Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Introdução: A Ataxia Espinocerebelar tipo 3 (SCA3), também chamada de Doença de Machado - Joseph (DMJ) é uma doença neurodegenerativa autossômica dominante causada pela expansão de uma sequência repetitiva CAG no gene ATXN3 localizado no cromossomo 14q32.1. Alelos normais contêm 12 a 43 repetições CAG; alelos causadores da doença contêm 51 ou mais repetições CAG (CAGexp). Existe uma forte correlação entre o tamanho da expansão e a gravidade da doença, e a antecipação é um fenômeno comum. Com idade de início média entre os 32 e os 40 anos, a SCA3/DMJ se apresenta com ataxia, disartria, disfagia com uma expressão clínica heterogênea e manifestações clínicas abrangendo mú ltiplos sistemas neurológicos como as vias piramidais e extrapiramidais, a sensibilidade e o neurônio motor inferior. O curso da SCA3/DMJ é invariavelmente progressivo, levando os pacientes à dependência funcional e ao retraimento social e não há tratamentos específicos para a condição. Estima-se que a sobrevida média após o início da doença seja de 21 anos. O inicio da doença na infância raramente foi relatado na literatura, e a progressão e característ icas da doença nesses casos não são conhecidas. Objetivo: caracterizar as manifestações clínicas e a progressão da doença em sujeitos portadores de SCA3/DMJ com idade de inicio da doença na infância. Métod os: casos com idade de início do primeiro sintoma igual ou anterior aos 12 anos (DMl-início-na-infância) foram identificados a partir do registro de casos diagnosticados com exame molecular e acompanhados no ambulatório de Neurogenética do Hospital ele Clínicas de Porto Alegre ele 2000 a 2014. Após consentimento, os pacientes foram entrevistados e examinados através das escalas cl ínicas SARA e NESSCA. As variáveis gênero, idade de início (ii), duração da doença (DD), as repetições CAG dos dois alelos, o gênero do genitor transmissor, a CAGexp do genitor e a antecipação foram obtidas e comparadas com os dados da coorte geral de casos da institu ição. Quando possível, essas comparações foram fe itas através de testes de qui-quadrado para variáveis categóricas, e de testes não paramétricas como Sperman e Mann-Whitney, para variáveis quantitativas. Doze meses após a primeira avaliação (baseline), as escalas NESSCA e SARA foram novamente aplicadas. As taxas de progressão da NESSCA e da SARA nesse intervalo foram medida através do teste modelo misto de coeficientes aleatórios sendo comparadas com as taxas obtidas em duas observações prospectivas prévias, realizadas em pacientes SCA3/DMJ com ii após os 12 anos: o estudo da história natural da SCA3/DMJ uti lizando a escala NESSCA (Jardim et ai, 2010) gerou o grupo Controle-I-IN, e o grupo placebo do e nsaio clín ico randomizado sobre o lítio (Saute et al,2014) gerou o grupo Controle-PL. Resultados: No período, 1317 pacientes (189 famílias) com SCA3/DMJ foram identificados na nossa instituição. Os 367 que fizeram o exame molecular fo ram analisados. Sua ii média ± dp (variação) foi de 34,1 ± 11 (5 a 59) anos. Oito pacientes de 6 fa mílias, todos heterozigotos para a CAGexp, foram identificados com DMI-início-na-infância (2,2% ). Sete dos oito DMJin ício-na-infância (87,5%) eram do sexo feminino (p = 0,057), a ii dos sintomas do grupo DMJ-início-na-infância variou entre 5 e 10 anos com mediana de 8 e intervalo inter-quartil 3 anos. A CAGexp variou de 80 a 91 repetições. A progressão da NESSCA foi de 0,8 pontos anuais em média no grupo Controle-PL e 2 pontos anuais em média no grupo DMJ-início-nainfância (p=O,OO L). A progressão da SARA foi de 0,6 pontos anuais em média no controle-PL e 2,3 pontos em média no grupo DMJ-início na infância (p=O,OOl). A escala NESSCA foi dividida em subescalas considerando sinais piramidais, extrapiramidais, ataxia, sinais de comprometimento periférico, disfagia, disartria e alterações oculomotoras. A progressão dessas subescalas foi comparada entre os dois grupos. Foi observado uma piora na progressão do grupo DMJ-início-na-infância nos sintomas extrapiramidais (p=0,006) e na disfagia (p=O,OOl). Conclusão: A DMI-início-na-infância está associada ao sexo feminino e apresenta uma velocidade de progressão maior do que os demais sujeitos com SCA3/MJD. / lntroduction: Spinocerebellar Ataxia 3 (SCA3), also known as Machado-Joseph Disease (MJD), is an autosomal dominant neurodegenerative disorder, associated to a CAG repeated expansion at the ATXN3 gene coding region, located at chromosome 14q32.1. Normal alteies contain 12-43 CAG repeats; alleles causing disease contain 51 or more CAG repetitions (CAGexp). There is a strong correlation between the expansion size anel the severity of disease, and the anticipation is a common phenomenon. The average age of onset is 32 to 40 years old anel the SCA3/MJD presents with ataxia, dysarthria, dysphagia with a heterogcneous clinicai expression anel clinicai manifestations affect multiple neurological systems as pyramidal anel extrapyramidal pathways, sensitivity anel motor neuron lower. The course of SCA3/MJD is invariably progressive, leading patients to fu nctional dependency anel social withdrawal and there are no specific treatments for the condition. lt is estimated that the avcrage survival after elisease onset is 21 years. The onset of disease in childhood was rarely reported in the literature, anel the disease progression anel characteristics in patients under 12 years is not known. Objcctive: To characterize patients with heterozygous SCA3/MJD with age of onsct before 12 ycars old. Methods: cases with age of onset before 12 years old were ident ificcl from the registry of cases eliagnosecl molecularly anel accompanied in the Neurogcnctics Clinic of Hospital de Clínicas de Porto Alegre (HCPA) from 2000 to 2014. After consent, patients were interviewed anel examined with the cl inicai scales SARA anel NESSCA. The variables gender, age at onset (ao), clisease cluration (DD), CAG repeats of the two ali cies, transmitter parent gender, CAGexp of parent anel anticipation were obtained anel compareci with data of the general cohort of cases from institution. These comparisons were macle using chi-square tests for categorical variables anel non-parametric tests as Spearman anel Mann-Whitney test for quantitative variables. Twelve months after the first evaluation (baseline), the SARA and NESSCA scales were again applied. Progression rates of SARA and NESSCA in that range were measured using the mixed model of random coefficient test were compareci with the rates obtained in two previous prospective observations, made in SCA3 I MJD patients with ao after 12 years: the Natural His tory Study of SCA3/DMJ using NESSCA (Jardim et al, 2010) generaLed the Control-HN group and Lhe placebo group randomized clinicai trial of lithium (Saute et ai, 2014) generated Control-PL group. Results: During the period, 1317 patienLs (189 fami lies) with SCA3/MJD were identified at HCPA. The 367 patients who dicl the molecular examinaLion were analyzecl. Its average ao ± SD (range) was 34.1 ± 11 (5-59) years. Six families of eight patients, ali heterozygous for CAGexp were identified MJD-chilclhood-onset group (2.2% ). Seven of the eight MJDchildhood- onset group (87.5%) were female (p = 0.057), ao of the symptoms of MJDchildhood- onset group ranged between 5 and 10 years wiLh a median of 8 and interquartile range 3 years. The CAGexp ranged 80-91 repetitions. The ann ual progression of NESSCA was 0.8 poinLs on average in Lhe Control-PL group anel 2 points on average in MJDchildhood- onset group (p = 0.001). The progression of SARA was 0.6 points per year on average in the Controi-PL group anel 2.3 points in average MJD group in childhood-onset (p = 0.001). The scale NESSCA was divided in subscales considering pyramidal signs, extrapyramidal signs, ataxia, peripheral signs, dysphagia, dysarthria and oculomotor and compareci to progression in both groups. The progression of these subscales was compareci between the two groups.

Introdução: A Ataxia Espinocerebelar tipo 3 (SCA3), também chamada de Doença de Machado - Joseph (DMJ) é uma doença neurodegenerativa autossômica dominante causada pela expansão de uma sequência repetitiva CAG no gene ATXN3 localizado no cromossomo 14q32.1. Alelos normais contêm 12 a 43 repetições CAG; alelos causadores da doença contêm 51 ou mais repetições CAG (CAGexp). Existe uma forte correlação entre o tamanho da expansão e a gravidade da doença, e a antecipação é um fenômeno comum. Com idade de início média entre os 32 e os 40 anos, a SCA3/DMJ se apresenta com ataxia, disartria, disfagia com uma expressão clínica heterogênea e manifestações clínicas abrangendo mú ltiplos sistemas neurológicos como as vias piramidais e extrapiramidais, a sensibilidade e o neurônio motor inferior. O curso da SCA3/DMJ é invariavelmente progressivo, levando os pacientes à dependência funcional e ao retraimento social e não há tratamentos específicos para a condição. Estima-se que a sobrevida média após o início da doença seja de 21 anos. O inicio da doença na infância raramente foi relatado na literatura, e a progressão e característ icas da doença nesses casos não são conhecidas. Objetivo: caracterizar as manifestações clínicas e a progressão da doença em sujeitos portadores de SCA3/DMJ com idade de inicio da doença na infância. Métod os: casos com idade de início do primeiro sintoma igual ou anterior aos 12 anos (DMl-início-na-infância) foram identificados a partir do registro de casos diagnosticados com exame molecular e acompanhados no ambulatório de Neurogenética do Hospital ele Clínicas de Porto Alegre ele 2000 a 2014. Após consentimento, os pacientes foram entrevistados e examinados através das escalas cl ínicas SARA e NESSCA. As variáveis gênero, idade de início (ii), duração da doença (DD), as repetições CAG dos dois alelos, o gênero do genitor transmissor, a CAGexp do genitor e a antecipação foram obtidas e comparadas com os dados da coorte geral de casos da institu ição. Quando possível, essas comparações foram fe itas através de testes de qui-quadrado para variáveis categóricas, e de testes não paramétricas como Sperman e Mann-Whitney, para variáveis quantitativas. Doze meses após a primeira avaliação (baseline), as escalas NESSCA e SARA foram novamente aplicadas. As taxas de progressão da NESSCA e da SARA nesse intervalo foram medida através do teste modelo misto de coeficientes aleatórios sendo comparadas com as taxas obtidas em duas observações prospectivas prévias, realizadas em pacientes SCA3/DMJ com ii após os 12 anos: o estudo da história natural da SCA3/DMJ uti lizando a escala NESSCA (Jardim et ai, 2010) gerou o grupo Controle-I-IN, e o grupo placebo do e nsaio clín ico randomizado sobre o lítio (Saute et al,2014) gerou o grupo Controle-PL. Resultados: No período, 1317 pacientes (189 famílias) com SCA3/DMJ foram identificados na nossa instituição. Os 367 que fizeram o exame molecular fo ram analisados. Sua ii média ± dp (variação) foi de 34,1 ± 11 (5 a 59) anos. Oito pacientes de 6 fa mílias, todos heterozigotos para a CAGexp, foram identificados com DMI-início-na-infância (2,2% ). Sete dos oito DMJin ício-na-infância (87,5%) eram do sexo feminino (p = 0,057), a ii dos sintomas do grupo DMJ-início-na-infância variou entre 5 e 10 anos com mediana de 8 e intervalo inter-quartil 3 anos. A CAGexp variou de 80 a 91 repetições. A progressão da NESSCA foi de 0,8 pontos anuais em média no grupo Controle-PL e 2 pontos anuais em média no grupo DMJ-início-nainfância (p=O,OO L). A progressão da SARA foi de 0,6 pontos anuais em média no controle-PL e 2,3 pontos em média no grupo DMJ-início na infância (p=O,OOl). A escala NESSCA foi dividida em subescalas considerando sinais piramidais, extrapiramidais, ataxia, sinais de comprometimento periférico, disfagia, disartria e alterações oculomotoras. A progressão dessas subescalas foi comparada entre os dois grupos. Foi observado uma piora na progressão do grupo DMJ-início-na-infância nos sintomas extrapiramidais (p=0,006) e na disfagia (p=O,OOl). Conclusão: A DMI-início-na-infância está associada ao sexo feminino e apresenta uma velocidade de progressão maior do que os demais sujeitos com SCA3/MJD. / lntroduction: Spinocerebellar Ataxia 3 (SCA3), also known as Machado-Joseph Disease (MJD), is an autosomal dominant neurodegenerative disorder, associated to a CAG repeated expansion at the ATXN3 gene coding region, located at chromosome 14q32.1. Normal alteies contain 12-43 CAG repeats; alleles causing disease contain 51 or more CAG repetitions (CAGexp). There is a strong correlation between the expansion size anel the severity of disease, and the anticipation is a common phenomenon. The average age of onset is 32 to 40 years old anel the SCA3/MJD presents with ataxia, dysarthria, dysphagia with a heterogcneous clinicai expression anel clinicai manifestations affect multiple neurological systems as pyramidal anel extrapyramidal pathways, sensitivity anel motor neuron lower. The course of SCA3/MJD is invariably progressive, leading patients to fu nctional dependency anel social withdrawal and there are no specific treatments for the condition. lt is estimated that the avcrage survival after elisease onset is 21 years. The onset of disease in childhood was rarely reported in the literature, anel the disease progression anel characteristics in patients under 12 years is not known. Objcctive: To characterize patients with heterozygous SCA3/MJD with age of onsct before 12 ycars old. Methods: cases with age of onset before 12 years old were ident ificcl from the registry of cases eliagnosecl molecularly anel accompanied in the Neurogcnctics Clinic of Hospital de Clínicas de Porto Alegre (HCPA) from 2000 to 2014. After consent, patients were interviewed anel examined with the cl inicai scales SARA anel NESSCA. The variables gender, age at onset (ao), clisease cluration (DD), CAG repeats of the two ali cies, transmitter parent gender, CAGexp of parent anel anticipation were obtained anel compareci with data of the general cohort of cases from institution. These comparisons were macle using chi-square tests for categorical variables anel non-parametric tests as Spearman anel Mann-Whitney test for quantitative variables. Twelve months after the first evaluation (baseline), the SARA and NESSCA scales were again applied. Progression rates of SARA and NESSCA in that range were measured using the mixed model of random coefficient test were compareci with the rates obtained in two previous prospective observations, made in SCA3 I MJD patients with ao after 12 years: the Natural His tory Study of SCA3/DMJ using NESSCA (Jardim et al, 2010) generaLed the Control-HN group and Lhe placebo group randomized clinicai trial of lithium (Saute et ai, 2014) generated Control-PL group. Results: During the period, 1317 patienLs (189 fami lies) with SCA3/MJD were identified at HCPA. The 367 patients who dicl the molecular examinaLion were analyzecl. Its average ao ± SD (range) was 34.1 ± 11 (5-59) years. Six families of eight patients, ali heterozygous for CAGexp were identified MJD-chilclhood-onset group (2.2% ). Seven of the eight MJDchildhood- onset group (87.5%) were female (p = 0.057), ao of the symptoms of MJDchildhood- onset group ranged between 5 and 10 years wiLh a median of 8 and interquartile range 3 years. The CAGexp ranged 80-91 repetitions. The ann ual progression of NESSCA was 0.8 poinLs on average in Lhe Control-PL group anel 2 points on average in MJDchildhood- onset group (p = 0.001). The progression of SARA was 0.6 points per year on average in the Controi-PL group anel 2.3 points in average MJD group in childhood-onset (p = 0.001). The scale NESSCA was divided in subscales considering pyramidal signs, extrapyramidal signs, ataxia, peripheral signs, dysphagia, dysarthria and oculomotor and compareci to progression in both groups. The progression of these subscales was compareci between the two groups.

Introdução: A Ataxia Espinocerebelar tipo 3 (SCA3), também chamada de Doença de Machado - Joseph (DMJ) é uma doença neurodegenerativa autossômica dominante causada pela expansão de uma sequência repetitiva CAG no gene ATXN3 localizado no cromossomo 14q32.1. Alelos normais contêm 12 a 43 repetições CAG; alelos causadores da doença contêm 51 ou mais repetições CAG (CAGexp). Existe uma forte correlação entre o tamanho da expansão e a gravidade da doença, e a antecipação é um fenômeno comum. Com idade de início média entre os 32 e os 40 anos, a SCA3/DMJ se apresenta com ataxia, disartria, disfagia com uma expressão clínica heterogênea e manifestações clínicas abrangendo mú ltiplos sistemas neurológicos como as vias piramidais e extrapiramidais, a sensibilidade e o neurônio motor inferior. O curso da SCA3/DMJ é invariavelmente progressivo, levando os pacientes à dependência funcional e ao retraimento social e não há tratamentos específicos para a condição. Estima-se que a sobrevida média após o início da doença seja de 21 anos. O inicio da doença na infância raramente foi relatado na literatura, e a progressão e característ icas da doença nesses casos não são conhecidas. Objetivo: caracterizar as manifestações clínicas e a progressão da doença em sujeitos portadores de SCA3/DMJ com idade de inicio da doença na infância. Métod os: casos com idade de início do primeiro sintoma igual ou anterior aos 12 anos (DMl-início-na-infância) foram identificados a partir do registro de casos diagnosticados com exame molecular e acompanhados no ambulatório de Neurogenética do Hospital ele Clínicas de Porto Alegre ele 2000 a 2014. Após consentimento, os pacientes foram entrevistados e examinados através das escalas cl ínicas SARA e NESSCA. As variáveis gênero, idade de início (ii), duração da doença (DD), as repetições CAG dos dois alelos, o gênero do genitor transmissor, a CAGexp do genitor e a antecipação foram obtidas e comparadas com os dados da coorte geral de casos da institu ição. Quando possível, essas comparações foram fe itas através de testes de qui-quadrado para variáveis categóricas, e de testes não paramétricas como Sperman e Mann-Whitney, para variáveis quantitativas. Doze meses após a primeira avaliação (baseline), as escalas NESSCA e SARA foram novamente aplicadas. As taxas de progressão da NESSCA e da SARA nesse intervalo foram medida através do teste modelo misto de coeficientes aleatórios sendo comparadas com as taxas obtidas em duas observações prospectivas prévias, realizadas em pacientes SCA3/DMJ com ii após os 12 anos: o estudo da história natural da SCA3/DMJ uti lizando a escala NESSCA (Jardim et ai, 2010) gerou o grupo Controle-I-IN, e o grupo placebo do e nsaio clín ico randomizado sobre o lítio (Saute et al,2014) gerou o grupo Controle-PL. Resultados: No período, 1317 pacientes (189 famílias) com SCA3/DMJ foram identificados na nossa instituição. Os 367 que fizeram o exame molecular fo ram analisados. Sua ii média ± dp (variação) foi de 34,1 ± 11 (5 a 59) anos. Oito pacientes de 6 fa mílias, todos heterozigotos para a CAGexp, foram identificados com DMI-início-na-infância (2,2% ). Sete dos oito DMJin ício-na-infância (87,5%) eram do sexo feminino (p = 0,057), a ii dos sintomas do grupo DMJ-início-na-infância variou entre 5 e 10 anos com mediana de 8 e intervalo inter-quartil 3 anos. A CAGexp variou de 80 a 91 repetições. A progressão da NESSCA foi de 0,8 pontos anuais em média no grupo Controle-PL e 2 pontos anuais em média no grupo DMJ-início-nainfância (p=O,OO L). A progressão da SARA foi de 0,6 pontos anuais em média no controle-PL e 2,3 pontos em média no grupo DMJ-início na infância (p=O,OOl). A escala NESSCA foi dividida em subescalas considerando sinais piramidais, extrapiramidais, ataxia, sinais de comprometimento periférico, disfagia, disartria e alterações oculomotoras. A progressão dessas subescalas foi comparada entre os dois grupos. Foi observado uma piora na progressão do grupo DMJ-início-na-infância nos sintomas extrapiramidais (p=0,006) e na disfagia (p=O,OOl). Conclusão: A DMI-início-na-infância está associada ao sexo feminino e apresenta uma velocidade de progressão maior do que os demais sujeitos com SCA3/MJD. / lntroduction: Spinocerebellar Ataxia 3 (SCA3), also known as Machado-Joseph Disease (MJD), is an autosomal dominant neurodegenerative disorder, associated to a CAG repeated expansion at the ATXN3 gene coding region, located at chromosome 14q32.1. Normal alteies contain 12-43 CAG repeats; alleles causing disease contain 51 or more CAG repetitions (CAGexp). There is a strong correlation between the expansion size anel the severity of disease, and the anticipation is a common phenomenon. The average age of onset is 32 to 40 years old anel the SCA3/MJD presents with ataxia, dysarthria, dysphagia with a heterogcneous clinicai expression anel clinicai manifestations affect multiple neurological systems as pyramidal anel extrapyramidal pathways, sensitivity anel motor neuron lower. The course of SCA3/MJD is invariably progressive, leading patients to fu nctional dependency anel social withdrawal and there are no specific treatments for the condition. lt is estimated that the avcrage survival after elisease onset is 21 years. The onset of disease in childhood was rarely reported in the literature, anel the disease progression anel characteristics in patients under 12 years is not known. Objcctive: To characterize patients with heterozygous SCA3/MJD with age of onsct before 12 ycars old. Methods: cases with age of onset before 12 years old were ident ificcl from the registry of cases eliagnosecl molecularly anel accompanied in the Neurogcnctics Clinic of Hospital de Clínicas de Porto Alegre (HCPA) from 2000 to 2014. After consent, patients were interviewed anel examined with the cl inicai scales SARA anel NESSCA. The variables gender, age at onset (ao), clisease cluration (DD), CAG repeats of the two ali cies, transmitter parent gender, CAGexp of parent anel anticipation were obtained anel compareci with data of the general cohort of cases from institution. These comparisons were macle using chi-square tests for categorical variables anel non-parametric tests as Spearman anel Mann-Whitney test for quantitative variables. Twelve months after the first evaluation (baseline), the SARA and NESSCA scales were again applied. Progression rates of SARA and NESSCA in that range were measured using the mixed model of random coefficient test were compareci with the rates obtained in two previous prospective observations, made in SCA3 I MJD patients with ao after 12 years: the Natural His tory Study of SCA3/DMJ using NESSCA (Jardim et al, 2010) generaLed the Control-HN group and Lhe placebo group randomized clinicai trial of lithium (Saute et ai, 2014) generated Control-PL group. Results: During the period, 1317 patienLs (189 fami lies) with SCA3/MJD were identified at HCPA. The 367 patients who dicl the molecular examinaLion were analyzecl. Its average ao ± SD (range) was 34.1 ± 11 (5-59) years. Six families of eight patients, ali heterozygous for CAGexp were identified MJD-chilclhood-onset group (2.2% ). Seven of the eight MJDchildhood- onset group (87.5%) were female (p = 0.057), ao of the symptoms of MJDchildhood- onset group ranged between 5 and 10 years wiLh a median of 8 and interquartile range 3 years. The CAGexp ranged 80-91 repetitions. The ann ual progression of NESSCA was 0.8 poinLs on average in Lhe Control-PL group anel 2 points on average in MJDchildhood- onset group (p = 0.001). The progression of SARA was 0.6 points per year on average in the Controi-PL group anel 2.3 points in average MJD group in childhood-onset (p = 0.001). The scale NESSCA was divided in subscales considering pyramidal signs, extrapyramidal signs, ataxia, peripheral signs, dysphagia, dysarthria and oculomotor and compareci to progression in both groups. The progression of these subscales was compareci between the two groups.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

L'ataxie spinocérébelleuse de type 7 (SCA7) est une maladie neurodégénerative due à une expansion de CAG traduit en polyQ dans la protéine ataxine-7. La SUMOylation, modification post-traductionnelle que nous avons identifiée moduler l'agrégation de la protéine mutante, est facilitée par une SUMO E3 ligase.Nous avons identifié RanBP2, une nucléoporine appartenant au complexe du pore nucléaire en tant que SUMO E3 ligase, via SUMO-1 de l'ataxine-7. En effet, le silencing de RanBP2 induit l'agrégation de l'ataxine-7 mutante, ce qui démontre l'implication de RanBP2 dans la physiopathologie de SCA7. Nous montrons également que l'ataxine-7 endogène est une cible modifiée par SUMO-1 et -2. L'ataxine-7 poly-SUMOylée, grâce à la présence de chaines SUMO2/3, est capable de recruter RNF4. Cette protéine conduit à la dégradation de l'ataxine-7 mutante par la voie du protéasome. La dégradation est abolie en présence d'un mutant de RNF4.Dans un modèle murin KI SCA7, nous avons quantifié l'expression des gènes impliqués dans la voie de la SUMOylation au niveau des régions les plus touchées du cerveau. Le niveau d'expression des ARNs messagers montre des altérations dépendantes des répétitions CAG du gène SCA7. A 6 mois (avant le début de la pathologie), les premières dérégulations sont observées; à 12 mois (à un stade avancé de la maladie), on note une diminution statistiquement significative de Sumo-1 dans le cervelet des souris Atxn7100Q/5Q. Ces résultats, alliés à l'observation de l'accumulation anormale des protéines SUMO-1 et RanBP2 dans le cervelet d'un patient SCA7, suggèrent que les voies de la SUMOylation in vivo peuvent être perturbées dans SCA7. / Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by a CAG expansion (polyQ) in the protein ataxin-7. SUMOylation, a post-translational modification that we identified to modulate mutant protein aggregation in a SCA7 cellular model, is facilitated by a SUMO E3 ligase. Here, we identified RanBP2 (Nup358), a nucleoporin belonging to the nuclear pore complex, as the major E3 enzyme implicated in ataxin-7 modification by SUMO-1. Indeed, RanBP2 silencing renders mutant ataxin-7 more prone to aggregation, thus demonstrating the implication of RanBP2 in SCA7 pathophysiology. We also show that endogenous ataxin-7 is a target for both SUMO-1 and -2 modification. Poly-SUMOylated ataxin-7 presents a docking site composed of SUMO2/3 chains for the recruitment of RNF4: this protein is a SUMO E3 ubiquitin ligase that mediates degradation of mutant ataxin-7 by the proteasome pathway. The degradation is abolished in presence of a mutant form of RNF4. In a SCA7 knock-in mouse model we quantified expression of SUMO-pathway related genes in cerebellum and retina, the most affected regions using quantitative RT-PCR. SUMO-related genes show expanded repeat-dependent alterations in expression patterns. At 6 months (before onset), deregulations begin to occur; by 12 months (late stage of disease), there is a statistically significant impairment in Sumo-1 levels in Atxn7100Q/5Q cerebellum. These results, together with the observation that SUMO-1 and RanBP2 protein accumulate abnormally in the cerebellum of a SCA7 patient, suggest that in vivo SUMO-modifying pathways may be perturbed in SCA7.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19376号 / 工博第4121号 / 新制||工||1635(附属図書館) / 32390 / 新制||工||1635 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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