Biology subjects

Sharp, R. C.

Publications and source records attributed to Sharp, R. C..

4 recordsLinked to original sources

bioRxiv2026

Bidirectional disruption of Lrrk2 function drives T cell dysregulation and an exhaustion-like immune response

Background: Neurodegenerative diseases including Parkinson's disease (PD) are increasingly associated with dysfunction in both central and peripheral immune systems. Pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) represent a major cause of familial PD, while common polymorphisms are associated with inflammatory diseases. Methods: Here, using immunophenotyping flow cytometry and quantitative PCR (qPCR), we compared immune cell populations and function across both the central and peripheral immune systems in C57BL/6J wild type (WT), Lrrk2 p.G2019S knock-in (GKI) and Lrrk2 knock-out (LKO) models in basal and ex vivo immune-stimulated conditions. Results: With a focus on T cell biology, compared to their WT counterparts at baseline, GKI mice exhibit higher populations of Cd8+ and TH17 T cell subsets in the brain, whereas LKO mice exhibited unique central memory (TCM), follicular helper (TFH), and TH2 lineages. In the periphery, GKI mice demonstrated higher TH1, TH2, and TH17 subset expansions, whereas peripheral alterations in LKO are largely restricted to TH17 subsets. Within mutant genotypes, a striking discrepancy was observed between baseline gene expression and the translated proteins encoded, that reveals a fundamental loss of basal immune homeostasis. This phenomenon was further exposed following an acute (6-hour) ex vivo lipopolysaccharide (LPS) immune challenge. Following stimulation, GKI immune cells had reduced transcription, alongside stalled translation, for almost all effector molecules examined, while LKO immune cells had fewer transcriptional changes compared to wild type. Overall, both mutant lines had stalled or flatline effector molecule production after immune stimulation, suggesting a profound loss of functional responsiveness. This hypothesis was supported by a significant increase in surface protein of the inhibitory receptor Pd-1 on regulatory T cells (TREG) and TH1/TH2 Cd4+ T cell subsets in GKI mice. LKO immune landscapes trended toward similar exhaustion patterns, albeit less evident. Conclusions: These data suggest that bidirectional disruptions to normal Lrrk2 function break immune homeostasis. Immune cell function should be carefully considered when targeting LRRK2 kinase activity in patients with PD.

immunology↗

bioRxiv2025

Peripheral inflammation mediates midbrain Lrrk2 kinase activity via Rab32 expression

Mutations that increase leucine-rich repeat kinase 2 (LRRK2) activity confer significant risk for Parkinsons disease (PD), yet incomplete disease penetrance suggest additional factors are required to manifest disease. We recently identified RAB32 Ser71Arg as a Mendelian gene for PD. Here, we establish Rab32 as a key mediator linking peripheral inflammation to Lrrk2 activation. We show that Rab32 and Rab38 expression are modestly, but inversely, correlated with their homolog Rab29. In vivo, peripheral lipopolysaccharide (LPS)-induced inflammation selectively induced Rab32 expression in midbrain Iba1+ microglia but not dopaminergic neurons, where it localized to Lamp1+ lysosomal compartments and correlated with Lrrk2 kinase activity. LPS similarly induces Rab32 expression in human induced pluripotent stem cell-derived microglia, demonstrating a unified biological response to inflammation across species. Promoter analysis identified Tfe3, a master regulator of lysosomal biogenesis and autophagy, as a key driver of Rab32 expression induced Lrrk2 kinase activation. During inflammation, Tfe3 translocated to the nucleus of midbrain Iba1+ microglia to induce Rab32 expression and Lrrk2 kinase activity. Knockdown of Tfe3, but not Tfeb, mitigates these effects, establishing Rab32 as a physiological rheostat of Lrrk2 activity. This mechanistic pathway enables peripheral inflammation to modulate LRRK2 activity and highlights Rab32/Tfe3 as a therapeutic targeting for neuroprotection in PD.

cell biology↗

bioRxiv2023

Flow cytometry for immune characterization in homogenized mouse brain: A systematic review

ObjectiveTo systematically review the neuroimmunology literature to determine the average immune cell counts reported by flow cytometry in wild-type (WT) homogenized mouse brain. BackgroundMouse models of gene dysfunction are widely used to study age-associated neurodegenerative disorders, including Alzheimers disease and Parkinsons disease. The importance of the neuroimmune system in these multifactorial disorders has become increasingly evident, and methods to quantify resident and infiltrating immune cells in brain, including flow cytometry, are necessary. However, there appears to be no consensus on the best approach to perform flow cytometry or quantify/report immune cell counts. The development of more standardized methods would accelerate neuroimmune discovery/validation by meta-analysis. MethodsExamination of the PROSPERO registry confirmed a systematic review of neuroimmunology by flow cytometry has yet to be reported. A protocol for a systematic review was subsequently based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) using Studies, Data, Methods and Outcomes (SDMO) criteria. Literature searches were conducted in Google Scholar and PubMed databases. From that search, 900 candidate studies were identified and 437 studies were assessed for eligibility based on formal exclusion criteria. ResultsOut of the 437 studies reviewed, 58 were eligible for inclusion and comparative analysis. Each study assessed immune cell subsets within homogenized mouse brain and used flow cytometry. Nonetheless, there was considerable variability in methods, data analysis, reporting and results. Descriptive statistics are presented on study designs and results, including medians with interquartile ranges (IQRs) and overall means with standard deviations (SD) for specific immune cell counts and their relative proportions, within and between studies. Results derived from 58 studies reported the most abundant immune cells within the brains were TMEM119+ microglia, bulk CD4+ T cells, and bulk CD8+ T cells. ConclusionExperiments to conduct and report flow cytometry data, derived from WT homogenized mouse brains, would benefit from a more standardized approach. While within study comparisons are valid, variability in methods counts of immune cell populations are too broad for meta-analysis. Inclusion of a minimal protocol with more detailed methods, controls and standards could enable this nascent field to compare results across studies.

neuroscience↗

bioRxiv2019

Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment epithelia utilizes microtubule-associated protein 1 light chain 3B

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid the visual cycle, nutrient supply, ion exchange and waste removal. Each day the RPE removes the oldest [~]10% of photoreceptor outer segments through phagocytic uptake, which peaks in a synchronous burst following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation and cell death. OSs are rich in very long chain fatty acids which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblotting and catalase activity assays, we investigated peroxisome abundance and activity at 6 am, 7 am (at lights on), 8 am, and 3 pm, in WT mice and mice lacking microtubule-associated protein 1 light chain 3B (LC3B), that have impaired degradation of phagosomes. We found that catalase activity, but not protein expression, is 50% higher in the morning compared with 3 pm, in RPE of WT but not LC3B-/- mice. Surprisingly, we found that peroxisome abundance was stable during the day, however numbers are elevated overall in LC3B-/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.

biochemistry↗