Publicação
Unanswered Role of Cholesterol Homeostasis in Parkinson’s Disease
| Resumo: | Cholesterol has a key role in neuronal function and alterations in brain cholesterol homeostasis correlate with neurodegeneration. While disruptions in cholesterol homeostasis have been clearly associated with neurodegenerative disorders such as Alzheimer’s and Huntington’s disease, the role of cholesterol in Parkinson’s disease (PD) remains controversial. To address this question, we characterized changes in cholesterol intracellular localization and levels using N2a mouse neuroblastoma cells treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxic metabolite, 1-methyl-4-phenylpyridinium (MPP+). Filipin III staining showed an increase in lysosomal accumulation of free cholesterol 16 hours after treatment with 1mM MPP+, and quantification of cholesterol levels also showed a significant increase in total and free intracellular cholesterol. In agreement, we observed a significant decrease in the mRNA levels and transcriptional activity of sterol regulatory element-binding protein (SREBP) 1 and 2 proteins, which are the main regulators of fatty acids and cholesterol synthesis. Concomitantly, there is a down-regulation in the mRNA levels of SREBP-target genes, such as fatty acid synthase, hydroxymethylglutaryl-CoA reductase and a significant decreased in the mRNA levels of NPC1 and lipase A. In order to uncover signaling pathways activated upon neuronal mitochondria dysfunction, involved in mitochondria-lysosome crosstalk, we identified an early activation of the AMPK/ mTOR signaling pathway, after 6 hours of MPP+ treatment. Nevertheless, in contrary to what was expected, we could not detect any alterations in the mRNA levels of TFEB and TFEB-target genes for the same time-point, that could be responsible to the previously detected increase in LAMP2 levels. To corroborate our results, we proceeded to characterized changes in cholesterol homeostasis in the brain of MPTP-treated mice (40mg/mL, i.p). Interestingly, we saw a significant decrease in NPC1 and LIPA mRNA levels 6 hours after MPTP administration, in both the midbrain and striatum. Thus, our results show that the inhibition of mitochondrial complex I leads to lysosomal accumulation, reduced SREBPs transcriptional activity and reduction of NPC1 expression, further corroborating previous reports that suggest PD as lysosomal storage disorder. Moreover, the changes in cholesterol homeostasis observed, may further contribute to the propagation of toxicity following mitochondrial dysfunction. |
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| Autores principais: | Caria, Inês de Sousa |
| Assunto: | 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) brain cholesterol lysosomes neurodegeneration Parkinson’s Disease sterol regulatory element-binding protein (SREBP) |
| Ano: | 2020 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Cholesterol has a key role in neuronal function and alterations in brain cholesterol homeostasis correlate with neurodegeneration. While disruptions in cholesterol homeostasis have been clearly associated with neurodegenerative disorders such as Alzheimer’s and Huntington’s disease, the role of cholesterol in Parkinson’s disease (PD) remains controversial. To address this question, we characterized changes in cholesterol intracellular localization and levels using N2a mouse neuroblastoma cells treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxic metabolite, 1-methyl-4-phenylpyridinium (MPP+). Filipin III staining showed an increase in lysosomal accumulation of free cholesterol 16 hours after treatment with 1mM MPP+, and quantification of cholesterol levels also showed a significant increase in total and free intracellular cholesterol. In agreement, we observed a significant decrease in the mRNA levels and transcriptional activity of sterol regulatory element-binding protein (SREBP) 1 and 2 proteins, which are the main regulators of fatty acids and cholesterol synthesis. Concomitantly, there is a down-regulation in the mRNA levels of SREBP-target genes, such as fatty acid synthase, hydroxymethylglutaryl-CoA reductase and a significant decreased in the mRNA levels of NPC1 and lipase A. In order to uncover signaling pathways activated upon neuronal mitochondria dysfunction, involved in mitochondria-lysosome crosstalk, we identified an early activation of the AMPK/ mTOR signaling pathway, after 6 hours of MPP+ treatment. Nevertheless, in contrary to what was expected, we could not detect any alterations in the mRNA levels of TFEB and TFEB-target genes for the same time-point, that could be responsible to the previously detected increase in LAMP2 levels. To corroborate our results, we proceeded to characterized changes in cholesterol homeostasis in the brain of MPTP-treated mice (40mg/mL, i.p). Interestingly, we saw a significant decrease in NPC1 and LIPA mRNA levels 6 hours after MPTP administration, in both the midbrain and striatum. Thus, our results show that the inhibition of mitochondrial complex I leads to lysosomal accumulation, reduced SREBPs transcriptional activity and reduction of NPC1 expression, further corroborating previous reports that suggest PD as lysosomal storage disorder. Moreover, the changes in cholesterol homeostasis observed, may further contribute to the propagation of toxicity following mitochondrial dysfunction. |
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