Mark S. Moehle

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.

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

Significance Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of late-onset Parkinson disease, but the mechanisms underlying LRRK2 action in neurodegeneration are not clear. We demonstrate that rats deficient in LRRK2 expression are protected from dopaminergic neurodegeneration caused by overexpression of α-synuclein or exposure to LPS. LRRK2 expression is induced in proinflammatory brain myeloid cells under pathological conditions. Our results suggest that LRRK2 inhibition may have important neuroprotective effects by critically modulating neuroinflammatory responses. LRRK2 inhibition may therefore be a potentially efficacious approach to slow or stop the progression of brain disorders where myeloid cell activation drives aspects of dysfunction. 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.

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.

M1 and M2 immune activation in Parkinson’s Disease: Foe and ally?

Parkinsons Disease (PD) is a chronic and progressive neurodegenerative disorder of unknown etiology. Autopsy findings, genetics, retrospective studies, and molecular imaging all suggest a role for inflammation in the neurodegenerative process. However, relatively little is understood about the causes and implications of neuroinflammation in PD. Understanding how inflammation arises in PD, in particular the activation state of cells of the innate immune system, may provide an exciting opportunity for novel neuroprotective therapeutics. We analyze the evidence of immune system involvement in PD susceptibility, specifically in the context of M1 and M2 activation states. Tracking and modulating these activation states may provide new insights into both PD etiology and therapeutic strategies.

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

Background: LRRK2 kinase activity has been implicated in Parkinson disease (PD). Results: LRRK2 kinase inhibition attenuated neurodegeneration in LRRK2 transgenic and wild-type rats. Conclusion: Chronic inhibition of LRRK2 kinase activity is well tolerated in rats and provides neuroprotection from α-synuclein overexpression. Significance: These results warrant further studies that test the therapeutic potential of LRRK2 kinase inhibitors in additional PD models. 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.

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 Parkinsons 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. J. Comp. Neurol. 522:2465–2480, 2014.

Urinary LRRK2 phosphorylation predicts parkinsonian phenotypes in G2019S LRRK2 carriers.

Objective: To test whether phosphorylated Ser-1292 LRRK2 levels in urine exosomes predicts LRRK2 mutation carriers (LRRK2+) and noncarriers (LRRK2−) with Parkinson disease (PD+) and without Parkinson disease (PD−). Methods: LRRK2 protein was purified from urinary exosomes collected from participants in 2 independent cohorts. The first cohort included 14 men (LRRK2+/PD+, n = 7; LRRK2−/PD+, n = 4; LRRK2−/PD−, n = 3). The second cohort included 62 men (LRRK2−/PD−, n = 16; LRRK2+/PD−, n = 16; LRRK2+/PD+, n = 14; LRRK2−/PD+, n = 16). The ratio of Ser(P)-1292 LRRK2 to total LRRK2 was compared between LRRK2+/PD+ and LRRK2− in the first cohort and between LRRK2 G2019S carriers with and without PD in the second cohort. Results: LRRK2+/PD+ had higher ratios of Ser(P)-1292 LRRK2 to total LRRK2 than LRRK2−/PD− (4.8-fold, p < 0.001) and LRRK2−/PD+ (4.6-fold, p < 0.001). Among mutation carriers, those with PD had higher Ser(P)-1292 LRRK2 to total LRRK2 than those without PD (2.2-fold, p < 0.001). Ser(P)-1292 LRRK2 levels predicted symptomatic from asymptomatic carriers with an area under the receiver operating characteristic curve of 0.844. Conclusion: Elevated ratio of phosphorylated Ser-1292 LRRK2 to total LRRK2 in urine exosomes predicted LRRK2 mutation status and PD risk among LRRK2 mutation carriers. Future studies may explore whether interventions that reduce this ratio may also reduce PD risk.

The G2019S LRRK2 mutation increases myeloid cell chemotactic responses and enhances LRRK2 binding to actin-regulatory proteins

The Leucine rich repeat kinase 2 (LRRK2) gene is genetically and biochemically linked to several diseases that involve innate immunity. LRRK2 protein is highly expressed in phagocytic cells of the innate immune system, most notably in myeloid cells capable of mounting potent pro-inflammatory responses. Knockdown of LRRK2 protein in these cells reduces pro-inflammatory responses. However, the effect of LRRK2 pathogenic mutations that cause Parkinsons disease on myeloid cell function is not clear but could provide insight into LRRK2-linked disease. Here, we find that rats expressing G2019S LRRK2 have exaggerated pro-inflammatory responses and subsequent neurodegeneration after lipopolysaccharide injections in the substantia nigra, with a marked increase in the recruitment of CD68 myeloid cells to the site of injection. While G2019S LRRK2 expression did not affect immunological homeostasis, myeloid cells expressing G2019S LRRK2 show enhanced chemotaxis both in vitro in two-chamber assays and in vivo in response to thioglycollate injections in the peritoneum. The G2019S mutation enhanced the association between LRRK2 and actin-regulatory proteins that control chemotaxis. The interaction between G2019S LRRK2 and actin-regulatory proteins can be blocked by LRRK2 kinase inhibitors, although we did not find evidence that LRRK2 phosphorylated these interacting proteins. These results suggest that the primary mechanism of G2019S LRRK2 with respect to myeloid cell function in disease may be related to exaggerated chemotactic responses.

α-Synuclein fibril-induced inclusion spread in rats and mice correlates with dopaminergic Neurodegeneration

Proteinaceous inclusions in neurons, composed primarily of α-synuclein, define the pathology in several neurodegenerative disorders. Neurons can internalize α-synuclein fibrils that can seed new inclusions from endogenously expressed α-synuclein. The factors contributing to the spread of pathology and subsequent neurodegeneration are not fully understood, and different compositions and concentrations of fibrils have been used in different hosts. Here, we systematically vary the concentration and length of well-characterized α-synuclein fibrils and determine their relative ability to induce inclusions and neurodegeneration in different hosts (primary neurons, C57BL/6J and C3H/HeJ mice, and Sprague Dawley rats). Using dynamic-light scattering profiles and other measurements to determine fibril length and concentration, we find that femptomolar concentrations of fibrils are sufficient to induce robust inclusions in primary neurons. However, a narrow and non-linear dynamic range characterizes fibril-mediated inclusion induction in axons and the soma. In mice, the C3H/HeJ strain is more sensitive to fibril exposures than C57BL/6J counterparts, with more inclusions and dopaminergic neurodegeneration. In rats, injection of fibrils into the substantia nigra pars compacta (SNpc) results in similar inclusion spread and dopaminergic neurodegeneration as injection of the fibrils into the dorsal striatum, with prominent inclusion spread to the amygdala and several other brain areas. Inclusion spread, particularly from the SNpc to the striatum, positively correlates with dopaminergic neurodegeneration. These results define biophysical characteristics of α-synuclein fibrils that induce inclusions and neurodegeneration both in vitro and in vivo, and suggest that inclusion spread in the brain may be promoted by a loss of neurons.

Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket

Background: LRRK2 kinase activity is linked to neurodegeneration. Results: Novel small molecule inhibitors provide insight into the structure and function of the LRRK2 kinase domain. Conclusion: A unique ATP-binding pocket structure in LRRK2 allows for potent and specific activity-selective and mutant-selective small molecules. Significance: Novel structure-activity relationships can be exploited for the development of new classes of kinase inhibitors. Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.