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Additional file 5. Mitochondria are distributed between astrocytes via tunneling nanotubes. Arrows indicate transfer of MitoTracker™ Green labeled mitochondria from the top cell to the bottom cell via tunneling nanotubes. The top panel show fluorescent mitochondria in white and the bottom panel show the merged images (GFP and bright field) of the same time points.

Additional file 3. Mitochondrial fusion protein levels are not affected by Aβ exposure. Western blot analysis demonstrated that OPA1 levels remain unchanged by Aβ exposure at 7d and 7d+6d.

Additional file 8. Lipid droplets are found in both Aβ exposed and control astrocytes. Lipid droplets (white asterisks) are observed in Aβ exposed astrocytes (a,b) and in controls (c,c´).

Additional file 1. Astrocytic markers. hiPSC-derived astrocytes were stained with DAPI (blue) and the astrocytic markers: GLAST-1 (A), Vimentin (B) S100β (C), AQP4 (D), GFAP (E), and Nestin (F). Scale bar: 20 μm.

Additional file 2. Aβ exposure affects mitochondrial motility in astrocytes. The parameter displacement, distance, velocity and speed were analyzed with Mitometer (A). Motility analysis showed a significant increase in the mitochondrial speed (B) and velocity (C) in Aβ-exposed astrocytes.

Additional file 7. Aβ exposure does not affect basal respiration of mitochondria, cell viability or apoptosis in astrocytes. Seahorse OCR analysis showed no differences in mitochondrial basal respiration between control and Aβ-exposed astrocytes (A). Alamar blue assay showed no decrease in viability in the astrocyte cultures exposed to Aβ (B). Similarly, WB analysis showed no increase in apoptosis markers caspase-3 and BAX in the Aβ-exposedcultures (C and D).

Additional file 1: Figure S1. Characterization of differentiated SH-SY5Y cells. Figure S2. Cytotoxicity of compounds evaluated in the HTS. Figure S3. Effects of luteolin in respiratory capacity and ΔΨm. Figure S4. Effects of luteolin in mitochondrial biogenesis and cristae organization. Figure S5. Effects of luteolin on calcium levels and characterization of isolated mitochondrial fractions.

Recent findings have shown that the connectivity and crosstalk between mitochondria and the endoplasmic reticulum (ER) at mitochondria&ndash, ER contact sites (MERCS) are altered in Alzheimer&rsquo, s disease (AD) and in AD-related models. MERCS have been related to the initial steps of autophagosome formation as well as regulation of mitochondrial function. Here, the interplay between MERCS, mitochondria ultrastructure and function and autophagy were evaluated in different AD animal models with increased levels of A&beta, as well as in primary neurons derived from these animals. We start by showing that the levels of Mitofusin 1, Mitofusin 2 and mitochondrial import receptor subunit TOM70 are decreased in post-mortem brain tissue derived from familial AD. We also show that A&beta, increases the juxtaposition between ER and mitochondria both in adult brain of different AD mouse models as well as in primary cultures derived from these animals. In addition, the connectivity between ER and mitochondria are also increased in wild-type neurons exposed to A&beta, This alteration in MERCS affects autophagosome formation, mitochondrial function and ATP formation during starvation. Interestingly, the increment in ER&ndash, mitochondria connectivity occurs simultaneously with an increase in mitochondrial activity and is followed by upregulation of autophagosome formation in a clear chronological sequence of events. In summary, we report that A&beta, can affect cell homeostasis by modulating MERCS and, consequently, altering mitochondrial activity and autophagosome formation. Our data suggests that MERCS is a potential target for drug discovery in AD.

Tese de doutoramento em Ciências da Saúde, ramo Ciências Biomédicas apresentada à Faculdade de Medicina da Universidade de Coimbra Huntington’s disease (HD) is a neurodegenerative disorder that gradually affects memory, cognitive skills and normal movements of the affected individuals and for which no disease modifying treatments exist. The disease is caused by an expanded CAG trinucleotide repeat (of variable length) in the HTT gene that encodes the huntingtin protein (HTT). Mutant HTT (mHTT) exhibits an abnormal elongated polyglutamine stretch that confers a toxic gain of function and predisposes the protein to fragmentation and aggregation, resulting in neuronal dysfunction and selective death in striatum and cortex. There is strong evidence that transcriptional deregulation and altered mitochondrial function occurs early and acts causally in HD pathogenesis; importantly, these events may be related to altered acetylation of proteins. Therefore, pharmacological strategies that interfere with both nuclear and mitochondrial protein acetylation may be therapeutically useful to hinder neuronal dysfunction in the course of HD pathology. In the present work, distinct lysine deacetylases (a heterogeneous group of proteins that remove acetyl groups from histones and other proteins regulating their structure and function), were pharmacologically or genetically targeted with the main purpose of counteracting mitochondrial and metabolic deficits in in vitro and in vivo models expressing human full-length mHTT. We have previously shown that mitochondrial dysfunction in HD persists at the level of pyruvate dehydrogenase (PDH), which functionally links glycolysis to oxidative phosphorylation. In Chapter 3 we report that decreased PDH activity in HD striatal cells expressing 111 glutamines (STHdhQ111/Q111) occurs through enhanced PDH kinases (PDKs) and reduced PDH phosphatase 1 protein expression, which trigger inhibitory phosphorylation of catalytic PDH E1α subunit in three different serine sites (Ser293, Ser300 and Ser232). Classes I and IIa histone deacetylase inhibitors (HDACi), sodium butyrate (SB) and phenylbutyrate, increased histone H3 acetylation and enhanced metabolism and mitochondrial respiration, similar to PDH activator dichloroacetate, suggesting that HDACi may favor activation of PDH complex. Concordantly, SB significantly decreased the expression of PDK2 and PDK3 in STHdhQ111/Q111 cells, which led to decreased PDH E1α phosphorylation in all serine sites. This effect occurs since HDACi SB stimulates HIF-1α degradation, a transcription factor that actively suppresses metabolism by directly transactivating genes encoding for PDKs. Therefore, partial depletion of PDK3 enhanced mitochondrial respiration and ATP synthesis in both wild-type and mutant STHdh cells, similarly to the effects achieved with SB treatment. YAC128 transgenic HD mice also exhibited decreased PDH E1α phosphorylation and PDK1-3 expression after SB treatment, which positively influenced central and peripheral metabolism. Further studies were conducted using modulators of class III lysine deacetylases, classically known as sirtuins. SIRT1, the best studied member of the sirtuins family, has been shown to exert neuroprotective roles in several models of neurodegeneration, boosting the interest in the development of SIRT1 activators. However, recent reports on SIRT1 inhibitors have also shown protective effects, casting doubt regarding previous findings. To better understand this paradox we tested resveratrol (RESV, a SIRT1 activator) and nicotinamide (NAM, a SIRT1 inhibitor) in in vitro models and in an animal model expressing mHTT (Chapter 4). We found that RESV enhanced lysine deacetylation activity and recovered abnormal mitochondrial phenotype of lymphoblasts from affected HD patients and cortical and striatal primary neurons isolated from YAC128 mice, which was associated with increased mitochondrial transcription and biogenesis. NAM had no effects on mitochondrial biogenesis, however increased NAD+ levels following NAM treatment may explain the positive impact on mitochondrial function in vitro. In symptomatic YAC128 mice, RESV completely abrogated deficits in motor learning and coordination, two well-established features of this transgenic HD model, and increased transcription of mitochondrial-encoded electron transport chain genes. In turn, NAM supplementation in vivo proved to be deleterious. The positive effects achieved with the SIRT-activating compound RESV led us to specifically target SIRT3 (Chapter 5), the major mitochondrial deacetylase that has received much attention for its role in oxidative metabolism and aging. We found increased SIRT3 levels and activity in HD lymphoblasts and in STHdhQ111/Q111 cells. Considering its potential neuroprotective role in HD, we overexpressed (OE) SIRT3, which was shown to co-localize more in mitochondria from HD striatal cells than in wild-type counterparts. Cortical tissue from YAC128 mice also exhibited lower acetylation of superoxide dismutase 2, a recognized target of SIRT3, suggesting increased SIRT3 activity. When OE in STHdh, SIRT3 enhanced lysine deacetylation and mitochondrial transmembrane potential. SIRT3 OE also reverted the loss in mitochondrial-related transcription factors, PGC-1α and TFAM, induced by mHTT. Still, increased mitochondrial content was not observed. Ultimately, STHdhQ111/Q111-SIRT3 OE cells showed lower susceptibility to apoptotic cell death. Overall, this study provides novel insights into the molecular deficits underlying mitochondrial dysfunction in HD and explores promising drugs that effectively modulate acetylation landscape, further impacting on mitochondrial and mitochondrial-related transcription proteins activity. Finally, increased mitochondrial activity and transcription partially control HD-related motor disturbances and neuronal death. A Doença de Huntington (DH) é uma doença neurodegenerativa que afeta gradualmente as capacidade cognitivas e motoras dos indivíduos afetados, e para a qual não existe tratamento neuroprotetor ou cura. A DH é causada por uma expansão de trinucleótidos CAG (de tamanho variável) no gene HTT que codifica para a proteína huntingtina (HTT). A HTT mutante (mHTT) possui uma expansão anormal de poliglutaminas que lhe confere uma função tóxica, predispondo-a para a fragmentação e agregação, resultando em disfunção e morte seletiva de neurónios estriatais e corticais. Fortes evidências sugerem que alterações na regulação da transcrição e na função mitocondrial ocorrem em estádios iniciais da doença e são fatores causais para a patogénese da DH; estes eventos poderão estar relacionados com alterações na acetilação de proteínas. Assim, estratégias farmacológicas que interfiram com a acetilação de proteínas nucleares e mitocondriais poderão ser profícuas no combate à disfunção neuronal no decurso da DH. Neste trabalho diferentes desacetilases de lisinas (um grupo heterogéneo de proteínas que remove grupos acetil de histonas ou outras proteínas, regulando a sua estrutura e função) foram farmacologicamente ou geneticamente moduladas com o objetivo de neutralizar os défices mitocondriais e metabólicos em modelos que expressam a mHTT. Anteriormente o nosso grupo de investigação mostrou que a disfunção mitocondrial ocorre ao nível da piruvato desidrogenase (PDH), um complexo enzimático que faz a ligação entre a glicólise e a fosforilação oxidativa. No Capítulo 3 desta tese mostrámos que a diminuição da atividade da PDH em células estriatais que expressam 111 glutaminas (STHdhQ111/Q111) ocorre devido ao aumento das cinases da PDH (PDKs) e à diminuição da fosfatase 1 da PDH, desencadeando a fosforilação (inibitória) da subunidade catalítica PDH E1α em três resíduos de serina (Ser293, Ser300 e Ser232). Inibidores das classes I e IIa das desacetilases de histonas (HDACi), os compostos butirato de sódio (BS) e fenilbutirato, aumentaram a acetilação da histona H3 e melhoraram o metabolismo e a respiração mitocondrial, de forma semelhante ao observado com dicloroacetato, um reconhecido ativador da PDH, sugerindo que os HDACi poderão favorecer a atividade da PDH. Em concordância, o SB diminuiu a expressão da PDK2 e PDK3 nas células STHdhQ111/Q111, o que levou a uma diminuição da fosforilação da PDH E1α em todos os resíduos de serina. Este efeito mediado pelo SB parece ter resultado da estimulação da degradação do HIF-1α, um fator de transcrição que inibe o metabolismo celular através da transativação de genes que codificam para as PDKs. De forma semelhante ao SB, a redução parcial da expressão da PDK3 aumentou a respiração mitocondrial e a síntese de ATP em ambas as células STHdh wild-type e mutante. No murganho transgénico YAC128 verificou-se uma diminuição na fosforilação da PDH E1α e na expressão das PDK1-3 após tratamento com SB, influenciando positivamente o metabolismo energético. Nos estudos que se seguiram foram utilizados moduladores de desacetilases de lisinas de classe III, conhecidas como sirtuinas. A SIRT1, o membro da família mais estudado, tem vindo a mostrar um papel neuroprotetor em vários modelos de neurodegenerescência, aumentando o interesse no desenvolvimento de ativadores da SIRT1. No entanto, em estudos mais recentes os inibidores da SIRT1 também mostraram resultados protetores, colocando em dúvida as observações anteriores. Para compreender melhor este paradoxo, testámos o efeito do resveratrol (RESV, ativador da SIRT1) e da nicotinamida (NAM, inibidor da SIRT1) em modelos in vitro e num animal modelo que expressa a mHTT (Capítulo 4). O RESV aumentou a atividade desacetilase e reverteu a disfunção mitocondrial quando testado em linfoblastos de doentes de Huntington e em neurónios corticais e estriatais isolados de embriões do murganho YAC128; estas observações foram associadas ao aumento da transcrição e biogénese mitocondrial. O tratamento com NAM não teve qualquer efeito na biogénese mitocondrial; no entanto, o aumento dos níveis de NAD+ poderão justificar o impacto positivo observado na função mitocondrial testada in vitro. Quando administrado numa fase sintomática do murganho YAC128, o RESV anulou os défices de aprendizagem e coordenação motora, duas características deste animal transgénico, e aumentou a transcrição de genes da cadeia respiratória mitocondrial codificados pelo DNA mitocondrial. Por sua vez, a administração de NAM teve um efeito deletério in vivo. Os efeitos positivos obtidos com o ativador da SIRT1, resveratrol, levou-nos a modular especificamente a SIRT3 (Capítulo 5), uma desacetilase mitocondrial que tem recebido especial atenção pelo seu papel no metabolismo oxidativo e no processo de envelhecimento. Em linfoblastos DH e em células STHdhQ111/Q111 observámos um aumento dos níveis proteícos, de RNAm e da atividade da SIRT3. Considerando o potencial terapêutico da SIRT3 na DH, sobre-expressámos (SE) esta proteína em células estriatais e verificámos que se co-localiza preferencialmente nas mitocôndrias das células estriatais mutantes, comparativamente às células wild-type. O córtex do murganho YAC128 também exibiu menor acetilação da proteína superóxido dismutase 2, um alvo da SIRT3, sugerindo uma maior atividade desta sirtuína no contexto da DH. Quando SE em células STHdh, a SIRT3 aumentou a desacetilação e o potencial de membrana mitocondrial. A SE da SIRT3 também reverteu a diminuição dos fatores de transcrição associados à mitocôndria, PGC-1α e TFAM, induzida pela mHtt. Contudo, não se observou um aumento da massa mitocondrial. Por fim, as células STHdhQ111/Q111 que com SIRT3 SE apresentaram uma menor suscetibilidade à morte celular por apoptose. Em conclusão, este estudo fornece novas perspetivas sobre os défices moleculares subjacentes à disfunção mitocondrial na DH, e explora estratégias farmacológicas promissoras que modificam a acetilação e que têm posterior impacto na atividade da mitocôndria e nos fatores de transcrição associados a este organelo. Adicionalmente, o aumento da função e transcrição mitocondrial influenciam os distúrbios motores e a morte neuronal associada à DH. Palavras chave: Doença de Huntington, mitocôndria, piruvato desidrogenase, desacetilases de lisinas, neuroproteção. Neuroscience prize 2013, supported by Santa Casa da Misericórdia de Lisboa

Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde A doença de Huntington (HD) é uma doença autossómica neurodegenerativa progressiva, caracterizada pela perda de coordenação motora, défice cognitivo e distúrbios psiquiátricos, afectando cerca de um em cada 100,000 indivíduos. A HD envolve uma perda selectiva dos neurónios espinhosos médios do estriado, como consequência de uma repetição da expansão de trinucleótido CAG (que codifica para glutamina) na proteína huntingtina (Htt), formando-se agregados e inclusões intranucleares com a progressão da doença. Tendo em conta as evidências da literatura que referem que a insulina e o factor de crescimento insulínico de tipo 1 (IGF-1) podem ter um potencial terapêutico na HD, colocou-se a hipótese que ambos os factores tróficos poderiam prevenir alterações na glicólise e na sinalização intracelular associadas à HD, mediadas pelos receptores de insulina e de IGF-1 (IR e IGF-1R, respectivamente). Assim, o objectivo deste estudo consistiu na avaliação das alterações metabólicas e da sinalização celular induzidas pela expressão da Htt humana mutante e os efeitos da exposição da insulina versus IGF-1 em neurónios do sistema nervoso central e células do sistema periférico. Assim, utilizaram-se neurónios corticais e estriatais derivados do murganho transgénico YAC128 (contendo o gene HD humano com 128 repetições CAG) versus o murganho wild-type (WT) e linhas celulares de linfoblastos humanos de pacientes HD e indivíduos controlo. Observou-se uma elevada capacidade redutora dos neurónios corticais de murganhos YAC128, comparada com os murganhos WT, determinada pelo ensaio de Alamar blue (AB). A adição de 56 mM de glucose (24 h) aos neurónios WT aumentou a redução da resazurina, sugerindo um aumento da capacidade redutora neuronal. Contudo, estes resultados não foram observados nos neurónios mutantes, sugerindo a ocorrência de alterações nas vias metabólicas devido à expressão da Htt mutante. Nesta perspectiva, observou-se um decréscimo significativo dos níveis de ATP e um aumento dos níveis de AMP nos neurónios corticais do murganho YAC128. A disfunção da glicólise nos neurónios corticais de YAC128 foi também confirmada por uma diminuição significativa dos níveis de lactato. É de realçar que os níveis de ATP foram aparentemente normalizados nos neurónios de murganhos YAC128 relativamente aos WT após a exposição à insulina (1 nM) ou IGF-1 (1 nM), durante 48 h. Nos neurónios estriatais não ocorreram alterações nos níveis de ATP ou na capacidade redutora entre células obtidas de murganhos YAC128 e WT. Os linfoblastos dos pacientes HD também apresentaram uma tendência para uma diminuição nos níveis de ATP, comparativamente aos linfoblastos controlo. Não obstante, os níveis de lactato apresentaram-se significativamente elevados em linfoblastos de pacientes HD, sugerindo uma estimulação da glicólise, que normalmente ocorre em células que apresentam disfunção mitocondrial. O tratamento com insulina ou IGF-1 também pareceu restabelecer os níveis de ATP e lactato. Relativamente às vias de sinalização intracelular, verificou-se uma tendência para um decréscimo nos níveis de fosforilação e consequente activação dos IR/IGF-1R, 44p e 42p ERK em linfoblastos de pacientes HD, recuperada após adição de insulina ou IGF-1 (0.1 nM; 24 h). Estes resultados sugerem que a disfunção metabólica associada à HD possui algumas características paralelas em culturas primárias de neurónios corticais dos murganhos YAC128 e em linfoblastos de pacientes HD. Adicionalmente, o tratamento com insulina e IGF-1 poderá prevenir a disfunção metabólica na HD. Huntington’s disease (HD) is an autosomal progressive neurodegenerative disorder, characterized by an age-dependent loss in motor coordination, cognitive impairment and psychiatric disturbances. It affects about one in 100,000 individuals. HD neuropathology involves the selective death of striatal medium-sized spiny neurons, as a consequence of an expansion of CAG repeats (that encodes for glutamine) in the huntingtin (Htt) protein, forming aggregates and intranuclear inclusions as the disease progresses. Based on the literature suggesting that insulin and particularly insulin-like growth factor-1 (IGF-1) may offer potential therapeutic protection against HD, we hypothesized that both trophic factors may prevent HD-associated changes in glycolysis and intracellular signaling mediated by the insulin and/or IGF-1 receptors (IR and IGF-1R, respectively). So, the objective of this study was to evaluate the cellular metabolic and signaling changes induced by expression of human mutant Htt and the in vitro effect of insulin versus IGF-1 in both central neurons and peripheral cells. Thus, we used cortical and striatal neurons derived from YAC128 transgenic mice (containing the human HD gene with 128 CAG repeats) versus wild-type (WT) mice and human lymphoblast cell lines derived from control individuals or HD patients. We observed a higher reducing capacity of cortical neurons from YAC128, compared with WT mice, as determined by the Alamar blue (AB) assay. Addition of 56 mM glucose (24 h) to WT neurons increased the reduction of resazurin, suggesting increased neuronal reducing capacity. However, this was not observed in mutant neurons, suggesting the occurrence of changes in metabolic pathways due to expression of mutant Htt. Accordingly, ATP levels were significantly reduced and AMP levels were increased in cortical neurons from YAC128 mice. Impaired glycolysis in cortical neurons from YAC128 mice was further confirmed by a significant decrease in lactate levels. Importantly, ATP levels appeared to be normalized in YAC128 mice relatively to neurons from WT mice after incubation with insulin (1 nM) or IGF-1 (1 nM), during 48 h. In striatal neurons, no significant changes were observed in both ATP levels and reducing capacity, between YAC128 and WT cells. Lymphoblasts from HD patients also showed a tendency for a decrease in ATP levels, compared with control lymphoblasts. Nevertheless, lactate levels were significantly elevated in HD patients’ lymphoblasts, suggesting a stimulation of glycolysis, which usually occurs in cells with dysfunctional mitochondria. Insulin or IGF-1 treatment (0.1 nM; 24 h) of HD lymphoblasts also appeared to restore both ATP and lactate levels. Regarding the signaling pathways, there was a tendency for a decrease in phosphorylation levels and thus activity of IR/IGF-1R, 44p and 42p ERK in HD patients, which was recovered upon addition of insulin and IGF-1. Data presented herein suggest that HD-associated metabolic dysfunction presents some parallel features in primary cortical neurons from YAC128 mice and in human lymphoblasts from HD patients. Additionally, insulin and IGF-1 treatment may prevent metabolic impairment in HD.

Insulin resistance is a major predictor of the development of metabolic disorders. Sirtuins (SIRTs) have emerged as potential targets that can be manipulated to counteract age-related diseases, including type 2 diabetes. SIRT2 has been recently shown to exert important metabolic effects, but whether SIRT2 regulates insulin sensitivity in hepatocytes is currently unknown. The aim of this study is to investigate this possibility and to elucidate underlying molecular mechanisms. Here, we show that SIRT2 is downregulated in insulin-resistant hepatocytes and livers, and this was accompanied by increased generation of reactive oxygen species, activation of stress-sensitive ERK1/2 kinase, and mitochondrial dysfunction. Conversely, SIRT2 overexpression in insulin-resistant hepatocytes improved insulin sensitivity, mitigated reactive oxygen species production and ameliorated mitochondrial dysfunction. Further analysis revealed a reestablishment of mitochondrial morphology, with a higher number of elongated mitochondria rather than fragmented mitochondria instigated by insulin resistance. Mechanistically, SIRT2 was able to increase fusion-related protein Mfn2 and decrease mitochondrial-associated Drp1. SIRT2 also attenuated the downregulation of TFAM, a key mtDNA-associated protein, contributing to the increase in mitochondrial mass. Importantly, we found that SIRT2 expression in PBMCs of human subjects was negatively correlated with obesity and insulin resistance. These results suggest a novel function for hepatic SIRT2 in the regulation of insulin sensitivity and raise the possibility that SIRT2 activators may offer novel opportunities for preventing or treating insulin resistance and type 2 diabetes.