thapsigargin and Huntington-Disease

Impairments in mitochondria and transcription are important factors in the pathogenesis of Huntington disease (HD), a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. This study investigated the effect of different metabolic states and peroxisome proliferator-activated receptor γ (PPARγ) activation on sensitivity to cellular stressors such as H(2)O(2) or thapsigargin in HD. Striatal precursor cells expressing wild type (STHdh(Q7)) or mutant huntingtin (STHdh(Q111)) were prepared in different metabolic conditions (glucose vs. pyruvate). Due to the fact that STHdh(Q111) cells exhibit mitochondrial deficits, we expected that in the pyruvate condition, where ATP is generated primarily by the mitochondria, there would be greater differences in cell death between the two cell types compared to the glucose condition. Intriguingly, it was the glucose condition that gave rise to greater differences in cell death. In the glucose condition, thapsigargin treatment resulted in a more rapid loss of mitochondrial membrane potential (ΔΨm), a greater activation of caspases (3, 8, and 9), and a significant increase in superoxide/reactive oxygen species (ROS) in STHdh(Q111) compared to STHdh(Q7), while both cell types showed similar kinetics of ΔΨm-loss and similar levels of superoxide/ROS in the pyruvate condition. This suggests that bioenergetic deficiencies are not the primary contributor to the enhanced sensitivity of STHdh(Q111) cells to stressors compared to the STHdh(Q7) cells. PPARγ activation significantly attenuated thapsigargin-induced cell death, concomitant with an inhibition of caspase activation, a delay in ΔΨm loss, and a reduction of superoxide/ROS generation in STHdh(Q111) cells. Expression of mutant huntingtin in primary neurons induced superoxide/ROS, an effect that was significantly reduced by constitutively active PPARγ. These results provide significant insight into the bioenergetic disturbances in HD with PPARγ being a potential therapeutic target for HD.

Topics: Anilides; Animals; Blotting, Western; Cell Line; Cells, Cultured; Glucose; Huntington Disease; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Mice; Polymerase Chain Reaction; PPAR gamma; Pyruvic Acid; Rats; Reactive Oxygen Species; Rolipram; Rosiglitazone; Superoxides; Thapsigargin; Thiazolidinediones

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington's disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Topics: Animals; Apoptosis; Autophagy; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Hepatocytes; Humans; Huntington Disease; Immunosuppressive Agents; Lysosomes; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Mitochondria; Nerve Tissue Proteins; Neurons; Peptides; Serotonin Plasma Membrane Transport Proteins; Serum; Sirolimus; Subcellular Fractions; Thapsigargin; Time Factors; Vinca Alkaloids

Huntington's disease is a neurodegenerative disorder caused by an expansion of CAGs repeats and characterized by alterations in mitochondrial functions. Although changes in Ca(2+) handling have been suggested, the mechanisms involved are not completely understood. The aim of this study was to investigate the possible alterations in Ca(2+) handling capacity and the relationship with mitochondrial dysfunction evaluated by NAD(P)H fluorescence, reactive oxygen species levels, mitochondrial membrane potential (DeltaPsi(m)) measurements and respiration in whole brain slices from R6/1 mice of different ages, evaluated in situ by real-time real-space microscopy. We show that the cortex and striatum of the 9-month-old R6/1 transgenic mice present a significant sustained increase in cytosolic Ca(2+) induced by glutamate (Glu). This difference in Glu response was partially reduced in R6/1 when in the absence of extracellular Ca(2+), indicating that N-methyl-D-aspartate receptors participation in this response is more important in transgenic mice. In addition, Glu also lead to a decrease in NAD(P)H fluorescence, a loss in DeltaPsi(m) and a further increase in respiration, which may have evoked a decrease in mitochondrial Ca(2+) Ca(2+)(m) uptake capacity. Taken together, these results show that alterations in Ca(2+) homeostasis in transgenic mice are associated with a decrease in Ca(2+)(m) uptake mechanism with a diminished Ca(2+) handling ability that ultimately causes dysfunctions and worsening of the neurodegenerative and the disease processes.

Topics: Animals; Brain; Calcium; Calcium Signaling; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Enzyme Inhibitors; Glutamic Acid; Homeostasis; Humans; Huntington Disease; Male; Membrane Potential, Mitochondrial; Mice; Mice, Transgenic; Mitochondria; NADP; Oxygen Consumption; Reactive Oxygen Species; Thapsigargin; Uncoupling Agents

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.

Topics: Animals; Autophagy; Calcium Channels, L-Type; Clonidine; Cyclic AMP; Humans; Huntington Disease; Imidazoline Receptors; Minoxidil; Protein Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Type C Phospholipases; Verapamil

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the PPAR family of transcription factors. Synthetic PPARgamma agonists are used as oral anti-hyperglycemic drugs for the treatment of non-insulin-dependent diabetes. However, emerging evidence indicates that PPARgamma activators can also prevent or attenuate neurodegeneration. Given these previous findings, the focus of this report is on the potential neuroprotective role of PPARgamma activation in preventing the loss of mitochondrial function in Huntington disease (HD). For these studies we used striatal cells that express wild-type (STHdh(Q7/Q7)) or mutant (STHdh(Q111/Q111)) huntingtin protein at physiological levels. Treatment of mutant cells with thapsigargin resulted in a significant decrease in mitochondrial calcium uptake, an increase in reactive oxygen species production, and a significant decrease in mitochondrial membrane potential. PPARgamma activation by rosiglitazone prevented the mitochondrial dysfunction and oxidative stress that occurred when mutant striatal cells were challenged with pathological increases in calcium. The beneficial effects of rosiglitazone were likely mediated by activation of PPARgamma, as all protective effects were prevented by the PPARgamma antagonist GW9662. Additionally, the PPARgamma signaling pathway was significantly impaired in the mutant striatal cells with decreases in PPARgamma expression and reduced PPARgamma transcriptional activity. Treatment with rosiglitazone increased mitochondrial mass levels, suggesting a role for the PPARgamma pathway in mitochondrial function in striatal cells. Altogether, this evidence indicates that PPARgamma activation by rosiglitazone attenuates mitochondrial dysfunction in mutant huntingtin-expressing striatal cells, and this could be an important therapeutic avenue to ameliorate the mitochondrial dysfunction that occurs in HD.

Topics: Anilides; Animals; Corpus Striatum; Huntingtin Protein; Huntington Disease; Hypoglycemic Agents; Membrane Potentials; Mice; Mitochondria; Mutation; Nerve Tissue Proteins; Nuclear Proteins; PPAR gamma; Reactive Oxygen Species; Rosiglitazone; Signal Transduction; Thapsigargin; Thiazolidinediones

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

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.

Topics: Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Calcium; Calpain; Caspases; Cell Line; Cell Nucleus; Cells, Cultured; Cloning, Molecular; Cytoplasm; Disease Progression; DNA, Complementary; Epitopes; Humans; Huntingtin Protein; Huntington Disease; Mice; Mice, Transgenic; Microscopy, Fluorescence; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Peptides; Plasmids; Precipitin Tests; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Thapsigargin

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG expansion that results in elongation of the polyglutamine tract at the N terminus of huntingtin (Htt). Abnormal proteolytic processing of mutant Htt has been implicated as a critical step in the initiation of HD. The protease(s) involved in this process has not been fully characterized. Here we report that activated calpain was detected in the caudate of human HD tissue but not in age-matched controls. In addition, one of the major N-terminal Htt proteolytic fragments found in human HD tissue appears to be derived from calpain cleavage. Htt fragments in HD lysates were similar in size to those produced by exposure of in vitro-translated Htt to exogenous calpain. Incubation of in vitro-translated Htt with calpain generated a cascade of cleavage events with an initial intermediate cleavage product at 72 kDa and a final cleavage product at 47 kDa. The rate of cleavage of Htt by calpain was polyglutamine-length-dependent. These results suggest that cleavage of Htt in human HD tissue is mediated in part by the Ca2+-activated neutral protease, calpain.

Topics: Adult; Aged; Amino Acid Sequence; Calpain; Cell Line; Enzyme Activation; Female; Humans; Huntingtin Protein; Huntington Disease; Male; Middle Aged; Molecular Sequence Data; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Peptides; Thapsigargin