HAP40 is a conserved central regulator of Huntingtin and a potential modulator of Huntington’s disease pathogenesis

Abstract

Perturbation of huntingtin (HTT)’s physiological function is one postulated pathogenic factor in Huntington’s disease (HD). However, little is known how HTT is regulated in vivo. In a proteomic study, we isolated a novel ~40kDa protein as a strong binding partner of Drosophila HTT and demonstrated it was the functional ortholog of HAP40, an HTT associated protein shown recently to modulate HTT’s conformation but with unclear physiological and pathologic roles. We showed that in both flies and human cells, HAP40 maintained conserved physical and functional interactions with HTT. Additionally, loss of HAP40 resulted in similar phenotypes as HTT knockout. More strikingly, HAP40 strongly affected HTT’s stability, as depletion of HAP40 significantly reduced the levels of endogenous HTT protein while HAP40 overexpression markedly extended its half-life. Conversely, in the absence of HTT, the majority of HAP40 protein were degraded, likely through the proteasome. Further, the affinity between HTT and HAP40 was not significantly affected by polyglutamine expansion in HTT, and contrary to an early report, there were no abnormal accumulations of endogenous HAP40 protein in HD cells from mouse HD models or human patients. Lastly, when tested in Drosophila models of HD, HAP40 partially modulated the neurodegeneration induced by full-length mutant HTT while showed no apparent effect on the toxicity of mutant HTT exon 1 fragment. Together, our study uncovers a conserved mechanism governing the stability and in vivo functions of HTT and demonstrates that HAP40 is a central and positive regulator of endogenous HTT. Further, our results support that mutant HTT is toxic regardless of the presence of its partner HAP40, and implicate HAP40 as a potential modulator of HD pathogenesis through its multiplex effect on HTT’s function, stability and the potency of mutant HTT’s toxicity.

HAP40 modulates mutant Huntingtin aggregation and toxicity in Huntington’s disease mice

“…Mounting evidence suggests that HAP40 plays an important role in regulating HTT protein conformation and stability, as the addition of HAP40 is essential for forming a stable HTT complex that can be used to resolve HTT protein structure via cryo-electron microscopy [ 9 ]. Moreover, the expression of HAP40 alters the level of HTT in cultured cells [ 32 , 33 ]. Our current study also shows that the expression of HAP40 and HTT is positively correlated in different brain regions of mice.…”

“…This result is consistent with the previous finding that the knockdown of HAP40 using shRNA promotes the aggregation of mHTT fragments in striatal cells [ 34 ]. However, one study reported that deletion of the Drosophila homolog of HAP40 (dHAP40) in a fly model expressing human full-length mHTT partially rescued degenerative phenotypes and prolonged lifespan [ 33 ]. It is noteworthy that although dHAP40 is a 40 kDa protein that interacts with dHtt, its binding affinity to human HTT is significantly reduced [ 33 ].…”

“…However, one study reported that deletion of the Drosophila homolog of HAP40 (dHAP40) in a fly model expressing human full-length mHTT partially rescued degenerative phenotypes and prolonged lifespan [ 33 ]. It is noteworthy that although dHAP40 is a 40 kDa protein that interacts with dHtt, its binding affinity to human HTT is significantly reduced [ 33 ]. In addition, the protein sequence homology between dHAP40 and human HAP40 is less than 30%, whereas the homology between mouse HAP40 and human HAP40 is more than 80%.…”

“…Therefore, the fly model may not fully recapitulate human HAP40 function. Indeed, overexpressing human HAP40 only marginally changed the eye degeneration in the fly model expressing human mHTT [ 33 ], which further indicates that it is imperative to study the HTT-HAP40 relationship in a higher organism, such as mice. Among the numerous HD mouse models available, we chose HD140Q KI mice for our study.…”

ProteinCoLoc streamlines Bayesian analysis of colocalization in microscopic images

“…Moreover, the interaction of the two proteins is likely essential for their function, as both proteins have co-evolved and their interaction is conserved in various species 15 . Interestingly, the protein levels and stability of both HAP40 and HTT are reduced in tissues from HD patients and mouse models 9 , 16 – 18 , suggesting that the reduction of their levels in cells may contribute to the pathophysiology of HD 8 , 18 . However, although the HTT-HAP40 complex is well characterised in vitro 10 , 12 , 15 , 18 , little is known regarding the extent of their interaction in vivo.…”