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Tracking Machado-Joseph disease (MJD): identification of prodromal and early disease molecular biomarkers
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Abstract(s)
A doença de Machado-Joseph (DMJ) é uma doença neurodegenerativa de poliglutamina (poliQ) rara, causada pela expansão de uma repetição CAG no gene ATXN3 que codifica a proteína ataxina-3 (ATXN3). A DMJ é uma doença multissistémica de início tardio, envolvendo predominantemente os sistemas cerebeloso, piramidal, extrapiramidal, oculomotor e periférico. A ataxia da marcha é geralmente definida como o início da doença, por ser o sintoma inicial mais frequente (≈92%); no entanto, alguns doentes apresentam outros sinais/sintomas ainda antes das alterações da marcha, incluindo diplopia. A DMJ é caracterizada por uma longa fase pré-clínica durante a qual algumas manifestações clínicas não específicas, alterações no
encéfalo e modificações moleculares já estão presentes, antes do aparecimento da doença. A ATXN3 é uma desubiquitinase, sendo que quer a proteína ATXN3 nativa quer a mutada são ubiquamente expressas em vários tipos de células de tecidos neuronais e não neuronais. A presença da expansão (CAG)n/trato poliQ expandido acima de um limiar patológico no transcrito/proteína ATXN3 inicia uma cascata de eventos patogénicos, os quais incluem a falhas na homeostasia celular, disfunção mitocondrial e stress oxidativo, alterações da apoptose, bem como desregulação transcricional. Algumas destas alterações moleculares têm potencial de serem usadas como biomarcadores da doença. Uma vez que as medidas clínicas atuais se
revelam insuficientes para medir com rigor a eficácia de eventuais agentes terapêuticos modificadores da doença, em particular na fase pré-clínica, a identificação de biomarcadores para as fases iniciais da doença é extremamente importante no que diz respeito à sua monitorização da progressão, nomeadamente no contexto do desenvolvimento de terapêuticas. O principal objetivo desta tese é melhorar o conhecimento acerca das alterações moleculares associadas com a DMJ e contribuir para a identificação de novos biomarcadores
transcricionais periféricos para a DMJ, particularmente nas fases iniciais da doença. No total, foram utilizadas amostras de RNA de sangue de 101 indivíduos molecularmente confirmados para a DMJ (20 indivíduos pré-clínicos, e 81 doentes incluindo 36 doentes da fase inicial – doentes com cinco ou menos anos de progressão) e 98 indivíduos controlo saudáveis dos Açores, bem como 20 doentes DMJ e 20 controlos saudáveis do Brasil. Foram também usadas amostras de cérebros post-mortem de doentes com confirmação molecular de DMJ (n=5) e de indivíduos controlo excluídos molecularmente para a DMJ (n=9); duas regiões severamente afetadas na DMJ (núcleos dentados do cerebelo e ponte) e uma região menos afetada (córtex frontal) foram
analisadas. Foram também usadas amostras de sangue e cérebro (ponte e córtex cerebral) de ratinhos transgénicos YACMJD84.2 (n=11) e wild-type (n=11) de 9 meses de idade, assim como de amostras de cérebro (ponte e córtex cerebral) de ratinhos transgénicos (n=4) e wild-type (n=5) de 18 meses de idade. Uma caracterização demográfica, genética e clínica detalhada dos sujeitos DMJ açorianos confirmou que os indivíduos pré-clínicos já apresentam alterações antes do aparecimento da doença, tais como nistagmo e/ou disfagia; para além disso, na coorte total de doentes, e tal como seria expectável, a pontuação total da NESSCA (Neurological Examination Score for Spinocerebellar Ataxia) aumenta com a progressão da doença. No subgrupo de doentes da fase inicial com dois momentos de avaliação disponíveis, a análise dos subitens da NESSCA revelou que as alterações não cerebelosas desta escala deram o contributo principal para o aumento anual da pontuação total, indicando que ambos os parâmetros cerebelosos e não cerebelosos dos doentes DMJ são úteis para avaliar a progressão da doença, nas suas fases iniciais. Sendo a PCR quantitativa em tempo real a principal técnica experimental escolhida para avaliar a expressão transcricional, no âmbito deste trabalho, os genes a utilizar como genes de referência foram primeiro selecionados com base na sua estabilidade de expressão. Todos os cinco genes candidatos testados (PPIB, TRAP1, B2M, DECR1 e FPGS) mostraram expressão estável em amostras de sangue de indivíduos DMJ e controlos açorianos, indicando que todos eles podem ser usados para normalizar dados de PCR quantitativa em tempo real em estudos
utilizando amostras de sangue de indivíduos DMJ; o TRAP1 foi identificado como o gene de referência mais estável de entre os cinco analisados. Para melhor elucidar o envolvimento da via mitocondrial da apoptose na patogénese da DMJ, foram analisados os níveis de expressão de três genes relacionados com este mecanismo (BCL2, BAX e TP53) em amostras de sangue e de cérebro post-mortem de indivíduos da DMJ e controlos. Os nossos resultados sugerem que a ativação da via mitocondrial da apoptose pode estar favorecida na periferia (amostras de sangue) dos indivíduos DMJ, tal como anteriormente reportado. Da mesma forma, os dados do cérebro post-mortem dos doentes DMJ mostraram que as células do córtex frontal, que se sabe estar pouco afetado na DMJ, podem ser mais vulneráveis a estímulos apoptóticos, enquanto as
células nos núcleos dentados do cerebelo e ponte, que se sabem ser atingidas na DMJ, são mais propensas a sobreviver, sugerindo que as mesmas possam ativar mecanismos protetores de sobrevivência na fase final da doença. Com o objetivo de avaliar se os ratinhos transgénicos replicavam os resultados obtidos em indivíduos DMJ, analisou-se os níveis de expressão do Bcl2, Bax e Tpr53, e avaliou-se se a expressão destas moléculas variava com a progressão da doença. Os resultados indicam que quantidade das proteínas Blc2 e Bax está alterada no cérebro de ratinhos transgénicos pré-sintomáticos de 9 meses de idade e sintomáticos de 18 meses de idade, tendo estas alterações variado com a progressão da doença, indicando que a resposta
das células a estímulos apoptóticos depende da fase da doença. Os nossos resultados indicam, ademais, que os ratinhos transgénicos de 18 meses de idade e os cérebros post-mortem DMJ estão em diferentes fases da doença. Com o objectivo de identificar novos biomarcadores transcricionais periféricos para a DMJ, utilizámos dados de um whole genome microarray realizado em amostras de sangue total de indivíduos DMJ e controlos, bem como dados disponíveis a partir de um estudo de expressão já realizado, tendo identificado os genes DDIT4, TRIM13 e P2RY13 como estando consistentemente desregulados em sangue de indivíduos DMJ
em diferentes coortes (Açores e Brasil). Além disso, os níveis de expressão destes três genes correlacionam com características clínicas/genéticas dos doentes, sendo que quantidade das suas respetivas proteínas, estavam também alteradas nos cérebros dos doentes DMJ, ligando estas alterações transcricionais à patogénese da doença. É de salientar que os nossos resultados indicam que os níveis de DDIT4 e TRIM13 em sangue periférico possam constituir biomarcadores transcricionais fiáveis para a DMJ, em particular para as fases iniciais da doença. É ainda importante referir que o(s) mecanismo(s) da doença nos quais as proteínas TRIM13 e P2RY13 estão envolvidas – respetivamente, a eliminação de proteínas mal enroladas e agregadas
durante o stress do reticulo endoplasmático, bem como a resposta inflamatória – aparentam estar mantidos no sistema nervoso central e na periferia (sangue) dos doentes DMJ.
Globalmente o trabalho desenvolvido nesta tese demonstrou que os genes DDIT4 e TRIM13 são iomarcadores transcricionais periféricos em particular para as fases iniciais da DMJ; assim nós antecipamos que estes podem ser incluídos num conjunto de vários biomarcadores fluidos e/ou outros biomarcadores de neuroimagem e neurofisiologia para complementar as escalas clínicas para uma precisa monitorização da progressão da doença e medição da eficácia das terapias em ensaios clínicos. Analisando uma via que se sabe estar alterada na DMJ, este trabalho evidenciou também que diferentes regiões do cérebro têm uma vulnerabilidade específica à apoptose; tal foi verificado em amostras de indivíduos DMJ e de um modelo de ratinho DMJ.
ABSTRACT: Machado-Joseph disease (MJD) is a rare late-onset polyglutamine (polyQ) neurodegenerative disease caused by the expansion of a CAG repeat in the ATXN3 gene encoding the ataxin-3 (ATXN3) protein. MJD is a multisystem disease involving predominantly the cerebellar, pyramidal, extrapyramidal, motor neuron and oculomotor systems. Disease onset is usually defined by gait ataxia, as it is the most frequent initial symptom of MJD (≈92%), but some patients already show other signs/symptoms prior to gait disturbances, including diplopia. MJD is characterized by a long preclinical phase during which some unspecific clinical manifestations, brain abnormalities and molecular changes are known to be already present, preceding disease onset. ATXN3 is a deubiquitinase, and both native and mutant ATXN3 are ubiquitously expressed in several cell-types in neuronal and non-neuronal tissues. An abnormal expansion of the (CAG)n/polyQ tract above a pathological threshold in the ATXN3 transcript/protein is thought to initiate a cascade of pathogenic events, such as failure of cellular protein homeostasis, mitochondrial dysfunction and oxidative stress, alterations in apoptosis, as well as transcriptional impairment. Importantly, some of these molecular alterations have the potential to be used as biomarkers of disease, which are urgently needed, in the context of the emergent clinical trials for MJD. Because current clinical measures are insufficient to rigorously measure the efficacy of disease-modifying therapeutic agents, in particular in the preclinical phase, the identification of biomarkers for the early stages of the disease is extremely important to allow monitoring the disease progression, namely for the development of therapeutics. The main goal of this thesis is to improve our knowledge of molecular alterations associated with MJD and to contribute for the identification of novel blood-based transcriptional biomarkers for MJD, particularly of the initial stages of the disease. In total, blood RNA samples from 101 molecularly confirmed MJD carriers (20 preclinical subjects, and 81 patients including 36 patients with five or less years of disease duration – early patients) and 98 healthy individuals from the Azores, as well as 20 MJD patients and 20 controls from Brazil were used in this thesis. Furthermore, frozen samples from post-mortem brain regions of deidentified molecularly confirmed MJD patients (n=5) and of control individuals molecularly excluded for MJD (n=9) were used; for each subject, two brain regions severely affected in MJD (dentate cerebellar nucleus and pons), and a region with lower degree of disease affectation (frontal cortex) were analyzed. Blood and brain samples (pons and cerebral cortex) from 9-month-old (MO) YACMJD84.2 (Q84) (n=11) and wild-type mice (n=11), as well brain samples (pons and cerebral cortex) from 18 MO Q84 (n=4) and wild type mice (n=5) were also used. A detailed demographic, genetic and clinical characterization of the Azorean MJD subjects confirmed that preclinical subjects show alterations before disease onset, such as nystagmus and/or dysphagia, and that for the overall MJD patients, total NESSCA (Neurological Examination Score for Spinocerebellar Ataxia) score increased with disease progression. Importantly, the analysis of the NESSCA subscores in the subset of early patients with follow-up data revealed that non-cerebellar NESSCA features were the major contributors to the annual increase of the total score, indicating that the evaluation of both cerebellar and non-cerebellar features of MJD patients would be useful to assess disease progression in the early stages of the disease. Because quantitative real-time PCR was the major experimental technique chosen to evaluate transcriptional expression in this work, the genes to be used as reference in expression studies were firstly selected based on their expression stability. All the five selected candidate housekeeping genes (PPIB, TRAP1, B2M, DECR1, and FPGS) showed stable expression in blood samples from Azorean MJD carriers and healthy controls, indicating that all of them can be used for normalizing quantitative real-time PCR data in studies using peripheral blood samples from MJD subjects; noteworthy, TRAP1 was identified as the most stable reference gene among the five analyzed. To better elucidate the involvement of the mitochondrial apoptosis pathway in MJD pathogenesis, the expression levels of three major apoptosis-related genes (BCL2, BAX and TP53) were analyzed in blood and post-mortem brain samples from MJD carriers and controls. Our results suggest that in MJD carriers’ activation of the mitochondrial apoptotic pathway might be favored at the periphery, as previously reported. Similarly, the data from post-mortem MJD patients’ brains showed that cells in the less-affected frontal cortex might be more vulnerable to apoptotic stimuli, while the remaining cells in the degenerative dentate cerebellar nucleus and pons are more prone to survive, suggesting that they may activate protective survival mechanisms at the end-stage of the disease. In addition, we determined whether Q84 mice replicate the findings in MJD subjects and evaluated how Bcl2, Bax and Tpr53 expression levels changed with disease progression. Blc2 and Bax proteins were altered in the brains of pre-symptomatic 9 MO and symptomatic 18 MO transgenic Q84 mice, and these alterations fluctuated with the disease progression, indicating that the response of the cells to apoptotic stimuli depends on the stage of disease. Also, our results also indicate that 18 MO Q84 transgenic mice and post-mortem MJD brains are at different stages of the disease. Pursuing the goal to identify novel blood-based transcriptional biomarkers for MJD, building on data from a cross-sectional blood-based whole genome microarray of MJD subjects and controls and of a previous expression study, we identified DDIT4, TRIM13 and P2RY13 as being consistently dysregulated in blood of MJD subjects across different cohorts (Azores and Brazil). Additionally, the expression levels of these three genes correlated with patients’ clinical/genetic features and their respective protein abundance was altered in brains of MJD patients, linking these transcriptional changes with MJD pathogenesis. Noteworthy, our results indicate that DDIT4 and TRIM13 are reliable blood-based transcriptional biomarker for MJD, in particular for the early stages of the disease. Importantly, our results further show that the disease mechanism(s) in which TRIM13 and P2RY13 proteins are involved – respectively, elimination of misfolded and aggregates proteins during endoplasmic reticulum stress, and inflammatory response – could be preserved in the central nervous system and the periphery (blood) of MJD patients. Overall, the work developed in this thesis demonstrates that DDIT4 and TRIM13 are blood-based transcriptional biomarkers for the early stages of MJD; thus we anticipate that they can be included in a set of various fluid biomarkers and/or other neuroimaging and neurophysiology biomarkers to complement clinical scales for the accurate monitoring of disease progression and measurement of the efficacy of therapeutics in clinical trials. Targeting an important pathway known to be disrupted in MJD, this work also evidenced that there is tissue-specific vulnerability to apoptosis in MJD subjects and in a MJD mouse model.
ABSTRACT: Machado-Joseph disease (MJD) is a rare late-onset polyglutamine (polyQ) neurodegenerative disease caused by the expansion of a CAG repeat in the ATXN3 gene encoding the ataxin-3 (ATXN3) protein. MJD is a multisystem disease involving predominantly the cerebellar, pyramidal, extrapyramidal, motor neuron and oculomotor systems. Disease onset is usually defined by gait ataxia, as it is the most frequent initial symptom of MJD (≈92%), but some patients already show other signs/symptoms prior to gait disturbances, including diplopia. MJD is characterized by a long preclinical phase during which some unspecific clinical manifestations, brain abnormalities and molecular changes are known to be already present, preceding disease onset. ATXN3 is a deubiquitinase, and both native and mutant ATXN3 are ubiquitously expressed in several cell-types in neuronal and non-neuronal tissues. An abnormal expansion of the (CAG)n/polyQ tract above a pathological threshold in the ATXN3 transcript/protein is thought to initiate a cascade of pathogenic events, such as failure of cellular protein homeostasis, mitochondrial dysfunction and oxidative stress, alterations in apoptosis, as well as transcriptional impairment. Importantly, some of these molecular alterations have the potential to be used as biomarkers of disease, which are urgently needed, in the context of the emergent clinical trials for MJD. Because current clinical measures are insufficient to rigorously measure the efficacy of disease-modifying therapeutic agents, in particular in the preclinical phase, the identification of biomarkers for the early stages of the disease is extremely important to allow monitoring the disease progression, namely for the development of therapeutics. The main goal of this thesis is to improve our knowledge of molecular alterations associated with MJD and to contribute for the identification of novel blood-based transcriptional biomarkers for MJD, particularly of the initial stages of the disease. In total, blood RNA samples from 101 molecularly confirmed MJD carriers (20 preclinical subjects, and 81 patients including 36 patients with five or less years of disease duration – early patients) and 98 healthy individuals from the Azores, as well as 20 MJD patients and 20 controls from Brazil were used in this thesis. Furthermore, frozen samples from post-mortem brain regions of deidentified molecularly confirmed MJD patients (n=5) and of control individuals molecularly excluded for MJD (n=9) were used; for each subject, two brain regions severely affected in MJD (dentate cerebellar nucleus and pons), and a region with lower degree of disease affectation (frontal cortex) were analyzed. Blood and brain samples (pons and cerebral cortex) from 9-month-old (MO) YACMJD84.2 (Q84) (n=11) and wild-type mice (n=11), as well brain samples (pons and cerebral cortex) from 18 MO Q84 (n=4) and wild type mice (n=5) were also used. A detailed demographic, genetic and clinical characterization of the Azorean MJD subjects confirmed that preclinical subjects show alterations before disease onset, such as nystagmus and/or dysphagia, and that for the overall MJD patients, total NESSCA (Neurological Examination Score for Spinocerebellar Ataxia) score increased with disease progression. Importantly, the analysis of the NESSCA subscores in the subset of early patients with follow-up data revealed that non-cerebellar NESSCA features were the major contributors to the annual increase of the total score, indicating that the evaluation of both cerebellar and non-cerebellar features of MJD patients would be useful to assess disease progression in the early stages of the disease. Because quantitative real-time PCR was the major experimental technique chosen to evaluate transcriptional expression in this work, the genes to be used as reference in expression studies were firstly selected based on their expression stability. All the five selected candidate housekeeping genes (PPIB, TRAP1, B2M, DECR1, and FPGS) showed stable expression in blood samples from Azorean MJD carriers and healthy controls, indicating that all of them can be used for normalizing quantitative real-time PCR data in studies using peripheral blood samples from MJD subjects; noteworthy, TRAP1 was identified as the most stable reference gene among the five analyzed. To better elucidate the involvement of the mitochondrial apoptosis pathway in MJD pathogenesis, the expression levels of three major apoptosis-related genes (BCL2, BAX and TP53) were analyzed in blood and post-mortem brain samples from MJD carriers and controls. Our results suggest that in MJD carriers’ activation of the mitochondrial apoptotic pathway might be favored at the periphery, as previously reported. Similarly, the data from post-mortem MJD patients’ brains showed that cells in the less-affected frontal cortex might be more vulnerable to apoptotic stimuli, while the remaining cells in the degenerative dentate cerebellar nucleus and pons are more prone to survive, suggesting that they may activate protective survival mechanisms at the end-stage of the disease. In addition, we determined whether Q84 mice replicate the findings in MJD subjects and evaluated how Bcl2, Bax and Tpr53 expression levels changed with disease progression. Blc2 and Bax proteins were altered in the brains of pre-symptomatic 9 MO and symptomatic 18 MO transgenic Q84 mice, and these alterations fluctuated with the disease progression, indicating that the response of the cells to apoptotic stimuli depends on the stage of disease. Also, our results also indicate that 18 MO Q84 transgenic mice and post-mortem MJD brains are at different stages of the disease. Pursuing the goal to identify novel blood-based transcriptional biomarkers for MJD, building on data from a cross-sectional blood-based whole genome microarray of MJD subjects and controls and of a previous expression study, we identified DDIT4, TRIM13 and P2RY13 as being consistently dysregulated in blood of MJD subjects across different cohorts (Azores and Brazil). Additionally, the expression levels of these three genes correlated with patients’ clinical/genetic features and their respective protein abundance was altered in brains of MJD patients, linking these transcriptional changes with MJD pathogenesis. Noteworthy, our results indicate that DDIT4 and TRIM13 are reliable blood-based transcriptional biomarker for MJD, in particular for the early stages of the disease. Importantly, our results further show that the disease mechanism(s) in which TRIM13 and P2RY13 proteins are involved – respectively, elimination of misfolded and aggregates proteins during endoplasmic reticulum stress, and inflammatory response – could be preserved in the central nervous system and the periphery (blood) of MJD patients. Overall, the work developed in this thesis demonstrates that DDIT4 and TRIM13 are blood-based transcriptional biomarkers for the early stages of MJD; thus we anticipate that they can be included in a set of various fluid biomarkers and/or other neuroimaging and neurophysiology biomarkers to complement clinical scales for the accurate monitoring of disease progression and measurement of the efficacy of therapeutics in clinical trials. Targeting an important pathway known to be disrupted in MJD, this work also evidenced that there is tissue-specific vulnerability to apoptosis in MJD subjects and in a MJD mouse model.
Description
Tese de Doutoramento, Biologia, 11 de novembro de 2022, Universidade dos Açores.
Keywords
Machado-Joseph Disease (MJD) ATXN3 Transcriptional Biomarkers Molecular Biomarkers
Pedagogical Context
Citation
Ferreira, Ana Filipa Bartolomeu. (2022). "Tracking Machado-Joseph disease (MJD): identification of prodromal and early disease molecular biomarkers". 205 p. (Tese de Doutoramento em Biologia). Ponta Delgada: Universidade dos Açores, 2022. Disponível em http://hdl.handle.net/10400.3/7109