RESUMEN
Cell physiology is impaired before protein aggregation and this may be more relevant than inclusions themselves for neurodegeneration. The present study aimed to characterize an animal model to enable the analysis of the cell biology before and after protein aggregation. Ten-month-old Lewis rats were exposed either to 1 or 2 mg/kg/day of rotenone, delivered subcutaneously through mini-pumps, for one month. Hyperphosphorylated TAU, alpha-synuclein, amyloid-beta peptide and protein carbonylation (indicative of oxidative stress) were evaluated in the hippocampus, substantia nigra and locus coeruleus through immunohistochemistry or western blot. It was found that 2 mg/kg/day rotenone increased amyloid-beta peptide, hyperphosphorylation of TAU and alpha-synuclein. Rotenone at 1mg/kg/day did not alter protein levels. Protein carbonylation remained unchanged. This study demonstrated that aged Lewis rats exposed to a low dose of rotenone is a useful model to study cellular processes before protein aggregation, while the higher dose makes a good model to study the effects of protein inclusions.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Agregación Patológica de Proteínas/inducido químicamente , Agregación Patológica de Proteínas/patología , Rotenona/administración & dosificación , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Animales , Western Blotting , Sistema Nervioso Central/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Inmunohistoquímica , Locus Coeruleus/efectos de los fármacos , Locus Coeruleus/metabolismo , Locus Coeruleus/patología , Masculino , Estrés Oxidativo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Agregación Patológica de Proteínas/metabolismo , Carbonilación Proteica , Ratas Endogámicas Lew , Reproducibilidad de los Resultados , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología , alfa-Sinucleína/efectos de los fármacos , alfa-Sinucleína/metabolismoRESUMEN
ABSTRACT Cell physiology is impaired before protein aggregation and this may be more relevant than inclusions themselves for neurodegeneration. The present study aimed to characterize an animal model to enable the analysis of the cell biology before and after protein aggregation. Ten-month-old Lewis rats were exposed either to 1 or 2 mg/kg/day of rotenone, delivered subcutaneously through mini-pumps, for one month. Hyperphosphorylated TAU, alpha-synuclein, amyloid-beta peptide and protein carbonylation (indicative of oxidative stress) were evaluated in the hippocampus, substantia nigra and locus coeruleus through immunohistochemistry or western blot. It was found that 2 mg/kg/day rotenone increased amyloid-beta peptide, hyperphosphorylation of TAU and alpha-synuclein. Rotenone at 1mg/kg/day did not alter protein levels. Protein carbonylation remained unchanged. This study demonstrated that aged Lewis rats exposed to a low dose of rotenone is a useful model to study cellular processes before protein aggregation, while the higher dose makes a good model to study the effects of protein inclusions.
RESUMO A fisiologia celular está prejudicada antes da agregação proteica podendo ser mais importante para a neurodegeneração do que as próprias inclusões. Assim, o objetivo deste estudo é caracterizar um modelo animal para analisar os mecanismos e efeitos da agregação proteica. Ratos Lewis com 10 meses de idade foram expostos a rotenona (1 ou 2 mg/kg/dia), administrada subcutaneamente, utilizando minibombas osmóticas. Os níveis de peptídeo beta-amiloide, TAU hiperfosforilada, alfa-sinucleína e proteínas carboniladas (indicativo de estresse oxidativo) foram avaliados por imunohistoquímica e western blot no hipocampo, substância negra e locus coeruleus. Foi demonstrado que 2 mg/kg/dia de rotenona promoveu aumento do peptídeo beta-amiloide, hiperfosforilação da TAU e alfa-sinucleína. Já 1 mg/kg/dia de rotenona não alterou os níveis dessas proteína nessas regiões. As proteínas carboniladas não se alteraram. Foi demonstrado que ratos Lewis idosos expostos a baixas doses de rotenona são modelo de estudo dos processos celulares antes da agregação proteica, enquanto 2 mg/kg/dia de rotenona permite estudos sobre os efeitos da agregação proteica.
Asunto(s)
Animales , Masculino , Rotenona/administración & dosificación , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Agregación Patológica de Proteínas/inducido químicamente , Agregación Patológica de Proteínas/patología , Ratas Endogámicas Lew , Sustancia Negra/efectos de los fármacos , Inmunohistoquímica , Sistema Nervioso Central/metabolismo , Western Blotting , Reproducibilidad de los Resultados , Péptidos beta-Amiloides/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Estrés Oxidativo , alfa-Sinucleína/efectos de los fármacos , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patologíaRESUMEN
Neurodegeneration is often accompanied by protein inclusions which may interfere with cell physiology. On the other hand, alteration in intracellular trafficking may precede impairment of neurotransmission and therefore trigger cell death. In view of this, it is hypothesized that changes in mitochondrial traffic may occur before neurodegeneration triggered by rotenone exposure and could favor this process. The effects of low concentrations of rotenone on the expression of dynein c1h1, dynactin and syntaphilin, which are proteins related to mitochondria transport and anchoring, were evaluated in cell cultures of substantia nigra, locus coeruleus and hippocampus as well as in these same brain areas in Lewis aged rats. The results indicate that low concentrations of rotenone decrease dynein c1h1 protein levels in cell cultures and brain areas of aged rats. Dynactin is decreased after exposure to 0.1 and 0.3 nM of rotenone, and increased after exposure to 0.5 nM of rotenone in cell cultures. Aged rats present increased dynactin expression. Syntaphilin expression decreased in vitro and increased in vivo after rotenone exposure. These findings suggest that changes in protein expression related to mitochondrial retrograde transport and anchoring occur before neurodegeneration induced by rotenone exposure, which may be a primary factor to trigger neurodegenerative mechanisms.
Asunto(s)
Proteínas Portadoras/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/efectos de los fármacos , Rotenona/farmacología , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Complejo Dinactina , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
The presence of protein aggregates is common in neurodegenerative disorders; however, the real cause and effect of these aggregates during neurodegeneration is still a matter of investigation. We hypothesize that impairment of intracellular traffic may appear in the absence of protein inclusions and might trigger protein aggregation. In the present study, we aimed to evaluate mitochondria mobility as well as protein and messenger RNA expression of KIF1B and KIF5 that are molecular motors for neuronal anterograde traffic, in hippocampus, substantia nigra, and locus coeruleus of 10-month-old Lewis rats and cultured cells, from these same areas, following exposure to low doses of rotenone that do not lead to protein inclusions. The present study showed alteration in KIF1B and KIF5 expression, as well as in mitochondria mobility prior to protein aggregation involved in neurodegenerative disorders. These findings suggest that change in intracellular trafficking might be critical and one of the primary events for impairment of cell physiology during neurodegeneration associated with protein inclusions.