Post-translational modifications clustering within proteolytic domains decrease mutant huntingtin toxicity

Abstract

Huntington's disease (HD) is caused in large part by a polyglutamine expansion within the huntingtin (Htt) protein. Post-translational modifications (PTMs) control and regulate many protein functions and cellular pathways, and PTMs of mutant Htt are likely important modulators of HD pathogenesis. Alterations of selected numbers of PTMs of Htt fragments have been shown to modulate Htt cellular localization and toxicity. In this study, we systematically introduced site-directed alterations in individual phosphorylation and acetylation sites in full-length Htt constructs. The effects of each of these PTM alteration constructs were tested on cell toxicity using our nuclear condensation assay and on mitochondrial viability by measuring mitochondrial potential and size. Using these functional assays in primary neurons, we identified several PTMs whose alteration can block neuronal toxicity and prevent potential loss and swelling of the mitochondria caused by mutant Htt. These PTMs included previously described sites such as serine 116 and newly found sites such as serine 2652 throughout the protein. We found that these functionally relevant sites are clustered in protease-sensitive domains throughout full-length Htt. These findings advance our understanding of the Htt PTM code and its role in HD pathogenesis. Because PTMs are catalyzed by enzymes, the toxicity-modulating Htt PTMs identified here may be promising therapeutic targets for managing HD.

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

“…Moreover, a recent study using a Drosophila model of HD expressing HTTex1 97Q via phosphomimetics showed that both S13D and S16D increase aggregation (Branco-Santos et al, 2017), in contrast to observations in mammalian cell models of HD showing that expression of the N-terminal fragments of HTT 1-171 142Q, HTTex1 97Q or HTTex1 82Q with S13D and S16D decreases mutant HTT-induced toxicity and aggregation (Arbez et al, 2017;Atwal et al, 2011;Branco-Santos et al, 2017). Although these findings show that PTMs, such as single or double phosphorylation, are sufficient to modify mutant HTT levels, aggregation, and toxicity, they highlight the limitations of using phosphomimetics.…”

“…HTT N17 domain was shown to contain a translocated promoter region (TPR)-dependent nuclear export signal (Atwal et al, 2007;Benn et al, 2005;Cornett et al, 2005;Maiuri et al, 2013;Rockabrand et al, 2007;Steffan et al, 2004). Mimicking phosphorylation at S13 and S16 (S13D/S16D) increased the localization of the HTT N17 peptide, HTTex1 or longer N-terminal HTT fragments to the nucleus (Arbez et al, 2017;Atwal et al, 2011;Havel et al, 2011;Thompson et al, 2009;Zheng et al, 2013). Moreover, we recently reported that single or double phosphorylation at S13 and/or S16 of HTTex1 significantly enhanced the targeting of mutant HTTex1 fibrils to the nuclear compartment (Deguire et al, 2018).…”

“…Previous studies indicated that phosphorylation or the introduction of phosphomimetic mutations at both S13 and S16 (Anne et al, 2007;Arbez et al, 2017;Branco-Santos et al, 2017;Chiki et al, 2017;Deguire et al, 2018) could suppress mutant HTT aggregation in vitro and in cellular models.…”

“…The expression of mutant HTT has been shown to induce cytotoxicity in neuronal models of HD (Arbez et al, 2017;Atwal et al, 2011). Having shown that TBK1 overexpression reduced mutant HTTex1 aggregates in HEK293T cells, we sought to determine whether there would also be suppression of mutant HTTex1 cytotoxicity.…”

“…Having shown that TBK1 overexpression reduced mutant HTTex1 aggregates in HEK293T cells, we sought to determine whether there would also be suppression of mutant HTTex1 cytotoxicity. We used a previously established cytotoxicity (cell death) assay based on the measurement of nuclear condensation (Cummings and Schnellmann, 2004) during neuronal cell death induced by the expression of mutant HTT (Arbez et al, 2017).…”