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Mutations within the LRRK2 gene have been identified in Parkinson's disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.

Converging evidence suggests that the Parkinson's disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cellular function by regulating actin dynamics. In the present study we investigate the role of LRRK2 in functional synaptic terminals of adult LRRK2-knockout and LRRK2(R1441G)-transgenic mice as well as in primary fibroblasts of LRRK2(G2019S) mutation carriers. We show that lack of LRRK2 decreases and overexpression of mutant LRRK2 age-dependently increases the effect of the actin depolymerizing agent Latrunculin A (LatA) on the synaptic cytoskeleton. Similarly, endogenous mutant LRRK2 increases sensitivity to LatA in primary fibroblasts. Under basal conditions however, these fibroblasts show an increase in F-actin bundles and a decrease in filopodial length which can be rescued by LatA treatment. Our data suggest that LRRK2 alters actin dynamics and F-actin structure both in brain neurons and skin fibroblasts. We hypothesize that increased F-actin bundling represents a compensatory mechanism to protect F-actin from the depolymerizing effect of mutant LRRK2 under basal conditions. Our data further indicate that LRRK2-dependent changes in the cytoskeleton might have functional consequences on postsynaptic NMDA receptor localization., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Increasing evidence suggests that Parkinson's disease (PD)-linked Leucine-rich repeat kinase 2 (LRRK2) has a role in peripheral and brain-resident immune cells. Furthermore, dysregulation of the anti-inflammatory, neurotrophic protein progranulin (PGRN) has been demonstrated in several chronic neurodegenerative diseases. Here we show that PGRN levels are significantly reduced in conditioned medium of LRRK2(R1441G) mutant mouse fibroblasts, leukocytes, and microglia, whereas levels of proinflammatory factors, like interleukin-1β and keratinocyte-derived chemokine, were significantly increased. Decreased PGRN levels were also detected in supernatants of cultured human fibroblasts isolated from presymptomatic LRRK2(G2019S) mutation carriers, while mitochondrial function was unaffected. Furthermore, medium levels of matrix metalloprotease (MMP) 2 increased, whereas MMP 9 decreased in LRRK2(R1441G) mutant microglia. Increased proteolytic cleavage of the MMP substrates ICAM-5 and α-synuclein in synaptoneurosomes from LRRK2(R1441G) mutant mouse brain indicates increased net synaptic MMP activity. PGRN levels were decreased in the cerebrospinal fluid of presymptomatic LRRK2 mutant mice, whereas PGRN levels were increased in aged symptomatic mutant mice. Notably, PGRN levels were also increased in the cerebrospinal fluid of PD patients carrying LRRK2 mutations, but not in idiopathic PD patients and in healthy control donors. Our data suggest that proinflammatory activity of peripheral and brain-resident immune cells may particularly contribute to the early stages of Parkinson's disease caused by LRRK2 mutations., (Copyright © 2014 Wiley Periodicals, Inc.)

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause familial as well as sporadic Parkinson's disease (PD) that is characterized by an age-dependent degeneration of dopaminergic neurons. LRRK2 is strongly expressed in neural stem cells (NSCs), but still the exact molecular function of LRRK2 in these cells remains unknown. By performing a systemic analysis of the gene expression profile of LRRK2-deficient NSCs, we found that the expression of several PD-associated genes, such as oxidation and reduction in mitochondria, are deregulated on LRRK2 absence. Our data, indeed, indicate that LRRK2 regulates the level of cellular oxidative stress and thereby influences the survival of NSCs. Furthermore, the lack of LRRK2 leads to an up-regulation of neuronal differentiation-inducing processes, including the Let-7a pathway. On the other hand, the constitutive mutant of LRRK2(R1441G), known to cause PD, leads to down-regulation of the same pathway. In agreement with the function of Let-7a during neuronal differentiation, LRRK2-deficient NSCs differentiate faster than wild-type cells, while LRRK2(R1441G)-expressing NSCs show impaired neuronal differentiation. These results might help better characterize the molecular mechanisms underlying the role of LRRK2 in NSCs and would further improve potential cell-replacement strategies as well as drug discovery approaches.

Recent studies indicate that the Parkinson's disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cytoskeletal functions by regulating actin and tubulin dynamics, thereby affecting neurite outgrowth. By interactome analysis we demonstrate that the binding of LRRK2 to tubulins is significantly enhanced by pharmacological LRRK2 inhibition in cells. Co-incubation of LRRK2 with microtubules increased the LRRK2 GTPase activity in a cell-free assay. Destabilization of microtubules causes a rapid decrease in cellular LRRK2(S935) phosphorylation indicating a decreased LRRK2 kinase activity. Moreover, both human LRRK2(G2019S) fibroblasts and mouse LRRK2(R1441G) fibroblasts exhibit alterations in cell migration in culture. Treatment of mouse fibroblasts with the selective LRRK2 inhibitor LRRK2-IN1 reduces cell motility. These findings suggest that LRRK2 and microtubules mutually interact both in non-neuronal cells and in neurons, which might contribute to our understanding of its pathogenic effects in Parkinson's disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. LRRK2 kinase activity is required for toxicity in neuronal cell cultures suggesting that selective kinase inhibitors may prevent neurodegeneration in patients. Directly monitoring LRRK2 activity in cells would be advantageous for the development of small molecule LRRK2 inhibitors. Here, we demonstrate that a monoclonal anti-LRRK2 antibody directed against the activation segment binds less efficiently to native LRRK2 protein in the presence of ATP-competitive LRRK2 inhibitors. Since kinase inhibitors prevent autophosphorylation and refolding of the activation segment, we hypothesize that the antibody preferentially binds to the active conformation of LRRK2 under native conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Adult neurogenesis, the formation of new neurons in the mammalian forebrain, is one important mechanism maintaining lifelong neuronal plasticity. The generation and maturation of adult neural stem and progenitor cells is impaired in models of neurodegenerative diseases, in particular Parkinson's disease (PD). Monogenetic forms of PD were identified and associated with several genes including the leucine-rich-repeat kinase 2 (LRRK2). Some of the underlying mechanisms in neurodegenerative diseases are closely linked to neuronal plasticity, and induce changes in adult neurogenesis, neuritic maintenance, synaptic transmission, and neural connectivity. We investigated adult neurogenesis and neuritic development of newly formed neurons in the hippocampal dentate gyrus of LRRK2 knockout mice. Proliferation and survival of newly generated cells were unchanged. However, the expression profile of maturation markers in surviving newly generated cells was altered. While immature neuronal phenotypes were significantly increased, the mature neuronal phenotype of surviving cells remained unchanged. Importantly, the absolute number of immature doublecortin positive neuroblasts was significantly increased in the hippocampus of LRRK2 knockout mice. These neuroblasts presented extended dendritic length with a more complex arborization. Furthermore, LRRK2 deletion resulted in an increased volume of the axonal mossy fiber bundle projecting from dentate granule cells to CA3 pyramidal neurons. Our findings suggest that LRRK2 influences neurogenesis and particularly neuronal morphogenesis. As neurogenesis and the pre-/post- synaptic compartments are significantly altered in PD, our data advance LRRK2 as a potent candidate in addressing neuroregenerative processes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been causally linked to neuronal cell death in Parkinson's disease. LRRK2 expression has also been detected in B lymphocytes and macrophages, suggesting a role in immune responses. In the present study, we demonstrate that LRRK2 is expressed in primary microglial cells isolated from brains of adult mice. Moreover, lipopolysaccharide (LPS)-activated microglial cells from mice overexpressing the Parkinson's disease-linked LRRK2(R1441G) mutation exhibit increased expression and secretion of proinflammatory cytokines compared with wild-type control microglia. Expression of the LPS receptor Toll-like receptor 4 (TLR4) and downstream signaling proteins did not differ between LRRK2(R1441G) transgenic microglia and wild-type controls. Consistently, conditioned medium from LPS-stimulated LRRK2(R1441G) transgenic microglia induced significant cell death when added to neuronal cultures. These findings indicate that enhanced neuroinflammation may contribute to neurodegeneration in Parkinson's disease patients carrying LRRK2 mutations., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment., Methodology/principal Findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture., Conclusion/significance: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro., Methodology/principal Findings: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7., Conclusions/significance: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson's disease pathogenesis.

Background: Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, however, the underlying pathogenic mechanisms are poorly understood. Several in vitro studies have shown that the most frequent mutation, LRRK2(G2019S), increases kinase activity and impairs neuronal survival. LRRK2 has been linked to the mitogen-activated protein kinase kinase kinase family and the receptor-interacting protein kinases based on sequence similarity within the kinase domain and in vitro substrate phosphorylation., Methodology/principal Findings: We used an unbiased proteomic approach to identify the kinase signaling pathways wherein LRRK2 may be active. By incubation of protein microarrays containing 260 signal transduction proteins we detected four arrayed Ste20 serine/threonine kinase family members (TAOK3, STK3, STK24, STK25) as novel LRRK2 substrates and LRRK2 interacting proteins, respectively. Moreover, we found that protein kinase C (PKC) zeta binds and phosphorylates LRRK2 both in vitro and in vivo., Conclusions/significance: Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations.

Mutations within the LRRK2 (leucine-rich repeat kinase 2) gene predispose humans to develop late-onset Parkinson's disease (PD). The most prevalent of these mutations, G2019S, has been shown to increase LRRK2 kinase activity. Therefore, the discovery of small molecule inhibitors of LRRK2(G2019S) through high-throughput screening (HTS) may provide a novel therapeutic strategy for treating PD. Current biochemical assays monitoring the activity of LRRK2(G2019S) either are radioactive or use short peptidic substrates. Here we describe the development and optimization of a novel HTS AlphaScreen assay for measuring the catalytic activity of full-length LRRK2(G2019S) using its putative physiological protein substrate moesin. The high sensitivity of this optimized 384-well assay allowed the use of enzyme concentrations as low as 0.75nM. The estimated apparent K(m) value for adenosine triphosphate (6 microM) using the glutathione S-transferase-moesin substrate was much lower than the one previously reported using LRRKtide, a synthetic peptide derived from moesin. Testing of nonselective kinase inhibitors (staurosporine, H-1152, and Y-27632) generated potencies consistent with published data. Finally, robotic validation of the assay yielded an average Z' factor of 0.80. Overall, these results indicate that the present HTS AlphaScreen assay might provide a more relevant biochemical approach for the discovery of novel LRRK2(G2019S) inhibitors., (2010 Elsevier Inc. All rights reserved.)

Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset Parkinson's disease. However, the regulators/effectors contributing to the (patho-)physiological functions of LRRK2 remain poorly defined. Here we show that human LRRK2 co-purifies/co-immunoprecipitates with elongation factor 1-alpha (EF1A). Co-incubation of recombinant LRRK2 and EF1A significantly reduces the kinase activity of LRRK2, whereas its GTPase activity remains unchanged. In addition to its canonical role in mRNA translation, EF1A maintains stability of the microtubule cytoskeleton. In the present study, EF1A promotes microtubule assembly in an in vitro tubulin polymerization assay which is impaired by co-incubation with LRRK2 at sub-stoichiometric concentrations. These findings suggest that the interaction between LRRK2 and EF1A may reciprocally modulate their physiological function.

Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset Parkinson's disease. The most prevalent LRRK2(G2019S) mutation has repeatedly been shown to enhance kinase activity and neurotoxicity, however, the molecular mechanisms leading to neurodegeneration remain poorly defined. Here we show that recombinant human LRRK2 preferentially phosphorylates tubulin-beta purified from bovine brain and that phosphorylation is three-fold enhanced by the LRRK2(G2019S) mutation. By tandem mass spectrometry, Thr107 was identified as phosphorylation site which is highly conserved between tubulin-beta family members and also between tubulin-beta genes of different species. LRRK2 was co-immunoprecipitated with tubulin-beta both from wild-type mouse brain and from LRRK2 over-expressing, non-neuronal human embryonic kidney 293 cells. However, an effect of LRRK2 on tubulin phosphorylation and assembly was only detectable in mouse brain samples. In vitro co-incubation of bovine brain tubulins with LRRK2 increased microtubule stability in the presence of microtubule-associated proteins which may explain the reduction in neurite length in LRRK2-deficient neurons in culture. These findings suggest that LRRK2(G2019S)-induced neurodegeneration in Parkinsonian brains may be partly mediated by increased phosphorylation of tubulin-beta and constraining of microtubule dynamics.

Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.