Altered Phase Separation and Cellular Impact in C9orf72-Linked ALS/FTD

Abstract

Since the discovery of the C9orf72 repeat expansion mutation as causative for chromosome 9-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in 2011, a multitude of cellular pathways have been implicated. However, evidence has also been accumulating for a key mechanism of cellular compartmentalization—phase separation. Liquid-liquid phase separation (LLPS) is fundamental for the formation of membraneless organelles including stress granules, the nucleolus, Cajal bodies, nuclear speckles and the central channel of the nuclear pore. Evidence has now accumulated showing that the formation and function of these membraneless organelles is impaired by both the toxic arginine rich dipeptide repeat proteins (DPRs), translated from the C9orf72 repeat RNA transcript, and the repeat RNA itself. Both the arginine rich DPRs and repeat RNA themselves undergo phase separation and disrupt the physiological phase separation of proteins involved in the formation of these liquid-like organelles. Hence abnormal phase separation may explain a number of pathological cellular phenomena associated with C9orf72-ALS/FTD. In this review article, we will discuss the principles of phase separation, phase separation of the DPRs and repeat RNA themselves and how they perturb LLPS associated with membraneless organelles and the functional consequences of this. We will then discuss how phase separation may impact the major pathological feature of C9orf72-ALS/FTD, TDP-43 proteinopathy, and how LLPS may be targeted therapeutically in disease.

Role of the Ubiquitin System in Stress Granule Metabolism

“…Another source of aggregation-prone proteins at SGs are misfolded proteins that evade or overwhelm cellular PQC systems. Hexanucleotide repeat expansions in the C9ORF72 gene are the most frequent genetic alteration found in ALS/FTD patients [ 102 , 103 ]. Importantly, they were shown to cause the accumulation of SGs in an induced pluripotent stem cell model of ALS/FTD in the absence of exogenous stress [ 104 ].…”

“…Importantly, they were shown to cause the accumulation of SGs in an induced pluripotent stem cell model of ALS/FTD in the absence of exogenous stress [ 104 ]. Among other potentially pathogenic effects, the hexanucleotide repeat expansions lead to the non-canonical translation of dipeptide repeat proteins, which have a high propensity to undergo LLPs in vitro and, at least upon ectopic overexpression, accumulate at SGs in cells, thus perturbing SG dynamics [ 31 , 103 , 105 , 106 ]. Similar to dipeptide repeat proteins, misfolding-prone variants of proteins that are not normally present at SGs can change the properties of SGs.…”

p53 Transactivation Domain Mediates Binding and Phase Separation with Poly-PR/GR

“…DPRs undergo phase separation in the presence of a crowding agent in vitro [ 37 ]. Overexpression of poly-PR/GR in cells increases the formation of stress granules, whereas they disrupt nucleolus structure and function [ 35 , 36 , 37 ] which in turn is associated with p53 stabilization during DNA damage response [ 38 ]. Additionally, overexpression of poly-PR/GR DPRs in neuronal cells triggers DNA damage [ 39 ].…”

p53 Transactivation Domain Mediates Binding and Phase Separation With poly-PR/GR

“…DPRs undergo phase separation in the presence of a crowding agent in vitro [37]. Overexpression of poly-PR/GR in cells increases formation of stress granule whereas they disrupt nucleolus function [35][36][37].…”

“…DPRs undergo phase separation in the presence of a crowding agent in vitro [37]. Overexpression of poly-PR/GR in cells increases formation of stress granule whereas they disrupt nucleolus function [35][36][37]. Besides that, forcing nucleolar disruption by anti-upstream binding factor microinjection stabilizes p53 [38].…”