Target Genes of Neuron-Restrictive Silencer Factor Are Abnormally Up-Regulated in Human Myotilinopathy
2007; Elsevier BV; Volume: 171; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2007.070520
ISSN1525-2191
AutoresMarta Barrachina, Jesús Rodríguez Moreno, Salvador Juvés, Dolores Moreno, Montse Olivé, Isidró Ferrer,
Tópico(s)Muscle Physiology and Disorders
ResumoMyotilinopathy is a subgroup of myofibrillar myopathies caused by mutations in the myotilin gene in which there is aggregation of abnormal cytoskeletal proteins and ubiquitin. We report here on the accumulation of neuron-related proteins such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1), synaptosomal-associated protein 25, synaptophysin, and α-internexin in aberrant protein aggregates in myotilinopathy. We have determined that the neuron-restrictive silencer factor (NRSF)/RE1 silencing transcription factor (REST), a transcription factor expressed in non-neuronal tissues repressing the expression of several neuronal genes, is reduced in myotilinopathies. Moreover, NRSF transfection reduces UCHL1, synaptosomal-associated protein 25, synaptophysin, and α-internexin mRNA levels in DMS53 cells, whereas short interferring NRSF transfection increases UCHL1 and synaptophysin mRNA levels in U87-MG cells. Chromatin immunoprecipitation assays have shown that NRSF interacts with the UCHL1 promoter in U87-MG and HeLa cells. In silico analysis of the UCHL1 gene promoter sequence using the MatInspector software has predicted three potential neuron-restrictive silencer elements (NRSEs): NRSE1 located in the complementary DNA chain and NRSE2 and NRSE3 in intron 1, in the coding and complementary chains, respectively. Together, these findings show, for the first time, abnormal regulation of NRSF/REST as a mechanism associated with the aberrant expression of selected neuron-related proteins, which in turn accumulate in abnormal protein aggregates, in myotilinopathy. Myotilinopathy is a subgroup of myofibrillar myopathies caused by mutations in the myotilin gene in which there is aggregation of abnormal cytoskeletal proteins and ubiquitin. We report here on the accumulation of neuron-related proteins such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1), synaptosomal-associated protein 25, synaptophysin, and α-internexin in aberrant protein aggregates in myotilinopathy. We have determined that the neuron-restrictive silencer factor (NRSF)/RE1 silencing transcription factor (REST), a transcription factor expressed in non-neuronal tissues repressing the expression of several neuronal genes, is reduced in myotilinopathies. Moreover, NRSF transfection reduces UCHL1, synaptosomal-associated protein 25, synaptophysin, and α-internexin mRNA levels in DMS53 cells, whereas short interferring NRSF transfection increases UCHL1 and synaptophysin mRNA levels in U87-MG cells. Chromatin immunoprecipitation assays have shown that NRSF interacts with the UCHL1 promoter in U87-MG and HeLa cells. In silico analysis of the UCHL1 gene promoter sequence using the MatInspector software has predicted three potential neuron-restrictive silencer elements (NRSEs): NRSE1 located in the complementary DNA chain and NRSE2 and NRSE3 in intron 1, in the coding and complementary chains, respectively. Together, these findings show, for the first time, abnormal regulation of NRSF/REST as a mechanism associated with the aberrant expression of selected neuron-related proteins, which in turn accumulate in abnormal protein aggregates, in myotilinopathy. Myofibrillar myopathies (MFMs) are a clinically and genetically heterogeneous group of inherited or sporadic muscle diseases characterized morphologically by the presence of nonhyaline structures corresponding to foci of dissolution of myofibrils and hyaline lesions composed of protein aggregates. Immunohistochemical studies have demonstrated intracytoplasmic accumulation of several muscle-related proteins, including cytoskeletal and myofibrillary proteins, as well as αB-crystallin and ubiquitin.1De Bleecker JL Engel AG Ertl BB Myofibrillar myopathy with abnormal foci of desmin positivity: II. Immunocytochemical analysis reveals accumulation of multiple other proteins.J Neuropathol Exp Neurol. 1996; 55: 563-577Crossref PubMed Scopus (158) Google Scholar, 2Nakano S Engel AG Waclawik AJ Emslie-Smith AM Busis NA Myofibrillar myopathy with abnormal foci of desmin positivity: I. Light and electron microscopy analysis of 10 cases.J Neuropathol Exp Neurol. 1996; 55: 549-562Crossref PubMed Scopus (159) Google Scholar, 3Nakano S Engel AG Akiguchi I Kimura J Myofibrillar myopathy: III. Abnormal expression of cyclin-dependent kinases and nuclear proteins.J Neuropathol Exp Neurol. 1997; 56: 850-856Crossref PubMed Scopus (46) Google Scholar, 4Goebel HH Goldfarb L Desmin-related myopathies.in: Karpati G Structural and Molecular Basis of Skeletal Muscle Diseases. ISN Neuropath Press, Basel, Switzerland2002: 70-73Google Scholar, 5Goldfarb LG Vicart P Goebel HH Dalakas MC Desmin myopathy.Brain. 2004; 127: 723-734Crossref PubMed Scopus (205) Google Scholar, 6Olivé M Goldfarb L Moreno D Laforet E Dagvadorj A Sambuughin N Martinez-Matos JA Martinez F Alio J Farrero E Vicart P Ferrer I Desmin-related myopathy: clinical, electrophysiological, radiological, neuropathological and genetic studies.J Neurol Sci. 2004; 219: 125-137Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 7Selcen D Ohno K Engel AG Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.Brain. 2004; 127: 439-451Crossref PubMed Scopus (238) Google Scholar, 8Fischer D Clemen CS Olive M Ferrer I Goudeau B Roth U Badorf P Wattjes MP Lutterbey G Kral T van der Ven PF Furst DO Vicart P Goldfarb LG Moza M Carpen O Reichelt J Schroder R Different early pathogenesis in myotilinopathy compared to primary desminopathy.Neuromuscul Disord. 2006; 16: 361-367Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Mutations in several genes have been identified as causing MFMs: desmin, αB-crystallin, selenoprotein N, myotilin, ZASP, and filamin C.5Goldfarb LG Vicart P Goebel HH Dalakas MC Desmin myopathy.Brain. 2004; 127: 723-734Crossref PubMed Scopus (205) Google Scholar, 7Selcen D Ohno K Engel AG Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.Brain. 2004; 127: 439-451Crossref PubMed Scopus (238) Google Scholar, 9Goldfarb LG Park KY Cervenakova L Gorokhova S Lee HS Vasconcelos O Nagle JW Semino-Mora C Sivakumar K Dalakas MC Missense mutations in desmin associated with familial cardiac and skeletal myopathy.Nat Genet. 1998; 19: 402-403Crossref PubMed Scopus (454) Google Scholar, 10Muñoz-Mármol AM Strasser G Isamat M Coulombe PA Yang Y Roca X Vela E Mate JL Coll J Fernandez-Figueras MT Navas-Palacios JJ Ariza A Fuchs E A dysfunctional desmin mutation in a patient with severe generalized myopathy.Proc Natl Acad Sci USA. 1998; 95: 11312-11317Crossref PubMed Scopus (235) Google Scholar, 11Vicart P Caron A Guicheney P Li Z Prevost MC Faure A Chateau D Chapon F Tome F Dupret JM Paulin D Fardeau M A missense mutation in the alphaB-crystallin chaperone gene causes desmin-related myopathy.Nat Genet. 1998; 20: 92-95Crossref PubMed Scopus (984) Google Scholar, 12Selcen D Engel AG Myofibrillar myopathy caused by novel dominant negative alpha B-crystallin mutations.Ann Neurol. 2003; 54: 804-810Crossref PubMed Scopus (236) Google Scholar, 13Ferreiro A Ceuterick-de Groote C Marks JJ Goemans N Schreiber G Hanefeld F Fardeau M Martin JJ Goebel HH Richard P Guicheney P Bonnemann CG Desmin-related myopathy with Mallory body-like inclusions is caused by mutations of the selenoprotein N gene.Ann Neurol. 2004; 55: 676-686Crossref PubMed Scopus (164) Google Scholar, 14Selcen D Engel AG Mutations in myotilin cause myofibrillar myopathy.Neurology. 2004; 62: 1363-1371Crossref PubMed Scopus (249) Google Scholar, 15Olivé M Goldfarb LG Shatunov A Fischer D Ferrer I Myotilinopathy: refining the clinical and myopathological phenotype.Brain. 2005; 128: 2315-2326Crossref PubMed Scopus (133) Google Scholar, 16Selcen D Engel AG Mutations in ZASP define a novel form of muscular dystrophy in humans.Ann Neurol. 2005; 57: 269-276Crossref PubMed Scopus (230) Google Scholar, 17Vorgerd M van der Ven PF Bruchertseifer V Lowe T Kley RA Schroder R Lochmuller H Himmel M Koehler K Furst DO Huebner A A mutation in the dimerization domain of filamin c causes a novel type of autosomal dominant myofibrillar myopathy.Am J Hum Genet. 2005; 77: 297-304Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar The causes of protein aggregation in MFMs are not fully understood, but our previous studies have shown that impaired protein degradation probably plays a crucial role, as suggested by abnormal expression levels and aberrant localization of several subunits of the proteasome 19S and 26S and by the up-regulation of immunoproteasomal subunits in muscle fibers containing abnormal protein deposits.18Ferrer I Martin B Castano JG Lucas JJ Moreno D Olive M Proteasomal expression, induction of immunoproteasome subunits, and local MHC class I presentation in myofibrillar myopathy and inclusion body myositis.J Neuropathol Exp Neurol. 2004; 63: 484-498PubMed Google Scholar Moreover, protein accumulations are enriched in clusterin and γ-tubulin, whereas p62 and mutant ubiquitin colocalize with protein aggregates, thus suggesting p62 involvement in protein aggregation and mutant ubiquitin in protein degradation in MFMs.19Ferrer I Carmona M Blanco R Moreno D Torrejón-Escribano B Olivé M Involvement of clusterin and the aggresome in abnormal protein deposits in myofibrillar myopathies and inclusion body myositis.Brain Pathol. 2005; 15: 101-108Crossref PubMed Scopus (38) Google Scholar, 20Olivé M van Leeuwen FW Janué A Moreno D Torrejón-Escribano B Ferrer I Expression of mutant ubiquitin (UBB+1) and p62 in myotilinopathies and desminopathies.Neuropathol Appl Neurobiol. 2007; (in press)PubMed Google Scholar Preliminary work in our laboratory identified the presence of ubiquitin carboxy-terminal hydrolase L3 (UCHL3) in normal and diseased muscle, but UCHL1 was also present in the setting of abnormal protein deposits in MFMs. This was unexpected, because UCHL1 is abundant in brain and testis, whereas other members of the UCHL family, but not UCHL1, are expressed in other organs.21Wilkinson KD Lee KM Deshpande S Duerksen-Hughes P Boss JM Pohl J The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase.Science. 1989; 246: 670-673Crossref PubMed Scopus (779) Google Scholar, 22Solano SM Miller DW Augood SJ Young AB Penney Jr, JB Expression of alpha-synuclein, parkin, and ubiquitin carboxy-terminal hydrolase L1 mRNA in human brain: genes associated with familial Parkinson's disease.Ann Neurol. 2000; 47: 201-210Crossref PubMed Scopus (185) Google Scholar, 23Kurihara LJ Semenova E Levorse JM Tilghman SM Expression and functional analysis of Uch-L3 during mouse development.Mol Cell Biol. 2000; 20: 2498-2504Crossref PubMed Scopus (67) Google Scholar UCHLs are enzymes involved in the hydrolysis of polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin-proteasome system favoring protein degradation.24Liu Y Fallon L Lashuel HA Liu Z Lansbury PT The UCHL1 gene encodes two opposing enzymatic activities that affect α-synuclein degradation and Parkinson's disease susceptibility.Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar In the nervous system, UCHL1 associates with ubiquitin and maintains free ubiquitin levels in neurons. Loss of UCHL1 reduces free ubiquitin and leads to inadequate ubiquitylation and protein accumulation in neurons.25Osaka H Wang YL Takada K Takizawa S Setsuie R Li H Sato Y Nishikawa K Sun YJ Sakurai M Harada T Hara Y Kimura I Chiba S Namikawa K Kiyama H Noda M Aoki S Wada K Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.Hum Mol Genet. 2003; 12: 1945-1958Crossref PubMed Scopus (330) Google Scholar Based on these findings, the present work was designed to study the localization and distribution of UCHL1 in MFMs, the expression of other neuronal proteins in MFMs that are not normally expressed in adult muscle fibers, and the mechanisms that modulate the abnormal expression of neuronal proteins in MFMs. For practical purposes, the study was focused on the MFM subgroup of myotilinopathies, disorders associated with mutations in the myotilin gene. We have determined that neuron-restrictive silencer factor (NRSF)/RE1 silencing transcription factor (REST), a transcription factor expressed in non-neuronal tissues repressing the expression of several neuronal genes, is reduced in myotilinopathies.26Chong JA Tapia-Ramirez J Kim S Toledo-Aral JJ Zheng Y Boutros MC Altshuller YM Frohman MA Kraner SD Mandel G REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons.Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 27Schoenherr CJ Anderson DJ The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes.Science. 1995; 267: 1360-1363Crossref PubMed Scopus (941) Google Scholar This reduction is accompanied by aberrant expression of the neuronal proteins synaptosomal-associated protein 25 (SNAP25) and synaptophysin, which are encoded by NRSF/REST target genes, and α-internexin.28Bruce AW Donaldson IJ Wood IC Yerbury SA Sadowski MI Chapman M Gottgens B Buckley NJ Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes.Proc Natl Acad Sci USA. 2004; 101: 10458-10463Crossref PubMed Scopus (391) Google Scholar Finally, we have shown that NRSF/REST is also involved in the regulation of UCHL1 and α-internexin gene expression. Muscle biopsies from 10 patients with myotilinopathy were included in the present study. In addition, muscle samples from five age-matched patients who were considered to be free of any neuromuscular disease after detailed clinical and pathological studies were used as controls. A detailed description of the clinical, pathological, and molecular studies of seven of the cases here included has been presented elsewhere.15Olivé M Goldfarb LG Shatunov A Fischer D Ferrer I Myotilinopathy: refining the clinical and myopathological phenotype.Brain. 2005; 128: 2315-2326Crossref PubMed Scopus (133) Google Scholar A summary of the patients included in the present study is shown in Table 1.Table 1Summary of the Main Clinical Findings in the Present SeriesCaseAge/genderAge at onsetSite of muscle biopsyMutationPatients152/M50Lateral gastrocnemiusS55F256/M52SoleusS55F370/M58Vastus lateralisS60C469/F62DeltoidS60C580/M70DeltoidS60F682/M62Vastus lateralisS60C773/F69DeltoidS55F880/F77Vastus lateralisK36E960/F50DeltoidS60C1054/M50Lateral gastrocnemiusS55FControls1175/MDeltoid1264/MDeltoid1365/FVastus lateralis1480/MVastus lateralis1558/FLateral gastrocnemiusM, male; F, female. Open table in a new tab M, male; F, female. For immunohistochemical studies, 6-μm-thick cryostat sections were stained with hematoxylin and eosin (H&E) and modified Gomori trichrome and processed for myotilin, ubiquitin, UCHL1, SNAP25, synaptophysin, and α-internexin immunohistochemistry. Briefly, cryostat sections were incubated with 1% hydrogen peroxide and 10% methanol for 30 minutes at room temperature, followed by 5% normal serum for 2 hours, and then incubated overnight with one of the primary antibodies. Mouse monoclonal anti-myotilin (Novocastra, Newcastle, UK), anti-synaptophysin (Dako, Glostrup, Denmark), anti-SNAP25 (Chemicon, Temecula, CA), and anti-α-internexin (Zymed, South San Francisco, CA) antibodies were used at dilutions of 1:200, 1:100, 1:2000, and 1:100, respectively. Rabbit polyclonal anti-ubiquitin (Dako) and anti-UCHL1 (Abcam, Cambridge, UK) antibodies were used at dilutions of 1:100 and 1:200, respectively. After washing, the sections were processed with the streptavidin-biotin Super Sensitive IHC detection system (BioGenex, San Ramon, CA). The peroxidase reaction was visualized with diaminobenzidine and hydrogen peroxide in phosphate-buffered saline (PBS). For double-labeling immunofluorescence, 6-μm-thick cryostat sections were stained with a saturated solution of Sudan black B (Merck, Darmstadt, Germany) for 30 minutes to block autofluorescence of lipofuscin granules, rinsed in 70% ethanol, and washed in distilled water. Sections were incubated at 4°C overnight with mouse monoclonal anti-myotilin antibody (Novocastra) at a dilution of 1:200 and rabbit polyclonal anti-UCHL1 antibody (Abcam) at a dilution of 1:200 in PBS. Secondary antibodies were Alexa488 anti-mouse (green) and Alexa546 anti-rabbit (red) (Molecular Probes, Leiden, The Netherlands), used at a dilution of 1:400. After washing with PBS, the sections were incubated in a cocktail of secondary antibodies in the same vehicle solution for 3 hours at room temperature. After washing in PBS, the sections were mounted in Immuno-Fluore mounting medium (ICN Biomedicals, Solon, CA), sealed, and dried overnight. Nuclei were stained with TO-PRO-3-iodide (Molecular Probes) diluted 1:1000. Sections incubated with the secondary antibodies only were used as controls. Samples were examined under a Leica TCS-SL confocal microscope (Leica, Wetzlar, Germany). Frozen muscles were directly homogenized in a final volume of 1:20 (w/v) in lysis buffer (75 mmol/L Tris-HCl, pH 6.8, 0.001% bromphenol blue, 15% sodium dodecyl sulfate, 20% glycerol, 5% β-mercaptoethanol, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin, leupeptin, and pepstatin). Samples were boiled at 95°C for 3 minutes and centrifuged at 9500 rpm for 5 minutes at room temperature. Samples were loaded in sodium dodecyl sulfate-polyacrylamide gels with Tris-glycine running buffer. Proteins were electrophoresed using a miniprotean system (Bio-Rad, Alcobendas, Spain) and transferred to nitrocellulose membranes with a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) for 45 minutes at 40 mA. Cell lines grown in six-well plates were homogenized with radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Lysates were maintained in agitation for 30 minutes at 4°C and then centrifuged at 12,000 rpm for 20 minutes at 4°C. Protein concentration was determined with BCA (Pierce, Woburn, MA) method. Twenty micrograms of total protein was boiled at 95°C for 3 minutes and loaded in sodium dodecyl sulfate-polyacrylamide gels with Tris-glycine running buffer. Proteins were electrophoresed using a miniprotean system (Bio-Rad) and transferred to nitrocellulose membranes with a Mini Trans-Blot electrophoresis transfer cell (Bio-Rad) for 1 hour at 100 V. Nitrocellulose membranes were blocked with PBS containing 5% skim milk for 30 minutes. Subsequently, the membranes were incubated at 4°C overnight with one of the primary antibodies in PBS containing 5% skim milk. The following antibodies were used: mouse monoclonal myotilin (Novocastra) used at a dilution of 1:10,000, rabbit polyclonal UCHL1 (Chemicon) diluted 1:500, anti-REST (Abcam) used at a dilution of 1:500 for muscle biopsies and diluted 1:250 for cell lines, anti-α-internexin (Zymed) at a dilution of 1:500, and mouse monoclonal anti-β-actin (clone AC-74; Sigma) diluted 1:30,000. After primary antibody incubation, the membranes were washed three times with PBS containing 5% skim milk for 5 minutes at room temperature and then incubated with the corresponding anti-rabbit or anti-mouse antibodies labeled with horseradish peroxidase (Dako) at a dilution of 1:1000 for 1 hour at room temperature. The membranes were subsequently washed four times for 5 minutes each with PBS at room temperature and developed with the chemiluminescence ECL Western blotting system (Amersham/Pharmacia, Buckinghamshire, UK) followed by apposition of the membranes to autoradiographic films (Hyperfilm ECL; Amersham). Control of protein loading was tested with Coomassie blue on the band of 205 kd corresponding to myosin. The selection of cell lines was based on preliminary studies in our lab directed to sample cells with different basal expression of UCH-L1 and NRSF/REST. The cell lines selected on this basis were as follows. Small cell lung cancer DMS53 (American Type Culture Collection no. CRL-2062; Manassas, VA) cells were maintained in Waymouth's MB 752/1 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. HeLa cells were maintained in Dulbecco's minimal essential medium (Invitrogen, El Prat de Llobregat, Spain) supplemented with 10% fetal bovine serum. U87-MG cells (American Type Culture Collection no. HTB-14) were maintained in minimal essential medium (Eagle's) with 2 mmol/L l-glutamine and supplemented with 10% fetal bovine serum and 1 mmol/L sodium pyruvate. Human neuroblastoma SH-SY5Y cell line was maintained in Dulbecco's minimal essential medium (Invitrogen) (European Collection of Cell Cultures no. 94030304; Salisbury, UK) supplemented with 10% fetal bovine serum. All cell lines were grown at 37°C in a humidified atmosphere of 5% CO2. DMS53 and SH-SY5Y cells were plated in six-well dishes at a concentration of 105 cells/well and cultured overnight before transfection. One microgram of REEX1 vector (kindly provided by Dr. Gail Mandel, Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY) was transfected using Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. After 5 hours of transfection, the medium was replaced by fresh medium. U87-MG cells were plated in six-well dishes at a concentration of 50,000 cells/well and cultured overnight before transfection. Following the instructions of the manufacturers, 100 nmol/L NRSF/REST short interferring (si)RNA 1 (5′-GCUUAUUAUGCUGGCAAAUTT-3′; Ambion, Madrid, Spain), NRSF/REST siRNA 2 (5′-GCCUUCUAAUAAUGUGUCATT-3′; Ambion), and a negative control or scramble siRNA (Ambion) were transfected using Lipofectamine 2000 (Invitrogen). After 5 hours of transfection, the medium was replaced by fresh medium. The analysis of siRNA was performed 48 hours later. RNA obtained from muscle biopsies was purified as previously described.29Barrachina M Castano E Ferrer I TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue.Neurochem Int. 2006; 49: 276-284Crossref PubMed Scopus (68) Google Scholar The purification of RNA from cell lines was performed with RNeasy Midi kit (Qiagen, Hilden, Germany) following the protocol provided by the manufacturer. The concentration of each sample was obtained from A260 measurements. RNA integrity was tested using the Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA). The retrotranscriptase reaction (100 ng RNA/μl) was performed using the High capacity cDNA Archive kit (Applied Biosystems, Madrid, Spain) following the protocol provided by the supplier. Parallel reactions for each RNA sample were run in the absence of MultiScribe Reverse Transcriptase to assess the degree of contaminating genomic DNA. The NRSF/REST TaqMan assay (Hs00194498_m1, TaqMan probe 5′-AGGAAGGCCGAATACAGTTATGGCC-3′) (Applied Biosystems) generates an amplicon of 79 bp and is located at position 341 between the 1 and 2 exon boundary of NM_005612.3 transcript sequence. The TaqMan assay for UCHL1 (Hs00188233_m1, TaqMan probe 5′-CCTGCTGAAGGACGCTGCCAAGGTC-3′) (Applied Biosystems) is located at position 648 between the 8 and 9 exon boundary of NM_004181.3 transcript sequence. It generates an amplicon of 100 bp. The TaqMan assay for Synaptophysin (Hs00300531_m1, TaqMan probe 5′-CGAGTACCCCTTCAGGCTGCACCAA-3′) (Applied Biosystems) generates an amplicon of 63 bp and is located at position 241 of NM_003179.2 transcript sequence. The TaqMan assay for α-internexin (Hs00190771_m1, TaqMan probe 5′-AGCAGCTTACAGGAAACTGCTGGAA-3′) (Applied Biosystems) generates an amplicon of 92 bp and is located at position 1240 of NM_032727.2 transcript sequence. The TaqMan assay for SNAP25 (Hs00268296_m1, TaqMan probe 5′-GAAGCCCAGGTCCAGAGCCAAACCC-3′) (Applied Biosystems) generates an amplicon of 63 bp and is located at position 153 of NM_003081.2 transcript sequence. TaqMan polymerase chain reaction (PCR) assays for all genes analyzed were performed in duplicate on cDNA samples in 96-well optical plates using an ABI Prism 7900 Sequence Detection system (Applied Biosystems). The plates were capped using optical caps (Applied Biosystems). For each 20-μl TaqMan reaction, 9 μl of cDNA (diluted 1/50 for studies with DMS53 and 1/10 for muscle biopsies) was mixed with 1 μl of 20× TaqMan Gene Expression Assays and 10 μl of 2× TaqMan Universal PCR Master Mix (Applied Biosystems). Parallel assays for each sample were performed with β-glucuronidase (GUSB) (Hs99999908_m1, TaqMan probe 5′-GACTGAACAGTCACCGACGAGAGTG-3′), for normalization. The reactions were performed using the following parameters: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Standard curves were prepared for all genes analyzed using serial dilutions of cDNA from DMS53 cells, U87-MG cells, and human control muscle biopsies. Finally, all TaqMan PCR data were captured using the Sequence Detector Software (SDS version 1.9; Applied Biosystems). For each experimental sample, the amount of target and endogenous reference (GUSB) was determined from the appropriate standard curve, which was plotted showing the cycle threshold (y) versus the log of nanograms of total control RNA. Then the amount of each target gene was divided by the endogenous reference GUSB amount to obtain a normalized target value, permitting determination of the relative mRNA levels of every gene in normal muscle and in myotilinopathy. TaqMan PCR assays for α-internexin and SNAP25 from human muscle biopsies were performed using the TaqMan PreAmp Master Kit (Applied Biosystems). Chromatin immunoprecipitation (ChIP) assay was performed according to the manufacturer's protocol (Upstate, Madrid, Spain) using 106 U87-MG, HeLa, and DMS53 cells. Ten micrograms of anti-NRSF/REST (P-18X, sc-15118X; Santa Cruz Biotechnology, Santa Cruz, CA) and 10 μg of anti-acetylated H3 (residue Lys9; Cell Signaling, Danvers, MA) were used for immunoprecipitation. Purified DNA was resuspended in 20 μl of DNase-free water, and 2 μl was used as a template in 25 μl of PCR reaction using GoTaq Flexi DNA Polymerase (Promega, Madrid, Spain). Primer concentration was 200 nmol/L. PCR primers were 5′-ACAAATCCCgTCTCCACAAC-3′ and 5′-GCCTAGGGAAGACGAAAAACA-3′ for the amplification of neuron-restrictive silencing element 1 (NRSE1) sequence of the UCHL1 gene promoter. The reaction was performed using the following parameters: 95°C for 2 minutes and 35 cycles of 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and a last hold of 72°C for 5 minutes. Muscle biopsies were characterized by the presence of nonhyaline and hyaline polymorphic inclusions of varying shapes and sizes. Hyaline inclusions stained bright pink with hematoxylin and eosin and blue-red or red-purple with the modified trichrome Gomori stain. Rimmed and unrimmed vacuoles were found in every case. Cytoplasmic bodies and collections of dense spheroid bodies were observed in the majority of cases. Immunohistochemical studies showed strong myotilin (Figure 1) and ubiquitin immunoreactivity in hyaline inclusions. Additional immunohistochemical studies demonstrated the presence of UCHL1, α-internexin, synaptophysin, and SNAP25 in parallel with myotilin aggregates in abnormal muscle fibers in myotilinopathy (Figure 2). Double-labeling immunofluorescence and confocal microscopy disclosed UCHL1 immunoreactivity in damaged muscle fibers in myotilinopathy, whereas no traces of UCHL1 occurred in control muscles. Aberrant UCHL1 deposition colocalized with myotilin aggregates in damaged fibers. These changes were specific, because no immunoreaction was elicited after incubation with the secondary antibodies alone (Figure 3). Synaptophysin and SNAP25 have been described as being regulated by NRSF/REST.28Bruce AW Donaldson IJ Wood IC Yerbury SA Sadowski MI Chapman M Gottgens B Buckley NJ Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes.Proc Natl Acad Sci USA. 2004; 101: 10458-10463Crossref PubMed Scopus (391) Google Scholar Following up on these data, we tested whether the expression levels of REST were modified in diseased muscles. Protein levels were analyzed by gel electrophoresis and Western blotting. Analysis of protein levels and densitometric quantifications are shown in Figure 4. UCHL1 protein levels were significantly increased in myotilinopathies [P < 0.01, analysis of variance with post hoc least significant difference (LSD) test], whereas NRSF/REST protein levels were reduced in diseased muscles (P < 0.05, analysis of variance with post hoc LSD test). Although the modifications in UCHL1 and REST levels were variable from one case to another, the density of the bands of UCHL1 inversely correlated with REST expression in every individual case. Interestingly, the higher expression of UCHL1 was associated with the higher deposition of myotilin (Figure 4). A significant increase in UCHL1 and SNAP25 mRNA levels was found in myotilinopathy when compared with control muscles (P < 0.05, analysis of variance with post hoc LSD test). This was associated with a parallel decrease in REST mRNA expression levels in diseased muscles (Figure 5A). α-Internexin mRNA levels were undetected in normal muscles, but they were clearly expressed in myotilinopathy (Figure 5B). As mentioned above, some of the up-regulated neuronal proteins detected in myotilinopathy have been described as being NRSF/REST target genes.28Bruce AW Donaldson IJ Wood IC Yerbury SA Sadowski MI Chapman M Gottgens B Buckley NJ Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes.Proc Natl Acad Sci USA. 2004; 101
Referência(s)