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Role of tomm40’523 – apoe haplotypes in alzheimer’s disease etiology – from clinics to mitochondria
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RESUMO: TOMM40’523 é um polimorfismo poli-T do gene TOMM40 que foi descrito por Roses
et al. como estando associado com o risco e idade de início da doença de Alzheimer
(DA). Entretanto, e com base na distribuição do número de timinas, três tipos de alelos
foram estabelecidos: “short” (S, ≤ 19), “long” (L, 20– 29) e “very long” (VL, ≥ 30).
Desde a descoberta inicial de Roses et al. vários estudos encontraram associações
entre o polimorfismo TOMM40’ 523 e características relacionadas com a DA entre elas,
idade de início e risco de DA, alterações na morfologia cerebral e cognição, enquanto
outros estudos não conseguiram replicar estas associações. Além do mais, este
polimorfismo foi pouco estudado em populações com défice cognitivo ligeiro (DCL) e
tanto quanto sabemos nenhum destes estudos se debruçou na relação entre o
polimorfismo TOMM40’ 523 e o risco e tempo de conversão de DCL para DA.
Assim, na primeira parte do trabalho aqui apresentado o nosso principal objetivo foi
investigar a relação do polimorfismo TOMM40’ 523 com o risco e tempo de conversão
de DCL para DA, bem como replicar a associação inicial descrita por Roses et al. com o
risco e idade de início da DA. Em segundo lugar, explorámos a relação entre este
polimorfismo e os níveis de biomarcadores de DA no líquido cefalorraquidiano (LCR),
nomeadamente Ab42, Tau total e fosfo-Tau. Para este efeito, procedemos à
genotipagem para o polimorfismo TOMM40’ 523 em 147 pacientes com DA, 102
pacientes com DCL e 105 controlos cognitivamente normais. Os pacientes DCL foram
subdivididos em dois grupos distintos: um grupo de pacientes DCL que converteram
para AD (DCL-C) e um grupo de pacientes que permaneceu cognitivamente estável (DCLNC).
Começámos neste estudo por demonstrar que os doentes DCL que não converteram
(DCL-NC) e os doentes que converteram (DCL-C) apresentavam uma distribuição distinta
do número de timinas neste polimorfismo, sendo o alelo L significativamente mais
frequente no grupo DLC-C. Posteriormente, avaliámos como é que esta diferença
afetava o risco de conversão de DCL para DA e verificámos que a presença de pelo
menos um alelo L aumentava significativamente o risco de conversão de DCL para DA.
Contudo, quando tido em conta o alelo ε4 do gene APOE, verificámos que tanto o alelo
L como alelo ε4 perdiam significância no modelo de conversão (p > 0.05). Então decidimos estudar o haplótipo APOE ε4-TOMM40ʹ 523 L e observámos que doentes com
este haplótipo apresentavam um risco significativamente maior de conversão de DCL
para DA e menores tempos de conversão. Verificou-se também uma associação
significativa entre este haplótipo e um perfil de biomarcadores compatível com DA,
nomeadamente menores níveis de Ab42 e níveis mais altos de Tau total e fosfo-Tau.
Adicionalmente, verificamos que o haplótipo APOE ε4-TOMM40ʹ 523 L estava
significativamente associado com um maior risco de desenvolver DA e com menor idade
de início da doença.
O gene TOMM40 codifica a proteína TOM40 (Translocase da membrana externa, 40
kD) que forma o canal na membrana externa da mitocôndria através do qual a maioria
das proteínas codificadas no citoplasma entram na mitocôndria. Sendo a importação de
proteínas essencial para a biogénese e funcionamento da mitocôndria, não é de
surpreender que a proteína TOM40 seja essencial para os seres eucarióticos, e que
modificações nesta proteína possam eventualmente levar a disfunção mitocondrial. A
disfunção mitocondrial é um evento bem caracterizado na DA. Tendo isto em conta,
colocámos a hipótese de que o polimorfismo TOMM40’ 523 poderia ter um papel
importante na disfunção mitocondrial e patogénese da DA. Utilizando células
mononucleares do sangue periférico (PBMCs) de doentes com DA, estudámos o impacto
deste polimorfismo em diferentes parâmetros mitocondriais como função, estrutura e
apoptose. As PBMCs foram obtidas de doentes homozigóticos para os alelos S, L e VL,
sendo denominados posteriormente como grupos S,L e VL.
Neste estudo, podémos observar que o grupo VL apresentava níveis
significativamente mais elevados da proteína TOM40, semelhante potencial de
membrana mitocondrial, maiores níveis de proteínas de fissão mitocondrial e uma
menor ativação da caspase 3 quando comparado com o grupo S. Apesar destes dois
últimos parâmetros não alcançarem significância estatística, estes dados reforçam a
hipótese de que o aumento nos níveis de TOM40, tal como observado também por
Zeitlow et al., poderão ser protetores para mitocôndria. Contudo estes dados têm de
ser replicados em experiências futuras.
Em jeito de sumário, neste trabalho pretendemos estudar o potencial papel do
polimorfismo TOMM40’523 no risco de desenvolver DA, bem como o seu envolvimento na fisiopatologia desta doença via disfunção mitocondrial, numa perspetiva “ da clinica
para a mitocôndria”.
ABSTRACT: TOMM40’523 is a poly-T polymorphism of the gene TOMM40 which was reported by Roses et al. to be associated with risk and age of onset (AOO) of Alzheimer’s Disease (AD) nearly a decade ago. Meanwhile, based on the distribution behavior of the number of thymine (T) residues, three categories (alleles) of repeat length were established: short (S, ≤ 19), long (L, 20– 29) and very long (VL, ≥ 30). Since the original discovery by Roses et al. multiple studies found associations between TOMM40’ 523 and LOAD-related features, such as AOO and risk of AD, brain structure and cognition, while other studies could not replicate these associations. Moreover, this polymorphism has been poorly addressed in mild cognitive impairment cohorts (MCI) and as far as we know, none of these studies fully addressed the connection between TOMM40’ 523 polymorphism and the risk and time of conversion from MCI to AD. Therefore, in the first part of this work our aim was to investigate the relationship between TOMM40’ 523 polymorphism with the risk and conversion time from MCI to AD, and replicate the association of TOMM40’523 polymorphism with AOO and risk of AD. Secondly, the association between TOMM40’ 523 genotype and AD cerebrospinal fluid (CSF) biomarkers, particularly Ab42, t-Tau and p-Tau, was also explored. For this purpose, 147 AD patients, 102 MCI patients and 105 cognitively normal controls were genotyped for poly-T polymorphism. MCI patients were subdivided into 2 groups, the group of patients that converted to AD (MCI-AD) and the group of those that remained stable (MCI-S). We first demonstrated that MCI non-converters (MCI-S) and converters (MCI-AD) had a different poli-T distribution, where the L allele was significantly more frequent in the MCI-AD group. We further evaluated how this difference impacted the risk of conversion and found that having at least one L allele significantly increased the risk of conversion from MCI to AD. However, when adjusted for the presence of APOE ε4 allele, both the L allele and ε4 allele lost significance in the model (p > 0.05). We then analysed the APOE ε4-TOMM40ʹ 523 L haplotype and observed that patients carrying this haplotype had significantly higher risk and mean lower times of conversion to AD. This haplotype was also significantly associated with a biomarker profile compatible with AD namely, significantly lower levels of Ab42 and higher levels of t-Tau and p-Tau. Similar results were observed for AD where ε4-L haplotype carriers where associated with a significantly higher risk of AD and lower AOO of AD patients. TOMM40 gene encodes the Tom40 protein (translocase of the outer mitochondrial membrane, 40 kD) which forms the channel subunit of the outer mitochondrial membrane protein complex through which the majority of nuclear-encoded proteins enter mitochondria. As the import of mitochondrial proteins into mitochondria is essential for biogenesis and functioning of mitochondria, it is not surprising that TOM40 is essential for life in eukaryotic organisms and that modifications in this protein could lead to mitochondrial dysfunction. Mitochondria dysfunction is a well characterized event in AD. Considering this, it was hypothesized that TOMM40’ 523 polymorphism could have a role on AD through mitochondrial dysfunction. However, the few studies performed so far did not reach clear conclusions. Using Peripheral Blood Mononuclear Cells (PBMCs) from AD patients, we addressed the impact of this polymorphism on different mitochondrial features, such as function, structure and apoptosis. We obtained PBMCs from patients homozygous for S, L and VL TOMM40’ 523 polymorphisms, which are thereafter called S, L and VL groups. In this study, we observed that VL group had significant higher levels of TOM40 than S group, similar mitochondrial membrane potential, higher mitochondrial fission protein levels and lower caspase activation. Although these two last parameters did not reach statistical significance, our data reinforce the hypothesis thatincreased levels of TOM40, as have been observed by Zeitlow et al.seem to be protective to mitochondria, however these experiments should be replicated. In summary in this work we aimed to study the potential role of TOMM40’523 as a risk gene for AD and its involvement in AD pathophysiology through mitochondrial dysfunction, under the perspective “from clinics to mitochondria”.
ABSTRACT: TOMM40’523 is a poly-T polymorphism of the gene TOMM40 which was reported by Roses et al. to be associated with risk and age of onset (AOO) of Alzheimer’s Disease (AD) nearly a decade ago. Meanwhile, based on the distribution behavior of the number of thymine (T) residues, three categories (alleles) of repeat length were established: short (S, ≤ 19), long (L, 20– 29) and very long (VL, ≥ 30). Since the original discovery by Roses et al. multiple studies found associations between TOMM40’ 523 and LOAD-related features, such as AOO and risk of AD, brain structure and cognition, while other studies could not replicate these associations. Moreover, this polymorphism has been poorly addressed in mild cognitive impairment cohorts (MCI) and as far as we know, none of these studies fully addressed the connection between TOMM40’ 523 polymorphism and the risk and time of conversion from MCI to AD. Therefore, in the first part of this work our aim was to investigate the relationship between TOMM40’ 523 polymorphism with the risk and conversion time from MCI to AD, and replicate the association of TOMM40’523 polymorphism with AOO and risk of AD. Secondly, the association between TOMM40’ 523 genotype and AD cerebrospinal fluid (CSF) biomarkers, particularly Ab42, t-Tau and p-Tau, was also explored. For this purpose, 147 AD patients, 102 MCI patients and 105 cognitively normal controls were genotyped for poly-T polymorphism. MCI patients were subdivided into 2 groups, the group of patients that converted to AD (MCI-AD) and the group of those that remained stable (MCI-S). We first demonstrated that MCI non-converters (MCI-S) and converters (MCI-AD) had a different poli-T distribution, where the L allele was significantly more frequent in the MCI-AD group. We further evaluated how this difference impacted the risk of conversion and found that having at least one L allele significantly increased the risk of conversion from MCI to AD. However, when adjusted for the presence of APOE ε4 allele, both the L allele and ε4 allele lost significance in the model (p > 0.05). We then analysed the APOE ε4-TOMM40ʹ 523 L haplotype and observed that patients carrying this haplotype had significantly higher risk and mean lower times of conversion to AD. This haplotype was also significantly associated with a biomarker profile compatible with AD namely, significantly lower levels of Ab42 and higher levels of t-Tau and p-Tau. Similar results were observed for AD where ε4-L haplotype carriers where associated with a significantly higher risk of AD and lower AOO of AD patients. TOMM40 gene encodes the Tom40 protein (translocase of the outer mitochondrial membrane, 40 kD) which forms the channel subunit of the outer mitochondrial membrane protein complex through which the majority of nuclear-encoded proteins enter mitochondria. As the import of mitochondrial proteins into mitochondria is essential for biogenesis and functioning of mitochondria, it is not surprising that TOM40 is essential for life in eukaryotic organisms and that modifications in this protein could lead to mitochondrial dysfunction. Mitochondria dysfunction is a well characterized event in AD. Considering this, it was hypothesized that TOMM40’ 523 polymorphism could have a role on AD through mitochondrial dysfunction. However, the few studies performed so far did not reach clear conclusions. Using Peripheral Blood Mononuclear Cells (PBMCs) from AD patients, we addressed the impact of this polymorphism on different mitochondrial features, such as function, structure and apoptosis. We obtained PBMCs from patients homozygous for S, L and VL TOMM40’ 523 polymorphisms, which are thereafter called S, L and VL groups. In this study, we observed that VL group had significant higher levels of TOM40 than S group, similar mitochondrial membrane potential, higher mitochondrial fission protein levels and lower caspase activation. Although these two last parameters did not reach statistical significance, our data reinforce the hypothesis thatincreased levels of TOM40, as have been observed by Zeitlow et al.seem to be protective to mitochondria, however these experiments should be replicated. In summary in this work we aimed to study the potential role of TOMM40’523 as a risk gene for AD and its involvement in AD pathophysiology through mitochondrial dysfunction, under the perspective “from clinics to mitochondria”.
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Palavras-chave
Doença de Alzheimer Etiologia Alzheimer’s Disease Etiology