Potential function for the Huntingtin protein as a scaffold for selective autophagy

Abstract

Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51-like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.

Keywords: Huntingtin; Huntington disease; neurodegeneration; polyglutamine; selective autophagy.

Fig. 1.

Loss of HTT function in Drosophila larvae or conditional knockout of HTT in the mouse CNS inhibits autophagy. (A) Loss of Drosophila HTT function results in an inhibition of autophagy in starved larvae as shown by significant reduction in numbers of acidic vesicles monitored by LysoTracker Red in fat body cells. (Scale bars, 30 μm.) (B) Drosophila p62/SQSTM1, Ref(2)P, significantly accumulates in the cytoplasm of fat body cells with HTT LOF in starved larvae. (Scale bars, 30 μm.) (C) Just before pupariation, the locomotion of the HTT LOF larvae exhibit a statistically significant climbing deficit. (D) Htt^flox/−;nestin-cre-tg CNS conditional Htt knockout mice significantly accumulate p62/SQSTM1 in the striatal insoluble fraction with aging. n = 3 for each genotype.

Fig. 2.

HTT copurifies with proteins required for regulation of autophagy. (A) HTT shares similarity with yeast proteins Atg23, Vac8, and Atg11 (15); its C-terminal Atg11-like domain may interact with mammalian homologs of yeast Atg11-binding proteins. A flexible hinge within the N-terminal domain of HTT may be modulated by HTT phosphorylation by IKK, AKT, and CDK5, activating the C-terminal Atg11-like domain of HTT to function in selective autophagy. (B) HTT constructs used in this study: Venus-HTT(2146–3144) (red) and Venus-HTT(1651–3144) (green). (C) Components of the Atg1 kinase complex and receptor proteins p62, BNIP3, and BNIP3L/NIX coimmunoprecipitate with HTT(2416–3144). HEK293T cells were cotransfected with Venus-HTT(2416–3144) and plasmids expressing HA-ULK1, MYC-RB1CC1/FIP200, MYC-ATG13, MYC-p62, MYC-BNIP3, and MYC-BNIP3L/NIX. Cell lysates were subjected to immunoprecipitation with (+) and without (−) antibodies against HA and MYC. (D) BECN-1 co-immunoprecipitation with full length HTT. HEK293T cells were cotransfected with pARIS-HTT (23Q or 100Q) and BECN1-GFP. Cell lysates were subjected to immunoprecipitation using antibodies against GFP. (E) 25QP and 97QP N-terminal HTT exon 1 fragments do not coimmunoprecipitate with Venus-HTT(2416–3144), but 586 amino acid HTT fragments do copurify. HEK293T cells were cotransfected with Venus-HTT(2416–3144) and with exon1 and 586 HTT fragment constructs. Cell lysates were subjected to immunoprecipitation using antibodies against Venus.

Fig. 3.

The C-terminal domain of HTT interacts with GABARAPL1 and LC3B. Venus-HTT(2416–3144) coimmunoprecipitates with mammalian Atg8s. HEK293T cells were cotransfected with Venus-HTT(2416–3144) and MYC-LC3B or MYC-GABARAPL1. Cell lysates were subjected to immunoprecipitation (IP) using anti-MYC. The resulting precipitates were examined by immunoblot analysis with the indicated antibodies. W3037A mutation significantly reduces the coimmunoprecipitation of Venus-HTT(2416–3144) with MYC-GABARAPL1 but not with MYC-LC3B.