Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

Nathan Riguet,Alice Patin,Veronika Reiterer,Sergey Nasarov,Johannes Burtscher,Hilal A Lashuel,Janna Hastings,Anne-Laure Mahul-Mellier,Hesso Farhan,Marie Croisier,Niran Maharjan,Graham Knott

doi:10.1038/s41467-021-26684-z

Abstract

Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington’s disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon1 of the Htt protein. Our findings provide unique insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve the polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; the recruitment and accumulation of remodeled or dysfunctional membranous organelles, and the impairment of the protein quality control and degradation machinery. We also show that nuclear and cytoplasmic Htt inclusions exhibit distinct biochemical compositions and ultrastructural properties, suggesting different mechanisms of aggregation and toxicity.

Highlights

Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington’s disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood
It has been recently demonstrated that Lewy bodies (LB) isolated from Parkinson’s disease brains or LB-like inclusions in primary neurons are composed of filamentous and aggregated forms of alpha-synuclein and a complex milieu of lipids, cytoskeletal proteins, and other proteins and membranous organelles, including mitochondria and autophagosomes[119,120,121,122]
In line with these findings, our electron microscopy (EM) data, together with 3D reconstructions, revealed the presence of membrane fragments and vesicles entrapped in the core of the Httex[1] inclusions. Consistent with these observations, our proteomic analysis revealed that 24% of the proteins enriched in the inclusions fraction belong to the endolysosomal compartments, the Golgi apparatus, and the trans-Golgi network (Fig. 3)

Summary

Introduction

Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington’s disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. We investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon[1] of the Htt protein. The mutant Httex1-based models are useful to study the pathogenesis of HD as they reproduce different aspects of Htt aggregation and have been instrumental in advancing our understanding of the sequence, molecular, and structural determinants of Htt aggregation and inclusion formation[10,26,27,28,29,35,36]. Given that a large body of published cellular studies on the mechanisms of Httex[1] aggregation and inclusion formation is based on constructs in which Httex[1] is fused to the GFP9,11,12,37–40, we compared, for the first time, the composition, ultrastructural properties, and toxicity of inclusions formed by native (tag-free) and GFP-tagged mutant Httex[1] proteins

Methods

Results

Conclusion