Immiscible inclusion bodies formed by polyglutamine and poly(glycine-alanine) are enriched with distinct proteomes but converge in proteins that are risk factors for disease and involved in protein degradation

Abstract

Poly(glycine-alanine) (polyGA) is one of the polydipeptides expressed in Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 1 caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ)-expanded Huntingtin exon 1 (Httex1) in Huntington's disease. Both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms and therefore recruit the same repertoire of endogenous proteins. When co-expressed in the same cell, polyGA 101 and Httex1(Q 97) inclusions adopted immiscible phases suggesting different endogenous proteins would be enriched. Proteomic analyses identified 822 proteins in the inclusions. Only 7 were specific to polyGA and 4 specific to Httex1(Q 97). Quantitation demonstrated distinct enrichment patterns for the proteins not specific to each inclusion type (up to~8-fold normalized to total mass). The proteasome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in Httex1(Q 97) aggregates. Both structures revealed a collection of folding and degradation machinery including proteins in the Httex1(Q 97) aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.

Synaptic disruption and CREB‐regulated transcription are restored by K + channel blockers in ALS

“…Notably, we did not detect GA aggregates in ALS C9orf72 MN, and the poly(GA) overexpression did not induce CBP aggregation. Nevertheless, Vasp, which interacts with CREB (Tateya et al, 2019) and controls synaptic dynamics (Lin et al, 2010), has been found to be highly enriched within GA aggregates (Radwan et al, 2020). Thus, these alterations seem to be triggered by the accumulation of aggregates, independently from their specific nature and composition.…”

Spatial Distribution of Intracellular Ion Concentrations in Aggregate‐Forming HeLa Cells Analyzed by μ‐XRF Imaging

“…Despite significantly higher levels of P, K, S, and Zn in the transfected cells, we did not find significant local accumulation of P, S, K, and Zn in Htt‐ex1 or PIC aggregates compared to the surrounding cytosol. This is still a remarkable result to report, since Htt‐ex1‐containing IBs constitute a rather distinct heterogenous subcellular environment containing a wide range of different proteins and biomolecules, for example associated with the proteasome complex or the chaperone machinery [41] . Our hypothesis was that the change in environmental conditions compared to the cytoplasm could in fact lead to a heterogenous portioning of the elemental concentrations, as observed for the amyloid β, despite the absence of metal binding sites within amyloids.…”

“…This is still a remarkable result to report, since Htt‐ex1‐containing IBs constitute a rather distinct heterogenous subcellular environment containing a wide range of different proteins and biomolecules, for example associated with the proteasome complex or the chaperone machinery. [41] Our hypothesis was that the change in environmental conditions compared to the cytoplasm could in fact lead to a heterogenous portioning of the elemental concentrations, as observed for the amyloid β, despite the absence of metal binding sites within amyloids. As such, the lack of specific ion‐binding sites in Htt‐ex1 appears to lead to Zn concentrations in regions containing aggregates that are indistinguishable from the surrounding cytosol.…”

A biosensor of protein foldedness identifies increased “holdase” activity of chaperones in the nucleus following increased cytosolic protein aggregation

“…Consistent with prior findings that protein aggregation can sequester quality control resources away from “housekeeping” activities and lead to metastably folded proteins aggregating ( 15 ), we found that the pool of resources binding to unfolded barnase biosensor decreased in the cytosol. Prior studies have found that Hsp70 and Hsp40 proteins are recruited into inclusions formed by Httex1 97Q and similar proteins with expanded polyglutamine sequences ( 16 , 17 , 18 , 19 ). One function for this recruitment may be disaggregation, in light of recent findings showing the Hsp70-based chaperone machinery can dissociate amyloid fibrils ( 20 ).…”