In this regard, whether ASK1 deficiency mitigates polyQ disease phenotype in appropriate mouse models will be an interesting issue to be examined. Another important issue to be elucidated is the relation between the ASK1CJNK pathway and caspase12, which has PST-2744 (Istaroxime) been reported to be essential for ER stress-induced apoptosis and neurotoxicity by amyloid- proteins (Nakagawa et al. shown) in primary neurons. em ASK1 /em ?/? neurons were also insensitive to thapsigargin and MG132 in JNK3 activation (Fig. ?(Fig.4D,4D, lanes 2,3,8,9). These results indicate that ASK1 is required for JNK3 activation by pathogenic polyQ. PolyQ-induced cell death was determined by MTT assay (Fig. ?(Fig.4E).4E). Q79, but not Q35, induced cell death in em ASK1 /em +/+ neurons. em ASK1 /em ?/? neurons were clearly more resistant to Q79-induced cell death than em ASK1 /em +/+ neurons (Fig. ?(Fig.4E),4E), indicating that ASK1 is required for polyQ-induced neuronal cell death. Discussion We have identified ASK1 as an essential component in the neuronal death signaling induced by expanded polyQ. Expanded polyQ has been known to cause various cellular events, including alteration of gene expression, abnormal protein interaction, and activation of caspase (Lin et al. 1999; Paulson et al. 2000), and all of these abnormal alterations potentially trigger neuronal cell death (Fig. ?(Fig.5).5). However, direct genetic evidence has never been provided to the hypothetical involvement of these events in the polyQ-induced cell death pathway. In the present study, we have shown that polyQ induces ER stress, which in turn activates the IRE1CTRAF2CASK1CJNK-signaling pathway leading to neuronal cell death (Fig. ?(Fig.5).5). The almost complete loss of JNK activation (Figs. ?(Figs.3A,3A, ?A,4D)4D) and cell death (Figs. ?(Figs.3E,F,3E,F, 4A,B) induced by ER stressors in em ASK1 /em ?/? cells strongly argues that ASK1 constitutes an essential and nonredundant cell death pathway in response to ER stress. Open in a separate window Figure 5 Schematic representation of the role of IRE1CTRAF2CASK1 cascade PST-2744 (Istaroxime) in the pathogenesis of polyQ disease. See text for details. Recent studies have suggested that polyQ disturbs UPS (Bence et al. 2001; Jana et al. 2001; Waelter et al. 2001), and thus compromise its ability to degrade not only polyQ but also unfolded proteins produced at a normal protein turnover. UPS dysfunction has been implicated in the pathogenesis of various neurodegenerative diseases (Sherman and Goldberg 2001) such as amyotrophic lateral sclerosis (Johnston et al. 2000) and Parkinson’s disease (Imai et al. 2001). However, the molecular mechanism by which polyQ-induced UPS dysfunction leads to neuronal cell death has remained unknown. Although polyQ-induced inhibition of the proteasome activity in primary neurons were incomplete (30% at most by Ad-Q79 at m.o.i. of 100; Fig. ?Fig.1E),1E), such an extent of proteasome dysfunction was sufficient to cause ER stress (Fig. ?(Fig.1C,D)1C,D) and neuronal cell death (Fig. ?(Fig.4C).4C). These results suggested that polyQ-induced proteasomal dysfunction and neuronal cell death can be linked by the ER stress-dependent cell death pathway. Consistently, activations of JNK by polyQ or proteasome inhibitors were HB5 completely abrogated in em ASK1 /em ?/? cells (Figs. ?(Figs.3B,3B, ?B,4D),4D), and em ASK1 /em ?/? cells were refractory to polyQ- and proteasome inhibitor-induced apoptosis (Figs. ?(Figs.3G,3G, ?G,4C,E).4C,E). This indicates that ASK1 is a key PST-2744 (Istaroxime) component in the polyQ-initiated cell death signaling cascade involving UPS dysfunction and ER stress (Fig. ?(Fig.5).5). On the other hand, resistance to proteasome inhibitor-induced apoptosis in em ASK1 /em ?/? cells was not as dramatic as that to ER stress-induced apoptosis (Figs. ?(Figs.3G,3G, ?G,4C).4C). These results suggest that ASK1CJNK- and ER stress-independent cell death pathway may also exist in the downstream of PST-2744 (Istaroxime) proteasome dysfunction. This may also reflect, in part, the incomplete resistance of em ASK1 /em ?/? neurons to polyQ-induced cell death (Fig. ?(Fig.44E). It has been suggested that neuronal cell death may not be necessary for the trigger of atrophy and ataxia. This hypothesis is supported by a recent study that showed the reversal motor dysfunction in a conditional HD model mouse in which expression of polyQ proteins were regulated by tetracycline-repressor (Yamamoto et al. 2000). In this mouse model, expression of polyQ induced PST-2744 (Istaroxime) HD-like phenotype without a significant.