Dipeptide repeat (DPR) pathology in the skeletal muscle of ALS patients with C9ORF72 repeat expansion
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C9ORF72 expansion is the most common genetic alteration in both familial and sporadic ALS . Dipeptide repeat (DPR) proteins are generated from repeat-associated non-ATG (RAN) translation of mutant C9ORF72 transcripts and may be toxic to cells [4, 14] in addition to toxicity resulting from RNA foci . DPRs in postmortem tissue are a pathologic hallmark of C9ORF72-associated amyotrophic lateral sclerosis (C9ALS), being identified in small neurons of hippocampus and cerebellum, as well as in neocortical neurons  and rarely in motor neurons . DPRs may be identified by antibodies to poly-glycine–alanine (GA) (generated via sense translation), as well as to poly-glycine–proline (GP) (sense and antisense) and glycine–arginine (GR) (sense). Poly-GA and poly-GP inclusions are the most abundant DPRs in postmortem tissue  and are also recognized by p62 and ubiquitin .
Several studies have also suggested that DPRs are not limited to neurons. One study of human tissue, applying antibodies against GGGGCC RAN-translated peptides, showed DPRs predominantly in neurons, but also in Sertoli cells . Subsequent work showed DPRs in C9ALS patient ependymal cells . A recent study of a zebrafish C9ORF72 model demonstrated DPRs in skeletal muscle , as well as motor neuron loss and muscle atrophy. Similarly, a transgenic fly C9ORF72 model showed distinct perinuclear poly-GP inclusions in muscle . This suggests that DPR pathology in skeletal muscle may also contribute to the ALS phenotype in disease models. It is not known whether DPR pathology is present in human ALS skeletal muscle, and thus potentially a contributing factor in C9ALS pathogenesis.
To address this question, we examined DPR inclusion pathology in 68 C9ALS skeletal muscle samples obtained from autopsies at Houston Methodist Hospital (HMH) and Mayo Clinic Jacksonville (MCJ). We assessed samples for the most common DPRs in human disease (poly-GA, poly-GP), poly-GR, as well as p62, N-terminal TDP-43, and phospho-TDP-43 (pTDP-43) pathology, using previously described methods [2, 5] (please also see Supplemental file for complete Methods). We also examined whether inclusion pathologies were associated with salient disease features, including the degree of muscle fiber atrophy, age at patient death, and disease duration.
DPRs were significantly associated with more severe atrophy in samples (Wilcoxon rank-sum Z = 4.4, P = 1.12E − 05) (see Supplemental Table 2). DPR pathology did not significantly associate with age at death, disease duration, or pTDP-43 pathology. Among samples with any inclusion pathology, 40% of patients had DPRs and pTDP-43, 32% had DPRs only, and 28% had pTDP-43 only. DPR-positive muscle samples were from patients with lower extremity (44.4%), upper extremity (22.2%) and bulbar (22.2%) onset sites. pTDP-43-positive and negative muscle samples did not significantly differ with respect to disease duration (P = 0.37), though a trend was seen for pTDP-43 pathology in younger C9ALS patients (P = 0.06). DPR-positive and pTDP-43-positive samples did not significantly differ in frequency by axial muscle group.
These results expand the spectrum of dipeptide repeat pathology in C9ALS to include patient skeletal muscle. Significantly greater atrophy was identified in muscle samples with poly-GA and poly-GP inclusions, suggesting a potential contribution of DPRs to C9ALS muscle pathology. Poly-GR inclusions were not seen. These may be absent in C9ALS muscle, or as in brain, are too sparse in muscle to be identified without further sampling (poly-GA and poly-GP inclusions themselves were sparse). RAN-translated peptides were not seen in three patient muscle samples in a prior study . A separate case report of a C9ALS patient muscle biopsy reported pleomorphic, ubiquitin and p62-positive cytoplasmic inclusions in muscle fibers  that more closely resemble the cytoplasmic p62 and pTDP-43-positive inclusions demonstrated here and shown previously . Although this is the first demonstration of DPRs in C9ALS patient muscle, earlier studies have shown DPRs in the skeletal muscle of transgenic zebrafish  and fly models of C9ALS . RNA foci have also been shown in iPSC-derived skeletal muscle from patients with C9ORF72 expansion . Future studies are needed to determine whether skeletal muscle-restricted expression of mutant C9ORF72 transcripts is sufficient to drive an ALS phenotype, and if so, whether there is a beneficial response in these models to antisense oligonucleotide therapy .
Funding was provided by a Clinician-Scientist Recruitment and Retention Program Award from the Houston Methodist Research Institute and an Investigator-initiated research award from the ALS Association. The authors appreciate the efforts of the HMH electron microscopy core and especially Ms. Claire Haueter. We are grateful to the ALS patients and their families for making this research possible.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in this study were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was also carried out with IRB approval from Houston Methodist Hospital (IRB-(3 N)-0114–0013).
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