LRRK2 G2019S kinase activity triggers neurotoxic NSF aggregation.

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta and the presence of protein aggregates in surviving neurons. The LRRK2 G2019S mutation is one of the major determinants of familial Parkinson's disease cases and leads to late-onset Parkinson's disease with pleomorphic pathology, including α-synuclein accumulation and deposition of protein inclusions. We demonstrated that LRRK2 phosphorylates N-ethylmaleimide sensitive factor (NSF). We observed aggregates containing NSF in basal ganglia specimens from patients with Parkinson's disease carrying the G2019S variant, and in cellular and animal models expressing the LRRK2 G2019S variant. We found that LRRK2 G2019S kinase activity induces the accumulation of NSF in toxic aggregates. Of note, the induction of autophagy cleared NSF aggregation and rescued motor and cognitive impairment observed in aged hG2019S bacterial artificial chromosome (BAC) mice. We suggest that LRRK2 G2019S pathological phosphorylation impacts on NSF biochemical properties, thus causing the formation of cytotoxic protein inclusions.

Divergent Effects of G2019S and R1441C LRRK2 Mutations on LRRK2 and Rab10 Phosphorylations in Mouse Tissues.

Mutations in LRRK2 cause familial Parkinson's disease and common variants increase disease risk. LRRK2 kinase activity and cellular localization are tightly regulated by phosphorylation of key residues, primarily Ser1292 and Ser935, which impacts downstream phosphorylation of its substrates, among which Rab10. A comprehensive characterization of LRRK2 activity and phosphorylation in brain as a function of age and mutations is missing. Here, we monitored Ser935 and Ser1292 phosphorylation in midbrain, striatum, and cortex of 1, 6, and 12 months-old mice carrying G2019S and R1441C mutations or murine bacterial artificial chromosome (BAC)-Lrrk2-G2019S. We observed that G2019S and, at a greater extent, R1441C brains display decreased phospho-Ser935, while Ser1292 autophosphorylation increased in G2019S but not in R1441C brain, lung, and kidney compared to wild-type. Further, Rab10 phosphorylation, is elevated in R1441C carrying mice, indicating that the effect of LRRK2 mutations on substrate phosphorylation is not generalizable. In BAC-Lrrk2-G2019S striatum and midbrain, Rab10 phosphorylation, but not Ser1292 autophosphorylation, decreases at 12-months, pointing to autophosphorylation and substrate phosphorylation as uncoupled events. Taken together, our study provides novel evidence that LRRK2 phosphorylation in mouse brain is differentially impacted by mutations, brain area, and age, with important implications as diagnostic markers of disease progression and stratification.

Levetiracetam treatment ameliorates LRRK2 pathological mutant phenotype.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). The LRRK2 physiological and pathological function is still debated. However, different experimental evidence based on LRRK2 cellular localization and LRRK2 protein interactors suggests that LRRK2 may be part and regulate a protein network modulating vesicle dynamics/trafficking. Interestingly, the synaptic vesicle protein SV2A is part of this protein complex. Importantly, SV2A is the binding site of the levetiracetam (LEV), a compound largely used in human therapy for epilepsy treatment. The binding of LEV to SV2A reduces the neuronal firing by the modulation of vesicle trafficking although by an unclear molecular mechanism. In this short communication, we have analysed the interaction between the LRRK2 and SV2A pathways by LEV treatment. Interestingly, LEV significantly counteracts the effect of LRRK2 G2019S pathological mutant expression in three different cellular experimental models. Our data strongly suggest that LEV treatment may have a neuroprotective effect on LRRK2 pathological mutant toxicity and that LEV repositioning could be a viable compound for PD treatment.

Superoxide Dismutases SOD1 and SOD2 Rescue the Toxic Effect of Dopamine-Derived Products in Human SH-SY5Y Neuroblastoma Cells.

The preferential loss of dopaminergic neurons in the substantia nigra pars compacta is one of the pathological hallmarks characterizing Parkinson's disease. Although the pathogenesis of this disorder is not fully understood, oxidative stress plays a central role in the onset and/or progression of Parkinson's disease and dopamine itself has been suggested to participate in the preferential neuronal degeneration through the induction of oxidative conditions. In fact, the accumulation of dopamine into the cytosol can lead to the formation of reactive oxygen species as well as highly reactive dopamine-quinones. In the present work, we first analyzed the cellular damage induced by the addition of dopamine (DA) in the culture medium of SH-SY5Y cells, discriminating whether the harmful effects were related to the generation of reactive oxygen species or to the toxicity associated to dopamine-derived quinones. Then, we tested and demonstrated the capability of the antioxidant enzymes SOD1 and SOD2 to protect cells from the noxious effects induced by DA treatment. Our results support further exploration of superoxide dismutating molecules as a therapeutic strategy against Parkinson's disease.

Transcriptome analysis of LRRK2 knock-out microglia cells reveals alterations of inflammatory- and oxidative stress-related pathways upon treatment with α-synuclein fibrils.

Several previous studies have linked the Parkinson's disease (PD) gene LRRK2 to the biology of microglia cells. However, the precise ways in which LRRK2 affects microglial function have not been fully resolved. Here, we used the RNA-Sequencing to obtain transcriptomic profiles of LRRK2 wild-type (WT) and knock-out (KO) microglia cells treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS) as a general inflammatory insult. We observed that, although α-synuclein PFFs and LPS mediate overlapping gene expression profiles in microglia, there are also distinct responses to each stimulus. α-Synuclein PFFs trigger alterations of oxidative stress-related pathways with the mitochondrial dismutase Sod2 as a strongly differentially regulated gene. We validated SOD2 at mRNA and protein levels. Furthermore, we found that LRRK2 KO microglia cells reported attenuated induction of mitochondrial SOD2 in response to α-synuclein PFFs, indicating a potential contribution of LRRK2 to oxidative stress-related pathways. We validate several genes in vivo using single-cell RNA-Seq from acutely isolated microglia after striatal injection of LPS into the mouse brain. Overall, these results suggest that microglial LRRK2 may contribute to the pathogenesis of PD via altered oxidative stress signaling.

The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A.

Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are a common cause of hereditary Parkinson's disease. LRRK2 regulates various intracellular vesicular trafficking pathways, including endolysosomal degradative events such as epidermal growth factor receptor (EGFR) degradation. Recent studies have revealed that a subset of RAB proteins involved in secretory and endocytic recycling are LRRK2 kinase substrates in vivo However, the effects of LRRK2-mediated phosphorylation of these substrates on membrane trafficking remain unknown. Here, using an array of immunofluorescence and pulldown assays, we report that expression of active or phosphodeficient RAB8A variants rescues the G2019S LRRK2-mediated effects on endolysosomal membrane trafficking. Similarly, up-regulation of the RAB11-Rabin8-RAB8A cascade, which activates RAB8A, also reverted these trafficking deficits. Loss of RAB8A mimicked the effects of G2019S LRRK2 on endolysosomal trafficking and decreased RAB7A activity. Expression of pathogenic G2019S LRRK2 or loss of RAB8A interfered with EGFR degradation by causing its accumulation in a RAB4-positive endocytic compartment, which was accompanied by a deficit in EGFR recycling and was rescued upon expression of active RAB7A. Dominant-negative RAB7A expression resulted in similar deficits in EGF degradation, accumulation in a RAB4 compartment, and deficits in EGFR recycling, which were all rescued upon expression of active RAB8A. Taken together, these findings suggest that, by impairing RAB8A function, pathogenic G2019S LRRK2 deregulates endolysosomal transport and endocytic recycling events.

The role of LRRK2 in cytoskeletal dynamics.

Leucine-rich repeat kinase 2 (LRRK2), a complex kinase/GTPase mutated in Parkinson's disease, has been shown to physically and functionally interact with cytoskeletal-related components in different brain cells. Neurons greatly rely on a functional cytoskeleton for many homeostatic processes such as local and long-distance vesicle transport, synaptic plasticity, and dendrites/axons growth and remodeling. Here, we will review the available data linking LRRK2 and the cytoskeleton, and discuss how this may be functionally relevant for the well-established roles of LRRK2 in intracellular trafficking pathways and outgrowth of neuronal processes in health and disease conditions.

Dopamine Oxidation Products as Mitochondrial Endotoxins, a Potential Molecular Mechanism for Preferential Neurodegeneration in Parkinson's Disease.

The preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta is responsible for the motor impairment associated with Parkinson's disease. Dopamine is a highly reactive molecule, which is usually stored inside synaptic vesicles where it is stabilized by the ambient low pH. However, free cytosolic dopamine can auto-oxidize, generating reactive oxygen species, and lead to the formation of toxic quinones. In the present work, we have analyzed the mechanisms through which the dysfunction of dopamine homeostasis could induce cell toxicity, by focusing in particular on the damage induced by dopamine oxidation products at the mitochondrial level. Our results indicate that dopamine derivatives affect mitochondrial morphology and induce mitochondrial membrane depolarization, leading to a reduction of ATP synthesis. Moreover, our results suggest that opening of the mitochondrial transition pore induced by dopamine-derived quinones may contribute to the specific Parkinson's disease-associated vulnerability of dopamine containing neurons.

Superoxide dismutating molecules rescue the toxic effects of PINK1 and parkin loss.

Reactive oxygen species exert important functions in regulating several cellular signalling pathways. However, an excessive accumulation of reactive oxygen species can perturb the redox homeostasis leading to oxidative stress, a condition which has been associated to many neurodegenerative disorders. Accordingly, alterations in the redox state of cells and mitochondrial homeostasis are established hallmarks in both familial and sporadic Parkinson's disease cases. PINK1 and Parkin are two genes which account for a large fraction of autosomal recessive early-onset forms of Parkinson's disease and are now firmly associated to both mitochondria and redox homeostasis. In this study we explored the hypothesis that superoxide anions participate in the generation of the Parkin and PINK1 associated phenotypic effect by testing the capacity of endogenous and exogenous superoxide dismutating molecules to rescue the toxic effects induced by loss of PINK1 or Parkin, in both cellular and fly models. Our results demonstrate the positive effect of an increased level of superoxide dismutase proteins on the pathological phenotypes, both in vitro and in vivo. A more pronounced effectiveness for mitochondrial SOD2 activity points to the superoxide radicals generated in the mitochondrial matrix as the prime suspect in the definition of the observed phenotypes. Moreover, we also demonstrate the efficacy of a SOD-mimetic compound, M40403, to partially ameliorate PINK1/Parkin phenotypes in vitro and in vivo. These results support the further exploration of SOD-mimetic compounds as a therapeutic strategy against Parkinson's disease.

Diabetes Mellitus as a Risk Factor for Parkinson's Disease: a Molecular Point of View.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by elevated concentrations of glucose in the blood. The chronic hyperglycemic state accounts for most of the vascular complications associated to the disease and the prevalent mechanism proposed is related to the glycating chemistry mediated by methylglyoxal (MG), which accumulates in T2DM. In recent years, a higher risk of Parkinson's disease (PD) onset in people affected by T2DM has become evident, but the molecular mechanisms underlying the interplay between T2DM and PD are still unknown. The oxidative chemistry of dopamine and its reactivity towards the protein α-Synuclein (aS) has been associated to the pathogenesis of PD. Recently, aS has also been described to interact with MG. Interestingly, MG and the dopamine oxidation products share both structural similarity and chemical reactivity. The ability of MG to spread over the site of its production and react with aS could represent the rationale to explain the higher incidence of PD in T2DM-affected people and may open opportunities for the development of novel strategies to antagonize the raise of PD.

The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1.

Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

Recent findings on the physiological function of DJ-1: Beyond Parkinson's disease.

Several mutations in the gene coding for DJ-1 have been associated with early onset forms of parkinsonism. In spite of the massive effort spent by the scientific community in understanding the physiological role of DJ-1, a consensus on what DJ-1 actually does within the cells has not been reached, with several diverse functions proposed. At present, the most accepted function for DJ-1 is a neuronal protective role against oxidative stress. However, how exactly this function is exerted by DJ-1 is not clear. In recent years, novel molecular mechanisms have been suggested that may account for the antioxidant properties of DJ-1. In this review, we critically analyse the experimental evidence, including some very recent findings, supporting the purported neuroprotective role of DJ-1 through different mechanisms linked to oxidative stress handling, as well as the relevance of these processes in the context of Parkinson's disease.

Role of LRRK2 in the regulation of dopamine receptor trafficking.

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD.

LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S) pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S) shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

GTPase activity regulates kinase activity and cellular phenotypes of Parkinson's disease-associated LRRK2.

Mutations in the LRRK2 gene cause autosomal dominant Parkinson's disease. LRRK2 encodes a multi-domain protein containing a Ras-of-complex (Roc) GTPase domain, a C-terminal of Roc domain and a protein kinase domain. LRRK2 can function as a GTPase and protein kinase, although the interplay between these two enzymatic domains is poorly understood. Although guanine nucleotide binding is critically required for the kinase activity of LRRK2, the contribution of GTP hydrolysis is not known. In general, the molecular determinants regulating GTPase activity and how the GTPase domain contributes to the properties of LRRK2 remain to be clarified. Here, we identify a number of synthetic missense mutations in the GTPase domain that functionally modulate GTP binding and GTP hydrolysis and we employ these mutants to comprehensively explore the contribution of GTPase activity to the kinase activity and cellular phenotypes of LRRK2. Our data demonstrate that guanine nucleotide binding and, to a lesser extent, GTP hydrolysis are required for maintaining normal kinase activity and both activities contribute to the GTP-dependent activation of LRRK2 kinase activity. Guanine nucleotide binding but not GTP hydrolysis regulates the dimerization, structure and stability of LRRK2. Furthermore, GTP hydrolysis regulates the LRRK2-dependent inhibition of neurite outgrowth in primary cortical neurons but is unable to robustly modulate the effects of the familial G2019S mutation. Our study elucidates the role of GTPase activity in regulating kinase activity and cellular phenotypes of LRRK2 and has important implications for the validation of the GTPase domain as a molecular target for attenuating LRRK2-mediated neurodegeneration.

Brain microdialysis in freely moving animals.

Brain microdialysis is an analytical technique used for the dynamic monitoring of brain neurochemistry in awake, freely moving animals. This technique requires the insertion of a small dialysis catheter, called a microdialysis probe, into a specific brain region, and its perfusion with an artificial extracellular fluid. The microdialysate samples, obtained from the probe outlet, can be analysed using high-performance liquid chromatography with electrochemical detection for the quantification of oxidizable molecules recovered from the extracellular space. In this chapter, we describe a protocol for performing a microdialysis setup and experiment in freely moving rats and mice. Furthermore, the high-performance liquid chromatographic determination of ascorbic acid, uric acid, catecholamines, indolamines and derivatives is described in detail.

Dual asymmetric-flow microdialysis for in vivo monitoring of brain neurochemicals.

Microdialysis is an extensively used technique for both in vivo and in vitro experiments, applicable to animal and human studies. In neurosciences, the in vivo microdialysis is usually performed to follow changes in the extracellular levels of substances and to monitor neurotransmitters release in the brain of freely moving animals. Catecholamines, such as dopamine and their related compounds, are involved in the neurochemistry and in the physiology of mental diseases and neurological disorders. It is generally supposed that the brain's energy requirement is supplied by glucose oxidation. More recently, lactate was proposed to be the metabolic substrate used by neurons during synaptic activity. In our study, an innovative microdialysis approach for simultaneous monitoring of catecholamines, indolamines, glutamate and energy substrates in the striatum of freely moving rats, using an asymmetric perfusion flow rate on microdialysis probe, is described. As a result of this asymmetric perfusion, two samples are available from the same brain region, having the same analytes composition but different concentrations. The asymmetric flow perfusion could be a useful tool in neurosciences studies related to brain's energy requirement, such as toxin-induced models of Parkinson's disease.