A07 Huntingtin-dependent stability of HAP40 and decreased HAP40 levels in huntington’s disease

Abstract

<h3>Background</h3> The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In a complex of the two proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains, rich in HEAT repeats, and a smaller bridge domain that connects the two. When generating HEK293-based cell lines for producing the HTT-HAP40 complex for structural studies (Guo et al. 2020, Nature), enhancing HTT expression resulted in increased HAP40 steady-state levels. <h3>Aims/Methods</h3> By Western Blot analysis, we investigated whether HAP40 steady-state protein levels are directly dependent on HTT levels and whether HAP40 protein levels are decreased in samples (lymphocytes and fibroblasts) from Huntington’s disease (HD) patients and mouse models alongside HTT levels. Moreover, we assessed by cycloheximide-chase assay and qRT-PCR, respectively, if HAP40 protein stability or HAP40/F8A mRNA levels are dependent on HTT levels. <h3>Results</h3> We show that steady-state protein levels of HAP40 strongly correlated with and were directly dependent on HTT protein expression levels. While HAP40 alone had a very short half-life, HAP40 was stabilized upon HTT overexpression. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of HD patients and in brain tissue of a well-characterized HD mouse model, concomitantly with decreased HTT levels in these cell types. <h3>Conclusion</h3> This data, together with our previous demonstration of likely coevolution of the two proteins and evolutionary conservation of their interaction in metazoans (Seefelder et al. 2020, BMC Evolutionary Biology) suggests that HAP40 is an obligate and likely functionally important interaction partner of HTT. Our observation of reduced HAP40 levels in HD indicates that loss-of-function phenotypes due to lowered HAP40 levels might contribute to the pathology in HD.