Soluble and insoluble dipeptide repeat protein measurements in C9orf72-frontotemporal dementia brains show regional differential solubility and correlation of poly-GR with clinical severity

Annelies Quaegebeur,Idoia Glaria,Tammaryn Lashley,Adrian M Isaacs

doi:10.1186/s40478-020-01036-y

Abstract

A C9orf72 repeat expansion is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. One of the suggested pathomechanisms is toxicity from dipeptide repeat proteins (DPRs), which are generated via unconventional translation of sense and antisense repeat transcripts with poly-GA, poly-GP and poly-GR being the most abundant dipeptide proteins. Animal and cellular studies highlight a neurotoxic role of poly-GR and poly-PR and to a lesser degree of poly-GA. Human post-mortem studies in contrast have been much less clear on a potential role of DPR toxicity but have largely focused on immunohistochemical methods to detect aggregated DPR inclusions. This study uses protein fractionation and sensitive immunoassays to quantify not only insoluble but also soluble poly-GA, poly-GP and poly-GR concentrations in brain homogenates of FTD patients with C9orf72 mutation across four brain regions. We show that soluble DPRs are less abundant in clinically affected areas (i.e. frontal and temporal cortices). In contrast, the cerebellum not only shows the largest DPR load but also the highest relative DPR solubility. Finally, poly-GR levels and poly-GP solubility correlate with clinical severity. These findings provide the first cross-comparison of soluble and insoluble forms of all sense DPRs and shed light on the distribution and role of soluble DPRs in the etiopathogenesis of human C9orf72-FTD.

Highlights

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are considered to be part of a disease spectrum most often caused by a non-coding hexanucleotide GGGGCCrepeat expansion in the C9orf72 gene [10, 14, 24]
Sense and Quaegebeur et al acta neuropathol commun (2020) 8:184 antisense expanded repeat transcripts undergo unconventional repeat-associated non-AUG dependent (RAN) translation generating five different proteins of repeating dipeptides referred to as dipeptide repeat proteins (DPRs) [2, 12, 22, 23]. These Dipeptide repeat protein (DPR) accumulate in aggregated form, which in post-mortem brains of C9orf72-mutant frontotemporal dementia (FTD) and/or ALS patients are visible on immunohistochemistry as p62-positive, predominantly neuronal cytoplasmic inclusions [1, 27]
Patient demographics Soluble and insoluble fractions of dipeptide repeat proteins (DPRs) poly-GP, poly-GA and poly-GR were measured in post-mortem brain from 13 subjects carrying a hexanucleotide repeat expansion mutation in the C9orf72 gene

Summary

Introduction

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are considered to be part of a disease spectrum most often caused by a non-coding hexanucleotide GGGGCCrepeat expansion in the C9orf gene [10, 14, 24]. Sense and Quaegebeur et al acta neuropathol commun (2020) 8:184 antisense expanded repeat transcripts undergo unconventional repeat-associated non-AUG dependent (RAN) translation generating five different proteins of repeating dipeptides (poly-GA, poly-GP, poly-GR, poly-PR and poly-PA) referred to as dipeptide repeat proteins (DPRs) [2, 12, 22, 23]. These DPRs accumulate in aggregated form, which in post-mortem brains of C9orf72-mutant FTD and/or ALS patients are visible on immunohistochemistry as p62-positive, predominantly neuronal cytoplasmic inclusions [1, 27]. Two recent studies showed a relationship between poly-GR inclusions and neurodegeneration [25, 26] with additional evidence that methylated poly-GR correlates with clinical severity [15]

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