A mutation in the C9ORF72 gene causes amyotrophic lateral sclerosis (ALS) through the accumulation of G4C2 RNA. Therapeutics that target G4C2 RNA are thus being developed. Testing these therapeutics in patients with “c9ALS” will depend on finding a marker to monitor the effect of treatments on G4C2 RNA. Gendron et al. demonstrate that poly(GP) proteins produced from G4C2 RNA are present in cerebrospinal fluid from c9ALS patients. Furthermore, using patient cell models and a mouse model of c9ALS, they report that poly(GP) proteins correlate with G4C2 RNA, suggesting that poly(GP) could be used to test potential treatments for c9ALS in upcoming clinical trials.
There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G4C2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G4C2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72-associated ALS (c9ALS). Therapeutics that target G4C2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G4C2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G4C2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G4C2 RNA and downstream G4C2 RNA–mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G4C2 RNA–based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention.
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.
To test our hypothesis, we used an international sampling of subjects (table S1) to (i) replicate our finding that poly(GP) is present in CSF from C9ORF72 mutation carriers (17), (ii) compare poly(GP) proteins in CSF between asymptomatic and symptomatic carriers, and (iii) examine the longitudinal profile of CSF poly(GP). Given these three primary analyses, P ≤ 0.017 was considered significant after Bonferroni adjustment.
Our CSF series comprised samples from 83 c9ALS patients [71 with c9ALS alone and 12 with comorbid FTD (c9ALS-FTD)] and 27 asymptomatic C9ORF72 repeat expansion carriers. CSF collected longitudinally was available for 33 of these subjects. Also included were samples from 24 C9ORF72 repeat expansion carriers clinically diagnosed with diseases other than c9ALS or c9ALS-FTD [c9FTD (n = 20), Alzheimer’s disease (n = 2), bipolar disease (n = 1), and dementia with Lewy bodies (n = 1)] and from 120 individuals without the C9ORF72 mutation. The latter encompassed patients with ALS (n = 57) or other neurological diseases [FTD (n = 4), Alzheimer’s disease (n = 10), and primary lateral sclerosis (n = 1)], as well as healthy controls (n = 48). Subject characteristics are provided in Table 1.