Biology subjects

West, A. B.

Publications and source records attributed to West, A. B..

4 recordsLinked to original sources

bioRxiv2020

Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

The mechanisms by which Parkinson disease-linked parkin confers neuroprotection of human dopamine cells remain elusive. We hypothesized that its cysteines mediate multiple anti-oxidant effects in the midbrain. By studying >60 control specimens, we found that in adult human brain - but not in skeletal muscle- parkin is mostly aggregated and insoluble due to oxidative modifications, such as at C253. In vitro, parkins oxidation directly reduces hydrogen peroxide (H2O2) to water. In parkin-deficient human brain, H2O2 concentrations are elevated. In dopamine toxicity studies, wild-type parkin -but not disease-associated mutants-prevents neural death by lowering H2O2 and sequestering radicals within insoluble aggregates. Parkin conjugates dopamine metabolites at the human-specific residue C95 and augments melanin formation in vitro. Using epitope-mapped antibodies, we found that in adult Substantia nigra neurons parkin localizes to neuromelanin within LAMP-3/CD63-positive lysosomes. We conclude that parkins own oxidation, previously considered a loss-of-function event, underlies three neuroprotective effects in adult midbrain: its cysteines participate in H2O2 reduction, dopamine radical conjugation and the formation of neuromelanin.

neuroscience↗

bioRxiv2020

Rab10 Regulates Macropinocytosis in Phagocytes

Genetic variation in LRRK2 associates with susceptibility to Parkinsons disease, Crohns disease, and mycobacteria infection, with high expression of LRRK2, and the LRRK2 kinase substrate Rab10, in phagocytic cells in the immune system. In mouse and human primary monocyte-derived macrophages, dendritic cells, and microglia-like cells, we find that Rab10 specifically regulates a specialized form of endocytosis known as macropinocytosis, without affecting phagocytosis or clathrin-mediated endocytosis. LRRK2 phosphorylates cytoplasmic PI(3,4,5)P3-positive GTP-Rab10 early macropinosomes, before EEA1 and Rab5 recruitment occurs. Macropinosome cargo in macrophages includes CCR5, CD11b, and MHCII, with LRRK2-phosphorylation of Rab10 potently blocking EHBP1L1-mediated recycling tubules and cargo turnover. EHBP1L1 over-expression competitively inhibits LRRK2-phosphorylation of Rab10, mimicking the effects of LRRK2 kinase inhibition in promoting cargo recycling. Both Rab10 knockdown and LRRK2 kinase inhibition potently suppresses the maturation of macropinosome-derived CCR5-loaded signaling endosomes important for CCL5-induced AKT-activation and chemotaxis. These data support a novel axis in the endolysosomal system whereby LRRK2-mediated Rab10 phosphorylation stalls vesicle fast-recycling to promote PI3K-AKT signal transduction.

cell biology↗

bioRxiv2020

Predictive Markers of LRRK2 Inhibition in Biofluids

Hyper-activated LRRK2 is linked to Parkinson’s disease susceptibility and progression. Quantitative measures of LRRK2 inhibition, especially in the brain, may be critical in the clinical development of successful LRRK2-targeting therapeutics. In this study, three different brain-penetrant and selective LRRK2 small-molecule kinase inhibitors (PFE-360, MLi2, and RA283) were orally administered to groups of cynomolgus macaques at different doses. Biofluid markers with proposed pharmacodynamic properties for assessing LRRK2 inhibition were measured from samples of blood, urine, and cerebral-spinal fluid (CSF). LRRK2 kinase inhibition led to consistent reduced pS935-LRRK2 and pRab10 proteins in blood mononuclear cells, reduced exosome LRRK2 protein and di-docosahexaenoyl (22:6) bis (monoacylglycerol) phosphate in urine, and reduced exosome LRRK2 and autophosphorylated pS1292-LRRK2 protein in CSF. Incomplete LRRK2 kinase inhibition reduced LRRK2 protein secretion in exosomes whereas high drug exposures may reduce both exosome and tissue levels of LRRK2 protein. These orthogonal markers for LRRK2 inhibition in urine and CSF can be used in combination with blood markers to non-invasively monitor the potency of LRRK2-targeting therapeutics in the brain and periphery.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsPDParkinson’s diseaseLRRK2Leucine-rich repeat kinase 2 NHPs: Non-human primatesdi-22:6-BMPdi-docosahexaenoyl (22:6) bis (monoacylglycerol) phosphate CSF: Cerebrospinal fluidATPadenosine-tri phosphateView Full Text

neuroscience↗

bioRxiv2019

Dopaminergic Neurodegeneration Induced by Parkinson's Disease-Linked G2019S LRRK2 is Dependent on Kinase and GTPase Activity

AO_SCPLOWBSTRACTC_SCPLOWMutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinsons disease (PD) and represent the most common known cause of PD. LRRK2 can function as both a protein kinase and GTPase and PD-linked mutations are known to influence both of these enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage and toxicity in cellular models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models based upon familial LRRK2 mutations often lack the hallmark features of PD and robust neurodegenerative phenotypes in general. Here, we develop a robust pre-clinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of full-length human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or in-diet dosing with the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the selective and robust destabilization of human LRRK2 protein in the rat brain relative to endogenous LRRK2. Our study further demonstrates that dopaminergic neurodegeneration induced by G2019S LRRK2 critically requires normal GTPase activity. The introduction of hypothesis-testing mutations that increase GTP hydrolysis or impair GTP binding activity provide neuroprotection against G2019S LRRK2 via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

neuroscience↗