Absence of Gem1 (mammalian Miro/Rhot) mitigates alpha-synuclein toxicity in a yeast model of Parkinson's disease.

Alpha-synuclein aggregation is a hallmark of Parkinson's disease (PD). Mutants A30P and A53T alpha-synuclein are known to exacerbate the toxicity of alpha-synuclein, which includes oxidative stress, mitochondrial and endoplasmic reticulum (ER) dysfunction. Saccharomyces cerevisiae (budding yeast) is a cellular model widely used to investigate mechanisms underlying neurodegenerative disorders, such as PD. In yeast, Gem1 (Miro/Rhot mammalian orthologue) coordinates mitochondrial dynamics and ER homeostasis, which is impaired in the presence of mutant alpha-synuclein and can lead to cell death. In this study, A30P or A53T alpha-synuclein were expressed in wild type or ΔGem (deletion of Gem1 gene) yeast strains. ΔGem cells presented decreased viability and increased mitochondrial H2O2 production and ER stress compared to wild type cells. However, in the presence of mutant alpha-synuclein, ΔGem cells showed increased growth compared to cells that do not express mutant alpha-synuclein. ΔGem cells expressing A53T alpha-synuclein also presented reduced ER stress and increased ability to deal with oxidative stress. Together, our results suggest that deletion of Gem1 activates pathways that strengthen cells against other stressful agents such as the presence of mutant alpha-synuclein.

BAG2 prevents Tau hyperphosphorylation and increases p62/SQSTM1 in cell models of neurodegeneration.

BACKGROUND: Protein aggregates are pathological hallmarks of many neurodegenerative diseases, however the physiopathological role of these aggregates is not fully understood. Protein quality control has a pivotal role for protein homeostasis and depends on specific chaperones. The co-chaperone BAG2 can target phosphorylated Tau for degradation by an ubiquitin-independent pathway, although its possible role in autophagy was not yet elucidated. In view of this, the aim of the present study was to investigate the association among protein aggregation, autophagy and BAG2 levels in cultured cells from hippocampus and locus coeruleus as well as in SH-SY5Y cell line upon different protein aggregation scenarios induced by rotenone, which is a flavonoid used as pesticide and triggers neurodegeneration. METHODS AND RESULTS: The present study showed that rotenone exposure at 0.3 nM for 48 h impaired autophagy prior to Tau phosphorylation at Ser199/202 in hippocampus but not in locus coeruleus cells, suggesting that distinct neuron cells respond differently to rotenone toxicity. Rotenone induced Tau phosphorylation at Ser199/202, together with a decrease in the endogenous BAG2 protein levels in SH-SY5Y and hippocampus cell culture, which indicates that rotenone and Tau hyperphosphorylation can affect this co-chaperone. Finally, it has been shown that BAG2 overexpression, increased p62/SQSTM1 levels in cells from hippocampus and locus coeruleus, stimulated LC3II recycling as well as prevented the raise of phosphorylated Tau at Ser199/202 in hippocampus. CONCLUSIONS: Results demonstrate a possible role for BAG2 in degradation pathways of specific substrates and its importance for the study of cellular aspects of neurodegenerative diseases.

Effects of Magnetite Nanoparticles and Static Magnetic Field on Neural Differentiation of Pluripotent Stem Cells.

Neurodevelopmental processes of pluripotent cells, such as proliferation and differentiation, are influenced by external natural forces. Despite the presence of biogenic magnetite nanoparticles in the central nervous system and constant exposure to the Earth's magnetic fields and other sources, there is scant knowledge regarding the role of electromagnetic stimuli in neurogenesis. Moreover, emerging applications of electrical and magnetic stimulation to treat neurological disorders emphasize the relevance of understanding the impact and mechanisms behind these stimuli. Here, the effects of magnetic nanoparticles (MNPs) in polymeric coatings and the static external magnetic field (EMF) were investigated on neural induction of murine embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs). The results show that the presence of 0.5% MNPs in collagen-based coatings facilitates the migration and neuronal maturation of mESCs and hiPSCs in vitro. Furthermore, the application of 0.4 Tesla EMF perpendicularly to the cell culture plane, discernibly stimulates proliferation and guide fate decisions of the pluripotent stem cells, depending on the origin of stem cells and their developmental stage. Mechanistic analysis reveals that modulation of ionic homeostasis and the expression of proteins involved in cytostructural, liposomal and cell cycle checkpoint functions provide a principal underpinning for the impact of electromagnetic stimuli on neural lineage specification and proliferation. These findings not only explore the potential of the magnetic stimuli as neural differentiation and function modulator but also highlight the risks that immoderate magnetic stimulation may affect more susceptible neurons, such as dopaminergic neurons.

Mitochondria-ER Tethering in Neurodegenerative Diseases.

Organelles juxtaposition has been detected for decades, although only recently gained importance due to a pivotal role in the regulation of cellular processes dependent on membrane contact sites. Endoplasmic reticulum (ER) and mitochondria interaction is a prime example of organelles contact sites. Mitochondria-associated membranes (MAM) are proposed to harbor ER-mitochondria tether complexes, mainly when these organelles are less than 30 nm apart. Dysfunctions of proteins located at the MAM are associated with neurodegenerative diseases such as Parkinson's, Alzheimer's and amyotrophic lateral sclerosis, as well as neurodevelopmental disorders; hence any malfunction in MAM can potentially trigger cell death. This review will focus on the role of ER-mitochondria contact sites, regarding calcium homeostasis, lipid metabolism, autophagy, morphology and dynamics of mitochondria, mainly in the context of neurodegenerative diseases. Approaches that have been employed so far to study organelles contact sites, as well as methods that were not used in neurosciences yet, but are promising and accurate ways to unveil the functions of MAM during neurodegeneration, is also discussed in the present review.

Parkin is downregulated among autophagy-related proteins prior to hyperphosphorylation of Tau in TS65DN mice.

Autophagy is a pathway through which cells execute a plethora of functions, such as macromolecules and organelles quality control, recycling of building blocks and apoptosis. Numerous studies have shown in the past that autophagy is an important mechanism associated with the pathology of various neurodegenerative diseases, whose impairment may lead to several disease-characteristic phenotypes (e.g. misfolded protein and defective organelles accumulation). With this in mind, we aimed to investigate whether alterations in expression of autophagy-related proteins would show before hyperphosphorylation of Tau, a hallmark of Alzheimer's disease (AD). After analyzing 7 different proteins, we observed that, while Pink1 and p62 show an age-related reduction in the Ts65Dn mice respectively in the locus coeruleus and hippocampus, Parkin shows an age-genotype interaction-associated reduction in both brain areas. This suggests potential outcomes in pathways associated with Parkin that could relate to later stages of the disease development.

Epigenetic regulation of retinal development.

In the developing vertebrate retina, retinal progenitor cells (RPCs) proliferate and give rise to terminally differentiated neurons with exquisite spatio-temporal precision. Lineage commitment, fate determination and terminal differentiation are controlled by intricate crosstalk between the genome and epigenome. Indeed, epigenetic regulation plays pivotal roles in numerous cell fate specification and differentiation events in the retina. Moreover, aberrant chromatin structure can contribute to developmental disorders and retinal pathologies. In this review, we highlight recent advances in our understanding of epigenetic regulation in the retina. We also provide insight into several aspects of epigenetic-related regulation that should be investigated in future studies of retinal development and disease. Importantly, focusing on these mechanisms could contribute to the development of novel treatment strategies targeting a variety of retinal disorders.

microRNAs expression correlates with levels of APP, DYRK1A, hyperphosphorylated Tau and BDNF in the hippocampus of a mouse model for Down syndrome during ageing.

Down syndrome (DS) patients are more susceptible to Alzheimer's disease (AD) due to the presence of three copies of genes on chromosome 21 such as DYRK1A, which encodes a broad acting kinase, and APP (amyloid precursor protein), leading to formation of amyloid beta (Aß) peptide and hyperphosphorylation of Tau. In this study, we investigated the association among miRNAs miR-17, -20a, -101, -106b, -199b, -26a, 26b and some of their target mRNAs such as APP, DYRK1A and BDNF, as well as the levels of hyperphosphorylated Tau in the hippocampus of a 2 and 5 months old mice model of trisomy 21 (Ts65Dn). Results indicated that increased APP expression in the hippocampus of 5 months old DS mice might be correlated with decrease in miR-17, -20a, -101 and -106b. Whereas at 2 months of age normal levels of APP expression in the hippocampus was correlated with increased levels of miR-17, -101 and -106b in DS mice. DYRK1A mRNA also increased in the hippocampus of 5 months old DS mice and it is associated with decreased levels of miR-199b. Increased levels of DYRK1A in 5-month old mice are associated with increased phosphorylation of Tau at Thr212 residue but not at Ser199-202. Tau pathology is accompanied by decreased expression of BDNF and increased miR-26a/b in mice of 5 months of age. Taken together, data indicate that miR-17, -20a, -26a/b, -101, -106b and -199b might be interesting targets to mitigate Tau and Aß pathology in DS.

Altered in vitro muscle differentiation in X-linked myopathy with excessive autophagy.

X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.This article has an associated First Person interview with the first author of the paper.

Restoration of Rab1 Levels Prevents Endoplasmic Reticulum Stress in Hippocampal Cells during Protein Aggregation Triggered by Rotenone.

Disrupted neuronal intracellular trafficking is often related with protein aggregates present in the brain during neurodegenerative diseases such as Alzheimer's. Impairment of intracellular transport may be related to Rab proteins, a class of small GTPases responsible for trafficking of organelles and vesicles. Deficit in trafficking between the endoplasmic reticulum (ER) and Golgi apparatus mediated by Rab1 and 6 may lead to increased unfolded protein response (UPR) and ER stress and remodeling. Thus, the objective of this study is to analyze the levels of Rabs 1 and 6 in the hippocampus of aged rats and in vitro during protein aggregation promoted by exposure to rotenone. Levels of Rabs 1 and 6, ATF6 and CHOP were measured by western blotting. PDI immunolabeling and ER-Tracker were employed to study ER morphology. MTT was used to analyze cell metabolism. Rab1 levels and cell viability decreased, whereas Rab6, UPR proteins and ER remodeling increased during protein aggregation, which were restored to normal levels after exogenous expression of Rab1.These results suggest that decrease of Rab1 levels contributes to ER stress and remodeling, while maintaining the elevated expression of Rab1 prevented impairment of cell viability during protein aggregation. In conclusion, Rab1 is a significant player to maintain intracellular homeostasis and its expression may mitigate ER dysfunction in the context of neurodegeneration-related protein inclusions.

Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells.

The work with midbrain dopaminergic neurons (mDAN) differentiation might seem to be hard. There are about 40 different published protocols for mDAN differentiation, which are eventually modified according to the respective laboratory. In many cases, protocols are not fully described, failing to provide essential tips for researchers starting in the field. Considering that commercial kits produce low mDAN percentages (20-50%), we chose to follow a mix of four main protocols based on Kriks and colleagues' protocol, from which the resulting mDAN were engrafted with success in three different animal models of Parkinson's disease. We present a differential step-by-step methodology for generating mDAN directly from human-induced pluripotent stem cells cultured with E8 medium on Geltrex, without culture on primary mouse embryonic fibroblasts prior to mDAN differentiation, and subsequent exposure of neurons to rock inhibitor during passages for improving cell viability. The protocol described here allows obtaining mDAN with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening.

Mild Exercise Differently Affects Proteostasis and Oxidative Stress on Motor Areas During Neurodegeneration: A Comparative Study of Three Treadmill Running Protocols.

Proteostasis and oxidative stress were evaluated in motor cortex and spinal cord of aged Lewis rats exposed to 1 mg/kg/day of rotenone during 4 or 8 weeks, prior or after practicing three protocols of mild treadmill running. Results demonstrated that exercise done after the beginning of neurodegeneration reverted the increased oxidative stress (measured by H2O2 levels and SOD activity), increased neuron strength, and improved proteostasis in motor cortex. Spinal cord was not affected. Treadmill running practiced before neurodegeneration protected cortical motor neurons of the rotenone-exposed rats; but in this case, oxidative stress was not altered, whereas proteasome activity was increased and autophagy decreased. Spinal cord was not protected when exercise was practiced before neurodegeneration. Prolonged treadmill running (10 weeks) increased oxidative stress, autophagy, and proteasome activity, whereas neuron viability was decreased in motor cortex. In spinal cord, this protocol decreased oxidative stress and increased proteasome activity. Major conclusions were that treadmill running practiced before or after the beginning of neurodegeneration may protect motor cortex neurons, whereas prolonged mild running seems to be beneficial for spinal cord.

Mild exercise differently affects proteostasis and oxidative stress on motor areas during neurodegeneration: a comparative study of three treadmill running protocols

Proteostasis and oxidative stress were evaluated in motor cortex and spinal cord of aged Lewis rats exposed to 1mg/kg/day of rotenone during 4 or 8weeks, prior or after practicing three protocols of mild treadmill running. Results demonstrated that exercise done after the beginning of neurodegeneration reverted the increased oxidative stress (measured by H2O2 levels and SOD activity), increased neuron strength, and improved proteostasis in motor cortex. Spinal cord was not affected. Treadmill running practiced before neurodegeneration protected cortical motor neurons of the rotenone-exposed rats; but in this case, oxidative stress was not altered, whereas proteasome activity was increased and autophagy decreased. Spinal cord was not protected when exercise was practiced before neurodegeneration. Prolonged treadmill running (10weeks) increased oxidative stress, autophagy, and proteasome activity, whereas neuron viability was decreased in motor cortex. In spinal cord, this protocol decreased oxidative stress and increased proteasome activity. Major conclusions were that treadmill running practiced before or after the beginning of neurodegeneration may protect motor cortex neurons, whereas prolonged mild running seems to be beneficial for spinal cord.

Mild exercise differently affects proteostasis and oxidative stress on motor areas during neurodegeneration: a comparative study of three treadmill running protocols

Proteostasis and oxidative stress were evaluated in motor cortex and spinal cord of aged Lewis rats exposed to 1mg/kg/day of rotenone during 4 or 8weeks, prior or after practicing three protocols of mild treadmill running. Results demonstrated that exercise done after the beginning of neurodegeneration reverted the increased oxidative stress (measured by H2O2 levels and SOD activity), increased neuron strength, and improved proteostasis in motor cortex. Spinal cord was not affected. Treadmill running practiced before neurodegeneration protected cortical motor neurons of the rotenone-exposed rats; but in this case, oxidative stress was not altered, whereas proteasome activity was increased and autophagy decreased. Spinal cord was not protected when exercise was practiced before neurodegeneration. Prolonged treadmill running (10weeks) increased oxidative stress, autophagy, and proteasome activity, whereas neuron viability was decreased in motor cortex. In spinal cord, this protocol decreased oxidative stress and increased proteasome activity. Major conclusions were that treadmill running practiced before or after the beginning of neurodegeneration may protect motor cortex neurons, whereas prolonged mild running seems to be beneficial for spinal cord.

Alpha-Synuclein Toxicity on Protein Quality Control, Mitochondria and Endoplasmic Reticulum.

Parkinson's disease (PD) is characterized by the presence of insoluble protein clusters containing α-synuclein. Impairment of mitochondria, endoplasmic reticulum, autophagy and intracellular trafficking proper function has been suggested to be caused by α-synuclein toxicity, which is also associated with the higher levels of ROS found in the aged brain and in PD. Oxidative stress leads to protein oligomerization and aggregation that impair autophagy and mitochondrial dynamics leading to a vicious cycle of organelles damage and neurodegeneration. In this review we focused on the role of α-synuclein dysfunction as a cellular stressor that impairs mitochondria, endoplasmic reticulum, autophagy and cellular dynamics culminating with dopaminergic depletion and the pathogenesis of PD.

Effects of mild running on substantia nigra during early neurodegeneration.

Moderate physical exercise acts at molecular and behavioural levels, such as interfering in neuroplasticity, cell death, neurogenesis, cognition and motor functions. Therefore, the aim of this study is to analyse the cellular effects of moderate treadmill running upon substantia nigra during early neurodegeneration. Aged male Lewis rats (9-month-old) were exposed to rotenone 1mg/kg/day (8 weeks) and 6 weeks of moderate treadmill running, beginning 4 weeks after rotenone exposure. Substantia nigra was extracted and submitted to proteasome and antioxidant enzymes activities, hydrogen peroxide levels and Western blot to evaluate tyrosine hydroxylase (TH), alpha-synuclein, Tom-20, PINK1, TrkB, SLP1, CRMP-2, Rab-27b, LC3II and Beclin-1 level. It was demonstrated that moderate treadmill running, practiced during early neurodegeneration, prevented the increase of alpha-synuclein and maintained the levels of TH unaltered in substantia nigra of aged rats. Physical exercise also stimulated autophagy and prevented impairment of mitophagy, but decreased proteasome activity in rotenone-exposed aged rats. Physical activity also prevented H2O2 increase during early neurodegeneration, although the involved mechanism remains to be elucidated. TrkB levels and its anterograde trafficking seem not to be influenced by moderate treadmill running. In conclusion, moderate physical training could prevent early neurodegeneration in substantia nigra through the improvement of autophagy and mitophagy.

Effects of mild running on substantia nigra during early neurodegeneration

Moderate physical exercise acts at molecular and behavioural levels, such as interfering in neuroplasticity, cell death, neurogenesis, cognition and motor functions. Therefore, the aim of this study is to analyse the cellular effects of moderate treadmill running upon substantia nigra during early neurodegeneration. Aged male Lewis rats (9-month-old) were exposed to rotenone 1mg/kg/day (8weeks) and 6weeks of moderate treadmill running, beginning 4weeks after rotenone exposure. Substantia nigra was extracted and submitted to proteasome and antioxidant enzymes activities, hydrogen peroxide levels and Western blot to evaluate tyrosine hydroxylase (TH), alpha-synuclein, Tom-20, PINK1, TrkB, SLP1, CRMP-2, Rab-27b, LC3II and Beclin-1 level. It was demonstrated that moderate treadmill running, practiced during early neurodegeneration, prevented the increase of alpha-synuclein and maintained the levels of TH unaltered in substantia nigra of aged rats. Physical exercise also stimulated autophagy and prevented impairment of mitophagy, but decreased proteasome activity in rotenone-exposed aged rats. Physical activity also prevented H2O2 increase during early neurodegeneration, although the involved mechanism remains to be elucidated. TrkB levels and its anterograde trafficking seem not to be influenced by moderate treadmill running. In conclusion, moderate physical training could prevent early neurodegeneration in substantia nigra through the improvement of autophagy and mitophagy.

Aß42-mediated proteasome inhibition and associated tau pathology in hippocampus are governed by a lysosomal response involving cathepsin B: Evidence for protective crosstalk between protein clearance pathways.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer's disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aß42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aß42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aß42 (0.5-1.5 µM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aß42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aß42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aß42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aß42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aß42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways.

Pericytes Extend Survival of ALS SOD1 Mice and Induce the Expression of Antioxidant Enzymes in the Murine Model and in IPSCs Derived Neuronal Cells from an ALS Patient.

Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset motor neuron disease causing a progressive, rapid and irreversible degeneration of motor neurons in the cortex, brain stem and spinal cord. No effective treatment is available and cell therapy clinical trials are currently being tested in ALS affected patients. It is well known that in ALS patients, approximately 50% of pericytes from the spinal cord barrier are lost. In the central nervous system, pericytes act in the formation and maintenance of the blood-brain barrier, a natural defense that slows the progression of symptoms in neurodegenerative diseases. Here we evaluated, for the first time, the therapeutic effect of human pericytes in vivo in SOD1 mice and in vitro in motor neurons and other neuronal cells derived from one ALS patient. Pericytes and mesenchymal stromal cells (MSCs) were derived from the same adipose tissue sample and were administered to SOD1 mice intraperitoneally. The effect of the two treatments was compared. Treatment with pericytes extended significantly animals survival in SOD1 males, but not in females that usually have a milder phenotype with higher survival rates. No significant differences were observed in the survival of mice treated with MSCs. Gene expression analysis in brain and spinal cord of end-stage animals showed that treatment with pericytes can stimulate the host antioxidant system. Additionally, pericytes induced the expression of SOD1 and CAT in motor neurons and other neuronal cells derived from one ALS patient carrying a mutation in FUS. Overall, treatment with pericytes was more effective than treatment with MSCs. Our results encourage further investigations and suggest that pericytes may be a good option for ALS treatment in the future. Graphical Abstract ᅟ.

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.

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.

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 / 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

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.