Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure "autophagic flux" in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the "autophagometer." In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. The addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a "colchicine block." Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an "in vivo autophagometer" study using colchicine in skeletal muscle.
We hypothesized that AMP-activated protein kinase-related kinase 5 (ARK5)/novel kinase family 1 (NUAK1), an AMP-activated protein kinase (AMPK)-related kinase that has been found to be stimulated by protein kinase B (Akt), would be expressed in rat skeletal muscle and activated by electrically elicited contractions, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), or insulin. We verified expression of ARK5 in muscle through RT-PCR and Western blot. Cross-reactivity of ARK5 immunoprecipitates with antibodies against phospho-AMPK was increased by approximately 30% by muscle contractions and approximately 60% by incubation of muscle with AICAR. AMPK was not detected in the ARK5 immunoprecipitates. Despite the apparent increase in phosphorylation of ARK5 at a site essential to its activation, neither contractions nor AICAR increased ARK5 activity. For muscles from animals injected with saline or insulin, we probed nonimmunoprecipitated samples in sequence for phosphotyrosine (P-Tyr), ARK5, and phosphorylated substrates of Akt (P-AS) and found that the ARK5 band could be precisely superimposed on phosphoprotein bands from the P-Tyr and P-AS blots. In the band corresponding to ARK5, insulin increased P-Tyr content by approximately 45% and cross-reactivity with the antibody against P-AS by approximately threefold. We also detected ARK5 in phosphotyrosine immunoprecipitates. Our data suggest that increased phosphorylation of ARK5 by muscle contractions or exposure to AICAR is insufficient to activate ARK5 in skeletal muscle, suggesting that some other modification (e.g., phosphorylation on tyrosine or by Akt) may be necessary to its activity in muscle.
Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation. Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation. Ubiquitinated inclusions (UBIs) 2The abbreviations used are: UBI, ubiquitinated inclusion; UPS, ubiquitin proteasome system; IBMPFD, inclusion body myopathy, Paget disease of the bone and frontotemporal dementia; ERAD, endoplasmic reticulum-associated degradation; GFP, green fluorescent protein; CFP, cyan fluorescent protein; LSD, least significant difference; WT, wild type; PBS, phosphate-buffered saline; TA, tibialis anterior; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS, tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] inner salt; FRAP, fluorescence recovery after photobleaching. 2The abbreviations used are: UBI, ubiquitinated inclusion; UPS, ubiquitin proteasome system; IBMPFD, inclusion body myopathy, Paget disease of the bone and frontotemporal dementia; ERAD, endoplasmic reticulum-associated degradation; GFP, green fluorescent protein; CFP, cyan fluorescent protein; LSD, least significant difference; WT, wild type; PBS, phosphate-buffered saline; TA, tibialis anterior; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS, tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] inner salt; FRAP, fluorescence recovery after photobleaching. are a common pathology in many degenerative disorders associated with brain and muscle (1Schwartz A.L. Ciechanover A. Annu. Rev. Med. 1999; 50: 57-74Crossref PubMed Scopus (373) Google Scholar). In many cases the molecular constituents of the UBIs are known and are a result of the misfolding of an aggregate prone or mutant protein such as α-synuclein in autosomal dominant Parkinson disease or expanded polyglutamine containing proteins in Huntington disease. UBIs in other degenerative disorders may be due to a global impairment in protein degradation. For example, after a misfolded substrate overwhelms the ubiquitin proteasome system (UPS) or as a result of mutations in proteins involved in specific protein degradation pathways. One example is the autosomal dominantly inherited disorder inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD) associated with mutations in the UPS chaperone p97/VCP (2Watts G.D. Wymer J. Kovach M.J. Mehta S.G. Mumm S. Darvish D. Pestronk A. Whyte M.P. Kimonis V.E. Nat. Genet. 2004; 36: 377-381Crossref PubMed Scopus (1098) Google Scholar). Affected tissue in IBMPFD contains prominent cytoplasmic and intranuclear UBIs (3Forman M.S. Mackenzie I.R. Cairns N.J. Swanson E. Boyer P.J. Drachman D.A. Jhaveri B.S. Karlawish J.H. Pestronk A. Smith T.W. Tu P.H. Watts G.D. Markesbery W.R. Smith C.D. Kimonis V.E. J. Neuropathol. Exp. Neurol. 2006; 65: 571-581Crossref PubMed Scopus (197) Google Scholar, 5Schroder R. Watts G.D. Mehta S.G. Evert B.O. Broich P. Fliessbach K. Pauls K. Hans V.H. Kimonis V. Thal D.R. Ann. Neurol. 2005; 57: 457-461Crossref PubMed Scopus (152) Google Scholar). In some cases these UBIs contain tubulofilamentous inclusions and insoluble protein aggregates (4Hubbers C.U. Clemen C.S. Kesper K. Boddrich A. Hofmann A. Kamarainen O. Tolksdorf K. Stumpf M. Reichelt J. Roth U. Krause S. Watts G. Kimonis V. Wattjes M.P. Reimann J. Thal D.R. Biermann K. Evert B.O. Lochmuller H. Wanker E.E. Schoser B.G. Noegel A.A. Schroder R. Brain. 2006; 130: 381-393Crossref PubMed Scopus (136) Google Scholar). p97/VCP belongs the AAA+ (ATPases associated with a variety of activities) protein family and participates in the degradation of proteins via the UPS (6Halawani D. Latterich M. Mol. Cell. 2006; 22: 713-717Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). p97/VCP has a clear role in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins (6Halawani D. Latterich M. Mol. Cell. 2006; 22: 713-717Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In association with co-factors derlin-1, Ufd1, and Npl4, p97/VCP participates in the retrotranslocation of misfolded endoplasmic reticulum lumen and transmembrane proteins facilitating their delivery to the UPS. We have previously shown that IBMPFD mutant p97/VCP fails to degrade the misfolded ERAD substrate ΔF508-CFTR as efficiently as wild type p97/VCP in cultured myoblasts (7Weihl C.C. Dalal S. Pestronk A. Hanson P.I. Hum. Mol. Genet. 2006; 15: 189-199Crossref PubMed Scopus (149) Google Scholar). p97/VCP may also facilitate the degradation of soluble cytosolic proteins. Recently, IBMPFD mutations were found to impair the degradation of the cytosolic protein, Unc-45 (8Janiesch P.C. Kim J. Mouysset J. Barikbin R. Lochmuller H. Cassata G. Krause S. Hoppe T. Nat. Cell Biol. 2007; 9: 379-390Crossref PubMed Scopus (118) Google Scholar). Whether IBMPFD mutations in p97/VCP affect the degradation of all UPS substrates resulting in UBIs is unknown.UBIs can also form in the setting of impaired macroautophagy, herein referred to as autophagy (9Rubinsztein D.C. Nature. 2006; 443: 780-786Crossref PubMed Scopus (1306) Google Scholar). Autophagy is a nonselective mechanism for degrading long-lived proteins and organelles, whereas the UPS is a regulated means of targeting short-lived proteins to the proteasome. Genetic inactivation of autophagy in mouse central nervous system tissue results in prominent UBIs and neurodegeneration (10Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Nature. 2006; 441: 885-889Crossref PubMed Scopus (3060) Google Scholar). Autophagy and the UPS have traditionally been thought to serve complementary yet parallel functions in protein homeostasis, and the inter-relationship between these two degradation systems is unclear (11Ding W.X. Ni H.M. Gao W. Yoshimori T. Stolz D.B. Ron D. Yin X.M. Am. J. Pathol. 2007; 171: 513-524Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). One potential point of intersection between autophagy and the UPS is the "aggresome" or inclusion body (12Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar). An aggresome is a microtubule-dependent pericentriolar region of the cell that contains sequestered misfolded or aggregated proteins (13Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1763) Google Scholar). Aggresome formation occurs in the setting of UPS dysfunction because of decreased proteasome activity or the overwhelming accumulation of misfolded proteins (13Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1763) Google Scholar). The aggresome also contains proteins such as LC3 and p62 along with lysosomes, suggesting that these are areas of active autophagic degradation (14Bjorkoy G. Lamark T. Brech A. Outzen H. Perander M. Overvatn A. Stenmark H. Johansen T. J. Cell Biol. 2005; 171: 603-614Crossref PubMed Scopus (2458) Google Scholar, 15Iwata A. Riley B.E. Johnston J.A. Kopito R.R. J. Biol. Chem. 2005; 280: 40282-40292Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). Ubiquitinated and aggregated proteins are trafficked to the aggresome via interactions with HDAC6 and dynein (13Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1763) Google Scholar, 15Iwata A. Riley B.E. Johnston J.A. Kopito R.R. J. Biol. Chem. 2005; 280: 40282-40292Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 16Kawaguchi Y. Kovacs J.J. McLaurin A. Vance J.M. Ito A. Yao T.P. Cell. 2003; 115: 727-738Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar). The molecular machinery involved in triaging degradation destined proteins to the UPS or autophagic pathways remains to be elucidated. This putative "segregase" would need to interact with ubiquitinated substrates as well as with both UPS and autophagic machinery.The current study examines the role of p97/VCP and its mutants in inclusion body formation and protein aggregate clearance. Although it is clear that p97/VCP plays a critical role in the ERAD degradation of misfolded proteins (6Halawani D. Latterich M. Mol. Cell. 2006; 22: 713-717Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), the role of p97/VCP in the degradation of cytosolically derived misfolded and aggregated proteins is less clear. Loss of p97/VCP function in mammalian cells leads to the accumulation of insoluble ubiquitinated proteins (17Dalal S. Rosser M.F. Cyr D.M. Hanson P.I. Mol. Biol. Cell. 2004; 15: 637-648Crossref PubMed Scopus (139) Google Scholar, 18Wojcik C. Rowicka M. Kudlicki A. Nowis D. McConnell E. Kujawa M. Demartino G.N. Mol. Biol. Cell. 2006; 17: 4006-4618Crossref Google Scholar, 19Wojcik C. Yano M. DeMartino G.N. J. Cell Sci. 2004; 117: 281-292Crossref PubMed Scopus (205) Google Scholar), similarly IBMPFD mutant p97/VCP expression in myoblasts and transgenic mouse muscle leads to the accumulation of UBIs (7Weihl C.C. Dalal S. Pestronk A. Hanson P.I. Hum. Mol. Genet. 2006; 15: 189-199Crossref PubMed Scopus (149) Google Scholar, 20Weihl C.C. Miller S.E. Hanson P.I. Pestronk A. Hum. Mol. Genet. 2007; 16: 919-928Crossref PubMed Scopus (90) Google Scholar). p97/VCP may be involved in inclusion body formation. Loss of p97/VCP or expression of a dominant negative ATP hydrolysis-deficient mutant impairs aggresome formation (19Wojcik C. Yano M. DeMartino G.N. J. Cell Sci. 2004; 117: 281-292Crossref PubMed Scopus (205) Google Scholar, 21Kitami M.I. Kitami T. Nagahama M. Tagaya M. Hori S. Kakizuka A. Mizuno Y. Hattori N. FEBS Lett. 2006; 580: 474-478Crossref PubMed Scopus (22) Google Scholar, 22Song C. Xiao Z. Nagashima K. Li C.C. Lockett S.J. Dai R.M. Cho E.H. Conrads T.P. Veenstra T.D. Colburn N.H. Wang Q. Wang J.M. Toxicol. Appl. Pharmacol. 2008; 228: 351-363Crossref PubMed Scopus (29) Google Scholar). p97/VCP associates with the aggresome essential protein, HDAC6 (23Boyault C. Gilquin B. Zhang Y. Rybin V. Garman E. Meyer-Klaucke W. Matthias P. Muller C.W. Khochbin S. EMBO J. 2006; 25: 3357-3366Crossref PubMed Scopus (218) Google Scholar, 24Seigneurin-Berny D. Verdel A. Curtet S. Lemercier C. Garin J. Rousseaux S. Khochbin S. Mol. Cell. Biol. 2001; 21: 8035-8044Crossref PubMed Scopus (270) Google Scholar). Overexpression of HDAC6 facilitates the degradation of expanded polyglutamine containing androgen receptor or ubiquitinated proteins in an autophagy-specific manner (25Pandey U.B. Nie Z. Batlevi Y. McCray B.A. Ritson G.P. Nedelsky N.B. Schwartz S.L. DiProspero N.A. Knight M.A. Schuldiner O. Padmanabhan R. Hild M. Berry D.L. Garza D. Hubbert C.C. Yao T.P. Baehrecke E.H. Taylor J.P. Nature. 2007; 447: 859-863Crossref PubMed Scopus (976) Google Scholar). We propose that p97/VCP associates with aggregated proteins and triages them to an inclusion body via interactions with HDAC6. IBMPFD mutants fail to release aggregated proteins, resulting in failed inclusion body formation and protein aggregate clearance.EXPERIMENTAL PROCEDURESPlasmid Constructs and Cell Culture—The following plasmids are previously described: pcDNA4.0/TO p97/VCP-His/Myc and pcDNA3.1p97/VCP-Myc/His (17Dalal S. Rosser M.F. Cyr D.M. Hanson P.I. Mol. Biol. Cell. 2004; 15: 637-648Crossref PubMed Scopus (139) Google Scholar). IBMPFD mutations were introduced into these vectors using site-directed mutagenesis (Stratagene) and sequenced for the integrity of the p97/VCP sequence and verification of their respective point mutations. For lentiviral constructs, a BamHI-AgeI-digested fragment from pcDNA3.1Myc/His vector with the murine p97/VCP-fused to a Myc tag (containing wild type and E578Q, R95G, R155H, and A232E point mutations) was cloned into the BamHI-AgeI site of CCIV FM1 (lentiviral shuttle vector with a cytomegalovirus promoter) to generate the lentiviral constructs p97/VCP-Myc-IRES-VenusGFP. Lentiviruses are prepared as previously described (26Lee C.S. Tee L.Y. Warmke T. Vinjamoori A. Cai A. Fagan A.M. Snider B.J. J. Neurochem. 2004; 91: 996-1006Crossref PubMed Scopus (62) Google Scholar). Lentiviral particles released into tissue culture serum are titered by counting the number of GFP fluorescing cells following transduction into HEK293 cells. polyQ80-CFP was created from the parent vector polyQ80-GFP obtained from Dr. Gene Johnson (Washington University). Expression constructs containing p97/VCP-DsRed-WT, E305Q/E578Q, R155H, R95G, and A232E were a gift from Dr. Nigel Cairns (Washington University), pCMV-GFP-LC3 was gift from Dr. Robert Baloh (Washington University), and mcherry-ATG5 and HDAC6-FLAG constructs were obtained via Addgene. pcDNA4.0/TO-polyQ80-CFP was generated by digesting the polyQ80-CFP containing plasmid with BamHI-NotI and ligating to the BglII-NotI sites in pcDNA4.0/TO.U2OS TRex cells stably transfected with a tetracycline repressor plasmid (Invitrogen) were transfected with pcDNA4.0/TO vectors to generate cell lines expressing p97/VCP. Medium containing 50 μg/ml hygromycin B (Invitrogen) and 125 μg/ml zeocin (Invitrogen) was added to select the stable cell lines. Approximately 12 days later, colonies of zeocin-resistant cells appeared. On average 25-30 colonies for each desired cell line were transferred into separate wells and screened. Immunofluorescence and Western blot analysis were used to identify lines that expressed protein only in the presence of tetracycline. The cells were maintained with 50 μg/ml hygromycin B and 65 μg/ml zeocin, with the exception of nonstably transfected U20S cells, which were maintained with hygromycin B. Protein expression was induced by the addition of 1 μg/ml tetracyline for the indicated times. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics at 37 °C with 5% CO2 and seeded in appropriate plates and grown to ∼80% confluence on the day of transfection. Transfections were performed with Lipofectamine 2000 (Invitrogen). For stable cell lines, tetracycline was added 16 h prior to plasmid transfection.Confocal and Electron Microscopy—U20S cells were plated on glass coverslips and transiently transfected with plasmid DNA or induced with tetracycline as above. 24-48 h post-transfection, the cells were washed twice with PBS, fixed with 3% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed, and then blocked with 3% goat serum for 1 h. After primary antibody incubation, the cells were incubated with Alexa Fluor-conjugated secondary antibody for 1 h, washed several times, and mounted in antifade solution. The samples were observed using a confocal microscope (Carl Zeiss, Jena, Germany).For thin section electron microscopy, transiently transfected cells were grown to 70% confluence in a 100-mm dish for 36 h. For sample preparation, trypsinized cells were collected, washed in phosphate-buffered saline, fixed in 2.5% glutaraldehyde in sodium cacodylate, embedded, sectioned, and stained with uranyl acetate according to standard procedures.Fluorescence Recovery after Photobleaching—The cells were plated and transfected 36 h prior to imaging on glass-bottomed dishes. Immediately before imaging, the medium was replaced with medium containing 10 mm Hepes buffer, pH 7.5. Imaging and photobleaching were performed using a Plan NEOFLUAR 63×/1.30 oil objective on an inverted confocal microscope (model LSM510 Meta; Carl Zeiss MicroImaging, Inc.). The cells transfected with CFP fusion proteins were imaged with 488-nm light and DsRed with 516-nm light, using 2% laser power and a pin hole of 1 airy unit. After two imaging scans, a selected area of the inclusion (region of interest) was bleached using maximal laser power for 10 iterations, and then the photobleached cell was imaged at 20-s intervals for 10 min. The collected images were analyzed using Zeiss LSM 5 software to calculate the mean fluorescence intensity in the region of interest as a function of time after photobleaching. The relative fluorescence intensity values are corrected by percentage of prebleaching values.Quantification of Inclusions—Normal U2OS cells and U2OS cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutants were induced with tetracycline and transfected with polyQ80-CFP for 48 h and then processed for fluorescence microscopic analysis of inclusion formation. The cells were counted. For inclusions, 10 random fields of each sample were selected, and the cells containing inclusions were manually counted for the number of inclusions/cell. ∼100 cells/experiment were counted and averaged among three replicates totaling ∼300 cells/condition. Counted cells were grouped into two categories: ≤3 inclusions/cell or >3 inclusions/cell, and the data were subjected to statistical analysis. Inclusion clearance was performed in a similar manner, except that total transfected cells were counted and the percentage of inclusion containing cells/total transfected cells were plotted for each time point and condition.Filter Trap Assay—Normal U2OS cells and U2OS cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutants were induced and transfected with polyQ80-CFP for 48 h. The cells were harvested using 1× PBS including protease inhibitors and brief sonication. Twenty μg of protein were mixed with 900 μl of PBS + 1% SDS. Subsequently, the samples were applied to a slot blot unit (Bio-Rad, Bio-Dot) and filtered through a nitrocellulose membrane. The membrane was probed with anti-GFP antibody.Immunoprecipitation and Western Blotting—Immunoprecipitation was performed using Seize Classic(G) immunoprecipitation kit (Pierce). The cells were washed with cold PBS, scraped, pelleted by centrifugation, and lysed on ice for 30 min with radioimmune precipitation assay buffer containing protease inhibitors. The cell lysate were briefly sonicated, centrifuged for 10 min at 12,000 × g at 4 °C, and the supernatants were used for immunoprecipitation. One hundred microliters of cell lysate was incubated with 2 μg of mouse monoclonal anti-Myc antibody. After overnight incubation at 4 °C with rotation, immune complex, and 400 μl of protein G (50% slurry) beads were placed into a spin cup column and incubated for 2 h at room temperature. The bound proteins were eluted from the beads, boiled for 5 min, and analyzed by Western blotting. Western blot analysis was performed as described previously (7Weihl C.C. Dalal S. Pestronk A. Hanson P.I. Hum. Mol. Genet. 2006; 15: 189-199Crossref PubMed Scopus (149) Google Scholar). The following antibodies were used: anti-p97 (BD Biosciences), anti-LC3 (NanoTools), anti-p62 (Abgent), anti-HDAC6, anti-Myc, and anti-tubulin (Cell Signaling Technology), anti-actin (sigma), anti-FK2 (Biomol), and rabbit polyclonal anti-GFP (B5).Electroporation into Mouse Tibialis Anterior (TA)—3-month-old female mice were anesthesized with pentobarbital, and their hindlimb was shaved. 20 μg of polyQ80-CFP expression construct resuspended in 50 μl of 1× PBS was injected into the TA with a 27-gauge needle. A 5-mm gap BTX 2 needle array attached to a BTX ECM 830 electroporator was set for 100 V, pulse length of 50 ms, pulse interval of 200 ms, and six pulses. The animals were allowed to recover, and TA muscle was harvested 7 days later.MTT/MTS Assays—U2OS stable cells were seeded in 6-well plates and allowed to attach overnight, and then the cells were induced. Transfection with 1.5 μg of polyQ80-CFP or polyQ19-CFP DNA was carried out using Lipofectamine 2000 (Invitrogen). Twelve hours post-transfection, the cells were harvested and replated into 96-well plates. A CellTiter 96™ AQueous nonradioactive cell proliferation assay (Promega) kit containing the tetrazolium compound MTS was used according to the manufacturer's instructions to measure the number of cells. MTS color change was monitored by using an ELX-800 universal plate reader (Bio-Tek, Winooski, VT) set at an absorbance reading of 490 nm. For proteasome sensitivity assays, the cells were plated in 96-well plates and treated with vehicle or 10-fold dilutions of 1 mm MG132 or lactacystin. MTS assays were performed 16 h later, and the ratio of cells was determined via comparison with vehicle-treated controls for each.Statistical Analysis—The data were evaluated by analysis of variance followed by Fishers LSD post hoc comparisons at p < 0.05.RESULTSIBMPFD Mutant p97/VCP Impairs Inclusion Body Formation—We generated U20S cell lines that stably express tetracycline-inducible p97/VCP-WT, non-disease-associated ATPase inactive p97/VCP-E578Q or one of four different IBMPFD mutants (R155H (RH), R95G (RG), A232E (AE), and L198W (LW)) with a C-terminal Myc tag (Fig. 1A). Consistent with our previous studies (7Weihl C.C. Dalal S. Pestronk A. Hanson P.I. Hum. Mol. Genet. 2006; 15: 189-199Crossref PubMed Scopus (149) Google Scholar), IBMPFD mutant p97/VCP and ATPase inactive p97/VCP-E578Q expression resulted in an increase in high molecular weight ubiquitinated proteins as seen via immunoblot or immunofluorescence 16 h after tetracycline induction (Fig. 1, B and C). When these cells were stressed with sublethal doses of the proteasome inhibitors MG132 or lactacystin, IBMPFD-expressing cells had fewer cells following treatment consistent with enhanced cell death as measured via MTT/MTS assay (Fig. 1D). Immunohistochemistry of MG132-treated cells with an anti-ubiquitin antibody (FK2) showed that only control U20S and p97/VCP-WT-expressing cells generated small perinuclear ubiquitin-positive inclusions consistent with an aggresome (Fig. 1C). In contrast, IBMPFD mutant-expressing cells had a perinuclear increase in ubiquitinated proteins but no clear aggresome (Fig. 1C). p97/VCP-E578Q-expressing cells had an increase in punctate endoplasmic reticulum-associated ubiquitinated inclusions as previously reported (17Dalal S. Rosser M.F. Cyr D.M. Hanson P.I. Mol. Biol. Cell. 2004; 15: 637-648Crossref PubMed Scopus (139) Google Scholar). This finding suggests that IBMPFD mutant cells have impaired ubiquitinated protein aggregate handling to inclusion bodies, resulting in increased sensitivity to proteasome inhibition. Alternatively, the absence of ubiquitin-positive perinuclear inclusions may more generally reflect an overwhelming of the UPS system in IBMPFD mutant-expressing cells.To see whether IBMPFD mutants mishandled aggregated proteins without exogenous proteasome inhibitors, we co-expressed an expanded polyglutamine containing fusion protein (polyQ80-CFP) in U20S cells. PolyQ80-CFP intrinsically aggregates and forms inclusion bodies consistent with an aggresome. Aggresomes are a cellular response to increased levels of misfolded or aggregated proteins (13Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1763) Google Scholar). They are localized to the microtubule organizing center and are surrounded by an intermediate filament network (13Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1763) Google Scholar). Although aggresomes contain aggregated protein, they are distinct from other protein aggregates because they are formed via an active sequestration of misfolded and aggregated proteins that requires an intact microtubule network. Therefore a protein or protein aggregate that fails to be rapidly degraded via the UPS will coalesce into these larger inclusion bodies or aggresomes (13Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1763) Google Scholar). Once in the aggresome, undegraded ubiquitinated and aggregated proteins are thought to be degraded via autophagy (27Taylor J.P. Tanaka F. Robitschek J. Sandoval C.M. Taye A. Markovic-Plese S. Fischbeck K.H. Hum. Mol. Genet. 2003; 12: 749-757Crossref PubMed Scopus (359) Google Scholar).Following co-expression of polyQ80-CFP in cells expressing p97/VCP-WT fused to a DSred fluorescent tag, we saw p97/VCP-WT surrounding the inclusion body. Inclusion bodies in p97/VCP-WT-expressing cells were predominantly single or <3 inclusions/transfected cell (Fig. 2A) and had properties consistent with an aggresome (not shown). In contrast, there was a decrease in single perinuclear polyQ80-CFP inclusion bodies (≤3 inclusions/cell) in IBMPFD mutant-expressing cells. Instead there was an increase in smaller cytosolic inclusions (>3 inclusions/cell) that did not always co-localize with the microtubule organizing center. p97/VCP-R155H, R95G, and A232E proteins co-localized with polyQ80-CFP, although there were both polyQ80-CFP and p97/VCP-IBMPFD inclusions that did not co-localize (Fig. 2A, see arrows). An aggresome is traditionally defined as a single perinuclear inclusion body (13Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1763) Google Scholar); however, for purposes of quantitation, we counted the number of cells with ≤3 inclusions (aggresomal) or >3 inclusions (non-aggresomal). These data are graphically represented in Fig. 2B and by a low magnification field of p97/VCP-WT or IBMPFD mutant-expressing cells 48 h after transfection with polyQ80-CFP (Fig. 2C). Impaired inclusion body or aggresome formation was seen following expression of other proteins known to accumulate in aggresomes such as ΔF508-CFTR (7Weihl C.C. Dalal S. Pestronk A. Hanson P.I. Hum. Mol. Genet. 2006; 15: 189-199Crossref PubMed Scopus (149) Google Scholar) and CD3δ-YFP (Fig. 2D). PolyQ80 inclusions in both p97/VCP-WT and IBMPFD mutant p97/VCP co-localized with ubiquitin (Fig. 2E). These data suggest that IBMPFD mutants mishandle ubiquitinated and aggregated proteins by failing to send them to a single inclusion body.FIGURE 2Aggresome formation is impaired in IBMPFD mutant-expressing cells and tissue. A, representative live cell images of cells co-expressing polyQ80-CFP (blue) with DsRed tagged p97/VCP-WT (WT) or IBMPFD mutants R155H (RH), A232E (AE), and R95G (RG). Note that although p97/VCP co-localizes with polyQ80-CFP, there is impaired inclusion formation in IBMPFD mutant-expressing cells. B, control U20S or U20S cells stably expressing tetracycline-inducible p97/VCP-WT, ATPase inactive p97/VCP-E578Q (EQ) or IBMPFD mutant p97/VCP R155H, A232E, and L198W (LW) were transfected with polyQ80-CFP for 48 h. The cells were fixed with 3% paraformaldyhyde and processed for fluorescence microscopic analysis of inclusion bodies. The cells were scored for the presence of ≤ 3 inclusions or >3 inclusions. *, p < 0.05 versus U20S control containing less than three aggregated proteins. C, representative low power fields from U20S cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutant p97/VCP R155H and A232E transfected with polyQ80-CFP for 48 h. The filled arrows denote cells with ≤3 inclusions, and the open arrows denote cells with >3 inclusions. D, confocal micrograph of p97/VCP-dsRed and CD3δ-YFP co-expression. Note that overexpressed CD3δ-YFP forms a perinuclear inclusion in p97/VCP-WT-expressing cells and generates multiple smaller inclusions in IBMPFD mutant cells (R155H and A232E). E, control or U20S cells expressing p97/VCP-WT or IBMPFD mutant p97/VCP R155H or A232E a
Inclusion body myopathy associated with Paget's disease of the bone and fronto-temporal dementia (IBMPFD) is a progressive autosomal dominant disorder caused by mutations in p97/VCP (valosin-containing protein). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and participates in multiple cellular processes. One particularly important role for p97/VCP is facilitating intracellular protein degradation. p97/VCP has traditionally been thought to mediate the ubiquitin-proteasome degradation of proteins; however, recent studies challenge this dogma. p97/VCP clearly participates in the degradation of aggregate-prone proteins, a process principally mediated by autophagy. In addition, IBMPFD mutations in p97/VCP lead to accumulation of autophagic structures in patient and transgenic animal tissue. This is likely due to a defect in p97/VCP-mediated autophagosome maturation. The following review will discuss the evidence for p97/VCP in autophagy and how a disruption in this process contributes to IBMPFD pathogenesis.
본 연구는 생강 추출물의 전립선비대증에 대한 억제 효과를 in vitro와 in vivo에서 확인하였다. 본 실험에서 사용된 생강 에탄올 추출물(ZOET)은 99.9% 에탄올을 사용하여 추출하였고, 수율은 5%로 나타났다. In vitro에서 ZOET의 총폴리페놀 및 플라보노이드 함량이 161.58±6.00 mg GAE/g, 71.47±1.85 mg CE/g으로 측정되었으며, ABTS 및 DPPH 라디칼 소거능은 ZOET 1 mg/mL의 농도에서 약 57.15%, 58.46%의 활성을 나타내었다. ZOET의 전립선비대세포증식 억제 효과를 평가하기 위하여 전립선비대 세포주인 BPH-1을 대상으로 ZOET를 다양한 농도로 처리하였고, 그 결과 유의적으로 세포증식이 감소하였다(P0.05). In vivo에서 Control군, 음성대조군인 BPH군, ZOET를 50 mg/kg과 150 mg/kg을 급여한 군 총 4군으로 나누어 실험하였다. ZOET-High군은 음성대조군에 비하여 체중당 전립선 비율이 약 16.77%, 혈청의 DHT 수준 30.97%, 전립선 조직의 5αR2 수준이 24.59% 감소하였다. 또한 ZOET의 세포고사에 미치는 영향을 알아보기 위하여 Bax/Bcl-2 비율을 확인하였으며, ZOET-High군의 Bax/Bcl-2 비율이 음성대조군인 BPH군에 비하여 약 1.6배 증가한 것으로 나타났다. 본 연구에 사용된 생강 에탄올 추출물의 급여는 전립선 무게, 혈청의 DHT, 전립선 조직의 5αR2 수준 감소와 Bcl-2의 mRNA 발현 억제 및 Bax/Bcl-2 비율 증가 효과를 보였으며, 이러한 결과로 보아 다양한 폴리페놀과 플라보노이드가 포함된 생강은 전립선비대증 개선에 도움을 줄 수 있을 것으로 생각된다.
Autophagy is the process of forming vesicle structure by response to nutritional deficiency or outside stress and then surrounding and decomposing cell organelles to make energy. Autophagy, which is activated by being given diverse stimuli of stress including exercise, plays an important role in generating and maintaining energy in diverse muscles as well as skeletal muscle. However, there are very few studies on the physiological mechanism to explain the effects of such autophagy on cardiac muscle both at home and abroad. Thus, this experiment was conducted through acute swimming exercise to learn what effect autophagy makes on cardiac muscle. For lab animal, 24 experimental rats were bred in groups of 6 mice (control + saline solution, control + colchicines, exercise + saline solution, exercise + colchicines) using plastic cages, with temperature of 24-25℃, 70-80% humidity and light control in 12-hour cycle for breeding room. The selected animals were supplied with pellet and sufficient water for the whole period of experiment. Before this exercise, all the subject mice were given training for adaptation for two days, 10 minutes for each, while at this exercise, 12 rats in exercise group were given exercise in the water of 35 to 36℃ in two divided sessions (1-hour swimming + 15-minute rest + 1-hour swimming). For colchicine and saline solution, three times of treatment in total were given including the two days right after adaption training and right after the main training. Samples of cardiac muscle were abstracted 24 hours after exercise while conducting Western blot by use of LC3 and p62 as antibodies to observe the change of cardiac muscle caused by autophagy. Group in treatment with colchicine showed a clearer difference in change compared to group in treatment with saline solution, while results were drawn among groups in treatment with colchicine. Compared to control group, change in LC3-II showed a significant difference for the whole group of ‘exercise + colchicine’ with a significant difference in Gel-2 and Gel-3, except Gel-1, for the analysis by gel. However, visible increase was also found in Gel-1. As to p62, compared to control group, the whole group of exercise + colchicine treatment showed significant difference. As with LC3-II, however, there was no significant difference found in Gel-1, but with significant difference in Gel-2 and Gel-3. Through this, it was found that acute swimming exercise makes significant effect on autophagy of cardiac muscle, meaning the effect of stimuli like exercise on cardiac muscle. Furthermore, supposing autophagy as apoptosis or energy source, it is considered possible to help in solving the problems of modern society by applying it to aging, sarcopenia, discontinuance in training, long-term exercise, etc.
The purpose of this study was to investigate the potential role of creatine in GLUT4 gene expression in rat skeletal muscle. Female Wistar rats were fed normal rat chow (controls) or chow containing 2% creatine monohydrate ad libitum for 3 wk. GLUT4 protein levels of creatine-fed rats were significantly increased in extensor digitorum longus (EDL), triceps, and epitrochlearis muscles compared with muscles from controls (P < 0.05), and triceps GLUT4 mRNA levels were approximately 100% greater in triceps muscles from creatine-fed rats than in muscles from controls (P < 0.05). In epitrochlearis muscles from creatine-fed animals, glycogen content was approximately 40% greater (P < 0.05), and insulin-stimulated glucose transport rates were higher (P < 0.05) than in epitrochlearis muscles from controls. Despite no changes in [ATP], [creatine], [phosphocreatine], or [AMP], creatine feeding increased AMP-activated protein kinase (AMPK) phosphorylation by 50% in rat EDL muscle (P < 0.05). Creatinine content of EDL muscle was almost twofold higher for creatine-fed animals than for controls (P < 0.05). Creatine feeding increased protein levels of myocyte enhancer factor 2 (MEF2) isoforms MEF2A ( approximately 70%, P < 0.05), MEF2C ( approximately 60%, P < 0.05), and MEF2D ( approximately 90%, P < 0.05), which are transcription factors that regulate GLUT4 expression, in creatine-fed rat EDL muscle nuclear extracts. Electrophoretic mobility shift assay showed that DNA binding activity of MEF2 was increased by approximately 40% (P < 0.05) in creatine-fed rat EDL compared with controls. Our data suggest that creatine feeding enhances the nuclear content and DNA binding activity of MEF2 isoforms, which is concomitant with an increase in GLUT4 gene expression.
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