Aged Lewis rats exposed to low and moderate doses of rotenone are a good model for studying the process of protein aggregation and its effects upon central nervous system cell physiology

Abstract

ARTICLEAged Lewis rats exposed to low and moderate doses of rotenone are a good model for studying the process of protein aggregation and its effects upon central nervous system cell physiology Ratos Lewis idosos expostos a baixa e moderada doses de rotenona são um bom modelo para estudar o processo de agregação proteica e seus efeitos sobre a fisiologia celular do sistema nervoso central Increase in lifespan is the reality for a number of countries around the world and is frequently associated with a rise in chronic diseases such as hypertension, diabetes, cancer, chronic obstructive pulmonary disease, visual impairment and neurodegeneration ABSTRACTCell 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.Keywords: protein aggregates; rats; neurodegeneration. RESUMOA 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.Palavras-chave: agregados proteicos; ratos; neurodegeneração.

Presence of insoluble Tau following rotenone exposure ameliorates basic pathways associated with neurodegeneration

“…In our previous study, we showed that exposure to rotenone (1 nM) can induce Aβ and alpha-synuclein aggregation in hippocampal cell cultures, however Tau aggregation required 50% less rotenone to be triggered (0.5 nM), suggesting that rotenone effect upon Tau aggregation is stronger and take place early than Aβ and α-synuclein aggregation. Furthermore, our group also found that in hippocampus of aged Lewis rats, exposed to 2 mg/kg/day of rotenone during 4 weeks, Tau hyperphosphorylation and aggregation is detected in the absence of α-synuclein dysfunction supporting our data ( Almeida et al., 2016 ). In this study, we extend our understanding of Tau pathology analyzing the interplay between oxidative stress and protein degradation pathways during the progression of Tau aggregation.…”

“…Rotenone can also promote Tau hyperphosphorylation and aggregation in a time and concentration- dependent manner in aged rats resembling our experimental protocol for hippocampal cell cultures ( Almeida et al., 2016 ). Then to further expand our findings, we evaluated whether parameters such as H 2 O 2 levels and proteasome activity are also disrupted before the formation of Tau aggregates in the hippocampus of aged rats.…”

“…We showed previously that low concentrations (0.5–1 nM) of rotenone, a mitochondrial complex I inhibitor widely employed in studies related to neurodegeneration ( Radad et al., 2008 , Ullrich and Humpel, 2009 ), were able to induce hyperphosphorylation and aggregation of constitutive Tau in the absence of cell death in primary hippocampus cell cultures ( Chaves et al., 2010 ). Chronic administration of rotenone to rats also induces cerebral tauopathy ( Höglinger et al., 2005 , Almeida et al., 2016 ) revealing rotenone as a reliable method to induce constitutive Tau aggregation in in vivo and in vitro systems.…”

Multiple neuroprotective features of Scutellaria pinnatifida–derived small molecule

“…One of the most well-known toxins is rotenone, a natural compound used as a pesticide [ 4 ], which can pass through the blood-brain barrier (BBB) [ 5 ]. It causes apoptotic cell death of tyrosine hydroxylase-positive neurons in substantia nigra (SN) of rats [ 6 ]. It induces aggregation of αSN, Aβ, and Tau in rotenone-treated mice, inhibits mitochondrial complex I, and eventually leads to dopaminergic neurodegeneration [ 7 , 8 , 9 ].…”

Effects of mild running on substantia nigra during early neurodegeneration

“…Animal models are reliable to understand the mechanisms of neurodegeneration (Jagmag, Tripathi, Shukla, Maiti, & Khurana, 2015). In this way, rotenone, a high affinity specific inhibitor of mitochondrial NADH dehydrogenase within complex I of the respiratory chain, have been used as a model of PD (Betarbet et al, 2000;Cannon et al, 2009), since it is able to promote α-synuclein aggregation in vitro (Chaves, Melo, Martins, & Ferrari, 2010;Radad, Gille, & Rausch, 2008;Ullrich & Humpel, 2009) and in vivo (Almeida, Silva, D'Unhao, & Ferrari, 2016;Hoglinger et al, 2005). Additionally, rotenone exposure leads to impairment of proteasome activity, oxidative/nitrosative stress, dysfunction of cytoskeleton; reduction of axonal transport and autophagic flux (Almeida, Chaves, et al, 2016;Chaves et al, 2016;Chaves, Melo, D'Unhao, Farizatto, & Ferrari, 2013;Henchcliffe & Beal, 2008), prior to formation of protein aggregates.…”