Dipeptide repeat (DPR) pathology in the skeletal muscle of ALS patients with C9ORF72 repeat expansion

C9ORF72 expansion is the most common genetic alteration in both familial and sporadic ALS [9]. Dipeptide repeat (DPR) proteins are generated from repeat-associated nonATG (RAN) translation of mutant C9ORF72 transcripts and may be toxic to cells [4, 14] in addition to toxicity resulting from RNA foci [13]. 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 [8] and rarely in motor neurons [6]. 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 [8] and are also recognized by p62 and ubiquitin [10]. Several studies have also suggested that DPRs are not limited to neurons. One study of human tissue, applying antibodies against GGG GCC RAN-translated peptides, showed DPRs predominantly in neurons, but also in Sertoli cells [1]. Subsequent work showed DPRs in C9ALS patient ependymal cells [10]. A recent study of a zebrafish C9ORF72 model demonstrated DPRs in skeletal muscle [11], as well as motor neuron loss and muscle atrophy. Similarly, a transgenic fly C9ORF72 model showed distinct perinuclear poly-GP inclusions in muscle [3]. 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, polyGP), 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 identified in 28 of 68 samples (41.2%) from 18 of 37 patients (48.6%). Figure 1 shows representative poly-GA, poly-GP, and p62 images from patient samples from MCJ (ALS02, ALS05) and HMH (ALS28, ALS35). DPR inclusions were comparable in size and shape to those in small neurons of dentate gyrus. DPRs were typically perinuclear and most conspicuous in atrophic fibers and were revealed by both poly-GA and poly-GP immunohistochemistry. Poly-GR immunohistochemistry, performed in a subset of 18 cases, was negative. Figure 1d, g, and j show MCJ staining with the remaining studies shown performed at HMH. Rare DPRs appeared intranuclear (please see Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0040 1-019-02050 -8) contains supplementary material, which is available to authorized users.

Supplemental Figure 1. DAPI (a, e), TRITC (pTDP-43) (b, f) and FITC (p62) (c, g) channel data, as well as the merged images (d, h), for two study cases from MCJ (ALS08, ALS06). Please see Supplemental Methods section for detail on immunofluorescence preparations and imaging. Merged images were generated in Olympus cellSens software from DAPI, TRITC, and FITC channel data. For panels i-l, immunohistochemical preparations on sequential five-micron sections show p62 and pTDP-43 inclusions in the same position and muscle fiber in study cases ALS06 (i, j) and ALS16 (k, l). All images taken using a 60x objective and further enlarged for detail. Scale bars in panels a, e, i, and k apply for panels a-d, e-h, i-j, and k-l, respectively.
Supplemental Figure 2. Rare intranuclear (b, d, and f) inclusions with poly-GA immunohistochemistry and more typical perinuclear (g, h) inclusions as shown in two study cases from HMH (ALS35, ALS37). Samples shown are from paraspinous muscle ("Para") or diaphragm ("DPG"). All photomicrographs were taken with a 60x objective and enlarged for detail (the scale bar shown in panel a applies to all images). Black arrows in panels b, d, and f indicate the subsarcolemmal nuclei (b, f), or centrally located (d) nucleus, and white arrows in panels b, d, f, g, and h indicate the poly-GA inclusion. For approximate reference, H&E sections from the same focus in the sample are shown for panels a, c, and e, but the H&E and poly-GA IHC slides shown are not consecutive sections and are separated by two five-micron thick sections.

METHODS
Sample collection and preparation: For Houston cases, all of the ALS patients came to autopsy at Houston Methodist Hospital (HMH) and were representative of the C9ALS patients seen at our institution. No C9ALS patients were excluded. These patients were evaluated in the clinic of study author SHA and autopsies were performed by study authors MDC, SZP, or ALR. For Houston cases, autopsy blocks were, blocked, and processed after a period of fixation of one week in 20% neutral buffered formalin. Muscles routinely sampled in our autopsy protocol include paraspinous (cervical, thoracic, lumbar, and sacral levels), diaphragm, deltoid, biceps, and quadriceps. This project utilized only one paraspinous and one diaphragm sample per patient. For these anatomic sites, formalin-fixed, paraffin-embedded tissue was sectioned at 5 µm, mounted on positively charged slides, and dried at 60°C. Autopsy samples collected at Mayo Clinic Jacksonville (MCJ) were collected per their autopsy protocol under the supervision of author DWD. MCJ tissues used in this study were diaphragm and iliopsoas muscle to most closely match the axial muscle samples studied at HMH. Unstained sections of muscles of interest from MCJ were sent to the lab of study author MDC at HMH. All work on this project was performed with the approval of the Institutional Review Board at HMH (IRB 2-0114-0013).
Immunohistochemical procedures: Deparaffinization and rehydration steps were carried out using sequential washes of reagent grade xylene, graded alcohols and water. Heat-based antigen retrieval was performed using a 1X antigen retrieval solution at pH 9 (Agilent Technologies; Santa Clara, CA) for one hour (95°C for 30 min followed by 30 min on ice). All washing steps were carried out using a commercial Tris-buffered saline solution (1×) containing Tween 20, pH 7.6 (Agilent Technologies). Endogenous peroxide was blocked using a 3% hydrogen peroxide solution (VWR International; Radnor, PA). Primary antibody was applied for at least one hour at 4°C following a one hour blocking step at room temperature with 2.5% horse serum (Vector Laboratories; Burlingame, CA). Slides were thoroughly washed and the ImmPress horseradish peroxidase (HRP) anti-rabbit and anti-mouse IgG detection kits (Vector Laboratories) were applied as appropriate for 1 hour at room temperature. After additional washing steps, the target antigen was visualized using DAB chromogen in substrate buffer (Agilent Technologies). Hematoxylin counterstain was applied after additional washing steps and slides were brought to xylene and mounted with Permount (ThermoFisher Scientific; Waltham, MA).
Immunofluorescence procedures: Immunofluorescence preparations were prepared in select samples for pTDP-43 and p62 co-labeling (primary review of these antibodies was done by immunohistochemistry). Slides were incubated overnight with primary antibodies at 4°C following deparaffinization, rehydration, and antigen retrieval procedures and a blocking step (2.5% horse serum), all as described above (washing steps were performed with fresh phosphate-buffered saline). Secondary antibodies were applied for 1 hour at room temperature, including Alexa Fluor 555 Anti-Rabbit IgG (1:200; A21429) and Alexa Fluor 488 anti-Mouse IgG (1:200; A11001) (Alexa Fluor are products of ThermoFisher). Primary and secondary