Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity

Abstract

IntroductionIntronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored.MethodsTo study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs).ResultsWe found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs.DiscussionThis study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

Functional Implications of Protein Arginine Methyltransferases (PRMTs) in Neurodegenerative Diseases

“…Therefore, we conclude that these two diseases may share some pathological mechanisms [ 58 ]. The mean survival of FTD ranges between 8 and 12 years, depending on the specific phenotype, while in ALS, mean survival is 1–5 years, with most patients dying due to respiratory paralysis [ 59 , 60 , 61 , 62 ].…”

“…Various roles of protein arginine methylation as a response to cell stress have also been described, such as chromatin remodeling, splicing, and stress granule dynamics in C9orf72 mutations. A possible association between the C9orf72 protein and PRMTs in regulating pathways in neurodegenerative disorders has been detected [ 61 ]. Moreover, PRMTs may regulate autophagy [ 61 ].…”

“…A possible association between the C9orf72 protein and PRMTs in regulating pathways in neurodegenerative disorders has been detected [ 61 ]. Moreover, PRMTs may regulate autophagy [ 61 ]. This is important, as symmetric dimethylation of stress granule proteins by PRMT5 has been found to reduce stress granules by autophagy [ 63 ].…”

Atypical Neuropsychiatric Presentation of FTD-ALS Caused by a Pathogenic Repeat Expansion in C9orf72: A Case Report

“…However, the development of these has been challenging, in that there may be both a toxic gain of function effect from the accumulation of expanded RNA foci and the accumulation of abnormal dipeptide repeat proteins (DPRs), as well as loss of function effect from a reduction in translation of C9orf72. 31 Despite the challenges, antisense oligonucleotide therapeutics to bind and remove toxic RNA are being clinically developed. 32,33 Small molecule approaches, such as repurposing Metformin to reduce toxic C9orf72 translation products, and new molecules such as TPN101 to reduce CNS inflammation are also in development.…”

Translation of dipeptide repeat proteins in C9ORF72 ALS/FTD through unique and redundant AUG initiation codons

“…The hexanucleotide GGGGCC repeat expansion in the first intron of C9ORF72 is the most common monogenic cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) ( DeJesus-Hernandez et al, 2011 ; Renton et al, 2011 ). This mutation is predicted to cause ALS/FTD via three non-mutually exclusive mechanisms: (1) a loss-of-function mechanism due to reduced C9ORF72 protein expression ( Liu et al, 2022b ; Banerjee et al, 2023 ; Dane et al, 2023 ; Zhu et al, 2020 ), (2) a gain-of-function mechanism due to toxicity from repeat-containing sense (GGGGCC) and antisense (CCCCGG) RNA ( McEachin et al, 2020 ; Parameswaran et al, 2022 ), and (3) toxicity from dipeptide repeat (DPR) proteins produced from these transcripts ( Loveland et al, 2022 ; Taylor et al, 2016 ; Kwon et al, 2014 ; Wen et al, 2014 ). However, loss of C9ORF72 protein by itself does not cause neurodegeneration ( Koppers et al, 2015 ).…”