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 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.

A FRET-based method for monitoring structural transitions in protein self-organization

“…To further confirm the identity of the final aggregate, a higher resolution confocal fluorescence image ( Figure 1 D) shows a star-like appearance that is very similar to GFP-fused mHttex1 proteins, as observed by cryo transmission electron microscopy (cryo-TEM) ( Riguet et al., 2021 ), thus suggesting that the Q71-VC eventually forms assemblies consisting of fibrils. While the aggregate quickly depletes the cytoplasm of Q71-VC, the free Q71-VC that is not yet incorporated retains the low FRET/donor ratio of the VC control ( Figures 1 B, 1E, and S1 D) with a small dependence of the FRET/donor ratio on the probe concentration due to intermolecular FRET.…”

“…Fluorescent protein fusions influence the aggregation behavior of proteins ( Riguet et al., 2021 ) ( Laine et al., 2019 ; Peskett et al., 2018 ; Xu et al., 2020 ). However, the VC-tagged proteins mostly recapitulate known mutation-dependent transitions and assembly behavior: (1) the glutamine-length dependence of the VC-Q series follows the established glutamine dependence, (2) intermediate phases in mHttex1 aggregation also have been shown to occur in a buffer without fluorescent protein fusions ( Posey et al., 2018 ), (3) the VC does not dominate VC-DNAJB6b binding to Q71 because the VC control does not bind Q71, (4) the VC-DNAJB6b retains its activity by preventing Q71 aggregation, and (5) the tag influences FUS-condensate formation depending on the terminus tagged, while the 521C mutation always induces expected assemblies.…”

Protein interaction networks in neurodegenerative diseases: From physiological function to aggregation

“…The etiology of HD is determined by the combined result of the emerging toxic properties of mutHTT protein, and potentially its mRNA, and the associated loss of normal HTT functions, with an inverse relationship between the expanded repeat length and the age of disease onset that can be modulated by additional gene modifiers ( 213 ). PolyQ expansions in mutHTT have been shown to drive the formation of pathological amyloid fibrils that are able to perturb the intracellular proteostasis network or deplete factors crucial for the basic neuronal cell functionality ( 214 , 215 ). Although mutHTT is typically thought to accumulate and exert its toxicity from within the cell, there is also recent evidence for an extracellular localization and transfer to neighboring cells or other tissues via the blood stream, supporting the idea that mutHTT can propagate in a prion-like fashion ( 216 , 217 ).…”

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation

“…Recent advances in protein chemistry, biophysics, imaging, and proteomics have begun to be applied in research to decipher and embrace the complexity of the processes underlying protein aggregation, in a context where a fibril-centric approach has dominated the field over the past decades [ 65 ]. The system becomes even more complex if the inclusion formation, or rather the assessment of the types and distribution of non-proteinaceous components of amyloid plaques, is considered, as recently revealed by correlative light electron microscopy (CLEM) in LBs [ 66 , 67 ] and Huntingtin inclusions [ 68 ]. These works stress the urgent need for model systems that capture the entire process from protein misfolding to inclusion formation and maturation, and not only fibril formation.…”