INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive motor neuron disease that typically results in muscle atrophy, paralysis, and eventually death within 5 years of onset. Up to 50% of ALS patients develop cognitive and behavioral impairments, and ~15% fulfill the criteria for frontotemporal dementia (FTD), which is characterized by changes in personality, behavior, and language (1).

Only one minimally effective drug, riluzole, is approved for ALS despite more than 30 clinical trials conducted since 1995. The dearth of ALS therapeutics stems partly from an incomplete understanding of the causative pathomechanisms. However, discovery of a G₄C₂ repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS and FTD (2, 3) has resulted in impressive efforts toward elucidating how this mutation causes c9ALS (C9ORF72-associated ALS) or c9FTD, collectively referred to as c9ALS/FTD. Although the normal number of C9ORF72 G₄C₂ repeats is lower than 30, c9ALS/FTD patients have several hundred to several thousand (4). Putative pathomechanisms associated with G₄C₂ repeat expansions include loss of C9ORF72 function as well as toxicity stemming from the accumulation of sense and antisense transcripts of the expanded repeats. These RNA transcripts assemble into structures called foci, aberrantly interact with RNA binding proteins, and cause defects in nucleocytoplasmic transport (5, 6). They additionally serve as templates for the synthesis of proteins of repeating dipeptides through repeat associated non-ATG (RAN) translation (7–11). Poly(GP), poly(GA), and poly(GR) proteins are produced from sense G₄C₂-containing transcripts, whereas poly(GP), poly(PA), and poly(PR) proteins are produced from antisense G₂C₄-containing transcripts. Neuronal inclusions of these so-called c9RAN proteins are pathognomonic to c9ALS/FTD, and studies show that certain c9RAN proteins, such as poly(GA), poly(GR), and poly(PR), are toxic in in vitro and in vivo overexpression models (5, 6). Potential mechanisms of toxicity associated with c9RAN proteins include nucleolar stress, impaired proteasomal function, and, as with G₄C₂ repeat RNA, impaired nucleocytoplasmic transport (5, 6).

On the basis of the expanding body of evidence supporting the role of G₄C₂ repeat RNA and c9RAN proteins in c9ALS/FTD pathogenesis, therapeutic approaches that target G₄C₂ RNA are being actively pursued. For example, antisense oligonucleotides (ASOs) complementary to G₄C₂ RNA or C9ORF72 transcripts (c9ASOs) decrease G₄C₂-containing RNA and consequently decrease the number of cells with RNA foci, as well as mitigate abnormalities in gene expression and nucleocytoplasmic transport in neurons differentiated from c9ALS patient-derived induced pluripotent stem cells (iPSCs) (12–14). In primary neurons and brain tissues from c9BAC mice expressing expanded G₄C₂ repeats, c9ASOs decrease G₄C₂ repeat–containing RNA, foci formation, and production of poly(GP) proteins (15, 16). Moreover, small-molecule binders of G₄C₂ RNA inhibit foci formation and RAN translation in patient-derived cell models (17).

Because possible therapeutics for c9ALS/FTD are being developed for clinical trials, it is paramount to address barriers in moving a treatment from bench to bedside. Chief among these is the lack of markers capable of predicting disease progression, monitoring the response to therapy, and confirming target engagement. Given that c9RAN proteins are synthesized from G₄C₂ repeat RNA, the target of therapeutic interventions under investigation, we anticipate that c9RAN proteins in cerebrospinal fluid (CSF) will reflect target engagement and biochemical responses to treatment. Although three c9RAN proteins are produced from G₄C₂ RNA, namely, poly(GP), poly(GA), and poly(GR), we believe that poly(GP) may be an especially suitable marker candidate. Both poly(GP) and poly(GA) are more highly expressed in the central nervous system (CNS) of c9ALS/FTD patients than poly(GR) (18). However, poly(GP) is more likely to be accurately measured in biospecimens because it is more soluble than poly(GA) (19). Indeed, in a small cohort of c9ALS patients, we established that poly(GP) can be detected in CSF (17). Thus, to prepare for upcoming clinical trials for c9ALS, the present study used patient CSF and several preclinical models to investigate the hypothesis that poly(GP) proteins could serve as an urgently needed pharmacodynamic marker for developing and testing therapies for treating c9ALS.