Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease

Transcriptional deregulation and changes in mitochondrial bioenergetics, including pyruvate dehydrogenase (PDH) dysfunction, have been described in Huntington's disease (HD). We showed previously that the histone deacetylase inhibitors (HDACIs) trichostatin A and sodium butyrate (SB) ameliorate mitochondrial function in cells expressing mutant huntingtin. In this work, we investigated the effect of HDACIs on the regulation of PDH activity in striatal cells derived from HD knock-in mice and YAC128 mice. Mutant cells exhibited decreased PDH activity and increased PDH E1alpha phosphorylation/inactivation, accompanied by enhanced protein levels of PDH kinases 1 and 3 (PDK1 and PDK3). Exposure to dichloroacetate, an inhibitor of PDKs, increased mitochondrial respiration and decreased production of reactive oxygen species in mutant cells, emphasizing PDH as an interesting therapeutic target in HD. Treatment with SB and sodium phenylbutyrate, another HDACI, recovered cell viability and overall mitochondrial metabolism in mutant cells. Exposure to SB also suppressed hypoxia-inducible factor-1 (HIF-1α) stabilization and decreased the transcription of the two most abundant PDK isoforms, PDK2 and PDK3, culminating in increased PDH activation in mutant cells. Concordantly, PDK3 knockdown improved mitochondrial function, emphasizing the role of PDK3 inactivation on the positive effects achieved by SB treatment. YAC128 mouse brain presented higher mRNA levels of PDK1-3 and PDH phosphorylation and decreased energy levels that were significantly ameliorated after SB treatment. Furthermore, enhanced motor learning and coordination were observed in SB-treated YAC128 mice. These results suggest that HDACIs, particularly SB, promote the activity of PDH in the HD brain, helping to counteract HD-related deficits in mitochondrial bioenergetics and motor function.SIGNIFICANCE STATEMENT The present work provides a better understanding of mitochondrial dysfunction in Huntington's disease (HD) by showing that the pyruvate dehydrogenase (PDH) complex is a promising therapeutic target. In particular, the histone deacetylase inhibitor sodium butyrate (SB) may indirectly (through reduced hypoxia-inducible factor 1 alpha stabilization) decrease the expression of the most abundant PDH kinase isoforms (e.g., PDK3), ameliorating PDH activity and mitochondrial metabolism and further affecting motor behavior in HD mice, thus constituting a promising agent for HD neuroprotective treatment.FCT, Santa Casa da Misericórdia de Lisboa (SCML), Fundação Luso-Americana para o Desenvolvimento (FLAD) Life Scienc

The NAD+-dependent deacetylase SIRT2 attenuates oxidative stress and mitochondrial dysfunction and improves insulin sensitivity in hepatocytes

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.European Regional Development Fund (ERDF), Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000012: HealthyAging2020); COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia (POCI-01-0145-FEDER-007440, SFRH/BPD/109347/2015 to R.M.O., SFRH/BD/86655/2012 to L.N. and SFRH/BPD/111815/2015 to P.G.); FLAD Life Science 2020 Grant to A.C.R.; European Molecular Biology Organization (EMBO Installation Grant to T.F.O.); DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) to T.F.O

The NAD+-dependent deacetylase SIRT2 attenuates oxidative stress and mitochondrial dysfunction and improves insulin sensitivity in hepatocytes

Funding Information: European Regional Development Fund (ERDF), Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000012: HealthyAging2020); COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia (POCI-01-0145-FEDER-007440, SFRH/BPD/109347/ 2015 to R.M.O., SFRH/BD/86655/2012 to L.N. and SFRH/BPD/ 111815/2015 to P.G.); FLAD Life Science 2020 Grant to A.C.R.; European Molecular Biology Organization (EMBO Installation Grant to T.F.O.); DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) to T.F.O.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.publishersversionpublishe

Mitochondrial hypermetabolism precedes impaired autophagy and synaptic disorganization in App knock-in Alzheimer mouse models.

Accumulation of amyloid β-peptide (Aβ) is a driver of Alzheimer's disease (AD). Amyloid precursor protein (App) knock-in mouse models recapitulate AD-associated Aβ pathology, allowing elucidation of downstream effects of Aβ accumulation and their temporal appearance upon disease progression. Here we have investigated the sequential onset of AD-like pathologies in AppNL-F and AppNL-G-F knock-in mice by time-course transcriptome analysis of hippocampus, a region severely affected in AD. Strikingly, energy metabolism emerged as one of the most significantly altered pathways already at an early stage of pathology. Functional experiments in isolated mitochondria from hippocampus of both AppNL-F and AppNL-G-F mice confirmed an upregulation of oxidative phosphorylation driven by the activity of mitochondrial complexes I, IV and V, associated with higher susceptibility to oxidative damage and Ca2+-overload. Upon increasing pathologies, the brain shifts to a state of hypometabolism with reduced abundancy of mitochondria in presynaptic terminals. These late-stage mice also displayed enlarged presynaptic areas associated with abnormal accumulation of synaptic vesicles and autophagosomes, the latter ultimately leading to local autophagy impairment in the synapses. In summary, we report that Aβ-induced pathways in App knock-in mouse models recapitulate key pathologies observed in AD brain, and our data herein adds a comprehensive understanding of the pathologies including dysregulated metabolism and synapses and their timewise appearance to find new therapeutic approaches for AD

Amyloid Β-Peptide Increases Mitochondria-Endoplasmic Reticulum Contact Altering Mitochondrial Function and Autophagosome Formation in Alzheimer's Disease-Related Models.

Recent findings have shown that the connectivity and crosstalk between mitochondria and the endoplasmic reticulum (ER) at mitochondria-ER contact sites (MERCS) are altered in Alzheimer'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 Aβ 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 Aβ 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 Aβ. This alteration in MERCS affects autophagosome formation, mitochondrial function and ATP formation during starvation. Interestingly, the increment in ER-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 Aβ 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

Limited effect of chronic valproic acid treatment in a mouse model of Machado-Joseph disease

Machado-Joseph disease (MJD) is an inherited neurodegenerative disease, caused by a CAG repeat expansion within the coding region of ATXN3 gene, and which currently lacks effective treatment. In this work we tested the therapeutic efficacy of chronic treatment with valproic acid (VPA) (200mg/kg), a compound with known neuroprotection activity, and previously shown to be effective in cell, fly and nematode models of MJD. We show that chronic VPA treatment in the CMVMJD135 mouse model had limited effects in the motor deficits of these mice, seen mostly at late stages in the motor swimming, beam walk, rotarod and spontaneous locomotor activity tests, and did not modify the ATXN3 inclusion load and astrogliosis in affected brain regions. However, VPA chronic treatment was able to increase GRP78 protein levels at 30 weeks of age, one of its known neuroprotective effects, confirming target engagement. In spite of limited results, the use of another dosage of VPA or of VPA in a combined therapy with molecules targeting other pathways, cannot be excluded as potential strategies for MJD therapeuticsPM received funding from Ataxia UK Grant (Project: Pharmacologic therapy for Machado-Joseph disease: from a C. elegans drug screen to a mouse model validation). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

Role of lysine deacetylases on transcription regulation and mitochondrial function in Huntington's disease

Tese de doutoramento em Ciências da Saúde, ramo Ciências Biomédicas apresentada à Faculdade de Medicina da Universidade de CoimbraHuntington’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 Lisbo

Mitochondria-Endoplasmic Reticulum Interplay Regulates Exo-Cytosis in Human Neuroblastoma Cells

Mitochondria–endoplasmic reticulum (ER) contact sites (MERCS) have been emerging as a multifaceted subcellular region of the cell which affects several physiological and pathological mechanisms. A thus far underexplored aspect of MERCS is their contribution to exocytosis. Here, we set out to understand the role of these contacts in exocytosis and find potential mechanisms linking these structures to vesicle release in human neuroblastoma SH-SY5Y cells. We show that increased mitochondria to ER juxtaposition through Mitofusin 2 (Mfn2) knock-down resulted in a substantial upregulation of the number of MERCS, confirming the role of Mfn2 as a negative regulator of these structures. Furthermore, we report that both vesicle numbers and vesicle protein levels were decreased, while a considerable upregulation in exocytotic events upon cellular depolarization was detected. Interestingly, in Mfn2 knock-down cells, the inhibition of the inositol 1,4,5-trisphosphate receptor (IP3R) and the mitochondrial calcium (Ca2+) uniporter (MCU) restored vesicle protein content and attenuated exocytosis. We thus suggest that MERCS could be targeted to prevent increased exocytosis in conditions in which ER to mitochondria proximity is upregulated

Mitochondrial Alterations in Neurons Derived from the Murine AppNL-F Knock-In Model of Alzheimer's Disease

Background: Alzheimer’s disease (AD) research has relied on mouse models overexpressing human mutant A βPP; however, newer generation knock-in models allow for physiological expression of amyloid-β protein precursor (AβPP) containing familial AD mutations where murine AβPP is edited with a humanized amyloid-β (Aβ) sequence. The AppNL-F mouse model has shown substantial similarities to AD brains developing late onset cognitive impairment. Objective: In this study, we aimed to characterize mature primary cortical neurons derived from homozygous AppNL-F embryos, especially to identify early mitochondrial alterations in this model. Methods: Primary cultures of AppNL-F neurons kept in culture for 12–15 days were used to measure Aβ levels, secretase activity, mitochondrial functions, mitochondrial-ER contacts, synaptic function, and cell death. Results: We detected higher levels of Aβ42 released from AppNL-F neurons as compared to wild-type neurons. AppNL-F neurons, also displayed an increased Aβ42/Aβ40 ratio, similar to adult AppNL-F mouse brain. Interestingly, we found an upregulation in mitochondrial oxygen consumption with concomitant downregulation in glycolytic reserve. Furthermore, AppNL-F neurons were more susceptible to cell death triggered by mitochondrial electron transport chain inhibition. Juxtaposition between ER and mitochondria was found to be substantially upregulated, which may account for upregulated mitochondrial-derived ATP production. However, anterograde mitochondrial movement was severely impaired in this model along with loss in synaptic vesicle protein and impairment in pre- and post-synaptic function. Conclusion: We show that widespread mitochondrial alterations can be detected in AppNL-F neurons in vitro, where amyloid plaque deposition does not occur, suggesting soluble and oligomeric Aβ-species being responsible for these alterations