Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson disease, and common genetic variation in LRRK2 modifies susceptibility to Crohn 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 pro-inflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with 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 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 iNOS induction, suggesting 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)-1␣ 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-1␣ 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-1␣; double-labeling immunofluorescence assays were analyzed with confocal microscopy to identify cellular sources of MIP-1␣. Immunocytochemistry assays were also used to detect 2 MIP-1␣ receptors, CCR1 and CCR5. Results-We found marked increases in tissue concentrations of MIP-1␣ in the HI cerebral hemisphere, peaking from 8 to 72 hours after lesioning. Immunocytochemistry assays revealed that MIP-1␣ was constitutively expressed in physiologically activated microglia; from 8 to 120 hours after lesioning, MIP-1␣ immunoreactive monocytes and microglia accumulated in the lesion territory. In immunoreactive cells, MIP-1␣ was diffusely distributed throughout the cytoplasm at early post-HI time intervals; by 72 hours, MIP-1␣ immunoreactivity was typically concentrated adjacent to the nucleus, a pattern indicative of active MIP-1␣ production. In P7 to P12 brain, many cells expressed MIP-1␣ 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

The transcriptional coactivator peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) is a master regulator of metabolism in peripheral tissues, and it has been proposed that PGC-1␣ plays a similar role in the brain. Recent evidence suggests that PGC-1␣ is concentrated in GABAergic interneurons, so we investigated whether male and female PGC-1␣ Ϫ/Ϫ mice exhibit abnormalities in interneuron gene expression and/or function. We found a striking reduction in the expression of the Ca 2ϩ -binding protein parvalbumin (PV), but not other GABAergic markers, throughout the cerebrum in PGC-1␣ ϩ/Ϫ and Ϫ/Ϫ mice. Furthermore, PGC-1␣ overexpression in cell culture was sufficient to robustly induce PV expression. Consistent with a reduction in PV rather than a loss of PV-expressing interneurons, spontaneous synaptic inhibition was not altered in PGC-1␣ Ϫ/Ϫ mice. However, evoked synaptic responses displayed less paired-pulse depression and dramatic facilitation in response to repetitive stimulation at the gamma frequency. PV transcript expression was also significantly reduced in retina and heart of PGC-1␣ Ϫ/Ϫ animals, suggesting that PGC-1␣ is required for proper expression of PV in multiple tissues. Together these findings indicate that PGC-1␣ is a novel regulator of interneuron gene expression and function and a potential therapeutic target for neurological disorders associated with interneuron dysfunction.

Background Our previous studies indicated that NMDA receptor (NMDAR) deletion from a subset of corticolimbic interneurons in the mouse brain during early postnatal development is sufficient to trigger several behavioral and pathophysiological features resembling the symptoms of human schizophrenia. Interestingly, many of these behavioral phenotypes are exacerbated by social isolation stress. However, the mechanisms underlying the exacerbating effects of social isolation are unclear. Methods Using GABAergic interneuron-specific NMDAR hypofunction mouse model (Ppp1r2-cre/fGluN1 KO mice), we investigated whether oxidative stress is implicated in the social isolation-induced exacerbation of schizophrenia-like phenotypes and further explored the underlying mechanism of elevated oxidative stress in KO mice. Results The reactive oxygen species (ROS) level in the cortex of group-housed KO mice was normal at eight weeks although increased at 16 weeks old. Post-weaning social isolation (PWSI) augmented the ROS levels in KO mice at both ages, which was accompanied by the onset of behavioral phenotype. Chronic treatment with apocynin, an ROS scavenger, abolished markers of oxidative stress and partially alleviated schizophrenia-like behavioral phenotypes in KO mice. Markers of oxidative stress following PWSI were especially prominent in cortical parvalbumin (PV)-positive interneurons. The vulnerability of PV interneurons to oxidative stress was associated with down-regulation of peroxisome proliferator-activated receptor α coactivator-13 (PGC-1α), a master regulator of mitochondrial energy metabolism and antioxidation. Conclusions These results suggest that a PWSI-mediated impairment in antioxidant defense mechanisms, presumably mediated by PGC-1α downregulation in the NMDAR-deleted PV-positive interneurons, results in oxidative stress, which, in turn, may contribute to exacerbation of schizophrenia-like behavioral phenotypes.