Microtubule destabilization and nuclear entry are sequential steps leading to toxicity in Huntington's disease

Abstract

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is ''downstream'' of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival. The primary and potentially reversible steps of toxicity mediated by mutant Huntington's disease (HD) protein [mutant huntingtin (mhtt)] are controversial. A major model suggests that short cleavage products of mhtt migrate to the nucleus and cause transcriptional dysfunction (1). The N-terminal truncated form of mhtt is known to bind and interfere with nuclear factors such as cAMP response element-binding protein (CREB) (2), CREB-binding protein (3), corepressor (4), and transcriptional activator Sp1 (5). Restoration of transcription by histone deacetylase inhibitors improves survival of affected neurons in a Drosophila model for HD (6), indicating that mhtt can cause deleterious alterations in transcription. However, transcriptional dysfunction is caused in cell, mouse, or fly models by expression of only the short truncated fragment of the mhtt (6-10). Short fragments can freely migrate into the nucleus and cause dysfunction there (10). In vivo, however, fragments must arise from cleavage of full-length mhtt, a large cytoplasmic protein with no nuclear localization signal (NLS) (10). Yet neither the timing nor the extent of cleavage of the full-length mhtt in vivo is known. Recent data suggest that cleavage of mhtt may be slow in human HD tissue (11) and raise the possibility that N-terminal cleavage may not be an early or primary event in toxicity.In fact, several lines of evidence suggest that nuclear events may not be sufficient to account for the initial toxic effects of mhtt. In presymptomatic or late-onset disease tissue, nuclear inclusions (NI) are often absent in patients, whereas cytoplasmic inclusions can exceed NIs (12). Second, many mhtt-interacting proteins identified by yeast two-hybrid are cytoplasmic proteins (13). Microarray analysis of HD mice reveals that expression of many trafficking proteins and cytoplasmic metabolizing enzymes is affected early in the toxic progression (14). Moreover, Sp1 has been shown to be a major target of mhtt, presumably mediating toxicity by repression of Sp1-dependent genes (15). However, many Sp1-dependent genes are unaffected by exp...

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