Dissertations / Theses on the topic 'SCA28'

Autosomal dominant spinocerebellar ataxias (SCAs) are genetically heterogeneous neurological disorders characterized by cerebellar dysfunction mostly due to Purkinje cell degeneration. Here we show that AFG3L2 mutations cause SCA type 28. Along with paraplegin, which causes recessive spastic paraplegia, AFG3L2 is a component of the conserved m-AAA metalloprotease complex involved in the maintenance of the mitochondrial proteome. We identified heterozygous missense mutations in five unrelated SCA families and found that AFG3L2 is highly and selectively expressed in human cerebellar Purkinje cells. m-AAA–deficient yeast cells expressing human mutated AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment and deficiency of respiratory chain complex IV. Structure homology modeling indicates that the mutations may affect AFG3L2 substrate handling. This work identifies AFG3L2 as a novel cause of dominant neurodegenerative disease and indicates a previously unknown role for this component of the mitochondrial protein quality control machinery in protecting the human cerebellum against neurodegeneration.

Autosomal dominant spinocerebellar ataxias (SCA) are a heterogeneous group of neurological disorders characterized by cerebellar dysfunction. We recently showed that AFG3L2 mutations cause dominant ataxia SCA28. AFG3L2 and its partner protein paraplegin, which causes recessive spastic paraparesis SPG7, are components of the m-AAA complex, involved in mitochondrial protein quality control. Since yeast functional studies showed that paraplegin coexpression can modulate AFG3L2 mutations, we investigated the possible coinheritance of AFG3L2 and SPG7 mutations in patients with spinocerebellar syndromes. We identified 3 probands with heterozygous mutations in both the AFG3L2 and the SPG7 genes. Two ataxic patients carry an AFG3L2 mutation affecting highly conserved amino acids located in the ATPase or in the proteolytic domains of the protein along with the parapleginA510V. The third proband carries a de novo AFG3L2 mutation in the highly conserved SRH region of the ATPase domain along with the inherited deletion of SPG7 exons 4-6. The clinical presentation of this patient is characterized by early onset optic atrophy and a L-dopa-responsive spastic-ataxic syndrome with extrapyramidal signs. A muscle biopsy revealed an isolated complex I deficiency. Moreover, evaluation of substrates processing in patient’s fibroblasts showed abnormal processing pattern of OPA1. In conclusion, our data indicate that the presence of a loss-of-function mutation in paraplegin may act as a disease modifier for heterozygous AFG3L2 mutations. Concurrent mutations in both components of the mitochondrial m-AAA complex may result in a complex phenotype, thus expanding the clinical spectrum of AFG3L2-associated mutations. Moreover, biochemical and cell biology studies revealed a crucial role of the m-AAA complex in the processing of OPA1 and the maintenance of mitochondrial morphology.

Des analyses d'ADN sur les restes des soldats de la Grande Armée de Napoléon (1812) ont révélé la présence entre autre de Rickettsia prowazekii. Pourtant la rickettsiologie ne commençera qu'avec les travaux de Ricketts et von Prowazek en 1910, et ne cessera de s'alimenter d'espèces et de pathologies nouvelles. En tant que premières bactéries intracellulaires strictes décrites, la taxonomie des rickettsies rassemblait initialement sur la base de ce critère, un grand nombre de genres bactériens ultérieurement reclassés avec l'avènement du séquençage et la découverte d'horloges moléculaires telle que la sous-unité 16S de ARN ribosomique ou le cytochrome C. Pour l'identification des espèces de Rickettsia, de nombreux critères phénotypiques dont la morphologie, les tests de fixation du complément, de neutralisation de toxines, de sérotypage et les profils protéiques ont longtemps été utilisés. Mais c'est la comparaison des séquences de gènes, dont ompA, ompB et sca4, qui ont permis d'identifier très précisément les espèces du genre Rickettsia et de proposer une classification phylogénique fiable. Cependant, la position phylogénique d'espèces telles que Rickettsia helvetica, Rickettsia canadensis et Rickettsia bellii n'a pu être déterminée avec certitude. Aussi, l'analyse basée sur la concaténation de plusieurs gènes, associée aux caractères phénotypiques peut constituer une meilleure alternative
The history of rickettsioses is probably as ancient as human civilisation. The first documented cases of rickettsioses dates back to 1812. In early part of the last century (1910) Ricketts and von Prowazek laid the foundation of modern rickettsiology. Their pioneering works eventually led to the recognition of new species and Rickettsiales infections. As soon as Rickettsia are the first strictly intracellular bacteria described, its taxonomy gathered on the basis of this criterion, and a great number of kinds of bacteria which will be identified only with the advent of the sequencing and the discovery of molecular clocks such as ribosomal 16S RNA and cytochrome C. Many phenotypic criterion such as morphology, tests of complement, neutralization of toxins, mousse serotyping and SDS-page proved reliable. However, gene comparison (ompA, ompB and sca4) will make it possible to very precisely determine the species containing of the genus Rickettsia and to suggest a classification supported by high bootstrap values as well as antibiotics tests. Nevertheless, the phylogenetic position of species such Rickettsia helvetica, Rickettsia canadensis and Rickettsia bellii could not be given with precision, and the polyphasic analysis of the classification of the Rickettsia species based on genes concatenation associated with phenotypic characters available might be alternatives for Rickettsia phylogeny

Objectifs: D’une part étudier les effets de la SCP à moyen et long terme chez les patients de notre centre ayant une forme génétique de MP, d’autre part effectuer une étude clinique, génétique et transcriptomique d’un groupe de patients parkinsoniens ayant une mutation du gène SCA2 (dits patients SCA2).Méthode: 1/ Effet de la SCP chez des patients ayant une forme génétique de MP: cinq patients ayant une forme génétique de MP, appartenant à une cohorte de 52 patients parkinsoniens ayant bénéficié d’une stimulation à haute fréquence du noyau sous-thalamique entre 1998 et 2000, ont été examinés avant la chirurgie puis à 1 et 5 ans. Les patients ont été évalués avec et sans L-dopa par plusieurs échelles: UPDRS II et III,dyskinésies, Schwab et England, Mattis et MADRS. Les résultats ont été comparés aux patients de la même cohorte ayant une forme sporadique de MP.2/ Etude des patients parkinsoniens SCA2 : la description clinique est rapportée rétrospectivement. Les études génétique et transcriptomique ont été effectuées chez 7 patients parkinsoniens et 8 patients cérébelleux SCA2, sur des cellules mononuclées sanguines. Le séquençage de l’ADN a permis de déterminer la longueur de la répétition de triplets CAG et d’identifier les interruptions par des triplets CAA. Le transcriptome de ces patients ainsi que de 13 témoins (sujets sains appariés sur le sexe et l’âge) a été réalisé sur deux plateformes de puces à ADN (Agilent et Illumina). L’analyse de l’expression des gènes chez les patients parkinsoniens et cérébelleux comparés à leurs contrôles respectifs a été réalisée avec le logiciel Genespring GX. Les gènes ayant une expression significativement différente (variation d’expression >1,3 et Welch t-test p< 0,05) ont été analysés à l’aide du logiciel Ingenuity Pathways Analysis qui identifie les voies canoniques significativement dérégulées.Résultats: 1/ Effet de la SCP chez des patients ayant une forme génétique de MP: les résultats de l’ensemble des parkinsoniens étaient comparables à la littérature. Les mouvements involontaires compliquant la dopathérapie s’amélioraient au cours du temps. Les patients ayant une forme génétique bénéficiaient d’un meilleur résultat que les autres parkinsoniens sur les signes dopasensibles et sur les complications dopa-induites.2/ Etude des patients parkinsoniens SCA2 : cliniquement, la MP était tout à fait classique, l’âge moyen de début était de 55,2 ans, tous les patients étaient dopasensibles et les complications typiques de la MP étaient constatées. Le séquençage de l’ADN a montré des expansions légèrement plus longues chez les patients cérébelleux (37-41 triplets) que chez les patients parkinsoniens (35-39). Les patients cérébelleux n’avaient pas d’interruptions CAA sur leur allèle muté. Tous les patients parkinsoniens avaient en revanche une séquence d’interruptions CAA inhabituelle. Pour ce qui concerne l’étude transcriptomique, nous avons constaté chez les patients cérébelleux et chez les parkinsoniens la dérégulation de l’expression de gènes connus pour interagir avec l’ataxine 2 (DDX6, PABP, gènes de la voie du métabolisme des inositol phosphates), ainsi que de gènes impliqués dans le métabolisme du cancer et dans l’immunité. Les patients parkinsoniens avaient un dérèglement des voies de signalisation de la sclérose latérale amyotrophique, du VEGF et de HIF1. Chez ces patients, l’expression de SNCA était diminuée, y compris chez les patients les moinsVIIsymptomatiques, alors qu’elle ne l’était pas chez les cérébelleux. Chez les patients cérébelleux, plusieurs voies concernant le métabolisme des ARN étaient dérégulées, ainsi que le métabolisme du phosphate inositol. Plusieurs voies canoniques impliquant l’apoptose étaient dans les 2 groupes de patients, avec une expression de gènes pro- et antiapoptotiques en faveur de l’apoptose chez les cérébelleux et en sa défaveur chez les parkinsoniens
Objectives: First, to study the mid- and long term effects of DBS in patients with a genetic form of PD from our clinic, and second, to achieve a clinical, genetic and transcriptomic study of a group of parkinsonian patients bearing a mutation in the SCA2 gene (so called SCA2 patients)Methods: 1/ Effects of DBS in patients with a genetic form of PD: five patients with a genetic form of PD, belonging to a cohort of 52 PD patients who underwent a subthalamic nucleus high frequency stimulation between 1998 and 2000, were evaluated before surgery and then after 1 and 5 years with and without L-dopa, using several scales: UPDRS II and III, dyskinesia, Schwab and England, Mattis and MADRS. The results were compared with the patients of the same cohort having a sporadic form of PD. 2/ Study of the parkinsonian SCA2 patients: the clinical picture is related retrospectively. Genetic and transcriptomic studies were performed on blood mononuclear cells from 7 parkinsonian and 8 cerebellar SCA2 patients. DNA sequencing allowed to determine the length of the CAG triplets repeat and to identify the interruptions by CAA triplets. Transcriptomes of these patients and of 13 matched controls (healthy subjects paired according to gender and age) were profiled using 2 platforms of whole human genome expression micro-arrays (Agilent and Illumina). Analyses of differential expression in cerebellar and parkinsonian patients vs their respective controls were performed with GeneSpring GX software. Genes with significant differences (fold change >1.3 and Welch t-test p< 0.05) were analyzed using Ingenuity Pathway Analysis software which identified significantly deregulated canonical pathways.Results: 1/ Effects of DBS in patients with a genetic form of PD: the results concerning the whole cohort of PD patients were similar to the literature. L-dopa-induced involuntary movements improved over time. Patients with a genetic form of PD had a best result than other patients on dopa-responsive signs and dopa-induced complications.2/ study of the parkinsonian SCA2 patients: clinical features were very typical of PD, with a mean age of onset of 55.2 years, a good L-dopa responsiveness, and classical complications of PD. DNA sequencing showed slightly longer expansions in cerebellar (37-41 triplets) than in parkinsonian patients (35-39). Cerebellar patients had no CAA interruption on their mutated allele. All parkinsonian patients had an unusual pattern of CAA interruptions. Concerning the transcriptomic study, cerebellar and parkinsonian patients had a deregulation in the expression of genes known to interact with ataxin-2 (DDX6, PABP, genes in the inositol phosphates metabolism pathway), as well as genes involved in the metabolism of cancer and in immunity. Parkinsonian patients had a deregulation of amyotrophic lateral sclerosis, VEGF and HIF1 signaling pathways. In these patients, including the least symptomatic ones, SNCA expression was down-regulated, whereas it was not in cerebellar patients. In cerebellar patients, several pathways concerning the metabolism or RNAs were deregulated, as well as p53 signaling. Several canonical pathways involving apoptosis were deregulated in both groups of patients, with an expression of pro- and antiapoptotic genes in favor of apoptosis in cerebellar patients and going against apoptosis in parkinsonian patients.

As ataxias espinocerebelares (SCAs) são doenças neurodegenerativas com herança autossômica dominante que apresentam grande heterogeneidade clínica e genética. O diagnóstico é realizado pela detecção da mutação no gene causador, que, na sua maioria, é uma expansão de repetições trinucleotídicas CAG. O objetivo deste estudo foi analisar os polimorfismos de repetições trinucleotídicas nos genes associados as SCAs tipo 1, tipo 2, tipo 3 e tipo 6 através de PCR-multiplex e eletroforese capilar, visando a melhoria do diagnóstico molecular e a determinação da distribuição das regiões polimórficas nos alelos normais. As análises foram realizadas em 124 pacientes não-aparentados que apresentavam sintomas de ataxia. Nessa amostra, foram identificados 10 pacientes com SCA2, 39 pacientes com SCA3 e 2 pacientes com SCA6. Não encontramos amostras com uma expansão CAG no gene SCA1. Os polimorfismos de cada loci foram estudados nos cromossomos normais desses pacientes (n=209-248). A freqüência dos alelos normais grandes no locus SCA1 (>32 repetições CAG) foi estabelecida em 0,05 e no locus SCA2 (>22 repetições CAG) foi 0,11, enquanto que no locus SCA3 (alelos >28 repetições) a freqüência foi 0,11. A freqüência de alelos normais grandes para o locus SCA6 (>13 repetições) foi 0,04. Concluindo, este estudo proporcionou a primeira análise detalhada da distribuição de repetições CAG nos loci SCA1, SCA2, SCA3 e SCA6 por amplificação multiplex e eletroforese capilar em pacientes brasileiros. A freqüência dos alelos normais grandes nos genes SCA3 e SCA6 nessa amostra reflete a prevalência destas duas doenças na nossa população, concordando com a hipótese que alelos patogênicos podem ser originados pela expansão de alelos normais grandes.
Spinocerebellar ataxias (SCAs) are neurodegenerative disorders inherited as an autosomal dominant trait that present large genetic and clinical heterogeneity. An accurate diagnosis relies on mutation detection in a specific causative gene, which is typically an abnormal number of CAG trinucleotide repeats. The aim of this study was to analyze polymorphic regions of trinucleotide repeats in SCA1, SCA2, SCA3, and SCA6 associated genes through multiplex PCR and capillary electrophoresis, aiming the improvement of molecular diagnosis and distribution of CAG repeats number in normal alleles. Analyses were carried out in 124 unrelated Brazilian patients who presented symptoms of progressive ataxia. To date, we identified 10 patients with SCA2, 39 patients with SCA3, and 2 patients with SCA6. No alleles were identified with a CAG expansion tract in the SCA1 gene. Normal CAG repeats length range was established using data from normal chromosomes (n=209-248). Frequency of large normal alleles in SCA1 locus (>32 CAG repeats) was determined to be 0.05. Frequency of large normal alleles at the SCA2 locus (>22 CAG repeats) was shown to be 0.11 while at the SCA3 locus (>28 CAG repeats) frequency of large normal alleles was 0.11. At the SCA6 locus, frequency of large normal alleles (>13 CAG repeats) was found to be 0.04. Moreover, this study provides the first detailed analysis, to our knowledge, of the distribution of CAG repeats at the SCA1, SCA2, SCA3, and SCA6 loci by multiplex-PCR and automated capillary electrophoresis in Brazilian patients. Frequency of large normal alleles in SCA3 and SCA6 genes established in this sample reflects the prevalence of these two diseases in our population, supporting the hypothesis that disease alleles emerge from expansion of large normal alleles.

A doença de Parkinson é freqüente no mundo todo, atingindo indivíduos de todas as idades e etnias. Embora comum e muito estudada, seus mecanismos causais ainda não são plenamente conhecidos e ainda não há tratamento curativo. Atualmente, são conhecidos alguns fatores ambientais e genéticos associados ao desenvolvimento da doença de Parkinson. Dentre os fatores genéticos, foram identificados diversos genes que podem, ou determinar a ocorrência da doença de forma mendeliana (genes principais), ou apenas aumentar o risco de seu surgimento (genes de suscetibilidade). Embora os fatores genéticos, em conjunto, sejam responsáveis por uma minoria dos casos, permanece relevante esta investigação, para promover um aconselhamento genético adequado para os portadores de formas mendelianas, para adequar medidas de tratamento e reconhecer características clínicas e de prognóstico, oportunizando, inclusive, ampliar o entendimento dessa condição. O presente estudo analisou pacientes portadores de doença de Parkinson em acompanhamento no Hospital de Clínicas de Porto Alegre, que apresentavam baixa idade de início dos sintomas, história familiar positiva ou presença de manifestações atípicas da doença. Essas características foram utilizadas como critério de seleção dos pacientes por estarem associadas com maior probabilidade de detecção de causas genéticas. Os pacientes foram submetidos à avaliação clínica e à testagem molecular para diversos genes principais e de suscetibilidade. Foram, posteriormente, comparadas as características clínicas dos pacientes positivos com relação aos demais pacientes estudados. Os resultados são apresentados sob a forma de três artigos, que descrevem, respectivamente, os achados moleculares da investigação para os genes causais autossômicos recessivos (genes PARK2, PARK6, PARK7 e PARK8), autossômicos dominantes (genes SCA1, SCA2, SCA3, SCA6 e SCA7) e o gene de suscetibilidade (gene GBA).

Objectifs: D'une part étudier les effets de la SCP à moyen et long terme chez les patients de notre centre ayant une forme génétique de MP, d'autre part effectuer une étude clinique, génétique et transcriptomique d'un groupe de patients parkinsoniens ayant une mutation du gène SCA2 (dits patients SCA2).Méthode: 1/ Effet de la SCP chez des patients ayant une forme génétique de MP: cinq patients ayant une forme génétique de MP, appartenant à une cohorte de 52 patients parkinsoniens ayant bénéficié d'une stimulation à haute fréquence du noyau sous-thalamique entre 1998 et 2000, ont été examinés avant la chirurgie puis à 1 et 5 ans. Les patients ont été évalués avec et sans L-dopa par plusieurs échelles: UPDRS II et III,dyskinésies, Schwab et England, Mattis et MADRS. Les résultats ont été comparés aux patients de la même cohorte ayant une forme sporadique de MP.2/ Etude des patients parkinsoniens SCA2 : la description clinique est rapportée rétrospectivement. Les études génétique et transcriptomique ont été effectuées chez 7 patients parkinsoniens et 8 patients cérébelleux SCA2, sur des cellules mononuclées sanguines. Le séquençage de l'ADN a permis de déterminer la longueur de la répétition de triplets CAG et d'identifier les interruptions par des triplets CAA. Le transcriptome de ces patients ainsi que de 13 témoins (sujets sains appariés sur le sexe et l'âge) a été réalisé sur deux plateformes de puces à ADN (Agilent et Illumina). L'analyse de l'expression des gènes chez les patients parkinsoniens et cérébelleux comparés à leurs contrôles respectifs a été réalisée avec le logiciel Genespring GX. Les gènes ayant une expression significativement différente (variation d'expression >1,3 et Welch t-test p< 0,05) ont été analysés à l'aide du logiciel Ingenuity Pathways Analysis qui identifie les voies canoniques significativement dérégulées.Résultats: 1/ Effet de la SCP chez des patients ayant une forme génétique de MP: les résultats de l'ensemble des parkinsoniens étaient comparables à la littérature. Les mouvements involontaires compliquant la dopathérapie s'amélioraient au cours du temps. Les patients ayant une forme génétique bénéficiaient d'un meilleur résultat que les autres parkinsoniens sur les signes dopasensibles et sur les complications dopa-induites.2/ Etude des patients parkinsoniens SCA2 : cliniquement, la MP était tout à fait classique, l'âge moyen de début était de 55,2 ans, tous les patients étaient dopasensibles et les complications typiques de la MP étaient constatées. Le séquençage de l'ADN a montré des expansions légèrement plus longues chez les patients cérébelleux (37-41 triplets) que chez les patients parkinsoniens (35-39). Les patients cérébelleux n'avaient pas d'interruptions CAA sur leur allèle muté. Tous les patients parkinsoniens avaient en revanche une séquence d'interruptions CAA inhabituelle. Pour ce qui concerne l'étude transcriptomique, nous avons constaté chez les patients cérébelleux et chez les parkinsoniens la dérégulation de l'expression de gènes connus pour interagir avec l'ataxine 2 (DDX6, PABP, gènes de la voie du métabolisme des inositol phosphates), ainsi que de gènes impliqués dans le métabolisme du cancer et dans l'immunité. Les patients parkinsoniens avaient un dérèglement des voies de signalisation de la sclérose latérale amyotrophique, du VEGF et de HIF1. Chez ces patients, l'expression de SNCA était diminuée, y compris chez les patients les moinsVIIsymptomatiques, alors qu'elle ne l'était pas chez les cérébelleux. Chez les patients cérébelleux, plusieurs voies concernant le métabolisme des ARN étaient dérégulées, ainsi que le métabolisme du phosphate inositol. Plusieurs voies canoniques impliquant l'apoptose étaient dans les 2 groupes de patients, avec une expression de gènes pro- et antiapoptotiques en faveur de l'apoptose chez les cérébelleux et en sa défaveur chez les parkinsoniens.

碩士
國立臺灣師範大學
生命科學研究所
98
Spinocerebellar ataxia type 8 (SCA8) is an autosomal dominant late-onset neurodegenerative disease. The cause of SCA8 was originally proposed associated with CTG trinucleotide repeat at 3’UTR (untranslated region) of ATXN8OS gene, lying on chromosome 13q21. However, recent studies suggest that bidirectional transcription of ATXN8OS occurs, with its anti-strand, ataxin8 (ATXN8), which encodes a polyQ protein in the CAG orientation, and ATXN8OS transcribed into potentially pathogenic CUG transcripts. SCA8 may thus has both RNA and protein gain of function mechanisms. To understand the molecular pathogenic mechanism of SCA8, we have established inducible PC12 cells with ATXN8OS-22R (normal 22 CTG repeats) or -150R (expanded 150 CTG repeats). Our results show that the viability and neurite outgrowth were significantly reduced in cells with ATXN8OS-150R after induction. Furthermore, the proliferation rate of the cells with ATXN8OS-150R (clones 1 and 4) was obviously decreased by flow cytometry. In addition, the presence of RNA foci was identified in cells with ATXN8OS-22R and -150R. Further investigation in impairment of neurite outgrowth revealed that the neuron differenciation signal transduction pathways of PC12 cells might not be the major targets directly affected by ATXN8OS gene. Whether the mechanism of ATXN8OS gene resulted in hypoplasia of neurite outgrowth related to RNA level associated with alternative splicing needs further investigation.

碩士
國立臺灣師範大學
生命科學研究所
93
Abstract The spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders characterized by cerebellar dysfunction alone or in combination with other neurological abnormalities. SCA type 8 (SCA8) has been attributed to the expanded CTG repeat at 3’ end of SCA8 gene on chromosome 13q21. To unravel the pathogenic mechanisms underlying SCA8 we tried to establish SCA8 Drosophila models in this study. The photoreceptor cells were degenerated when SCA8 gene with expanded CTG repeats were expressed in the transgenic flies, suggesting the repeat sequence exhibits pathogenic effect. The repeat containing transcripts were found accumulated in nuclei as RNA foci using in situ hybridization. As sequence analysis of SCA8 gene did not reveal that SCA8 encodes any significant ORFs in previous study, suggesting that SCA8 will not encode any protein product. Nevertheless, expression constructs with EGFP fused at 3’ end of SCA8 were able to express in the eye discs of transgenic flies, suggesting that SCA8 may contain translatable ORF. Expressing the repeat containing ORF in transgenic flies cause severe degenerative phenotype. However the transcripts were aggregated in RNA foci and can not be transported to cytosol to be translated into polyLeu containing polypeptide. From these results we have learned that SCA8 can exert its cytotoxcity effect at RNA level. Previous studies demonstrated that RNA binding proteins, such as muslcle-blind and PKAAP, were associated with CUG containing transcripts to form RNA foci, which sequesters the expression level of these RNA binding proteins and enhance the pathogenic effect. We found our SCA8 fly model displayed different degenerative phenotype when placed at mbl, and PKAAP mutant background. Interestingly, the chaperone protein, Hsp70, was found alleviated SCA8 disease presentation. It is very likely that mis-folded proteins may also play a role in SCA8 pathogenesis. Since 5’ end of SCA8 is overlapped with a nearby gene KLHL1, we would like to know whether KLHL1 is also play a role in SCA8 pathogenesis. Retinal expression of KLHL1 did not cause obviously eye degeneration. Co-expression of both SCA8 and KLHL1 did not enhance the disease phenotype. This has demonstrated that KLHL1 may not participate in the pathogenesis of SCA8.

碩士
國立臺灣師範大學
生命科學研究所
95
Spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders characterized by cerebellar dysfunction alone or in combination with other neurological abnormalities. More than 28 SCA types have been described. Among them, SCA2 and SCA17 were caused by the expansions of coded CAG trinucleotide repeats and SCA14 cause by mutations in the protein kinase Cγ. We examined the CAG repeat range of SCA2 Ataxin-2 gene in 179 normal controls and in patients with various neurodegenerative diseases, including 15 ataxia, 137 Parkinson's disease, 124 dementia, 84 essential tremor, and 41 chorea and dystonia. No SCA2 expanded allele was found. In the screening of SCA14 PRKCG gene mutation, we sequenced the exons 4 and 5-containing DNA fragment from 30 patients with parkinsonism and again no mutation was found. To study the protein-protein interaction between HMGB1 and TBP, GST fused HMGB1, nTBP-Q20/Q45/Q61 (N-terminal TBP) and fTBP-Q20/Q45/Q61 (full-length TBP) were overexpressed in E. coli BL21 cells and purified by affinity chromatography. Quartz Crystal Microbalance (QCM) was used to study the interactions between HMGB1 and TBP protein carrying 20, 45 or 61 polyQ track. While the interaction between HMGB1 and full-length or N-terminal TBP was similar, the length of polyQ track affects the binding of HMGB1 to TBP, with strongest binding of nTBP-Q45 (ΔHz = 13.0) and weakest binding of nTBP-Q61 (ΔHz = 4.3).

碩士
國立海洋大學
食品科學系
90
Abstract Neisseria sp. strain SCA38 was isolated from the biofouling material suspended in sea water. Four metal ions or three buffering agents was added individually into the MSBB to testing the EAS production of strain SCA38, the results indicated that the EAS yields were increased to the range of 0.13-0.28 g/L. And as yeast extract or polypeptone/yeast extract was used to replace the original nitrogen source, the EAS yields of strain SCA38 were higher than the inorganic nitrogen replacement groups. Five monosaccharide and three disaccharides were used individually to replace the carbon source of MSBB, the results indicated that the substitution of mannose in the MSBB performed higher EAS yield (0.81 g/L) than others. In the study of incubation conditions for the maximum EAS production of strain SCA38, the results showed that while the initial pH at 8.2, shaking speed with 150 rpm, or incubation temperature at 26oC the higher EAS yields ranged from 0.71 to 0.83 g/L were observed. Strain SCA38 was cultured in the M-MPB-G with the addition of substratum such as actived carbon flake, chitin flake, wood piece, stainless steel beads, glass beads, or polypropylene beads. The EAS production results indicated that the chitin flake added to M-MPB-G performed the highest EAS yield as 0.98 ± 0.27 g/L. To test the different agitating speed on the EAS yield for strain SCA38, the higher EAS yield of this strain were observed while 200 rpm was employed. The proximate compositions of lyophilized EAS produced by strain SCA38 in various media are primarily carbohydrate 63.1-71.0%, and 8.7-15.9% the second highest content was crude protein. The results observed from gel permeation chromatography implied that the EAS derived from strain SCA38 was a complex compound with polysaccharide and protein. The solubility of rehydrated aqueous solution with strain SCA38 EAS powder was dissolved in distilled water, 1.0-4.0% NaCl, 80% formic acid, and 99% dimethyl sulphoxide. The relative viscosity (RV) of strain SCA38 EAS rehydrated aqueous solution was increased while the concentration of EAS was increased in the reacting solution. However, EAS solutions with the addition of increasing NaCl concentration were performed the decreasing RV. The 0.2% lyophilized EAS powder rehydrated is solution showed the higher and more stable RV over a pH range from 6.0 to 8.0. The RV of 0.2% lyophilized EAS powder rehydrated solution more stabled at 40oC, and then declined while the temperature is either raising or falling. The emulsion activity (EA) of EAS obtained from strain SCA38 was examined as the EAS concentration ranged from 0.2% to 0.8%, the results showed that the EAS standing among 42% to 48%. As 0.4% or 1.0% EAS was added to the testing solution, the resultant emulsion stabilities (ES) were both 53%, which is better than the rest testing groups. The EAS recovered from the cultivation of strain SCA38 in MSBB M-MPB-M, and M-MPB-G were used to test their capability on antioxidation and antimutagenicity in the concentration ranged from 10 to 50 mg/mL. The antioxidative performance of those EAS were not remarkable on the scavenging DPPH free radical, but the lyophilized EAS (derived from M-MPB-M) showed a good result on the inhibition of hemoglobin catalyzing linoleic acid. While in such 1% EAS solution, the inhibition rate could reach 93.040.58%. As to the EAS recovered from the strain SCA38 cultured MSBB and M-MPB-G, the inhibition performances on the hemoglobin catalyzing linoleic acid were ranged from 5.51% to 79.81% as the added concentration of EAS was increased. As to the antimutagenicity of EAS (5%) that produced from strain SCA38 cultured MSBB, the inhibition rate to the mutation caused by MMNG or B[a]P could be 37.1% and 5.5%, respectively. In the same testing system, the EAS of strain SCA38 that derived from M-MPB-G had inhibition rates while against the mutation caused by among 85.7-88.6% MMNG.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant ataxia caused by an expansion of ‘CAG’ repeats in the exon 1 of the gene ATXN2, conferring a gain of toxic function that triggers the appearance of the disease phenotype. The disease is characterized by several symptoms including progressive gait ataxia and dysarthria, slow saccadic eye movements, sleep disturbances, cognitive impairments and psychological dysfunctions such as insomnia and depression, among others. The available treatments rely on palliative care alone, which mitigate some of the major symptoms but ultimately fails to block the disease progression. This current lack of effective therapies highlight the need to create new platforms, allowing the study of the disease molecular basis and the development of new therapeutic strategies. For many years, this depended upon the analysis of postmortem tissues from deceased patients, which can provide data with great biological significance but are limited to the terminal stages of this disease. However, the identification of the causative mutation related to SCA2 and the development of state-of-the-art molecular technologies paved the way for the creation of some models in yeast, C. elegans, D. melanogaster and mice. These models have provided the researchers with greater insight into the molecular pathways of the disorder to reinforce the human patient’s data. Furthermore, a set of 4 transgenic and 1 knock-in mouse successfully recreated many of the SCA2 major symptoms, including gait ataxia, motor incoordination and neuropathological features such as ataxin-2 inclusions and cerebellar degeneration. This set of characteristics make these mice invaluable to deepen our understanding of the human disease and also reliable platforms to test for new therapeutic alternatives. Notwithstanding this, the available models have focused mainly on the cerebellar dysfunctions that characterize SCA2, while other brain regions and their contributions to the pathophysiology of this disease remain generally unexplored. Indeed, the anomalies in the cerebellum cannot explain all of the symptoms displayed by the human patients, suggesting that other brain areas might be affected as well. One of these areas is the striatum, which has been reported to display mutant ataxin-2 inclusions and neurodegeneration, that might be responsible for the cognitive impairments and the parkinsonism often observed in patients. In this work, we have used lentiviral vectors to overexpress a full-length mutated version of ataxin-2 in the striatum of C57BL/6 mice, successfully inducing a neuronal dysfunction phenotype. However, no ubiquitinated inclusions were detected nor a neuroinflammatory response was observable at 8 weeks of age. Interestingly, this time point was enough to detect a clear synaptic dysfunction in the striatum, which could be the cause for the hyperactive exploratory behavior displayed by these mice. Furthermore, the unilateral injection of ATXN2MUT in the striatum induced a mild ipsilateral turning behavior that suggests impairment in the dopaminergic transmission. In conclusion, we present an alternative modeling strategy for SCA2 that might disclose the contribution of the striatum to disease progression and study particular characteristics of this disorder that were not possible with the existing models.
A ataxia espinocerebelosa do tipo 2 (SCA2) é uma doença genética autossómica dominante que tem como causa uma expansão do trinucleótido ‘CAG’ no exão 1 do gene ATXN2. Este número anormal de repetições confere propriedades tóxicas à proteína ataxina-2, resultando em morte e disfunção neuronal e induzindo assim o desenrolar da patologia. Esta doença é caracterizada por uma série de sintomas de carácter progressivo, como por exmplo: ataxia locomotora, disartria, distúrbio dos movimentos oculares sacádicos, perturbações do sono, disfunções cognitivas e perturbações psiquiátricas como depressão, insónias, entre outros. Até à data, não existe nenhum tratamento que modifique a progressão da doença, sendo que as únicas opções terapêuticas consistem em medidas de cuidados paliativos e de melhoria da qualidade de vida. Esta situação apela à criação de novas plataformas de estudo da SCA2 que permitam estudar em maior detalhe o desenvolvimento da doença, bem como testar novas alternativas terapêuticas. Durante vários anos, estes estudos estavam limitados à análise de tecidos post-mortem de doentes, que naturalmente estavam limitados ao estádio terminal da doença. Este facto impossibilitava o estudo dos primeiros mecanismos moleculares que levam ao seu desenvolvimento. No entanto, esta situação foi revertida com a descoberta da mutação causadora de SCA2, e a consequente criação de vários modelos celulares e animais em leveduras (Saccharomyces cerevisiae), nemátodes (Caenorhabditis elegans), mosca-dafruta (Drosophila melanogaster) e ratinho (Mus musculus). Estes estudos permitiram aprofundar os mecanismos moleculares por detrás da doença e também as funções celulares da proteína ataxina-2, reforçando assim os dados provenientes de tecido humano. Por fim, o desenvolvimento de vários modelos transgénicos e de knock-in em ratinhos possibilitou recriar vários sintomas de SCA2 nos animais, incluindo descoordenação motora, degenerescência do cerebelo e a formação de agregados de ataxina-2. Estes animais constituem assim um recurso valioso para aprofundar o nosso conhecimento desta patologia bem como para testar o efeito de novos fármacos e estratégias terapêuticas. No entanto, os modelos descritos até ao momento focam-se quase exclusivamente numa única zona do cérebro – o cerebelo – relegando para segundo plano outras regiões que também estão envolvidas em SCA2. Uma destas zonas é o estriado, no qual já foi descrito em doentes a presença de inclusões ubiquitinadas bem como neurodegenerescência. O facto de muitos doentes sofrerem de Parkinsonismo, chegando até a responder positivamente ao tratamento com Levodopa, parece reforçar a tese de que o estriado contribui para o desenvolvimento da patologia, apesar deste tópico não estar de todo esclarecido. No decorrer deste projeto, usámos lentivirus recombinantes para induzir a expressão de ataxina-2 mutante no estriado de ratinhos WT, e gerámos com sucesso um fenótipo de disfunção neuronal nesta região cerebral. Apesar de não termos observado a presença de inclusões ubiquitinadas nem de uma resposta neuroinflamatória clara, descrevemos uma série de alterações nos níveis de proteína e RNA de vários constituintes sináticos, bem como alterações comportamentais exibidas por estes animais. Por fim, também observámos que os animais injetados unilateralmente com ataxina-2 mutante possuem um comportamento rotativo tendencioso, indicando uma possível disfunção na transmissão dopaminérgica do estriado. Em resumo, nesta dissertação apresentamos uma estratégia alternativa para modelar a ataxia espinocerebelosa do tipo 2 em ratinho, que contribuiu para aprofundar o papel do estriado no desenvolvimento da patologia. Também acreditamos que no futuro será um modelo valioso para estudar determinadas características desta doença que não seriam possíveis com outros modelos existentes

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative polyglutamine disease caused by an aberrant expansion of the trinucleotide CAG in the coding region of the ATXN2 gene. As a result, ataxin-2, the protein product resulting from this gene, harbors an abnormally expanded polyglutamine tract, which renders it more prone to aggregation and consequent formation of inclusion bodies within the brain of SCA2 patients. Moreover, a toxic gain-of-function of ataxin-2, accompanied by the reported toxicity of an ATXN2 antisense RNA, are thought to induce cellular alterations with subsequent neuronal death. Although only some neuroanatomical regions are primarily affected in SCA2, the neurodegeneration process seems to progress towards other brain regions, in latter stages of the disease. Nevertheless, the mechanisms responsible for this apparent disease spreading phenomenon have never been addressed. In the last decade, recurrent evidence has shown that extracellular vesicles (EVs) can mediate neuron-neuron transfer of aggregate-prone proteins associated with many neurodegenerative diseases. In some cases, it was also reported that this mechanism can induce toxicity in healthy recipient cells, suggesting an explanation for disease spreading. This study aimed to investigate the possibility of ataxin-2, in its protein and/or mRNA forms, being intercellularly transferred, similarly to what has been reported for proteins involved in neurodegenerative diseases. We show that eGFP-tagged ataxin-2 can be detected in non-transfected neuroblastoma cells when these are cultured in conditioned medium derived from eGFP-ataxin-2-transfected cells. Moreover, we show that ataxin-2 mRNA was found within two different types of extracellular vesicles, which were able to induce the formation of ataxin-2 aggregate-like structures within recipient cells, both in vitro and in vivo. Overall, this study suggests for the first time that EV-mediated transport of ataxin-2 mRNA can induce the formation of ataxin-2 aggregates in healthy recipient cells, possibly representing a novel mechanism involved in SCA2 pathogenesis.
A ataxia espinocerebelosa tipo 2 (SCA2, do inglês spinoceberellar ataxia type 2) é uma doença neurodegenerativa que pertence ao grupo das doenças de poliglutaminas. A SCA2 é causada por uma expansão aberrante de uma região repetida de codões CAG presente na região codificante do gene ATXN2. Esta mutação resulta na síntese de uma proteína denominada ataxina-2, que contém uma sequência repetitiva de glutaminas anormalmente expandida, na sua região N-terminal. Os monómeros de ataxina-2 mutante tendem a agregar, levando à formação de oligómeros e, eventualmente, de agregados proteicos de natureza amilóide. De facto, uma das características da SCA2, à semelhança de outras doenças de poliglutaminas, é a presença de agregados proteicos constituídos por diversas proteínas no tecido nervoso dos doentes, denominados de corpos de inclusão. Contudo, existe uma grande incerteza no que concerne à possibilidade de estes agregados serem a causa direta da toxicidade observada na SCA2, uma vez que alguns estudos sugerem a ausência de correlação entre a presença de agregados e neurodegeneração. De facto, à semelhança do que acontece no contexto de outras doenças neurodegenerativas, alguns autores sugerem a possibilidade de pequenas espécies proteicas, como os oligómeros de ataxina-2, constituírem a verdadeira causa de toxicidade, enquanto a agregação macromolecular destas espécies poderá constituir um mecanismo celular de proteção, que pode contrariar a morte neuronal. Por outro lado, a ataxina-2 mutante poderá interagir de forma aberrante com certas proteínas ou perder a sua capacidade intrínseca de se associar com outras, resultando num quadro geral de disfunção da ataxina-2. Consequentemente, a falta de ataxina-2 funcional poderá levar a mecanismos de patogénese que incluem disfunção da autofagia, efeitos deletérios resultantes do processamento aberrante de pré-mRNA, toxicidade mediada por RNA, aumento de stress oxidativo, alterações na sinalização celular e perturbações na homeostasia de cálcio. Tanto a presença de agregados de ataxina-2 como o processo de neurodegeneração são especialmente marcados em regiões particulares do encéfalo, nomeadamente no cerebelo e ponte dos doentes de SCA2. Contudo, a neurodegeneração aparenta estender-se a outras regiões neuroanatómicas, concomitantemente à progressão da doença. Apesar da aparente seletividade regional onde a morte neuronal ocorre, os mecanismos responsáveis pela sua propagação até diferentes áreas anatómicas interconectadas permanecem pouco compreendidos na SCA2, bem como no contexto de outras doenças neurodegenerativas. Durante a última década, diversos estudos têm vindo a demonstrar a participação de vesículas extracelulares na propagação de proteínas com conformações aberrantes, associadas a diversos processos neurodegenerativos. Em alguns casos, verificou-se que este tipo de mecanismo de transporte intercelular constituía uma verdadeira causa de propagação de toxicidade para células saudáveis, sustentando a hipótese da existência de um mecanismo de disseminação de doença. As vesículas extracelulares são estruturas delimitadas por uma bicamada lipídica, que são secretadas, virtualmente, por todos os tipos celulares. Existem diversos tipos de vesículas extracelulares que diferem fisicamente, molecularmente e funcionalmente entre si. Contudo, existe um conjunto de sobreposições de caraterísticas entre os diferentes grupos de vesículas extracelulares que dificultam a sua correta caracterização e classificação. Apesar das diferenças, na sua generalidade, as vesículas extracelulares são capazes de transportar um conjunto de cargas variadas entre células, nomeadamente proteínas, lípidos e diversos tipos de RNAs, que podem, potencialmente, influenciar o comportamento da célula que as internaliza, seja num contexto fisiológico ou patológico. O facto de as vesículas extracelulares serem recorrentemente implicadas na propagação de proteínas com conformação aberrante em diversas doenças neurodegenerativas levou-nos a questionar se este potencial mecanismo de disseminação de doença poderia existir na SCA2 e, assim, explicar, pelo menos em parte, a progressiva neurodegeneração que é observável nos doentes. Deste modo, o objetivo principal deste trabalho consistiu em determinar se a ataxin-2 ou o seu transcrito de mRNA são transportados intercelularmente e se este processo contribui para a propagação de um fenótipo de doença. Na primeira parte deste estudo, observámos que a incubação de células de neuroblastoma de murganho (N2a) com meio condicionado obtido a partir de células que expressam ataxina-2 humana conduz à deteção da proteína humana naquelas células. Adicionalmente, verificámos que o mRNA da forma humana da ataxina-2 mutante se encontra presente em dois tipos diferentes de vesículas extracelulares (exossomas e microvesículas) obtidas a partir de células N2a que expressam a proteína humana. Observámos, ainda, que ambos os tipos de vesículas extracelulares isolados foram capazes de induzir, em células N2a, a formação de estruturas intracelulares semelhantes a agregados de ataxina-2, que aumentaram em número e volume de forma progressiva. Na segunda parte deste trabalho, averiguámos a possibilidade de as vesículas extracelulares serem capazes de propagar a forma humana da ataxina-2 mutante por diferentes regiões do encéfalo de murganhos. Para isso, isolámos exossomas e microvesículas de células N2a que expressavam a ataxina-2 mutante e injetámos, separadamente, as vesículas extracelulares no ventrículo lateral direito dos animais, através de cirurgia estereotáxica. Vinte e quatro horas depois, verificámos a presença de foci contendo ataxina-2 em diversas regiões anatómicas do encéfalo, em diferentes graus de abundância. Por exemplo, as células de Purkinje do cerebelo, que se sabem estarem especialmente afetadas na SCA2, apresentavam uma maior quantidade de foci, comparativamente ao caudoputamen. Por outro lado, as células das camadas granular e molecular do cerebelo não mostravam qualquer presença destas estruturas. Estas discrepâncias observadas poderão ser explicadas pela especificidade de internalização das vesículas extracelulares, que poderá variar de acordo com o tipo celular que as internaliza. Em conclusão, este estudo sugere pela primeira vez que a ataxina-2 mutante é transportada intercelularmente, pelo menos na forma de mRNA, através de vesículas extracelulares que promovem a formação de agregados de ataxina-2 em células saudáveis. Este processo poderá contribuir para a patofisiologia da SCA2 e para a seletividade regional da neurodegeneração que se verifica na doença.