INTRODUÇÃO: A distonia é caracterizada por contrações musculares sustentadas, frequentemente gerando movimentos de torção, repetitivos ou posturas anormais. A classificação de distonia é baseada em três eixos: idade de início, distribuição e etiologia. A definição criada pelo Comitê da Fundação de Pesquisa Médica em Distonia, em 1984, ainda está em uso atualmente. Questionamentos têm sido feitos para uma nova proposta de classificação de distonia. MATERIAIS E MÉTODOS: Trata-se de um estudo transversal descritivo de uma série de pacientes com diagnóstico de distonia primária e distonia-plus acompanhados em uma clínica brasileira especializada em distúrbios do movimento, no período de março de 2015 até fevereiro de 2016. Foi realizada a coleta de dados demográficos, clínicos e da gravidade da distonia. Os pacientes foram reavaliados e reclassificados segundo a nova proposta de classificação para as distonias. Utilizamos métodos descritivos para apresentarmos as características da amostra, o teste de Shapiro-Wilk para distribuição normal, o teste de Kruskal-Wallis para comparar grupos independentes e variáveis sem distribuição normal e o teste do X2 para comparar variáveis nominais. RESULTADOS: Dos 289 pacientes, foram 235 pacientes avaliados segundo a distribuição corporal, sendo blefaroespasmo 60 pacientes (26%), distonia cervical 51 pacientes (22%), distonia de membro 28 pacientes (12%), distonia laríngea 10 pacientes (4%), distonia oromandibular 3 pacientes (1%), distonia segmentar 46 pacientes (20%), distonia multifocal 23 pacientes (9%) e distonia generalizada 14 pacientes (6%). Foram 227/235 (97%) pacientes com distonia primária e 8 (3%) pacientes com distonia-plus. Houve maior prevalência no sexo feminino com 144 pacientes. Os pacientes com distonia generalizada, multifocal, distonia dopa-responsiva e distonia-parkinsonismo tiveram a idade de início da distonia inferior a todos os demais grupos (p=0,0001). Os pacientes com distonia do membro superior e distonia tarefa-específica tiveram a idade de início da distonia inferior às outras distonias focais e distonias segmentares (p=0,0001). Os pacientes com distonia tarefa-específica e distonia focal do membro superior têm escolaridade superior aos demais grupos (p=0,0001). Em média, foram 5±7,8 anos para o diagnóstico definitivo de distonia e 2±1,5 médicos até que o diagnóstico de distonia fosse confirmado. O truque sensorial foi mais frequente na distonia cervical 30/51 (59%) pacientes. Segundo a nova classificação, encontramos apenas mudança de resultados na distonia multifocal com 21/235 (9%) pacientes e na distonia generalizada com 16/235 (6%) pacientes. Foram 164/235 (70%) pacientes na faixa etária maior que 40 anos. Todos os pacientes tiveram curso da doença com padrão estático e a maioria com variabilidade persistente em 186/235 (79%) pacientes. Foram 227/235 (97%) pacientes com distonia isolada. Nenhum paciente apresentou lesão adquirida ou degenerativa. Foram 211/235 (90%) pacientes que apresentaram diagnóstico etiológico idiopático esporádico. CONCLUSÃO: A nova classificação de distonia não apresentou novos benefícios na prática clínica diária, comparada com classificações anteriores. O benefício maior foi uma reorganização e formação de novas definições e critérios para atividade de pesquisas científicas e identificação de casos mais raros. / INTRODUCTION: Dystonia is characterized by sustained muscle contractions, frequently causing twisting movements, repetitive or abnormal postures. Dystonia rating is based on three axes: age of onset, distribution and etiology. The definition established by the Committee of the Medical Research Foundation in dystonia in 1984, is still in use today. Claims have been made for a new proposal for dystonia rating. MATERIALS AND METHODS: This is a descriptive cross-sectional study of a number of patients with primary dystonia and dystonia-plus assisted in a Brazilian clinic specializing in movement disorders, from March 2015 to February 2016 data collection was performed demographic, clinical and severity of dystonia. The patients were re-evaluated and reclassified according to the new proposed classification for dystonia. We used descriptive methods to present the characteristics of the sample, the Shapiro-Wilk test for normal distribution, the Kruskal-Wallis test to compare independent groups and variables without normal distribution and the X2 test to compare nominal variables. RESULTS: Of the 289 patients, there were 235 patients evaluated according to body distribution, with blepharospasm 60 patients (26%), cervical dystonia 51 patients (22%), a member of dystonia 28 patients (12%), laryngeal dystonia 10 patients (4%), oromandibular dystonia 3 patients (1%), segmental dystonia 46 patients (20%), dystonia multifocal 23 patients (9%) and generalized dystonia 14 patients (6%). Were 227/235 (97%) patients with primary dystonia and 8 (3%) patients with dystonia-plus. There was a higher prevalence in females with 144 patients. Patients with generalized dystonia, multifocal, dopa-responsive dystonia and dystonia-parkinsonism had the age of onset of dystonia lower than all other groups (p = 0.0001). Patients with dystonia of the upper limb and task-specific dystonia had the early age of less dystonia other focal dystonias and segmental dystonia (p=0.0001). Patients with task-specific dystonia and focal upper limb dystonia have higher education to other groups (p=0.0001). On average, there were 5 ± 7.8 years for the definitive diagnosis of dystonia and 2 ± 1.5 doctors until the diagnosis of dystonia were confirmed. The sensory trick was more frequent in cervical dystonia 30/51 (59%) patients. According to the new classification, we found only change results in multifocal dystonia with 21/235 (9%) patients and generalized dystonia with 16/235 (6%) patients. Were 164/235 (70%) patients aged greater than 40 years. All patients had disease course with static standard and most with persistent variability in 186/235 (79%) patients. Were 227/235 (97%) patients with dystonia isolated. No patient had acquired or degenerative injury. Were 211/235 (90%) patients with sporadic idiopathic etiologic diagnosis. CONCLUSION: The new dystonia rating showed no new benefits in clinical practice compared with previous ratings. The greatest benefit was a reorganization and training of new definitions and criteria for scientific research activity and identification of rare cases.

Introdução: A cãibra do escrivão (CE) é uma distonia focal com contração sustentada e involuntária das mãos, dedos e/ou braços durante a escrita. Poucos estudos avaliaram desempenho motor e cognição na CE a partir de testes neuropsicológicos. Os achados do estudo podem colaborar na reabilitação da doença. Objetivo: Avaliar os desempenhos motor e cognitivo dos pacientes com CE. Métodos: Foram avaliados 21 pacientes e 21 controles pareados por sexo e idade. Dados clínicos foram colhidos e combinados com avaliação da presença de sintomas de depressão, ansiedade e apatia por meio, respectivamente, dos Inventários de Depressão e Ansiedade de Beck e da Escala de Apatia. A gravidade da distonia foi quantificada pelas escalas Writer\'s Cramp Rating Scale e The Arm Dystonia Disabiliity Scale. O desempenho motor foi avaliado pelo Purdue Pegboard Test (PPT). Demais testes neuropsicológicos aplicados foram: Mini Exame do Estado Mental (MEEM), Frontal Assessment Battery (FAB), Symbol Digit Modalities Test (SDMT), Stroop Test (ST) e Trail Making Test (TMT). Resultados: Pacientes com CE apresentaram desempenho inferior aos controles nos testes: MEEM, FAB e ST parte \"W\". Após correção para anos de estudo, variável demográfica com diferença estatística significativa entre os grupos, manteve-se diferença significativa apenas na FAB. Pacientes apresentaram desempenho inferior aos controles nas tarefas com a mão dominante, com ambas as mãos e no teste de montagem do PPT. Após correção para variável anos de estudo, não houve diferença significativa, apenas tendência estatística à performance inferior de pacientes na tarefa montagem do PPT. O desempenho da tarefa montagem do PPT em pacientes com CE teve forte correlação positiva com testes neuropsicológicos, e não com gravidade motora da distonia. Conclusões: O estudo sugere que pacientes com CE apresentem disfunção executiva. As aterações motoras encontradas na tarefa montagem do PPT dos pacientes com CE estão possivelmente relacionadas ao déficit no funcionamento executivo destes indivíduos. / Introduction: The writer\'s cramp (WC) is a focal dystonia with sustained and involuntary contraction of the hands, fingers and/or arms during writing. Few studies have evaluated motor performance and cognition of these patients using neuropsychological tests. The findings may contribute to rehabilitation of this disease. Objective: evaluate motor and cognitive performance of patients with WC. Methods: Twenty-one patients and 21 matched controls by sex and age were evaluated. Clinical data were collected, with assessment of symptoms of depression, anxiety, and apathy using the respective clinical scales: Beck Depression Inventory, Beck Anxiety Inventory and Apathy Scale. Severity of dystonia was quantified by Writer\'s Cramp Rating Scale and The Arm Dystonia Disability Scale. Motor performance was assessed by Purdue Pegboard Test (PPT). Other neuropsychological tests applied were: Mini Mental State Examination (MMSE), Frontal Assessment Battery (FAB), Symbol Digit Modalities Test (SDMT), Stroop Test (ST) and Trail Making Test (TMT). Results: Patients with WC had inferior performance to the controls in MMSE, FAB and ST part \"W\". After correction for years of study, a demographic variable with significant statistical difference between the groups, a significant difference was maintained only in FAB. Patients had inferior performance to the controls in these tasks of PPT: task with the dominant hand, with both hands and in PPT assembly. After correction for the variable \'years of study\', there was no significant difference, only statistical tendency to inferior performance of patients in PPT assembly. The performance of PPT assembly task in patients with WC had a strong positive correlation with neuropsychological tests, and not with motor severity of dystonia. Conclusions: The study suggests that patients with WC have executive dysfunction. The motor alterations in patients with WC is possibly related to deficit in executive functioning

INTRODUÇÃO: A distonia é caracterizada por contrações musculares sustentadas, frequentemente gerando movimentos de torção, repetitivos ou posturas anormais. A classificação de distonia é baseada em três eixos: idade de início, distribuição e etiologia. A definição criada pelo Comitê da Fundação de Pesquisa Médica em Distonia, em 1984, ainda está em uso atualmente. Questionamentos têm sido feitos para uma nova proposta de classificação de distonia. MATERIAIS E MÉTODOS: Trata-se de um estudo transversal descritivo de uma série de pacientes com diagnóstico de distonia primária e distonia-plus acompanhados em uma clínica brasileira especializada em distúrbios do movimento, no período de março de 2015 até fevereiro de 2016. Foi realizada a coleta de dados demográficos, clínicos e da gravidade da distonia. Os pacientes foram reavaliados e reclassificados segundo a nova proposta de classificação para as distonias. Utilizamos métodos descritivos para apresentarmos as características da amostra, o teste de Shapiro-Wilk para distribuição normal, o teste de Kruskal-Wallis para comparar grupos independentes e variáveis sem distribuição normal e o teste do X2 para comparar variáveis nominais. RESULTADOS: Dos 289 pacientes, foram 235 pacientes avaliados segundo a distribuição corporal, sendo blefaroespasmo 60 pacientes (26%), distonia cervical 51 pacientes (22%), distonia de membro 28 pacientes (12%), distonia laríngea 10 pacientes (4%), distonia oromandibular 3 pacientes (1%), distonia segmentar 46 pacientes (20%), distonia multifocal 23 pacientes (9%) e distonia generalizada 14 pacientes (6%). Foram 227/235 (97%) pacientes com distonia primária e 8 (3%) pacientes com distonia-plus. Houve maior prevalência no sexo feminino com 144 pacientes. Os pacientes com distonia generalizada, multifocal, distonia dopa-responsiva e distonia-parkinsonismo tiveram a idade de início da distonia inferior a todos os demais grupos (p=0,0001). Os pacientes com distonia do membro superior e distonia tarefa-específica tiveram a idade de início da distonia inferior às outras distonias focais e distonias segmentares (p=0,0001). Os pacientes com distonia tarefa-específica e distonia focal do membro superior têm escolaridade superior aos demais grupos (p=0,0001). Em média, foram 5±7,8 anos para o diagnóstico definitivo de distonia e 2±1,5 médicos até que o diagnóstico de distonia fosse confirmado. O truque sensorial foi mais frequente na distonia cervical 30/51 (59%) pacientes. Segundo a nova classificação, encontramos apenas mudança de resultados na distonia multifocal com 21/235 (9%) pacientes e na distonia generalizada com 16/235 (6%) pacientes. Foram 164/235 (70%) pacientes na faixa etária maior que 40 anos. Todos os pacientes tiveram curso da doença com padrão estático e a maioria com variabilidade persistente em 186/235 (79%) pacientes. Foram 227/235 (97%) pacientes com distonia isolada. Nenhum paciente apresentou lesão adquirida ou degenerativa. Foram 211/235 (90%) pacientes que apresentaram diagnóstico etiológico idiopático esporádico. CONCLUSÃO: A nova classificação de distonia não apresentou novos benefícios na prática clínica diária, comparada com classificações anteriores. O benefício maior foi uma reorganização e formação de novas definições e critérios para atividade de pesquisas científicas e identificação de casos mais raros. / INTRODUCTION: Dystonia is characterized by sustained muscle contractions, frequently causing twisting movements, repetitive or abnormal postures. Dystonia rating is based on three axes: age of onset, distribution and etiology. The definition established by the Committee of the Medical Research Foundation in dystonia in 1984, is still in use today. Claims have been made for a new proposal for dystonia rating. MATERIALS AND METHODS: This is a descriptive cross-sectional study of a number of patients with primary dystonia and dystonia-plus assisted in a Brazilian clinic specializing in movement disorders, from March 2015 to February 2016 data collection was performed demographic, clinical and severity of dystonia. The patients were re-evaluated and reclassified according to the new proposed classification for dystonia. We used descriptive methods to present the characteristics of the sample, the Shapiro-Wilk test for normal distribution, the Kruskal-Wallis test to compare independent groups and variables without normal distribution and the X2 test to compare nominal variables. RESULTS: Of the 289 patients, there were 235 patients evaluated according to body distribution, with blepharospasm 60 patients (26%), cervical dystonia 51 patients (22%), a member of dystonia 28 patients (12%), laryngeal dystonia 10 patients (4%), oromandibular dystonia 3 patients (1%), segmental dystonia 46 patients (20%), dystonia multifocal 23 patients (9%) and generalized dystonia 14 patients (6%). Were 227/235 (97%) patients with primary dystonia and 8 (3%) patients with dystonia-plus. There was a higher prevalence in females with 144 patients. Patients with generalized dystonia, multifocal, dopa-responsive dystonia and dystonia-parkinsonism had the age of onset of dystonia lower than all other groups (p = 0.0001). Patients with dystonia of the upper limb and task-specific dystonia had the early age of less dystonia other focal dystonias and segmental dystonia (p=0.0001). Patients with task-specific dystonia and focal upper limb dystonia have higher education to other groups (p=0.0001). On average, there were 5 ± 7.8 years for the definitive diagnosis of dystonia and 2 ± 1.5 doctors until the diagnosis of dystonia were confirmed. The sensory trick was more frequent in cervical dystonia 30/51 (59%) patients. According to the new classification, we found only change results in multifocal dystonia with 21/235 (9%) patients and generalized dystonia with 16/235 (6%) patients. Were 164/235 (70%) patients aged greater than 40 years. All patients had disease course with static standard and most with persistent variability in 186/235 (79%) patients. Were 227/235 (97%) patients with dystonia isolated. No patient had acquired or degenerative injury. Were 211/235 (90%) patients with sporadic idiopathic etiologic diagnosis. CONCLUSION: The new dystonia rating showed no new benefits in clinical practice compared with previous ratings. The greatest benefit was a reorganization and training of new definitions and criteria for scientific research activity and identification of rare cases.

Introdução: A cãibra do escrivão (CE) é uma distonia focal com contração sustentada e involuntária das mãos, dedos e/ou braços durante a escrita. Poucos estudos avaliaram desempenho motor e cognição na CE a partir de testes neuropsicológicos. Os achados do estudo podem colaborar na reabilitação da doença. Objetivo: Avaliar os desempenhos motor e cognitivo dos pacientes com CE. Métodos: Foram avaliados 21 pacientes e 21 controles pareados por sexo e idade. Dados clínicos foram colhidos e combinados com avaliação da presença de sintomas de depressão, ansiedade e apatia por meio, respectivamente, dos Inventários de Depressão e Ansiedade de Beck e da Escala de Apatia. A gravidade da distonia foi quantificada pelas escalas Writer\'s Cramp Rating Scale e The Arm Dystonia Disabiliity Scale. O desempenho motor foi avaliado pelo Purdue Pegboard Test (PPT). Demais testes neuropsicológicos aplicados foram: Mini Exame do Estado Mental (MEEM), Frontal Assessment Battery (FAB), Symbol Digit Modalities Test (SDMT), Stroop Test (ST) e Trail Making Test (TMT). Resultados: Pacientes com CE apresentaram desempenho inferior aos controles nos testes: MEEM, FAB e ST parte \"W\". Após correção para anos de estudo, variável demográfica com diferença estatística significativa entre os grupos, manteve-se diferença significativa apenas na FAB. Pacientes apresentaram desempenho inferior aos controles nas tarefas com a mão dominante, com ambas as mãos e no teste de montagem do PPT. Após correção para variável anos de estudo, não houve diferença significativa, apenas tendência estatística à performance inferior de pacientes na tarefa montagem do PPT. O desempenho da tarefa montagem do PPT em pacientes com CE teve forte correlação positiva com testes neuropsicológicos, e não com gravidade motora da distonia. Conclusões: O estudo sugere que pacientes com CE apresentem disfunção executiva. As aterações motoras encontradas na tarefa montagem do PPT dos pacientes com CE estão possivelmente relacionadas ao déficit no funcionamento executivo destes indivíduos. / Introduction: The writer\'s cramp (WC) is a focal dystonia with sustained and involuntary contraction of the hands, fingers and/or arms during writing. Few studies have evaluated motor performance and cognition of these patients using neuropsychological tests. The findings may contribute to rehabilitation of this disease. Objective: evaluate motor and cognitive performance of patients with WC. Methods: Twenty-one patients and 21 matched controls by sex and age were evaluated. Clinical data were collected, with assessment of symptoms of depression, anxiety, and apathy using the respective clinical scales: Beck Depression Inventory, Beck Anxiety Inventory and Apathy Scale. Severity of dystonia was quantified by Writer\'s Cramp Rating Scale and The Arm Dystonia Disability Scale. Motor performance was assessed by Purdue Pegboard Test (PPT). Other neuropsychological tests applied were: Mini Mental State Examination (MMSE), Frontal Assessment Battery (FAB), Symbol Digit Modalities Test (SDMT), Stroop Test (ST) and Trail Making Test (TMT). Results: Patients with WC had inferior performance to the controls in MMSE, FAB and ST part \"W\". After correction for years of study, a demographic variable with significant statistical difference between the groups, a significant difference was maintained only in FAB. Patients had inferior performance to the controls in these tasks of PPT: task with the dominant hand, with both hands and in PPT assembly. After correction for the variable \'years of study\', there was no significant difference, only statistical tendency to inferior performance of patients in PPT assembly. The performance of PPT assembly task in patients with WC had a strong positive correlation with neuropsychological tests, and not with motor severity of dystonia. Conclusions: The study suggests that patients with WC have executive dysfunction. The motor alterations in patients with WC is possibly related to deficit in executive functioning

DYT1 dystonia is an early onset central nervous system-based movement disorder characterized by uncontrolled sustained muscle contractions that can lead to debilitating abnormal postures. Though a genetic mutation in the gene TOR1A is responsible for most DYT1 cases, the low penetrance of the disease implicates additional genetic and environmental modifiers. Current therapeutic options for DYT1 dystonia are limited to symptomatic treatments with variable effectiveness. Currently, the underlying pathogenesis of this disease and the role of torsinA (torA), the protein product of TOR1A, in the development of this disease have yet to be established. In the first part of this thesis we aimed to further understand the effects of the TOR1A mutation at the molecular, cellular and organismal level in order to identify disease associated biomarkers that can be later used to measure the effectiveness of novel therapies. We found that expression of mutant torsinA (torA(ÄE)) in a cellular and an animal model of DYT1 had no significant effect on global transcription, despite its interaction with nuclear envelope proteins. Recent research has unearthed a role for microRNAs (miRNAs) in neuronal development and maturation. Consequently we explored whether torA(ÄE) expression in murine neural tissue was associated with changes in miRNA expression in young DYT1 knockin (KI) mice. Since the primary sight of dysfunction is still being debated, we profiled miRNA expression of the two strongest candidates, the striatum and cerebellum, both of which have well established roles in the control and coordination of muscle movements. We have identified several microRNAs that were uniquely altered in either the striatum or cerebellum and further research will be conducted to determine their usability as disease biomarkers. Finally, we were unable to identify motor phenotypes in either a DYT1 (KI) mice or a novel DYT1 transgenic model in open field, rotarod or staircase forepaw reaching tests. In the second part of this thesis we aimed to develop and evaluate the safety and efficacy of viral therapeutic RNAi constructs for in DYT1 murine models. DYT1 is an ideal candidate for this form of therapy due to its dominant inheritance, common mutation and potentially reversible phenotype. Virally delivered short-hairpin RNAs (shRNA) designed to knockdown torA(ÄE) in either an allele-specific or nonallele-specific manner were injected into the striatum of DYT1 transgenic or KI mice respectively. Unexpectedly, we found widespread lethal toxicity and behavioral abnormalities in mice injected with either therapeutic or control shRNAs that weren't observed in mice injected with no shRNAs. Further studies found that regions where toxic shRNAs were expressed corresponded with neuronal loss and glial activation. Finally, we found evidence that the severity of toxicity was influenced in part by the genetic background of the mice. In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.

DYT1 dystonia (DYT1) is a disabling inherited neurological disorder with juvenile onset. The genetic mutation in DYT1 leads to the deletion of a glutamic acid (E) residue in the protein torsinA. The function of torsinA and how the mutation leads to DYT1 is poorly understood. We hypothesize that how efficiently the disease-linked mutant protein is cleared may be critical for DYT1 pathogenesis. Therefore we explored mechanisms of torsinA catabolism, employing biochemical, cellular, and animal-based approaches. We asked if torsinA(wt) and torsinA(DE) are degraded preferentially through different catabolic mechanisms, specifically the ubiquitin proteasome pathway (UPP) and autophagy. We determined that torsinA(wt) is cleared by autophagy while torsinA(DE) is efficiently degraded by the UPP suggesting degradation processes can modulate torsinA(DE) levels. Proteins implicated in recognizing motifs on torsinA(DE) for targeting to the UPP represent candidate proteins that may modify DYT1 pathogenesis. We examined how removal of the hydrophobic domain and mutation of glycosylated asparagine residues on torsinA altered stability and catabolic mechanism. We found the glycosylation sites on torsinA are important for stability modulate its degradation through the UPP. F-box G-domain protein 1 (FBG1) has been implicated in degradation of glycosylated ER proteins. We hypothesized that FBG1 would promote torsinA degradation and demonstrated that FBG1 modulates levels of torsinA in a non-canonical manner through the UPP and autophagy. We examined if lack of FBG1 in a torsinA(DE) mouse model altered motor phenotypes. We saw no effect which suggests FBG1 does not alter DYT1 pathogenesis despite its promotion of torsinA(DE) degradation. In addition, we explored a potential mechanism for the previously described role of torsinA in modulating cytoplasmic protein aggregation. We hypothesized this endoplasmic reticulum (ER) resident protein would indirectly alter cytoplasmic protein aggregation through modulation of ER stress. We employed a poly-glutamine expanded repeat protein and pharmacological ER stressors to determine that torsinA does not alter poly-glutamine protein aggregation nor ER stress in a mammalian system. In summary, this thesis suggests proteins involved in the catabolism of torsinA(DE) may modify DYT1 pathogenesis and that torsinA and its DYT1-linked mutant are model proteins for investigating ER protein degradation by the UPP and autophagy.

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Botulinum toxin (BTX) is used in various fields of medicine, including the treatment of hyperhidrosis and cervical dystonia. Botox®, Dysport®, Xeomin® and NeuroBloc® are commercially available BTX products, which are formulated differently and their dosing units are unique. Dosage and concentration of the prepared solution for injection varies considerably among studies comparing the products. Improved guidelines on concentration and dosing when changing from one product to another are warranted. This would ensure the use of the lowest effective doses for good effect, minimal risk of antibody formation and side-effects as well as reduced costs. The aim of the present work was to find the most appropriate BTX concentration for each of the four products to achieve the highest sweat reducing effect and to investigate dose conversion ratios between Botox and Dysport in the treatment of cervical dystonia when the products are diluted to the same concentration, 100 U/ml. Paper I and II clearly confirm that it is crucial to consider the BTX concentration in a treatment regimen, especially when changing between different products. The optimal concentration to reduce sweating varies among the products and was found to be 25 U/ml for Botox and Xeomin, approximately 100 U/ml for Dysport and 50 U/ml for NeuroBloc. However, for NeuroBloc the optimal concentration might be even lower. In Paper III, which is a retrospective study using casebook notes from 75 patients with cervical dystonia, it was found that the most appropriate dose conversion ratio to use when switching from Botox to Dysport was 1:1.7. In Paper IV, Botox and Dysport were prospectively compared in a double-blind, randomized clinical trial in two different dose conversion ratios (1:3 and 1:1.7) when diluted to the same concentration (100 U/ml). No statistically significant difference was seen between Botox (1:3) and Dysport nor between Botox (1:1.7) and Dysport four weeks after treatment. Some of the secondary outcome observations, however, did indicate that the ratio 1:3 resulted in suboptimal efficacy of Botox but this must be further validated in a larger patient material.

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).