Autosomal-dominant Familial Parkinson's Disease Research Articles

VPS35, the core component of the retromer complex, and Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease that is common in middle-aged and elderly people, and its onset is related to multiple factors, such as heredity, environment, and age. The vesicle protein sorting 35 (VPS35) gene was found to be a late-onset autosomal dominant familial PD (PARK17) causative gene. The protein encoded by this gene is located in the endosome and aggregates with other membrane proteins to form a retromer complex, which participates in the membrane protein cycle between the endosome and the Golgi network. Increasing evidence shows that VPS35 may participate in the pathogenesis of PD by affecting autophagy, mitochondria, neurosynaptic transmission, dopamine signaling pathways, and so forth, and it can interact with other disease-causing genes of familial PD. This article aimed to review the functions of VPS35 and the mechanism of its mutations in PD that have been discovered in recent years.

Dopaminergic neurodegeneration induced by Parkinson's disease-linked G2019S LRRK2 is dependent on kinase and GTPase activity

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of late-onset, autosomal-dominant familial Parkinson's disease (PD). LRRK2 functions as both a kinase and GTPase, and PD-linked mutations are known to influence both enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage in culture models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models containing familial LRRK2 mutations often lack robust PD-like neurodegenerative phenotypes. Here, we develop a robust preclinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or administration of the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the robust destabilization of human LRRK2 protein in the brain relative to endogenous LRRK2. Our study further demonstrates that G2019S LRRK2-induced dopaminergic neurodegeneration critically requires normal GTPase activity, as hypothesis-testing mutations that increase GTP hydrolysis or impair GTP-binding activity provide neuroprotection although via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent preclinical model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

The Emerging Functions of LRRK2 and Rab GTPases in the Endolysosomal System.

The leucine-rich repeat kinase 2 (LRRK2), the most common causative gene for autosomal-dominant familial Parkinson’s disease, encodes a large protein kinase harboring multiple characteristic domains. LRRK2 phosphorylates a set of Rab GTPases in cells, which is enhanced by the Parkinson-associated LRRK2 mutations. Accumulating evidence suggests that LRRK2 regulates intracellular vesicle trafficking and organelle maintenance including Golgi, endosomes and lysosomes. Furthermore, genetic knockout or inhibition of LRRK2 cause lysosomal abnormalities in rodents and primates, and cells from Parkinson’s patients with LRRK2 mutations also exhibit altered lysosome morphology. Cell biological studies on LRRK2 in a diverse cellular context further strengthen the potential connection between LRRK2 and regulation of the endolysosomal system, part of which is mediated by Rab phosphorylation by LRRK2. We will focus on the latest advances on the role of LRRK2 and Rab in relation to the endolysosomal system, and discuss the possible link to the pathomechanism of Parkinson’s disease.

Polo-like kinase 2 modulates α-synuclein protein levels by regulating its mRNA production

Variations in the α-synuclein-encoding SNCA gene represent the greatest genetic risk factor for Parkinson's disease (PD), and duplications/triplications of SNCA cause autosomal dominant familial PD. These facts closely link brain levels of α-synuclein with the risk of PD, and make lowering α-synuclein levels a therapeutic strategy for the treatment of PD and related synucleinopathies.In this paper, we corroborate previous findings on the ability of overexpressed Polo-like kinase 2 (PLK-2) to decrease cellular α-synuclein, but demonstrate that the process is independent of PLK-2 phosphorylating S129 in α-synuclein because a similar reduction is achieved with the non-phosphorable S129A mutant α-synuclein. Using a specific PLK-2 inhibitor (compound 37), we demonstrate that endogenous PLK-2 phosphorylates S129 only in some cells, but increases α-synuclein protein levels in all tested cell cultures and brain slices. PLK-2 is found to regulate the transcription of α-synuclein mRNA from both the endogenous mouse SNCA gene and transgenic vectors that only contain the open reading frame. Moreover, we are the first to show that regulation of α-synuclein by PLK-2 is of physiological importance since 10days' inhibition of endogenous PLK-2 in wt C57BL/6 mice increases endogenous α-synuclein protein levels.Our findings collectively demonstrate that PLK-2 regulates α-synuclein levels by a previously undescribed transcription-based mechanism. This mechanism is active in cells and brain tissue, opening up for alternative strategies for modulating α-synuclein levels and thereby for the possibility of modifying disease progression in synucleinopaties.

Overexpression of Parkinson's Disease-Associated Mutation LRRK2 G2019S in Mouse Forebrain Induces Behavioral Deficits and α-Synuclein Pathology.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an unambiguous cause of late-onset, autosomal-dominant familial Parkinson's disease (PD) and LRRK2 mutations are the strongest genetic risk factor for sporadic PD known to date. A number of transgenic mice expressing wild-type or mutant LRRK2 have been described with varying degrees of LRRK2-related abnormalities and modest pathologies. None of these studies directly addressed the role of the kinase domain in the changes observed and none of the mice present with robust features of the human disease. In an attempt to address these issues, we created a conditional LRRK2 G2019S (LRRK2 GS) mutant and a functionally negative control, LRRK2 G2019S/D1994A (LRRK2 GS/DA). Expression of LRRK2 GS or LRRK2 GS/DA was conditionally controlled using the tet-off system in which the presence of tetracycline-transactivator protein (tTA) with a CAMKIIα promoter (CAMKIIα-tTA) induced expression of TetP-LRRK2 GS or TetP-LRRK2 GS/DA in the mouse forebrain. Overexpression of LRRK2 GS in mouse forebrain induced behavioral deficits and α-synuclein pathology in a kinase-dependent manner. Similar to other genetically engineered LRRK2 GS mice, there was no significant loss of dopaminergic neurons. These mice provide an important new tool to study neurobiological changes associated with the increased kinase activity from the LRRK2 G2019S mutation, which may ultimately lead to a better understanding of not only the physiologic actions of LRRK2, but also potential pathologic actions that underlie LRRK2 GS-associated PD.

Leucine-rich repeat kinase 2 exacerbates neuronal cytotoxicity through phosphorylation of histone deacetylase 3 and histone deacetylation.

Parkinson's disease (PD) is characterized by slow, progressive degeneration of dopaminergic neurons in the substantia nigra. The cause of neuronal death in PD is largely unknown, but several genetic loci, including leucine-rich repeat kinase 2 (LRRK2), have been identified. LRRK2 has guanosine triphosphatase (GTPase) and kinase activities, and mutations in LRRK2 are the major cause of autosomal-dominant familial PD. Histone deacetylases (HDACs) remove acetyl groups from lysine residues on histone tails, promoting transcriptional repression via condensation of chromatin. Here, we demonstrate that LRRK2 binds to and directly phosphorylates HDAC3 at Ser-424, thereby stimulating HDAC activity. Specifically, LRRK2 promoted the deacetylation of Lys-5 and Lys-12 on histone H4, causing repression of gene transcription. Moreover, LRRK2 stimulated nuclear translocation of HDAC3 via the phoshorylation of karyopherin subunit α2 and α6. HDAC3 phosphorylation and its nuclear translocation were increased in response to 6-hydroxydopamine (6-OHDA) treatment. LRRK2 also inhibited myocyte-specific enhancer factor 2D activity, which is required for neuronal survival. LRRK2 ultimately promoted 6-OHDA-induced cell death via positive modulation of HDAC3. These findings suggest that LRRK2 affects epigenetic histone modification and neuronal survival by facilitating HDAC3 activity and regulating its localization.

Clinical heterogeneity of LRRK2 p.I2012T mutation

IntroductionLeucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD). However, only few cases carrying LRRK2 mutations have been reported in Taiwanese PD patients. MethodsWe used targeted next generation sequencing (NGS), covering 24 candidate genes involved in neurodegenerative disorders, to analyze 40 probands with familial PD, and 10 patients with mixed neurodegenerative disorders. Sanger sequencing of the identified mutation in the first set of the study was performed in additional 270 PD patients, including 139 familial PD and 131 early-onset PD (onset age less than 50 years old), and 300 age/gender matched control subjects. ResultsWe found a missense variant, p.I2012T, in the LRRK2 gene in one sporadic patient having early-onset frontotemporal dementia with parkinsonism and dystonia. Sanger sequencing this substitution in additional 270 PD patients in the second set of the study revealed two additional variant carriers: one having autosomal-dominant familial PD, and one with sporadic PD. The p.I2012T substitution was absent in 300 normal control subjects. Analyzing family members of the proband with p.I2012T revealed co-segregation of the variant and parkinsonism. Clinical presentations, levodopa responses, and Tc99mTRODAT-SPECT imaging findings of this index family were similar to idiopathic PD. ConclusionsOur results revealed clinical heterogeneity of the LRRK2 p.I2012T substitution, and demonstrated the use of targeted NGS for genetic diagnosis in multiplex families with PD or mixed neurodegenerative disorders.

Differential Effects of Familial Parkinson Mutations in LRRK2 Revealed by a Systematic Analysis of Autophosphorylation

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified in pedigrees of autosomal-dominant familial Parkinson's disease (PARK8). It has been shown that the kinase activity of LRRK2 is required for its neuronal toxicity, although how familial Parkinson mutations affect the function of LRRK2 has not been well characterized. In the present study, we systematically characterized the autophosphorylation of LRRK2 by phosphopeptide mapping and identified Thr1348, Thr1349, and Thr1357 as the major autophosphorylation sites. We found that the autophosphorylation at Thr1357 is downregulated by the Y1699C mutation, possibly through a conformational alteration of the ROC domain. We also found that I2020T mutant LRRK2 undergoes excessive autophosphorylation in cell lysates in vitro at a low concentration of ATP. These results highlight the differential effects of familial mutations in LRRK2 on its conformation and enzymatic properties.

LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.

LRRK2 I2020T mutation is associated with tau pathology

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal-dominant familial Parkinson's disease (FPD). The variable pathological features of LRRK2-linked FPD include Lewy bodies, degeneration of anterior horn cells associated with axonal spheroids, neurofibrillary tangles (NFTs) and TAR DNA-binding protein of 43 kDa (TDP-43) positive inclusion bodies. Furthermore, abnormal hyperphosphorylation of microtubule associated protein tau, in part generated by catalysis of protein kinases, has been reported to be involved in progressive neurodegeneration in a number of diseases, including FPD. Thus, we examined six patients carrying the LRRK2 I2020T mutation, a pathogenic mutation associated with PARK8, and found abnormal tau phosphorylation depositions in the brainstem. Additionally, we found LRRK2 I2020T enhanced tau phosphorylation in cultured cells co-expressing LRRK2-I2020T and 3 or 4-repeated tau. This is the first report describing the relationship between hyperphosphorylation of tau and LRRK2 I2020T.

Phosphorylation of α-Synuclein Protein at Ser-129 Reduces Neuronal Dysfunction by Lowering Its Membrane Binding Property in Caenorhabditis elegans

α-Synuclein is causative for autosomal dominant familial Parkinson disease and dementia with Lewy bodies, and the phosphorylation of α-synuclein at residue Ser-129 is a key posttranslational modification detected in Parkinson disease/dementia with Lewy bodies lesions. However, the role of Ser-129 phosphorylation on the pathogenesis of Parkinson disease/dementia with Lewy bodies remains unclear. Here we investigated the neurotoxicity of Ser-129-substituted α-synuclein in the transgenic Caenorhabditis elegans (Tg worm) model of synucleinopathy. Tg worms pan-neuronally overexpressing nonphosphorylatable (S129A) α-synuclein showed severe defects including motor dysfunction, growth retardation, and synaptic abnormalities. In contrast, Tg worms expressing phosphorylation mimic (S129D) α-synuclein exhibited nearly normal phenotypes. Biochemical fractionation revealed that the level of membrane-bound α-synuclein was significantly increased in S129A-α-synuclein Tg worms, whereas S129D- as well as A30P-α-synuclein displayed lower membrane binding properties. Furthermore, A30P/S129A double mutant α-synuclein did not cause neuronal dysfunction and displayed low membrane binding property. In human neuroblastoma SH-SY5Y cells, localization of S129A-α-synuclein to membranes was significantly increased. Finally, gene expression profiling of S129A-Tg worms revealed a dramatic up-regulation of Daf-16/FOXO pathway genes, which likely act against the dysfunction caused by S129A-α-synuclein. These results imply a role of Ser-129 phosphorylation of α-synuclein in the attenuation of α-synuclein-induced neuronal dysfunction and downstream stress response by lowering the membrane binding property.

Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phospho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity.

LRRK2 Modulates Vulnerability to Mitochondrial Dysfunction in Caenorhabditis elegans

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines of Caenorhabditis elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.

Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity

α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

LRRK2 mutation in familial Parkinson’s disease in a Taiwanese population: clinical, PET, and functional studies

Pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease (PD). We performed clinical, imaging, and molecular functional studies in one family with the R1441H and six families with the G2385R variants of Lrrk2. To determine the contribution of these variants to familial PD in Taiwanese, we screened 32 Taiwanese or ethnic Chinese patients with familial PD for four pathogenic substitutions (R1441H, I2012T, I2020T, and G2019S) and one susceptibility polymorphism (G2385R). The frequencies of R1441H and G2385R were 3.7% and 22.2%, respectively. G2019S, I2012T, and I2020T were not detected. The clinical phenotypes and [(18)F]-dopa PET findings for subjects with R1441H or G2385R resembled those of patients with idiopathic PD; however, their lymphoblastoid cell lines showed increased apoptosis following exposure to a proteosome inhibitor. Thus, LRRK2 mutations are rare in Taiwanese with familial PD. Further study is needed to identify causative genes or unique biomarkers for familial PD.

Demonstration of a role for alpha-synuclein as a functional microtubule-associated protein.

Alpha-synuclein is a major constituent of pathological intracellular inclusion bodies, a common feature of several neurodegenerative diseases. Two missense mutations in the alpha-synuclein gene have been identified in confirmed autosomal-dominant familial Parkinson's disease, which segregate with the illness. However, the physiological function of alpha-synuclein remains unknown. After biochemical investigations we have revealed tubulin to be an alpha-synuclein associated/binding protein. Here, we show that alpha-synuclein induces polymerization of purified tubulin into microtubules. Mutant forms of alpha-synuclein lose this potential. The binding site of alpha-synuclein to tubulin is identified, and co-localization of alpha-synuclein with microtubules is shown in cultured cells. To our knowledge, this is the first demonstration of microtubule-polymerizing activity of alpha-synuclein. Now we can see a striking resemblance between alpha-synuclein and tau: both have the same physiological function and pathological features, making abnormal structures in diseased brains known as synucleinopathies and tauopathies. The discovery of a physiological role for alpha-synuclein may provide a new dimension in researches into the mechanisms of alpha-synuclein-associated neurodegenerative diseases.

Molecular findings in familial Parkinson disease in Spain.

Several genetic errors in alpha-synuclein (Park1) and ubiquitin carboxyl-terminal-hydrolase L1(Park5) genes cause autosomal dominant familial Parkinson disease. Mutations in the parkin gene (Park2) are the major cause of autosomal recessive Parkinson disease. To analyze the clinical and molecular data of 19 Spanish kindreds (13 with recessive, 4 with dominant, and 2 with uncertain inheritance) who have familial Parkinson disease. We searched for the previously described mutations in Park1 and Park5 genes and for new or described mutations in Park2. We used single-strand conformation polymorphism, direct sequencing, and restriction digestion of polymerase chain reaction (PCR)-amplified genomic DNA for this study. None of these families have either Park1 or Park5 mutations. We found 5 different mutations in Park2 gene in 5 of the families with recessive inheritance. To our knowledge, 2 of these mutations, V56E and C212Y, have not been previously reported. The other mutations found (deletion of exons 3 and 5 and 225delA) have been described in other ethnic groups. Heterozygous carriers of a single Park2 mutation either were asymptomatic or developed clinical symptoms in late adulthood or after brief exposure to haloperidol therapy. Mutations in Park2 gene account for 38% of the families with recessive parkinsonism in Spain. We found 2 cases of simple heterozygous Park2 mutation carriers that developed clinical symptoms, either in late adulthood or after brief exposure to parkinsonizing agents. Thus, hereditary Parkinson disease has more variable clinical phenotype and molecular defects than previously thought since heterozygous mutations could be a risk factor for parkinsonism.