

cell biology↗

Regulation of thymocyte β-selection, development and positive selection by glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase, that exists as two isoforms in mammals, GSK-3 and GSK-3{beta}, that are key downstream mediators of the phosphatidylinositol 3 kinase, Wnt, Notch and other pathways. Here, we report that simultaneous inactivation of both GSK-3 and GSK-3{beta} during early thymocyte ontogeny has profound effects on both {beta}-selection and positive selection, key checkpoints essential to producing functionally mature {beta} T cells. Conditional GSK-3/{beta} knockout animals (LckCre+ GSK-3{beta}fl/fl) possessed pre-double positive (pre-DP) thymocytes (CD4-CD8-CD117-CD25-) with compromised TCR{beta} chain expression along with elevated levels of {beta}-catenin and reduced Notch activity. {beta}-selection was impaired allowing pre-DP thymocytes to differentiate to DP thymocytes (CD4+CD8+) while bypassing strict requirements for productive TCR{beta} chain rearrangements and functional expression. Also impaired was the requisite pre-TCR and Notch-mediated expansion that normally precedes differentiation to the DP stage. Consequently, LckCre+ GSK-3{beta}fl/fl mice initially generated fewer DP thymocytes that expressed significantly reduced levels of mature TCR. The aberrant DP thymocytes expressed high levels of the pro-survival Bcl-2 family member Mcl-1, failed positive selection and accumulated as CD4hiCD8lo positive selection intermediates resulting in loss of both mature CD4 and CD8 lineages. LckCre+ GSK-3{beta}fl/fl mice succumbed to oligoclonal peripheral lymphomas with high penetrance. These data reveal essential roles for GSK-3 in several checkpoints of early T cell development.

immunology↗