RESEARCH ARTICLE

Isoform-Specific Antibodies Reveal Distinct Subcellular Localizations of C9orf72 in Amyotrophic Lateral Sclerosis Shangxi Xiao,1 Laura MacNair, B.Sc. (Hons),1,2 Philip McGoldrick, PhD,1 Paul M. McKeever, B.Sc. (Hons),1,2 Jesse R. McLean, PhD,1 Ming Zhang, PhD,1 Julia Keith, MD,2,3 Lorne Zinman, MD,2,3 Ekaterina Rogaeva, PhD,1 and Janice Robertson, PhD1 Objective: A noncoding hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). It has been reported that the repeat expansion causes a downregulation of C9orf72 transcripts, suggesting that haploinsufficiency may contribute to disease pathogenesis. Two protein isoforms are generated from three alternatively spliced transcripts of C9orf72; a long form (C9L) and a short form (C9-S), and their function(s) are largely unknown owing to lack of specific antibodies. Methods: To investigate C9orf72 protein properties, we developed novel antibodies that recognize either C9-L or C9-S. Multiple techniques, including Western blot, immunohistochemistry, and coimmunoprecipitation, were used to determine the expression levels and subcellular localizations of C9-L and C9-S. Results: Investigation of expression of C9-L and C9-S demonstrated distinct biochemical profiles, region-specific changes, and distinct subcellular localizations in ALS tissues. In particular, C9-L antibody exhibited a diffuse cytoplasmic staining in neurons and labeled large speckles in cerebellar Purkinje cells. In contrast, C9-S antibody gave very specific labeling of the nuclear membrane in healthy neurons, with apparent relocalization to the plasma membrane of diseased motor neurons in ALS. Coimmunoprecipitation experiments revealed an interaction of the C9-isoforms with both Importin b1 and Ran-GTPase, components of the nuclear pore complex. Interpretation: Using these antibodies, we have shown that C9orf72 may be involved in nucleocytoplasmic shuttling and this may have relevance to pathophysiology of ALS/FTLD. Our antibodies have provided improved detection of C9orf72 protein isoforms, which will help elucidate its physiological function and role in ALS/FTLD. ANN NEUROL 2015;78:568–583

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exanucleotide (G4C2) repeat expansions within a noncoding region of C9orf72 are the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), accounting for up to 50% of ALS, 29% of FTLD, and 88% of ALS/ FTLD patients, including familial and sporadic cases.1–4 The majority of normal human C9orf72 alleles carry less than 20 repeats, whereas large expansions consisting of hundreds or thousands of repeats lead to disease, though the precise pathological cutoff is not well defined.1,2,5,6 Patients carrying the expansion can show heterogeneous clinical pre-

sentation, and the expansion has been identified in patients with other neurological diseases. 1,2,7–19 Three potential pathomechanisms have been proposed to arise from the repeat expansion in C9orf72: RNAmediated toxicity through generation of RNA foci and sequestration of RNA-binding proteins from their normal targets; expression of dipeptide repeat proteins by repeatassociated non-ATG (RAN) translation; and haploinsufficiency.1,2,20–23 C9orf72 generates three transcripts through alternative splicing that encode 2 protein isoforms; a long isoform of approximately 54 kDa (termed C9-L),

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24469 Received Feb 27, 2015, and in revised form Jun 30, 2015. Accepted for publication Jun 30, 2015. Address correspondence to: Professor Janice Robertson, Tanz Center for Research in Neurodegenerative Diseases, Krembil Discovery Tower, University of Toronto, 60 Leonard Avenue, Toronto, Ontario, Canada ON M5T 2S8. E-mail: [email protected] From the 1Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; and 3Sunnybrook Health Sciences Center, Toronto, Ontario, Canada C 2015 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association. 568 V This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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TABLE. ALS and Control Cases Used in This Study

Case No.

Sex

Age (years)

Diagnosis

Gene Mutation

A1

F

49

sALS

c9-ALS

A2

M

70

ALS

c9-ALS

A3

M

64

ALS/FTLD

c9-ALS

A4

F

58

fALS

c9-ALS

A5

M

51

sALS

c9-ALS

A6

M

60

fALS/FTLD

c9-ALS

A7

M

60

ALS/FTLD

c9-ALS

A8

F

47

ALS/FTLD

c9-ALS

A9

M

57

fALS/FTLD mild AD

c9-ALS

A10

M

61

ALS, mild AD

c9-ALS

A11

F

59

fALS/FTLD

c9-ALS

A12

F

73

ALS

Non-c9-ALS

A13

F

71

ALS

Non-c9-ALS

A14

M

64

ALS

Non-c9-ALS

A15

M

58

ALS

Non-c9-ALS

A16

F

79

ALS/FTLD

Non-c9-ALS

A17

M

57

ALS

Non-c9-ALS

A18

M

52

ALS

Non-c9-ALS

A19

F

71

ALS

Non-c9-ALS

C1

F

91

AD/DLBD

N/A

C2

M

62

NN

N/A

C3

M

67

AD/DLBD

N/A

sALS 5 sporadic ALS; fALS 5 familial ALS; FTLD 5 frontotemporal degeneration; C9orf72 5 chromosome 9 open reading frame 72; N/A 5 not applicable; AD 5 Alzheimer’s disease, DLBD 5 diffuse Lewy body disease; NN 5 non-neurological.

corresponding to variants 2 (V2) and 3 (V3), and a short isoform of approximately 24 kDa (termed C9-S) corresponding to variant 1 (V1).24 Haploinsufficiency was initially suggested as a disease mechanism owing to the decreased abundance of V2 and V3 transcripts in c9-ALS cases, and consistent with this hypothesis, reduced expression of selected or total C9orf72 transcripts in C9orf72 repeat expansion carrier-derived cells or tissues have been widely reported.1,24–31 Although there is little known about the effects of C9orf72 haploinsufficiency, the development of motor phenotypes in zebrafish and Caenorhabditis elegans owing to downregulation of C9orf72 orthologues supports its role as a disease pathomechanism.30,32 It has been predicted that C9orf72 proteins may belong to the DENN-like protein family and play roles in Rabmediated cellular trafficking; however, the precise functions and properties of C9orf72 protein are largely unknown owing to a lack of specific antibodies.33,34 Currently available October 2015

commercial antibodies show a high degree of nonspecific binding and few discriminate between C9-L and C9-S, preventing detailed study of the two isoforms.34 To date, there is a single report of an in-house–generated C9orf72 antibody that was used to detect a significant downregulation of C9-L in the frontal cortex, but not cerebellum, of c9-ALS cases.29 However, this antibody was generated against the Nterminus of C9orf72, which is common to both C9-L and C9-S, therefore preventing discrimination of C9-L and C9-S isoforms in applications such as immunohistochemistry. To investigate the properties of C9orf72 isoforms, we have generated two antibodies that specifically recognize either C9-L or C9-S. Using these antibodies, we have characterized C9-L and C9-S isoforms in ALS patient tissue and demonstrated differential biochemical profiles, region-specific changes in expression levels, and distinct subcellular localizations in neuronal cells. Further investigation into the potential function(s) of C9orf72 isoforms revealed interaction with 569

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Importin b1 and Ran-GTPase, proteins involved in nucleocytoplasmic import, suggesting that C9orf72 isoforms have distinct functions that may be relevant to ALS pathogenesis.

Materials and Methods Patient information, Tissue Collection, and Consent Informed consent was obtained from all participants with approval from the local ethical review board. Patients were diagnosed at the ALS Clinic at Sunnybrook Health Sciences Center in Toronto, using the revised El Escorial Criteria.35 C9orf72 expansion carriers were identified as described previously.3 The list of cases used for the study is shown in the Table.

Generation of C9-Isoform Specific Antibodies To detect C9-L and C9-S proteins, rabbit polyclonal antisera were generated to synthetic peptides corresponding to a unique sequence in C9-L (HEHIYNQRRYMRSE) located at residues 321 to 334 of the full-length protein, and the last 12 amino acids of C9-S, which included a unique lysine residue at the C-terminus derived from partial read through into intron 5 (AAGLysTAAstop), which is partially retained in V1 (IGDSSHEGFLLK), each linked to keyhole limpet hemocyanin. The antisera were subsequently affinity purified using the respective peptide immunogens.

Constructs and Stable Cell Lines The coding sequences of C9-L or C9-S were cloned into peGFP-C2 plasmid at EcoRI and BamHI sites and verified by sequencing. For stable cell lines, the plasmids containing Nterminally-eGFP-tagged C9-L and C9-S, and peGFP-C2 empty vector were linearized using EcoO109I restriction enzyme and transfected into N2a cells using Lipofectamine LTX (15338; Invitrogen, Carlsbad, CA) with 200 mg/ml of neomycin (10131-035; Gibco, Grand Island, NY) used for selection. For antibody characterization, HEK293T cells were transiently transfected with N-terminally-eGFP-tagged C9-L or C9-S using Lipofectamine LTX reagents. After 24 hours of transfection, cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (50mM of Tris [pH 7.5], 150mM of NaCl, 0.1% sodium dodecyl sulphate [SDS], 0.5% sodium deoxycholate, and 1% NP-40) with protease inhibitors (P8340; SigmaAldrich, St. Louis, MO). Cells were maintained in Dulbecco’s modified Eagle’s medium, (11995-065; Gibco) and 10% heatinactivated fetal bovine serum (10082147; Gibco).

Sequential Protein Extraction Approximately 100mg of frozen tissue (frontal cortex, temporal cortex, motor cortex, cerebellum, and lumbar spinal cord) was homogenized using a Tenbroeck Tissue Homogenizer in 1ml of low-salt buffer (10mM of Tris [pH 7.5], 150mM of NaCl, 5mM of ethylenediaminetetraacetic acid [EDTA]). Homogenates were spun at 20,000g for 20 minutes at 4 8C and supernatant was saved as “low-salt fraction.” The remaining pellet was homogenized in 1ml of high-salt buffer (50mM of Tris [pH 7.5], 750mM of NaCl, and 5mM EDTA) before centrifugation

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using the same settings. After centrifugation, the supernatant was taken as the “high-salt fraction” and the remaining pellet was homogenized with 1ml of high-salt buffer containing 1% Triton X-100 and centrifuged as described above. The resultant supernatant was kept as the “high-salt Triton fraction” and the pellet homogenized with 1ml of high-salt buffer with 30% sucrose. After centrifugation, floating myelin and supernatant were removed and the remaining pellet was solubilized with 100 ll of urea buffer (7M of urea, 2M of thiourea, 4% CHAPS, and 30mM of Tris-HCl [pH 8.5]) and saved as the “urea fraction.” Protease inhibitors were added to each buffer before homogenization. All homogenates were stored at –80 8C.

Western blotting SDS (33) sample buffer (187.5mM of Tris-HCl [pH 6.8], 6% SDS, 30% glycerol, 0.03% bromophenol blue, and 15% bmercaptoethanol) was added to homogenates before heating at 95 8C for 5 minutes. For central nervous system (CNS) tissue, approximately 50 lg of samples were then loaded onto 10% Novex Tris-Glycine Gel (EC6078BOX; Life Technologies, Carlsbad, CA) gels and electrophoresed with Novex TrisGlycine SDS Running Buffer (LC2675; Life Technologies). Gels were transferred using Tris-glycine transfer buffer (48mM of Tris, 39mM of glycine, 0.04% SDS, and 20% methanol) to 0.2 lm of PVDF (polyvinylidene fluoride) membrane, then placed in blocking solution 5% skimmed milk powder in Trisbuffered saline (TBS; 50mM of Tris-HCl [pH 7.6] and 150mM of NaCl) for 1 hour at ambient temperature. Primary antibodies were diluted in blocking solution as follows: rabbit anti-C9-S (in-house; 1:1,000); rabbit anti-C9-L (in-house; 1:1,000); rabbit anti-C9orf72 (sc-138763; 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti–glyceraldehyde 3phosphate dehydrogenase (GAPDH; ab8245; 1:2,000; Abcam, Cambridge, MA); mouse anti-b-actin (ab8226; 1:2,000; Abcam); mouse anti-enhanced green fluorescent protein (eGFP; 632569; 1:2,000; Clontech Laboratories, Mountain View, CA); goat anti-Lamin-B (sc-6217; 1:500; Santa Cruz Biotechnology); mouse anti-Ran-GTPase (610340; 1:1,000; BD Biosciences, San Jose, CA); and mouse anti-Importinb-1 (sc-137016; 1:2,000; Santa Cruz Biotechnology), then incubated with the PVDF membranes at 4 8C overnight. After 5 3 10-minute washes with TBS containing 0.05% Tween-20 (Sigma-Aldrich), membranes were incubated with secondary antibodies; antimouse horseradish peroxidise (HRP) conjugated (NA931; 1:5,000; VWR International, Radnor, PA), anti-rabbit HRP conjugated (NA934; 1:5,000; VWR), anti-goat HRP conjugated (R21459; 1:5,000; Molecular Probes, Eugene, OR) diluted in blocking solution for 1 hour at room temperature. Antibody labelling was visualized using Western Lightning Plus ECL (PerkinElmer, Waltham, MA). For quantification, chemiluminescence detection on immunoblots was carried out using a LI-COR Odyssey Imaging System. Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD). Densitometric values for C9-L and C9-S were normalized to b-actin and GAPDH, respectively. Unpaired

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two-tailed t tests were used for statistical analysis (GraphPad Software Inc., La Jolla, CA).

Coimmunoprecipitation of eGFP-Tagged C9-L and C9-S From Mammalian Cell Line N2a cell lines stably expressing eGFP-tagged C9-L, eGFPtagged C9-S, or eGFP empty vector were plated on 15-cm plates. When approximately 85% confluent, cells were harvested in phosphate-buffered saline, pelleted at 500g for 5 minutes, and lysed with 1.6ml of lysis buffer (10mM of TrisHCl [pH 7.5], 150mM of NaCl, 0.5mM of EDTA, and 0.5% NP-40 with protease inhibitors). After lysates were centrifuged at 20,000g for 10 minutes at 4 8C, 1.6ml of the supernatants were collected and diluted with 0.8ml of dilution buffer (10mM of Tris-HCl [pH 7.5], 150mM of NaCl, and 0.5mM EDTA with protease inhibitors). Diluted supernatants were then precleared with magnetic agarose beads for 30 minutes at 4 8C and precipitated using GFP-Trap_magnetic agarose beads (gtma-20; ChromoTek, Hauppauge, NY) for 60 minutes at 4 8C. After thorough washing with dilution buffer (10mM of Tris-HCl [pH 7.5], 150mM of NaCl, 0.5mM of EDTA with protease inhibitors), beads were resuspended in RIPA buffer and boiled for 10 minutes at 958C to dissociate immunocomplexes. Finally, beads were magnetically separated and the supernatants collected and subjected to Western blot analysis.

Immunohistochemistry with 3,30 Diaminobenizidine and Immunofluorescence Staining Six-micrometer sections of paraffin-embedded human ALS and control spinal cord or cerebellum were deparaffinized at 60 8C for 20 minutes on a heat block and incubated in 2 3 5-minute washes of xylene and rehydrated through a series of washes in graded ethanol and finally in water. Antigen retrieval was carried out by pretreating the sections with TE9 buffer (10mM of Trizma base, 1mM of EDTA, and 0.0005% Tween 20 [pH 9]), at 110 8C in a pressure cooker for 15 minutes. For 3,30 -diaminobenizidine (DAB) staining, endogenous peroxidases were depleted by incubation with 3% H2O2 for 10 minutes at room temperature. Slides were blocked for 30 minutes in 5% (w/v) bovine serum albumin, 2.5% normal horse serum, and 0.3% Triton X-100 in TBS. For primary antibodies, rabbit anti-C9S (in-house; 1:2,500 for spinal cord and 1:10,000 for cerebellum) and rabbit anti-C9-L (in-house; 1:3,000 for spinal cord and 1:10,000 for cerebellum) were diluted in Dako antibody diluent (S0809; Agilent Technologies, Santa Clara, CA) and incubated overnight at 4 8C. After 3 3 5-minute washes in TBS-0.1% Tween 20 (TBST), slides were treated with ImmPRESS (MP7401; Vector Laboratories, Burlingame, CA) anti-rabbit peroxidase for 30 minutes at room temperature. Staining was developed under a light microscope with the ImmPACT DAB peroxidase substrate kit (SK-4105; Vector Laboratories) for 2 to 10 minutes and counterstained with Hematoxylin Solution, Gill No.1 (GHS132; Sigma-Aldrich). Slides were then dehydrated and mounted using Cytoseal 60 (8310-16; Thermo Fisher Scientific, Waltham, MA). For antibody competition

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assay, 3-lm serial sections were used and incubated with C9-L and C9-S antibody alone or C9-L and C9-S antibody combined with 5 times excess (w/v) of respective blocking peptide. For immunofluorescence, tissues on slides were permeabilized and blocked with blocking solution (10% donkey serum, 3% bovine serum albumin, and 0.3% Triton X-100 in TBS) at ambient temperature for 1 hour. For primary antibodies, rabbit anti-C9S (in-house; 1:200), rabbit anti-C9-L (in-house; 1:200), goat anti-Importin b1 (sc-1919 and sc-1863; 1:50; Santa Cruz Biotechnology), mouse anti-Importin b1 (sc-1156; 1:600 Santa Cruz Biotechnology), goat anti-RAN-GTPase (sc-1156; 1:50; Santa Cruz Biotechnology), mouse anti-Ran-GTPase (610340; 1:50; BD Biosciences), goat anti-Lamin B (sc-6216, sc-6217, and sc-30264; 1:50; Santa Cruz Biotechnology), and mouse anti–TAR DNA-binding protein 43 (TDP-43; ab104223 and ab57105; 1:100; Abcam) were diluted in Dako antibody diluent and incubated with sections overnight at 4 8C. After 3 3 5minute washes with TBST, slides were incubated with the appropriate Alexa Fluor 488, 594, or 647 secondary antibodies (1:500; Invitrogen) in Dako antibody diluent for 40 minutes at room temperature. After 3 3 5-minute washes with TBST, slides were mounted with ProLong Gold antifade reagent with DAPI (P36931; Life Technologies). One-way analysis of variance using post-hoc analysis with Turkey’s multiple comparisons test was used for statistical analysis (GraphPad Software).

Results Generation and Characterization of C9orf72 Splice Isoform Antibodies Two protein isoforms of C9orf72 are generated from three alternatively spliced transcripts. C9-L is expressed from V2 and V3 and consists of 481 amino acids encoded by exons 2 to 11 (Fig 1A, B), whereas C9-S, generated from V1, consists of 222 amino acids encoded by exons 2 to 5, which has the same sequence as C9-L except for a unique amino acid (lysine) at the Cterminus encoded by partial retention of intron 5 (Fig 1A, B). To investigate C9orf72 protein properties, synthetic peptides corresponding to unique amino acid sequences (residues 321–334) within the C-terminal region of C9-L, and the last 12 amino acids of C9-S including the unique lysine at the C-terminus, were used to generate antibodies specific for C9-L and C9-S, respectively (Fig 1B). A BLAST search using these sequences showed no homology with other proteins. To verify antibody specificity, immunoblots of lysates from HEK293T cells expressing N-terminal-eGFP-tagged C9L or similarly tagged C9-S were probed using the C9-L and C9-S antibodies, as well as a commercial antibody against eGFP. The C9-L antibody only recognized eGFPC9-L, but not eGFP-C9-S (Fig 1C), whereas C9-S antibody only detected eGFP-C9-S, but not eGFP-C9-L (Fig 1D). The eGFP antibody recognized both eGFP-C9-L and eGFP-C9-S (Fig 1E). 571

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FIGURE 1: (A) Schema represent three known alternative transcript variants of C9orf72 with location of the G4C2-repeat expansions indicated. V1 is a short transcript with noncoding exon 1a as the 50 -untranslated region (UTR; black), exons 2 to 5 (green), and partial retention of intron 5 (red) comprising the coding sequence for C9-S. V2 and V3 differ in their inclusion of noncoding exon 1a or 1b at the 50 -UTR, respectively, and share exons 2 to 11 (green) as coding sequence. (B) Partial retention of intron 5 in V1 adds a unique lysine residue at the C-terminus of C9-S, with a synthetic peptide corresponding to the final 12 amino acids used to generate the C9-S-specific antibody. A synthetic peptide corresponding to amino acid residues 321 to 334, a unique sequence absent from C9-S, was used to generate the C9-L-specific antibody. (C–E) Antibody specificity was confirmed by western blot analyses of lysates from transfected HEK293T cells expressing eGFP-C9-L (lane 1), eGFP-C9-S (lane 2), or nontransfected cells (lane 3). The C9-L antibody specifically recognized eGFP-C9-L, but not eGFP-C9-S, in the respective cell lysates (C, arrow). The C9-S antibody specifically recognized eGFP-C9-S, but not eGFP-C9-L, in the respective cell lysates (D, arrow). An eGFP antibody detected both eGFP-C9-L and eGFP-C9-S (E, arrows). The lower-molecular-weight species running just below C9-L and C9-S is owing to partial cleavage of the eGFP tag (asterisks in C, D, and E). eGFP 5 enhanced green fluorescent protein.

Differential Biochemical Profile and Distinct Subcellular Localization of C9-L and C9-S After characterization of C9orf72 isoform antibodies, we investigated the solubility of C9-L and C9-S in cerebellar tissue of randomly selected control and ALS cases. Protein was sequentially extracted with a series of buffers of increasing stringency and partitioned into different fractions: low-salt fraction; high-salt fraction; high-salt Triton fraction; and urea fraction. The predicted molecular weight of C9-L is 54.3 kDa, and it ran at this approximate molecular weight on SDS/polyacrylamide gel electrophoresis (PAGE; Fig 2A). C9-L was detected in the high-salt Triton fraction and urea fraction, and this labeling was abrogated by competition with the peptide immunogen (Fig 2A, B). Interestingly, C9-L appeared as a doublet, with the lower band at a higher intensity compared to the upper band in the high-salt Triton fraction, and conversely in the urea fraction (Fig 2A, asterisks). The predicted molecular weight for C9-S is 24.8 kDa, and it ran at this approximate molecular weight on 572

SDS-PAGE (Fig 2C). C9-S was detected mainly in the low-salt fraction and at low levels in the high-salt Triton fraction, and this labeling was abrogated by competition with the peptide immunogen (Fig 2C, D). A strongly immunoreactive band of 48 kDa was labeled in the high-salt Triton fraction with the C9-S antibody, the labeling of which was also ablated by peptide competition (Fig 2C, D). This band was labeled by two other commercially available C9-antibodies. The first from Santa Cruz Biotechnology (SC-14; Fig 2E) was raised to residues 165 to 215; the other from Abcam (ab121779; data not shown) was raised to residues 110 to 199, showing no overlap with the peptide sequence used to generate the C9-S antibody. These results indicate that the band of 48 kDa is a C9orf72 species and, based on its molecular weight, could be a dimer of C9-S, although this remains to be verified. There were no apparent differences in the biochemical profile of C9-L or C9-S between control, c9-ALS, and non-c9-ALS cases (not shown). Volume 78, No. 4

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FIGURE 2: Protein was sequentially extracted from the cerebellum of control and amyotrophic lateral sclerosis (ALS) cases using a series of buffers of increasing stringency: low salt (LS), high salt (HS), high-salt Triton (HST), and urea buffer and analyzed by western blot. The results shown are of two non-c9 ALS cases (even and odd numbers), but the same findings were made using control and c9-ALS patient tissues (not shown). (A) C9-L isoform was detected using C9-L antibody in HST and urea fractions (lanes 5–8) and absent in LS and HS fractions (lanes 1–4). Of note, C9-L appeared to run as a doublet (lanes 5–8; indicated with asterisks), with the lower band stronger in the HST fraction (lanes 5 and 6) and the upper band stronger in the urea fraction (lanes 7 and 8). (B) Labeling of C9-L was abrogated with blocking peptide. (C) C9-S was detected with C9-S antibody mainly in the LS fraction and to a lesser extent in the HST fraction. A higher-molecular-weight species observed in the HST fraction could be a C9-S doublet (lanes 5 and 6, arrow and asterisk). (D) Labeling of C9-S was abrogated with blocking peptide. (E) Labeling of the same samples with a commercial antibody also showed C9-S in the LS fraction (lanes 1 and 3) and also labeled the possible C9-S doublet in the HST fraction (lanes 5 and 6, arrow and asterisk). A species corresponding to the molecular weight of C9-L was apparent in the urea fraction, as were additional bands of 60 and 90 kDa, the identity of which are unknown.

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FIGURE 3: Levels of C9-L in urea-soluble fractions from different regions of the central nervous system of c9-ALS and non-c9ALS cases were quantified by densitometric analysis of western blots probed with C9-L antibody and normalized to b-actin. There was no significant difference in C9-L levels in the motor cortex (A), cerebellum (D), or lumbar spinal cord (E) of c9-ALS cases compared to non-c9-ALS cases (n 5 3 per group). A significant downregulation of C9-L levels was found in the temporal (B) and frontal cortex (C) of c9-ALS cases compared to non-c9-ALS cases (n 5 3 per group: p < 0.01; p < 0.05). Of note, a C9-L doublet was apparent in some of the samples analyzed (asterisks in B and C). Data are mean 6 standard error of the mean. ALS 5 amyotrophic lateral sclerosis.

Altered Levels of C9-L and C9-S in CNS Regions of c9-ALS and Non-c9-ALS Cases Given that it has been reported that the repeat expansion can lead to decreased abundance of C9orf72 transcripts, C9-L and C9-S levels were examined in the urea and low-salt fractions of CNS tissues (frontal 574

cortex, temporal cortex, motor cortex, cerebellum, and lumbar spinal card) from randomly selected c9-ALS (n 5 3) and non-c9-ALS (n 5 3) cases, respectively. Quantification of C9-L in the temporal cortex (Fig 3B; p 5 0.0045) and frontal cortex (Fig 3C; p 5 0.034) revealed significantly decreased levels of C9-L in c9Volume 78, No. 4

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FIGURE 4: C9-S levels in different regions of the central nervous system were quantified by densitometric analysis of western blots probed with C9-S antibody in the LS fraction of c9-ALS and non-c9-ALS cases and normalized to GAPDH. There was no significant difference in C9-S levels in the motor cortex (A), cerebellum (D), or lumbar spinal cord (E) of c9-ALS cases compared to non-c9-ALS cases (n 5 3 per group). A significant upregulation of C9-S levels was found in the temporal (B) and frontal cortex (C) of c9-ALS cases compared to non-c9-ALS cases (n 5 3 per group: both p < 0.05). Data are mean 6 standard error of the mean. ALS 5 amyotrophic lateral sclerosis; GAPDH 5 glyceraldehyde 3-phosphate dehydrogenase.

ALS cases compared to non-c9-ALS cases. No significant differences were observed in motor cortex (Fig 3A; p 5 0.052) and cerebellum (Fig 3D; p 5 0.1279), with only marginal detection of C9-L in lumbar spinal cord of either c9- or non-c9-ALS cases (Fig 3E; p 5 0.7927). Interestingly, the C9-L doublet observed in Figure 2A was also observed in some of the samples of both c9 and non-c9 cases (Fig 3B, C, asterisks). Using the same October 2015

samples as in Figure 3, a relative increase in C9-S was observed in c9-ALS cases, compared to non-c9-ALS cases, in both the temporal cortex (Fig 4B; p 5 0.036) and frontal cortex (Fig 4C; p 5 0.031), with no significant differences observed in the motor cortex (Fig 4A; p 5 0.34) or cerebellum (Fig 4D; p 5 0.571) and only marginal detection of C9-S in the lumbar spinal cord (Fig 4E; p 5 0.985). 575

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Distinct Subcellular Localization of C9-L and C9-S in Cerebellar Purkinje Cells Coincident with the differing C9orf72 biochemical profiles, C9-L and C9-S also displayed distinct subcellular localizations in cerebellar Purkinje cells of both c9-ALS and nonc9-ALS cases. DAB immunohistochemistry (Fig 5A, B) and immunofluorescence staining (Fig 5C, D, E) using the isoform-specific antibodies revealed that C9-L was spread diffusely throughout the cytoplasm and labeled numerous large speckles within the cytoplasm and dendritic processes of Purkinje cells. Specificity of this labeling was confirmed by peptide competition (Fig 5F, G). A range of different antibodies were tested to identify these speckles, including endosomal, lysosomal, autophagosomes, and polysomal markers, with none giving positive immunoreactivity. As such, these speckles remain to be identified. In contrast, C9S was localized to the nuclear membrane of Purkinje cells of both c9-ALS and non-c9-ALS cases, as observed both by DAB labeling (Fig 5H, I, arrows) and immunofluorescence labeling (Fig 5J, K, L). Specificity of this labeling was confirmed by peptide competition (Fig 5M, N, arrows). Variable Levels of C9-L and Altered Subcellular Localization of C9-S in Spinal Motor Neurons of c9-ALS and Non-c9-ALS Cases Compared to Control Immunohistochemical analysis of spinal motor neurons from control cases with C9-L antibody revealed a diffuse labeling throughout the neuronal cytoplasm (Fig 6A, D). A similar distribution was also observed in ALS cases (Fig. 6B, C, E, F); however, staining was variable, with some neurons showing a stronger level of staining relative to control, irrespective of c9-genetic status (compare Fig 6E with 6F). Moreover, there was no apparent labeling of intracytoplasmic inclusion bodies with C9-L antibody (Fig 6E, arrow). Labeling of control motor neurons with C9-S antibody revealed nuclear membrane labeling, akin to that observed in cerebellar Purkinje cells (Fig 6G, J, arrows). However, in ALS cases, this nuclear membrane labeling was diminished and/or completely absent (Fig 6H, I, K, L, arrows) with instead a stronger immunoreactivity observed at the plasma membrane (Fig 6I, K, L, arrowheads). The number of motor neurons showing this loss of C9-S immunoreactivity from the nuclear membrane was quantified and shown to be equally prevalent in c9-ALS cases and non-c9-ALS cases (Fig 6M). As with C9-L, there was no apparent labeling of intracytoplasmic inclusions with C9-S antibody (Fig 6L, arrow). C9-S Is Associated With Nucleocytoplasmic Transport Proteins Because C9-S was localized to the nuclear membrane of Purkinje cells and spinal motor neurons, this suggested 576

that it may have a role(s) in nucleocytoplasmic shuttling. Indeed, both C9-L and C9-S showed localization to the nuclear membrane in transfected mouse N2a cells (data not shown). To investigate this, triple immunofluorescence labeling was performed to detect the relative subcellular distributions of C9-S, nuclear membrane marker Lamin B, and either Importin-b1 or Ran-GTPase in spinal motor neurons of ALS cases (Fig 7). In control cases, C9-S colocalized with Lamin B on the nuclear membrane and also showed diffuse labeling in the nucleus (Fig 7A, B, K, L, arrows). Similar colocalizations were also observed with antibodies to Importin-b1 (Fig 7C, arrow) and Ran-GTPase (Fig 7M, arrow). In spinal motor neurons of c9-ALS cases, Lamin-B retained its localization to the nuclear membrane (Fig 7G, Q, arrows), whereas there was loss of C9-S immunoreactivity from the nuclear membrane (Fig 7F, P, arrows) that also correlated with a loss of Importin-b1 (Fig 7H, arrow) and Ran-GTPase (Fig 7R, arrow) labeling. This suggested that C9-S might directly interact with Importin-b1 or Ran-GTPase. To test this, we performed coimmunoprecipitation experiments from N2a cells stably expressing eGFP-C9-L, eGFP-C9-S, or eGFP using commercially available eGFP antibody covalently coupled to magnetic beads. Western blots of immunoprecipitates confirmed that both C9-L (lane 7) and C9-S (lane 8) interacted with Importin-b1 and Ran-GTPase, but not with LaminB in N2a cells (Fig 7U). Loss of C9-S, Importin-b1, and Ran-GTPase Nuclear Membrane Labeling Correlates With Cytoplasmic Mislocalization of TDP-43 Mislocalization of TDP-43 from the nucleus to the cytoplasm of affected neurons is a hallmark pathology of the majority of ALS and FTLD cases, including those caused by C9orf72 repeat expansions.36 Triple immunofluorescence labeling of spinal motor neurons of c9-ALS cases showed that TDP-43 mislocalization and formation of cytoplasmic aggregates correlated with the loss of C9-S, Importin-b1, and Ran-GTPase nuclear membrane labeling (Fig 8).

Discussion Haploinsufficiency of C9orf72 was originally suggested as a pathomechanism in c9-ALS/FTLD owing to reduced transcript abundance in repeat expansion carriers.1,2 Subsequent reports have confirmed downregulation of C9orf72 transcripts in c9-ALS/FTLD cases, either in a transcript-specific manner or of total transcripts; however, other reports have failed to find any differences in C9orf72 transcript levels.1,2,24–31 Although degenerative effects of C9orf72 haploinsufficiency are supported by Volume 78, No. 4

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FIGURE 5: 3,30 -diaminobenzidine immunohistochemistry of (A) non-c9-ALS and (B) c9-ALS cases with C9-L antibody revealed that C9-L was spread diffusely throughout the neuronal cytoplasm and also labeled distinct speckles within the perikarya and dendritic processes (arrows). (C–E) Labeling of granular structures was confirmed by immunofluorescence labeling (arrows). (F and G) Serial sections showing C9-L antibody labeling were ablated with competing peptide (arrows). Immunohistochemistry of (H) non-c9-ALS and (I) c9-ALS cases with C9-S antibody revealed labeling of the nuclear membrane (arrows). (J–L) This localization of C9-S to the nuclear membrane was confirmed by immunofluorescence (arrow). (M and N) Specificity of labeling was confirmed by peptide blocking using serial sections (arrows). Scale bar 5 25 lm (A, B, and H–L), 30 lm (C–E), and 50 lm (F, G, M, and N). ALS 5 amyotrophic lateral sclerosis.

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FIGURE 6: 3,30 -diaminobenzidine immunohistochemistry using the isoform-specific antibodies was used to examine the subcellular localization of C9-L and C9-S in spinal motor neurons of control, non-c9-ALS, and c9-ALS cases. (A and D) C9-L labeling of control motor neurons appeared diffuse and granular within the perikaryon and neurites. C9-L antibody labeling of (B and E) non-c9-ALS and (C and F) c9-ALS cases was similar to control, but some cases showed increased labeling of motor neurons (B and F) whereas others showed reduced labeling (E). Of note, there was no apparent labeling of cytoplasmic inclusions with C9-L antibody (E, arrow). (G and J) Labeling of control motor neurons with C9-S antibody revealed staining of the nuclear membrane (arrows). Labeling of (H and K) non-c9-ALS or (I and L) c9-ALS cases showed a diminishment (H) or complete absence of C9-S labeling (I, K, and L) of the nuclear membrane with an apparent coincident increase in labeling of the plasma membrane (I, K, and L, arrowheads). As with C9-L antibody, there was no apparent labeling of cytoplasmic inclusions with C9-S antibody (L, arrow). (M) There was a significant decrease in the percentage of C9-S staining of motor neuron nuclei in non-c9ALS (n 5 7) and c9-ALS (n 5 12) compared to control (n 5 3; p < 0.001). Data are mean 6 standard error of the mean. Scale bar 5 20 lm (A, D, G, and J) and 10 lm (B, C, E, F, H, I, K, and L). ALS 5 amyotrophic lateral sclerosis.

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the development of motor deficits in zebrafish and C. elegans with reduced levels of their C9orf72 orthologs, very little is known about their properties or functions.30,32 To further our understanding of the normal functions of C9orf72, we have generated antibodies that differentiate between the C9-L and C9-S isoforms. These antibodies have shown that C9-L has a mainly diffuse cytoplasmic localization with variable levels of intensity that does not

appear be specific to c9-cases. Antibody to C9-S revealed a very striking localization to the nuclear membrane, with an apparent redistribution to the plasma membrane in ALS motor neurons again occurring irrespective of the genetic cause of disease. Importantly, we also discovered an interaction of the C9-isoforms with components of the nuclear pore complex, suggesting that C9orf72 may have roles in nucleocytoplasmic shuttling.

FIGURE 7:

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For biochemical analysis of the C9-L and C9-S isoforms, cerebellar tissue was chosen given that previous reports have noted that this region has the highest levels of C9orf72 mRNA and protein.2,29 Biochemical fractionation of cerebellar tissue showed that C9-L was present mainly in the high-salt Triton and urea-soluble fractions, whereas C9-S was abundant in both the low- and high-salt Triton fractions, suggesting that C9-L and C9-S may have differing subcellular localizations. There was no apparent variation in the biochemical profiles of C9-L or C9-S in control versus c9-ALS or non-c9-ALS cases. Investigation of C9-isoform levels in different regions of the CNS revealed a significant downregulation of C9-L in the temporal cortex and frontal cortex of c9-ALS cases, compared to non-c9 ALS cases, whereas no significant differences were observed in motor cortex or cerebellar tissues. There was negligible detection of C9-L in the lumbar spinal cord of either c9-ALS or non-c9-ALS cases. In contrast to downregulation of C9-L, there was a coincident upregulation of C9-S in the temporal cortex and frontal cortex of c9-ALS cases relative to non-c9 cases, with no significant differences apparent in the other regions. This regionspecific downregulation of C9-L is consistent with a recently published study that used a pan-C9orf72 antibody developed in-house that revealed a decrease in C9-L levels in the frontal cortex, but not cerebellum, of c9-ALS/FTLD cases; however C9-S levels were not assessed.29 At present, it is unclear whether downregulation of C9-L and coincident upregulation of C9-S are associated or occur independently. The finding of a reduction of C9-L protein is consistent with previous reports showing a decrease of C9orf72 transcripts in c9-linked ALS/FTLD. This downregulation could be linked to the location of the expansion repeats in the promoter region of V2.39 Alternatively, or in combination, epigenetic modifications, such as hypermethylation of CpG islands juxtaposing the repeats or binding of trimethylated histones, may underlie decreased abundance of C9orf72 transcripts.24,31,38–40 At the immunohistochemical level, C9-L antibody showed a diffuse labeling in the cytoplasm of cerebellar

Purkinje cells, with a striking labeling of numerous speckles that were observed both in the neuronal perikarya and dendritic processes. The identity of these speckles remains unknown, and it should be noted that they were observed in both c9-ALS and non-c9-ALS cases. In contrast, C9-S antibody gave a very specific labeling of the nuclear membrane, clearly showing that C9-L and C9-S have different subcellular localizations in Purkinje cells. Previously published reports using commercial antibodies against C9orf72 protein have noted diffuse and granular staining in neuronal cytoplasm and neurites; however, these antibodies show high levels of nonspecific staining and are either unable to discriminate between C9-L and C9-S or are solely directed against C9-L.11,12,15,16,40,44 Thus, our antibodies provide greatly improved detection of C9orf72 protein isoforms and have revealed distinct subcellular localizations of C9-L and C9-S. Immunohistochemical labeling of spinal cord tissue showed that the subcellular localization of C9-L in diseased motor neurons appeared unchanged in c9-ALS and non-c9-ALS cases compared to controls. However, changes in the relative intensity of staining between cases were apparent, with some c9-ALS cases showing a relative increase in labeling and others a decrease. The reasons for this are unclear, but could be related to differences in transcript levels between cases owing to varying lengths of expansion repeats and/or methylation status, which could affect expression levels. In addition, unlike staining of Purkinje cells, there was no labeling of speckles with C9-L antibody in motor neurons of the spinal cord in either disease or control cases. However, as observed in Purkinje cells, C9-S antibody labeled the nuclear membrane of spinal motor neurons in control cases, but this labeling was diminished or completely absent in motor neurons of both c9-ALS and non-c9-ALS cases. Remarkably, this loss of C9-S nuclear membrane labeling in ALS motor neurons also correlated with a loss of Importin-b1 and RanGTPase labeling, whereas Lamin-B labelling was sustained, indicating that the nuclear membrane was still intact. This suggested association between C9-S, Importin-b1 and

FIGURE 7: Triple immunofluorescence labeling was performed on lumbar spinal cord tissue from control and c9-ALS cases to assess colocalization of C9-S with proteins involved in nucleocytoplasmic transport. (A, F, K, and P) is C9-S antibody labeling, (B, G, L, and Q) is Lamin-B antibody labeling, (C and H) is Importin-b1 antibody labeling, and (M and R) is Ran-GTPase antibody labeling. (A–E) Labeling of control motor neurons showed colocalization of C9-S (green), Lamin-B (red), and Importin-b1 (far red) to the nuclear membrane (arrows). In c9-ALS cases, C9-S (F) and Importin-b1 (H) labeling was lost from the nuclear membrane, whereas Lamin-B (G) labeling was retained (arrows). (F) Loss of C9-S nuclear labeling correlated with an increase in plasma membrane labeling (arrowhead). (K–O) Labeling of control motor neurons showed colocalization of C9-S (green), Lamin-B (red), and Ran-GTPase (far red) to the nuclear membrane (arrows). (P-T) In c9-ALS motor neurons, C9-S (P) and RanGTPase (R) labeling was lost from the nuclear membrane, whereas Lamin-B (Q) labeling was retained. (P) Loss of C9-S nuclear labeling correlated with an increase in plasma membrane labeling (arrowhead). (U) Coimmunoprecipitation from N2a cells stably expressing eGFP-C9-L, eGFP-C9-S, or eGFP only using eGFP antibody showed that both C9-L and C9-S interacted with Importin-b1 and RanGTPase, but not with Lamin-B (U, lanes 7 and 8). Scale bar 5 20 lm (A–E and K–O) and 15 lm (F–J and P–T). ALS 5 amyotrophic lateral sclerosis; DAPI 5 40 ,6-diamidino-2-phenylindole; eGFP 5 enhanced green fluorescent protein.

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FIGURE 8: Triple immunofluorescence staining of spinal motor neurons of control and c9-ALS cases with antibodies to (A, F, and K) C9-S; (B, G, and I) Ran-GTPase; (C, H, and M) TDP-43; and (D, I, and N) DAPI stain. (A–E) Note nuclear membrane labeling of control motor neuron with C9-S and RanGTPase antibodies, and nuclear localization of TDP-43 (arrows). Note loss of C9S (F and K) and Ran-GTPase (G and L) nuclear membrane labeling in c9-ALS cases (arrows) and coincident cytoplasmic mislocalization of TDP-43 appearing either diffusely (H) or as inclusions (M, arrows). Note the increased intensity of C9-S antibody labeling of the plasma membrane in c9-ALS cases (arrowheads). Triple immunofluorescence staining of spinal motor neurons of control and c9-ALS cases with antibodies to (A0 , F0 , and K0 ) C9-S; (B0 , G0 , and I0 ) Importin-b1; (C0 , H0 , and M0 ) TDP-43; and (D0 , I0 , and N0 ) DAPI stain. (A0 –E0 ) Note nuclear membrane labeling of control motor neuron with C9-S and Lamin B antibodies, and nuclear localization of TDP-43 (arrows). Note loss of C9-S (F0 and G0 ) and Importin-b1 (G0 and H0 ) nuclear membrane labeling in c9-ALS cases (arrows) and coincident cytoplasmic mislocalization of TDP-43 (H0 and M0 , arrows). Note the increased intensity of C9-S antibody labeling of the plasma membrane in c9-ALS cases (arrowheads). Scale bar 5 20 lm (A–E, K–O, A0 -E0 , and K0 –O0 ) and 10 lm (F-J and F0 –J0 ). ALS 5 amyotrophic lateral sclerosis; DAPI 5 40 ,6-diamidino-2-phenylindole; TDP-43 5 TAR DNA-binding protein 43.

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Ran-GTPase was confirmed by coimmunoprecipitation studies in transfected cells. Despite subcellular localization in spinal motor neurons and Purkinje cells suggesting otherwise, C9-L was also shown to interact with Importin-b1 and Ran-GTPase in N2a cells. This may be owing to lack of a short isoform of mouse C9orf72 ortholog in N2a cells; therefore, C9-L may behave differently in these cells; moreover, the differing distributions of C9-L in Purkinje versus spinal motor neurons may reflect distinct functions in the different cell types. Nevertheless, these results suggest that nucleocytoplasmic transport may be disrupted in c9-ALS and non-c9-ALS cases, and that C9orf72 proteins may function in this pathway. Given that TDP-43 pathology is common to the majority of ALS cases, and TDP-43 requires nuclear trafficking, we addressed whether nuclear depletion and coincident cytoplasmic mislocalization of TDP-43 was associated with disruption of nuclear membrane proteins in c9-ALS cases.43,46 In all motor neurons examined, we found that loss of C9-S, Importin-b1, and Ran-GTPase from the nuclear membrane correlated with a loss of nuclear TDP43 and mislocalization to the cytoplasm, forming a spectrum of TDP-43 pathologies, including diffuse, skein-like, and aggregated inclusions (data not shown). Published data have shown that nuclear import of TDP-43 involves interaction with Importin-b1 and is dependent on RanGTPase, a master regulator of nuclear transport.43–46 Disruption of Importin-b1 causes cytoplasmic accumulation of TDP-43, and impairment of Ran-GTPase-mediated nuclear trafficking has been shown to correlate with nuclear depletion of TDP-43, preceding neurodegeneration in progranulin knockout mice.44,46 Furthermore, TDP-43 has been identified as a regulator of Ran-GTPase expression, suggesting that disruption of either nuclear TDP-43 localization or Ran-GTPase expression can have a negative outcome on the other, thereby enhancing any toxic effects of TDP-43 nuclear depletion.46 Therefore, our results further implicate disruption of nucleocytoplasmic transport with depletion of nuclear TDP-43. This pathway has been previously implicated in ALS pathogenesis, and we provide confirmatory evidence that nucleocytoplasmic transport may be disrupted in c9-ALS cases, and novel evidence that C9orf72 isoforms may function in this pathway.43,47,48

Canada Research Chair. P. McGoldrick holds a Milton Safenowitz Postdoctoral Fellowship from the American ALS Association, P. McKeever holds a doctoral award from the Alzheimer Society of Canada, and J.M. is the James Hunter and Family ALS Senior Research Fellow. We are profoundly thankful to the patients and families for the generous donation of tissues for the study, as well as Shawn Hobbs, Myrna Moore, Olive Grozelle, and Denise Miletic for their assistance.

Authorship S.X., L.M., P.M., P.M.M., and J.R. contributed to conception and design of the study; S.X, L.M, P.M. P.M.M, J.R.M, M.Z, J.K, L.Z, and J.R. contributed to data collection and analysis; S.X., L.M., P.M., and J.R. wrote the manuscript.

Potential Conflicts of Interest Nothing to report.

References 1.

DeJesus-Hernandez M, Mackenzie IR, Boeve BF, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 2011;72:245–256.

2.

Renton AE, Majounie E, Waite A, et al. A Hexanucleotide Repeat Expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 2011;72:257–268.

3.

Xi Z, Zinman L, Grinberg Y, et al. Investigation of C9orf72 in 4 neurodegenerative disorders. Arch Neurol 2012;69:1583.

4.

Cruts M, Gijselinck I, Van Langenhove T, et al. Current insights into the C9orf72 repeat expansion diseases of the FTLD/ALS spectrum. Trends Neurosci 2013;36:450–459.

5.

Mackenzie IR, Frick P, Neumann M. The neuropathology associated with repeat expansions in the C9ORF72 gene. Acta Neuropathol 2014;127:347–357.

6.

Hardy J, Rogaeva E. Motor neuron disease and frontotemporal dementia: sometimes related, sometimes not. Exp Neurol 2014; 262:75–83.

7.

Rutherford NJ, Heckman MG, DeJesus-Hernandez M, et al. Length of normal alleles of C9orf72 GGGGCC repeat do not influence disease phenotype. Neurobiol Aging 2012;33:2950.e5–2950.e7.

8.

Beck J, Poulter M, Hensman D, et al. Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population. Am J Hum Genet 2013;92:345–353.

9.

Boeve BF, Boylan KB, Graff-Radford NR, et al. Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72. Brain 2012;135:765–783.

10.

Chi o A, Borghero G, Restagno G, et al. Clinical characteristics of patients with familial amyotrophic lateral sclerosis carrying the pathogenic GGGGCC hexanucleotide repeat expansion of C9ORF72. Brain 2012;135:784–793.

11.

Cooper-Knock J, Hewitt C, Highley JR, et al. Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72. Brain 2012;135:751–764.

Acknowledgment This work was funded by grants from the Canadian Institutes of Health Research (JNM-90963; to J.R.), Krembil Scientific Development Seed Fund (to E.R., J.R., and L.Z.), W. Garfield Weston Foundation (to E.R.), Temerty Family Foundation (to L.Z.), and James Hunter and Family ALS Initiative (to J.R. and L.Z.). J.R. holds a Tier 2 582

Volume 78, No. 4

Xiao et al: C9orf72 Isoforms in ALS

12.

Hsiung GYR, DeJesus-Hernandez M, Feldman HH, et al. Clinical and pathological features of familial frontotemporal dementia caused by C9ORF72 mutation on chromosome 9p. Brain 2012; 135:709–722.

13.

Mahoney CJ, Beck J, Rohrer JD, et al. Frontotemporal dementia with the C9ORF72 hexanucleotide repeat expansion: clinical, neuroanatomical and neuropathological features. Brain 2012;135:736– 750.

30.

Ciura S, Lattante S, Le Ber I, et al. Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis. Ann Neurol 2013;74:180-187.

31.

Liu EY, Russ J, Wu K, et al. C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD. Acta Neuropathol 2014;128:525–541.

32.

Murray ME, DeJesus-Hernandez M, Rutherford NJ, et al. Clinical and neuropathologic heterogeneity of c9FTD/ALS associated with hexanucleotide repeat expansion in C9ORF72. Acta Neuropathol 2011;122:673–690.

Therrien M, Rouleau GA, Dion PA, Parker JA. Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans. PLoS ONE 2013;8:e83450.

33.

Levine TP, Daniels RD, Gatta AT, et al. The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs. Bioinformatics 2013;29:499–503.

15.

Sim on-S anchez J, Dopper EGP, Cohn-Hokke PE, et al. The clinical and pathological phenotype of C9ORF72 hexanucleotide repeat expansions. Brain 2012;135:723–735.

34.

Farg MA, Sundaramoorthy V, Sultana JM, et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Hum Mol Genet 2014;23:3579–3595.

16.

Snowden JS, Rollinson S, Thompson JC, et al. Distinct clinical and pathological characteristics of frontotemporal dementia associated with C9ORF72 mutations. Brain 2012;135:693–708.

35.

17.

Majounie E, Abramzon Y, Renton AE, et al. Repeat expansion in C9ORF72 in Alzheimer’s disease. N Engl J Med 2012;366:283– 284.

Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2000;1:293– 299.

36.

Mackenzie IR, Arzberger T, Kremmer E, et al. Dipeptide repeat protein pathology in C9ORF72 mutation cases: clinicopathological correlations. Acta Neuropathol 2013;126:859–879.

37.

Haeusler AR, Donnelly CJ, Periz G, et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 2014;507:195–200.

38.

Xi Z, Rainero I, Rubino E, et al. Hypermethylation of the CpGisland near the C9orf72 G4C2-repeat expansion in FTLD patients. Hum Mol Genet 2014;23:5630–5637.

39.

Xi Z, Zinman L, Moreno D, et al. Hypermethylation of the CpG island near the G4C2 repeat in ALS with a C9orf72 expansion. Am J Hum Genet 2013;92:981–989.

40.

Belzil VV, Bauer PO, Gendron TF, et al. Characterization of DNA hypermethylation in the cerebellum of c9FTD/ALS patients. Brain Res 2014;1584:15–21.

41.

Stewart H, Rutherford NJ, Briemberg H, et al. Clinical and pathological features of amyotrophic lateral sclerosis caused by mutation in the C9ORF72 gene on chromosome 9p. Acta Neuropathol 2012;123:409–417.

42.

Satoh JI, Tabunoki H, Ishida T, et al. Dystrophic neurites express C9orf72 in Alzheimer’s disease brains. Alzheimers Res Ther 2012;4:33.

14.

18.

Lesage S, Le Ber I, Condroyer C, et al. C9orf72 repeat expansions are a rare genetic cause of parkinsonism. Brain 2013;136:385–391.

19.

Coon EA, Daube JR, DeJesus-Hernandez M, et al. Clinical and electrophysiologic variability in amyotrophic lateral sclerosis within a kindred harboring the C9ORF72repeat expansion. Amyotroph Lateral Scler Frontotemporal Degener 2013;14:132–137.

20.

Gendron TF, Belzil VV, Zhang YJ, Petrucelli L. Mechanisms of toxicity in C9FTLD/ALS. Acta Neuropathol 2014;127:359–376.

21.

Stepto A, Gallo JM, Shaw CE, Hirth F. Modelling C9ORF72 hexanucleotide repeat expansion in amyotrophic lateral sclerosis and frontotemporal dementia. Acta Neuropathol 2013;127:377–389.

22.

Ash PE, Bieniek KF, Gendron TF, et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 2013;77:639–646.

23.

Mori K, Weng SM, Arzberger T, et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 2013;339:1335–1338.

24.

Belzil VV, Bauer PO, Prudencio M, et al. Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood. Acta Neuropathol 2013; 126:895–905.

43.

25.

Gijselinck I, Van Langenhove T, van der Zee J, et al. A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study. Lancet Neurol 2011;11:54–65.

Ito D, Suzuki N. Conjoint pathologic cascades mediated by ALS/ FTLD-U linked RNA-binding proteins TDP-43 and FUS. Neurology 2011;77:1636–1643.

44.

Fratta P, Poulter M, Lashley T, et al. Homozygosity for the C9orf72 GGGGCC repeat expansion in frontotemporal dementia. Acta Neuropathol 2013;126:401–409.

Nishimura AL, Zupunski V, Troakes C, et al. Nuclear import impairment causes cytoplasmic trans-activation response DNAbinding protein accumulation and is associated with frontotemporal lobar degeneration. Brain 2010;133:1763–1771.

45.

Melchior F, Guan T, Paschal B, Gerace L. Biochemical and structural analysis of nuclear protein import. Cold Spring Harb Symp Quant Biol 1995;60:707–716.

46.

Ward ME, Taubes A, Chen R, et al. Early retinal neurodegeneration and impaired Ran-mediated nuclear import of TDP-43 in progranulin-deficient FTLD. J Exp Med 2014;211:1937–1945.

47.

Zhang J, Ito H, Wate R, et al. Altered distributions of nucleocytoplasmic transport-related proteins in the spinal cord of a mouse model of amyotrophic lateral sclerosis. Acta Neuropathol 2006; 112:673–680.

48.

Tran D, Chalhoub A, Schooley A, et al. A mutation in VAPB that causes amyotrophic lateral sclerosis also causes a nuclear envelope defect. J Cell Sci 2012;125:2831–2836.

26.

27.

Almeida S, Gascon E, Tran H, et al. Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons. Acta Neuropathol 2013;126: 385–399.

28.

Donnelly CJ, Zhang P-W, Pham JT, et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 2013;80:415–428.

29.

Waite AJ, Ba€ umer D, East S, et al. Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion. Neurobiol Aginh 2014;35:1779.e5–1779.e13.

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