München, Techn. Univ., Diss., 2005.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2017. / Made available in DSpace on 2018-01-09T03:25:17Z (GMT). No. of bitstreams: 1 348740.pdf: 1813084 bytes, checksum: 9fa3d648d4ccff8a20e8857951c99b25 (MD5) Previous issue date: 2017 / A doença de Huntington (DH) é uma doença neurodegenerativa causada por uma expansão de repetições CAG no gene que codifica a proteína huntingtina. O estágio sintomático é definido pelo aparecimento de sintomas motores. Entretanto, sintomas psiquiátricos, incluindo humor deprimido, são características comuns da DH e podem ocorrer até uma década antes da manifestação dos sintomas motores. Os camundongos transgênicos YAC128 são um modelo da DH e apresentam comportamento tipo-depressivo e déficits cognitivos antes dos sintomas motores, similar ao que ocorre em humanos. O que nos permite estudar as alterações comportamentais emocionais sem confundir com os efeitos causados pela deficiência motora. Neste estudo os camundongos YAC128 foram utilizados para testar os efeitos do tratamento intranasal (i.n.) por 15 dias com o fator neurotrófico derivado do encéfalo (BDNF) na dose de 1,66 µg/kg na prevenção dos comportamentos tipo-depressivo. Os camundongos YAC128 tratados com BDNF apresentaram uma diminuição do comportamento anedônico nos testes de preferência a sacarose e no teste de borrifagem de sacarose. Além disso, o tratamento com BDNF i.n. foi capaz de prevenir o aumento do tempo de imobilidade no teste de suspensão pela cauda e no teste do nado forçado sem alterar a locomoção no campo aberto. Investigou-se posteriormente se o efeito do tipo-antidepressivo do BDNF estava associado com uma modulação da neurogênese hipocampal. Entretanto, o tratamento i.n. com BDNF por 15 dias aumentou a proliferação celular (Ki-67) e a diferenciação neuronal (DCX) no giro denteado (GD) hipocampal dos camundongos selvagens sem alterar estes parâmetros nos animais YAC128. Com o objetivo de verificar se as alterações comportamentais ocorreram devido a um aumento na neuroplasticidade avaliou-se a densidade dendrítica destes animais. Nossos resultados mostram que o tratamento com BDNF na dose utilizada não foi capaz de modificar a plasticidade dendrítica. Em conclusão, nossos resultados sugerem que a administração não invasiva de BDNF por via i.n. pode ter um importante potencial terapêutico para tratar sintomas tipo-depressivos em pacientes em estágios iniciais da DH sem alterar a neurogênese e a plasticidade dendrítica. / Abstract : Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the Huntington?s disease gene. The symptomatic stage of the disease is defined by the onset of motor symptoms. However, psychiatric symptoms, including depressive humor, are common features of HD and can occur a decade before the manifestation of motor symptoms. The YAC128 HD transgenic mouse model develops motor deficits at a later stage, allowing more time to study the occurrence of early-stage depressive behaviors without the confounding effects of motor impairment. We used this HD mouse model to test the effects of intranasal (i.n.) brain-derived neurotrophic factor (BDNF) treatment for 15 days, with a dose of 1.66 µg/kg, in the occurrence of depressive-like behaviors during the early-stage of disease progression. The BDNF-treated YAC128 mice showed a decrease in anhedonic behavior in the sucrose preference test and in the splash test. In addition, i.n. treatment with BDNF was able to prevent the increase in the immobility time in the tail suspension test and in the forced swim test without altering the locomotion investigated in the open field test. Furthermore, we also investigated whether the antidepressant-like effects of BDNF were associated with an increase in adult hippocampal neurogenesis. However, BDNF treatment only increased cell proliferation (ki-67) and neuronal differentiation (DCX) in the hippocampal dentate gyrus (DG) of wild-type (WT) mice without altering these parameters in their YAC128 counterparts. To verify if the behavioral changes were caused by an increase in the neuroplasticity, we analyzed dendritic density in the hippocampal DG of WT and YAC128 mice treated with BDNF, using Sholl Analyses. Our results show that BDNF i.n. administration for 15 days was not able of modifying dendritic plasticity. In conclusion, our results suggest that non-invasive administration of BDNF via the i.n. route may have a therapeutic potential for the treatment of mood disorders in early symptomatic HD patients without altering neurogenesis and dendritic plasticity.

Introdução: A doença de Huntington é uma doença neurodegenerativa autossômica dominante causada pela expansão de um segmento repetitivo CAG no gene HTT. Caracteriza-se por transtornos do movimento, em especial coreia, alterações comportamentais e declínio cognitivo. A doença tem um curso progressivo e inexorável. Objetivos: Este estudo tem os seguintes objetivos: (1) revisar de forma sistemática os estudos sobre doença de Huntington na América Latina; (2) determinar a proporção de casos brasileiros com fenótipo doença de Huntington que são portadores da expansão no gene HTT; (3) caracterizar as transmissões CAGexp na coorte HTT e determinar se idade do genitor está associado à piora das instabilidades; (4) descrever a progressão das manifestações neurológicas, IMC e concentrações séricas de carnitina livre, valina, leucina e isoleucina durante o período de seguimento; (5) definir se as progressões desses candidatos a biomarcadores acompanham a piora neurológica e podem superar a sensibilidade da escala UHDRS na descrição da progressão da doença. Métodos: Realizamos uma revisão sistemática dos aspectos genéticos da doença de Huntington na América Latina. Em seguida recrutamos pacientes com o fenótipo doença de Huntington de vários centros do país através da Rede Neurogenética e avaliamos a proporção de diagnósticos confirmados. Nas famílias provenientes de nosso centro e dos hospitais São Paulo (UNIFESP) e Gaffrée e Guinle (UNIRIO), recrutamos indivíduos sintomáticos e em risco para caracterizar as transmissões CAGexp e avaliar fatores que determinam as instabilidades. Nos mesmos centros que avaliamos as transmissões, selecionamos um grupo de indivíduos sintomáticos e em risco para determinar as concentrações séricas de carnitina livre, valina, leucina e isoleucina e as correlacionar com variáveis de gravidade da doença (IMC, escala UHDRS). O comportamento desses potenciais biomarcadores foi avaliado de forma prospectiva em um grupo de indivíduos sintomáticos. Resultados: Constatamos a escassez de estudos sobre aspectos moleculares da doença de Huntington na América Latina e Brasil. Das 104 famílias com fenótipo doença de Huntington recrutadas em diversos centros do Brasil, 93 (89,4%) apresentavam expansão CAG no gene HTT; 4 (3,8%) apresentam o diagnóstico de HDL-2 (Huntington’s disease-like 2); 1 (1%) apresentam expansão CAG no gene ATXN2 (SCA2); e 6 (5,8%) ficaram sem diagnóstico. Um número substancial de portadores foi recrutado e permitiu estimar a prevalência mínima da doença, ao menos no Rio Grande do Sul. Além disso, trinta e duas transmissões de genitor para filho (13 paternas e 19 maternas) foram obtidas nessa coorte. As transmissões paternas foram mais instáveis do que as maternas (p=0.005, Mann- Whitney). As transmissões paternas foram mais frequentemente expansões (69,2% expansões) e as maternas foram mais estáveis (57,9% estáveis) (p=0,004, Fisher). Em 51 pares de irmãos incluídos, a idade do progenitor no momento da concepção não pareceu estar relacionada com aumento da instabilidade do CAGexp. Na avaliação dos potenciais biomarcadores, incluímos 116 indivíduos (74 sintomáticos, 20 portadores assintomáticos e 22 não portadores). No baseline, os níveis (mediana/intervalo interquartil) de valina estavam reduzidos tanto em indivíduos sintomáticos (110 / 88,4-131) como nos portadores assintomáticos (101,25 / 79,6-123,5) em comparação com não portadores (123 / 98.65– 164.25) (p=0.018 e p=0.042, Mann-Whitney). Não houve diferença entre os grupos nos níveis de carnitina e isoleucina+leucina (ns, Kruskal-Wallis). Na avaliação de seguimento em 43 indivíduos sintomáticos (mediana=1,08 anos), o escore motor total da UHDRS aumentou 4,8 pontos (p = 0.001, GEE) e a escala funcional TFC reduziu 0,89 pontos (p <0.0001, GEE). O IMC (p=0,52, GEE) e os níveis de valina (p=0,43, GEE) permaneceram estáveis e os níveis de carnitina (p=0,039, GEE) e isoleucina+leucina (p=0,037, GEE) aumentam durante o seguimento. Conclusão: Os resultados desses estudos conseguiram traçar um perfil mais acurado da doença de Huntington no Brasil, tanto do ponto de vista epidemiológico quanto no comportamento das transmissões CAG nas famílias identificadas. A progressão da doença, medida pela UHDRS, foi semelhante à observada em coortes do Hemisfério Norte. Além disso, verificamos que a carnitina livre e os aminoácidos de cadeia ramificada não são bons biomarcadores de progressão na doença de Huntington. / Introduction: Huntington's disease is an autosomal dominant neurodegenerative disease caused by an expansion of a repetitive CAG segment in the HTT gene. Movement disorders (especially chorea), behavioral problems and cognitive decline characterize the disease, which has an inexorable progression. Up to now, there is no disease modifying treatment for Huntington's disease. Objectives: This study aimed to address the following objectives: (1) review, in a systematic manner, studies of Huntington's disease in Latin America; (2) determine the proportion of cases with Huntington's disease phenotype that are carriers of the expansion in the HTT gene; (3) characterize CAGexp transmissions in the HTT cohort and determine a possible association of the age of the parent with further instability of CAGexp; (4) describe the progression of neurological manifestations, BMI and serum concentrations of free carnitine, valine, leucine and isoleucine during a follow-up period; (5) determine whether changes in time of these compounds are correlated with the simultaneous neurological worsening and if they may overcome the sensitivity of the UHDRS scale in describing the progression of the disease. Methods: We performed a systematic review of the genetic aspects of Huntington's disease in Latin America. We then recruit patients with the Huntington's disease phenotype from various centers throughout the country through the Rede Neurogenética and evaluate the proportion of confirmed diagnoses. In families from our center and hospitals São Paulo (UNIFESP) and Gaffrée and Guinle (UNIRIO), we recruit symptomatic and at-risk individuals to characterize CAGexp transmissions and evaluate factors that determine instabilities. In the same centers that evaluated the transmissions, we selected a group of symptomatic and at-risk individuals to determine serum free carnitine, valine, leucine, and isoleucine levels and to correlate them with disease severity variables (BMI, UHDRS scale). The behavior of these potential biomarkers was evaluated prospectively in a group of symptomatic individuals. Results: We found a lack of studies on genetic aspects of Huntington's disease in Latin America and Brazil. Ninety-three (89.4%) out of 104 families with Huntington's disease phenotype recruited in several centers in Brazil had CAG expansion in the HTT gene; 4 (3.8%) had the diagnosis of HDL-2 (Huntington's disease-like 2); 1 (1%) a CAG expansion in the ATXN2 gene (SCA2); and 6 (5.8%) remained undiagnosed. Two hundred and seventy-nine carriers were included in our cohort, allowing to estimate the minimal prevalence of disease in Rio Grande do Sul as 1.85/100,000. Thirty-two transmissions (13 paternal and 19 maternal) were obtained. Parental transmission was more unstable than maternal transmission (p = 0.005, Mann-Whitney). Parental transmission most often expanded (69.2%), and maternal was more often stable (57.9% stable) (p = 0.004, Fisher). In 51 pairs of siblings included, age of the progenitor at conception did not appear to be related to increased instability of CAGexp. A total of 116 individuals (74 symptomatic, 20 asymptomatic carriers and 22 non-carriers) were included in the biomarkers analysis. At baseline, valine (median / interquartile range) levels were reduced in both symptomatic (110 / 88.4-131) and asymptomatic carriers (101.25 / 79.6-123.5) when compared to non-carriers (123 / 98.65- 164.25) (p = 0.018, p = 0.042, Mann-Whitney). There was no difference between groups in their levels of carnitine and isoleucine + leucine (ns, Kruskal-Wallis). In the follow-up evaluation in 43 symptomatic individuals (median = 1.08 years), the total motor score of the UHDRS increased by 4.8 points (p = 0.001, GEE) and the functional TFC scale decreased 0.89 points (p <0.0001, GEE). BMI (p = 0.52, GEE) and valine levels (p = 0.43, GEE) remained stable and levels of carnitine (p = 0.039, GEE) and isoleucine + leucine (p=0.037, GEE) increased during follow-up. Conclusion: The results of these studies drew a more accurate profile of Huntington's disease in Brazil, since they generated both a minimal prevalence in our region and the behavior of CAG transmissions in the Brazilian families. Disease progression as measured by UHDRS was similar to the progression rate observed in cohorts from the North Hemisphere. In addition, we have found that free carnitine and branched-chain amino acids are not suitable to be considered good biomarkers of HD progression.

Introdução: A doença de Huntington é uma doença neurodegenerativa autossômica dominante causada pela expansão de um segmento repetitivo CAG no gene HTT. Caracteriza-se por transtornos do movimento, em especial coreia, alterações comportamentais e declínio cognitivo. A doença tem um curso progressivo e inexorável. Objetivos: Este estudo tem os seguintes objetivos: (1) revisar de forma sistemática os estudos sobre doença de Huntington na América Latina; (2) determinar a proporção de casos brasileiros com fenótipo doença de Huntington que são portadores da expansão no gene HTT; (3) caracterizar as transmissões CAGexp na coorte HTT e determinar se idade do genitor está associado à piora das instabilidades; (4) descrever a progressão das manifestações neurológicas, IMC e concentrações séricas de carnitina livre, valina, leucina e isoleucina durante o período de seguimento; (5) definir se as progressões desses candidatos a biomarcadores acompanham a piora neurológica e podem superar a sensibilidade da escala UHDRS na descrição da progressão da doença. Métodos: Realizamos uma revisão sistemática dos aspectos genéticos da doença de Huntington na América Latina. Em seguida recrutamos pacientes com o fenótipo doença de Huntington de vários centros do país através da Rede Neurogenética e avaliamos a proporção de diagnósticos confirmados. Nas famílias provenientes de nosso centro e dos hospitais São Paulo (UNIFESP) e Gaffrée e Guinle (UNIRIO), recrutamos indivíduos sintomáticos e em risco para caracterizar as transmissões CAGexp e avaliar fatores que determinam as instabilidades. Nos mesmos centros que avaliamos as transmissões, selecionamos um grupo de indivíduos sintomáticos e em risco para determinar as concentrações séricas de carnitina livre, valina, leucina e isoleucina e as correlacionar com variáveis de gravidade da doença (IMC, escala UHDRS). O comportamento desses potenciais biomarcadores foi avaliado de forma prospectiva em um grupo de indivíduos sintomáticos. Resultados: Constatamos a escassez de estudos sobre aspectos moleculares da doença de Huntington na América Latina e Brasil. Das 104 famílias com fenótipo doença de Huntington recrutadas em diversos centros do Brasil, 93 (89,4%) apresentavam expansão CAG no gene HTT; 4 (3,8%) apresentam o diagnóstico de HDL-2 (Huntington’s disease-like 2); 1 (1%) apresentam expansão CAG no gene ATXN2 (SCA2); e 6 (5,8%) ficaram sem diagnóstico. Um número substancial de portadores foi recrutado e permitiu estimar a prevalência mínima da doença, ao menos no Rio Grande do Sul. Além disso, trinta e duas transmissões de genitor para filho (13 paternas e 19 maternas) foram obtidas nessa coorte. As transmissões paternas foram mais instáveis do que as maternas (p=0.005, Mann- Whitney). As transmissões paternas foram mais frequentemente expansões (69,2% expansões) e as maternas foram mais estáveis (57,9% estáveis) (p=0,004, Fisher). Em 51 pares de irmãos incluídos, a idade do progenitor no momento da concepção não pareceu estar relacionada com aumento da instabilidade do CAGexp. Na avaliação dos potenciais biomarcadores, incluímos 116 indivíduos (74 sintomáticos, 20 portadores assintomáticos e 22 não portadores). No baseline, os níveis (mediana/intervalo interquartil) de valina estavam reduzidos tanto em indivíduos sintomáticos (110 / 88,4-131) como nos portadores assintomáticos (101,25 / 79,6-123,5) em comparação com não portadores (123 / 98.65– 164.25) (p=0.018 e p=0.042, Mann-Whitney). Não houve diferença entre os grupos nos níveis de carnitina e isoleucina+leucina (ns, Kruskal-Wallis). Na avaliação de seguimento em 43 indivíduos sintomáticos (mediana=1,08 anos), o escore motor total da UHDRS aumentou 4,8 pontos (p = 0.001, GEE) e a escala funcional TFC reduziu 0,89 pontos (p <0.0001, GEE). O IMC (p=0,52, GEE) e os níveis de valina (p=0,43, GEE) permaneceram estáveis e os níveis de carnitina (p=0,039, GEE) e isoleucina+leucina (p=0,037, GEE) aumentam durante o seguimento. Conclusão: Os resultados desses estudos conseguiram traçar um perfil mais acurado da doença de Huntington no Brasil, tanto do ponto de vista epidemiológico quanto no comportamento das transmissões CAG nas famílias identificadas. A progressão da doença, medida pela UHDRS, foi semelhante à observada em coortes do Hemisfério Norte. Além disso, verificamos que a carnitina livre e os aminoácidos de cadeia ramificada não são bons biomarcadores de progressão na doença de Huntington. / Introduction: Huntington's disease is an autosomal dominant neurodegenerative disease caused by an expansion of a repetitive CAG segment in the HTT gene. Movement disorders (especially chorea), behavioral problems and cognitive decline characterize the disease, which has an inexorable progression. Up to now, there is no disease modifying treatment for Huntington's disease. Objectives: This study aimed to address the following objectives: (1) review, in a systematic manner, studies of Huntington's disease in Latin America; (2) determine the proportion of cases with Huntington's disease phenotype that are carriers of the expansion in the HTT gene; (3) characterize CAGexp transmissions in the HTT cohort and determine a possible association of the age of the parent with further instability of CAGexp; (4) describe the progression of neurological manifestations, BMI and serum concentrations of free carnitine, valine, leucine and isoleucine during a follow-up period; (5) determine whether changes in time of these compounds are correlated with the simultaneous neurological worsening and if they may overcome the sensitivity of the UHDRS scale in describing the progression of the disease. Methods: We performed a systematic review of the genetic aspects of Huntington's disease in Latin America. We then recruit patients with the Huntington's disease phenotype from various centers throughout the country through the Rede Neurogenética and evaluate the proportion of confirmed diagnoses. In families from our center and hospitals São Paulo (UNIFESP) and Gaffrée and Guinle (UNIRIO), we recruit symptomatic and at-risk individuals to characterize CAGexp transmissions and evaluate factors that determine instabilities. In the same centers that evaluated the transmissions, we selected a group of symptomatic and at-risk individuals to determine serum free carnitine, valine, leucine, and isoleucine levels and to correlate them with disease severity variables (BMI, UHDRS scale). The behavior of these potential biomarkers was evaluated prospectively in a group of symptomatic individuals. Results: We found a lack of studies on genetic aspects of Huntington's disease in Latin America and Brazil. Ninety-three (89.4%) out of 104 families with Huntington's disease phenotype recruited in several centers in Brazil had CAG expansion in the HTT gene; 4 (3.8%) had the diagnosis of HDL-2 (Huntington's disease-like 2); 1 (1%) a CAG expansion in the ATXN2 gene (SCA2); and 6 (5.8%) remained undiagnosed. Two hundred and seventy-nine carriers were included in our cohort, allowing to estimate the minimal prevalence of disease in Rio Grande do Sul as 1.85/100,000. Thirty-two transmissions (13 paternal and 19 maternal) were obtained. Parental transmission was more unstable than maternal transmission (p = 0.005, Mann-Whitney). Parental transmission most often expanded (69.2%), and maternal was more often stable (57.9% stable) (p = 0.004, Fisher). In 51 pairs of siblings included, age of the progenitor at conception did not appear to be related to increased instability of CAGexp. A total of 116 individuals (74 symptomatic, 20 asymptomatic carriers and 22 non-carriers) were included in the biomarkers analysis. At baseline, valine (median / interquartile range) levels were reduced in both symptomatic (110 / 88.4-131) and asymptomatic carriers (101.25 / 79.6-123.5) when compared to non-carriers (123 / 98.65- 164.25) (p = 0.018, p = 0.042, Mann-Whitney). There was no difference between groups in their levels of carnitine and isoleucine + leucine (ns, Kruskal-Wallis). In the follow-up evaluation in 43 symptomatic individuals (median = 1.08 years), the total motor score of the UHDRS increased by 4.8 points (p = 0.001, GEE) and the functional TFC scale decreased 0.89 points (p <0.0001, GEE). BMI (p = 0.52, GEE) and valine levels (p = 0.43, GEE) remained stable and levels of carnitine (p = 0.039, GEE) and isoleucine + leucine (p=0.037, GEE) increased during follow-up. Conclusion: The results of these studies drew a more accurate profile of Huntington's disease in Brazil, since they generated both a minimal prevalence in our region and the behavior of CAG transmissions in the Brazilian families. Disease progression as measured by UHDRS was similar to the progression rate observed in cohorts from the North Hemisphere. In addition, we have found that free carnitine and branched-chain amino acids are not suitable to be considered good biomarkers of HD progression.

In neurons, modification by the lipid palmitate regulates trafficking and function of signaling molecules, neurotransmitter receptors and associated synaptic scaffolding proteins. HIP14 (huntingtin interacting protein 14) is the first identified and characterized mammalian palmitoyl transferase that regulates this process. I have shown that HIP14 has striking effects on modulating trafficking and function of many proteins important for synapse formation and plasticity such as PSD-95, a postsynaptic scaffolding molecule. The importance of the finding that HIP14 is a neuronal palmitoyl transferase is further emphasized by our recent discovery that huntingtin protein folding, trafficking and function are regulated by the enzyme HIP14. Expansion of the polyglutamine tract in huntingtin as seen in Huntington Disease (HD) results in reduced association with HIP14 and decreased palmitoylation of huntingtin, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity. By manipulating HIP14 levels through expression or knockdown, we can manipulate the number of huntingtin inclusion bodies and neuronal cell viability. Overall, these discoveries offer novel mechanism for HD pathogenesis and provide new approaches to therapy for HD. The tight association of HIP14 with wild-type huntingtin, which differs from other known enzyme-substrate interactions, indicates that huntingtin serves other functions beyond being a substrate of HIP14. I have discovered that, in vitro, wild-type huntingtin may facilitate activity of HIP14 to palmitoylate other neuronal substrates such as SNAP25, PSD95 and GAD65. By contrast, mutant htt does not act this way, probably due to lack of interaction with HIP14. Furthermore, immunoprecipitated HIP14 from huntingtin+/- mice also exhibits less enzyme activity in palmitoylating GST-SNAP25 in vitro, suggesting that decreased huntingtin expression compromises HIP14 activity. In vivo, using Acyl Biotin Exchange assay, I have also found that palmitoylation of a number of presynaptic and postsynaptic proteins that are involved in neurotransmission are reduced in huntingtin+/- mice. This study not only ascribes an important biochemical function to wild-type huntingtin, but also suggests that defects in protein palmitoylation in general due to mutant huntingtin lack of ability to facilitate HIP14 activity may contribute to the pathogenesis of HD. / Medicine, Faculty of / Graduate

Compared with saline injected controls, rats with bilateral injections of kainic acid (KA) in the dorsal striatum showed temporary aphagia and adipsia, long-lasting body weight decreases, increased locomotor response to d-amphetamine, increased spontaneous nocturnal locomotor activity, increased resistance to extinction, impaired acquisition and retention of avoidance behavior and increased latencies to leave start boxes in various mazes. The KA injections resulted in loss of local neurons in the dorsal striatum, with no appreciable damage either to dopaminergic terminals or to extrinisic myelinated axons, thus supporting both the selective neurotoxic action of KA on neuronal perikarya and the proposed similarity of KA-induced striatal lesions with those found in the caudate-putamen of patients with Huntington's disease (HD). The present results demonstrate that KA striatal lesioned rats also show behavioral and pharmacological similarities with HD patients. In addition, they support the view that HD is characterized by a "subcortical dementia syndrome". A review of HD is also presented. / Medicine, Faculty of / Graduate

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a mutation involving an expansion of the CAG trinucleotide repeats in the gene encoding for huntingtin (HTT) protein. The discovery of the disease-causing faulty gene (mutant huntingtin; mHTT) has enabled valid presymptomatic gene assessment for HD with results being categorized as ‘gene-expanded (GE; CAG repeats ≥ 36)' or ‘gene non-expanded (GNE)'. Individuals tested to be gene-expanded are destined to develop HD symptoms and will receive clinical diagnosis at an average age of 40 years when abnormal motor symptoms manifest. Those who are GNE will not develop HD. The availability of genetic testing has also provided a valuable research opportunity to study the pathoetiology of HD in PreHD subjects (those tested to be ‘gene-expanded' but are in the prediagnostic stage of HD). The genetic mutation results in widespread neuronal degeneration preferentially within the striatum. The clinical manifestations of HD include a triad of motor, cognitive and psychiatric symptoms. Challenging the classical view of HD as a neurodegenerative disease, recent studies have brought about a conceptual shift to include abnormal neurodevelopmental aspects in the etiology of HD1, based on the notion that lifelong HTT gene mutation may compromise HTT's crucial role in normal brain development. The fronto-striatal circuitry has been a main interest in HD research for its profound pathological association with symptom manifestation and marked neuroanatomical change. However, no study to date has investigated the neuropathological alteration of the fronto-striatal circuitry during childhood in mHTT carriers. In line with the proposed new perspective, the overall hypothesis of the current proposal is that the deteriorating effect of mHTT on the fronto-striatal circuits stems from abnormal development of these circuits. Therefore, the main goal of the current study was to enhance our understanding of how mHTT alters the evolving capacity of the fronto-striatal circuitry from a developmental perspective. To this end, the study examined the fronto-striatal circuit structure (using magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI)) and function (using resting state functional MRI and cognitive/behavior tests) in children (6-18 years of age) at risk for HD. Healthy control children with no family history of HD were also evaluated. For research purposes only, children at risk for HD were genotyped and designated as GE or GNE based the result of their genetic testing. While the assessment of the fronto-striatal circuit development focused on anatomical delineations, the tests of the fronto-striatal circuit functionality were carried out separately for the three distinct cognitive, motor and affective loops within the fronto-striatal circuitry. The overall aim of the project was to evaluate the effect of mHTT on subcortical brain structures, white matter connection development and functional integrity of the cognitive, motor and affective loops within the fronto-striatal circuitry in Gene-Expanded (GE) children compared to those of Healthy Control (HC) children and again to those who are Gene Non-Expanded (GNE). Test of the hypothesized mHTT-associated developmental alteration of the fronto-striatal circuitry was addressed in two specific aims: Specific Aim #1: To observe subcortical gray matter volumes and white matter integrity of the fronto-striatal circuitry in children at risk for HD Decline in corpus striatal volume as well as aberrant fronto-striatal circuit connectivity has been reported in manifest HD patients and also in preHD adults.2-4 However, there is no experimental evidence of the onset or pattern of the pathophysiological change in the fronto-striatal circuitry. Therefore, volumes of subcortical structures and white matter integrity were measured in order to assess the development of the fronto-striatal circuitry in children at risk for HD. Specific Aim #2: To assess the functionalities of specific fronto-striatal circuit loops in children at risk for HD Functional abnormalities of the fronto-striatal circuitry have been observed in HD patients as well as in preHD adults.5 In order to closely examine the mHTT effect on fronto-striatal circuit function, resting state functional connectivity as well as performances on cognitive and behavioral tasks tapping into the functionality of the three fronto-striatal loops were evaluated in children at risk for HD. It was hypothesized that GE children would have diminished fronto-striatal circuit function. The GE children were predicted to show statistically significant deficits in 1) resting state functional connectivity between specific frontal lobe areas and striatal sub-regions, 2) cognitive control, 3) motoric control and 4) behavior control when compared to healthy controls and children without the HTT CAG expansion. The current study reports the baseline profile of the fronto-striatal structure and function in children at risk for HD. We have found that children who are on average 30 years ahead of HD diagnosis to have developmental alterations in the brain structure and function directly linked to the effect of mHTT. Brain morphology analysis revealed specific subcortical gray and white matter changes. The changes include disproportionately smaller caudate and putamen volumes and increased radial diffusivity localized to the external capsule which were more evident in males with HTT gene expansion. Fronto-striatal circuit functional assessment revealed a drop in motor functionality (both at rest and active performance) and externalizing behavior problems indicative of compromised inhibition than aggression. Children from HD families but do not have the genetic mutation also showed development aberrations in both brain structure and function when compared to healthy controls. Importantly, the altered structural morphology and functional profile seen in the GNE group differed from that of the GE children, emphasizing the impact of mHTT. The findings from those who share similar household environment but differ in genetic expansion status are important in highlighting the potential interaction of gene-environment effect on the manifestation of mHTT related changes seen in the children with the genetic expansion for HD. Investigation of subtle but persistent effects of mHTT on normal neural developmental processes may further our understanding of the pathogenesis of HD. Continuous longitudinal comprehensive assessments of the mHTT associated neurophenotype would aid in prognostic scenario estimation and thereby lead to effective clinical decision making to maximize the benefit of early intervention.

L'atrophie du striatum causée par la dégénérescence massive des neurones épineux de projection de cette composante des ganglions de la base est la caractéristique neuropathologique la plus frappante de la maladie d'Huntington (MH). Même si cette pathologie épargne la plupart des interneurones striataux, nous ne disposons que de très peu d'information concernant le sort des interneurones géants exprimant la calrétinine dans la MH. Notre objectif était donc de caractériser le phénotype chimique de ces neurones, de les dénombrer et de cartographier leur distribution dans le striatum de sujets normaux et de patients atteints de la MH. Les techniques de double immunofluorescence utilisant des anticorps dirigés contre la calrétinine (CR) et la choline acétyltransférase (ChAT), ont permis de visualiser les neurones à calrétinine géants et d'étudier leur niveau de colocalisation avec la ChAT. Trois types d'interneurones géants ont été visualisés dans le striatum d'individus sains : a) Les neurones exprimant uniquement la CR; b) les neurones exprimant la CR et la ChAT ; et c) les neurones exprimant uniquement la ChAT. Les neurones CR+/ChAT+ étaient plus nombreux que les neurones exprimant ChAT seulement, eux-mêmes plus nombreux que les neurones exprimant CR uniquement. La densité des neurones CR+/ChAT+ et CR-/ChAT- était diminuée de moitié dans le striatum des patients atteints de la MH comparés aux contrôles. Cette diminution suggère que ces neurones sont affectés par la MH. Cependant des études effectuées à l'aide d'un anticorps contre le récepteur à substance P, neurokinine-1 (NK-1R), comme marqueur des interneurones géants exprimant la CR et ChAT, indique que ces neurones sont sélectivement épargnés par le processus dégénératif de la MH. Ainsi, l'apparente diminution du nombre de neurones CR+/ChAT+ et CR-/ChAT+ ne semble pas être la conséquence d'une dégénérescence neuronale, mais plutôt d'une diminution de l'expression de ces deux protéines. Nos résultats indiquent que les processus dégénératifs impliqués dans la MH affectent l'expression des protéines CR et ChAT par les interneurones géants, sans causer la mort de ceux-ci. / The severe atrophy of the striatum that characterizes Huntington's disease (HD) is due to massive losses of striatal medium spiny projection neurons. In contrast, interneurons are relatively spared in HD, but little is known of the fate of the large interneurons that express calretinin (CR) in HD. We addressed this issue by applying a double immunofluorescent labeling technique to post-mortem material obtained from HD patients and age-matched controls. Antibodies against CR and choline acetyltransferase (ChAT) were used to compare the distribution, density and degree of ChAT co-localization of the CR+ striatal interneurons in normal and HD cases. The normal human striatum was found to contain 3 types of large interneurons: a) neurons expressing CR only; b) neurons displaying ChAT only; and c) neurons co-expressing CR and ChAT. The CR+/ChAT+ neurons outnumbered neurons expressing ChAT only, which were themselves more numerous than neurons displaying CR only. A two-fold decrease in the density of CR+/ChAT+, CR-/ChAT+ occurred in the striatum of HD patients compared to that of controls, suggesting that these neurons are affected in HD. However, studies undertaken with neurokinine-1 receptor NK1R as a marker of large CR+ and ChAT+ neurons revealed that these striatal neurons are selectively spared in HD patients. Hence, the apparent decrease in the number of CR+/ChAT+, CR-/ChAT+ neurons in HD does not appear to result from a degeneration of these cells, but rather from a marked diminution of the expression of CR and ChAT. Our data indicate that the neurodegenerative processes at play in HD affect the expression of CR and ChAT proteins without causing the death of these large striatal interneurons.

La maladie de Huntington (MH) est une maladie neurodégénérative caractérisée par plusieurs symptômes moteurs, cognitifs et psychiatriques, causés par une expansion du trinucléotide CAG présent dans le gène huntingtine. L’expression de la protéine correspondante mène éventuellement à un dysfonctionnement et une mort cellulaire. OBJECTIFS: À ce jour, il n’existe pas de thérapies efficaces afin de traiter ou ralentir la MH, mais les essais actuels suggèrent qu’une réduction de la quantité de protéines mutantes pourrait être bénéfique. Dans cette étude, il a été question d’utiliser le système CRISPR/Cas9. Grâce à l’utilisation de l’enzyme Cas9, cette technologie d’édition du génome bien précise permet de cibler et corriger une mutation génétique; ce qui permettrait dans le cas de la MH de réduire le gène causant la pathologie. MÉTHODE : Un modèle de souris MH et un modèle contrôle sauvage, âgés de 12 mois, ont reçu des injections stéréotaxiques intracérébrales de virus AAV9 contenant la Cas9D10A et/ou l’ARN guide (SgCTG/eGFP). Des tests cognitifs et moteurs ont été effectués pendant 3 mois après les injections virales. Des tissus et du sang ont été récoltés lors du sacrifice des animaux. Les échantillons de cerveaux ont été analysés par immunofluorescence et par western blot pour déterminer le degré de contraction ainsi que le niveau d’expression et de colocalisation de la Cas9D10A et de l’ARN guide. RÉSULTATS: Une amélioration de certains comportements est notable après 3 mois post-injection chez les souris MH traitées avec les deux constructions. Les analyses biochimiques ont permis de détecter l’expression des deux vecteurs et une réduction de la quantité de protéines mutées. CONCLUSION : CRISPR/Cas9 pourrait potentiellement être utilisé pour réduire la quantité de protéines mutées et améliorer certains comportements cognitifs et moteurs. Une optimisation de l’utilisation de cette technologie pourrait mener à une thérapie intéressante pour la MH / Huntington’s disease (HD) is a neurodegenerative disorder characterized by a triad of motor, cognitive and psychiatric symptoms which results from an expansion of the CAG tract in the huntingtin gene. This mutation encodes for a longer polyglutamine stretch in the mutant huntingtin protein. Expression of the mutant protein eventually leads to cell dysfunction and death. OBJECTIVE: To this day, there is no disease-modifying therapy available for HD, but ongoing trials suggest that reducing the amount of the mutant protein could be beneficial. In this study we used the CRISPR/Cas9 system, a precise gene editing technology which can be directly employed with its partner enzyme, Cas9, to specifically target and correct genetic mutations, to shrink the causative mutation associated with HD. METHODS: A mice model of HD and littermate controls were aged to 12 months prior to undergoing stereotaxic intracerebral injections of AAV9 viruses containing either Cas9 D10A nickase and/or guide RNA (SgCTG/eGFP) targeted to the CAG expansion. Cognitive and motor testing were performed for 3 months after viral infection. Tissues and blood were collected at the completion of the protocol. Brain samples were analysed using immunofluorescence and western blotting to determine the degree of contraction achieved and the degree of expression and colocalization of GFP (guide RNA) and HA tags (Cas9D10A). RESULTS: Mild behavioural improvements were detected 3 months after co-administration of the two viral constructs to HD mice. Importantly, biochemical studies detected expression of both the guide RNA and the Cas9 in addition to a reduction in the amount of mHTT detected in HD mice receiving both viruses. CONCLUSION: CRISPR/Cas9 can be used to reduce the amount of mHTT and this is associated with improvements in cognitive and fine motor deficits. Further optimization of this technology could lead to a clinically relevant therapy.

O objetivo deste trabalho foi caracterizar as alterações de linguagem na doença de Huntington e correlacioná-las com os transtornos motores, cognitivos, psiquiátricos e, também, com o tempo de doença. Foram estudados 26 indivíduos, divididos em grupo leve (11 doentes) e moderado (15 doentes), comparados com dois grupos controle. Foram encontradas alterações em provas de compreensão e expressão da linguagem oral e gráfica para os dois grupos de doentes. Evidências sugerem que não há prejuízo nas representações semânticas, e que as dificuldades de linguagem estão relacionadas com o declínio cognitivo global e, especialmente, com o prejuízo das funções executivas. As alterações de linguagem se correlacionaram com o desempenho em tarefas cognitivas, mas não com as alterações motoras ou psiquiátricas da doença. Também não foi encontrada correlação entre o desempenho de linguagem e o tempo de doença / The objective of this study was to characterize the language alterations in Huntington's disease and how they relate to severity of motor, cognitive, psychiatric disturbances and also with the disease duration. Twenty-six (26) individuals were divided into groups characterized as lightly (11) and moderately ill (15) and compared with two control groups. Alterations in exams of language comprehension and expression were noticed for both groups of sick individuals. The result indicates no evident loss in semantic representation. The language difficulties are related to a global cognitive decline and, principally, to loss of executive functions. The language alterations were significantly correlated to performance in cognitive tasks, but not to the motor or psychiatric alterations of the disease. There was also no correlation observed between the language performance and duration of illness