The dopaminergic neurodegeneration in the substantia nigra pars compacta (SNpc) is responsible for the marked motor impairment observed in Parkinson's disease (PD). However, the molecular mechanisms underlying this are not completely understood. Since by the time of diagnosis, 50-70% of the dopaminergic neurons of the nigrostriatal pathway have already been degenerated, it is difficult to investigate the early-stage events of disease pathogenesis. Due to inaccessibility of the human brain to study initial pathogenic mechanisms of the disease, experimental models have been developed in an attempt to elucidate PD etiology and its progression. Nevertheless, PD models are a controversial issue in neuroscience research since it is challenging to mimic human neuronal complexity. Therefore, the lack of optimal models that recreate disease pathology is one of the causes of failure of clinical trials that have attempted to find new/better PD therapies. Taking this in consideration, the development of more suitable models is necessary to improve our knowledge regarding PD etiological mechanisms. Additionally, the understanding of the advantages and disadvantages of models already established would also be beneficial for PD research, which our group addressed by reviewing this subject. Considering this, we chose SH-SY5Y cells as a PD model for our studies. To investigate the initial stages of PD-induced neurodegeneration, our work focused in the role of cofilin-1, a protein involved in mitochondrial dysfunction caused by oxidant-induced-apoptosis, which are two pathogenic processes strongly related to PD. Hence, in the thesis, we aimed to validate the use of retinoic-acid-(RA)-differentiated SH-SY5Y cells as an in vitro model and use it to investigate the potential role of cofilin-1 in the initial molecular and cellular mechanisms of PD. Although SH-SY5Y cells are widely used in PD research, their major drawback is their lack of important neuronal features, such as low levels of proliferation and stellate morphology. On the other hand, SH-SY5Y cells can acquire a neuronal phenotype when treated with differentiation agents such as RA. Since several protocols have been described, the consequence of which may be the discrepancies observed among studies regarding neuronal and dopaminergic features. In Chapter I, we aimed to validate a RA-differentiation protocol for SH-SY5Y cells previously established by our research group, focusing upon characterization of neuronal features and its subsequent response to 6-hydroxydopamine (6-OHDA), a toxin widely used to induce dopaminergic degeneration. RA-differentiated SH-SY5Y cells have low proliferative rates, a pronounced neuronal morphology and high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and dopamine transporter (DAT). After exploring phenotypic differences between these two models, we verified that RA-differentiated cells were more sensitive to 6-OHDA toxicity than undifferentiated cells, which could be related to an increase of DAT immunocontent. Many lines of evidence have showed that DAT is responsible for 6-OHDA uptake in vivo. Once inside the neuron, 6-OHDA underwent auto-oxidation causing a significant increase in oxidative stress. However, toxin uptake is not an essential step in undifferentiated SH-SY5Y cells, as auto-oxidation occurs extracellularly. We showed here, for the first time, that RA-differentiated SH-SY5Y cells can mimic, at least in part, an important mechanism of the 6-OHDA-induced cell death found in previous in vivo studies. Hence, the cellular model established by our research group presents essential neuronal features, being a suitable model for PD research. In Chapter II, RA-differentiated SH-SY5Y cells were used as cellular model to investigate disease molecular mechanisms, focusing upon cofilin-1. Our previous data have shown that oxidation of non-phosphorylate (activated) cofilin-1 leads to mitochondrial dysfunction and cell death induced by apoptosis in tumour cells. Here we found that cofilin-1 played a role in early stages of neuronal apoptosis induced by 6-OHDA in our cellular model since cofilin-1 mitochondrial translocation precedes organelle dysfunction. Overexpression of wild type CFL1 resulted in increased sensitivity of SH-SY5Y cells to 6-OHDA-induced neuronal cell death. Furthermore, overexpression of non-oxidizable CFL1 containing Cys-to-Ala mutations (positions 39, 80 and 139) increased neuronal resistance to this toxin, suggesting that oxidation is an important step in 6-OHDA toxicity. Follow-up experiments were performed in order to evaluate clinically whether cofilin-1 pathway proteins content is altered in PD post mortem human brain. Our findings showed a significant decrease in p-cofilin-1/cofilin-1 ratio in PD patients, which indicates an increase in the amount of activated cofilin-1 available for oxidation. Moreover, through principal component analysis, the immunodetection of cofilin-1 pathway proteins were able to discriminate controls and PD individuals during the early-stage of neuropathological changings. Hence, we demonstrated, for the first time, a possible role for cofilin-1 in PD pathogenesis and its potential use as biomarker. Taken together, our data showed that RA-differentiated SH-SY5Y cells present terminally-differentiated dopaminergic neuron features, that are essential to mimic dopaminergic neurons. By using this cellular model and post mortem brain tissue, we also demonstrated a possible role for cofilin-1 in early steps of the neurodegeneration process found in PD, which it could impact drug and biomarker discovery researches.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Acetaminophen (APAP) is the analgesic most used in world, in therapeutic doses does not show toxicity, but in elevated doses can cause hepatic damage due the formation of his metabolic toxic N-acetyl-p-benzoquinonimine (NAPQI). The brain damage can occurs because hepatic damage a condition called hepatic encephalopathy, since the liver already show his function altered, like transform ammonia in urea, causing accumulation of ammonia in the brain, which is toxic for this organ. Furthermore, the NAPQI can cause oxidative damage and mitochondrial dysfunction on brain tissue. The diphenyl diselenide [(PhSe)2] is an organoselenium compound that exhibit antioxidant activity and potential pharmacological. The aim of this study is investigated the ability of (PhSe)2 in reversing the oxidative brain damage and mitochondrial dysfunction induced by a toxic dose of APAP. Mice received a APAP (600mg/kg), followed by a dose of (PhSe)2 (15,6 mg/kg) 1 hour latter. Four hours after APAP administration, plasma was withdrawn from the mice and used for biochemical assays of aspastate aminotransferase (AST) and alanine aminotransferase (ALT) confirming the hepatic damage. The APAP administration increase lipid peroxidation, production of reactive oxygen species and decrease in activity of Na+, K+ - ATPase enzyme. Similary, APAP caused alteration on parameters of mitochondrial function. The treatment with (PhSe)2 revert the cerebral damage induced by a single dose of APAP. / O paracetamol é o analgésico mais usado no mundo, em doses terapêuticas não apresenta nenhuma toxicidade, porém em doses elevadas pode causar dano hepático pela formação do seu metabólito tóxico N-acetil-p-benzoquinoneimina (NAPQI). O dano cerebral pode ocorrer em decorrência ao dano no fígado, uma condição chamada de encefalopatia hepática, já que o fígado pode apresentar um comprometimento das suas funções, como transformar amônia em uréia, causando um acúmulo de amônia no organismo, sendo esta tóxica para o cérebro. Além disso, o NAPQI pode causar estresse oxidativo e disfunção mitocondrial no cérebro. O disseleneto de difenila [(PhSe)2] é um composto orgânico de selênio que apresenta atividade antioxidante e potencial farmacológico. O objetivo desse estudo é verificar a capacidade do (PhSe)2 em reverter o dano cerebral e disfunção mitocondrial causada por uma dose tóxica de paracetamol. Os camundongos receberam paracetamol (600 mg/kg) e 1h após, disseleneto de difenila (15,6 mg/kg). Após 4h da administração do paracetamol, coletou-se o soro para as análises bioquímicas de aspartato aminotransferase (AST) e alanina aminotransferase (ALT) que confirmaram o dano hepático. A administração de APAP aumentou a peroxidação lipídica, a produção de espécies reativas de oxigênio e diminuiu a atividade da enzima Na+, K+ - ATPase. Da mesma forma, o APAP alterou os parâmetros de funcionalidade mitocondrial. O tratamento com (PhSe)2 reverteu o dano cerebral induzido por uma dose única de APAP.

No description available.

No description available.

No description available.

Le syndrome de Leigh, type canadien français (LSFC) est une maladie infantile orpheline causée par une mutation du gène lrpprc. Elle se caractérise par une déficience tissu spécifique de cytochrome c oxydase (COX), une dysfonction mitochondriale et la survenue de crises d’acidose lactique fatales dans plus de 80% de cas. Selon les familles des patients, ces crises apparaissent lors d’une demande excessive d’énergie. Malheureusement, les mécanismes sous-jacents à l’apparition des crises et notamment la physiopathologie du LSFC demeurent inconnus. Afin de mieux comprendre les mécanismes de régulation du métabolisme énergétique chez les patients LSFC, nous avons examiné la régulation de la protéine kinase activée par l’AMP (AMPK), une enzyme clé de l'homéostasie énergétique, de même que certaines de ses voies cibles (SIRT1/PGC1α et Akt/mTOR) dans les fibroblastes de patients LSFC et de témoins en conditions basales et conditions de stress. En conditions basales, l’activité de l’AMPK était similaire dans les cellules LSFC et les témoins. Par contre, les cellules LSFC montraient une surexpression significative des voies Akt/mTOR et SIRT1/PGC1α comparativement aux cellules témoins. Nous avons aussi examiné ces voies de signalisation suite à une incubation de 4h avec 10 mM de lactate et 1 mM de palmitate (LP), nous permettant de mimer les conditions de « crise ». Nos résultats ont démontré que le LP augmentait les niveaux de phosphorylation de l’AMPK de 90% (p<0,01) dans les cellules témoins mais pas dans les cellules LSFC. Pourtant, l’AMPK est activée dans les cellules LSFC en réponse à une hypoxie chimique induite par le 2,4 dinitrophénol. Dans les cellules témoins, le LP augmentait aussi les niveaux d’expression de SIRT1 (57%, p<0,05), de LRPPRC (23%, p=0,045) et de COXIV (19%, p<0,05). Un prétraitement de 48h au ZMP, un activateur pharmacologique de l’AMPK, a eu un effet additif avec le LP et des augmentations de SIRT1 phosphorylée (120%, p<0,05), de SIRT1 total (75%, p<0,01), de LRPPRC (63%, p<0,001) et de COXIV (38%, p<0,001) ont été observées. Tous ces effets étaient aussi abolis dans les cellules LSFC. En conclusion, nos résultats ont démontré des altérations importantes de la régulation du métabolisme énergétique dans les fibroblastes de patients LSFC. / Leigh syndrome French Canadian type (LSFC) is an orphan infantile disease caused by mutations in the LRPPRC gene. It is characterized by a tissue-specific cytochrome c oxidase deficiency (COX), mitochondrial dysfunction and fatal lactic acidosis crises which occur in more than 80% of cases. According to parents, these crises occur during stressful situations. The pathophysiology underlying this disease and the factors that precipitate these crises remain unknown. To better understand the regulation of energy metabolism in LSFC patients, we examined the activity of AMP activated protein kinase (AMPK), a key regulator of energy balance, and its downstream targets (SIRT1/PGC1α and Akt/mTOR) in LSFC and control fibroblasts under basal and stress conditions. Our results showed that AMPK activity was similar in LSFC and control cells under basal conditions. On the other hand, Akt/mTOR and SIRT1/PGC1α pathways were up regulated in LSFC cells compared to controls. We next examined AMPK activity in cells treated with 10 mM lactate and 1mM palmitate (LP) for 4h, thus mimicking the conditions of “crisis”. Following this treatment, AMPK phosphorylation levels increased significantly (90%, p<0.01) in control cells but not in LSFC cells. Nevertheless, AMPK seems functional in LSFC cells because the enzyme was activated in response to chemical hypoxia induced by 2,4 dinitrophenol. LP also increased the expression of SIRT1 (57%, p<0.05), LRPPRC (23%, p=0.045) and COXIV (19%, p<0.05), in controls cells. Furthermore, pretreatment with ZMP, a pharmacological activator of AMPK, had an additive effect with LP leading to a further increase in the activity of SIRT1 (120%, p<0.05), as well as the expression levels of SIRT1 (75%, p<0.01), LRPPRC (63%, p<0.001) and COXIV (38%, p<0.001). All these effects were abolished in LSFC cells and thus, our data highlight alterations in the regulation of key enzymes of energy metabolism, including the activation of AMPK, in LFSC fibroblasts.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-05-09T19:13:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Efeito neuroprotetor de Anacardium Microcapum-duke em dano induzido por 6-ohda em córtex cerebral de Pintainhos.pdf: 1865633 bytes, checksum: 3c3083e33b14387c66dd69ed3da41472 (MD5) / Made available in DSpace on 2017-05-09T19:13:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Efeito neuroprotetor de Anacardium Microcapum-duke em dano induzido por 6-ohda em córtex cerebral de Pintainhos.pdf: 1865633 bytes, checksum: 3c3083e33b14387c66dd69ed3da41472 (MD5) Previous issue date: 2017-04-05 / A Doença de Parkinson (DP) é uma doença degenerativa, crônica e progressiva, acomete o sistema nervoso central e é responsável pela degeneração dos neurônios dopaminérgicos. Sabese que alterações genéticas e ambientais como exposição a agroquímicos, estresse oxidativo e disfunção mitocondrial estão associados à progressão da doença. A neurotoxina 6hidroxidopamina (6-OHDA), é um análogo estrutural da catecolamina dopamina, servindo como um modelo de neurotoxicidade por mecanismos semelhantes ao observado na DP. O mecanismo de citotoxicidade atribuído a 6-OHDA está diretamente ligado com a produção de espécies reativas de oxigênio (EROs) oriundas da inibição da respiração mitocondrial e sua auto-oxidação. A busca por terapias alternativas como os antioxidantes tem crescido ao longo dos anos, buscando atenuar a progressão da DP através de compostos bioativos de plantas. Neste estudo buscou-se avaliar o efeito neuroprotetor de Anacardium microcarpum frente ao dano induzido pela neurotoxina 6-OHDA em fatias corticais de pintainhos. Fatias foram incubada por 2h na presença da neurotoxina e diferentes concentrações do extrato hidroalcoólico (AMHE) e frações acetato de etila (AMEAF) e metanólica (AMMF) de A. microcarpum. AMHE, AMMF e AMEAF (1-1000 µg/mL) não apresentaram citotoxicidade per se nas fatias corticais. AMMF e AMEAF restauraram a queda da viabilidade induzida por 6OHDA (500 µM) a partir da concentração de 100 µg/mL, sendo a AMMF nesta mesma concentração, capaz de reverter a peroxidação lipídica causada pelo composto. 6-OHDA aumentou a atividade de GST e TrxR e diminuiu a atividade da GPx, além de diminuir os níveis de GSH total e aumentar a razão GSH/GSSG. Tais efeitos não foram observados na presença de AMME. Ainda, 6-OHDA inibiu o complexo I da cadeia respiratória mitocondrial, sendo que a fração não reverteu este efeito. Além disso, a auto-oxidação de 6-OHDA não foi revertida pela planta. A fosforilação de p38, JNK1/2, ERK1/2 e AKT bem como clivagem de PARP foi avaliada frente ao tratamento com neurotoxina e fração metanólica. A fração não levou a aumentos significativos na fosforilação das MAPKs bem como na expressão destas, também não levou à clivagem da proteína PARP. Entretanto, na presença de fração e 6-OHDA houve aumento significativo na fosforilação de ERK1/2 sem alterar sua expressão. Averiguou-se o envolvimento de ERK1/2 e AKT, proteínas envolvidas nos mecanismos de sobrevivência, na neurotoxicidade induzida por 6-OHDA através do uso de inibidores. Observou-se que na presença de inibidor, o extrato não foi capaz de proteger contra o dano promovido por 6-OHDA. Nossos resultados sugerem que o extrato tem ação antioxidante contra o estresse oxidativo decorrente da inibição da respiração mitocondrial e auto-oxidação da neurotoxina, sem no entanto interferir nestes processos. Além disso, sugere-se que as proteínas anti-apoptóticas ERK1/2 e AKT estejam envolvidas no efeito neuroprotetor da fração por mecanismos ainda não conhecidos. Nossos dados mostram pela primeira vez a ação neuroprotetora de A. microcarpum frente ao dano neuronal induzido pela 6-OHDA em fatias cerebrais e / Parkinson's disease (PD) is a degenerative, chronic and progressive disease, which affects the central nervous system and it is responsible for degeneration of dopaminergic neurons. It is known that genetic and environmental factors such as exposure to agrochemical, oxidative stress and mitochondrial dysfunction are associated with progression of the disease. 6hydroxydopamine (6-OHDA) is a structural analogue of catecholamine dopamine, used as a model of neurotoxicity by similar mechanism in PD. The mechanism of cytotoxicity attributed to 6-OHDA is linked to the production of reactive oxygen species (ROS) from inhibition of mitochondrial respiration and its autoxidation. The search for alternative therapies such as antioxidants has grown over the years, seeking to mitigate the progress of PD through bioactive plant compounds. This study aimed to evaluate the neuroprotective effect of Anacardium microcarpum on the damage induced by neurotoxin 6-OHDA in cortical slices of chicks. Slices were incubated for 2 h in the presence of neurotoxin and different concentrations of hydroalcoholic extract (AMHE) and ethyl acetate (AMEAF) and methanol (AMMF) fractions of A. microcarpum. AMHE, AMMF and AMEAF (1-1000 μg/mL) did not show cytotoxicity per se in the cortical slices. AMMF and AMEAF restored the drop in viability caused by 6OHDA (500 μM) from the concentration of 100 μg/mL. 6-OHDA increased GST and TrxR activity while GPx activity and total GSH levels was decreased with an augmented ratio GSH / GSSG. These effects were not observed in the presence of AMMF and 6-OHDA. Furthermore, 6-OHDA inhibited the complex I of the mitochondrial respiratory chain but this effect was not reversed by fraction as well as the self-oxidation of 6-OHDA was not avoided by the plant. Phosphorylation of p38, JNK1/2, ERK1/2 and AKT as well as PARP cleavage was evaluated against treatment with neurotoxin and methanolic fraction. The methanolic fraction and 6OHDA did not alter phosphorylation of MAPKs, as well as expression of these proteins, not did it result in the cleavage of the PARP protein in the time studies. However, in the presence of fraction and 6-OHDA there was a significant increase in ERK1/2 phosphorylation without altering its expression. The involvement of ERK1/2 and AKT proteins in protective mechanism of fraction was analyzed through the use of inhibitors. It was observed that in presence of inhibitor, the extract was not able to protect against the damage promoted by 6-OHDA. Our results suggest that the extract presented antioxidant action against oxidative stress resulted from the inhibition of mitochondrial respiration and neurotoxin autoxidation. In addition, it is suggested that antiapoptotic proteins ERK1/2 and AKT are involved in neuroprotective effect of the fraction. Our data show for the first time a neuroprotective action of A. microcarpum against neuronal damage induced by 6-OHDA in cerebral slices and highlights the potential of this plant as a source of bioactive compounds with therapeutic potential

A disfunção mitocondrial pode ser um mecanismo chave associado à ocorrência de doenças metabólicas como o diabetes. Neste contexto, é importante obeservar os mecanismos envolvidos nesse processo. MicroRNAs (miRs) são conhecidos por regular a expressão de genes em vários processos fisiológicos, incluindo o metabolismo de glicose e ácidos graxos, biogênese mitocondrial, proliferação, diferenciação e morte celular no músculo esquelético. Usando análise \"in silico\" (Sfold2.2) identificamos 219 microRNAs que, potencialmente, se ligam à região 3 \'UTR do PGC-1?, um gene envolvido na biogênese mitocondrial e no metabolismo de glicose. Dos 219 candidatos, encontramos um alto valor de energia livre de hibridização entre o microRNA miR-696 e PGC-1? (-29,8 kcal / mol), sugerindo que o miR-696 poderia estar envolvido na regulação negativa do PGC-1? resultando em disfunção mitocondrial. Consistente com esta hipótese, observamos que a expressão do miR-696 apresentou-se aumentada nos músculos esqueléticos de dois modelos de camundongos com diabetes: camundongos diabéticos induzidos por STZ e camundongos alimentados com dieta hiperlipídica. Para compreender se o miR-696 regula a disfunção mitocondrial utilizamos células musculares C2C12 expostas a uma alta dose de ácido palmítico (700 µM) durante 24 horas, o que causou uma redução na expressão de genes mitocondriais, bem como no consumo de oxigênio. Vale destacar que a inibição do miR-696 através da transfecção de oligonucleotídeos antisenso (ASO) preveniu, parcialmente, a perda da função mitocondrial de células C2C12 tratadas com ácido palmítico. Curiosamente, não houve nenhuma alteração nos níveis de miR-696 em modelos envolvidos com a proteína AMPK, tal como em células C2C12 incubadas com uma droga ativadora de AMPK (AICAR) e no músculo esquelético de camundongos transgênicos superexpressando AMPK?2 com o domínio quinase inativo ou AMPK?3 com mutação de ativação crônica (R70Q). Em contraste, a expressão alterada de uma quinase relacionadas com a AMPK, SNF1-AMPK-related kinase (SNARK), recentemente demonstrada por ter sua expressão aumentada em virtude do envelhecimento, exerceu efeitos significativos sobre a expressão do miR- 696, como por exemplo sua redução dependente do knockdown de SNARK em células C2C12. Consistente com estes resultados, a superexpressão de SNARK em células C2C12 resultou no aumento da expressão do miR-696 e redução na expressão do PGC-1?, bem como no consumo de oxigénio. Nossos resultados demonstram que o estresse metabólico aumenta a expressão do miR-696 no músculo esquelético, que por sua vez inibe a sinalização da PGC-1? e a função mitocondrial. Ainda, apesar da AMPK não se apresentar como mediadora da expressão do miR-696, SNARK pode desempenhar um papel neste processo através do mecanismo de sinalização SNARKmiR-696-PGC-1?. / Mitochondrial dysfunction may be a key underlying mechanism for occurrence of metabolic disease and diabetes; thus elucidating how this process occurs is of great value. MicroRNAs (miRs) are known to regulate gene expression in several physiological processes including metabolism, mitochondrial biogenesis, proliferation, differentiation and cell death in multiple tissues including adipose tissue and skeletal muscle. Using \"in silico\" analysis (Sfold2.2) we identified 219 unique microRNAs that potentially bind to the 3\'UTR region of PGC-1?, a gene involved in mitochondrial biogenesis and glucose metabolism. Out of the 219 candidates, there was a high value of hybridization free energy between the microRNA miR-696 and PGC-1? (- 29.8 kcal/mol), suggesting that miR-696 could be involved in the downregulation of PGC-1?, which in turn could cause mitochondrial dysfunction. Consistent with this hypothesis we found that miR-696 expression was increased in the skeletal muscles of two mouse models of diabetes that have impaired mitochondrial function: STZ-induced diabetic mice and chronic high fat fed mice. To understand if miR-696 regulates mitochondrial dysfunction we used C2C12 muscle cells exposed to a high dose of palmitic acid (700 µM) for 24 hours, which caused a decrease in mitochondrial gene expression and in oxygen consumption. Importantly, inhibition of miR-696 using an antisense oligo approach rescued the mitochondrial function by restoration of mitochondrial-related genes and increased oxygen consumption in the palmitic acid-treated C2C12 cells. Interestingly, there was no change in miR-696 levels in models involved with AMPactivated protein kinase such as C2C12 cells incubated with AICAR, skeletal muscle from AMPK?2 dominant-negative transgenic mice, and transgenic mice overexpressing the activating R70Q AMPK mutation. In contrast, altered expression of the AMPK-related kinase, SNF1- AMPK-related kinase (SNARK), recently shown to increase with aging, had significant effects on miR-696 expression. Knockdown of SNARK in C2C12 cells significantly decreased miR-696. Consistent with these findings, SNARK overexpression in C2C12 cells increased miR-696 concomitant with a decrease in PGC-1? expression and decreased oxygen consumption. Our findings demonstrate that metabolic stress increases miR-696 expression in skeletal muscle which in turn inhibits PGC-1? signaling and mitochondrial function. While AMPK does not mediate miR-696 expression, SNARK may play a role in this process through a SNARK-miR- 696-PGC-1? signaling mechanism.

Abstract Cardiac and skeletal muscle cell contraction is a result of excitation-contraction coupling (ECC), where an electrical signal leads to a rise in intracellular calcium levels and contraction. This process is carefully regulated to meet physiological demand and heavily dependent on an adequate energy supply. Disturbed ECC can have severe consequences on muscle cell function and underlies many cardiac and skeletal muscle pathologies. Cell stress, changing intracellular Ca2+ concentrations, and calcium signal dynamics can all play a role in the transcriptional regulation of genes involved in myocyte Ca2+-handling. In this thesis project, the transcriptional control of ECC was studied in skeletal and cardiac myocytes. Skeletal myocyte calsequestrin (CASQ1) was downregulated in a mouse model of mitochondrial myopathy and it contributed to the decreased SR Ca2+ load and impaired Ca2+ handling in Tfam-/- skeletal myocytes. In cultured neonatal cardiomyocytes, mitochondrial uncoupler FCCP-induced mitochondrial dysfunction led to downregulation of cardiac calsequestrin (CASQ2) and similarly impaired Ca2+ handling. Whereas there was no increase in reactive oxygen species (ROS) levels in Tfam-/- myocytes, cultured cells exposed to FCCP did display increased ROS, an effect that was counteracted by coexposure with the ROS scavenger (NAC). NAC attenuated FCCP-induced CASQ2 downregulation and restored Ca2+ handling. Therefore, mitochondrial dysfunction led to CASQ1/2 downregulation and impaired Ca2+ handling in these two cell types, but by different mechanisms. This project also looked at the role of Ca2+ dynamics on the transcriptional regulation of Ca2+ handling genes. Increased intracellular Ca2+ levels and &#946;-adrenergic stimulation of cardiomyocytes activate Ca2+-calmodulin kinase II (CaMKII) and can trigger hypertrophic remodeling. It was found that CaMKII downregulated expression of the L-type Ca2+ channel &#945;1c-subunit (Cacna1c) in cultured cardiomyocytes. Analysis of the Cacna1c promoter revealed that the transcriptional repressor DREAM bound to a putative downstream regulatory element. The results shed light on the complex interplay between muscle cell energetics and transcriptional regulation of SR Ca2+ handling proteins. A unique pathway for Cacna1c transcriptional regulation by CaMKII and DREAM was also described. / Tiivistelmä Sydän- ja luustolihassolujen supistuminen on seurausta ärsytys-supistuskytkennästä (ECC), jossa sähköinen ärsytys kohottaa solunsisäistä kalsiumpitoisuutta ja aiheuttaa supistuksen. Tätä säädellään tarkasti fysiologisen tarpeen mukaan, ja se riippuu riittävästä energian saannista. Häiriintynyt ECC voi aiheuttaa vakavia seurauksia lihassolujen toiminnalle, ja se on mukana monien sydän- ja luustolihasten sairauksien synnyssä. Tässä tutkimuksessa ECC:n transkriptionaalista säätelyä tutkittiin luustolihasten ja sydämen lihassoluissa. Luustolihassolujen kalsekvestriinin (CASQ1) väheneminen pienensi SR:n Ca2+-määrää mitokondrioiden myopatian hiirimallissa ja heikensi Ca2+-tasapainon ylläpitoa Tfam-/--luustolihassoluissa. Viljellyissä vastasyntyneiden kammio-sydänlihassoluissa mitokondrio-irtikytkijän FCCP:n aiheuttama mitokondrioiden toimintahäiriö johti sydämen kalsekvestriinin (CASQ2) vähenemiseen ja heikensi samalla tavalla Ca2+-tasapainon ylläpitoa. Vaikka Tfam-/--myosyyteissä reaktiivisten happilajien (ROS) tasot eivät olleet koholla, FCCP:lle altistetuissa viljellyissä soluissa ROS kuitenkin lisääntyi. Vaikutusta esti ROS-puhdistaja NAC, joka heikensi FCCP:n aiheuttamaa CASQ2:n laskua ja palautti Ca2+-säätelyn normaaliksi. Mitokondrioiden toimintahäiriö siis johti CASQ1/2:n vähenemiseen ja Ca2+-säätelyn heikentymiseen molemmissa solutyypeissä, mutta eri mekanismeilla. Tässä tutkimuksessa tarkasteltiin myös Ca2+-dynamiikan osuutta Ca2+-tasapainoon osallistuvien geenien transkription säätelyssä. Lisääntynyt solunsisäinen Ca2+-taso ja sydänlihassolujen &#946;-adrenerginen stimulointi aktivoivat Ca2+-kalmoduliinikinaasi II:n (CaMKII), ja ne voivat laukaista sydämen hypertrofisen uudelleenmuovautumisen. Havaittiin, että CaMKII vähensi L-tyypin Ca2+-kanavan a1c-alayksikön (Cacna1c) ilmentymistä viljellyissä sydänlihassoluissa. Promoottorianalyysi osoitti tämän johtuvan transkription repressorin DREAM:n sitoutumisesta oletettuun DRE:hen (alavirrassa sijaitseva säätelyelementti). Nämä tulokset tuovat uutta tietoa lihassolujen energiatalouden ja SR:n Ca2+:n vaikuttavien proteiinien transkription säätelyn vuorovaikutuksesta. Lisäksi havaittiin ainutlaatuinen Cacna1c-transkription säätelyn reitti, johon osallistuvat CaMKII ja DREAM.

Methamphetamine (METH) is a potent psychostimulant known to cause cognitive abnormalities and neurodegenerative changes in the brains of METH abusers. One approach for developing therapies for METH abuse is to understand the molecular mechanisms of toxicity of the drug. Investigations in our laboratory and elsewhere have shown that single intraperitoneal injections of METH (30-40 mg/kg of body weight) can cause damage to striatal and cortical monoaminergic systems and induce neuronal apoptosis in the striatum of rodents via activation of endoplasmic reticulum (ER) and mitochondrial death pathways. Hence, the purpose of this thesis was to investigate if toxic binge METH injections can cause ER- and mitochondria-induced stress in the rat striatum. Recent studies have suggested that dopamine (DA) D1 and D2 receptors might mediate neuronal apoptosis in the striatum after single toxic METH doses. We therefore hypothesized that signaling through these two types of DA receptors might activate toxic effects of the binge METH regimen. The role of DA D1 or D2 receptors in METH-induced cell death pathways was thus examined by using pharmacological inhibitors of these receptors. In this dissertation, I report that binge METH regimen caused differential changes in immediate early genes (IEGs) that are known to influence synaptic changes in the brain. METH-induced changed in the expression of the IEGs were dependent on DA D1 receptor stimulation. The second study examined the effects of binge METH on the expression of ER stress- and mitochondrial dysfunction-responsive genes. Pretreatment with the DA D1 receptor antagonist, SCH23390, caused complete inhibition of METH-induced ER and mitochondrial stresses whereas the DA D2 receptor antagonist, raclopride, provided only partial blockade. SCH23390 also blocked METH-induced hyperthermia whereas raclopride failed to do so. Interestingly, both antagonists attenuated METH-induced dopaminergic and serotonergic deficits in the striatum. Moreover, SCH23390 but not raclopride blocked METH-induced serotonergic deficits in cortical tissues. I also found that METH treatment induced upregulation of activin βA mRNA, increased TGF-β and phosphorylated Smad2 proteins in the rat striatum. SCH23390 pretreatment completely blocked all these effects whereas raclopride did not block METH-induced increases in TGF-β expression.