In the translation of discoveries from the laboratory to the clinic, the track record in developing disease-modifying therapies in neurodegenerative disease is poor. A carefully designed development ...pipeline built from discoveries in both pre-clinical models and patient populations is necessary to optimize the chances for success. Genetic variation in the leucine-rich repeat kinase two gene (LRRK2) is linked to Parkinson disease (PD) susceptibility. Pathogenic mutations, particularly those in the LRRK2 GTPase (Roc) and COR domains, increase LRRK2 kinase activities in cells and tissues. In some PD models, small molecule LRRK2 kinase inhibitors that block these activities also provide neuroprotection. Herein, the genetic and biochemical evidence that supports the involvement of LRRK2 kinase activity in PD susceptibility is reviewed. Issues related to the definition of a therapeutic window for LRRK2 inhibition and the safety of chronic dosing are discussed. Finally, recommendations are given for a biomarker-guided initial entry of LRRK2 kinase inhibitors in PD patients. Four key areas must be considered for achieving neuroprotection with LRRK2 kinase inhibitors in PD: 1) identification of patient populations most likely to benefit from LRRK2 kinase inhibitors, 2) prioritization of superior LRRK2 small molecule inhibitors based on open disclosures of drug performance, 3) incorporation of biomarkers and empirical measures of LRRK2 kinase inhibition in clinical trials, and 4) utilization of appropriate efficacy measures guided in part by rigorous pre-clinical modeling. Meticulous and rational development decisions can potentially prevent incredibly costly errors and provide the best chances for LRRK2 inhibitors to slow the progression of PD.
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•LRRK2 small molecule kinase inhibitor studies are revealing the type of therapeutic benefit associated with blocking LRRK2•LRRK2 mutation carriers provide an ideal clinical population for biomarker optimization and initial efficacy trials.•Empirical measurements of LRRK2 inhibition in clinical populations should guide development pipelines and efficacy trials.
Abstract
Human genetic studies implicate LRRK2 and RAB7L1 in susceptibility to Parkinson disease (PD). These two genes function in the same pathway, as knockout of Rab7L1 results in phenotypes ...similar to LRRK2 knockout, and studies in cells and model organisms demonstrate LRRK2 and Rab7L1 interact in the endolysosomal system. Recently, a subset of Rab proteins have been identified as LRRK2 kinase substrates. Herein, we find that Rab8, Rab10, and Rab7L1 must be membrane and GTP-bound for LRRK2 phosphorylation. LRRK2 mutations that cause PD including R1441C, Y1699C, and G2019S all increase LRRK2 phosphorylation of Rab7L1 four-fold over wild-type LRRK2 in cells, resulting in the phosphorylation of nearly one-third the available Rab7L1 protein in cells. In contrast, the most common pathogenic LRRK2 mutation, G2019S, does not upregulate LRRK2-mediated phosphorylation of Rab8 or Rab10. LRRK2 interaction with membrane and GTP-bound Rab7L1, but not Rab8 or Rab10, results in the activation of LRRK2 autophosphorylation at the serine 1292 position, required for LRRK2 toxicity. Further, Rab7L1 controls the proportion of LRRK2 that is membrane-associated, and LRRK2 mutations enhance Rab7L1-mediated recruitment of LRRK2 to the trans-Golgi network. Interaction studies with the Rab8 and Rab10 GTPase-activating protein TBC1D4/AS160 demonstrate that LRRK2 phosphorylation may block membrane and GTP-bound Rab protein interaction with effectors. These results suggest reciprocal regulation between LRRK2 and Rab protein substrates, where Rab7L1-mediated upregulation of LRRK2 kinase activity results in the stabilization of membrane and GTP-bound Rab proteins that may be unable to interact with Rab effector proteins.
Genetic variation in LRRK2 associates with the susceptibility to Parkinson's disease, Crohn's disease, and mycobacteria infection. High expression of LRRK2 and its substrate Rab10 occurs in ...phagocytic cells in the immune system. In mouse and human primary 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, before EEA1 and Rab5 recruitment to early macropinosomes occurs. Macropinosome cargo in macrophages includes CCR5, CD11b, and MHCII, and LRRK2‐phosphorylation of Rab10 potently blocks EHBP1L1‐mediated recycling tubules and cargo turnover. EHBP1L1 overexpression 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 suppress the maturation of macropinosome‐derived CCR5‐loaded signaling endosomes that are critical for CCL5‐induced immunological responses that include Akt activation and chemotaxis. These data support a novel signaling axis in the endolysosomal system whereby LRRK2‐mediated Rab10 phosphorylation stalls vesicle fast recycling to promote PI3K‐Akt immunological responses.
Synopsis
How the Parkinson's disease‐associated kinase LRRK2 modulates the function of Rab‐family small GTPases is ill‐defined. In primary human and mouse phagocytic cells, LRRK2‐dependent phosphorylation of GTP‐bound Rab10 blocks macropinosome fast‐recycling and boosts CCL5‐induced AKT signalling.
Rab10 regulates early macropinosome trafficking.
LRRK2 phosphorylates GTP‐Rab10 bound to macropinosomes.
Rab10 phosphorylation stalls macropinosomes by inhibiting vesicle fast‐recycling.
LRRK2‐dependent phosphorylation of Rab10 potentiates CCL5‐induced AKT signaling.
Rab10 phosphorylation by Parkinson's disease‐linked LRRK2 blocks macropinosome recycling and boosts CCL5‐induced AKT signalling in primary human and mouse phagocytic cells.
Recent studies of genomic variation associated with autism have suggested the existence of extreme heterogeneity. Large-scale transcriptomics should complement these results to identify core ...molecular pathways underlying autism. Here we report results from a large-scale RNA sequencing effort, utilizing region-matched autism and control brains to identify neuronal and microglial genes robustly dysregulated in autism cortical brain. Remarkably, we note that a gene expression module corresponding to M2-activation states in microglia is negatively correlated with a differentially expressed neuronal module, implicating dysregulated microglial responses in concert with altered neuronal activity-dependent genes in autism brains. These observations provide pathways and candidate genes that highlight the interplay between innate immunity and neuronal activity in the aetiology of autism.
The accumulation of aggregated α-synuclein in susceptible neurons in the brain, together with robust activation of nearby myeloid cells, are pathological hallmarks of Parkinson's disease (PD). While ...microglia represent the dominant type of myeloid cell in the brain, recent genetic and whole-transcriptomic studies have implicated another type of myeloid cell, bone-marrow derived monocytes, in disease risk and progression. Monocytes in circulation harbor high concentrations of the PD-linked enzyme leucine-rich repeat kinase 2 (LRRK2) and respond to both intracellular and extracellular aggregated α-synuclein with a variety of strong pro-inflammatory responses. This review highlights recent findings from studies that functionally characterize monocytes in PD patients, monocytes that infiltrate into cerebrospinal fluid, and emerging analyses of whole myeloid cell populations in the PD-affected brain that include monocyte populations. Central controversies discussed include the relative contribution of monocytes acting in the periphery from those that might engraft in the brain to modify disease risk and progression. We conclude that further investigation into monocyte pathways and responses in PD, especially the discovery of additional markers, transcriptomic signatures, and functional classifications, that better distinguish monocyte lineages and responses in the brain from other types of myeloid cells may reveal points for therapeutic intervention, as well as a better understanding of ongoing inflammation associated with PD.
Broadly neutralizing antibodies (bNAbs) to HIV-1 envelope glycoprotein (Env) can prevent infection in animal models. Characterized bNAb targets, although key to vaccine and therapeutic strategies, ...are currently limited. We defined a new site of vulnerability by solving structures of bNAb 8ANC195 complexed with monomeric gp120 by X-ray crystallography and trimeric Env by electron microscopy. The site includes portions of gp41 and N-linked glycans adjacent to the CD4-binding site on gp120, making 8ANC195 the first donor-derived anti-HIV-1 bNAb with an epitope spanning both Env subunits. Rather than penetrating the glycan shield by using a single variable-region CDR loop, 8ANC195 inserted its entire heavy-chain variable domain into a gap to form a large interface with gp120 glycans and regions of the gp120 inner domain not contacted by other bNAbs. By isolating additional 8ANC195 clonal variants, we identified a more potent variant, which may be valuable for therapeutic approaches using bNAb combinations with nonoverlapping epitopes.
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•Broadly neutralizing antibody 8ANC195 recognizes a new epitope on HIV-1 Env•8ANC195 epitope bridges gp120 and gp41 subunits of HIV-1 Env•8ANC195 inserts a heavy-chain variable domain into a gap in the Env glycan shield•8ANC195 epitope involves gp120 glycans and protein residues of the gp120 inner domain
Characterized targets of broadly neutralizing antibodies (bNAbs) to HIV-1, although key to vaccine and therapeutic strategies, are currently limited. In this study, Scharf et al. have now solved the structures of the patient-derived bNAb 8ANC195 complexed with monomeric gp120 (by X-ray crystallography) and trimeric Env (by electron microscopy). The structures uncover a vulnerable site spanning both HIV-1 envelope protein subunits bound by 8ANC195. A more potent variant isolated from the same patient may be valuable for therapeutic approaches using bNAb combinations with nonoverlapping epitopes.
Extracellular vesicles (EVs) harbor thousands of proteins that hold promise for biomarker development. Usually difficult to purify, EVs in urine are relatively easily obtained and have demonstrated ...efficacy for kidney disease prediction. Herein, we further characterize the proteome of urinary EVs to explore the potential for biomarkers unrelated to kidney dysfunction, focusing on Parkinson's disease (PD).
Using a quantitative mass spectrometry approach, we measured urinary EV proteins from a discovery cohort of 50 subjects. EVs in urine were classified into subgroups and EV proteins were ranked by abundance and variability over time. Enriched pathways and ontologies in stable EV proteins were identified and proteins that predict PD were further measured in a cohort of 108 subjects.
Hundreds of commonly expressed urinary EV proteins with stable expression over time were distinguished from proteins with high variability. Bioinformatic analyses reveal a striking enrichment of endolysosomal proteins linked to Parkinson's, Alzheimer's, and Huntington's disease. Tissue and biofluid enrichment analyses show broad representation of EVs from across the body without bias towards kidney or urine proteins. Among the proteins linked to neurological diseases, SNAP23 and calbindin were the most elevated in PD cases with 86% prediction success for disease diagnosis in the discovery cohort and 76% prediction success in the replication cohort.
Urinary EVs are an underutilized but highly accessible resource for biomarker discovery with particular promise for neurological diseases like PD.
Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), and common genetic variation in LRRK2 modifies susceptibility to Crohn's disease and leprosy. ...High levels of LRRK2 expression in peripheral monocytes and macrophages suggest a role for LRRK2 in these cells, yet little is known about LRRK2 expression and function in immune cells of the brain. Here, we demonstrate a role for LRRK2 in mediating microglial proinflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with toll-like receptor 4 (TLR4)-stimulated rat primary microglia show that inflammation increases LRRK2 activity and expression, while inhibition of LRRK2 kinase activity or knockdown of protein attenuates TNFα secretion and nitric oxide synthase (iNOS) induction. LRRK2 inhibition blocks TLR4 stimulated microglial process outgrowth and impairs ADP stimulated microglial chemotaxis. However, actin inhibitors that phenocopy inhibition of process outgrowth and chemotaxis fail to modify TLR4 stimulation of TNFα secretion and inducible iNOS induction, suggesting that LRRK2 acts upstream of cytoskeleton control as a stress-responsive kinase. These data demonstrate LRRK2 in regulating responses in immune cells of the brain and further implicate microglial involvement in late-onset PD.
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