Delineation of Htt clearance mechanisms is of great significance because an accumulation of mutant Htt is implicated in HD pathogenesis. We demonstrate that the IKK complex phosphorylates Htt at S13 and may activate its degradation, similar to IKK-mediated degradation of IκBα and FOXO3a (Karin and Ben-Neriah, 2000
; Karin et al., 2002
; Hu et al., 2004
). The proposed selective degradation of phosphorylated wt Htt, which involves both the proteasome and lysosome, may include a transient nuclear localization mediated by phosphorylation of Htt, where it is subsequently acetylated, ubiquitinated, and SUMOylated in an order that remains to be established. Proteins involved in lysosomal degradation pathways, Hsc70, LAMP-2A, and Atg7, appear to modulate the levels of these modified forms of Htt in mammalian cells. Our data also suggest that IKK-β–mediated Htt S13 phosphorylation is more efficient for wt than for expanded polyQ truncated Htt polypeptides ( and ), which may result in reduced clearance of mutant Htt by inhibiting this phosphorylation-driven mechanism, and ultimately contribute to disease. Finally, modified species recognized by phospho- and acetyl-specific antibodies are present in mouse brain.
Mimicking phosphorylation of Htt serines 13 and 16 increases soluble Httex1p-GFP nuclear localization (). In previous studies, we showed that Htt interacts with the acetyltransferase CBP (Steffan et al., 2000
); therefore CBP, a nuclear protein, is a candidate acetyltransferase for Htt lysine 9, as was demonstrated for lysine 444 (Jeong et al., 2009
). CBP/p300 contains ubiquitin ligase activity which regulates protein degradation (Grossman et al., 2003
); therefore, this E3 ligase activity of CBP could also be involved in regulating Htt ubiquitination. A futher connection between the IKK-mediated phosphorylation of Htt and CBP activites may exist, as CBP interacts directly with IKK-γ in the nucleus (Verma et al., 2004
) and is a substrate for IKK-α (Huang et al., 2007
), suggesting that CBP and the IKK complex could function together to modulate Htt stability.
Relevant to the potential role for S13 and S16 phosphorylation in nuclear localization, nuclear caspase-6 cleavage of mutant Htt has been implicated in its pathogenic potential (Graham et al., 2006
; Warby et al., 2008
). We find that IKK activates phosphorylation of Htt fragments, one of which is consistent with the predicted size of a wt Htt caspase-6 cleavage product () and the fragment recently shown to be generated in neurons with activation of IKK-β (Khoshnan et al., 2009
). It is therefore an intriguing possibility that phosphorylation by IKK ultimately promotes Htt nuclear localization, poly-SUMOylation, acetylation by CBP, and subsequent caspase-6 cleavage, which all facilitate a regulated form of clearance.
Phosphorylation of Htt by IKK appears to activate its degradation at least in part by the lysosome, dependent on LAMP-2A levels (), the integral membrane receptor protein that can directly import proteins across the lysosomal membrane for CMA (Massey et al., 2006b
). The CMA chaperone Hsc70 preferentially interacts with phosphomimetic wt Httex1p and reduces Htt-mediated toxicity (). Combined, this data may suggest that phosphorylated Htt is degraded in a LAMP-2A–dependent mechanism through CMA. Because phosphorylation can trigger nuclear localization and acetylation of specific Htt species () and overexpression of Hsc70 increases levels of acetylated and phosphorylated endogenous full-length and fragmented Htt (Fig. S5), the findings implicate Hsc70 in either mediating an interaction of Htt with the IKK complex, or alternatively activating the IKK complex, as has been demonstrated for the ubiquitin ligase parkin, which is mutated in Parkinson's disease (Henn et al., 2007
CMA activity declines with age due to a gradual decrease of LAMP-2A levels in lysosomes (Cuervo and Dice, 2000
), whereas artificially maintaining LAMP-2A levels in aging rat liver similar to those in young animals can restore CMA activity to youthful levels and improve organ function (Zhang and Cuervo, 2008
). Because HD is a neurodegenerative disease associated with aging and we have found clearance of phosphorylated Htt dependent on LAMP-2A, a reduction in LAMP-2A levels over time may be tied to HD pathogenesis. We propose a hypothetical model for the progression of HD at the molecular level (), where IKK phosphorylates Htt and activates a cascade of Htt post-translational modifications and caspase cleavage (Khoshnan et al., 2009
) associated with rapid Htt degradation by the proteasome and the lysosome in unaffected neurons. In a presymptomatic HD neuron, IKK could be induced by the presence of the mutant protein (Khoshnan et al., 2004
), thus stimulating an IKK-mediated mechanism of Htt clearance, consistent with the innate immune activation that occurs in premanifest patients well before symptom onset (Björkqvist et al., 2008
). As long as LAMP-2A levels remain high, patients can degrade mutant Htt before it can cause toxicity, despite progressively inhibited proteasome activity and reduced efficiency of mutant Htt phosphorylation. As aging and mutant Htt together progressively impair proteasome and overall lysosomal activity, and as LAMP-2A levels decline with age, modified Htt may accumulate, enhancing HD pathogenesis.
Figure 8. Proposed molecular mechanism for the development of HD. Normal neuronal function: IKK phosphorylates wt Htt activating its post-translational modification, caspase cleavage, and clearance by the proteasome and lysosome. Presymptomatic HD neuronal function: (more ...)
From this model, it follows that increasing the efficiency of Htt clearance by the lysosome, or increasing levels or mobility (Kaushik et al., 2006
) of functional LAMP-2A in the lysosomal membrane early in the disease process, could delay HD onset and serve as a therapeutic strategy. Treatment choice may vary depending on the stage of HD. In mammals, early treatment to increase Htt phosphorylation and acetylation may be useful when levels of LAMP-2A are adequate, as suggested by the reduced toxicity in acutely transfected rat slice cultures () and complete lack of neurotoxicity in BACHD mice when Htt is mutated to mimic the phosphorylated form (Gu et al., 2009
). However, when LAMP-2A levels are low or its function is impaired, drugs that activate the formation of the post-translationally modified Htt species normally targeted for degradation by the lysosome might actually increase HD pathogenesis through increased nuclear accumulation and aggregation of the mutant protein. At this stage of disease, serine-modified Htt would accumulate, leading to increased pathology as a result of the intrinisic toxicity of the modified Htt. Consistent with this, we find that mimicking Httex1p phosphorylation increases its toxicity and aggregation in Drosophila
photoreceptor neurons where components of the mammalian machinery to degrade phosphorylated Htt, specifically LAMP-2A, are not present (unpublished data). Thus, during late-stage HD, it may be harmful to increase pathways involved in IKK activation, SUMOylation, and acetylation, whereas in presymptomatic stages, these pathways may be protective.
The Htt protein itself may play an integral role in autophagic clearance of proteins. Conditional knockout of Htt in the mouse central nervous system results in an accumulation of neuropil protein aggregates containing ubiquitin and p62/SQSTM1 (unpublished data). Htt has been shown to associate with autophagosomes (Atwal and Truant, 2008
) and lysosomes (unpublished data) and may therefore play a functional and regulatory role in a selective protein clearance mechanism ultimately involved in its own processing. Extensive investigation will be necessary to test this possibility and elucidate the precise mechanisms involved.