Dissertations / Theses on the topic 'LRRK2 GENE'

Microbiota’s influence on human health and disease is a growing research field including neurodegenerative diseases such as Parkinson’s disease (PD). The disease symptoms involve movement disorder, manifesting tremor, rigidity, bradykinesia and instability. At the molecular level, the disease exhibits; aggregated alfa-synuclein trapped inside neurons in the brain, in so called Lewy bodies, and loss of dopaminergic neurons in substantia nigra.The working hypothesis of this project is that human microbiome composition and interactions mediate environment and lifestyle influences on disease expression of PD. To validate this hypothesis, a mouse model (C57BL/J6 mice) was used. Two knock-in mouse lines were used; one carrying the wild type, human Leucine-Rich-Repeat-Kinase 2 (LRRK2) and the second carrying the most common Caucasian LRKK2/G2019S mutant. LRRK2 is a tyrosine kinase known to interact with Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a cytosolic microbe peptide sensing receptor. To establish the tools and knowledge required for the analyses, the initial part of the project was to analyze the expression levels of LRRK2 and NOD2 in wild-type C57BL/J6 mice in specific pathogen free (SPF), and mice devoid of exposure to living microbes, so called germ-free (GF) mice. Along with this analyse, expression levels of the transgenic LRKK2 proteins in the genetically modified mice was monitored. The focus was on the following tissues: striatum, midbrain, hippocampus, small intestine and large intestine and applied immune-histochemistry (IHC) combined with Western blot analysis.Results; significantly higher expression levels of LRRK2 were observed in microbe exposed mice versus GF mice with the exception of the large intestine which showed the opposite. Moreover, NOD2 showed a trend of lower expression levels in all brain GF areas tested with the exception to striatum. For the transgenic human knock-in LRKK2 proteins, increased expression of hLRKK2 were observed in striatum and large intestine compared to G2019S. Reduced hLRKK2 expression was observed in midbrain. The results suggest a strong correlation between LRRK2 expression and the gut microbiota and a need for continued research to better understand the role our indigenous microbiome may play in onset/progression of PD.
Mikroflorans betydelse för människors hälsa och sjukdomar är ett framväxande och banbrytande forskningsfält. Forskning har inte bara visat på mikroflorans betydelse för friska tillstånd utan också för utveckling av sjukdomar, så som Parkinsons sjukdom (PD). PD är en neurodegenerativ sjukdom med symptom som innefattar rörelsestörningar; tremor, stelhet, bradykinesi och instabilitet. På molekylär nivå ses aggregerat alfa-synuclein inuti neuroner i hjärnan, i så kallade Lewy-kroppar samt förlust av dopaminerga neuroner i substantia nigra.Hypotesen som utformats i detta projekt utgick ifrån att mikroflorans sammansättning och interaktioner, medierar miljö- och livsstilsfaktorer vilket leder till utveckling av PD. För att testa hypotesen användes musmodellen C57BL / J6 i vildtyp form samt i transgen form. De transgena formerna bestod av två olika knock-in modeller; en som bär den vilda typen av humant Leucin-Rich-Repeat-Kinase 2 (hLRRK2) och en som bär den vanligaste kaukasiska mutationen av samma protein, G2019S. LRRK2 är ett tyrosin kinas som interagerar med Nucleotide-binding-oligomerization-domain-containing-protein 2 (NOD2), en cytosolisk mikrobpeptidreceptor. Analyser av LRRK2 och NOD2 utfördes på vildtypen av C57BL / J6-möss i specifikt patogenfria (SPF) förhållanden samt på möss som saknar exponering för levande mikrober, så kallade bakteriefria (GF). I de transgena mössen analyserades de genetiskt modifierade LRKK2-proteinerna, hLRRK2 och G2019S, samt NOD2 i möss i SPF förhållanden. Följande vävnader undersöktes; striatum, mellanhjärnan, hippocampus, tunntarmen och tjocktarmen med immunhistokemi (IHC) i kombination med Western blot-analys.Resultaten visade på en betydligt högre uttrycksnivå av LRRK2 i mikrobexponerade möss jämfört med GF möss med undantag för tjocktarmen där resultatet visade det motsatta. Dessutom visade resultaten en trend på lägre uttrycksnivåer av NOD2 i alla analyserade områden i hjärnan med undantag för striatum. För de transgena humana knock-in-LRKK2-proteinerna observerades ökat uttryck av LRKK2 i striatum och tjocktarm jämfört med G2019S, samt reducerat LRKK2 uttryck i mellanhjärnan. Resultaten visar på en stark korrelation mellan LRRK2-uttryck och tarmens mikroflora och implicerar förbättrad förståelse av mikroflorans roll i början och under progression av PD.

Introdução: A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum. Os sintomas motores são decorrentes da morte de neurônios dopaminérgicos da Substância Nigra mesencefálica e por inclusões intracitoplasmáticas de ?-sinucleína, os corpúsculos de Lewy (CL). A doença pode ser o resultado de fatores ambientais agindo sobre um indivíduo geneticamente susceptível. O objetivo desse estudo foi verificar a frequência de mutações no gene PARK8/LRRK2 em uma amostra de pacientes brasileiros portadores de DP e descrever as principais correlações clínicas encontradas nos pacientes com mutações. Metodologia: Estudo transversal baseado no protocolo padronizado pelo projeto LARGE-PD (Latin American Research Consortium on The Genetics of PD) aplicado em 282 pacientes com DP recrutados de ambulatórios especializados em Distúrbios do Movimento do Hospital das Clínicas de Ribeirão Preto/USP e do Hospital São Paulo/UNIFESP, entre os anos de 2007 e 2014. O material genético colhido foi enviado para Seattle, com análise genética realizada no laboratório do Dr. Cyrus Zabetian da Universidade de Washington. Resultados: Realizado pesquisa genética para o LRRK2 em 229 pacientes de 282 pacientes que preencheram o protocolo. Quatro (1,74%) pacientes foram positivos para a mutação. Nos casos de inicio precoce, a frequência foi de apenas um caso (2,43% - 1/41). Três pacientes tinham história familiar positiva para DP (3,7% - 3/81). A idade de inicio dos sintomas variou entre 38 e 55 anos. A mutação G2019S esteve presente em 1,31% (3/229). Foi encontrado também um caso de mutação para R1441C. Conclusões: O LRRK2 se mostrou um importante gene correlacionado a DP, tendo como principal mutação a G2019S. O início dos sintomas variou entre 38 e 55 anos, sempre unilateral, com boa resposta a Levodopa.
Introduction: Parkinson\'s disease (PD) is the second most common neurodegenerative disease. Motor symptoms are due to the death of dopaminergic neurons in the midbrain Substance Nigra and intracytoplasmic inclusions known as Lewy bodies (CL), rich in a protein called ?-synuclein. The disease can be the result of environmental factors acting on an individual genetically susceptible, multifactorial etiology. The aim of this study was to determine the frequency of mutations in the gene PARK8 / LRRK2 in a sample of brazilian patients with PD and describe the main clinical correlations in patients with mutations. Methodology: This is a crosssectional study based on a standardized protocol for LARGE-PD project (Latin American Research Consortium on The Genetics of PD) applied in 282 patients with PD recruited from specialized clinics in Movement Disorders seen at Hospital das Clínicas de Ribeirão Preto/USP and Hospital São Paulo/UNIFESP, between the years 2007 and 2014. The genetic material was sent to Seattle, and genetic analysis was performed in the laboratory of Dr. Cyrus Zabetian at the University of Washington. Results: Realized genetic research for LRRK2 in 229 patients of 282 patients who met the LARGE-PD protocol. Observed four (1,74%) patients positive for the mutation. In cases of early-onset, the frequency was only one case (2,43% - 1/41). Three patients had a family history of PD (3,7% - 3/81). The age of onset of symptoms in patients with mutations varied between 38 and 55 years. A total of PD patients with the DNA analyzed, G2019S was present in 1,31% (3/229). It was also found one case to mutation R1441C. Conclusions: The LRRK2 had great influence gene correlated with PD, the main mutation G2019S. The onset of symptoms varied between 38 and 55 years, always one-sided, with good response to levodopa.

The leucine-rich repeat kinase 2 mutation (LRRK2) G2019S in the kinase-domain is the most common genetic cause of late-onset autosomal dominant Parkinson’s Disease (PD), occurring in >85% of patients carrying this LRRK2 mutation. LRRK2-related PD is clinically indistinguishable from the classic idiopathic form, being characterized by classic neuropathological hallmarks such as progressive degeneration of the substantia nigra pars compacta (SNpc) dopaminergic neurons, gliosis and α-synuclein and ubiquitine-positive intraneuronal cytoplasmic inclusions. The main goal of this thesis work was to evaluate the role played by the kinase function of LRRK2 in the expression of motor phenotype and dopamine transmission in mice, since transgenic models reported so far failed to recapitulate the parkinsonian phenotype and its neuropathology. To directly explore the impact of the kinase-enhancing G2019S mutation on motor activity in vivo, a longitudinal phenotyping approach was developed. We enrolled two cohorts of G2019S knock-in (KI) mice and wild-type littermates (WT) and analyzed their motor activity at different ages (3, 6, 10, 15 and 19 months) using a set of complementary behavioral tests, specific for akinesia, bradykinesia and overall gait ability. Our study revealed that G2019S KI mice motor performance remained stable up to the age of 19 months and did not show the typical age-related decline in immobility time and stepping activity of WT. To confirm that enhanced kinase activity accounts for this phenotype, we adopted a combined genetic and pharmacological approach. On one hand we performed a parallel longitudinal study in mice carrying a LRRK2 mutation (D1994S) that impairs kinase activity (kinase-dead, D1994S KD), on the other hand we administered two LRRK2 kinase inhibitors (H-1152 and Nov-LRRK2-11) in G2019S mice. We found that i) KD mice were not phenotypic and ii) LRRK2 inhibitors reversed the hyperkinetic phenotype of G2019S KI mice, while being ineffective in WT or in D1994S KD animals. In vivo LRRK2 targeting of kinase inhibitors was further substantiated by the reduction of LRRK2 phosphorylation at Ser935 in the striatum and/or cortex at efficacious doses of LRRK2 inhibitors. In order to investigate whether the hyperkinetic phenotype of G2019S mice was associated with dysfunction of striatal dopamine neurotransmission, we carried out a series of behavioral, biochemical, and neurochemical experiments. No changes in nigral dopamine cell counts or dopamine striatal density were observed in G2019S mice. However, the overall pattern of responses to a D2/D3 receptor agonist or antagonists and to D1/D5 receptor antagonists suggested an elevated tonic activation of dopamine receptors in G2019S KI mice. Furthermore, blockade of the dopamine transporter (DAT) resulted in an enhancement of motor performance of WT but not G2019S KI mice. Results from in vitro binding assays revealed a reduction in the DAT protein levels which was associated with an increased dopamine reuptake in G2019S KI mice. In vivo microdialysis showed a reduced metabolites/dopamine ratio in in the striatum of G2019S mice, suggesting a reduced dopamine turnover. Overall the data provide genetic and pharmacological evidence that the kinase activity of LRRK2 is highly implicated in the modulation of motor activity along with the striatal dopaminergic system. However, whether and how the observed changes in motor phenotype and dopamine transmission translate into the overt parkinsonian pathology remains a matter for speculation. It is also possible that G2019S KI mice reflect a pre-symptomatic stage of the disease, as observed in other genetic models of PD. Nonetheless, the present thesis work proposes G2019S KI mice as a valuable in vivo model to investigate the effects of LRRK2 inhibitors.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Parkinsons disease (PD) is the second most common neurodegenerative disease after Alzheimers disease, affecting nearly 1% of people above 65 years of age. The major clinical symptoms of this disease are: resting tremor, bradykinesia, rigidity, postural instability and a positive response to dopamine replacement therapy. Pathological findings include selective degeneration of dopaminergic neurons within the substantia nigra, with proteinaceous Lewy body inclusions in surviving cells. The pathogenesis of PD is not yet completely understood, however, both genetic and environmental factors contribute to the disease phenotype. Mutations in the leucine-rich repeat kinase 2 gene (LRRK2; OMIM 609007) represent the most frequent genetic known cause of familial and sporadic PD. The LRRK2 gene encodes a protein, member of the ROCO protein family, that contains both GTPase (ROC) domain and kinase (MAPKKK) domain, as well as, other motifs. In this study, we have screened the main domains of the LRRK2 in a group of 204 PD Brazilian patients. The screening was performed by direct sequencing of the PCR products. By the analysis of 14 exons corresponding to ROC, COR and MAPKKK domains, we identified 31 sequence variations. The novel variants, p.C1770R and p.C2139S, may play a role in the PD pathogenesis. Three exonic alterations (p.R1398R, p.T1410M and p.Y2189C) and nine intronic variants (c.4317+16C>T, c.5317+59A>C, c.5509+20A>C, c.5509+52T>C, c.5509+122A>G, c.5657-46C>T, c.6382-36G>A, c.6382-37C>T and c.6576+44T>C) seem to be not pathogenic. A total of 17 exonic and intronic alterations were previously described in the literature as non-pathogenic polymorphisms (p.R1398H, p.K1423K, p.R1514Q, p.P1542S, c.4828-31T>C, p.G1624G, p.K1637K, p.M1646T, p.S1647T, c.5015+32A>G, c.5170+23T>A, c.5317+32C>T, p.G1819G, c.5948+48C>T, p.N2081D, p.E2108E and c.6381+30A>G). The frequency of pathogenic mutations or potentially pathogenic variants was 3.4% (including the p.G2019S mutation, previously described in our previous report: Pimentel et al., 2008; Abdalla-Carvalho et al., 2010). In familial cases (11.1%) this frequency was approximately six times higher than in sporadic cases (1.8%). Our results suggest that LRRK2 mutations have an important contribution to PD development among Brazilian population.
A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente depois da Doença de Alzheimer, afetando aproximadamente 1% da população com idade superior a 65 anos. Clinicamente, esta doença caracteriza-se pela presença de tremor em repouso, bradicinesia, rigidez muscular e instabilidade postural, os quais podem ser controlados com a administração do levodopa. As características patológicas da DP incluem a despigmentação da substância nigra devido à perda dos neurônios dopaminérgicos e a presença de inclusões proteicas denominadas corpos de Lewy nos neurônios sobreviventes. As vias moleculares envolvidas com esta patologia ainda são obscuras, porém a DP é uma doença complexa, resultante da interação entre fatores ambientais e causas genéticas. Mutações no gene leucine-rich repeat kinase 2 (LRRK2; OMIM 609007) constituem a forma mais comum de DP. Este gene codifica uma proteína, membro da família de proteínas ROCO, que possui, entre outros domínios, dois domínios funcionais GTPase (ROC) e quinase (MAPKKK). Neste estudo, os principais domínios do gene LRRK2 foram analisados em 204 pacientes brasileiros com DP por meio de sequenciamento dos produtos da PCR. Através da análise de 14 exons correspondentes aos domínios ROC, COR e MAPKKK foram identificadas 31 variantes. As alterações novas, p.C1770R e p.C2139S, possuem um potencial papel na etiologia da DP. Três alterações exônicas (p.R1398R, p.T1410M e p.Y2189C) e nove intrônicas (c.4317+16C>T, c.5317+59A>C, c.5509+20A>C, c.5509+52T>C, c.5509+122A>G, c.5657-46C>T, c.6382-36G>A, c.6382-37C>T e c.6576+44T>C) são potencialmente não patogênicas. Ao todo, dezessete variantes exônicas e intrônicas constituem polimorfismos já relatados na literatura (p.R1398H, p.K1423K, p.R1514Q, p.P1542S, c.4828-31T>C, p.G1624G, p.K1637K, p.M1646T, p.S1647T, c.5015+32A>G, c.5170+23T>A, c.5317+32C>T, p.G1819G, c.5948+48C>T, p.N2081D, p.E2108E e c.6381+30A>G). A frequência total de alterações potencialmente patogênicas ou patogênicas detectadas em nossa amostra foi de 3,4% (incluindo a mutação p.G2019S, anteriormente descrita em 2 artigos publicados por nosso grupo: Pimentel et al., 2008; Abdalla-Carvalho et al., 2010), sendo a frequência de mutações nos casos familiares (11,1%) cerca de seis vezes maior do que a encontrada nos casos isolados da DP (1,8%). Os resultados alcançados neste estudo revelam que mutações no gene LRRK2 desempenham um papel significativo como fator genético para o desenvolvimento da DP em pacientes brasileiros.

Parkinson’s disease (PD) is one of the most common long-term degenerative disorders that affect the nervous system. Clinical symptoms are bradykinesia, resting tremor and postural imbalance due to the loss of dopaminergic neurons in the substantia nigra pars compacta. Heterozygous mutations in the Leucine Rich Repeat Kinase 2 gene (LRRK2) have been identified both in familial and sporadic cases of PD. The most common variant is the p.Gly2019Ser substitution (c.6055G>A). To date there is no effective treatment available. The genome editing tool CRISPR/Cas9 has recently transformed the field of biotechnology and biomedical discovery, posing the basis for the development of innovative treatments. Using CRISPR/Cas9 technology and Homology Directed Repair, our project aims to validate gene editing as an alternative therapeutic approach for PD through the genetic correction of the pathogenic p.Gly2019Ser LRRK2 mutation restoring the wild-type sequence both in human and mouse models. Specifically, we tested various strategies, based on the CRISPR/Cas9-based genome editing technique, for the correction of LRRK2 p.Gly2019Ser (c.6055G>A) variant in primary mouse and human fibroblasts with promising results. If the correction experiments in in vitro models will confirm the good efficiency of the approach, these experiments will represent a fundamental step for the subsequent evaluation of the potential of gene therapy for the treatment of PD as well as other brain disorders for which no therapy is currently available.

Parkinson’s disease is an insidious neurodegenerative disease, for which there are no reliable pre-symptomatic biomarkers or preventative therapies. While mostly sporadic, approximately 15% of PD cases report an affected first-degree relative. The study of these patients, and those that belong to larger multi-incident families, have previously identified a number of rare genetic causes of disease. The characterisation of these genes and proteins have uncovered novel underlying molecular mechanisms involved in disease, which has facilitated the investigation of novel therapeutic interventions. Further, the study of affected and asymptomatic carriers of these mutations benefit biomarker research. To this end, this study has screened and reported on subjects with known genetic causes of disease from the Queensland Parkinson’s Project (QPP). These include two VPS35 p.D620N kindreds, eleven LRRK2 p.G2019S kindreds and one phenocopy, two SNCA duplication kindreds and four kindreds with PARK2 mutations, two of which had seemingly dominant inheritance. Additionally, this study also conducted gene hunting analyses to identify novel high impact genetic mutations that are sufficient to cause parkinsonism. Initially, a prioritisation and genetic pre-screening model was established to select families that had the highest potential to identify novel causes of disease. The model considered the number of, and the relationships between, affected members, as well as the likelihood of recruiting more family cases. Then genetic pre-screening investigated copy number variations, polymorphic nucleotide expansions as well as employing initial whole exome sequencing to identify possible causes of disease. The results from this analysis identified eleven multi-incident families suitable for next generation sequencing in multiple affected members. Whole exome sequencing was coupled with supportive evidence such as variant frequency, differential expression in an in vitro disease model and genotype analysis of other cases to identify the most likely candidates for disease. While more evidence of causality is required, the KCNJ15 p.R28C mutation was found to segregate with disease in a kindred consisting of nine affected members, as well as being identified in two additional patients, one sporadic case from Queensland, Australia, and one Italian familial case. Further, the SIPA1L1 p.R236Q mutation was identified in members from one multiincident family and one familial case. While, analysis of de novo arising mutations in an early onset case identified the novel FAM134B p.D381V mutation as a candidate for disease. Analysis of a family with multiple movement disorders identified a number of possible candidates, which require further investigation in other multiple systems atrophy, essential tremor, dystonic and PD cases. Furthermore, this study has also collated putative PD genes from the literature, and examined their segregation within the multi-incident families of the QPP. A number of mutations in these genes were identified to segregate, at least partially, with disease. However, considerable more evidence is required before their role in disease can be determined. The study of multi-incident families provides valuable insights into genetic aberrations that are sufficient to cause disease. The investigation of these monogenic forms of disease allow for the characterisation of molecular processes that may lead to disease. Indeed, the putative genetic targets identified in this study may improve our understanding of molecular pathways involved in parkinsonism, which may consequently aid in the development of novel therapies or biomarkers.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Background: Although Parkinson’s disease (PD) is commonly described as a motor disorder, non-motor symptoms and cardiovascular dysautonomia are important factors of disability and impaired quality of life in PD patients. To date, the definite correlation of cardiovascular symptoms with motor and other non-motor symptoms of PD are still largely unclear. Besides, the discovery of genetic mutations related to PD, such as LRRK2 mutations, provides better possibility to identify specific phenotypes both for motor and non-motor symptoms. Obiectives: The main objective of our study was directed to investigate the presence and the correlation of cardiovascular dysautonomia with other non-motor symptoms in PD patients, with with or without LRRK2 mutations. Finally, we performed a study of heart rate variability (HRV) and exercise stress echocardiography with the attempt to investigate dysautonomia in these groups of patients. Methods: Forty fifteen Sardinian PD patients (240 men and 165 women) were included in the study. PD patients were screened for the presence of LRRK2 mutations. Motor impairment and disability were assessed using the Hoehn & Yahr staging and the Unified PD Rating Scale (UPDRS) part-III. Cardiovascular symptoms and other non-motor symptoms were assessed with the Non-Motor Symptoms Scale (NMSS). Correlations between cardiovascular symptoms and other PD features were studied. Heart rate variability (HRV) and exercise stress echocardiography were performed in two subgroups of patients (with or without LRRK2 mutations) and in a control group, matched for age and gender. Results: Cardiovascular symptoms were among the five non-motor symptoms more reported and were correlated with multiple non motor symptoms, especially with fatigue and loss of weight. PD patients with LRRK2 mutations reported less severity of cardiovascular symptoms. HRV revealed reduced LF/HF ratio both in PD patients with or without LRKK2 mutations, with enhancement of HF in LRRK2 patients. Cardiovascular symptoms detected at NMSS correlated with LF/HF ratio. Echocardiography show a Δ Strain rate reduced in parkinsonian patients. Δ Strain rate was also correlated with the LF/HF ratio in the LRRK2 group. Discussion: The better assessment of non-motor and cardiovascular symptoms in PD patients with and without LRRK2 mutation, both with the systematic administration of symptomatic scales, such as the NMSS, and with a combined approach with HRV and exercise stress echocardiography, might lead to the detection of better monitoring systems and to the possible improvement of current strategies used in the treatment of these disabling conditions.

Inter-organelle communication is a key feature of eukaryotic cells and has been found to be fundamental in many different cellular processes. One of the best characterized interorganelle cross talk due to membrane contact sites is that between Endoplasmic Reticulum (ER) and mitochondria. Also referred to as mitochondria associated ER-membranes (MAMs) or Mitochondria-ER contact sites (MERCs), their existence was discovered 50 years ago through electron microscopic studies, but their functional significance started to emerge only in late 90s when the role of MERCs in calcium exchange from ER to mitochondria was demonstrated. Despite the importance of these contacts sites in physiology and pathology, only few proteins have so far been identifed involved in the structural maintenace of the distance between the two organelles in mammals. Mitofusin 2 (MFN2) was the first structural tether to be identified. MFN2 has been found on both OMM and ER cytosolic face and is able to form homo and heterotypic interactions with MFN1, thus tethering the two organelles. As residual juxtaposition between the two organelles is still observed in Mfn2-/- cells, additional tethering proteins have to exist. To identify them, we set out to perform two replicates of a genome wide screening in mouse embryonic fibroblasts (MEFs). In order to perform the genome wide screening, we capitalized on the FRET based biosensor, where CFP fused with FRB domain and YFP fused with FKBP domain were targeted to ER (by a Sac 1 signaling sequence) and mitochondria (by an Akap signaling sequence) respectively (Csordas et al., 2010). We modified this probe by introducing between the cDNAs of the two fluorescent proteins a self-cleaving Tav2A peptide in order to have a single mRNA construct that allows the expression of equimolar level of the proteins. FKBP and FRB binding domain are able to heterodymerize upon addition of rapamycin, thus allowing the measurement not only of the basal level of juxtaposition between the two organelles, but also of the maximum level of contacts that can occur in a cell. We called this new construct FRET ER-mitochondria probe (FEMP). FEMP unique features allow us to discriminate between proteins whose role is keeping the two organelles closer, termed as "tethers", and proteins that keep the two organelles apart, defined as "spacers". We analyzed raw images from the screen and calculated two indexes, namely basal and maximum MERC index, mirroring the level of contacts observed at any given timepoint and the maximum possible level of contacts respectively. Following automated image analysis and statistical analysis performed on ~10,000 genes, after candidate selection we identified 205 genes as ER-mitochondria tethers (i.e., genes that once ablated increase the distance between the two organelles) and 59 genes as spacers (i.e., genes that once ablated decrease the distance between the two organelles) affecting both basal and maximum MERC index in both replicates. Moreover, we identified 625 tehters and 696 spacers affecting only the basal MERC index; 519 tethers and 67 spacers affecting only the maximum MERC indexes. Protein classes analysis of these three groups of genes by Panther predicted both already known and new protein classes that are yet to explored in terms of ER-mitochondria communication. Subcellular localization analysis to identify predicted proteins to be present in both ER and outer mitochondrial membrane (OMM) of the gene lists detailed before, revealed 13 proteins among the common tethers and spacers, 30 proteins affecting only the basal MERC index and 16 proteins affecting only the maximum MERC index localized on both organelles. One of the protein present in the last group is Leucine Rich Repeat Kinase 2 (LRRK2) and we have further characterized it as ER-mitochondria tether. Subcellular fractionation experiments showed that LRKK2 localized mostly in MAMs. As expected for a tether, levels of ER-mitochondria juxtaposition, measured with FEMP, were decrased in LRRK2-/- MEF. ER-mitochondria proximity was fully restored by reintroduction in MEF LRRK2-/- of wt protein but not of the familial PD associated mutants. In conclusion, we have developed a new method to assess the proximity between ER and mitochondria and we have utilized this technology to perform two replicates of a high content screen identifying novel structural components of the ER-mitochondria contact sites.
La comunicazione tra organelli cellulari è una caratteristica fondamentale delle cellule eucariotiche ed esercita un ruolo fondamentale in molti processi cellulari. Uno dei processi di comunicazione tra organelli cellulari tra i più caratterizzati è quello dovuto ai siti di contatto tra le membrane di mitocondri e reticolo endoplasmatico (ER). Anche noti come "Mitochondria-associated ER membranes" (MAMs) o "Mitochondria-ER contact sites" (MERCs), la loro esistenza è stata scoperta 50 anni fa tramite studi di microscopia elettronica, ma il loro significato funzionale è iniziato ad emergere solo alla fine degli anni 90 quando è stato dimosdtrato il ruolo dei MERCs nello scambio di calcio dall'ER. Nonostante l'importanza di questi siti di contatto tra organelli sia in fisiologia sia in patologia, solo poche proteine coinvolte nel mantenimento strutturale della distanza tra i due organelli sono state finora identificate nei mammiferi. Mitofusina2 (MFN2) è stato il primo "tether" strutturale ad essere identificato. E' stato rilevato che MFN2 è localizzata sia nella membrana mitocondriale esterna (OMM) sia sulla superficie citosolica dell'ER ed ' in grado di formare intrazioni omo- ed eterotipiche con MFN1, mantenendo quindi la distanza tra i due organelli. Poiché una residua giustapposizione tra i due organelli è stata osservata in cellule MFN2-/-, ulteriori proteine che esercitano questo ruolo devono esistere. Per identificarle, abbiamo stabilito un protocollo ed eseguito due repliche di uno screening genomico su larga scala in fibroblasti embrionali di topo (MEF). Per eseguire questo screening, abbiamo sfruttato un biosensore basato sulla FRET, dove la proteina fluorescente CFP fusa con il dominio funzionale FRB e la proteina fluorescente YFP fusa con il dominio funzionale FKBP vengono fatte localizzare rispettivamente all'ER (grazie alla sequenza di segnale Sac1) ed ai mitocondri (grazie alla sequenza di segnale Akap1) (Csordas G. et al., 2010). Abbiamo modificato questo costrutto inserendo tra i cDNA delle due proteine il peptide autocatalitico Tav2A per ottenere un singolo mRNA e quindi l'espressione equimolare delle due proteine. I domini funzionali FKBP e FRB sono in grado di eterodimerizzare con l'aggiunta di Rapamicina, permettendo così la misurazione non solo dei livelli di giustapposizione basale tra i due organelli, ma anche del massimo livello di contatti che possono avvenire in una cellula. Abbiamo chiamato questo nuovo costrutto FRET ER-mitochondria probe (FEMP). Le caratteristiche uniche del FEMP ci consentono didiscriminare tra le proteine il cui ruolo è quello di mantenere i due organelli vicini, chiamate "tethers", e proteine che invece tengono i due organelli più distanti, definiti "spacers". Le immagini ottenute dallo screening sono state analizzate e sono stati calcolati due indici, chiamati "basal MERC index" e "maximum MERC index", che rappresentano rispettivamente il livello di contatti osservabili in qualsiasi momento in una cellula e il massimo livello di contatti possibile. A seguito di un'analisi delle immagini automatizzata e di un'analisi statistica effettutata su ~10,000 geni, dopo un processo di selezione abbiamo identificato 205 geni come "tethers" (geni che una volta eliminati aumentano la distanza tra i due organelli) tra mitocondri e ER e 59 geni come "spacers" (geni che una volta eliminati diminuiscono la distanza tra i due organelli) che influenzano sia il basal sia il maximum MERC index in entrambe le repliche. Inoltre, sono stati identificati 625 tethers e 696 spacers che influenzano solo il basal MERC index; e 519 tethers e 67 spacers che modificano solo il maximum MERC index. Analisi delle classi di proteine presenti in questi tre gruppi tramite Panther ha rivelato sia classi di proteine il cui ruolo in questo processo era noto, sia nuove classi di proteine il cui ruolo nella comunicazione tra ER e mitocondri deve ancora essere esplorato. Analisi della localizzazione cellulare per identificare proteine localizzate sia nell'ER sia nei mitocondri delle liste di geni esposte in precedenza, ha rivelato l'esistenza di 13 proteine tra i tethers e gli spacers comuni, 30 proteine che influenzano solo il basal MERC index e 16 proteine che influenzano solo il maximum MERC index localizzate in entrambi gli organelli. Una delle proteine presente nell'ultimo gruppo è "Leucine Rich Repeat Kinase 2" (LRRK2) che abbiamo ulteriormente caratterizzato come tether tra ER e mitocondri. Esperimenti di frazionamento cellulare dimostrano che LRRK2 è localizzata principalmente nelle MAMs. Come previsto per un tether, il livello di prossimità tra ER e mitocondri, misurato tramite FEMP, sono diminuiti in MEF LRRK2-/-. La prossimità tra i due organelli è pienamente recuperata dalla reintroduzione in MEF LRRK2-/- della proteina WT, ma non dei mutanti associati alle forme di Parkinson familiare. In conclusione, abbiamo sviluppato un nuovo metodo per determinare la prossimità tra ER e mitocondri e abbiamo utilizzato questa tecnologia per eseguire due repliche di uni screening gnomico su larga scala identificando nuovi componenti strutturali dei contatti tra mitocondri e ER.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente no mundo, afetando 1-2% da população acima de 65 anos, caracterizada clinicamente por tremor em repouso, bradicinesia, instabilidade postural e rigidez muscular. Essas manifestações surgem devido à degeneração neuronal progressiva e à presença de inclusões proteicas ricas em α-sinucleína. A DP é decorrente da interação entre fatores ambientais e genéticos, e entre os fatores genéticos, variantes exônicas de transmissão dominante nos genes LRRK2 (leucine-rich repeat kinase 2), VPS35 (vacuolar protein sorting 35) e EIF4G1 (eukaryotic translation initiation factor 4-gamma 1) têm sido associadas à etiologia da doença. Entretanto, estudos sobre o efeito dessas variantes na população brasileira são raros ou inexistentes. Por essa razão, neste trabalho rastreamos mutações nos genes VPS35 (p.D620N; p.R524W), EIF4G1 (p.R1205H; p.A502V) e LRRK2 (p.G2019S) em uma amostra de 582 pacientes brasileiros com DP não aparentados e 329 indivíduos controles saudáveis. Além disso, conduzimos o primeiro estudo caso-controle para análise de variantes exônicas raras (p.Q1111H, p.T1410M, p.M1646T, p.S1761R, p.Y2189C) e comuns (p.N551K, p.R1398H, p.K1423K) no gene LRRK2 em um subgrupo de 329 pacientes brasileiros com DP, não aparentados, naturais da região sudeste. Esse subgrupo foi analisado e comparado com 222 indivíduos controles saudáveis a fim de verificar associações dessas variantes e a DP. Em relação às mutações dos genes VPS35 e EIF4G1, não foram encontradas alterações nos pacientes com DP. A mutação p.G2019S no gene LRRK2 foi encontrada em 15 probandos (2,6%), dos quais 9 são do sexo feminino (64,3%). O tremor em repouso foi observado em 47,36% dos pacientes com a mutação p.G2019S como primeiro sintoma motor. As análises das variantes raras no gene LRRK2 não revelaram qualquer associação estatisticamente significante. Entre as variantes comuns, a p.K1423K mostrou evidência de associação de risco com a DP (p<0,05) na estratificação contendo o grupo de indivíduos com história familiar da doença e para as variantes p.N551K e p.R1398H não foram observadas associações. A análise do haplótipo p.N551K-p.R1398H-p.K1423K revelou associação de proteção na amostra sudeste e na estratificação Rio de Janeiro (p<0,05). Esse haplótipo não está em desequilíbrio de ligação na amostra de 222 indivíduos controles brasileiros analisados (r2≤45). Os resultados obtidos neste estudo representam contribuições valiosas ao entendimento da relação entre as variantes genéticas estudadas e o risco de desenvolvimento da doença de Parkinson, principalmente no que se refere aos endofenótipos associados.
Parkinsons disease (PD) is the second most common neurodegenerative disorder in the world, affecting 1-2% of population more than 65 years of age, clinically recognized by resting tremor, bradykinesia, postural instability and rigidity. These manifestations occur due to progressive neuronal degeneration and to the presence of protein inclusions enriched with α-synuclein. PD results from the interaction between environmental and genetic factors, and, among genetic factors, dominant exonic variants in LRRK2 (leucine-rich repeat kinase 2), VPS35 (vacuolar protein sorting 35) e EIF4G1 (eukaryotic translation initiation factor 4-gamma 1) genes have been described as causes of the disease. However, studies of the effect of these variants in Brazilian population are rare or do not exist. For this reason, in this study we decided screening mutations in VPS35 (p.D620N; p.R524W), EIF4G1 (p.R1205H; p.A502V) and LRRK2 (p.G2019S) genes in a cohort of 582 unrelated Brazilian patients with PD and 329 healthy individuals control. In additional, we carried on the first case-control study to analyze LRRK2 exonic rare (p.Q1111H, p.T1410M, p.M1646T, p.S1761R, p.Y2189C) and common (p.N551K, p.R1398H, p.K1423K) variants in a subgroup of 329 unrelated Brazilian patients with PD from Southeastern region. This group was analyzed and compared to 222 healthy individuals control in order to verify associations between these variants and PD. Regarding mutations of VPS35 and EIF4G1 genes, we have not found any alteration in Brazilian patients with PD. The mutation p.G2019S in LRRK2 gene was found in 15 probands (2.6%), 9 of them are female (64,3%). Resting tremor was observed in 47,36% of p.G2019S patients as the predominant initial symptom. Regarding the LRRK2 rare variants, the results showed no significant association. Among LRRK2 common variants, the p.K1423K showed evidence of risk association with PD (p<0,05) in the stratified analysis concerning the group of patients with family history of the disease, in contrast, p.N551K and p.R1398H variants showed no associations. The analysis of p.N551K-p.R1398H-p.K1423K revealed protection in Southeastern group and Rio de Janeiro stratification (p<0,05). This haplotype is not in disequilibrium linkage in 222 Brazilian healthy individuals control analyzed (r2≤45). Results obtained in this research represent valuable contributions for the understanding of association between the genetic variants studied and the risk of developing PD, particularly with regard to the associated endophenotypes.

碩士
國立清華大學
系統神經科學研究所
99
Leucinerichrepeat kinase 2(LRRK2) is a multifunctional domain protein, which molecular weight is 285kD.There have been identified that many mutation cause familial Parkinson disease.LRRK2 gene mutations are the most common genetic cause of Parkinson’s disease. However, little is known about the biological function of LRRK2 gene. In m ystudy, I use Drosophila as an animal model; overexpression of LRRK2 can increase Drosophila lifespan and resistance to oxidative stress. For example expression by pan neuronal driver Elav can extend lifespan and oxidative stress caused by paraquat and rotenone. Using GMR specific Gal4 driver to expression PolyQ and LRRK2 simultaneously can ameliorate 41Q induce eye degeneration, but not for 63Q and 108Q longer PolyQ induce toxicity. Moreover, I use three apoptosis related gene to examine the rescue efficiency by expression LRRK2 gene. I found that LRRK2 can reverse grim caused eye degeneration and down-size fly eyes, but there is little effect on hid or reaper induced eye abnormality. Ubiquitous expression of LRRK2 extends Drosophila lifespan and increase oxidative stress resistance. Toexamine which tissue is responsible for resistance to oxidative stress, we use Ddc(dopa-decarboxylase) Gal4 driver and its subset neuron drivers TH(dopaminergic) and TPH(serotoninergic)neurons, unlike our expectations, there is no protection on TH drive LRRK2 neurons ;however there is partially protection at lower concentration of paraquat on TPH neurons. To summarize my research, there is lifespan extension in Drosophila and oxidative stress by overexpression LRRK2 gene, and protection from 41Q induced eye degeneration. Finally expressions of LRRK2 show reduce the ability to grim cause down size of fly eyes and eye degeneration.

Millions of individuals worldwide are suffering from Parkinson‘s disease (PD) which is one of the most common neurodegenerative diseases. Clinically characterization of PD can be shown by rigidity, slow movement, rest tremor etc. On gradual progression of the disease, non-motor symptoms such as anxiety, dementia, constipation and depression can be observed. Mutations in these proteins like Parkin, α-synuclein, Leucine-Rich Repeat Kinase 2(LRRK2), PTEN-Induced Kinase (PINK1) can result in development of PD. Lewy bodies [LB] which is one of the major features of PD consists mainly of aggregates of α-synuclein. Numerous inhibitors of LRRK2 kinase are being identified, which can show protective behavior against LRRK2-induced degeneration of neurons. Thus LRRK2 kinase inhibition seems to be a potential new therapeutic agent for the treatment of PD. Natural plants and their derivatives are being utilized as an effective medication for treatment of various disorders since years as they possess many important biological activities such as anti-inflammatory, anti-oxidative, anti-cholinesterase. Desmodium gangeticum, Aloe vera, Ocimum sanctum, Moringa oliefera, Inonotus obliquus plants are being utilized as natural drugs for treatment of PD but their mechanism of action is yet not very clear. In this study we are going to study the interactions of these plants active constituents with LRRK2 kinase protein with the help of protein ligand docking. Therefore, the present study is an attempt for better understanding of the mechanism of action of these plants active constituents as well as comparative study of their effects in the treatment of PD.

碩士
國立臺灣師範大學
生命科學研究所
96
Abstract Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by resting tremor, rigidity, bradykinesia, and postural instability. The symptoms of PD in pathology are neuronal loss in the basal ganglia, especially in the substantia nigra pars compacta, and presence of intracellular Lewy body inclusions in surviving neurons. Mutations in leucine-rich repeat kinase 2 (LRRK2, encoding dardarin) are the most frequent genetic cause of PD known nowadays. Mutations in different functional regions of LRRK2 have been reported. Studies from China, Singapore, and Japan indicate that the G2385R polymorphism is an ancient and common risk factor for sporadic PD in the Asian population. In this study, we screened LRRK2 mutations in PD patients and identified a reported (R1441H) and two novel (R767H and S885N) mutations, in addition to 5 nonsynonymous and 8 synonymous amino acid substitutions. In the case-control study, the frequency of the heterozygous G2385R genotype was higher in PD compared to controls (8.4 vs. 4.2%, odds ratio = 2.08, 95% CI: 1.05-4.41, P = 0.044). Moreover, M2397T acting synergistically with G2385R to play role in PD susceptibility (4.2 vs. 0.4%, odds ratio = 10.63, 95% CI: 2.08-194.26, P = 0.024). Granulin (GRN) has been identified as one of the gene responsible for frontotemporal dementia (FTD) and mutations in GRN have been reported. Using cDNA sequencing, we found two novel mutations L57M and T487I, additionally to a synonymous D128 and a C>T variation in 3’UTR. The studies may provide a tool for clinical diagnosis and counseling.

碩士
國立臺灣師範大學
生命科學研究所
100
Mutations in leucine-rich repeat kinase 2 (LRRK2, encoding dardarin) is the most common cause of autosomal dominant PD. LRRK2 protein is expressed ubiquitously, particularly in the central nervous system, major organs and also in the lymphocytes. Previously we screened LRRK2 mutations in early-onset PD (EOPD) patients and identified two novel (R767H and S885N) mutations, in addition to 6 nonsynonymous amino acid substitutions. In this study, 21 newly recruited EOPD patients were screened using direct cDNA sequencing and one novel N2047 T>C variant was identified. PCR-RFLP analysis of the known mutation also identified a S885N mutation carrier. In addition, the results of a case-control study in a cohort of PD and ethnically matched controls revealed that G2385R GA genotype or A allele was significantly associated with the risk of PD. Finally, the EGFP- and Myc-tagged wild type, R767H and S885N LRRK2 constructs were transiently expressed in HEK-293T cells. Western blot analysis and immunofluorescence microscopy examination revealed that both wild type and mutant LRRK2 proteins exhibit similar cytoplasmic distribution and mitochondria and endoplasmic reticulum co-localization.

博士
國立臺灣大學
臨床醫學研究所
99
Background Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, with a prevalence close to 1% after age 65. Causal genes for Mendelian-inherited PD have been reported. The recent discovery of LRRK2 (Leucine-rich repeat kianse 2) as a causative PD gene has provided insights into the pathophysiology of the disease. Because the clinical phenotype of LRRK2 mutations resembles idiopathic PD, LRRK2 has emerged as the most relevant player in PD pathogenesis identified to date. Many LRRK2 gene mutations have been reported. The G2019S mutation is the hot spot mutation in Caucasians and the G2385R polymorphism is reported to be a genetic risk factor in Easten populations. Mutant Lrrk2 carrying human dominant mutations has enhanced kinase activity, resulting in cell toxicity and neurite shrinkage. Knock-down or ablation of LRRK2 in mammalian cultured neurons promotes neurite outgrowth through actin cytoskeletal rearrangment. However, the information regarding the LRRK2 mutation in the Asian population is rare and the functional relevance of these mutations (such as G2019S) or risk variant (G2385R) are unclear. In addition, very recently, aother gene for early onset-PD (PARK9/ATP13A2) was identified. The phenotype of affected individuals is juvenile onset of PD and may combine the phenotypes of dementia and pyramidal degeneration. The ATP13A2 protein is assumed to be the neuronal P-type ATPase and the intracellylar location is primarily in the lysosome. The mechanism by which loss of ATP13A2 causes parkinsonism and the possible function is unclear. To date there have been few studies examining the frequency of ATP13A2 mutations in parkinsonism, nor in different populations. The data in Asian populations are lack. Purpose We propose this research proposal with the following aims to evaluate the the frequency and functional significance of LRRK2 and ATP13A2 mutations in patients with PD in Taiwanese. 1. To determine the frequency of mutations of the ATP13A2 gene in PD patients of Taiwanese. 2. To determine the frequency of mutations of the LRRK2 gene in PD patients of Taiwanese. 3. To elucidate the functional relevance of LRRK2 genetic substitutions using lymphoblastoid cell lines derived from patients with LRRK2 substitutions. 4. Employing Drosophila da neurons as a model system, we aim to characterize the role of LRRK2 mutations in Drosophila dendrite morphogenesis by creating transgenic Drosophila models. Materials and methods We recruit more than 500 PD patients and control subjects to evaluate the frequency of ATP13A2 and LRRK2 mutations in Taiwanese populations using the method of direct sequencing in the first set of the study. In the second part, we use EBV transformed lymphoblastoid cell lines derived from patients carring LRRK2 mutations to elucidate the possible molecular functional changes. We also create transgenic Drosophila models carrying wild type LRRK2, G2019S, R1441C and G2385R to elucidate the molecular effects of these LRRK2 mutatuins in the in vivo model system. Results We identified one novel missense variant, Ala746Thr, in a single heterozygous state in three patients (1.7% in EOPD). The variant was not observed in 589 ethnicity matched controls. The frequency of this variant was significantly higher in PD cases than controls (p=0.01, relative risk 4.3, 95%CI 1.9-4.3). The clinical phenotype and 18F-dopa PET image of ATP13A2 Ala78Thr carriers are similar to that seen in idiopathic PD. The variant is located between the highly conserved phosphorylation region and the 5th transmembrane domain of the ATP13A2 protein. We also found the frequencies of R1441H and G2385R in familial PD patients were 3.7% and 22.2%, respectively. The clinical phenotypes and [18F]-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. By using Drosophila as a model system, we found that expression of G2019S mutant in Drosophila dendritic arborization neurons induces mislocalization of the axonal protein tau in dendrites and causes dendrite degeneration. G2019S-induced dendrite degeneration is suppressed by reducing the level of tau protein and aggravated by tau coexpression. Further genetic analyses suggest that G2019S and Tau function synergistically to cause microtubule fragmentation, inclusion formation and dendrite degeneration. Mechanistically, hyperactivated G2019S promotes tau phosphorylation at the T212 site by the Drosophila GSK3β homolog Shaggy (Sgg). G2019S increases the recruitment of autoactivated Sgg, thus inducing hyperphosphorylation and mislocalization of tau with resultant dendrite degeneration. Conclusions Our study not only provides a genetic epidemiology information regarding the muattaion frequency of ATP13A2 and LRRK2 in Taiwnaese PD patients but also provide a molecular and cellular mechanism in understanding the regulation of neurite degeneration by LRRK2. Our restuls will be stretched to understand the pathomechanism of LRRK2-linked PD and the transgenic LRRK2 Drosophila model could be a plateform for further drug screening.

碩士
國立臺北科技大學
生化與生醫工程研究所
101
According to the research, renal cell carcinoma and bladder cancer incidence in Taiwan are rising year by year. APTX plays a major role in single/double strand DNA repair. In recent years, APTX gene becomes a biomarker of the cancer for irinotecan chemotherapy. One of the mutation hotspot of LRRK2 is G2019S, which plays a major role in both neurodegenerative disease and cancer. In current study, we collected 8 RCC and 5 TCC cell lines. The abnormality of genes APTX, LRRK2 and MET were observed by fluorescence in situ hybridization (FISH). Q-PCR and western blot were used to observe APTX mRNA and protein expression. FISH analysis showed amplification of APTX gene, only presented in one RCC cell lines (RCC52) with four gene copies, however the rest of the cell lines showed normal gene copy number. We then further examined the mRNA expression level of APTX in all cell lines. Interestingly, the result of RCC52 cell line remained consistence with high expression of RNA when compared to the normal renal cells, on the other hand, other cell lines presented lower expression of APTX gene while compared to the normal renal cell. The result of protein level shows different expression of Aprataxin in clear cell subtype. We suggest that APTX gene most likely play as the function of tumor suppressor gene in RCCs. Once a mutation occurred on this gene sequence, it might indirectly trigger tumoral formation. Although LRRK2 gene copy number was normal, we suggest that mutation of gene LRRK2 can be identified by sequencing.

La maladie de Parkinson (MP) est une affection neurodégénérative invalidante et incurable. Il est maintenant clairement établi que d’importants déterminants génétiques prédisposent à son apparition. La recherche génétique sur des formes familiales de la MP a mené à la découverte d’un minimum de six gènes causatifs (SNCA, LRRK2, Parkin, PINK1, DJ-1 and GBA) et certains, par exemple LRRK2, contiennent des variations génétiques qui prédisposent également aux formes sporadiques. La caractérisation des protéines codées par ces gènes a mené à une meilleure compréhension des mécanismes moléculaires sousjacents. Toutefois, en dépit de ces efforts, les causes menant à l’apparition de la MP restent inconnues pour la majorité des patients. L’objectif général des présents travaux était d’identifier des mutations prédisposant à la MP dans la population canadienne-française du Québec à partir d’une cohorte composée principalement de patients sporadiques. Le premier volet de ce projet consistait à déterminer la présence de mutations de LRRK2 dans notre cohorte en séquençant directement les exons contenant la majorité des mutations pathogéniques et en effectuant une étude d’association. Nous n’avons identifié aucune mutation et l’étude d’association s’est avérée négative, suggérant ainsi que LRRK2 n’est pas une cause significative de la MP dans la population canadienne-française. La deuxième partie du projet avait pour objectif d’identifier de nouveaux gènes causatifs en séquençant directement des gènes candidats choisis à cause de leurs implications dans différents mécanismes moléculaires sous-tendant la MP. Notre hypothèse de recherche était basée sur l’idée que la MP est principalement due à des mutations individuellement rares dans un grand nombre de gènes différents. Nous avons identifié des mutations rares dans les gènes PICK1 et MFN1. Le premier code pour une protéine impliquée dans la régulation de la transmission du glutamate tandis que le second est un des acteurs-clés du processus de fusion mitochondriale. Nos résultats, qui devront être répliqués, suggèrent que le séquençage à grande échelle pourrait être une méthode prometteuse d’élucidation des facteurs de prédisposition génétiques à la MP ; ils soulignent l’intérêt d’utiliser une population fondatrice comme les canadiens-français pour ce type d’étude et devraient permettre d’approfondir les connaissances sur la pathogénèse moléculaire de la MP.
Parkinson’s disease (PD) is a complex neurological disorder with significant genetic predisposing factors which are extremely heterogeneous. Investigations of familial forms of the disorder revealed causative mutations in six different genes, namely SNCA, LRRK2, Parkin, PINK1, DJ-1 and GBA, and functional analyses of these gene products pinpointed dysfunction of key molecular pathways involved in the neurodegenerative process of the disorder. Further sequencing and genome-wide association studies indicated that some of these genes, including LRRK2, also contain variants predisposing to sporadic forms of PD. Despite these significant breakthroughs, the vast majority of PD genetic predisposing factors remain unknown. Our goal was to identify mutations predisposing to PD in the French Canadian (FC) population from a cohort mostly composed of late-onset sporadic cases. We therefore sequenced the two exons of LRRK2 that contain most of the pathogenic mutations and we performed a case-control association study. Sequencing analysis did not reveal any reported or novel mutations and the case-control association study did not provide any positive signal, thus indicating that common variants in LRRK2 are unlikely to be a significant cause of late-onset PD in the FC population. Because of the allelic and non-allelic genetic heterogeneity observed for PD, we hypothesized that dozens of genes may carry rare pathogenic mutations. The second part of this research project was therefore aimed at identifying new PD causative genes by direct sequencing of genes functionally associated with the known causative gene pathways. Our screening uncovered several rare mutations likely pathogenic in the PICK1 and the MFN1 genes. PICK1 is involved in internalization of AMPA receptors whereas MFN1 is one of the core components of the mitochondrial fusion/fission machinery. Although these observations will need to be replicated, our findings support the previously suspected pathogenic role for glutamate excitotoxicity and imbalanced mitochondrial dynamics in Parkinson’s disease. They further emphasize the value of inbred populations in genetic studies of PD and provide new clues to the molecular pathogenesis of the disorder.

Mutations in SNCA, LRRK2, PINK and DJ1 plays a very important part in pathological process of Parkinson’s disease therefore SNPs associated with these genes were picked for detailed examination of their unfavourable effects on human body. SNPs were taken from NCBI dbSNP and only missense and mutations with unknown significance were taken into consideration. To study the deleterious effect of these SNPs we followed sequence specific and sequence-structure specific methods in order to provide more accurate results. SIFT, PolyPhen2, SNP & Go and iMutant3.0 were used for detection of deleterious SNPs and MD simulations were performed using NAMD to validate the results. The study suggested that V1598E and P2119L of LRRK2 gene could indirectly or directly affect the Hydrogen bonding pattern and destabilize the amino acid interactions of gene to certain extent.