Effects of overexpression of Huntingtin proteins on mitochondrial integrity

Abstract

Huntington's disease (HD) is caused by an expansion of a CAG trinucleotide sequence that encodes a polyglutamine tract in the huntingtin (Htt) protein. Expansion of the polyglutamine tract above 35 repeats causes disease, with the age of onset inversely related to the degree of expansion above this number. Growing evidence suggests that mitochondrial function is compromised during HD pathogenesis, but how this occurs is not understood. We examined mitochondrial properties of HeLa cells that expressed green fluorescent protein (GFP)- or FLAG-tagged N-terminal portions of the Htt protein containing either, 17, 28, 74 or 138 polyglutamine repeats. Immunofluorescence staining of cells using antibodies against Tom20, a mitochondrion localized protein, revealed that cells expressing Htt proteins with 74 or 138 polyglutamine repeats were more sensitized to oxidative stress-induced mitochondria fragmentation and had reduced ATP levels compared with cells expressing Htt proteins with 17 or 28 polyglutamine repeats. By measuring changes in fluorescence of a photoactivated GFP protein targeted to mitochondria, we found that cells expressing red fluorescent protein (RFP)-tagged Htt protein containing 74 polyglutamine repeats had mitochondria that displayed reduced movement and fusion than cells expressing RFP-Htt protein with 28 polyglutamine repeats. Overexpression of Drp-1(K38A), a dominant-negative mitochondria-fission mutant, or Mfn2, a protein that promotes mitochondria fusion, suppressed polyglutamine-induced mitochondria fragmentation, the reduction of ATP levels and cell death. In a Caenorhabditis elegans model of HD, we found that reduction of Drp-1 expression by RNA interference rescued the motility defect associated with the expression of Htt proteins with polyglutamine repeats. These results suggest that the increase in cytotoxicity induced by Htt proteins containing expanded polyglutamine tracts is likely mediated, at least in part, by an alteration in normal mitochondrial dynamics, which results in increased mitochondrial fragmentation. Furthermore, our results suggest that it might be possible to reverse polyglutamine-induced cytotoxicity by preventing mitochondrial fragmentation.

Ordered Assembly of Heat Shock Proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on Expanded Polyglutamine Fragments Revealed by Chemical Probes

“…2B) (49). We also noted a number of mitochondrial proteins, consistent with an emerging role for that organelle in polyQ-associated processes (50). Although care must be taken to avoid over-interpreting these results, we also noticed that treatment with 115-7c for 24 h shifted the Q103-associated patterns to more closely resemble those of Q25, perhaps consistent with the ability of this compound to suppress aggregation (see Fig.…”

Bioenergetic dysfunction in Huntington's disease human cybrids

“…Mitochondrial dysfunction constitutes a cellular hallmark for neurodegeneration and occurs as a consequence of defective mitochondrial composition, trafficking to synapses, calcium handling, ATP production, transcription abnormalities and/or ETC impairment (Rosenstock et al, 2010). Overexpression of mutant huntingtin, but not the normal protein, increases oxidative stress-induced mitochondrial fragmentation in HeLa cells, which correlates with increased caspase-3 activation and cell death (Wang et al, 2009). Moreover, both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion have been described in moderate-to-severe grade HD patients' striatal neurons (Kim et al, 2010).…”

“…Moreover, both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion have been described in moderate-to-severe grade HD patients' striatal neurons (Kim et al, 2010). Furthermore, overexpression of proteins that stimulate mitochondrial fusion attenuates the toxicity of Htt proteins containing expanded polyglutamine tracts in both cells and animals (Wang et al, 2009). The protein expression levels and the phosphorylation of PDH subunit E1α were assessed using a kit, as described in Materials and Methods.…”

Coenzyme Q protects Caenorhabditis elegans GABA neurons from calcium-dependent degeneration

“…Inhibition of the fission-promoting dynamin GTPase, Drp-1, for example, blocks mitochondrial fragmentation and death induced by overexpression of the mutant Huntingtin protein (mtHtt) in cultured cells (8). The progressive onset of these diseases is correlated with an age-dependent decline in mitochondrial function, but the mechanistic link between diminished mitochondrial activity and neurodegeneration is poorly understood.…”