New Avenues for the Treatment of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HD gene. The disease is characterized by neurodegeneration, particularly in the striatum and cortex. The first symptoms usually appear in mid-life and include cognitive deficits and motor disturbances that progress over time. Despite being a genetic disorder with a known cause, several mechanisms are thought to contribute to neurodegeneration in HD, and numerous pre-clinical and clinical studies have been conducted and are currently underway to test the efficacy of therapeutic approaches targeting some of these mechanisms with varying degrees of success. Although current clinical trials may lead to the identification or refinement of treatments that are likely to improve the quality of life of those living with HD, major efforts continue to be invested at the pre-clinical level, with numerous studies testing novel approaches that show promise as disease-modifying strategies. This review offers a detailed overview of the currently approved treatment options for HD and the clinical trials for this neurodegenerative disorder that are underway and concludes by discussing potential disease-modifying treatments that have shown promise in pre-clinical studies, including increasing neurotropic support, modulating autophagy, epigenetic and genetic manipulations, and the use of nanocarriers and stem cells.

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.

Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.

Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Autophagy Dysfunction as a Phenotypic Readout in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Diseases.

Autophagy is an evolutionarily conserved catabolic pathway for the degradation of cytoplasmic constituents in eukaryotic cells. It is the primary disposal route for selective removal of undesirable cellular materials like aggregation-prone proteins and damaged organelles for maintaining cellular homeostasis, and for bulk degradation of intracellular macromolecules and recycling the breakdown products for providing energy homeostasis during starvation. These functions of autophagy are attributed to cellular survival and thus pertinent for human health; however, malfunction of this process is detrimental to the cells, particularly for post-mitotic neurons. Thus, basal autophagy is vital for maintaining neuronal homeostasis, whereas autophagy dysfunction contributes to neurodegeneration. Defective autophagy has been demonstrated in several neurodegenerative diseases wherein pharmacological induction of autophagy is beneficial in many of these disease models. Elucidating the mechanisms underlying defective autophagy is imperative for the development of therapies targeting this process. Disease-affected human neuronal cells can be established from patient-derived human induced pluripotent stem cells (hiPSCs) that provide a clinically relevant platform for studying disease mechanisms and drug discovery. Thus, modeling autophagy dysfunction as a phenotypic readout in patient-derived neurons provides a more direct platform for investigating the mechanisms underlying defective autophagy and evaluating the therapeutic efficacy of autophagy inducers. Toward this, several hiPSC-derived neuronal cell models of neurodegenerative diseases have been employed. In this review, we highlight the key methodologies pertaining to hiPSC maintenance and neuronal differentiation, and studying autophagy at an endogenous level in hiPSC-derived neuronal cells.

Analysis of Mitochondrial Dysfunction by Microplate Reader in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Disorders.

Mitochondria are responsible for many vital pathways governing cellular homeostasis, including cellular energy management, heme biosynthesis, lipid metabolism, cellular proliferation and differentiation, cell cycle regulation, and cellular viability. Electron transport and ADP phosphorylation coupled with proton pumping through the mitochondrial complexes contribute to the preservation of mitochondrial membrane potential (ΔΨm). Importantly, mitochondrial polarization is essential for reactive oxygen species (ROS) production and cytosolic calcium (Ca2+) handling. Thus, changes in mitochondrial oxidative phosphorylation (OXPHOS), ΔΨm, and ATP/ADP may occur in parallel or stimulate each other. Brain cells like neurons are heavily reliant on mitochondrial OXPHOS for its high-energy demands, and hence improper mitochondrial function is detrimental for neuronal survival. Indeed, several neurodegenerative disorders are associated with mitochondrial dysfunction. Modeling this disease-relevant phenotype in neuronal cells differentiated from patient-derived human induced pluripotent stem cells (hiPSCs) provide an appropriate cellular platform for studying the disease pathology and drug discovery. In this review, we describe high-throughput analysis of crucial parameters related to mitochondrial function in hiPSC-derived neurons. These methodologies include measurement of ΔΨm, intracellular Ca2+, oxidative stress, and ATP/ADP levels using fluorescence probes via a microplate reader. Benefits of such an approach include analysis of mitochondrial parameters on a large population of cells, simultaneous analysis of different cell lines and experimental conditions, and for drug screening to identify compounds restoring mitochondrial function.

Mitochondrial SIRT3 confers neuroprotection in Huntington's disease by regulation of oxidative challenges and mitochondrial dynamics.

SIRT3 is a major regulator of mitochondrial acetylome. Here we show that SIRT3 is neuroprotective in Huntington's disease (HD), a motor neurodegenerative disorder caused by an abnormal expansion of polyglutamines in the huntingtin protein (HTT). Protein and enzymatic analysis revealed that increased SIRT3 is a signature in several HD models, including human HD brain, which is regulated by oxidative species. While loss of SIRT3 further aggravated the oxidative phenotype, antioxidant treatment regularized SIRT3 levels. SIRT3 overexpression promoted the antioxidant effect in cells expressing mutant HTT, leading to enhanced mitochondrial function and balanced dynamics. Decreased Fis1 and Drp1 accumulation in mitochondria induced by SIRT3 expression favored mitochondrial elongation, while the SIRT3 activator ε-viniferin improved anterograde mitochondrial neurite transport, sustaining cell survival. Notably, SIRT3 fly-ortholog dSirt2 overexpression in HD flies ameliorated neurodegeneration and extended lifespan. These findings provide a link between oxidative stress and mitochondrial dysfunction hypotheses in HD and offer an opportunity for therapeutic development.

Calcium and cell death signaling in neurodegeneration and aging.

Transient increase in cytosolic (Cac2+) and mitochondrial Ca2+ (Ca m2+) are essential elements in the control of many physiological processes. However, sustained increases in Ca c2+ and Ca m2+ may contribute to oxidative stress and cell death. Several events are related to the increase in Ca m2+, including regulation and activation of a number of Ca2+ dependent enzymes, such as phospholipases, proteases and nucleases. Mitochondria and endoplasmic reticulum (ER) play pivotal roles in the maintenance of intracellular Ca2+ homeostasis and regulation of cell death. Several lines of evidence have shown that, in the presence of some apoptotic stimuli, the activation of mitochondrial processes may lead to the release of cytochrome c followed by the activation of caspases, nuclear fragmentation and apoptotic cell death. The aim of this review was to show how changes in calcium signaling can be related to the apoptotic cell death induction. Calcium homeostasis was also shown to be an important mechanism involved in neurodegenerative and aging processes.

In Vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells.

Autophagy is a vital homeostatic pathway essential for cellular survival and human health. It primarily functions as an intracellular degradation process for the turnover of aggregation-prone proteins and unwanted organelles. Dysregulation of autophagy underlying diverse human diseases reduces cell viability, whereas stimulation of autophagy is cytoprotective in a number of transgenic disease models including neurodegenerative disorders. Thus, therapeutic exploitation of autophagy is considered a potential treatment strategy in certain human diseases, and therefore, chemical inducers of autophagy have tremendous biomedical relevance. In this review, we describe the in vitro screening platforms to identify autophagy modulators in mammalian cells using various methodologies including fluorescence and high-content imaging, flow cytometry, fluorescence and luminescence detection by microplate reader, immunoblotting, and immunofluorescence. The commonly used autophagy reporters in these screening platforms are either based on autophagy marker like LC3 or autophagy substrate such as aggregation-prone proteins or p62/SQSTM1. The reporters and assays for monitoring autophagy are evolving over time to become more sensitive in measuring autophagic flux with the capability of high-throughput applications for drug discovery. Here we highlight these developments and also describe the stringent secondary autophagy assays for characterizing the autophagy modulators arising from the primary screen. Since autophagy is implicated in myriad human physiological and pathological conditions, these technologies will enable identifying novel chemical modulators or genetic regulators of autophagy that will be of biomedical and fundamental importance to human health.

Dysregulation of autophagy as a common mechanism in lysosomal storage diseases.

The lysosome plays a pivotal role between catabolic and anabolic processes as the nexus for signalling pathways responsive to a variety of factors, such as growth, nutrient availability, energetic status and cellular stressors. Lysosomes are also the terminal degradative organelles for autophagy through which macromolecules and damaged cellular components and organelles are degraded. Autophagy acts as a cellular homeostatic pathway that is essential for organismal physiology. Decline in autophagy during ageing or in many diseases, including late-onset forms of neurodegeneration is considered a major contributing factor to the pathology. Multiple lines of evidence indicate that impairment in autophagy is also a central mechanism underlying several lysosomal storage disorders (LSDs). LSDs are a class of rare, inherited disorders whose histopathological hallmark is the accumulation of undegraded materials in the lysosomes due to abnormal lysosomal function. Inefficient degradative capability of the lysosomes has negative impact on the flux through the autophagic pathway, and therefore dysregulated autophagy in LSDs is emerging as a relevant disease mechanism. Pathology in the LSDs is generally early-onset, severe and life-limiting but current therapies are limited or absent; recognizing common autophagy defects in the LSDs raises new possibilities for therapy. In this review, we describe the mechanisms by which LSDs occur, focusing on perturbations in the autophagy pathway and present the latest data supporting the development of novel therapeutic approaches related to the modulation of autophagy.

Comparative Mitochondrial-Based Protective Effects of Resveratrol and Nicotinamide in Huntington's Disease Models.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylase that regulates longevity and enhances mitochondrial metabolism. Both activation and inhibition of SIRT1 were previously shown to ameliorate neuropathological mechanisms in Huntington's disease (HD), a neurodegenerative disease that selectively affects the striatum and cortex and is commonly linked to mitochondrial dysfunction. Thus, in this study, we tested the influence of resveratrol (RESV, a SIRT1 activator) versus nicotinamide (NAM, a SIRT1 inhibitor) in counteracting mitochondrial dysfunction in HD models, namely striatal and cortical neurons isolated from YAC128 transgenic mice embryos, HD human lymphoblasts, and an in vivo HD model. HD cell models displayed a deregulation in mitochondrial membrane potential and respiration, implicating a decline in mitochondrial function. Further studies revealed decreased PGC-1α and TFAM protein levels, linked to mitochondrial DNA loss in HD lymphoblasts. Remarkably, RESV completely restored these parameters, while NAM increased NAD+ levels, providing a positive add on mitochondrial function in in vitro HD models. In general, RESV decreased while NAM increased H3 acetylation at lysine 9. In agreement with in vitro data, continuous RESV treatment for 28 days significantly improved motor coordination and learning and enhanced expression of mitochondrial-encoded electron transport chain genes in YAC128 mice. In contrast, high concentrations of NAM blocked mitochondrial-related transcription, worsening motor phenotype. Overall, data indicate that activation of deacetylase activity by RESV improved gene transcription associated to mitochondrial function in HD, which may partially control HD-related motor disturbances.

Insulin and IGF-1 regularize energy metabolites in neural cells expressing full-length mutant huntingtin.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to the expression of mutant huntingtin. Bioenergetic dysfunction has been described to contribute to HD pathogenesis. Thus, treatment paradigms aimed to ameliorate energy deficits appear to be suitable candidates in HD. In previous studies, we observed protective effects of insulin growth factor-1 (IGF-1) in YAC128 and R6/2 mice, two HD mouse models, whereas IGF-1 and/or insulin halted mitochondrial-driven oxidative stress in mutant striatal cells and mitochondrial dysfunction in HD human lymphoblasts. Here, we analyzed the effect of IGF-1 versus insulin on energy metabolic parameters using striatal cells derived from HD knock-in mice and primary cortical cultures from YAC128 mice. STHdh(Q111/Q111) cells exhibited decreased ATP/ADP ratio and increased phosphocreatine levels. Moreover, pyruvate levels were increased in mutant cells, most probably in consequence of a decrease in pyruvate dehydrogenase (PDH) protein expression and increased PDH phosphorylation, reflecting its inactivation. Insulin and IGF-1 treatment significantly decreased phosphocreatine levels, whereas IGF-1 only decreased pyruvate levels in mutant cells. In a different scenario, primary cortical cultures derived from YAC128 mice also displayed energetic abnormalities. We observed a decrease in both ATP/ADP and phosphocreatine levels, which were prevented following exposure to insulin or IGF-1. Furthermore, decreased lactate levels in YAC128 cultures occurred concomitantly with a decline in lactate dehydrogenase activity, which was ameliorated with both insulin and IGF-1. These data demonstrate differential HD-associated metabolic dysfunction in striatal cell lines and primary cortical cultures, both of which being alleviated by insulin and IGF-1.

Activation of IGF-1 and insulin signaling pathways ameliorate mitochondrial function and energy metabolism in Huntington's Disease human lymphoblasts.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Mitochondrial dysfunction associated with energy failure plays an important role in this untreated pathology. In the present work, we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin (at low nM) on signaling and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways linked to activation of insulin and IGF-1 receptors (IR,IGF-1R), Akt, and ERK was largely restored by IGF-1 and, at a less extent, by insulin in HD human lymphoblasts. Importantly, both neurotrophic factors stimulated huntingtin phosphorylation at Ser421 in HD cells. IGF-1 and insulin also rescued energy levels in HD peripheral cells, as evaluated by increased ATP and phosphocreatine, and decreased lactate levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential (Δψm) in HD lymphoblasts, which occurred concomitantly with increased levels of cytochrome c. Indeed, constitutive phosphorylation of huntingtin was able to restore the Δψm in lymphoblasts expressing an abnormal expansion of polyglutamines. HD lymphoblasts further exhibited increased intracellular Ca(2+) levels before and after exposure to hydrogen peroxide (H2O2), and decreased mitochondrial Ca(2+) accumulation, being the later recovered by IGF-1 and insulin in HD lymphoblasts pre-exposed to H2O2. In summary, the data support an important role for IR/IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts.

Current therapeutic advances in patients and experimental models of Huntington's disease.

Huntington's disease (HD) clinical manifestations begin insidiously and are progressively incapacitating. Symptomatic therapies, in particular dopamine blockers and neuroleptics, are presently the only treatment for HD. Identification of neuropathological mechanisms that underlie the selective striatal and cortical neurodegeneration has allowed for the development of novel neuroprotective therapies that may improve HD patients' quality of life and enhance their survival. In this review we describe the symptomatic and neuroprotective therapies in HD that are currently in a preclinical or clinical stage. Neuroprotective therapies can act at several stages of HD, namely through: i) transcription modulation, ii) regulation of neurotrophic factors levels, iii) inhibition of metabolic dysfunction through metabolic enhancers, iv) apoptosis inhibition, v) autophagy regulation, vi) transglutaminase inhibition, and/or vii) modulation of neurotransmitter receptors. Moreover, emerging therapies in HD, including gene therapy using siRNA and shRNA to silence CAG repeats or deep brain stimulation, have shown promising results. Although most of the therapies are at a pre-clinical stage, phase II-III clinical trials have been performed for each pathophysiological mechanism of the disease. Thus, efforts should continue to ensure that effective therapies are studied and tested to help mitigate HD.

Insulin and IGF-1 improve mitochondrial function in a PI-3K/Akt-dependent manner and reduce mitochondrial generation of reactive oxygen species in Huntington's disease knock-in striatal cells.

Oxidative stress and mitochondrial dysfunction have been described in Huntington's disease, a disorder caused by expression of mutant huntingtin (mHtt). IGF-1 was previously shown to protect HD cells, whereas insulin prevented neuronal oxidative stress. In this work we analyzed the role of insulin and IGF-1 in striatal cells derived from HD knock-in mice on mitochondrial production of reactive oxygen species (ROS) and related antioxidant and signaling pathways influencing mitochondrial function. Insulin and IGF-1 decreased mitochondrial ROS induced by mHtt and normalized mitochondrial SOD activity, without affecting intracellular glutathione levels. IGF-1 and insulin promoted Akt phosphorylation without changing the nuclear levels of phosphorylated Nrf2 or Nrf2/ARE activity. Insulin and IGF-1 treatment also decreased mitochondrial Drp1 phosphorylation, suggesting reduced mitochondrial fragmentation, and ameliorated mitochondrial function in HD cells in a PI-3K/Akt-dependent manner. This was accompanied by increased total and phosphorylated Akt, Tfam, and mitochondrial-encoded cytochrome c oxidase II, as well as Tom20 and Tom40 in mitochondria of insulin- and IGF-1-treated mutant striatal cells. Concomitantly, insulin/IGF-1-treated mutant cells showed reduced apoptotic features. Hence, insulin and IGF-1 improve mitochondrial function and reduce mitochondrial ROS caused by mHtt by activating the PI-3K/Akt signaling pathway, in a process independent of Nrf2 transcriptional activity, but involving enhanced mitochondrial levels of Akt and mitochondrial-encoded complex IV subunit.

Glutathione redox cycle dysregulation in Huntington's disease knock-in striatal cells.

Huntington's disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and γ-glutamyl transpeptidase (γ-GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to counterbalance increased ROS formation and emergent apoptotic features in HD striatal cells.

FK506 ameliorates cell death features in Huntington's disease striatal cell models.

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by striatal neurodegeneration, involving apoptosis. FK506, an inhibitor of calcineurin (or protein phosphatase 3, formerly known as protein phosphatase 2B), has shown neuroprotective effects in several cellular and animal models of HD. In the present study, we show the protective effects of FK506 in two striatal HD models, primary rat striatal neurons treated with 3-nitropropionic acid (3-NP) and immortalized striatal STHdh cells derived from HD knock-in mice expressing normal (STHdh(7/7)) or full-length mutant huntingtin (FL-mHtt) with 111 glutamines (STHdh(111/111)), under basal conditions and after exposure to 3-NP or staurosporine (STS). In rat striatal neurons, FK506 abolished 3-NP-induced increase in caspase-3 activation, DNA fragmentation/condensation and necrosis. Nevertheless, in STHdh(111/111) cells under basal conditions, FK506 did not prevent, in a significant manner, the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, or alter Bax/Bcl-2 ratio, but significantly reverted caspase-3 activation. In STHdh(111/111) cells treated with 0.3mM 3-NP or 25 nM STS, linked to high necrosis, exposure to FK506 exerted no significant effects on caspase-3 activation. However, treatment of STHdh(111/111) cells exposed to 10nM STS with FK506 effectively prevented cell death by apoptosis and moderate necrosis. The results suggest that FK506 may be neuroprotective against apoptosis and necrosis under mild cell death stimulus in the presence of FLmHtt.

Mitochondrial-associated metabolic changes and neurodegeneration in Huntington's disease - from clinical features to the bench.

Huntington's disease (HD) is a genetic neurodegenerative disease selectively leading to striatal neurodegeneration, but also affecting the cortex and the hypothalamus. Although it is hard to predict the sequence of cell-damaging events occurring in HD patients, several pathological mechanisms have been proposed to explain HD selective neurodegeneration and disease symptomatology. Abnormalities in mitochondrial function and bioenergetics contribute to cell death and have been reported in HD-affected individuals, both in central and peripheral tissues. Moreover, the latter has been characterized in several HD models. Thus, this review describes the converging mechanisms that lead to mitochondrial and metabolic abnormalities in thoroughly studied in vivo and in vitro HD models, including excitotoxicity, altered calcium handling, changes in mitochondrial structure and dynamics and transcription deregulation, which may represent important disease therapeutic targets. Furthermore, the review describes the current evidences of metabolic disturbances in the brain of HD-affected humans and of peripheral metabolic and mitochondrial changes, weight loss and endocrine abnormalities operating in the whole HD body.

Evaluation of some cell death features by real time real space microscopy.

In the past few years, the investigation of many cell death features, especially ones associated with changes in DeltaPsi(m), have gained an important insights with the development of high-resolution fluorescence microscopy. With the use of real time real space measurements, it was possible to perform dynamic studies not only to investigate the location of the organelles, but also to follow changes in transport mechanism, such as Ca(2+) concentration in different subcellular compartments. In addition, this technique has been used for the simultaneous tracking of organelle location, ion measurements, and DeltaPsi(m,) which clearly contributed to further understanding mechanisms related to the control of cell death. This chapter describes the methodology employed to study changes in DeltaPsi(m), Bax translocation, and Ca(2+) measurements upon apoptotic induction. It also details the new technique developed and employed in our laboratory to measure Ca(2+) signaling in brain slices by confocal microscopy. This method has been applied to investigate real time real space studies in different models of neurodegenerative processes, such as Huntington's disease and aging.

Calcium and cell death signaling in neurodegeneration and aging

Transient increase in cytosolic (Cac2+) and mitochondrial Ca2+ (Ca m2+) are essential elements in the control of many physiological processes. However, sustained increases in Ca c2+ and Ca m2+ may contribute to oxidative stress and cell death. Several events are related to the increase in Ca m2+, including regulation and activation of a number of Ca2+ dependent enzymes, such as phospholipases, proteases and nucleases. Mitochondria and endoplasmic reticulum (ER) play pivotal roles in the maintenance of intracellular Ca2+ homeostasis and regulation of cell death. Several lines of evidence have shown that, in the presence of some apoptotic stimuli, the activation of mitochondrial processes maylead to the release of cytochrome c followed by the activation of caspases, nuclear fragmentation and apoptotic cell death. The aim of this review was to show how changes in calcium signaling can be related to the apoptotic cell death induction. Calcium homeostasis was also shown to be an important mechanism involved in neurodegenerative and aging processes.

Aumentos transientes no cálcio citosólico (Ca c2+) e mitocondrial (Ca m2+) são elementos essenciais no controle de muitos processos fisiológicos. No entanto, aumentos sustentados do Ca c2+ e do Ca m2+ podem contribuir para o estresse oxidativo ea morte celular. Muitos eventos estão relacionados ao aumentono Ca c2+, incluindo a regulação e ativação de várias enzimas dependentes de Ca2+ como as fosfolipases, proteases e nucleases. A mitocôndria e o retículo endoplasmático têm um papel central na manutenção da homeostase intracellular de Ca c2+ e na regulação da morte celular. Várias evidências mostraram que, na presença de certos estímulos apoptóticos, a ativação dos processos mitocondriais pode promover a liberação de citocromo c, seguida da ativação de caspases, fragmentação nuclear e morte celular por apoptose. O objetivo desta revisão é mostrar como aumentos na sinalização de Ca2+ podem estar relacionados aos eventos de indução da morte celular apoptótica. Além disso, evidenciar como a homeostase de Ca2+ pode ser importante e está envolvida nos mecanismos presentes nos processos de neurodegeneração e envelhecimento.