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

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 inclusions in cells overexpressing mutant exon1 of the Htt protein. Our findings provide novel 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 1) polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; 2) recruitment and accumulation of remodeled or dysfunctional membranous organelles; and 3) impairement of the protein quality control and degradation machineries. Interestingly, nuclear and cytoplasmic Htt inclusions exhibited distinct biochemical composition and ultrastructural properties, suggesting different formation mechanisms and toxicity.Graphical AbstractSchematic depictions and original electron micrographs of cytoplasmic inclusions formed by native (tag-free) mutant Huntington exon1 proteins (Httex1 72Q, left) and the corresponding GFP fusion protein (Httex1 72Q-GFP).

The Nt17 domain and its helical conformation regulate the aggregation, cellular properties and neurotoxicity of mutant huntingtin exon 1

“…Similarly, the reduction in the number of cells with inclusions was much less pronounced after overexpression of the M8P-Httex1-GFP 72Q than M8P-Httex1 tag-free ( Figure 4B). This emphasizes our recent findings showing that the fusion of GFP proteins to Httex1 alters the structural and aggregation properties of the protein as well as its biological functions 52 .…”

“…These effects were examined in the context of Httex1 carrying polyQ tracts of various lengths (16Q, 39Q, or 72Q), which also allowed us to investigate the effect of the Nt17 domain as a function of increasing polyQ-repeat length. Given that recent studies from our group demonstrated that the addition of a GFP tag to the Httex1 72Q protein dramatically influenced the biochemical composition and ultrastructural properties of Httex1 inclusions 52 , we performed our studies using Httex1 or ΔNt17-Httex1 constructs with different polyQ-repeat lengths (16Q, 39Q, and 72Q, Figure S7A) and fused or not to GFP. The use of Httex1-GFP fusion proteins allows us to compare our findings to published studies, most of which are based on the use of mutant Httex1-GFP/YFP proteins [61][62][63][64][65][66][67][68][69][70] .…”

“…As all the Httex1 species do not run equally in a SDS-PAGE gel (e.g: Httex1 fibrils do not enter into SDS-PAGE gel and stay mainly in the wells of the stacking part; ΔNt17-Httex1 constructs have a low charge state and low SDS-binding capacity and are characterized by aberrant mobility in SDS-Gels), we have developed an unbiased semi-quantitative method based on confocal microscope imaging combined with an analytical pipeline using Image J and Durcupan (Electron Microscopy Sciences, Hatfield, PA, USA) as previously described 52,117,118 .…”

“…The ultrathin sections (50-60 nm) cut serially and collected onto 2 mm single-slot copper grids coated with formvar plastic support film were finally contrasted with uranyl acetate and lead citrate before being imaged with a transmission electron microscope (Tecnai Spirit EM, FEI, The Netherlands) operating at 80 kV acceleration voltage and equipped with a digital camera (FEI Eagle, FEI) 52,117,118 .…”

A Rationale for Hypoxic and Chemical Conditioning in Huntington’s Disease

“…Additionally, the disease-causing mHtt protein interacts with mitochondria and may specifically impair complex II [ 189 ]. mHtt has been demonstrated to induce mitochondrial fragmentation [ 190 ] and cytoplasmic inclusions of mHtt Exon1 have recently been shown to also recruit mitochondria and cause their fragmentation as well as mitochondrial respiratory abnormalities [ 191 ]. Hypoxic conditioning, on the other hand, may counteract mitochondrial fragmentation by enhancing mitochondrial fusion [ 192 ].…”