Egor Y Plotnikov

Anastasia A Chupyrkina

Irina B Pevzner

Nickolaj K Isaev

Dmitry B Zorov
email: zorov@belozersky.msu.ru

Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney's mitochondria

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis.

Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial disfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions released from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.

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Introduction

Rhabdomyolysis (muscle fiber dissolution) is the injury-induced pathological lysis of skeletal muscle tissue, leading to the release of intracellular components into the extracellular fluid and the circulation. This process explains approximately 10 to 15% of all acute renal failure (ARF) cases. The significance of such pathology may be clear from the fact that the major cause of rhabdomyolysis is the crush factor, i.e., myolysis due to traumatic compression of muscle as a result of accidents or disasters. There is a number of case reports on the development of rhabdomyolysis associated with drug use such as cocaine and ecstasy [START_REF] Roth | Acute rhabdomyolysis associated with cocaine intoxication[END_REF][START_REF] Cunningham | Ecstasy-induced rhabdomyolysis and its role in the development of acute renal failure[END_REF], venom poisoning [START_REF] Bush | Severe rattlesnake envenomation with anaphylaxis and rhabdomyolysis[END_REF] and alcohol abuse [START_REF] Vanholder | Rhabdomyolysis[END_REF]. Interestingly, while 0.1% of all patients with coronary artery disease using statins to lower blood cholesterol suffer from severe rhabdomyolysis that apparently results from altering the production of small proteins necessary for myocyte maintenance [START_REF] Evans | Effects of HMG-CoA reductase inhibitors on skeletal muscle: are all statins the same?[END_REF], 1-5% of all statin users complain on myalgias and muscle weakness [START_REF] Thompson | Statin-associated myopathy[END_REF].

Many researchers suggest the main toxic element to be myoglobin, an 18,800 Da oxygen carrier muscle cell protein. Myoglobin is quickly filtered by the glomeruli and then resides in the tubules, where it may cause obstruction and renal failure. The mechanisms of kidney injury during rhabdomyolysis are still not completely understood.

There is a lack of knowledge on the participation of mitochondria as well as of reactive oxygen and nitrogen species (ROS and RNS correspondingly) in the development of myoglobinuric renal dysfunction, whereas its importance in some other renal pathologies, such as kidney ischemia and others is well established [START_REF] Plotnikov | The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney[END_REF].

While in earlier reports it was a widely hold believe that the general cause of myoglobinuric ARF is the formation of myoglobin aggregates and renal tubular obstruction, leading to impairment of primary urine flow and reabsorption [START_REF] Oken | Glycerol-induced hemoglobinuric acute renal failure in the rat. I. Micropuncture study of the development of oliguria[END_REF][START_REF] Pelletier | Acute renal failure following kidney transplantation associated with myoglobinuria in patients treated with rapamycin[END_REF][START_REF] Visweswaran | Rhabdomyolysis[END_REF], recent studies have shown the development of oxidative stress under the conditions of myoglobinuria, accompanied by the increased production of ROS and lipid peroxidation [START_REF] Shah | Evidence suggesting a role for hydroxyl radical in glycerol-induced acute renal failure[END_REF][START_REF] Zager | Mitochondrial free radical production induces lipid peroxidation during myohemoglobinuria[END_REF]. The heme of a myoglobin molecule is thought to play the key role in this process. In one study the oxidative damage was caused by the direct effect of heme without releasing iron from the heme [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF]. However, the presence of hemoxygenase and ferrochelatase in the cell [START_REF] Barnes | Ferrochelatase and -aminolaevulate synthetase in brain, heart, kidney and liver of normal and porphyric rats. The induction of -aminolaevulate synthetase in kidney cytosol and mitochondria by allylisopropylacetamide[END_REF][START_REF] Zorov | Mitochondria revisited. Alternative functions of mitochondria[END_REF] may potentially result in the appearance of free iron in the cell with further ignition of Fenton reaction. At the same time, under rhabdomyolysis

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and myoglobin accumulation in tubules, their epithelial cells show elevated expression of hemoxygenase [START_REF] Ishizuka | Regulation and immunohistochemical analysis of stress protein heme oxygenase-1 in rat kidney with myoglobinuric acute renal failure[END_REF] which makes such process of iron release highly probable. We must admit that earlier reports suggested that myoglobin may release free ferrous (Fe 2+ ) ions, which could induce lipid peroxidation as a result of the generation of hydroxyl radicals via Fenton's reaction [START_REF] Rice-Evans | Oxidised low density lipoproteins induce iron release from activated myoglobin[END_REF]. An alternative mechanism could involve the heme moiety of myoglobin absorbed by a renal tubule cell [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Moore | 2nd: A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure[END_REF]. It can be realized either through endocytotic absorption of myoglobin [START_REF] Gburek | Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin[END_REF] by the tubular cells similar to what happens with albumin [START_REF] Birn | Renal albumin absorption in physiology and pathology[END_REF] or through the direct transport of heme (for example, by a specific transporter HCP1 (heme carrier transporter) which is known to facilitate heme transport into the cell and specifically participates in heme capture in kidney cells [START_REF] Sharma | Heme carrier protein 1 (HCP1) expression and functional analysis in the retina and retinal pigment epithelium[END_REF]. Indirect evidence of the activation of hemoxygenase after myoglobin accumulation in the tubules [START_REF] Ishizuka | Regulation and immunohistochemical analysis of stress protein heme oxygenase-1 in rat kidney with myoglobinuric acute renal failure[END_REF] is in support of such speculation, however, there is no doubt that the expression of this protein can be determined by other factors including oxidative stress [START_REF] Morse | Heme oxygenase-1, a critical arbitrator of cell death pathways in lung injury and disease[END_REF].

In renal tubules, oxyferrous myoglobin would undergo autooxidation to the ferric form (MetMb, Fe 3+ ), which is catalytically competent to promote a lipid peroxidation reactions. The redox cycle occurs between the ferric (Fe 3+ ) and ferryl ([Fe=O] 2+ ) myoglobin oxidation states, and the latter can directly initiate lipid oxidation [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Moore | 2nd: A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure[END_REF].

Until now, most researchers have proposed development of oxidative stress during rhabdomyolysis, after detecting the presence of lipid peroxidation products either in serum or in renal tissue. There is a consensus that one of the basic regulators of ROS production and cell death are mitochondria [START_REF] Zorov | The mitochondrion as janus bifrons[END_REF] but the knowledge on these organelles functioning under myoglobinuria is still not complete. There are limited evidences of mitochondria participation in development of pathological processes during rhabdomyolysis [START_REF] Zager | Mitochondrial free radical production induces lipid peroxidation during myohemoglobinuria[END_REF][START_REF] Zager | Effects of inorganic iron and myoglobin on in vitro proximal tubular lipid peroxidation and cytotoxicity[END_REF][START_REF] Zager | Myoglobin toxicity in proximal human kidney cells: roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport[END_REF].

Moreover, very little is known about the role of RNS in myoglobinuria. NO is an important modulator of oxidative stress and may be a predisposing as well as a protective factor. This issue has taken on special significance after the discovery of mitochondrial NO-synthase (NOS) [START_REF] Giulivi | Production of nitric oxide by mitochondria[END_REF], which enables mitochondria to directly regulate their ROS production [START_REF] Brown | Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase[END_REF].

Our study was focused on mitochondrial functioning, development of oxidative stress and increase of NO production in a glycerol-induced myoglobinuria in the rat as

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well as the study of myoglobin effect on isolated rat kidney mitochondria. The goal of this work was to investigate the mitochondrial status during myoglobin-induced ARF, to model the mitochondria dysfunction caused by myoglobin in vitro and to study the role of mitochondrial NOS in this pathology.

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Materials and methods

Modeling of glycerol-induced rhabdomyolysis in rat.

Experiments were performed on outbred white male rats (180-200 g) fed ad libitum. Animal protocols were approved by the institutional review ethical committee.

Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Animals in the rhabdomyolysis group were deprived of water for approximately 36 hr and then injected with glycerol (10 ml/ kg of a 50% water solution) intramuscular in two legs [START_REF] Zurovsky | Models of glycerol-induced acute renal failure in rats[END_REF]. Control animals were untreated. At regular intervals after rhabdomyolysis induction, blood samples were collected for measurement of biochemical parameters and kidney were excised for the mitochondria isolation. respiration rate of isolated mitochondria [START_REF] Chance | A simple and rapid assay of oxidative phosphorylation[END_REF]. Proper ratio (V 3 /V 4 ), also called respiratory control index defines proton fluxes through the inner mitochondrial membrane under the state of the ATP synthesis (V 3 ) versus the resting state (V 4 ).

Isolation of mitochondria from rat kidney

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Mitochondrial uncoupling was induced by adding 2,4-dinitrophenol (DNP) to a final concentration of 100 µM. Oxygen consumption rate for state 3 and 4 was calculated per amount of mitochondrial protein in the sample.

Measurement of MDA products and nitric oxide in serum and mitochondria of rat kidney

Malonic dialdehyde was determined in the sample by thiobarbituric acid assay according to the method of [START_REF] Mihara | Determination of malonaldehyde precursor in tissues by thiobarbituric acid test[END_REF] with minor modification. Each sample was mixed with 0.8% thiobarbituric acid and 1% H 3 PO 4 in the ratio 0.9: 1.0: 3.0. The mixture was boiled for 45 min and centrifuged after cooling at 15000g for 10 min. Absorbance of the resulting supernatant at 532 nm was measured by a Hitachi 557 spectrophotometer. 1,3,3tetraetoxipropan (Sigma, USA) was used as a standard solution.

Nitric oxide was measured by colorimetric method of Griess (Griess reagent, Sigma-Aldridge, USA). To remove protein serum, the samples were mixed with an equal volume of ice methanol, incubated for 5 min at 4 ºC and centrifuged at 20000g for 10 min. The resulting supernatant was combined with an equal volume of Griess reagent and incubated for 30 min at 4 ºC. The absorbance at 540 nm was measured using a plate reader (LabSystems Multiscan).

Incubation of mitochondria with myoglobin in vitro

Mitochondria were diluted to a final concentration of 4 mg/ml with the incubation buffer containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, 0.1% BSA, pH 7.4. 5 mM K-succinate and 10 µM rotenone were added to initiate the respiration. Myoglobin was added to the sample to final concentrations of 50, 100, 500 µM, accordingly. The control sample was supplemented either with the same amount of BSA or the incubation medium. Samples were incubated for 1 hr at room temperature with gentle shaking then some aliquots of mitochondria were used for oxygen consumption measurements and

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another were centrifuged (10500g) for 10 min. The pellet was suspended in 1.15% KCl with 10 mM EGTA and was used for MDA measurements. Supernatant was used for nitrite determination.

Western blot analysis.

Samples of serum or mitochondria, normalized for total protein content (50 mg/lane), were separated by Tris-tricine electrophoresis into 16.5% PAAG and transferred onto a PVDF membrane (Amersham Pharmacia Biotech, Rainham, UK).

Membranes were blocked with 3% (wt/vol) non-fat milk in PBS with 0.1% (v/v) Tween 20, subsequently incubated with primary antibodies: mouse monoclonal anti-cytochrome c 1:1000 (BD Biosciences Pharmingen, USA), mouse monoclonal anti-porin 1:1000 (BD Biosciences Pharmingen, USA) or rabbit monoclonal myoglobin 1:500 (Santa Cruz, USA) and stained with anti-mouse IgG or anti-rabbit IgG conjugated with horseradish peroxidase (Calbiochem, USA). The detection was made using ECL Western blotting kit (Amersham Pharmacia Biotech, Rainham, UK). Spots density analysis in the film was done by ImageJ software (NIH, Bethesda, MD, USA) after scanning.

Measurement of mitochondria membrane potential and ROS production in isolating tubules

Kidneys of 3-7 days old rats were excised and dissociated with 0.1% collagenase solution (20 min at 37ºC). Large fragments of tissue were removed, and tubules were precipitated by gentle centrifugation (50g) for 3 min. The pellet was suspended in DMEM/F-12 medium without bicarbonate supplemented with 20 mM HEPES. All further steps were done at 37 ºC.

The myoglobin solution was added to the tubules suspension to final concentration of 500 μM and incubated for 1 hr. Then tubules were washed out of myoglobin and incubated with fluorescent probes TMRE (200 nM) and 2',7'-dichlorodihydrofluorescein diacetate (DCF, 10 μM, Invitrogen, USA). Renal tubules were imaged with an LSM510 inverted confocal microscope (Carl Zeiss Inc., Jena, Germany) in a multi-channel mode as appropriate for the dyes loaded. Analysis of fluorochrome incorporation was performed in glass-bottom dishes with the excitation at 488 nm (for DCF) and 543 nm (for TMRE), and emission collected at 500-530 nm and >560 nm, respectively. To minimize the contribution of photo-induced mitochondria/cell damage in the relative fluorescence intensities we analyzed images used by averaging first four scans only. Images were processed by ImageJ software (NIH, Bethesda, MD, USA).

Statistics

All the experiments were performed more than in triplicate. All data are mean ± SEM. Comparisons within groups were made by Student t test, and P value less than 0.05 was taken to indicate statistical significance.

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Results

Mitochondrial damage in myoglobin-induced ARF

This study of ARF development under rhabdomyolysis demonstrates that the activities of principal enzyme-reporters of the tissue damage (such as lactate dehydrogenase (LDH)

and alanine aminotransferase (ALT)) increased greatly in the blood within the first 2 days (Fig. 1B). A basic index of severe ARF development was the increase in the blood of nitrogen metabolism products (urea and creatinine) in the blood, which reached its maximum at 1-2 days (Fig. 1A). Further, most parameters returned to a normal level that allows suggesting functional recovery of kidneys.

We found that myoglobin deposits in the kidney tubules become already detectable during very first hours after rhabdomyolysis induction (Fig. 1C). In control kidney, myoglobin was not detected (Fig. 1С). Myoglobin appeared in the renal tissue 3 hr after glycerol injection, and its maximal concentration was observed after 15 hr, and went down to a non-detectible level after 24 hr. The latter may be due to the possibility that the protein moiety of myoglobin molecule was at least partially degraded, resulting in the failure of specific antibodies to detect the antigen. Also myoglobin could be rapidly excreted in the urine already at first day of rhabdomyolysis.

We used the release of cytochrome c from kidney mitochondria as a marker of their damage [START_REF] Zager | Proximal tubular cytochrome c efflux: determinant, and potential marker, of mitochondrial injury[END_REF]. There is evidence that at cell apoptotic death, cytochrome c not only is released from mitochondria into the cytoplasm, but also appears in the extracellular space. It seems that blood plasma cytochrome c level reflects the degree of tissue injury.

In fact, we revealed that cytochrome c was detected in the blood of rhabdomyolytic rats (Fig. 1D). This demonstrated the extensive damage of renal cells and the induction of their apoptotic or necrotic death. Cytochrome c was not found in control serum.

More conclusive proof of apoptotic launching and mitochondria injury in renal cells was obtained by western blotting of mitochondria isolated from kidneys on different periods of rhabdomyolysis. We found that the amount of cytochrome c in mitochondria

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was decreased and maximum of cytochrome c release was observed at 4 hr after rhabdomyolysis induction.

Oxidative stress and dysfunction of mitochondria in rhabdomyolytic rats

We analyzed basic functional parameters of mitochondria isolated from kidney at different periods after rhabdomyolysis induction. In a first turn, the oxygen consumption rates with succinate in state 3 and 4 (V 3 U and V 4 S ) and MDA levels were measured and V 3 U /V 4 S ratio was determined in mitochondria. We revealed an essential increase of MDA level in mitochondria on the very first day of myoglobinuria (Fig. 2A), which demonstrated that a significant oxidative stress evolves, leading to a lipid peroxidation in mitochondria. The greatest rise was observed after 4 hr when MDA level rose twice as compared with control values. After 15 hr of rhabdomyolysis, MDA concentration in mitochondria stabilized and later on the same day began to drop.

Twenty-four hours after glycerol injection the oxygen consumption rate (at state 4) increased (Fig. 2B). This indirectly indicates some loss of a coupling between oxidation and phosphorylation in mitochondria, possibly due to a change of permeability in the inner mitochondrial membrane to ions. Although the state 3 respiration rate (with the uncoupler) did not change much (data not shown), the ratio V 3 U /V 4 S in rhabdomyolytic mitochondria was significantly diminished.

In vitro model of myoglobin influence on mitochondria functioning

For more comprehensive study of myoglobin effect on mitochondria, we used an in vitro model, where myoglobin directly interacts with isolated renal mitochondria.

Apart from the consideration that the renal tubule cells intake mainly heme or heme iron [START_REF] Zager | Mitochondrial free radical production induces lipid peroxidation during myohemoglobinuria[END_REF], the probability of absorption by the cells of intact myoglobin molecules from tubules lumen cannot be ruled out. Such transport is typical for most proteins as they are filtering into primary urine from blood plasma following by reabsorption in renal tubules [START_REF] Pollock | Albumin transport and processing by the proximal tubule: physiology and pathophysiology[END_REF].

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To study the direct effect of myoglobin on kidney mitochondria, the mitochondrial suspension was supplemented with a commercially available form of myoglobin, metmyoglobin (Fe 3+ ). It is known that mitochondria can reduce the iron in metmyoglobin molecule (Fe 3+ ) to a ferrous form (Fe 2+ ) using succinate as a respiratory substrate [START_REF] Tang | Mitochondrial reduction of metmyoglobin: dependence on the electron transport chain[END_REF], resulting in the presence of both forms of myoglobin in the incubation medium. Therefore, this ability of mitochondria has no need to artificially reduce myoglobin's heme, and apparently, the system more closely corresponds to the situation in kidney in vivo, where both forms of myoglobin are present.

When studying the interaction of myoglobin with mitochondria in vitro we analyzed the mitochondrial oxygen consumption rate after incubation with myoglobin and the behavior of markers of oxidative and nitrosyl stress, because these parameters were found to change in vivo. In a control system, we substituted myoglobin for appropriate concentration of BSA. Data on mitochondria function impairment are presented in Table 1.

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We found that mitochondrial respiration rate in state 4 increases twice and to about three-fold as referred to control values at myoglobin concentrations 100 and 500 μM correspondingly 1 hr after preincubation with myoglobin at room temperature.

Incubation with BSA did not affect the respiration rate, showing that the previous effect was not due to a change of oncotic pressure caused by the presence of protein in the incubation medium. The uncoupling respiration rate (state 3u) almost did not change during incubation with either myoglobin or BSA, which correlates with our data for function of mitochondria isolated from the kidneys of rats suffering myoglobinuria. The low respiratory control of mitochondria incubated with myoglobin may be explained by that on average the population of isolated mitochondria is less coupled and apparently there is a link between relative uncoupling and the presence of peroxidated lipids, suggesting a lipid peroxide-induced distortion of the ion permeability of the inner mitochondrial membrane. Further experiments revealed that 100 μM cyclosporine A (CsA) protects from the myoglobin-induced drop of the respiratory control (Table 1).

These results can be interpreted in the way that Mb causes the induction of the permeability transition in some part of the population of isolated mitochondria (potentially resulting in the absence of the membrane potential in these mitochondria and maximal activation of their respiration), which is misleading by giving an average lowering of the respiratory control with the tentative wrong interpretation of mitochondrial uncoupling.

Incubation of mitochondria with 100 μM myoglobin in the presence of 2 mM chelator of iron ions, deferoxamine (DFO) showed that this chelating significantly although not fully restored the respiratory control value (Table 1). When compared with the data on the prevention of the myoglobin-induced changes of mitochondrial functional parameters, these data show that chelation of iron ions does not prevent the mitochondrial permeability transition caused by myoglobin supplement. The administration of mitochondria-targeted antioxidant SkQ1 also did not improve the functional characteristics of uncoupled mitochondria (Table 1). Both DFO and antioxidant greatly reduced the generation of MDA-products and generation of nitrite as well (see below).

However we cannot exclude the possibility that the inhibitory effect of DFO is due to its ability to reduce ferryl myoglobin [START_REF] Cooper | A hydrogen-donating monohydroxamate scavenges ferryl myoglobin radicals[END_REF][START_REF] Reeder | Desferrioxamine inhibits production of cytotoxic heme to protein cross-linked myoglobin: a mechanism to protect against oxidative stress without iron chelation[END_REF] which is known to promote lipid

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peroxidation. We tried to add reducing equivalents (i.e. ascorbate, not shown) and observed lower amount of MDA products in isolated mitochondria after incubation with myoglobin. However we cannot exclude that ascorbate can significantly shift the entire redox state of mitochondrial matrix towards more reduced state thus increasing the antioxidative capacity of the system. Unfortunately, all possible experimental designs cannot provide the clean discrimination between these two possibilities.

Oxidative stress and NO production in mitochondria caused by myoglobin

The suggestion that the failure of the permeability barrier by the inner mitochondrial membrane after myoglobin exposure is due to lipid peroxidation of mitochondrial membranes was confirmed by the data that MDA products in mitochondria are doubled during incubation with 100 μM myoglobin for 1 hr and almost tripled with 500 μM (Fig. 3A). Simultaneous incubation with both myoglobin and 100 nM CsA reduced the MDA production markedly, however, the control values were not achieved.

This implied that myoglobin-induced PTP induction is at least partially responsible for oxidative stress and lipid peroxidation. Essential anti-oxidative effect was noticed for DFO, although again, as in case of cyclosporine A, full recovery was not observed (Fig. 3). The beneficial effect of SkQ1 was modest but significant (Fig. 3). It is reported that this new mitochondria-targeted antioxidant accumulates in mitochondria and efficiently prevents ROS production and mitochondria dysfunction in different types of oxidative stress [START_REF] Skulachev | A biochemical approach to the problem of aging: "megaproject" on membrane-penetrating ions. The first results and prospects[END_REF][START_REF] Antonenko | Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies[END_REF][START_REF] Bakeeva | Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS-and age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke)[END_REF][START_REF] Agapova | Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells[END_REF][START_REF] Neroev | Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals[END_REF][START_REF] Anisimov | Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 5. SkQ1 prolongs lifespan and prevents development of traits of senescence[END_REF][START_REF] Skulachev | An attempt to prevent senescence: A mitochondrial approach[END_REF][START_REF] Plotnikov | Interrelations of mitochondrial fragmentation and cell death under ischemia/reoxygenation and UV-irradiation: protective effects of SkQ1, lithium ions and insulin[END_REF]. Thus, it looks probable, that free iron released from the myoglobin's heme significantly contributed to the overall damaging effect of myoglobin on mitochondria, inducing lipid peroxidation, apparently mediated by a Fenton reaction. Mitochondriatargeted antioxidants apparently replenished mitochondrial antioxidative defense and significantly hampered deleterious Fe-mediated oxidative lipid damage somehow going with the involvement of the mitochondrial permeability transition.

Finally, we studied the change of NO-production during myoglobin-induced oxidative damage of mitochondria. The prerequisites for such a study were our findings of the rise of a nitrite (product of NO oxidation) level in the blood plasma of rhabdomyolytic rats. On the second day after onset of myoglobinuria, the concentration of nitrite in serum was 12.0±1.5 μM, whereas it was 4.5±0.5 μM in the control sample. In

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vitro model also revealed the growth of the nitrite concentration in the incubation medium of isolated mitochondria (Fig. 3B). Although the increase of nitrite level at 100 μM myoglobin was not significant, it reached almost 7-fold at 500 μM of myoglobin referred to a level observed to a control value. We proposed that myoglobin activates the mitochondrial NOS, resulting in the rise of NO level and received indirect support of this idea from the fact that the supplement of the system with NOS substrate L-arginine (5 mM) significantly enhanced the effect of myoglobin alone (Fig. 3B). Conversely, NOS inhibitor L-nitroarginine decreased the nitrite concentration to the control level. DFO reduced the myoglobin-induced growth of nitrite concentration at such conditions as well.

The tentative explanation of these data is that myoglobin supplement causes the activation of ROS production (possibly superoxide because it may be scavenged by hydrophobic mitochondria-targeted antioxidant SkQ, which in turn activates NOS). In this case, the rise of MDA products (possibly originating from the elevated ROS level) goes in parallel with the rise of nitrite (apparently originating from activated NOS).

Oxidative stress and dysfunction of renal tubules mitochondria induced by myoglobin.

Apparent signs of mitochondrial alterations induced by myoglobin found in isolated mitochondria were confirmed by using confocal microscopy of isolated renal tubules. The incubation of tubules with 100 μM myoglobin resulted in a rise of DCF fluorescence signal in tubules cells (Fig. 3) pointing to the induced increase of ROS production in these cells. At the same time, we revealed a significant reduction of TMRE fluorescence signal, pointing to the drop of the mitochondrial membrane potential (Fig. 3).

These facts support the conclusion that myoglobin causes the distortion of barrier function of the inner mitochondria membrane and provokes the development of oxidative stress. Interestingly, the simultaneous staining of tubules with DCF and TMRE revealed good colocalization of these probes, which points to dominating generation of ROS in mitochondria.

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In a given study, we pursued a goal to ascertain the link between provoked ARF development and changes in renal mitochondria functioning after rhabdomyolysis induction. We found that this pathology proceeds with peroxidation of mitochondrial lipids pointing to the involvement of the oxidative stress. It appears conceivable that such mitochondrial damage is one of principal causes of kidney dysfunction. The dynamics after the crash onset show that already during the first hours after muscle degradation myoglobin becomes detectable in renal tubules, and kidney mitochondria malfunctioning can be observed. Induced pathological changes in kidney resulting in the loss of mitochondrial cytochrome c and its appearance in urine, possibly associated with the induction of mitochondrial permeability transition and apoptotic challenge were indicated in earliest reports [START_REF] Zager | Proximal tubular cytochrome c efflux: determinant, and potential marker, of mitochondrial injury[END_REF][START_REF] Liu | Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c[END_REF].

It is possible that these changes were accompanied by development of oxidative stress, peroxidation of mitochondrial membranes and uncoupling of oxidative phosphorylation in mitochondria. Stimulation of these processes was proved by accumulation of MDA-products in kidney mitochondria and falling of the respiratory control within very first days after the induction of rhabdomyolysis. Kidney malfunctioning, increase of uremia and azotemia occur either simultaneously with these events or with some delay (Fig. 1), and is likely a consequence of mitochondrial dysfunction and oxidative stress.

Such set of abnormalities is typical for ARF provoked by ischemia/reperfusion [START_REF] Plotnikov | The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney[END_REF]. The mitochondrial dysfunction and hyperproduction of ROS tightly correlate with ischemic damage often resulting in a kidney cells death and further ARF. However, apparently under myoglobinuria another scenario may come into play also with the involvement of oxidative stress. In this case, redox-modified myoglobin and/or iron released from its heme are most likely the primary sources of radicals, and the damage of mitochondria could be a secondary process [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Moore | 2nd: A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure[END_REF]. According to this scenario, mitochondria cannot only transmit but also amplify the transmission of oxidative damage [START_REF] Zorov | Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes[END_REF]. But the detailed mechanism of myoglobin influence on mitochondria remained unresolved. In two studies, renal tubules [START_REF] Zager | Effects of inorganic iron and myoglobin on in vitro proximal tubular lipid peroxidation and cytotoxicity[END_REF] or cultivated cells [START_REF] Zager | Myoglobin toxicity in proximal human kidney cells: roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport[END_REF] were exposed to myoglobin, and mitochondrial functioning was evaluated through indirect effect of mitochondrial inhibitors on MDA-products formation and viability of cells. We still

Discussion

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cannot exclude the possibility that the primary target for myoglobin being in a native or modified form and for iron released from its heme may be upstream of mitochondria with further signaling to mitochondria.

Apparently, in such complex system as a cell, exogenous myoglobin may lead to a change in different pathways and mitochondrial contribution might be significantly obscured. No doubt that cellular model being more difficult in interpretation still is more advanced being more close to in vivo situation but more simplified model of direct effect of myoglobin on mitochondria was needed. We were aware of that quite often the data received in vitro (with isolated mitochondria) and in situ (with isolated cells) are conflicting. Particularly in the above mentioned study, when using cells, the inhibition of a respiratory chain by rotenone led to increase of MDA-products generation, while antimycin A caused their reduction, although in both cases the death of kidney cells virtually did not change. However, it is known that these inhibitors have the opposite effects on isolated mitochondria, for example rotenone decreases ROS generation by blocking formation of superoxide in the complex I of respiratory chain [START_REF] Boveris | The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen[END_REF] and antimycin increases this generation by blocking a respiratory chain at Q-cycle level [START_REF] Boveris | The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen[END_REF].

On the other hand, some effectors (i.e. hypoxia/reoxygenation) tested in isolated mitochondria [START_REF] Kuzminova | The permeability transition pore induced under anaerobic conditions in mitochondria energized with ATP[END_REF][START_REF] Krasnikov | The Ca2+ -induced pore opening in mitochondria energized by succinate-ferricyanide electron transport[END_REF][START_REF] Zenebe | Hypoxia/reoxygenation of isolated rat heart mitochondria causes cytochrome c release and oxidative stress; evidence for involvement of mitochondrial nitric oxide synthase[END_REF] demonstrate the responses similar to those observed in the cells with some peculiar distinctions. In addition, it has been shown that myoglobin in its native form can be uptaken by the renal cells by endocytosis [START_REF] Gburek | Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin[END_REF] thus getting a direct access to intracellular mitochondria. Considering this, the study of interaction of myoglobin with isolating mitochondria made good sense.

In fact, using isolated mitochondria we found that myoglobin induces the same changes in mitochondrial functions as was found for myoglobinuria in vivo. The rise of respiration rate in state 4 and the drop of respiratory control were arguments in proof of mitochondrial uncoupling and accumulation of MDA directly pointed to that deleterious effect of myoglobin apparently involved lipid peroxidation. Also, the development of oxidative stress and the loss of mitochondria potential in tubule cells loaded with myoglobin were observed as well. The key role in mitochondrial lipid peroxidation probably belongs to iron ions released from the myoglobin heme since this process was sensitive to Fe-chelator DFO (Fig. 3а). The lack of mitigating effect of DFO and

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antioxidant SkQ1 on the mitochondrial coupling after incubation with myoglobin was quite unexpected. Both compounds causing elevation of MDA products apparently via induced lipid peroxidation did not restore the respiration control, decreased during myoglobin action. Due to the ability of DFO to reduce ferryl myoglobin, we still cannot exclude the possibility that since lipid peroxidation does occur in this system, this might suggest that ferryl myoglobin is generated and the presence of DFO lowers the toxicity of myoglobin through its reducing capacity. Unfortunately, the usage of other reducing agents (such as ascorbate which lowered lipid peroxidation) seems to be not optimal since we cannot exclude the general shift of the redox state in the entire system (mitochondrial matrix) towards more reduced one which may affect the antioxidative capacity of the system.

We found that PTP inhibitor, cyclosporine A, prevents myoglobin-induced mitochondrial damage. It not only restored the respiratory control, being felt during incubation with myoglobin, but also reduced the accumulation of MDA products. Based on these facts one can suppose two tentative pathways of myoglobin effect on mitochondria. From one side, it could promote the lipid peroxidation and on the other hand, it could induce mitochondrial permeability transition and these processes within certain limits apparently proceed quite independently. On the other hand, the inhibition of PTP partially reduced the lipid peroxidation, proving the pore to be one of the key players in myoglobin-mediated oxidative stress in mitochondria. But based on the absence of restoring effects of DFO and antioxidant SkQ1 on the mitochondrial coupling we conclude that suppression of lipid peroxidation cannot prevent the mitochondria dysfunction.

Another important finding was the demonstration of NO synthesis activation in mitochondria exposed to myoglobin. NO participation in regulation of damage during rhabdomyolysis was suggested in earlier reports [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Moore | 2nd: A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure[END_REF] with later evidence of positive influence of NO-donors on kidney functional recovery after rhabdomyolysis [START_REF] Chander | Molsidomine, a nitric oxide donor and L-arginine protects against rhabdomyolysis-induced myoglobinuric acute renal failure[END_REF]. In our experiments, we revealed the increase of nitrite level in the blood of animals after rhabdomyolysis with the non-specific inhibitor of NOS, L-NAME being protective from this rise which was explained by the enhanced generation of NO by NOS under rhabdomyolysis. NO is known to be an important regulator of mitochondria functions,

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with respiratory components as primary targets possibly attenuating ROS generation [START_REF] Brown | Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase[END_REF].

Earlier we demonstrated the rise of NO synthesis in mitochondria of the kidney exposed to ischemia/reperfusion [START_REF] Plotnikov | The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney[END_REF]. Since in this study the nitrite abundance was blunted by DFO and specific mitochondria-targeted antioxidant SkQ1, we can speculate that myoglobin itself, iron ions released from it and induced ROS generation as well activate mitochondrial NOS.

In conclusion, we found mitochondria to play an important role in the development of kidney dysfunction under rhabdomyolysis with the involvement of ROS and RNS. In this case, mitochondria are not only the target for myoglobin and iron ions released from it but also a transmitter and induced source of these reactive molecules. All

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  of these factors affect mitochondrial respiration, which apparently contributes greatly to the kidney pathological changes, possibly resulting in kidney cells death and ARF development. Lipid peroxidation of mitochondrial membranes may become a crucial factor of myoglobin-induced damage of mitochondria, which can be developed after direct interaction of mitochondria with myoglobin. Participation of mitochondrial NOS in these processes opens new horizons in the system of renal cell interactions with myoglobin, which needs further investigation.Acknowledgements: This work was supported by Center of Mitoengineering and RFBR grants 08-04-01667, 09-04-01096 A C C E P T E D M A N U S C R I P T

Fig. 1 .Fig. 2 .

 12 Fig.1. ARF development and the damage of kidney mitochondria in rhabdomyolytic rats. The rise of blood urea and creatinine level (A), and ALT and LDH activity (B) are indices of ARF development on the first days after rhabdomyolysis induction. Appearance of myoglobin in the blood correlates with its accumulation in the kidney (C, western blotting of kidney homogenate with anti Mb antibodies where lane 1, rat skeletal muscle; lane 2, control kidney; lanes 3-5, the kidney 4, 15 and 24 hr after induction of rhabdomyolysis correspondingly). Cytochrome c appears in the blood (D, western blotting of blood serum with anti-cytochrome c antibodies where lane 1, control serum; lanes 2-5, 5, 16, 24 and 48 hr after induction of rhabdomyolysis correspondingly) with a corresponding loss of cytochrome c in isolated kidney mitochondria (E, lanes 1-4, 4, 16, 24, and 48 hr, and lane 5, control kidney mitochondria. F. The same samples as in E with anti-VDAC antibodies

Fig. 3 .

 3 Fig. 3. Oxidative stress and NO production in rat kidney mitochondria incubated with myoglobin. Generation of MDA-products (A) and nitrite (B) in mitochondria, incubated with different concentrations of myoglobin supplemented with: 5 mM of NOS substrate L-arginine; 2 mM of iron chelator, DFO; 100 nM of PTP inhibitor, CsA; 100 nM mitochondria-targeted antioxidant SkQ1, and 5 mM of NOS inhibitor L-NAME.

Fig. 4 .

 4 Fig. 4. ROS generation and disruption of mitochondrial potential in renal tubules cells incubated with myoglobin. Incubation of renal tubules with 500 μM myoglobin for 1 hr causes significant increase of ROS production (B) and the drop of the membrane potential as indicated by the rise of DCF (B) and TMRE (D) fluorescence. A, C, control

Table 1 .

 1 The

	Sample	V 4 S

influence of incubation with myoglobin on functional characteristics of renal mitochondria: the respiration rates in state 3 (V 3 s ) and 4 (V 4 u ) and the respiratory control. The effects of mitochondria-targeted antioxidant SkQ1, iron ions chelator DFO and PTP inhibitor cyclosporin A are presented.

(nmol О 2 min -1 mg -1 ) V 3 U (nmol О 2 min -1 mg -1 ) Respiratory control Control 11,5±2,1 55,1±5,3 4,8±0,6 100 μM Mb 22,7±1,7 54,5±4,2 2,4±0,3

	500 μM Mb	32,5±5,4	51,0±3,6	1,6±0,6
	100 μM Mb + 2 mM DFO	16,6±1,2	53,2±6,8	3,2±0,2
	500 μM Mb + 100 nM SkQ1	38,6±4,6	54,1±6,8	1,4±0,2
	100 μM Mb +100 nМ CsA	12,3±2,8	52,9±10,3	4,3±0,5
	100 μM BSA	12,6±1,3	52,1±9,1	4,1±0,3
	500 μM BSA	11,85±1,7	54,3±11,3	4,5±0,1

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renal tubules stained with DCF and TMRM correspondingly. Diagrams indicate average fluorescence intensity through 10 confocal images in each sample. Bar 100 mµ.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T


Dr Xiao-Jiang Li

Adam L Orr

Shihua Li

Impaired mitochondrial trafficking in Huntington's disease

Keywords: Mitochondria, Huntington, trafficking, neurodegeneration

Impaired mitochondrial function has been well documented in Huntington's disease. Mutant huntingtin is found to affect mitochondria via various mechanisms including the dysregulation of gene transcription and impairment of mitochondrial function or trafficking. The lengthy and highly branched neuronal processes constitute complex neural networks in which there is a large demand for mitochondria-generated energy. Thus, the impaired mitochondria trafficking in neuronal cells may play an important role in the selective neuropathology of Huntington's disease. Here we discuss the evidence for the effect of the Huntington's disease protein huntingtin on the intracellular trafficking of mitochondria and the involvement of this defective trafficking in the pathogenesis of Huntington's disease. 1.

Introduction

Huntington's disease (HD) is a dominant, progressive neurodegenerative genetic disease that is caused by a CAG triplet repeat expansion (>35 CAGs) in the first exon of the gene encoding the huntingtin (htt) protein. HD is characterized by uncontrolled movement, dementia, emotional disturbance, and early death, which often occur in middle age. These symptoms are associated with the neuronal loss that occurs preferentially in medium spiny neurons of the striatum and also extends to other brain regions during the late stages. The preferential degeneration of striatal neurons can be reproduced by administration of the mitochondrial toxins in different animal models, leading to the prevalent theory that mitochondrial dysfunction contributes to the pathogenesis of HD. In support of this idea, many recent studies have

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demonstrated that mutant htt affects the function of mitochondria in various HD cellular and animal models [START_REF] Bossy-Wetzel | Mutant huntingtin and mitochondrial dysfunction[END_REF]. The negative impact of mutant htt on mitochondria appears to result from the indirect effects of mutant htt on the nuclear expression of genes important for the biogenesis of mitochondria and the direct effects of htt on the respiration function and trafficking of mitochondria. In this review, we focus on the relationship between mutant htt and mitochondria trafficking as well as HD pathogenesis.

Mitochondrial trafficking

Mitochondria are the primary generators of ATP and are important regulators of intracellular calcium homeostasis. Although the human brain comprises 2% of total body mass, it consumes 20% of resting metabolic energy [START_REF] Silver | Oxygen and ion concentrations in normoxic and hypoxic brain cells[END_REF]. Neurons are highly specialized cell types and possess lengthy axons and dendrites that are highly branched and constitute complex neural networks via synapses. Synaptic connections are highly plastic and undergo continuous remodeling to modulate information processing [3.4]. Thus, neurons represent a particular anatomical and physiological challenge for mitochondrial trafficking within neuronal processes and nerve terminals.

Mitochondria are dynamically transported along lengthy neuronal processes to provide energy to nerve terminals and maintain the normal neuronal function. Mitochondria are highly dynamic organelles that fuse and divide in neurons. The movement of mitochondria also facilitates its fusion and fission events in neurons and retrieval of damaged mitochondria for degradation by autophagy. Although how mitochondria are transported and regenerated remains to be understood, the failure to move mitochondria or deliver mitochondria to appropriate sites in neurons would result in energy starvation and impairment of neuronal interactions or neural network function.

Mitochondrial movement in neurons is highly diverse and complex. There are stationary and motile mitochondria, which move with different speeds and in different directions

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. Three major groups of proteins are involved in transporting mitochondria in neurons: [START_REF] Bossy-Wetzel | Mutant huntingtin and mitochondrial dysfunction[END_REF] cytoskeletal microtubules and actin microfilaments, (2) molecular motors, and (3) adaptor and scaffolding proteins that link cargos to motors and the cytoskeleton. In neurons, microtubules are likely to be tracks for transport over long distances while actin microfilaments mediate travel over short distances. The molecular motors, kinesins and cytoplasmic dynein, which are ATPases, transport mitochondria toward (+)-and (-)-ends of microtubules, respectively. It remains unclear how the cytoskeletal substrates mediate the bidirectional and dynamic transport of mitochondria.

The association of motor proteins with their cargos is largely mediated by adaptor and scaffolding proteins. For example, Milton and syntabulin are implicated as scaffolding proteins for linking mitochondria with kinesin heavy chain [START_REF] Cai | Syntabulin-mediated anterograde transport of mitochondria along neuronal processes[END_REF][START_REF] Stowers | Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein[END_REF]. Reducing the expression of these proteins leads to abnormal distribution of mitochondria, as expected from disrupted anterograde transport. APLIP1 is another example of a scaffolding protein that may interact with both kinesin and dynein to regulate retrograde transport of mitochondria [START_REF] Horiuchi | APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila[END_REF]. Thus, complex mitochondrial movements are likely mediated by various and dynamic interactions between regulatory proteins, adaptors, motors, and cytoskeletal elements.

Mitochondrial energy impairment in HD

The hypothesis that cellular energy production and metabolism are compromised in HD was originally derived from clinical observations of both peripheral weight loss and central deficits in brain glucose utilization in HD patients [START_REF] Sanberg | Body weight and dietary factors in Huntington's disease patients compared with matched controls[END_REF][START_REF] Kuhl | Cerebral metabolism and atrophy in Huntington's disease determined by 18FDG and computed tomographic scan[END_REF]. Subsequent studies of peripheral tissues from HD patients confirmed the negative effect of mutant htt on the energetic status in various cell types [START_REF] Gu | Mitochondrial defect in Huntington's disease caudate nucleus[END_REF]. Analyses of metabolic enzyme activity in post-mortem HD brain tissue have consistently revealed deficits in enzymes of the mitochondrial TCA cycle and OXPHOS system. In particular, decreased complex II/III activity and complex II (SDH) expression are seen in HD striatum but not cortex or cerebellum [START_REF] Browne | Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia[END_REF][START_REF] Tabrizi | Biochemical abnormalities and excitotoxicity in Huntington's disease brain[END_REF][START_REF] Benchoua | Involvement of mitochondrial complex II defects in neuronal death produced by N-terminus fragment of mutated huntingtin[END_REF].

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Systemic administration of SDH inhibitors 3-nitropropionic acid and malonate in rodents and non-human primates causes striatal pathology and behavioral abnormalities reminiscent of HD [START_REF] Lin | Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases[END_REF]. Activities of several mitochondrial enzyme, including pyruvate dehydrogenase, aconitase, SDH, and cytochrome oxidase, are significantly reduced in brain tissue from HD patients [START_REF] Gu | Mitochondrial defect in Huntington's disease caudate nucleus[END_REF]. Following the generation of genetic animal models of HD, several more specific deficits in mitochondrial function have been consistently observed. ΔΨm, ATP production, Ca 2+ handling, reactive oxygen species (ROS) generation, and apoptotic induction are all altered in various HD models [START_REF] Trushina | Oxidative stress and mitochondrial dysfunction in neurodegenerative diseases[END_REF]. However, the primacy of mitochondrial energetic function in HD pathogenesis remains unclear. Of particular importance is the question of whether mitochondrial function is influenced by the direct effect of mutant htt or via an indirect mechanism (e.g. altered nuclear transcription).

It is known that mutant htt can accumulate in the nucleus to interact with a number of transcription factors, leading to gene transcriptional regulation [START_REF] Li | Huntingtin-protein interactions and the pathogenesis of Huntington's disease[END_REF]. Several recent studies highlight the potential relevance of nuclear mutant htt on mitochondrial function. Mutant htt has been shown to alter transcriptional activity of p53 and PGC-1α, two nuclear factors that are known to indirectly regulate mitochondrial function via their transcriptional activity [START_REF] Bae | p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease[END_REF][START_REF] Cui | Transcriptional repression of PGC-1alpha by mutant huntingtin leads to mitochondrial dysfunction and neurodegeneration[END_REF][START_REF] Weydt | Thermoregulatory and metabolic defects in Huntington's disease transgenic mice implicate PGC-1alpha in Huntington's disease neurodegeneration[END_REF].

In vitro studies indicate that ΔΨm and mitochondrial Ca 2+ regulation are directly impaired by mutant htt and that increased ROS generation is the driving force for these alterations [START_REF] Panov | Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines[END_REF][START_REF] Choo | Mutant huntingtin directly increases susceptibility of mitochondria to the calciuminduced permeability transition and cytochrome c release[END_REF][START_REF] Puranam | Polyglutamine expansion inhibits respiration by increasing reactive oxygen species in isolated mitochondria[END_REF]. Further, these studies demonstrate similar impairments in mitochondria isolated from HD patients or mouse models and normal mitochondria incubated with purified mutant proteins. These studies also suggest that other mitochondrial abnormalities observed in HD patients and model systems could be directly caused by mutant htt.

Mutant htt affects mitochondrial trafficking

There is increased evidence that mutant htt can also affect the trafficking of mitochondria in neurons. Cortical neurons transfected with mutant Htt display reduced

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mitochondrial trafficking specifically to cytoplasmic htt aggregates, and the degree of movement impairment is correlated with the size of aggregates [START_REF] Chang | Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons[END_REF]. However, abnormal mitochondrial motility was also observed in HD striatal neurons in the absence of aggregates [25]. The effects of mutant htt on mitochondrial movement support the early studies that htt plays an essential role in axonal transport in Drosophila [START_REF] Gunawardena | Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila[END_REF] and that polyQ expanded htt inhibits transport in squid axoplasm [START_REF] Szebenyi | Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport[END_REF]. The trafficking function of htt is also indicated by its association with HAP1 [START_REF] Li | A huntingtin-associated protein enriched in brain with implications for pathology[END_REF]. HAP1 is a neuronal protein that is essential for neuronal function and viability, as elimination of HAP1 leads to postnatal death of mice [START_REF] Chan | Targeted disruption of Huntingtin-associated protein-1 (Hap1) results in postnatal death due to depressed feeding behavior[END_REF][START_REF] Li | Lack of huntingtin-associated protein-1 causes neuronal death resembling hypothalamic degeneration in Huntington's disease[END_REF]. HAP1 interacts with microtubule transporters dynactin p150 [31,[START_REF] Engelender | Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin[END_REF] and kinesin light chain [START_REF] Mcguire | Interaction of Huntingtinassociated protein-1 with kinesin light chain: implications in intracellular trafficking in neurons[END_REF]. The complex containing htt and HAP1 may act as a docking platform to modulate vesicular cargo attachment to both dynein/dynactin and kinesin microtubule motors [START_REF] Caviston | Huntingtin facilitates dynein/dynactin-mediated vesicle transport[END_REF]. A HAP1 homolog in Drosophila, Milton, influences mitochondrial distribution in axons through its interaction with the mitochondrial protein Miro [START_REF] Fransson | The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking[END_REF][START_REF] Glater | Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent[END_REF]. Also, htt may directly interact with trafficking proteins and acts as a molecular switch for bidirectional transport in neurons [START_REF] Colin | Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons[END_REF].

Although there is compelling evidence that htt and its associated proteins, such as HAP1, are involved in intracellular trafficking, the mechanisms by which mutant htt affects intracellular organelle trafficking remain to be fully understood. It is likely that an abnormal interaction between mutant htt and HAP1 affects the trafficking of organelles in neurons by disrupting the formation of trafficking complexes and impairs vesicular transport in mammalian cells [START_REF] Gauthier | Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules[END_REF]. Mutant htt may also sequester wild type htt and trafficking proteins to impair neuronal trafficking [25,[START_REF] Gunawardena | Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila[END_REF]. In addition, loss of the normal function of htt can affect vesicular transport in neurons [START_REF] Gunawardena | Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila[END_REF][START_REF] Caviston | Huntingtin facilitates dynein/dynactin-mediated vesicle transport[END_REF].

It is also possible that different htt forms differentially affect intracellular trafficking. It is possible that large htt aggregates can physically block the neuronal trafficking if their size exceeds the narrow region of neuronal processes. However, it remains unclear which form of soluble mutant htt is more harmful to the vesicular transport in neurons. Htt is a large (350

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kDa), predominantly cytoplasmic protein that is a substrate for various proteolytic enzymes.

Proteolytic cleavage of full length mutant htt yields small N-terminal fragments containing the polyQ domain that readily misfold and aggregate in both neuronal nuclei and processes [START_REF] Gutekunst | Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology[END_REF][START_REF] Zhou | Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity[END_REF]. The strong toxic property of proteolytically processed mutant htt is evidenced by the more rapid disease progression of HD mice expressing smaller N-terminal mutant htt fragments than that of mice expressing full length mutant htt [START_REF] Li | Multiple pathways contribute to the pathogenesis of Huntington disease[END_REF]. This phenomenon has led to extensive study of the proteolysis of htt and the identification of a number cleavage sites in the N-terminal region of htt [START_REF] Ellerby | Neurodegenerative disease: cut to the chase[END_REF].

Using a knock-in mouse model of HD, we show that specific N-terminal fragments, likely smaller than the first 500 amino acids, of mutant htt preferentially associate with mitochondria in vivo and that N-terminal mutant htt fragments affect the trafficking of mitochondria [START_REF] Orr | N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking[END_REF]. This biochemical finding supports the recent subcellular localization evidence that the first 17 amino acids of htt are required for localization of exon1 htt to mitochondria [START_REF] Rockabrand | The first 17 amino acids of Huntingtin modulate its sub-cellular localization, aggregation and effects on calcium homeostasis[END_REF]. We also found age-dependent accumulation of mutant htt on mitochondria and that this accumulation directly correlates with disease progression. Finally, we demonstrate that mitochondrial function can be disrupted by soluble N-terminal mutant htt fragments independent of their nuclear accumulation or aggregation [START_REF] Orr | N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking[END_REF]. Our observation of impaired mitochondrial trafficking and decreased ATP level in synaptosomes caused by mutant htt suggests that impaired trafficking of mitochondria in neuronal processes can decrease mitochondrial ATP supply in nerve terminals [START_REF] Wang | Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice[END_REF]. The decreased ATP level can also affect the transport of mitochondria in neuronal processes. Together, these findings suggest that abnormal interaction between mutant htt and mitochondria may represent a cytoplasmic pathological event that can serve as a therapeutic target for HD.

Impaired mitochondrial trafficking and HD pathogenesis

Impaired mitochondrial transport probably has multiple consequences that increase in

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severity with the duration of impaired transport. Poor ATP distribution in nerve terminals is likely to be an initial outcome and can affect the cellular processes that require mitochondria in a rapid and dynamic manner. These processes include specific synaptic sites undergoing morphogenesis or potentiation and are likely to actively recruit mitochondria under normal conditions. Consistent with this idea, there are a reduced synaptic ATP level and impaired activity of ubiquitin-proteasomal system (UPS) in synapses of HD mice [43.45]. It is known that the UPS function is largely dependent on ATP. Synaptic UPS may be more vulnerable to impaired mitochondrial function or deficient ATP supplies in nerve terminals. Similarly, other nerve terminal functions, such as neurotransmitter release and synaptic vesicle biogenesis, could also be affected if mitochondria transport to nerve terminal is reduced by mutant htt. In addition, impairment of mitochondrial movement may impede fusion-mediated mtDNA complementation and autophagic degradation of damaged mitochondria [START_REF] Nakada | Inter-mitochondrial complementation: Mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA[END_REF][START_REF] Ono | Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria[END_REF].

As discussed above, neuronal function is particularly dependent on the intracellular trafficking of organelles and molecules over the long distance of axons or neuronal processes.

The unique anatomic structures of neuronal processes may make neurons more vulnerable to the impaired trafficking caused by mutant htt, which also contributes to the selective neurodegeneration in HD. However, other polyglutamine disease proteins that largely accumulate in the nucleus can also cause selective neurodegeneration via different mechanisms. Because misfolded proteins preferentially accumulates, in an age-dependent manner, in neuronal cells than in other types of cells in all polyQ diseases, neuronal ability to remove misfolded polyQ proteins is likely decreased by aging related factors. Indeed, agedependent decrease of UPS activity is more pronounced in neurons than in glial cells [START_REF] Tydlacka | Differential activities of the ubiquitin-proteasome system in neurons versus glia may account for the preferential accumulation of misfolded proteins in neurons[END_REF]. It is also possible that the clearance of mutant htt or damaged mitochondria by autophagy is decreased with age. Once the cellular capacity to clear mutant polyQ proteins is decreased, misfolded proteins can accumulate in the nucleus to affect gene expression and in the neuronal processes to cause cytoplasmic toxicity. In the case of HD, mutant htt has additional

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adverse effects on intracellular trafficking. Thus, the impaired trafficking of mitochondria in neuronal processes and reduced ATP supply to nerve terminals can lead to specific dysfunction of neurons.

Conclusion:

Recent studies have suggested several mechanisms for mitochondrial dysfunction in HD. These mechanisms can be classified as the indirect effects of mutant htt via transcriptional dysregulation of genes important for mitochondrial biogenesis and the direct effects of mutant htt on mitochondria. Biochemical and cell biology studies have provided strong evidence for the negative impact of mutant htt on mitochondrial movement. Moreover, inhibition of the distribution of mutant htt in neuronal processes by an intracellular antibody can alleviate HD neurological symptoms in HD transgenic mice [START_REF] Wang | Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin[END_REF]. Because neuronal function is largely dependent on intracellular trafficking in lengthy processes and nerve terminals, the impaired intracellular trafficking in HD neurons is likely to contribute to the specific neuropathology in HD. In addition, given that htt is predominantly distributed in the cytoplasm, the cytoplasmic toxicity of mutant htt could play an important role in HD pathology. Understanding how mutant htt affects the transport of mitochondria will help us to find an effective means to reduce the selective neuropathology in HD.

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Acknowledgements:

This work was supported by grants of National Institutes of Health NS036232, NS041669, AG019206 (XJL) and NS045016 (SHL).

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