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Human Molecular Genetics Advance Access originally published online on March 13, 2007
Human Molecular Genetics 2007 16(7):783-797; doi:10.1093/hmg/ddm023
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Extended polyglutamine repeats trigger a feedback loop involving the mitochondrial complex III, the proteasome and huntingtin aggregates

Hirokazu Fukui1 and Carlos T. Moraes1,2,3,*

1 Neuroscience Program, 2 Department of Neurology and 3 Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL 33136, USA

* To whom correspondence should be addressed at: Department of Neurology, University of Miami Miller School of Medicine, 1095 NW 14th Terrace, Miami, FL 33136, USA. Tel: +1 3052435858; Fax: +1 3052433914; Email: cmoraes{at}med.miami.edu

Received November 21, 2006; Revised January 22, 2007; Accepted February 9, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mitochondrial abnormalities represent a major cytopathology in Huntington's disease (HD), a fatal neurodegenerative disease caused by CAG repeat expansions in the gene encoding huntingtin (Htt). In the present study, we investigated whether defects in the mitochondrial respiratory function are consequences of the expression of mutant Htt or they promote the formation of Htt aggregates. To take advantage of existing mitochondrial DNA mutants, we developed human osteosarcoma 143B cells expressing mutant Htt in an inducible manner and found that cells expressing mutant Htt but not wild-type Htt exhibited a reduced activity of complex III and an increased activity of complex IV. Conversely, pharmacological treatments that inhibited complex III activity significantly promoted the formation of Htt aggregates. This complex III-mediated modulation of Htt aggregates was also observed in a neuronal progenitor RN33B cell line transduced by lentivirus carrying mutant Htt. This effect of complex III inhibition on the Htt aggregates appeared to be mediated by the inhibition of proteasome activity, but not by ATP depletion or production of reactive oxygen species. Accordingly, complex III mutant cells also showed decreased proteasome activity. These results suggest the presence of a feedback system connecting the mitochondrial respiratory complex III and the production of Htt aggregates. Our results suggest that therapeutic interventions targeting complex III and/or proteasome could ameliorate the progress of HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny GABAergic neurons in the striatum and the resulting abnormal involuntary movements and cognitive dysfunction (1–3). The incidence of this fatal disease is strictly determined by the inheritance of expanded CAG repeats (≥40) in exon 1 of the huntingtin (htt) gene or IT15 gene (4). The encoded full-length mutant protein comprising more than 3160 amino acids is prone to proteolysis, which involves caspases (5,6), cathepsins (7) and calpains (8,9), and the resulting N-terminal fragments containing expanded polyglutamine (polyQ) repeats tend to aggregate, being the major components of intracellular inclusions found in the affected areas of HD postmortem brains (10). These aggregates are likely to play a key role in the development of HD (11), although this remains controversial (12).

Despite the monogenetic nature of this disease, a recent study on the Venezuelan HD kindreds revealed that significant variability exists in the age of onset even within families and, most strikingly, even among individuals with same length of CAG repeats (13). In transgenic models of HD, it has been shown that environmental enrichment delays the progression and onset of disease (14), suggesting that there are additional genetic and/or environmental factors controlling the onset of HD aside from the length of CAG repeats, and identifying those factors would greatly contribute to extend the lifetime of HD patients with improved quality of life. Those factors may alter the accumulation of misfolded proteins and/or alter the resistance of neurons to the toxicity of mutant Htt.

Our hypothesis was that the decline in the mitochondrial respiratory function, which has been demonstrated to occur in various types of organisms ranging from Drosophila to humans during normal aging processes (15–18), could contribute to the progression of HD possibly by modifying the accumulation of misfolded mutant Htt, accounting for an age-dependent aspect of this disease. Similar contribution of mitochondrial dysfunction to disease onset was suggested for another motor neurodegenerative disease, Parkinson's disease (PD), in which the deficiency in mitochondrial complex I in the substantia nigra would promote the accumulation of protein inclusions (Lewy bodies) consisting of {alpha}-synuclein (19,20). As is the case for other neurodegenerative diseases including Alzheimer's disease (AD) (21,22), PD and amyotrophic lateral sclerosis (ALS) (23), the impairment in the mitochondrial respiratory chain, as well as hypometabolism of glucose, was also found in the brains of HD patients (24–26). Those metabolic abnormalities could play a triggering role in the pathogenesis of HD, supported by the findings that systemic and intrastriatal injections of 3-nitropropionic acid (3-NP), a selective complex II inhibitor, cause the selective degeneration of striatal neurons, producing HD-like symptoms in rats and primates (27–31).

Another possibility that accounts for the mitochondrial dysfunction in HD brains is that those alterations in mitochondrial complex activities are due to the toxic effects of mutant Htt. Several studies attempted to address this question by using animal and cellular models of HD. Milakovic and Johnson (32) observed impaired ATP production and complex I-driven and complex II-driven state 3 respirations in the clonal striatal cells immortalized from mutant htt knock-in mouse embryos carrying polyQ111 mutation. Seong et al. (33), in the same cell line, further confirmed the reduced ATP/ADP ratio, which was associated with the chronic activation of N-methyl-D-aspartate receptors. In these studies, however, no alterations in the respiratory complex activities were identified, possibly due to the chronic adaptation of cells to mutant Htt. Benchoua et al. (34) showed that the introduction of mutant htt into primary striatal neurons by lentivirus impaired the mitochondrial complex II, leading striatal neurons to cell death, stressing the important role of mitochondrial dysfunction in the neurodegenerative process. Although similar dysfunction of complex II has not been reported in the striatum of mouse models of HD (35,36), they also observed the decrease in the expression and the activity of complex II in the postmortem HD brains. Most recently, Solans et al. (37) reproduced the early defects in respiratory function and complex II and III activities in a yeast model of HD, which expresses the N-terminal fragment of Htt in an inducible manner.

To confirm and extend the above studies, we evaluated the mitochondrial toxicity of mutant Htt in human osteosarcoma cells (143B cells) that contained a transgene expressing GFP-tagged N-terminal fragment of pathogenic huntingtin (HttQ103-GFP) in an inducible manner. Although the proliferating nature of this cell line makes it not ideal to evaluate the cellular toxicity of Htt, as implicated from previous studies (38–41), it provides a suitable biochemical environment to study the effect of mutated mtDNA on Htt aggregation. The availability of mutant 143B cells in which the assembly and activity of complex III are impaired due to an out-of-frame mutation in cytochrome b gene (42), as well as a mtDNA-less derivative, allowed us to confirm the effect of respiratory complex defects in the accumulation of Htt aggregates. Furthermore, the inducible nature of the protein in this system allowed us to determine the time frame of such alterations.

The main purpose of this study was to investigate whether (i) the defects in the mitochondrial respiratory functions are caused by the expression of mutant Htt and/or (ii) these defects contribute to the progress of HD by promoting the formation of Htt aggregates. Our findings implicate the presence of a feedback system between mitochondrial complex III dysfunction and the accumulation of mutant Htt with the involvement of the proteasome system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Establishment of 143B cells expressing huntingtin proteins
A GFP-tagged DNA segment encoding exon 1 of human Htt with wild-type 25 polyglutamine repeat (Q25) or with pathogenic Q103 (37,43) was cloned into an inducible vector (44), in which the expression of the insert can be induced by the addition of the synthetic steroid RU486 (also known as mifepristone). Human osteosarcoma 143B cells were stably transfected with either construct, selected under hygromycin, and the inducibility of proteins was confirmed by optical detection of green fluorescence as well as western blotting (Fig. 1). Clones with inducible HttQ25-GFP expressed diffuse green-fluorescence throughout the cell upon induction (Fig. 1A), whereas a subpopulation of clones with inducible HttQ103-GFP gene (~30% of GFP-positive cells) formed protein aggregates visible under the microscope both in the cytosol and in the nucleus (inset of Fig. 1B). Cytosolic Htt aggregates partially colocalized with mitochondria stained by Mitotracker Red, consistent with the previous finding that Htt protein can interact with the outer membrane of the mitochondria (45). However, no major differences were observed in the mitochondrial network morphology after HttQ103-GFP induction. From Day 1 to Day 2 after induction, the proportion of cells having protein aggregates (out of total GFP-positive cells) significantly increased from ~10 to 30% (Fig. 1C). However, it reached a plateau after 2 days of induction: a subpopulation of cells did not form protein aggregates even after a long period of induction (5–6 days) and showed a more diffuse pattern of expression (Fig. 1C). Western blots confirmed that these clones expressed the appropriate sizes of GFP-tagged proteins upon induction with RU486 (Fig. 1D). Expression of proteins was not observed in the absence of RU486, and the expression level of proteins remained the same after 1-day induction in most of our western blots. Although some previous studies detected high molecular-weight Htt aggregates that barely entered the polyacrylamide gel by western blotting (39,46), our western blots did not show the presence of such high molecular weight complexes. We also evaluated whether the expression of mutant Htt affected the cellular growth of 143B cells. In contrast to the early cytotoxicity of mutant Htt in a yeast model of HD (37), evident impairment of cellular growth or cell death was observed in neither the Q103-GFP nor Q25-GFP line after induction (data not shown).


Figure 1
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  		Figure 1. Establishment of 143B cells with inducible Htt proteins. Selected clones, stably transfected with the inducible htt constructs were examined for their abilities to express GFP38 tagged HttQ25 or HttQ103 upon the addition of RU486 (10 nM). (A, B) Each clone was induced for 2–3 days, and inducibility and cellular localization of induced HttQ25-GFP (A) and HttQ103-GFP (B) proteins were examined by an inverted fluorescence microscope and by a confocal microscope. The main panels with lower magnification (10x) show the expression of each GFP-tagged Htt proteins captured after 3-day induction by a fluorescence microscope. The inset in each figure shows more detail cellular localization of GFP-tagged proteins (green) and mitochondria (red) captured by a confocal microscope with 100 x objective. For the confocal analysis, mitochondria were pre-stained with Mitotracker Red for 30 min prior to the examination. (C) The average proportions of aggregate-positive cells (out of total GFP-positive cells) were microscopically quantified after induction in the HttQ103-GFP clone (see Materials and Methods). (D) The inducibility of each protein was further examined by western blotting for these clones at 0, 1, 3 and 5 days after the addition of RU486. GFP-tagged proteins and ß-tubulin were visualized with a rabbit polyclonal anti-GFP antibody and a monoclonal anti ß-tubulin antibody, respectively.

 
Filter retardation assay selectively detects an aggregated form of Htt protein
To quantify the intracellular protein aggregates observed in our cell lines, we employed a filter-retardation assay (47): briefly, SDS-solubilized cell homogenates were filtered through a 0.2 µm cellulose acetate membrane. Then, the retained SDS-insoluble aggregates were immunodetected on the membrane. Different amounts of cell homogenates (6, 18 and 54 µg in protein) from 143B cells with HttQ103-GFP gave rise to dose-dependent signals (Fig. 2A and B). On the other hand, no signal was obtained from cell homogenates from the HttQ25 clone, indicating the discriminative and quantitative nature of this assay for aggregates in our system. Despite the relatively constant level of protein expression in the HttQ103 clone, which was revealed by western blotting, the signals obtained from the filter retardation assay increased with time during the initial period of induction (Fig. 2C).


Figure 2
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  		Figure 2. Quantification of Htt aggregates by filter retardation assays. (A) Three days after induction, 6, 18 and 54 µg of SDS-lysates of the HttQ25-GFP and HttQ103-GFP clones were filtered through cellulose acetate membrane to trap aggregate forms of Htt/GFP fusion protein. The trapped proteins were subsequently detected by a polyclonal anti-GFP antibody. (B) Quantification of signals obtained from each fraction revealed the quantitative and discriminative nature of this assay in our system. (C) The formation of Htt aggregates increased with time in the HttQ103-GFP clone after induction. The signals obtained from filter retardation assays (amount of Htt aggregates) were normalized by the signals from western blotting (expression level of total HttQ103-GFP protein) for 1-day, 2-day and 3-day-induced samples.

 
The expression of HttQ103-GFP inhibits complex III activities
It has been reported that clonal striatal cells with homozygous mutant htt gene exhibit reduced oxygen consumption accompanied by reduced ATP production (32). To confirm this finding, we initially investigated the oxygen consumption of the HttQ25-GFP clone and HttQ103-GFP clone by a polarographic method in the presence or absence of RU486. As shown in Figure 3A, the HttQ103-GFP clone exhibited slightly reduced endogenous oxygen consumption after 3-day induction, whereas the endogenous oxygen consumption of the HttQ25-GFP clone was not altered even after induction of the Htt protein. Noticeably, Complex IV-mediated oxygen consumption driven by the addition of ascorbate and TMPD was not altered in the HttQ103-GFP clone after induction, implicating that the deficiency of the respiratory chain was upstream of Complex IV. This relatively mild defect is not surprising considering that not all cells in the culture showed clear aggregates and that electron transport chain complexes need to be inhibited beyond a threshold for respiration to be impaired. These observations are in accordance with the previous finding that Complex I- and Complex II- driven but not Complex IV-driven State 3 respirations were reduced in the clonal striatal cells with mutant Htt compared to those with wild-type Htt (32).


Figure 3
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  		Figure 3. Analyses of mitochondrial respiratory functions in the HttQ25-GFP and HttQ103-GFP clones. (A) Endogenous and Complex IV-driven oxygen utilization was measured in triplicate for induced (+RU486) and non-induced (–RU486) populations of each clone as well as untransfected 143B cells. There was a tendency for the induction of HttQ103-GFP protein but not HttQ25-GFP to inhibit the endogenous oxygen consumption (P = 0.06). (B–D) Activities of Complex II + III (B), Complex IV (C), Complex II (D) and Complex III (E) were determined spectrophotometrically for total cell homogenates, and normalized by the amounts of protein and the activities of CS, which did not differ among samples (data not shown).

 
To critically evaluate the effects of mutant Htt on the respiratory complexes of the mitochondria, we have measured the enzymatic activities of Complex II, Complex II + III and Complex IV in addition to citrate synthase (CS), a matrix enzyme involved in the Krebs cycle (Fig. 3B–D). The activity of CS did not show significant alteration in both clones upon induction (data not shown), suggesting that the expression of Htt proteins did not affect the amount of mitochondria. On the other hand, the combined activity of Complex II + III normalized to the activity of CS was significantly reduced in the HttQ103-GFP clone after 1-day and 3-day inductions (Fig. 3B). Interestingly, the reduction in the Complex II + III activity was accompanied by an increase in the Complex IV activity in the HttQ103-GFP clone (Fig. 3C), which may reflect the cellular attempt to compensate for the deficiency upstream in the respiratory chain. Since we did not observe alterations in Complex II (Fig. 3D), we wondered whether the reduced activity of Complex III might be responsible for the reduced activity of Complex II + III in the HttQ103-GFP clone. To address this question, we measured the activity of Complex III in newly prepared cell homogenates. This assay revealed that the 3d-induction of HttQ103-GFP but not HttQ25-GFP significantly reduced Complex III activity down to 74% (Fig. 3E). Although it remains undetermined whether the expression of HttQ103-GFP alters the level of ubiquinone that medicates electron transfer between Complex II and Complex III, our data clearly demonstrated that the expression of HttQ103-GFP impairs the activity of Complex III.

The expression of HttQ103-GFP does not have a major effect on the steady-state levels of complex III subunits
To study whether altered expression of mitochondrial proteins underlies the reduction in the Complex III activity, we evaluated the steady-state levels of different mitochondrial proteins that comprise the mitochondrial respiratory chain. Western blots did not show major changes in steady-state levels of Complex III subunits in the HttQ103-GFP clone (Fig. 4). Blue native PAGE followed by western blotting for different respiratory complexes also did not show significant alterations in the levels of assembled Complex III (data not shown).


Figure 4
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  		Figure 4. Immunoblot analysis of respiratory complex subunits. Non-induced and 3-day induced HttQ25-GFP and HttQ103-GFP clones (40 µg in protein) were subjected to 4–20% SDS-PAGE. The steady-state levels of Complex III subunits (ISP, Core 1 and Core 2), Complex II subunit (SDH), and Complex IV subunit (COX I) were immunodetected along with cytochrome c, porin and ß-tubulin. ISP, Rieske Iron-sulfur protein; Cyt c, cytochrome c.

 
Inhibition of complex III increases the formation of Htt aggregates in 143B cells
The second objective of this study was to investigate whether the impairment of mitochondrial function, which could occur during the normal aging process (but is more pronounced in HD patients) could contribute to the accumulation of misfolded Htt protein, thereby contributing to the pathogenesis of HD. To examine whether drug-induced mitochondrial dysfunctions affect the kinetics of aggregate formation, the expression of HttQ103-GFP was induced for 2 days in the presence of rotenone (Rot, Complex I inhibitor), Antimycin A (AA, Complex III inhibitor), and potassium cyanide (KCN, Complex IV inhibitor) as well as a pro-oxidant, tert-butyl hydroperoxide (tBOOH). The concentrations of each drug used in this study were pre-titrated so that each inhibitor completely blocked cellular respiration but did not affect cell viability in a 3-day period. The filter retardation assay for the triplicates for each treatment revealed that AA consistently and significantly increased the aggregate formation (Fig. 5A), whereas rotenone and KCN did not have significant effects. This effect of AA on aggregates was dose-independent (which was in excess for complete complex III inhibition) and did not involve the increase in the steady-state level of SDS-soluble Htt protein (data not shown). Considering the previous findings that AA most potently induces the production of superoxide anion (O2–) (48,49), hydrogen peroxide (H2O2) and hydroxyl radical (OH) (50–52), the above data might indicate the involvement of reactive oxygen species (ROS) in the formation of Htt aggregates. However, neither H2O2 nor tBOOH affected the formation of aggregates (Fig. 5A and data not shown). Moreover, the application of an antioxidant, N-acetyl cysteine (NAC), that indirectly scavenges H2O2 via the production of the reduced form of glutathione (53) did not inhibit AA-mediated increase of aggregate formation (Fig. 5B). An alternative antioxidant, resveratrol, a polyphenol compound extracted from red wine, also failed to reduce the aggregate load (Fig. 5B). In addition, the measurement of ROS production by fluorogenic dye, H2DCF-DA, showed that the application of AA did not increase H2O2 production in our system (Fig. 5C).


Figure 5
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  		Figure 5. The effects of mitochondrial inhibitors, a pro-oxidant, and antioxidants on the formation of Htt aggregates in the HttQ103-GFP clone. (A) The expression of HttQ103-GFP was induced for 2 days in the presence of Rot, AA, KCN and tBOOH, and the formed Htt aggregates were detected and quantified by filter retardation assay. (B) The HttQ103-GFP clone was induced for 1 day in the presence of 40 nM AA and an antioxidant (5 mM NAC, 10 µM resveratrol or 50 µM resveratrol). The effects of those antioxidants on the Htt aggregates were evaluated by the filter retardation assay in triplicate. (C) The production of ROS in the HttQ103-GFP clone was monitored by H2DCF-DA in the presence of tBOOH (a positive control), different concentrations of AA and a combination of AA and NAC (a negative control). (D) The involvement of superoxide anion on the formation of Htt aggregates was evaluated by the addition of MnTBAP, a superoxide scavenger. Concurrently, myxothiazol, another Complex III inhibitor, that has been reported not to induce ROS production in many systems, and lactacystin, a proteasome inhibitor, were tested for their abilities to modulate the formation of Htt aggregates. The expression of HttQ103-GFP was induced for 1 day, and the quantification of aggregates was performed by the filter retardation assay for the triplicate of each group.

 
Since the application of pro-oxidants (H2O2 and tBOOH) and antioxidants (NAC and resveratrol) did not alter the kinetics of aggregate formation, it is unlikely that the increase in the endogenous H2O2 plays a role in the AA-associated increase in Htt aggregates. These observations, however, did not exclude the involvement of O2–. To test if AA-stimulated production of O2– may participate in the increase in the aggregate amount, the expression of HttQ103-GFP was induced in the presence of AA and MnTBAP, a O2– scavenger (54), or MnTBAP alone. Figure 5D shows that MnTBAP did not suppress AA-mediated increase in the formation of Htt aggregates, and MnTBAP alone did not have any effect, further confirming that the effect of AA is not mediated by ROS production. In agreement, the levels of total protein carbonyls (determined by oxyblot) between AA-treated and untreated HttQ103 samples were not significantly different (data not shown).

To study whether this positive effect of AA is through the inhibition of Complex III or another property unique to AA, another Complex III inhibitor, myxothiazol, was tested for its ability to modulate the formation of Htt aggregates in the 143B cells with inducible HttQ103- GFP. AA and myxothiazol inhibit Complex III by binding distinct sites within the Q-cycle of Complex III (Qi and Qo sites, respectively) (55,56). Although AA has been shown to increase the production of O2– in the isolated heart mitochondria (55,57) and liver mitochondria (58), this property was absent for myxothiazol in those systems. As shown in Figure 5D, 1-day incubation with myxothiazol also increased the formation of Htt aggregates as assessed by the filter retardation assay. The effect of myxothiazol on aggregate formation was even more pronounced than the effect of AA. Therefore, the inhibition of Complex III but not another unique property of AA would be responsible for the increase in the Htt aggregates.

Inhibition of complex III increases the formation of Htt aggregates in RN33B cells
To evaluate the modulation of Htt aggregates by Complex III inhibitors in a more appropriate cellular context, we generated lentivirus carrying wild-type HttQ25-GFP or pathogenic HttQ103-GFP and transduced neuronal progenitor cells (RN33B cells) established from rat raphe nuclei (59). 4 days after infection, RN33B cells transduced with HttQ103-GFP virus but not HttQ25-GFP formed microscopically visible Htt aggregates (Fig. 6A and B). A western blot confirmed the expression of appropriate sizes of GFP-tagged proteins in those populations (data not shown), and a filter retardation assay successfully detected Htt aggregates from RN33B cells expressing HttQ103-GFP but not HttQ25-GFP (data not shown). To evaluate the effects of respiratory inhibitors on Htt aggregates in this system, the cells transduced with HttQ103-GFP virus were expanded, and treated with Rot, AA and KCN. The alterations in the amount of Htt aggregates were evaluated by filter retardation assay. Consistent with the results in 143B cells with inducible HttQ103-GFP, AA selectively increased the amount of Htt aggregates (Fig. 6C). Therefore, the modulation of Htt aggregates by Complex III is not specific to 143B cells but also applicable to RN33B cells.


Figure 6
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  		Figure 6. The effects of mitochondrial inhibitors on the formation of Htt aggregates in RN33B cells stably expressing HttQ103-GFP. (A, B) The expression of HttQ25-GFP (A) and HttQ103-GFP (B) was microscopically assessed in lentivirally transduced RN33B cells 4 days after infection. (C) The effects of mitochondrial inhibitors (Rot, AA and KCN) on Htt aggregates were evaluated by filter retardation assay in RN33B cells transduced with lentivirus carrying HttQ103-GFP at the amount of MOI50 ~1 month after infection. Non-treated and treated samples were analyzed in triplicate, and the average amounts of Htt aggregates (arbitrary unit) were compared among different treatments.

 
Drug-induced complex III inhibition impairs the activity of proteasomes without altering its assembly
Previously, age-dependent inhibition of ubiquitin-proteasome system (UPS) has been proposed to be a possible mechanism underlying the age-dependent increase in the formation of Htt aggregates (60). Indeed, in many experimental systems including our system, the application of a proteasome inhibitor, lactacystin, increased the amount of Htt aggregates (61,62). Furthermore, the proteasome activity has been found to be reduced in multiple areas of the brain and skin fibroblasts from HD patients (63). To study if AA-mediated increase of Htt aggregation involved the inhibition of proteasomes, we examined proteasome activities of non-induced HttQ103-GFP clone treated with AA or other drugs used in the above experiments by using a fluorogenic peptide, Suc-LLVY-AMC, as a proteasome substrate (61,64). This experiment revealed that AA, but not other respiratory inhibitors, significantly inhibited proteasome activity (Fig. 7A). Furthermore, the application of antioxidants, NAC and resveratrol had no effects on the AA-mediated inhibition of proteasomes, and two different concentrations of tBOOH did not have significant effects on proteasome activity (Fig. 7B). A similar, but more robust trend was observed for the HttQ103-GFP clone induced for 48 h in the presence of RU486 (Fig. 7C): AA consistently inhibited the proteasome activity, and this inhibition was not rescued by the application of NAC. Interestingly, the AA inhibition of proteasome activity was more pronounced in this induced condition than in the non-induced condition (Fig. 7A and B), which may reflect the intensification of proteasome inhibition by induced Htt aggregates as discussed below (Fig. 8). These observations implicate the proteasome in the AA-mediated increase of Htt aggregation by an unknown mechanism.


Figure 7
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  		Figure 7. The measurement of proteasome activities in the presence of mitochondrial inhibitors and antioxidants. (A) Mitochondrial inhibitors (Rot, AA and KCN) were evaluated for their effect in the chymotrypsin-like activity of proteasomes in the non-induced HttQ103-GFP clone by using a fluorogenic substrate, Suc-LLVY-AMC. Darker bars in all panels indicate Complex III deficient samples. (B) Antioxidants (NAC and resveratrol) and a pro-oxidant (tBOOH) as well as AA were evaluated for their effect on proteasome activity in the non-induced HttQ103-GFP clone. (C) The effects of AA and NAC on the chymotrypsin-like activity of proteasomes were evaluated in the HttQ103 clone induced for 2 days. The effect of ethanol, the solvent of AA, was also evaluated in this experiment. Lactacystin was used as a negative control. (D) The effects of myxothiazol and MnTBAP were evaluated by the same method to study the involvement of superoxide anion on the proteasome activity in the non-induced HttQ103-GFP clone. (E) Cell extracts prepared from the HttQ103-GFP clone untreated or treated with AA for 24 h were separated by non-denaturing PAGE, transferred to PVDF membrane and blotted against {alpha}4 subunit of proteasome. Samples were analyzed in triplicate. (F) Proteasome activity was measured in the 4.1 clone carrying wild-type human mitochondria, the {Delta}4-cyt b 3.E clone carrying mutant mitochondria expressing a truncated (not functional) cytochrome b, and the 206-{rho}0 cells lacking mtDNA. All bars represent the average of fluorescence signals obtained from at least three measurements 45 min after the initiation of proteasome reactions.

 


Figure 8
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  		Figure 8. A model for the feedback loop enhancing the formation of Htt aggregates. Polyglutamine oligomers or aggregates affect the mitochondrial respiratory Complex III through an unknown mechanism. Our data also showed that the Complex III defect impairs proteasome function, which leads to further formation of expanded Htt aggregates. The latter also have been shown to impair the proteasomes (76,77).

 
To confirm that drug-induced Complex III inhibition leads to the impairment of proteasome function, we treated the non-induced HttQ103 clone with myxothiazol prior to the measurement of proteasome function (Fig. 7D). As expected, myxothiazol significantly inhibited the proteasome activity comparably to AA, providing further evidence that the inhibition of Complex III reduces the proteasome activity. As seen for the formation of Htt aggregates, the application of MnTBAP failed to inhibit the AA-mediated inhibition of proteasome activity (Fig. 7D), further supporting that the inhibition of proteasome is not mediated by ROS.

To investigate whether this inhibition of proteasome activity is mediated by the impairment of proteasome assembly, the levels of 26S proteasome holocomplex were analyzed using non-denaturing PAGE in the HttQ103-GFP clone untreated or treated with AA for 24 h. This experiment showed no marked alteration in the level of the assembled proteasome in AA-treated cells (Fig. 7E). Therefore, the inhibition of Complex III impairs the proteasome activity without affecting the assembly of proteasome complex.

Genetic inactivation of complex III impairs proteasome function
To further confirm the relationship between Complex III function and proteasome activity, we utilized mutant 143B cells with impaired Complex III assembly and activity. mtDNA-less 143B {rho}0 cells (206-{rho}0 cells) were repopulated with either the mitochondria harboring a 4 bp-deletion in mtDNA-encoded cytochrome b gene ({Delta}4-cyt b mutation) or wild-type mtDNA, both of which were obtained from a patient with parkinsonism (42). The former cell line ({Delta}4-cyt b 3.E clone) had a specific impairment in Complex III function, whereas the latter cell line (4.1 clone) had normal Complex III function, comparable to the wild-type 143B cells. The measurement of proteasome activity in those cells revealed that {Delta}4-cyt b 3.E clone and 206-{rho}0 cells had 29.5 and 48.8% less activity than 4.1 clone, respectively (Fig. 7F). Since respiratory-deficient mutant cells maintain their intracellular ATP levels by glycolysis in the presence of uridine and pyruvate in vitro (65), the observed difference in the proteasome activity in the above three cell lines would not be due to the difference in ATP levels, but rather would be attributable to the deficiency of Complex III in the 3.E clone and a deficiency of Complexes I, III, IV and V in the mtDNA-less 206-{rho}0 cells.

Collectively, the above findings suggest that Complex III inhibition leads to proteasome dysfunction, which, in turn, may contribute to the accumulation of misfolded Htt aggregates, thereby accelerating the progression of HD.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Mitochondrial dysfunctions have been associated with major neurodegenerative diseases including the AD, PD, HD and ALS. In PD, mitochondrial dysfunction, especially Complex I inhibition, appears to participate in the pathogenesis (19,20). Rodents and primates exposed to Complex I inhibitors exhibit selective dopaminergic neurons in the substantia nigra, resulting in irreversible Parkinsonism. Several mutations in the mitochondrial proteins encoded by mitochondrial and nuclear DNA were associated with familial and idiopathic forms of PD. The compelling evidence suggesting that mitochondrial dysfunction can be a primary instigator of PD comes from the findings that the inhibition of Complex I leads to the formation of {alpha}-synucleinpositive cytoplasmic inclusions in animal and cellular models of PD (66–68). Since the overexpression of {alpha}-synuclein also causes parkinsonism accompanied by the degeneration of nigrostriatal pathway, the dysfunction of Complex I is most likely contributing to the PD by modulating the accumulation of misfolded proteins possibly through the inhibition of proteasome (69,70).

Unlike PD and other major neurodegenerative diseases, polyglutamine diseases represented by HD are autosomal dominant neurodegenerative diseases. The individuals that inherit the mutant htt gene containing 40 or more CAG repeats invariably suffer from the devastating motor and cognitive dysfunctions, and initially it appeared no other factors modified the onset of HD. A recent study performed on Venezuelan HD kindreds over 23 years, however, shed light onto the possibility that there are other genetic or environmental factors that modulate the onset of HD (13).

Although the predominant hypotheses explaining the abnormalities in the mitochondrial respiratory chain found in the postmortem HD patients state that they are directly or indirectly caused by the expression of toxic mutant Htt (32–34,37,71), we wondered whether those mitochondrial dysfunctions could modulate the accumulation of mutant Htt, as demonstrated for the accumulation of {alpha}-synuclein-positive inclusions.

In the present study, we established a cellular model of HD, in which the expression of HttQ25-GFP or HttQ103-GFP is induced by the addition of RU486 in human osteosarcoma 143B cells. We chose to use these non-neural cells for two reasons: (a) the availability of mtDNA mutants and (b) the possibility of developing an inducible system. The toxicity of Htt does vary depending on the cell type, and therefore we did not focus this study on the toxic effects of Htt, but rather on the interplay between extended Htt, aggregates and proteasome function.

Our initial evaluations of different mitochondrial inhibitors, pro-oxidants and antioxidants on the formation of Htt aggregates revealed that Complex III inhibitors, AA and myxothiazol selectively promoted the accumulation of Htt aggregates. Because other respiratory inhibitors did not have significant effects on the formation of Htt aggregates, the effects of Complex III inhibitors cannot be simply explained by the depletion of ATP. Our data also showed that this phenomenon is not mediated by ROS. These observations are in agreement with a recent finding that overexpression of superoxide dismutase 1 (SOD1) does not improve the disease progression and life expectancy in a mouse model of HD (72). A hint explaining the effects of Complex III inhibitors on the Htt aggregates came from the findings that Complex III inhibitors but not other respiratory inhibitors impaired the chymotrypsin-like activity of proteasomes in a ROS-independent manner. This defect in activity was not accompanied by a decrease in the steady-state levels of the 26S proteasome. Furthermore, the 143B cells with Complex III deficiency also had reduced the proteasome activity.

Since the application of a proteasome inhibitor, lactacystin, also resulted in the increase in the aggregate load in our system, we suggest that the effects of Complex III inhibitors on the Htt aggregates are mediated by the inhibition of proteasomes. However, at this point, it is not clear how Complex III inhibition specifically affects the proteasome function in our system. It is not likely that the two distinct Complex III inhibitors tested, AA and myxothiazol, affect the proteasome function directly. There may exist a Complex III-specific downstream signaling pathway that affects the proteasome function. It is noteworthy that a Complex I inhibitor has also been shown to reduce the proteasome function in the rat dopaminergic neurons isolated from the ventral mesencephalon (69). The effects of mitochondrial inhibitors on the proteasomes could be cell-type specific, and it would be intriguing to evaluate the differential effects of mitochondrial inhibitors on the proteasomes in the primary striatal neurons and other types of neurons. In any case, our results provide evidence that the impairment of Complex III found in HD patients may participate in the accumulation of misfolded Htt proteins.

The second objective of this study was to determine whether mitochondrial dysfunctions found in HD patients are triggered by the expression of mutant Htt. Our assessment of respiratory activities showed that 143B cells with HttQ103-GFP exhibit slightly reduced total respiration after 3-day induction, which was accompanied by significantly reduced Complex II + III activities but increased Complex IV activity. The increased Complex IV activity would most likely reflect a cellular attempt to compensate for the deficiency of Complex III. The Complex III defect is probably related to a reduced activity rather than reduced enzyme levels, as we could not detect significant changes in Complex III holoenzyme or subunits levels. At a glance, it appears that this result conflicts with the clinical observation that Complex IV activity was reduced in the postmortem brains of HD patients (24,25). This discrepancy can be explained by the duration of exposure to mutant Htt; during the initial period of exposure, cells may attempt to upregulate Complex IV activity in response to Complex III dysfunction. However, chronic Complex III dysfunction may result in the collapse of oxidative phosphorylation system and eventual degeneration of mitochondrial membranes. Although Benchoua et al. observed the decrease in Complex II activity was accompanied by the downregulation of subunits of Complex II in the striatal neurons 6 weeks after the infection of lentivirus expressing mutant Htt (34), we have not observed the same deficit in our HttQ103- GFP clone 3 days after induction. This difference could be attributable to the durations of expression or cell types used.

What would be the underlying mechanism explaining the mitochondrial toxicity of mutant Htt? Interesting findings came from Panov et al. (73) who first demonstrated, by electron microscope, that mutant Htt can localize to mitochondria in brain of a HD transgenic model. Choo et al. (45) further showed that both wild type and mutant Htt are associated with the outer membrane of mitochondria isolated from wild type and mutant mouse striatal cells, respectively. The direct association of mutant Htt with the mitochondria could affect the respiratory complex activities of mitochondria (37). However, a recent study that evaluated the direct effects of purified mutant Htt on the mitochondrial respiratory chain of the isolated mitochondria failed to demonstrate this effect (71). Another possibility involves mutant Htt disrupting the proper intracellular arrangement of mitochondria by blocking normal intracellular trafficking of mitochondria (74). It has been shown that, in skeletal muscles of desmin-null mice, proper positioning of mitochondria inside the cells is critical for their respiratory activity (75). Recently, Solans et al. (37) proposed that misfolded or aggregated Htt can disturb the network of actin cytoskeleton, which in turn leads to the alteration of mitochondrial distribution and function. Further investigations will be required to clarify molecular pathways leading to respiratory deficits in HD.

In conclusion, we demonstrated that the expression of mutant Htt selectively impairs the Complex III activity. The deficits in the Complex III, in turn, promote the accumulation of Htt aggregates through the inhibition of proteasome activity (Fig. 8). Another feedback loop between aggregated/misfolded Htt and the UPS may also contribute to the enhancement of crosstalk between the mitochondria and mutant Htt (60,76,77). The presence of such feedback systems involving mitochondrial Complex III, the proteasome and misfolded/aggregated Htt implicates that augmenting the mitochondrial respiratory function, especially, Complex III activity, could slow down the progression of HD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell lines
Human osteosarcoma 143B cells obtained form ATCC were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 µg/ml sodium pyruvate, 50 µg/ml uridine and 20 µg/ml gentamycin. Mitochondrial DNA (mtDNA)-less {rho}0 osteosarcoma cells (143B/206-{rho}0 cells), those repopulated with the mitochondria harboring 4 bp cytochrome b gene deletion ({Delta}4-cyt b 3.E clone), and those repopulated with wild-type human mitochondria (4.1 clone) were reported previously (42), and were maintained in the above DMEM. Neuronal progenitor RN33B cells established from rat raphe nuclei (59) were kindly provided by Dr Eaton (University of Miami) and maintained at 33°C in DMEM/F12 (1:1) medium containing 10% fetal bovine serum and 100 units/ml penicillin-streptomycin. Human kidney 293T cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum, 2 mM L-Glutamine and 100 units/ml penicillin-streptomycin in the Viral Core facility (University of Miami).

Establishment of cell lines with inducible Htt
The exon 1 of wild type and pathogenic human Htt with GFP tagged at its 3'end and with FLAG tagged at its 5'end (HttQ25-GFP and HttQ103-GFP) was kindly provided by Dr Barrientos (University of Miami) (37,43), and was subcloned into an inducible vector previously described (44). Osteosarcoma 143B cells were transfected with 1 µg of either construct using FuGENE 6 Reagent (Roche Applied Science, Indianapolis, IN) in a six-well plate. Transfected cells were transferred to 10-cm culture dishes 2 days after transfection. The stably transfected clones were selected in the presence of 400 µg/ml hygromycin, isolated by cloning rings and maintained in the presence of 100 µg/ml hygromycin. The induction of the Htt proteins was achieved by adding 10 ng/ml of RU486 (Invitrogen-Molecular Probes, Carlsbad, CA) into the culture media. The inducibility of the Htt proteins was assessed by fluorescence microscopy and western blotting as below. All experiments were performed on representative HttQ25-GFP and HttQ103-GFP clones with tight control and high expression of Htt proteins.

Growth curves
To determine the rate of cell growth of the HttQ25-GFP and HttQ103-GFP clones in the presence and absence of RU486, 10 x 103 cells were plated in triplicates on 24-well plates. Cells were trypsinized and counted every 24 h on a Z1 Coulter Cell Counter (Beckman Coulter, Fullerton, CA) over 6 days. Medium (high-glucose DMEM with 400 µg/ml Hygromycin) was replaced every other day during the experiment.

Microscopic analysis of Htt expression
The selected hygromycin-resistant clones were induced for 2 or 3 days by adding RU486 in a culture medium, and inducibility and expression levels of Htt proteins were examined in culture plates by an Axiovert 200M inverted fluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with 10 x and 20 x objective lens. The average proportion of aggregate-positive cells out of total GFP-positive cells was quantified from randomly selected 10 microscopic fields 1-day, 2-days, 3-days and 5-days after induction of HttQ103-GFP protein in 143B cells using the fluorescence microscope. For confocal examination cells were preloaded with 200 nM of Mitotracker Red (Invitrogen-Molecular Probes) for 30 min. Then, cells were washed with phosphate-buffered saline (PBS) twice, mounted on glass slides using Prolong Antifade kit (Invitrogen-Molecular Probes), and subjected to microscopic examination by a Carl Zeiss Laser Scanning Microscope 510. Colocalization of GFP and Mitotracker signals was subsequently analyzed with the LSM software (Carl Zeiss, Thornwood, NY).

Generation of recombinant lentiviral constructs and transduction of RN33B cells
The above HttQ25-GFP and HttQ103-GFP were cloned into p156RRLsinPPThCMVMCSpre vector for lentiviral production. Plasmids were transfected into human kidney 293T cells, and viral particles were produced as described previously (78). For the transduction of RN33B cells with lentivirus carrying HttQ103-GFP or HttQ25-GFP, RN33B cells were plated at 5 x 104 cells/well in 24-well-plates 1 day before infection. Each type of lentivirus was applied to RN33B cells at multiplicity of infection (MOI, the ratio of T.U. to a number of cells) of 50, 100, 200 and 400. Four days after infection, the expression of GFP-tagged proteins were microscopically examined as above. The MOI of 50 was used to evaluate the effect of OXPHOS inhibitors on Htt aggregation.

Western blotting analysis
Cells were harvested after trypsinization, washed with PBS twice and cell pellets were stored in –80°C until use. Cell pellets were homogenized in PBS containing 1 x complete protease inhibitor cocktail (Roche) by using a pellet pestle® homogenizer (Kimble-Kontes, Vineland, NJ). The protein content was determined by a BioRad protein assay reagent (BioRad, Hercules, CA), and 40 µg of total protein was separated by 4–20% gradient SDS-PAGE. Proteins were transferred to Protran BA83 nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and probed with anti-GFP (1:10,000) (BD Clontech, Palo Alto, CA), anti-ß-tubulin (1:1000) (Sigma, Saint Louis, MO), anti-cytochrome c (1:5000) (BD Pharmigen, San Diego, CA), anti-succinate dehydrogenase (SDH) (1:1000), anti-COX I (1:1000), anti-Core1 (1:1000), anti-Core2 (1:1000), anti-ISP (1:1000) and anti-porin (1:1000) antibodies (Invitrogen-Molecular Probes). The probing of membranes with those primary antibodies was followed by the incubation with appropriate secondary antibodies conjugated with either IRDye 680 or 800 (1:3000) (Rockland, Gilbertsville, PA), which were subsequently detected by the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). The quantification of protein signals was performed by using the Odyssey software provided by LI-COR. Western blot of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) was performed to estimate the levels of respiratory holocomplexes as described (79).

Filter retardation assay
The HttQ103-GFP clone was incubated for 1–3 days in the presence of different mitochondrial inhibitors, pro-oxidants or/and antioxidants. Unless otherwise stated, the following concentration of each drug was used: Rotenone (100 nM), Antimycin A (40 nM), Myxothiazol (1 µM), Potassium cyanide (0.5 mM), tBOOH (0.25 mM) and hydrogen peroxide (0.5 mM), NAC (5 mM, pH 7.2), resveratrol (10 µM and 50 µM), MnTBAP (Manganese 5,10,15, 20-tetrakis (4-benzoic acid) porphyrin, 50 µM and 100 µM), and Lactacystin (10 µM). Lactacystin was applied for 8 h before harvesting cells due to the cytotoxicity of this drug.

Filter retardation assay to quantify the amount of SDS-insoluble protein aggregates was performed following the method described by Scherzinger et al. (47) with minor modifications. Unless otherwise indicated, 18 µg of total cell homogenate was solubilized in 200 µl of PBS containing 2% SDS and 1% ß-mercaptoethanol, boiled for 3 min and filtered through cellulose acetate membrane (Schleicher and Schuell, 0.2 µM pore size) by using Bio-Dot SF microfiltration apparatus (BioRad) with weak vacuum. Each well was washed twice with PBS. The membrane was blocked with Odyssey blocking solution (LI-COR), and the retained SDS insoluble aggregates were detected by a polyclonal anti-GFP antibody (1:10 000). The detection and quantification of signals were performed with the Odyssey system as described above.

Assessment of proteasome activity
The chymotrypsin-like activity of proteasomes from 143B cells treated with different drugs was measured by monitoring the cleavage of a fluorogenic substrate, Suc-Leu-Leu-Val-Tyr-AMC (Bachem Bioscience, King of Prussia, PA), following the protocol provided by Chemicon (Temecula, CA) with minor modifications. Briefly, 5 x 104 or 10 x 104 cells were incubated in PBS containing a drug indicated in each figure for 3 h at 37°C. The enzymatic reaction by proteasomes was initiated by adding 10 x assay buffer (250 mM HEPES pH 7.5, 5 mM EDTA, 0.5% NP-40, 0.01% SDS at final concentrations) and 50 µM of the proteasome substrate. Forty-five minutes after the addition of the substrate, fluorescence originating from released AMC was measured at 37°C in a Wallac Victor2 1420 Multilabel counter (Perkin-Elmer Life Sciences, Boston, MA) with excitation and emission wavelengths of 355 and 460 nM. For negative controls, cells were incubated with 10 µM lactacystin (Cayman Chemical, Ann Arbor, MI), a proteasome inhibitor.

For the measurement of proteasome activities from clones 4.1, {Delta}4-cyt b 3.E and 206-{rho}0, cells were homogenized in PBS containing 1 x complete protease inhibitor cocktail (Roche). Twenty-four microgram of protein, estimated by the BioRad protein assay reagent, was re-suspended in the above 1x assay buffer, and its proteasome activity was determined as above by using the same fluorogenic substrate. As a negative control, the clone 4.1 was pre-incubated with 10 µM lactacystin for 15 min at 37°C before the addition of the assay buffer and the fluorogenic substrate.

Detection of proteasome holocomplex by non-denaturing PAGE
The HttQ103-GFP clone untreated or treated with AA for 24 h was solubilized for 15 min on ice in a buffer containing 25 mm Tris-HCl (pH 7.5), 0.2% NP-40, 100 mM NaCl and 20% Glycerol. Following the centrifugation at 20 000 g for 30 min at 4°C, supernatant was transferred to a clear tube and the protein content was determined using BioRad Dc protein assay kit. Two hundred microgram of total protein was resuspended in a loading buffer containing 0.18 M Tris-borate (pH 8.3), 10% Glycerol, 1% 2-mercaptoethanol and 0.04% xylene cyanol, and were separated by a native non-denaturing PAGE as described previously (80,81) with modifications. Separating gels containing 4.5% acrylamide-bisacrylamide (37:1), 1 x running buffer (see below) and 3% RhinohideTM polyacrylamide strengthener (Invitrogen-Molecular Probes), were polymerized with 0.1% N,N,N',N'-tetramethylethylenediamine (TEMED) and 0.1% ammonium persulfate. Non-denaturing gels were run at 80 V for 30 min and then at 100 V for additional 4.5 h at 4°C in a running buffer containing 0.18 M Tris-borate (pH 8.3) and 5 mM MgCl2. Separated proteins were transferred to PVDF overnight at 30 V at 4°C in a transferring buffer containing 50 mm Tris-borate, 10% methanol and 0.05% SDS (pH 8.5). Transferred proteasome holocomplex was detected using an antibody against {alpha}4 subunit of proteasome (1:1000) (BIOMOL, Polymouth Meeting, PA) following the procedure of western blotting described above.

Assessment of oxidative stress
The production of ROS in the HttQ103-GFP clone was measured in the presence of tBOOH, AA or AA + NAC, by using a cell permeable fluorogenic dye, H2DCF-DA (Invitrogen-Molecular Probes), following the protocol described by Esposti and McLennan (82). In brief, 50 x 103 cells were resuspended into 100 µl of PBS. The cells were pre-incubated with 10 µM of H2DCF-DA and other drugs tested for 10 min in dark prior to the activation of reaction by 20 mM glucose. The fluorescence originating from an oxidized product, DCF, was detected at 37°C by the Wallac Victor2 1420 Multilabel counter with excitation and emission wavelengths of 485 and 520 nm, respectively.

Protein carbonyls were determined by the Oxyblot kit (Chemicon), as instructed by the manufacturer.

Analysis of mitochondrial respiratory functions
Polarographic studies measuring endogenous oxygen consumption were performed for the 143B cells, the HttQ25-GFP clone and the HttQ103-GFP clone untreated or pretreated with RU486 for 3 days (83). Cells (1.5 x 106) were resuspended in 0.5 ml of respiration medium (20 mM HEPES, 250 mM Sucrose, 10 mM MgCl2, 2 mM potassium phosphate, pH 7.1), and their endogenous oxygen consumption was measured with a Clark oxygen electrode in a micro water-jacketed cell, magnetically stirred, at 37°C (Hansatech Instruments Limited, Norfolk, UK). Endogenous oxygen consumption was inhibited by Antimycin A (10 nM). Subsequently, Complex IV-driven oxygen consumption was obtained by adding ascorbate (10 mM) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (0.2 mM), which was later inhibited by KCN (1 mM).

The activities of Complex II, Complex II + III and Complex IV as well as CS were measured spectrophotometrically essentially following the protocols provided by Barrientos et al. (83). The activity of Complex III was measured spectrophotometrically using reduced Coenzyme Q2 (CoQ2-H2) as an electron donor and cytochrome c as an electron acceptor as described (84) with modifications. Harvested cells were homogenized in PBS containing 1 x complete protease inhibitor cocktail as above, and an appropriate dilution of cell homogenates was applied to assay mixture containing 0.25 M sucrose, 1 mM EDTA (pH 7.4), 50 mM Tris-HCl (pH.7.4), 2 mM KCN, 10 µM rotenone, 0.04% (v/v) Tween-20, 0.02% (v/v) Triton-X and 50 µM cytochrome c. The reaction was initiated by adding 50 µM CoQ2-H2 at 30°C, and the linear increase in absorbance at 550 nM, which reflects reduction of cytochrome c, was monitored over a few minutes spectrophotometrically. The Complex III-specific reduction of cytochrome c was terminated by the addition of 400 nM stigmatellin, which blocks electron transfer from reduced ubiquinol to iron sulfur protein at the redox center of Complex III (55). The activity of Complex III was calculated as previously using an extinction coefficient of 19.1 mM–1 cm–1 (84). The reduction of CoQ2 to CoQ2-H2 was performed essentially as described (84), and the concentration of purified CoQ2-H2 was determined by scanning absorbance at 288–320 nm and by using an extinction coefficient of 4.14 mM–1 cm–1.

Statistical analysis
Statistical significance was determined for differences between a control group and an experimental group with two-tailed, unpaired Student t-test. ** indicates P < 0.01, and * indicates P < 0.05. The error bars in each figure represent standard deviations.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Antoni Barrientos for reagents and insightful discussions and to Dr Mary Eaton for the RN33B cells. This work was supported by grants from the PHS (R01- NS41777) and the University of Miami Neuroscience Center (to C.T.M.). H.F. was supported by Lois Pope LIFE Fellowship.

Conflict of Interest statement. The authors declare no conflict of interest.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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