Rita M. Cowell

LRRK2 Inhibition Attenuates Microglial Inflammatory Responses

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinsons disease (PD), and common genetic variation in LRRK2 modifies susceptibility to Crohns 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.

Localization of the transcriptional coactivator PGC-1α to GABAergic neurons during maturation of the rat brain

The transcriptional coactivator peroxisome proliferator activated receptor γ coactivator 1α (PGC‐1α) can activate a number of transcription factors to regulate mitochondrial biogenesis and cell‐specific responses to cold, fasting, and exercise. Recent studies indicate that PGC‐1α knockout mice exhibit behavioral abnormalities and progressive vacuolization in various brain regions. To investigate the roles for PGC‐1α in the nervous system, we evaluated the temporal and cell‐specific expression of PGC‐1α in the normal developing rat brain. Western blot of whole brain homogenates with a PGC‐1α‐specific antibody revealed that PGC‐1α protein was most abundant in the embryonic and early postnatal forebrain and cerebellum. Using quantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR), we determined that PGC‐1α mRNA expression increased most markedly between postnatal days 3 (P3) and 14 in the cortex, striatum, and hippocampus. Immunohistochemical and immunofluorescence analyses of brain tissue indicated that while PGC‐1α was found in most neuronal populations from embryonic day 15 to P3, it was specifically concentrated in GABAergic populations from P3 to adulthood. Interestingly, PGC‐1α colocalized with the developmentally regulated chemoattractant reelin in the cortex and hippocampus, and the survival‐promoting transcription factor myocyte enhancing factor 2 was highly concentrated in GABAergic populations in the striatum and cerebellum at times of PGC‐1α expression. These results implicate PGC‐1α as a regulator of metabolism and/or survival in GABAergic neurons during a phase of mitochondrial and synaptic changes in the developing brain and suggest that PGC‐1α may be a good target for increasing metabolism in GABAergic populations in neurodevelopmental and neurodegenerative disorders. J. Comp. Neurol. 502:1–18, 2007.

LRRK2 secretion in exosomes is regulated by 14-3-3

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinsons 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.

Dopamine transporter antagonists block phorbol ester-induced dopamine release and dopamine transporter phosphorylation in striatal synaptosomes

We have reported that inhibition of protein kinase C blocks the Ca(2+)-independent reverse transport of dopamine mediated by amphetamine. In this study we investigated whether activation of protein kinase C by 12-O-tetradecanoyl phorbol-13-acetate (TPA) would mediate dopamine release through the plasmalemmal dopamine transporter. TPA, at 250 nM, increased the release of dopamine from rat striatal slices and synaptosomes while the inactive phorbol ester, 4alpha-phorbol, was ineffective. The TPA-mediated dopamine release was independent of extracellular calcium and was blocked by a selective protein kinase C inhibitor, Ro31-8220. The dopamine transporter antagonists, cocaine and GBR 12935 blocked the TPA-mediated dopamine release. In addition, cocaine blocked TPA-mediated phosphorylation of the plasmalemmal dopamine transporter. These results suggest that activation of protein kinase C results in reverse transport of dopamine through the plasmalemmal dopamine transporter and the phosphorylated substrate could be the dopamine transporter.

Social Isolation Exacerbates Schizophrenia-Like Phenotypes via Oxidative Stress in Cortical Interneurons

BACKGROUND Our previous studies indicated that N-methyl-D-aspartate 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 With γ-aminobutyric acid-ergic interneuron-specific NMDAR hypofunction mouse model (Ppp1r2-Cre/fGluN1 knockout [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 8 weeks although increased at 16 weeks old. Postweaning 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 after PWSI were especially prominent in cortical parvalbumin (PV)-positive interneurons. The vulnerability of PV interneurons to oxidative stress was associated with downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (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, might contribute to exacerbation of schizophrenia-like behavioral phenotypes.

Parvalbumin Deficiency and GABAergic Dysfunction in Mice Lacking PGC-1α

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of metabolism in peripheral tissues, and it has been proposed that PGC-1α plays a similar role in the brain. Recent evidence suggests that PGC-1α is concentrated in GABAergic interneurons, so we investigated whether male and female PGC-1α −/− mice exhibit abnormalities in interneuron gene expression and/or function. We found a striking reduction in the expression of the Ca2+-binding protein parvalbumin (PV), but not other GABAergic markers, throughout the cerebrum in PGC-1α +/− and −/− mice. Furthermore, PGC-1α 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-1α −/− 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-1α −/− animals, suggesting that PGC-1α is required for proper expression of PV in multiple tissues. Together these findings indicate that PGC-1α is a novel regulator of interneuron gene expression and function and a potential therapeutic target for neurological disorders associated with interneuron dysfunction.

Convergence of genetic and environmental factors on parvalbumin-positive interneurons in schizophrenia.

Schizophrenia etiology is thought to involve an interaction between genetic and environmental factors during postnatal brain development. However, there is a fundamental gap in our understanding of the molecular mechanisms by which environmental factors interact with genetic susceptibility to trigger symptom onset and disease progression. In this review, we summarize the most recent findings implicating oxidative stress as one mechanism by which environmental insults, especially early life social stress, impact the development of schizophrenia. Based on a review of the literature and the results of our own animal model, we suggest that environmental stressors such as social isolation render parvalbumin-positive interneurons (PVIs) vulnerable to oxidative stress. We previously reported that social isolation stress exacerbates many of the schizophrenia-like phenotypes seen in a conditional genetic mouse model in which NMDA receptors (NMDARs) are selectively ablated in half of cortical and hippocampal interneurons during early postnatal development (Belforte et al., 2010). We have since revealed that this social isolation-induced effect is caused by impairments in the antioxidant defense capacity in the PVIs in which NMDARs are ablated. We propose that this effect is mediated by the down-regulation of PGC-1α, a master regulator of mitochondrial energy metabolism and anti-oxidant defense, following the deletion of NMDARs (Jiang et al., 2013). Other potential molecular mechanisms underlying redox dysfunction upon gene and environmental interaction will be discussed, with a focus on the unique properties of PVIs.

Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies

Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinsons disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinsons Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.

Neuronal Inactivation of Peroxisome Proliferator-activated Receptor γ Coactivator 1α (PGC-1α) Protects Mice from Diet-induced Obesity and Leads to Degenerative Lesions

Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a transcriptional coactivator that regulates diverse aspects of energy metabolism in peripheral tissues. Mice deficient in PGC-1α have elevated metabolic rate and are resistant to diet-induced obesity. However, it remains unknown whether this alteration in energy balance is due to the action of PGC-1α in peripheral tissues or the central nervous system. In this study, we generated neuronal PGC-1α knock-out mice (BαKO) using calcium/calmodulin-dependent protein kinase IIα (CaMKIIα)-Cre to address its role in the regulation of energy balance and neuronal function. Unlike whole body PGC-1α null mice, BαKO mice have normal adaptive metabolic response to starvation and cold exposure in peripheral tissues. In contrast, BαKO mice are hypermetabolic, and similar to whole body PGC-1α null mice, are also resistant to diet-induced obesity, resulting in significantly improved metabolic profiles. Neuronal inactivation of PGC-1α leads to striatal lesions that are reminiscent of neurodegeneration in whole body PGC-1α null brain and impairs nutritional regulation of hypothalamic expression of genes that regulate systemic energy balance. Together, these studies have demonstrated a physiological role for neuronal PGC-1α in the control of energy balance. Our results also implicate CaMKIIα-positive neurons as an important part of the neural circuitry that governs energy expenditure in vivo.

Developmental changes in the expression of chemokine receptor CCR1 in the rat cerebellum

Chemokines are small, soluble proteins that regulate leukocyte migration, adhesion, and proliferation. Recent evidence suggests that chemokine receptors are expressed in the central nervous system and that their functions extend beyond their roles in inflammation. Specific chemokines and their receptors are implicated in cerebellar development. In this study, we evaluated the expression of β‐chemokine receptor CCR1 in the immature and adult rat cerebellum and report striking developmental changes in CCR1 expression. Reverse transcriptase polymerase chain reaction assays of cerebellum revealed moderate increases in CCR1 mRNA expression from postnatal day (P) 3 to adulthood. Light and confocal microscopy were used to evaluate developmental changes in the neuroanatomical and cell‐specific distribution of CCR1 immunoreactivity. CCR1 immunoreactivity was detected as early as P3 and peaked between P7 and P21. The predominant CCR1‐immunoreactive neuronal cell types included granule cells of the internal granular layer, Purkinje cells, Golgi cells, and molecular layer interneurons; Bergmann glia, astrocytes, and resting microglia also expressed CCR1. In contrast, granule cells in the external germinal layer, descending granule cells, and activated microglia rarely expressed CCR1. We also evaluated the expression of the CCR1 ligand macrophage inflammatory protein‐1α (MIP‐1α/CCL3). Two cell populations expressed MIP‐1α: physiologically activated microglia in white matter (P7–P14) and Purkinje cells (P7–adult). MIP‐1α‐positive cells were frequently located near the processes and cell bodies of CCR1‐immunoreactive cells, during times of neuronal and glial maturation (second and third postnatal weeks). These findings provide support for the hypothesis that CCR1 plays a role in postnatal cerebellar development. J. Comp. Neurol. 457:7–23, 2003.