Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration.

Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.

Abrogation of α-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats.

Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene can cause late-onset Parkinson disease. Past studies have provided conflicting evidence for the protective effects of LRRK2 knockdown in models of Parkinson disease as well as other disorders. These discrepancies may be caused by uncertainty in the pathobiological mechanisms of LRRK2 action. Previously, we found that LRRK2 knockdown inhibited proinflammatory responses from cultured microglia cells. Here, we report LRRK2 knockout rats as resistant to dopaminergic neurodegeneration elicited by intracranial administration of LPS. Such resistance to dopaminergic neurodegeneration correlated with reduced proinflammatory myeloid cells recruited in the brain. Additionally, adeno-associated virus-mediated transduction of human α-synuclein also resulted in dopaminergic neurodegeneration in wild-type rats. In contrast, LRRK2 knockout animals had no significant loss of neurons and had reduced numbers of activated myeloid cells in the substantia nigra. Although LRRK2 expression in the wild-type rat midbrain remained undetected under nonpathological conditions, LRRK2 became highly expressed in inducible nitric oxide synthase (iNOS)-positive myeloid cells in the substantia nigra in response to α-synuclein overexpression or LPS exposures. Our data suggest that knocking down LRRK2 may protect from overt cell loss by inhibiting the recruitment of chronically activated proinflammatory myeloid cells. These results may provide value in the translation of LRRK2-targeting therapeutics to conditions where neuroinflammation may underlie aspects of neuronal dysfunction and degeneration.

Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are found in a significant proportion of late-onset Parkinson's disease (PD) patients. Elucidating the neuroanatomical localization of LRRK2 will further define LRRK2 function and the molecular basis of PD. Here, we utilize recently characterized monoclonal antibodies to evaluate LRRK2 expression in rodent brain regions relevant to PD. In both mice and rats, LRRK2 is highly expressed in the cortex and striatum, particularly in pyramidal neurons of layer V and in medium spiny neurons within striosomes. Overall, rats have a more restricted distribution of LRRK2 compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the substantia nigra pars compacta. Expression of the pathogenic LRRK2-G2019S protein from mouse bacterial artificial chromosome (BAC) constructs closely mimics endogenous LRRK2 distribution in the mouse brain. However, LRRK2-G2019S expression derived from human BAC constructs causes LRRK2 to be expressed in additional neuron subtypes in the rat such as striatal cholinergic interneurons and the substantia nigra pars compacta. The distribution of LRRK2 from human BAC constructs more closely resembles descriptions of LRRK2 in humans and nonhuman primates. Computational analyses of DNA regulatory elements in LRRK2 show a primate-specific promoter sequence that does not exist in lower mammalian species. These noncoding regions may be involved in directing neuronal expression patterns. Together, these studies will aid in understanding the normal function of LRRK2 in the brain and will assist in model selection for future studies.

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

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.

Effect of fenbendazole on three behavioral tests in male C57BL/6N mice.

Pinworms are highly contagious parasites of laboratory rodents that often are treated with fenbendazole. To our knowledge, the effect of fenbendazole at therapeutic dosages on behavioral tests in mice has not been evaluated. Here we studied 6-wk-old male C57BL/6N mice. We compared the behavior of control mice (fed regular diet) with 3 groups of mice treated with dietary fenbendazole. Treatment groups were 4 wk of fenbendazole, 2 wk of fenbendazole followed by 2 wk of regular diet, and 2 wk of regular diet followed by 2 wk of fenbendazole. At the end of dietary treatment all groups were tested by open field for central, peripheral and vertical activity; elevated plus maze for anxiety; and rotarod for motor ability and then evaluated by clinical pathology and selected histopathology. Treated and control groups showed no differences in open field or elevated plus maze testing, histopathology, or clinical pathology. However mice treated for 4 wk with fenbendazole or 2 wk of fenbendazole followed by 2 wk regular diet stayed on the rotarod for shorter periods than did controls, and mice treated with 2 wk of regular diet followed by 2 wk fenbendazole showed a trend toward shorter rotarod times. In light of this study, we suggest that open field and elevated plus maze testing is unlikely to be affected by 4 wk fenbendazole treatment in male C57BL/6 mice; however, behavioral tests of motor ability such as rotarod tests may be affected during and for at least 2 wk after fenbendazole treatment.