There are several hypothetical mechanisms that could underlie striatal cell death in HD. Main hypotheses include abnormal transcription (Sugars and Rubinsztein, 2003
), increased transglutaminase activity (Lesort et al., 2002
), early axonal transport dysfunction (Li and Li, 2004
), and disruption of Ca2+
homeostasis (Bezprozvanny and Hayden, 2004
). Protein misfolding, reduction of autophagy, and proteasome dysfunction play important roles in HD (Meriin and Sherman, 2005
). In addition, mitochondrial dysfunction could also contribute to neurodegeneration (Brouillet et al., 2005
). However, there existed no direct evidence that mitochondrial change could play a causal role in HD.
In the present study, we hypothesized that an early decrease in the levels of one or more of the four subunits constituting complex II/SDH could, at least in part, be responsible for mitochondrial dysfunction in HD. Taking advantage of a newly developed model of progressive striatal degeneration in primary cultures (Zala et al., 2005
), we examined this hypothesis using biochemical and cytological methods.
Our results show that expressing the N-terminus part of Htt with a pathological polyglutamine expansion in cultured striatal primary neurons reduced the levels of complex II/SDH subunits Ip and Fp and the dehydrogenase activity of the complex. We also found similar molecular defects in complex II/SDH in the HD striatum. No major changes in the expression of SDH subunits were found in the cerebral cortex and cerebellum, two regions less vulnerable to degeneration in HD patients. Our data are consistent with the reduced activity of complex II-III in HD patients reported in the literature. A decrease in succinate oxidation ranging from 39 to 59% has been observed in the caudate nucleus of HD patients (Butterworth et al., 1985
; Brennan et al., 1985
; Mann et al., 1990
; Gu et al., 1996
; Browne et al., 1997
; Tabrizi et al., 1999
). In the putamen, succinate oxidation showed an average 69% decrease (Browne et al., 1997
). In line with this, striatal cell lines expressing the full-length Htt with 111 polyglutamine (Knock-in 111) were found highly vulnerable to the complex II inhibitor 3NP (Ruan et al., 2004
). In the present study, neither striatal cells expressing Htt171-82Q nor the HD striatum showed profound alterations in α-subunit of complex V, cytochrome c, and BclXL. Levels of subunit IV of cytochrome oxidase (complex IV) and calbindin were slightly reduced in III/II HD patients, consistent with striatal degeneration. However, depletion of these proteins appeared less pronounced than that of SDH subunits. In cell expressing Htt171-82Q at 6 wk postinfection, we found no modification of subunit IV of cytochrome oxidase (complex IV). These observations support the view that HD may be preferentially associated with complex II defects. Our results obtained in cell culture also suggest that the effect of the short fragment of mutated Htt is likely not a nonspecific toxic effect but has some relevance to the human disorder. In addition, SDH/complex II alterations are probably not simply the consequence of neuronal loss because the severity of reduction of the complex is disproportionate in comparison with the extent of cell degeneration (~20% range; see present data and Zala et al., 2005
The mechanisms that could underlie the loss of complex II/SDH in our in vitro model have not been elucidated. Interestingly, the present results indicate that the short N-terminus fragment of mutated Htt alone is sufficient to produce complex II/SDH defects. This is in agreement with the hypothesis that, at least in part, N-terminus fragments of mutated Htt generated from the cleavage of the full-length protein contribute to the neurotoxicity caused by the mutation (Li and Li, 2004
; Wellington et al., 2002
). The present study shows that although protein levels and activity of complex II/SDH were markedly altered, the levels of mRNA of subunits were not decreased, suggesting the involvement of posttranscriptional regulation. Our results suggest that the loss of Ip protein precedes the loss of Fp protein. In human, mutations in any of the SDH subunits cause the complex II to fully disassemble (Rustin et al., 2002
). Thus, it is possible that short fragments of mutated Htt produce their effect preferentially on the Ip subunit, triggering destabilization of the entire complex, and in turn the loss of the Fp protein. Given that mutated Htt is located in the cytoplasm and can bind the outer mitochondrial membrane (Panov et al., 2002
; Choo et al., 2004
), many speculative mechanisms can be proposed to explain how mutated Htt reduces posttranscriptional Ip levels. For instance, mutated Htt could induce a decrease in the import of Ip into the mitochondria, an increase in degradation, or an abnormal assembly. One interesting mechanism may be related to p53. A role for the accumulation of p53 in mitochondrial abnormalities and degeneration in HD has been recently demonstrated (Bae et al., 2005
). The accumulation of p53 could possibly result from proteasome dysfunction induced by short fragments of mutated Htt (Jana et al., 2001
; Waelter et al., 2001
). Apart from its role as a transcription factor, p53 interacts in the cytosol with proteins (e.g., Bax and BclXL) involved in regulation of the mitochondrial pathway of apoptosis (Chipuk et al., 2005
). p53 might be even localized within the mitochondria (Heyne et al., 2004
). p53 is also involved in oxidative stress (Culmsee and Mattson, 2005
), and SDH Ip is often considered as a “sensor” of the ubiquinone pool, a major source of reactive oxygen species in the cells (Rustin et al., 2002
). Thus, it is possible that p53 could be involved in the reduction of complex II/SDH levels induced by mutated Htt.
Another interesting possibility is related to the Ip protein mRNA structure. Ip mRNA possesses an UTR named IRE (iron responsive element) sequence to which specific proteins (IRP-1 and IRP-2) can bind, regulating translation (Gray et al., 1996
). The presence of mutated Htt could interfere at this level to modify posttranscriptionally SDH expression. Supporting this hypothesis, huntingtin has been implicated in regulation of iron homeostasis (Hilditch-Maguire et al., 2000
). Of interest, IRP-2 levels are regulated by the ubiquitin-proteasome pathway (Guo et al., 1995
). Accumulation of IRP2 might reduce translation of mRNAs coding SDH Ip subunit, leading to depletion of the protein. Ongoing experiments will examine the role of IRPs.
The present results show that loss of complex II/SDH subunits and activity was accompanied by a significant decrease in mitochondrial membrane potential in striatal neurons expressing Htt171-82Q. Our observation is in agreement with the results obtained in mitochondria of HD patients and transgenic YAC72 mouse HD model that showed abnormal mitochondrial transmembrane potential (Sawa et al., 1999
; Panov et al., 2002
). Noticeably, Bae et al.
) showed reduced accumulation of Mitotracker Red in striatal neuronal cells expressing mutated Htt, along with changes in JC-1 fluorescence, indicating anomalies in mitochondrial membrane potential. Reduction of SDH activity could reduce the proton gradient produced by complex III and complex IV across the inner mitochondrial membrane. Interestingly, we found that overexpressing Ip or Fp proteins using lentivirus could prevent Htt171-82Q–induced loss of mitochondrial membrane potential. This suggests that loss of mitochondrial membrane potential in neurons expressing short N-terminus fragment of mutated Htt could be, at least in part, a direct consequence of complex II/SDH defects.
We showed that correcting the molecular defect of SDH complex blocked death of striatal cells induced by expressing low levels of the N-terminus part of mutated Htt. When Htt171-82Q expression levels were increased using the tetracycline-regulated promoter, overexpression of SDH-A or SDH-B modified neither the reduction of DARPP32 expression in striatal cultures nor the levels of ubiquitin-positive inclusions. However, the rescuing effect of SDH-A and SDH-B overexpression from cell death was seen. These results indicate that loss of complex II/SDH is probably not responsible for loss of a number of striatal markers and does not play a direct effect on sequestration/detoxification of mutated Htt fragment. Importantly our results provide evidence that complex II/SDH subunits are critical for the execution of cell death induced by toxicity of N-terminus fragment of mutated Htt.
The observation that overexpression of one SDH subunit leads to an increased expression of the entire complex may sound surprising at first. It is probable that this phenomenon is due to an increased “stabilization” of the SDH complex. In humans, mutations in any of the SDH subunits cause the complex II to fully disassemble (Rustin et al., 2002
). One elegant study showed that, in lymphoblast cells isolated from a patient with homozygous Fp mutations leading to major loss of SDH (Bourgeron et al., 1995
), overexpression of a wild-type Fp protein led to a fully functional complex (Parfait et al., 2000
In conclusion, the present study shows preferential loss of complex II/SDH subunits in the HD striatum and a cellular model of the disease and provides the first proof of concept that regulating complex II/SDH expression may be of therapeutic interest to slow down striatal degeneration in HD. Animal experiments are awaited to determine whether such an experimental therapy could be effective in vivo.