Missense mutations inleucine-rich repeat kinase 2 (LRRK2)cause late-onset Parkinson's disease (PD), and common genetic variation inLRRK2modifies 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.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials.

Background and Purpose — Macrophage inflammatory protein (MIP)-1α is a well-characterized monocyte chemoattractant; its role in regulating monocyte and microglial recruitment and activation in the injured neonatal brain is unknown. We evaluated the impact of acute hypoxic-ischemic (HI) brain injury on the expression of MIP-1α in neonatal rat brain. Methods — To elicit forebrain ischemic injury, 7-day-old (P7) rats underwent right carotid ligation, followed by 2.5 hours of 8% oxygen exposure. We used an enzyme-linked immunosorbent assay and immunohistochemistry to detect MIP-1α; double-labeling immunofluorescence assays were analyzed with confocal microscopy to identify cellular sources of MIP-1α. Immunocytochemistry assays were also used to detect 2 MIP-1α receptors, CCR1 and CCR5. Results — We found marked increases in tissue concentrations of MIP-1α in the HI cerebral hemisphere, peaking from 8 to 72 hours after lesioning. Immunocytochemistry assays revealed that MIP-1α was constitutively expressed in physiologically activated microglia; from 8 to 120 hours after lesioning, MIP-1α immunoreactive monocytes and microglia accumulated in the lesion territory. In immunoreactive cells, MIP-1α was diffusely distributed throughout the cytoplasm at early post-HI time intervals; by 72 hours, MIP-1α immunoreactivity was typically concentrated adjacent to the nucleus, a pattern indicative of active MIP-1α production. In P7 to P12 brain, many cells expressed MIP-1α receptors; both CCR1 and CCR5 immunoreactivity were localized to endothelium and ependyma; CCR1-immunoreactive astrocytes and neurons and CCR5-immunoreactive microglia were also identified. Conclusions — These data implicate MIP-1α as a mediator of the complex and sustained inflammatory response initiated by perinatal HI braininjury.

Conflicting data have emerged regarding the role of complement activation in the pathophysiology of cerebral ischemia. On the basis of considerable evidence implicating inflammatory mediators in the progression of neonatal brain injury, we evaluated the contribution of complement activation to cerebral hypoxic-ischemic (HI) injury in the neonatal rat. To elicit unilateral forebrain HI injury, 7-d-old rats underwent right carotid ligation followed by 1.5-2 hr of exposure to 8% oxygen. Using immunoprecipitation and Western blot assays, we determined that HI induces local complement cascade activation as early as 8 hr post-HI; there was an eightfold increase in the activation fragment inactivated C3b at 16 hr. With immunofluorescence assays and confocal microscopy, both C3 and C9 were localized to injured neurons 16 and 24 hr post-HI. To investigate the contribution of systemic complement to brain injury, we administered the complement-depleting agent cobra venom factor (CVF) 24 hr before HI lesioning and evaluated both acute HI-induced complement deposition and the extent of resulting tissue injury 5 d after lesioning. CVF depleted both systemic and brain C3 by the time of surgery and reduced infarct size. Analysis of lesioned, CVF-treated animals demonstrated minimal neuronal C3 deposition but no reduction in C9 deposition. C3-immunoreactive microglia were identified in injured areas. These results indicate that complement activation contributes to HI injury in neonatal rat brain, systemic administration of CVF does not eliminate complement deposition within injured brain, and microglia may represent an important local source of C3 after acute brain injury.