Patients and Samples
Muscle biopsy specimens from 50 patients with inflammatory myopathies (IBM N=23; polymyositis N=9; dermatomyositis N=18), 10 patients with genetically-determined myopathies (4 with hereditary inclusion body myopathies, 2 suspected and 1 confirmed VCP mutations, and 1 suspected GNE mutation; 2 with clinical and histopathological diagnoses of myofibrillar myopathy but with unconfirmed mutations; and 1 each with confirmed mutations in dystrophin, ZNF9, calpain, and ryanodine receptor), 3 patients with neurogenic atrophy, and 4 normals underwent immunohistochemical studies for TDP-43. Subsets of these and other samples were studied with additional methods. Patients with IBM fulfilled criteria for definite or possible IBM;14
patients with polymyositis (PM) or dermatomyositis (DM) fulfilled criteria for definite or probable PM or DM.15
No patient with IBM received corticosteroids for treatment of the myopathy at any time. Normal subjects had no symptoms, signs, laboratory findings, or pathological abnormalities of a neuromuscular disease. Muscle biopsies were performed for diagnostic purposes. Blood samples from 6 patients with IBM were analyzed for the presence of TDP-43 mutations. Patients provided informed consent for research studies, as approved by our institutional review boards.
Serial sections and counting methods used in studies
From muscle samples of 50 patients with inflammatory myopathies, we performed serial 10 μm sections and stained 1 section with hematoxylin and eosin (H&E) and an adjacent section for TDP-43 and DNA with fluorescent molecules in all samples. Varying numbers of further adjacent sections were stained for Congo red and the fluorescent combinations of TDP-43/SMI-31/DAPI, TDP-43/MHCf/DAPI, and TDP-43/R1282/DAPI. For quantitation of the number of myofibers with sarcoplasmic TDP-43, two investigators (MS and SG) independently examined microscopic sections at 400x, randomly choosing fields and counting all myofibers in each field until at least 150 myofibers per patient section were counted. Each investigator was blinded to the other's results.
Ten-micron cryostat sections were fixed in either cold (5°C) 4% paraformaldehyde (PFA) for 5 minutes and then soaked consecutively in cold (5°C) 0.05 M Tris buffer, pH 7.5, room temperature Tris buffer, or were fixed in cold acetone (−10° ± 5°C) for 5 minutes and soaked in Tris buffer at room temperature. Tissue sections were transferred to 0.05 M Tris-saline-Triton X-100 buffer (TBS-T), pH 7.5, supplemented with 4% porcine serum for immunohistochemistry or to TBS-T for immunofluorescence. The latter tissue sections were incubated for 30 minutes with Image-iT FX signal enhancer reagent (cat. no. I36933, Molecular Probes, Inc./Invitrogen, Eugene, OR), although omitting this step did not appear to diminish the fluorescence signal-to-noise ratio. These slides were rinsed and soaked in TBS-T, then soaked in 0.05 M Tris-Brij-35 buffer, pH 7.5, supplemented with 2% bovine serum albumin. Following all incubations, slides were rinsed and soaked in TBS-T, and soaked in the same Tris-porcine serum buffer or Tris-bovine serum albumin buffer, respectively, prior to a subsequent step.
The primary antibodies used were rabbit polyclonal anti-TDP-43 (antibody to TAR DNA-binding protein 43, cat. no. 10782−2-AP, ProteinTech Group, Inc., Chicago, IL), mouse monoclonal anti-myosin heavy chain-fast (MHC-fast, cat. no. NCL-MHCf, clone WB-MHCf, isotype IgG1, Novocastra/Vision BioSystems, Inc., Norwell, MA/Leica Microsystems), mouse monoclonal antibody (SMI-31, ascites fluid) to neurofilaments, phosphorylated epitope (cat. no. SMI-31R, clone SMI-31, isotype IgG1, Covance Research Products, Inc., Berkeley, CA), mouse monoclonal anti-emerin antibody (cat. no. VP-E602, clone 4G5, isotype IgG1, Novacastra Laboratories Ltd., Newcastle upon Tyne, UK; obtained from Vector Laboratories Inc., Burlingame, CA), and rabbit polyclonal antibody R1282 directed against beta-amyloid (provided by Dr. Dennis J. Selkoe).
IHC and IF studies with TDP-43 antibody (PFA fixation,1:2000, 0.27 μg/ml, overnight) were carried out in similar fashion. Secondary antibodies were horseradish peroxidase-conjugated polymer bound to goat anti-rabbit immunoglobulins (cat. no. DPVR-110HRP, 30 mins., anti-rabbit PowerVision, ImmunoVision Technologies, Co./Vision BioSystems, Inc./Leica Microsystems) and Alexa Fluor 555 (or 488)-labeled goat anti-rabbit immunoglobulins (1:400, 5 μg/ml, 65 mins., Molecular Probes, Inc./Invitrogen), respectively. Dual staining (IF) of PFA fixed tissue sections with TDP-43 (1:2000, 0.27 μg/ml, overnight) and MHC-fast (1:60 dilution of reconstituted lyophilized tissue culture supernatant, 1 hr.) was carried out in sequence, followed by incubation with an admixture of Alexa Fluor 555-labeled goat anti-rabbit immunoglobulins and Alexa Fluor 488-labeled goat anti-mouse immunoglobulins (each at 1:400 dilution and 5 μg/ml, 1 hr. incubation; Molecular Probes, Inc./Invitrogen). With the same protocol (IF), dual staining (PFA fixation) with TDP-43 and mouse monoclonal anti-Emerin (1:100, 0.84 μg/ml, 90 mins.) was followed by incubation with an admixture of Alexa Fluor 488-labeled goat anti-rabbit immunoglobulins and Alexa Fluor 555-labeled goat anti-mouse immunoglobulins (each at 1:400 dilution and 5 μg/ml, 1 hr. incubation; Molecular Probes, Inc./Invitrogen).
Similarly, IHC and IF staining with SMI-31 (PFA or no fixation, 1:10,000, overnight) utilized secondary antibody horseradish peroxidase-conjugated polymer bound to goat anti-mouse immunoglobulins (cat. no. DPVM-110HRP, 30 mins, anti-mouse PowerVision, ImmunoVision Technologies, Inc.) and Alexa Fluor 488-labeled goat anti-mouse immunoglobulins (1:400 dilution, 5 μg/ml, 65 mins., Molecular Probes, Inc./Invitrogen), respectively. Dual staining (IF) of PFA fixed tissue sections with TDP-43 antibody was carried out overnight. An admixture of SMI-31 and TDP-43 antibodies contained each antibody at a final dilution as previously used. Secondary antibodies were an admixture of Alexa Fluor 555-labeled goat anti-rabbit immunoglobulins and Alexa Fluor 488-labeled goat anti-mouse immunoglobulins (each at 1:400 dilution and 5 μg/ml, 65 mins. incubation; Molecular Probes, Inc./Invitrogen). IF staining with R1282 antibody (1:1000, 20 hrs.) utilized secondary antibody Alexa Fluor 555-labeled goat anti-rabbit immunoglobulins (1:400, 5 μg/ml, 2 hrs., Molecular Probes, Inc./Invitrogen).
Congo red histochemistry
Frozen muscle sections were stained with Congo red (cat. no. C-580, Certified Biological Stain, total dye content 98%, C. I. no. 22120, Fisher Scientific, Pittsburgh, PA) based on the procedure of Puchtler et al.25
as described by Mendell et al.18
Whole muscle lysates (WML) were prepared using 5 mg of cryostat sectioned muscle dounce homogenized in 200 μl of lysis buffer (containing 20 mM Tris pH 7.6, 2% SDS, 5 mM DTT), centrifuged at 10,000 g for 10 min at 4°C and the supernatant removed. The micro BCA assay (Pierce, Rockford, IL) was used to determine protein concentration, and the fractions were stored at −80°C. For SDS-PAGE, 30 μg of WML from each sample was diluted with NuPAGE LDS Sample Buffer (4X) (Invitrogen, Carlsbad, CA), reduced with 10 mM DTT, heated at 95°C for 10 minutes, centrifuged at 2000 g for 10 minutes, loaded onto 4−12% Bis-Tris Gels (Invitrogen), and resolved using MOPS running buffer (Invitrogen) at a voltage of 100−150 mV. The gels were transferred to a nitrocellulose membrane using NuPAGE Transfer Buffer (Invitrogen) at 30 mV for 1.5 hours, washed in PBS including 0.1% Tween-20 (PBST0.1%), blocked for 1 hr in 5% fat free milk in PBST0.1% (5%milk/PBST0.1%) and stored at 4°C.
Immunoblotting was carried out by incubating the membranes with rabbit anti-TDP-43 (cat# 10782−2-AP, ProteinTech Group; 1:1000 dilution overnight at 4°C), and after washing, with goat anti-rabbit HRP (cat # ab6721, Abcam, Cambridge, MA; 1:5000 dilution for 1 hour at room temperature). After stripping the blots using Restore Western Blot Stripping Buffer (cat #21062, Pierce) they were incubated with rabbit anti-actin (cat # sc-1616, Santa Cruz, Santa Cruz, CA; 1:10,000 dilution for 1 hour at room temperature), and after washing, with goat anti rabbit HRP (cat # ab6721, Abcam; 1:10,000 dilution for 1 hour at room temperature). SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford IL) and Kodak films were used for visualization of the bands.
TDP-43 transcript measurement by microarrays
A subset of patients had muscle samples available for additional microarray experiments. Microarray experiments were performed on 25 inflammatory myopathy (IBM N=9, PM N=6, and DM N=10) and 10 normal muscle samples as previously described using Affymetrix HU-133A arrays representing approximately 18,000 genes.11
Gene expression levels were calculated using GC-Content Robust Multichip Analysis (GCRMA).31
Affymetrix probeset 200020_at representing TARDBP was used for TDP-43 transcript abundance.
TDP-43 gene sequencing
DNA was purified from 50 μl of human peripheral blood mononuclear cells (PBMCs) from 6 patients with IBM using the Qiagen DNeasy Blood and Tissue kit (cat #69505). Purified DNA quality and concentration were assessed using a Beckman Coulter DU-800 spectrophotometer and requiring a 260/280 ratio of greater than 1.7. All the exons and the intron-exon boundaries of TARDBP gene were PCR amplified with intronic primers, and sequencing of the amplified fragments was performed using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Wellesley, MA) using standard protocols. Reactions were run on an ABI3130, and mutation analysis was performed using Sequencher software v4.6 (Gene Codes Corporation, Ann Arbor, MI).