cytochrome-c-t and Huntington-Disease

Protein folding is a natural phenomenon through which a linear polypeptide possessing necessary information attains three-dimension functionally active conformation. This is a complex and multistep process and therefore, the presence of several intermediary structures could be speculated as a result of protein folding. In in vivo, this folding process is governed by the assistance of other proteins called molecular chaperones and heat shock proteins. Due to the mechanism of protein folding, these intermediary structures remain major challenge for modern biology. Mutation in gene encoding amino acid can cause adverse environmental conditions which may result in misfolding of the linear polypeptide followed by the formation of aggregates and amyloidosis. Aggregation contributes to the pathophysiology of several maladies including diabetes mellitus, Huntington's and Alzheimer's disease. The propensity of native structure to form aggregated and fibrillar assemblies is a hallmark of amyloidosis. During aggregation of a protein, transition from α helix to β sheet is observed, and mainly β sheeted structure is visualised in a mature fibril. Heme proteins are very crucial for major life activities like transport of oxygen and carbon dioxide, synthesis of ATP, role in electron transport chain, and detoxification of free radicals formed during biochemical reactions. Any structural variation in the heme proteins may lead to a fatal response. Hence characterization of the folding intermediates becomes crucial. The characterization has been deciphered with the help of strong denaturants like acetonitrile and TFE. Moreover, possible role of elimination of these aggregates and prevention of protein denaturation is also discussed. Current review deals with the basic process and mechanism of the protein folding in general and the ultimate outcomes of the protein misfolding. Since Native conformation of heme proteins is essential for some vital activities as listed above, we have discussed possible prevention of denaturation and aggregation of heme proteins such as Hb, cyt c, catalase & peroxidase.

Topics: Alzheimer Disease; Amyloid; Amyloidosis; Catalase; Cytochromes c; Diabetes Mellitus; Gene Expression; Heat-Shock Proteins; Hemoglobins; Humans; Huntington Disease; Molecular Chaperones; Peroxidase; Protein Aggregates; Protein Conformation; Protein Folding

There is evidence of an imbalance of mitochondrial fission and fusion in patients with Huntington's disease (HD) and HD animal models. Fission and fusion are important for mitochondrial homeostasis including mitochondrial DNA (mtDNA) maintenance and may be relevant for the selective striatal mtDNA depletion that we observed in the R6/2 fragment HD mouse model. We aimed to investigate the fission/fusion balance and the integrity of the mitochondrial membrane system in cortex and striatum of end-stage R6/2 mice and wild-type animals. Mitochondrial morphology was determined using electron microscopy, and transcript and protein levels of factors that play a key role in fission and fusion, including DRP1, mitofusin 1 and 2, mitofilin and OPA1, and cytochrome c and caspase 3 were assessed by RT-qPCR and immunoblotting. OPA1 oligomerisation was evaluated using blue native gels. In striatum and cortex of R6/2 mice, mitochondrial cristae morphology was abnormal. Mitofilin and the overall levels of the fission and fusion factors were unaffected; however, OPA1 oligomerisation was abnormal in striatum and cortex of R6/2 mice. Mitochondrial and cytoplasmic cytochrome c levels were similar in R6/2 and wild-type mice with no significant increase of activated caspase 3. Our results indicate that the integrity of the mitochondrial cristae is compromised in striatum and cortex of the R6/2 mice and that this is most likely caused by impaired OPA1 oligomerisation.

Topics: Animals; Caspase 3; Cerebral Cortex; Corpus Striatum; Cytochromes c; Disease Models, Animal; DNA, Mitochondrial; Dynamins; Electron Transport Complex I; GTP Phosphohydrolases; Huntingtin Protein; Huntington Disease; Mice; Mice, Inbred BALB C; Mice, Transgenic; Mitochondria; Mitochondrial Dynamics; Trinucleotide Repeats

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.

Topics: Animals; Calcium; Cell Line; Cytochromes c; Electron Transport; Energy Metabolism; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Huntingtin Protein; Huntington Disease; Insulin; Insulin-Like Growth Factor I; Lymphocytes; Male; Membrane Potential, Mitochondrial; Mitochondria; Nerve Tissue Proteins; Oxygen Consumption; Phosphorylation; Receptor, IGF Type 1; Signal Transduction; Sus scrofa

Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD.

Topics: Calbindins; Cytochromes c; DNA-Binding Proteins; DNA, Mitochondrial; Dynamins; Electron Transport Complex IV; Energy Metabolism; Fluorescent Antibody Technique; Gene Expression; Gene Expression Profiling; GTP Phosphohydrolases; Humans; Huntingtin Protein; Huntington Disease; Membrane Potential, Mitochondrial; Membrane Transport Proteins; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Proteins; Neostriatum; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Peroxisome Proliferator-Activated Receptors; Polymerase Chain Reaction; S100 Calcium Binding Protein G; Superoxide Dismutase; Transcription Factors

Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Drosophila; Energy Metabolism; Enzyme Inhibitors; GTP-Binding Proteins; Heat-Shock Proteins; Histones; Humans; Huntington Disease; Mice; Mitochondria; Nitro Compounds; Peptides; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Promoter Regions, Genetic; Propionates; Protein Glutamine gamma Glutamyltransferase 2; Transcription Factors; Transcription, Genetic; Transglutaminases

Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, is accompanied by multiple mitochondrial alterations. Here, we show that mitochondrial fragmentation and cristae alterations characterize cellular models of HD and participate in their increased susceptibility to apoptosis. In HD cells, the increased basal activity of the phosphatase calcineurin dephosphorylates the pro-fission dynamin related protein 1 (Drp1), increasing its mitochondrial translocation and activation, and ultimately leading to fragmentation of the organelle. The fragmented HD mitochondria are characterized by cristae alterations that are aggravated by apoptotic stimulation. A genetic analysis indicates that correction of mitochondrial elongation is not sufficient to rescue the increased cytochrome c release and cell death observed in HD cells. Conversely, the increased apoptosis can be corrected by manoeuvres that prevent fission and cristae remodelling. In conclusion, the cristae remodelling of the fragmented HD mitochondria contributes to their hypersensitivity to apoptosis.

Topics: Animals; Apoptosis; Cell Line; Cells, Cultured; Cytochromes c; Dynamins; Female; GTP Phosphohydrolases; Humans; Huntington Disease; Male; Mice; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Neurons; Protein Transport

Polyglutamine expansions in huntingtin (Htt) are known to cause the profound neurodegenerative disorder, Huntington's disease (HD). Mitochondrial dysfunction has long been implicated in the pathophysiology of HD, but the underlying mechanism remains obscure. An article by Costa et al in this months edition describes a smooth mechanistic cascade from the well-accepted upstream event that mutant Htt is associated with Ca(2+) handling abnormalities, through to apoptotic neuronal death. The proposed cascade implicates calcineurin, activated by abnormal Ca(2+) levels, in the dephosphorylation of dynamin-1-like protein (Drp1), increasing its association with mitochondria and promoting fission, cristae disruption, cytochrome c release and apoptosis (Fig 1). Together with the recent reports of increased mitochondrial fission in striatal neurons from HD patients, the article by Costa et al provides a compelling case for the role of abnormal mitochondrial networking in HD pathogenesis.

Topics: Animals; Apoptosis; Cell Line; Cytochromes c; Dynamins; GTP Phosphohydrolases; Humans; Huntington Disease; Mice; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Neurons

Release of mitochondrial cytochrome c resulting in downstream activation of cell death pathways has been suggested to play a role in neurologic diseases featuring cell death. However, the specific biologic importance of cytochrome c release has not been demonstrated in Huntington's disease (HD). To evaluate the role of cytochrome c release, we screened a drug library to identify new inhibitors of cytochrome c release from mitochondria. Drugs effective at the level of purified mitochondria were evaluated in a cellular model of HD. As proof of principle, one drug was chosen for in depth evaluation in vitro and a transgenic mouse model of HD. Our findings demonstrate the utility of mitochondrial screening to identify inhibitors of cell death and provide further support for the important functional role of cytochrome c release in HD. Given that many of these compounds have been approved by the Food and Drug Administration for clinical usage and cross the blood-brain barrier, these drugs may lead to trials in patients.

Topics: Animals; Brain; Carbonic Anhydrase Inhibitors; Caspases; Cell Death; Cell Line, Transformed; Cytochromes c; Disease Models, Animal; Drug Evaluation, Preclinical; Huntington Disease; Longevity; Membrane Potential, Mitochondrial; Methazolamide; Mice; Mice, Transgenic; Mitochondria; Neuroprotective Agents; Treatment Outcome

Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt), is characterized by extensive loss of striatal neurons. The dysregulation of type 2 transglutaminase (TG2) has been proposed to contribute to the pathogenesis in HD as TG2 is up-regulated in HD brain and knocking out TG2 in mouse models of HD ameliorates the disease process. To understand the role of TG2 in the pathogenesis of HD, immortalized striatal cells established from mice in which mutant htt with a polyglutamine stretch of 111 Gln had been knocked-in and wild type (WT) littermates, were stably transfected with human TG2 in a tetracycline inducible vector. Overexpression of TG2 in the WT striatal cells resulted in significantly greater cell death under basal conditions as well as in response to thapsigargin treatment, which causes increased intracellular calcium concentrations. Furthermore, in WT striatal cells TG2 overexpression potentiated mitochondrial membrane depolarization, intracellular reactive oxygen species production, and apoptotic cell death in response to thapsigargin. In contrast, in mutant striatal cells, TG2 overexpression did not increase cell death, nor did it potentiate thapsigargin-induced mitochondrial membrane depolarization or intracellular reactive oxygen species production. Instead, TG2 overexpression in mutant striatal cells attenuated the thapsigargin-activated apoptosis. When in situ transglutaminase activity was quantitatively analyzed in these cell lines, we found that in response to thapsigargin treatment TG2 was activated in WT, but not mutant striatal cells. These data suggest that mutant htt alters the activation of TG2 in response to certain stimuli and therefore differentially modulates how TG2 contributes to cell death processes.

Topics: Apoptosis; Blotting, Western; Calcium; Caspase Inhibitors; Cell Death; Cytochromes c; GTP-Binding Proteins; Humans; Huntingtin Protein; Huntington Disease; L-Lactate Dehydrogenase; Membrane Potentials; Mitochondrial Membranes; Mutation; Neostriatum; Nerve Tissue Proteins; Nuclear Proteins; Protein Glutamine gamma Glutamyltransferase 2; Reactive Oxygen Species; Thapsigargin; Transfection; Transglutaminases

(1) Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of polymorphic CAG repeats beyond 36 at exon 1 of huntingtin gene (htt). To study cellular effects by expressing N-terminal domain of Huntingtin (Htt) in specific cell lines, we expressed exon 1 of htt that codes for 40 glutamines (40Q) and 16Q in Neuro2A and HeLa cells. (2) Aggregates and various apoptotic markers were detected at various time points after transfection. In addition, we checked the alterations of expressions of few apoptotic genes by RT-PCR. (3) Cells expressing exon 1 of htt coding 40Q at a stretch exhibited nuclear and cytoplasmic aggregates, increased caspase-1, caspase-2, caspase-8, caspase-9/6, and calpain activations, release of cytochrome c and AIF from mitochondria in a time-dependent manner. Truncation of Bid was increased, while the activity of mitochondrial complex II was decreased in such cells. These changes were significantly higher in cells expressing N-terminal Htt with 40Q than that obtained in cells expressing N-terminal Htt with 16Q. Expressions of caspase-1, caspase-2, caspase-3, caspase-7, and caspase-8 were increased while expression of Bcl-2 was decreased in cells expressing mutated Htt-exon 1. (4) Results presented in this communication showed that expression of mutated Htt-exon 1 could mimic the cellular phenotypes observed in Huntington's disease and this cell model can be used for screening the agents that would interfere with the apoptotic pathway and aggregate formation.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calpain; Caspase 2; Cell Survival; Cells, Cultured; Cytochromes c; DNA Fragmentation; Electron Transport Complex II; Enzyme Activation; Exons; Green Fluorescent Proteins; HeLa Cells; Humans; Huntingtin Protein; Huntington Disease; Mice; Mitochondria; Nerve Tissue Proteins; Nuclear Proteins; Recombinant Fusion Proteins; Transfection; Trinucleotide Repeats

To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Apoptosis Inducing Factor; Brain; Caspases; Cytochromes c; DNA-Binding Proteins; Electron Transport Complex I; Electron Transport Complex II; HeLa Cells; Humans; Huntington Disease; Intracellular Signaling Peptides and Proteins; Iron-Sulfur Proteins; Mice; Mitochondria; Nerve Degeneration; Neurons; Protein Subunits; Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Succinate Dehydrogenase

Mutated huntingtin (htt) is ubiquitously expressed in tissues of Huntington's disease (HD) patients. In the brain, the mutated protein leads to neuronal cell dysfunction and death, associated with formation of htt-positive inclusions. Given increasing evidence of abnormalities in HD skeletal muscle, we extensively analyzed primary muscle cell cultures from seven HD subjects (including two unaffected mutation carriers). Myoblasts from presymptomatic and symptomatic HD subjects showed cellular abnormalities in vitro, namely mitochondrial depolarization, cytochrome c release, increased caspase-3, -8, and -9 activities, and defective cell differentiation. Another notable feature was the formation of htt inclusions in differentiated myotubes. This study helps to advance current knowledge about the downstream effects of the htt mutation in human tissues. Further applications may include drug screening using this human cellular model.

Topics: Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Differentiation; Cells, Cultured; Cytochromes c; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Humans; Huntingtin Protein; Huntington Disease; Inclusion Bodies; Membrane Potential, Mitochondrial; Muscle, Skeletal; Mutation; Myoblasts; Nerve Tissue Proteins; Nuclear Proteins

An important aspect of Huntington's disease (HD) pathogenesis which may have important therapeutic implications is that the cellular events leading to cell death may be different in cortical and striatal neurons. In the present study, we characterized cellular changes in cortical and striatal neurons treated with the mitochondrial toxin 3-nitropropionic acid (3NP) in culture. Degeneration induced by 3NP was similar in both striatal and cortical neurons as observed using markers of cell viability and DNA fragmentation. However, in striatal neurons, 3NP produced a marked delocalization of Bad, Bax, cytochrome c and Smac while this was not observed in cortical neurons. Death of striatal neurons was preceded by activation of calpain and was blocked by calpain inhibitor I. In cortical neurons, calpain was not activated and calpain inhibitor I was without effect. In both cell types, caspase-9 and -3 were not activated by 3NP and the caspase inhibitor zVAD-fmk did not provide neuroprotective effect. Interestingly, treatment with staurosporine (STS) triggered caspase-9 and -3 in cortical and striatal cells, suggesting that the molecular machinery related to caspase-dependent apoptosis was functional in both cell types even though this machinery was not involved in 3NP toxicity. The present results clearly demonstrate that under mitochondrial inhibition, striatal and cortical neurons die through different pathways. This suggests that mitochondrial defects in HD may trigger the death of cortical and striatal neurons through different molecular events.

Topics: Animals; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-Associated Death Protein; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Death; Cell Respiration; Cells, Cultured; Cerebral Cortex; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Fetus; Huntington Disease; Mitochondria; Mitochondrial Proteins; Neostriatum; Nerve Degeneration; Neurotoxins; Nitro Compounds; Propionates; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Signal Transduction

Huntington's disease (HD) is initiated by an abnormally expanded polyglutamine stretch in the huntingtin protein, conferring a novel property on the protein that leads to the loss of striatal neurons. Defects in mitochondrial function have been implicated in the pathogenesis of HD. Here, we have examined the hypothesis that the mutant huntingtin protein may directly interact with the mitochondrion and affect its function. In human neuroblastoma cells and clonal striatal cells established from HdhQ7 (wild-type) and HdhQ111 (mutant) homozygote mouse knock-in embryos, huntingtin was present in a purified mitochondrial fraction. Subfractionation of the mitochondria and limited trypsin digestion of the organelle demonstrated that huntingtin was associated with the outer mitochondrial membrane. We further demonstrated that a recombinant truncated mutant huntingtin protein, but not a wild-type, directly induced mitochondrial permeability transition (MPT) pore opening in isolated mouse liver mitochondria, an effect that was prevented completely by cyclosporin A (CSA) and ATP. Importantly, the mutant huntingtin protein significantly decreased the Ca2+ threshold necessary to trigger MPT pore opening. We found a similar increased susceptibility to the calcium-induced MPT in liver mitochondria isolated from a knock-in HD mouse model. The mutant huntingtin protein-induced MPT pore opening was accompanied by a significant release of cytochrome c, an effect completely inhibited by CSA. These findings suggest that the development of specific MPT inhibitors may be an interesting therapeutic avenue to delay the onset of HD.

Topics: Adenosine Triphosphate; Animals; Calcium; Cell Line; Cyclosporine; Cytochromes c; Humans; Huntingtin Protein; Huntington Disease; Intracellular Membranes; Ion Channels; Mice; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Recombinant Proteins; Serotonin Plasma Membrane Transport Proteins