Mutant Huntingtin and Elusive Defects in Oxidative Metabolism and Mitochondrial Calcium Handling .

Elongation of a polyglutamine (polyQ) stretch in huntingtin protein (Htt) is linked to Huntington's disease (HD) pathogenesis. The mutation in Htt correlates with neuronal dysfunction in the striatum and cerebral cortex and eventually leads to neuronal cell death. The exact mechanisms of the injurious effect of mutant Htt (mHtt) on neurons are not completely understood but might include aberrant gene transcription, defective autophagy, abnormal mitochondrial biogenesis, anomalous mitochondrial dynamics, and trafficking. In addition, deficiency in oxidative metabolism and defects in mitochondrial Ca(2+) handling are considered essential contributing factors to neuronal dysfunction in HD and, consequently, in HD pathogenesis. Since the discovery of the mutation in Htt, the questions whether mHtt affects oxidative metabolism and mitochondrial Ca(2+) handling and, if it does, what mechanisms could be involved were in focus of numerous investigations. However, despite significant research efforts, the detrimental effect of mHtt and the mechanisms by which mHtt might impair oxidative metabolism and mitochondrial Ca(2+) handling remain elusive. In this paper, I will briefly review studies aimed at clarifying the consequences of mHtt interaction with mitochondria and discuss experimental results supporting or arguing against the mHtt effects on oxidative metabolism and mitochondrial Ca(2+) handling

Oxidative metabolism and Ca2+ handling in striatal mitochondria from YAC128 mice, a model of Huntington's disease

The mechanisms implicated in the pathology of Huntington's disease (HD) remain not completely understood, although dysfunction of mitochondrial oxidative metabolism and Ca2+ handling have been suggested as contributing factors. However, in our previous studies with mitochondria isolated from the whole brains of HD mice, we found no evidence for defects in mitochondrial respiration and Ca2+ handling. In the present study, we used the YAC128 mouse model of HD to evaluate the effect of mHtt on respiratory activity and Ca2+ uptake capacity of mitochondria isolated from the striatum, the most vulnerable brain region in HD. Isolated, Percoll-gradient purified striatal mitochondria from YAC128 mice were free of cytosolic and ER contaminations, but retained attached mHtt. Both nonsynaptic and synaptic striatal mitochondria isolated from early symptomatic 2-month-old YAC128 mice had similar respiratory rates and Ca2+ uptake capacities compared with mitochondria from wild-type FVB/NJ mice. Consistent with the lack of difference in mitochondrial respiration, we found that the expression of several nuclear-encoded proteins in striatal mitochondria was similar between wild-type and YAC128 mice. Taken together, our data demonstrate that mHtt does not alter respiration and Ca2+ uptake capacity in striatal mitochondria isolated from YAC128 mice, suggesting that respiratory defect and Ca2+ uptake deficiency most likely do not contribute to striatal pathology associated with HD

Mutant huntingtin fails to directly impair brain mitochondria

Although the mechanisms by which mutant huntingtin (mHtt) results in Huntington's disease (HD) remain unclear, mHtt‐induced mitochondrial defects were implicated in HD pathogenesis. The effect of mHtt could be mediated by transcriptional alterations, by direct interaction with mitochondria, or by both. In the present study, we tested a hypothesis that mHtt directly damages mitochondria. To test this hypothesis, we applied brain cytosolic fraction from YAC128 mice, containing mHtt, to brain non‐synaptic and synaptic mitochondria from wild‐type mice and assessed mitochondrial respiration with a Clark‐type oxygen electrode, membrane potential and Ca2+ uptake capacity with tetraphenylphosphonium (TPP+)‐ and Ca2+‐sensitive electrodes, respectively, and, reactive oxygen species production with Amplex Red assay. The amount of mHtt bound to mitochondria following incubation with mHtt‐containing cytosolic fraction was greater than the amount of mHtt bound to brain mitochondria isolated from YAC128 mice. Despite mHtt binding to wild‐type mitochondria, no abnormalities in mitochondrial functions were detected. This is consistent with our previous results demonstrating the lack of defects in brain mitochondria isolated from R6/2 and YAC128 mice. This, however, could be because of partial loss of mitochondrially bound mHtt during the isolation procedure. Consequently, we increased the amount of mitochondrially bound mHtt by incubating brain non‐synaptic and synaptic mitochondria isolated from YAC128 mice with mHtt‐containing cytosolic fraction. Despite the enrichment of YAC128 brain mitochondria with mHtt, mitochondrial functions (respiration, membrane potential, reactive oxygen species production, Ca2+ uptake capacity) remained unchanged. Overall, our results suggest that mHtt does not directly impair mitochondrial functions, arguing against the involvement of this mechanism in HD pathogenesis

Ca(2+) handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease

We investigated Ca(2+) handling in isolated brain synaptic and non-synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non-synaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca(2+) uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca(2+) capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca(2+) uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen because of mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca(2+) followed by recovery to near resting levels. Following recovery of cytosolic Ca(2+), mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca(2+), suggesting similar Ca(2+) release and, consequently, Ca(2+) loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca(2+) handling in brain mitochondria of YAC128 mice. We demonstrate that mutant huntingtin (mHtt) binds to brain synaptic and nonsynaptic mitochondria and the amount of mitochondria-bound mHtt correlates with increased mitochondrial Ca(2+) uptake capacity. We propose that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage

The Role of Adenine Nucleotide Translocase in the Mitochondrial Permeability Transition

The mitochondrial permeability transition, a Ca2+-induced significant increase in permeability of the inner mitochondrial membrane, plays an important role in various pathologies. The mitochondrial permeability transition is caused by induction of the permeability transition pore (PTP). Despite significant effort, the molecular composition of the PTP is not completely clear and remains an area of hot debate. The Ca2+-modified adenine nucleotide translocase (ANT) and F0F1 ATP synthase are the major contenders for the role of pore in the PTP. This paper briefly overviews experimental results focusing on the role of ANT in the mitochondrial permeability transition and proposes that multiple molecular entities might be responsible for the conductance pathway of the PTP. Consequently, the term PTP cannot be applied to a single specific protein such as ANT or a protein complex such as F0F1 ATP synthase, but rather should comprise a variety of potential contributors to increased permeability of the inner mitochondrial membrane

The effect of mitochondrial calcium uniporter and cyclophilin D knockout on resistance of brain mitochondria to Ca2+-induced damage

The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial depolarization and swelling, the major signs of Ca2+-induced mitochondrial damage. Mitochondrial depolarization inhibits ATP production, whereas swelling results in the release of mitochondrial pro-apoptotic proteins. The extent to which simultaneous deletion of MCU and CyD inhibits PTP induction and prevents damage of brain mitochondria is not clear. Here, we investigated the effects of MCU and CyD deletion on the propensity for PTP induction using mitochondria isolated from the brains of MCU-KO, CyD-KO, and newly created MCU/CyD-double knockout (DKO) mice. Neither deletion of MCU nor of CyD affected respiration or membrane potential in mitochondria isolated from the brains of these mice. Mitochondria from MCU-KO and MCU/CyD-DKO mice displayed reduced Ca2+ uptake and diminished extent of PTP induction. The Ca2+ uptake by mitochondria from CyD-KO mice was increased compared with mitochondria from WT mice. Deletion of CyD prevented mitochondrial swelling and resulted in transient depolarization in response to Ca2+, but it did not prevent Ca2+-induced delayed mitochondrial depolarization. Mitochondria from MCU/CyD-DKO mice did not swell in response to Ca2+, but they did exhibit mild sustained depolarization. Dibucaine, an inhibitor of the Ca2+-activated mitochondrial phospholipase A2, attenuated and bovine serum albumin completely eliminated the sustained depolarization. This suggests the involvement of phospholipase A2 and free fatty acids. Thus, in addition to induction of the classical PTP, alternative deleterious mechanisms may contribute to mitochondrial damage following exposure to elevated Ca2+

Oxidative metabolism and Ca2+ handling in isolated brain mitochondria and striatal neurons from R6/2 mice, a model of Huntington's disease

Alterations in oxidative metabolism and defects in mitochondrial Ca2+ handling have been implicated in the pathology of Huntington's disease (HD), but existing data are contradictory. We investigated the effect of human mHtt fragments on oxidative metabolism and Ca2+ handling in isolated brain mitochondria and cultured striatal neurons from the R6/2 mouse model of HD. Non-synaptic and synaptic mitochondria isolated from the brains of R6/2 mice had similar respiratory rates and Ca2+ uptake capacity compared with mitochondria from wild-type (WT) mice. Respiratory activity of cultured striatal neurons measured with Seahorse XF24 flux analyzer revealed unaltered cellular respiration in neurons derived from R6/2 mice compared with neurons from WT animals. Consistent with the lack of respiratory dysfunction, ATP content of cultured striatal neurons from R6/2 and WT mice was similar. Mitochondrial Ca2+ accumulation was also evaluated in cultured striatal neurons from R6/2 and WT animals. Our data obtained with striatal neurons derived from R6/2 and WT mice show that both glutamate-induced increases in cytosolic Ca2+ and subsequent carbonilcyanide p-triflouromethoxyphenylhydrazone-induced increases in cytosolic Ca2+ were similar between WT and R6/2, suggesting that mitochondria in neurons derived from both types of animals accumulated comparable amounts of Ca2+ Overall, our data argue against respiratory deficiency and impaired Ca2+ handling induced by human mHtt fragments in both isolated brain mitochondria and cultured striatal neurons from transgenic R6/2 mice

Deletion of mitochondrial calcium uniporter incompletely inhibits calcium uptake and induction of the permeability transition pore in brain mitochondria

Ca2+ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca2+ uptake machinery. The MCU is a Ca2+-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca2+ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca2+ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca2+ ionophore ETH129 significantly accelerated Ca2+ uptake. The residual Ca2+ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na+/Ca2+ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca2+ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca2+ uptake pathway in brain mitochondria that mediates residual Ca2+ influx and induction of PTP in a fraction of the mitochondrial population

Glucose-independent Acetate Metabolism Promotes Melanoma Cell Survival and Tumor Growth

Tumors rely on multiple nutrients to meet cellular bioenergetics and macromolecular synthesis demands of rapidly dividing cells. Although the role of glucose and glutamine in cancer metabolism is well understood, the relative contribution of acetate metabolism remains to be clarified. We show that glutamine supplementation is not sufficient to prevent loss of cell viability in a subset of glucose-deprived melanoma cells, but synergizes with acetate to support cell survival. Glucose-deprived melanoma cells depend on both oxidative phosphorylation and acetate metabolism for cell survival. Acetate supplementation significantly contributed to maintenance of ATP levels in glucose-starved cells. Unlike acetate, short chain fatty acids such as butyrate and propionate failed to prevent loss of cell viability from glucose deprivation. In vivo studies revealed that in addition to nucleo-cytoplasmic acetate assimilating enzyme ACSS2, mitochondrial ACSS1 was critical for melanoma tumor growth in mice. Our data indicate that acetate metabolism may be a potential therapeutic target for BRAF mutant melanoma