Biology subjects

Sharp, R.

Publications and source records attributed to Sharp, R..

8 recordsLinked to original sources

bioRxiv2026

Vps35 p.D620N causes Lrrk2 kinase hyperactivity, chronic microglial activation and inflammation

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2), RAB32, and vacuolar protein sorting 35 (VPS35) cause dominantly inherited, late-onset Parkinson's disease (PD). Mutations in all three genes constitutively activate LRRK2 kinase activity, enhancing innate immune defense against pathogens; however, chronic activation is linked to dopaminergic neuronal degeneration. LRRK2 and RAB32 are highly expressed in all myeloid cells including brain microglia, whereas VPS35, a core component of the retromer, is ubiquitously expressed. Nevertheless, the VPS35 p.D620N substitution induces the greatest constitutive increase in endogenous LRRK2 kinase activity, and its consequences for immune function remain unclear. We therefore examined the transcriptomic and functional effects of wild type (WT) and Vps35 p.D620N knock-in mice (VKI) from microglia, isolated from brains of naive animals at six-month of age, and initially analyzed by single-cell RNA sequencing (scRNAseq). Despite the absence of overt behavioral differences, differential gene expression revealed genotype-dependent changes in antimicrobial humoral immunity, lysosomal stress sensing, and phagocytosis. S100 family genes and lipocalin 2 were markedly upregulated, a finding subsequently validated by gene expression and immunohistochemistry. Downregulation was also observed in pathways governing microglial regulation of synaptic transmission, neuronal development and homeostatic immune signaling. Peripherally-administered lipopolysaccharide (LPS) stimulated microglial activation and phagocytic markers in WT brain, and further increased morphological activation and synaptic engulfment in VKI animals. Compared to WT, Vps35 p.D620N drives a chronic pro-inflammatory microglial state characterized by elevated innate immune signaling, lysosomal stress and enhanced phagocytic activity. Hence, heightened microglial sensitivity, further exacerbated by a peripheral immune challenge, offers a parsimonious, if multicellular, explanation for the multifactorial etiology of PD, and may help to account for its penetrance and variable expressivity. Together, these findings provide mechanistic insight into how retromer dysfunction and LRRK2 kinase hyperactivity intersect with microglial biology to influence PD pathogenesis, potentially promoting synaptic remodeling and neurodegenerative vulnerability.

neuroscience↗

bioRxiv2022

Vitamin B2 enables peroxisome proliferator-activated receptor α regulation of fasting glucose availability

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2 deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPAR, including those required for gluconeogenesis. Treatment with the PPAR agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. Overall, these findings reveal PPAR governs metabolic responses to FAD availability and nominate its pharmacologic activation as strategies for organic acidemias.

physiology↗

bioRxiv2022

Structure of a HIV-1 IN-Allosteric Inhibitor Complex at 2.93 Angstrom Resolution: Routes to Inhibitor Optimization

HIV integrase (IN) inserts viral DNA into the host genome and is the target of the strand transfer inhibitors (STIs), a class of small molecules currently in clinical use. Another potent class of antivirals is the allosteric inhibitors of integrase, or ALLINIs. ALLINIs promote IN aggregation by stabilizing an interaction between the catalytic core domain (CCD) and carboxy -terminal domain (CTD) that undermines viral particle formation in late replication. Ongoing challenges with inhibitor potency, toxicity, and viral resistance motivate research to understand their mechanism. Here, we report a 2.93 [A] X-ray crystal structure of the minimal ternary complex between CCD, CTD, and the ALLINI BI-224436. This structure reveals an asymmetric ternary complex with a prominent network of {pi}-mediated interactions that suggest specific avenues for future ALLINI development and optimization.

biochemistry↗

bioRxiv2021

Accumulation of auranofin in white adipose tissues lowers leptin levels and exerts anti-diabetic effects

Low-grade, sustained inflammation in white adipose tissue (WAT) characterizes obesity and frequently coincides with insulin resistance and type 2 diabetes (T2D). However, pharmacological targeting of WAT inflammation lacks durable therapeutic effects. Through a computational screen, we identified the FDA-approved rheumatoid arthritis drug auranofin is a putative small molecule for obesity treatment. We discovered that allometrically scaled safe auranofin doses homed to WAT and improved insulin sensitivity in obese wild-type mice. Auranofin treatment also normalized other obesity-associated abnormalities, including hepatic steatosis and hyperinsulinemia. Surprisingly, the anti-diabetic effects of auranofin required leptin lowering and beta-adrenergic receptors in WAT. These metabolic benefits of leptin reduction were superior to any immune impacts of auranofin in WAT. Our studies reveal important metabolic properties of anti-inflammatory treatments and contribute to the notion that leptin reduction in the periphery can be accomplished to treat obesity and T2D.

physiology↗

bioRxiv2021

Combined single cell proteomics and transcriptomics reveals discrete human tendon cells populations persist in vitro and on fibrous scaffolds

Chronic tendinopathy represents a growing burden to healthcare services in an active and ageing global population. The ability to identify, isolate and interrogate, in vitro, key pathogenic and reparative tendon cell populations is essential to developing precision therapies and implantable materials. Human hamstring tendon cells were cultured for 8 days on either tissue culture plastic or aligned electrospun fibres made of polydioxanone (absorbable polymer). Combined single cell surface proteomics and unbiased single cell transcriptomics (CITE-Seq) revealed six discrete cell clusters, four of which shared key gene expression determinants with ex vivo human cell clusters. These were PTX3_PAPPA, POST_SCX, DCN_LUM and ITGA7_NES cell clusters. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+, and POSTN_SCX cells were CD90+CD95+CD10+. Three clusters preferentially survived and proliferated on the aligned electrospun fibres; DCN_LUM, POSTN_SCX, and PTX3_PAPPA. They maintained high expression of tendon matrix associated genes, including COL1A1, COL1A2, COL3A1, ELN, FBLN1, and up-regulated genesets enriched for TNF-{gamma} signalling via NF{kappa}B, IFN-{gamma} signalling and IL-6/ STAT3 signalling. When cells were pre-selected based on surface protein markers, a similar up-regulation of pro-inflammatory signalling pathways was observed, particularly in PTX3 gene expressing CD10+CD26+CD54+ cells, with increased expression of genes associated with TNF- signalling and IFN-{gamma} signalling. Discrete human tendon cell sub populations persist in vitro culture and can be recognised by specific gene and surface protein signatures. Aligned PDO fibres promote the survival of three clusters, including pro-inflammatory PTX3 expressing CD10+CD26+CD54+ cells found in chronic tendon disease.

cell biology↗

bioRxiv2020

Assembly of higher-order SMN oligomers is essential for metazoan viability and requires an exposed structural motif present in the YG zipper dimer

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins, and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a variation of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, bioinformatic, biophysical, and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.

biochemistry↗

bioRxiv2020

Ubc9 deletion in adipocytes causes lipoatrophy in mice

ObjectiveWhite adipose tissue (WAT) expansion regulates energy balance and overall metabolic homeostasis. WAT absence or loss occurring through lipodystrophy and lipoatrophy contributes to the development of dyslipidemia, hepatic steatosis, and insulin resistance. We previously demonstrated the sole small ubiquitin-like modifier (SUMO) E2-conjuguating enzyme Ubc9 represses human adipocyte differentiation. Germline and other tissue-specific deletions of Ubc9 frequently cause lethality in mice. As a result, the role of Ubc9 during WAT development remains unknown. MethodsTo determine how Ubc9 impacts body composition and energy balance, we generated adipocyte-specific Ubc9 knockout mice (Ubc9a-KO). CRISPR/Cas9 gene editing inserted loxP sites flanking exons 3 and 4 at the Ubc9 locus. Subsequent genetic crosses to AdipoQ-Cre transgenic mice allowed deletion of Ubc9 in white and brown adipocytes. We measured multiple metabolic endpoints that describe energy balance and carbohydrate metabolism in Ubc9a-KO and littermate controls during postnatal growth. ResultsTo our surprise, Ubc9a-KO mice developed hyperinsulinemia and hepatic steatosis. Global energy balance defects emerged from dysfunctional WAT marked by pronounced local inflammation, loss of serum adipokines, hepatomegaly, and near absence of major adipose tissue depots. We observed progressive lipoatrophy that commences in the early adolescent period. ConclusionsOur results demonstrate that Ubc9 expression in mature adipocytes is essential for maintaining WAT expansion. Deletion of Ubc9 in fat cells compromised and diminished adipocyte function that provoked WAT inflammation and ectopic lipid accumulation in the liver. Our findings reveal an indispensable role for Ubc9 during white adipocyte expansion and endocrine control of energy balance.

developmental biology↗

bioRxiv2019

Identification of human tendon cell populations in healthy and diseased tissue using combined single cell transcriptomics and proteomics

The long-term morbidity of tendon disease in an increasingly active and ageing population represents a growing area of unmet clinical need. Tendon disorders commonly affect the lower limb, ranging from isolated tendon rupture to degenerative joint deformity. In the absence of valid animal models of chronic tendinopathy, traditional methods to isolate and identify crucial sub types involved in disease are limited by the heterogeneity of tendon cells, by their relative paucity in tissue and by the density of the surrounding collagen matrix. To overcome this, we have used next generation CITE-sequencing to combine surface proteomics with in-depth, unbiased gene expression analysis of single cells derived ex vivo from healthy and diseased tendon. For the first time we have been able to show that human tendon consists of at least eight sub-populations of cells. In addition to endothelial cells, Tc cells, and macrophages, there are five distinct tenocyte populations expressing COL1A genes. These consist of a population of resident cells expressing microfibril associated genes (FBN1, VCAN, DCN, EMILIN1, MFAP5), a group of SCX+ cells co-expressing high levels of pro-inflammatory markers, a population of APOD+ fibro-adipogenic progenitors (FAPs), TPPP3/PRG4+ chondrogenic cells (COMP, CILP, PRG4) and ITGA7+ Smooth Muscle-Mesenchymal Cells, recently described in mouse muscle but not, as yet, in human tendon. Surface proteomic analysis identified markers by which these sub-classes could be isolated and targeted in future. In comparison to healthy tendon, diseased tendon harboured a greater proportion of SCX+ tendon cells and these expressed high levels of pro-inflammatory markers including CXCL1, CXCL6, CXCL8, PDPN and previously undescribed PTX3. We were also able to show that whereas disease associated genes such as CD248 and PDPN were expressed by COL1+ tenocytes, IL33 was restricted to endothelial cells of chronically diseased tendon.

cell biology↗