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• W2300917170 abstract "Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys77 and Cys48 were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model. Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys77 and Cys48 were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model. We previously identified the formation of S-(2-succino)cysteine (2SC) 1The abbreviations used are:2SCS-(2-succino)cysteine2Dtwo-dimensionalBSbrainstemCBcerebellumCPEcysteine pyridylethylationCrus 1crus 1 ansiform lobuleC2SCcysteine succination by fumarateCtxcortexERendoplasmic reticulumFNfastigial nucleusGCgas chromatographyGigigantocellular reticular nucleusGSHglutathioneHLRCChereditary leiomyomatosis and renal cell carcinomaHNE4-hydroxy-2-nonenalIOinferior oliveMDmitochondrial diseasesMOXmethionine oxidationMtmicrotubular pelletOBolfactory bulbOXPHOSoxidative phosphorylationPVDFpolyvinylidene difluorideRIPAradioimmuno precipitation assaySMtsupernatant after tubulin polymerizationSNsciatic nerveStrstriatumSupcleared supernatantVDACvoltage-dependent anion channelVNvestibular nuclei. (protein succination) as a result of the irreversible reaction of fumarate with reactive cysteine thiols (1.Alderson N.L. Wang Y. Blatnik M. Frizzell N. Walla M.D. Lyons T.J. Alt N. Carson J.A. Nagai R. Thorpe S.R. Baynes J.W. S-(2-Succinyl)cysteine: a novel chemical modification of tissue proteins by a Krebs cycle intermediate.Arch. Biochem. Biophys. 2006; 450: 1-8Crossref PubMed Scopus (129) Google Scholar, 2.Nagai R. Brock J.W. Blatnik M. Baatz J.E. Bethard J. Walla M.D. Thorpe S.R. Baynes J.W. Frizzell N. Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress.J. Biol. Chem. 2007; 282: 34219-34228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Fumarate concentrations are increased during adipogenesis and adipocyte maturation (2.Nagai R. Brock J.W. Blatnik M. Baatz J.E. Bethard J. Walla M.D. Thorpe S.R. Baynes J.W. Frizzell N. Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress.J. Biol. Chem. 2007; 282: 34219-34228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 3.Lai R.K. Goldman P. Organic acid profiling in adipocyte differentiation of 3T3-F442A cells: increased production of Krebs cycle acid metabolites.Metabolism. 1992; 41: 545-547Abstract Full Text PDF PubMed Scopus (10) Google Scholar), and the excess of glucose and insulin leads to augmented protein succination in the adipose tissue of type 2 diabetic mice (4.Frizzell N. Rajesh M. Jepson M.J. Nagai R. Carson J.A. Thorpe S.R. Baynes J.W. Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin.J. Biol. Chem. 2009; 284: 25772-25781Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 5.Thomas S.A. Storey K.B. Baynes J.W. Frizzell N. Tissue distribution of S-(2-succino)cysteine (2SC), a biomarker of mitochondrial stress in obesity and diabetes.Obesity. 2012; 20: 263-269Crossref PubMed Scopus (30) Google Scholar). Protein succination is also specifically increased in fumarate hydratase deficient hereditary leiomyomatosis and renal cell carcinoma (HLRCC), because of the decreased conversion of fumarate to malate (6.Bardella C. El-Bahrawy M. Frizzell N. Adam J. Ternette N. Hatipoglu E. Howarth K. O'Flaherty. L. Roberts I. Turner G. Taylor J. Giaslakiotis K. Macaulay V.M. Harris A.L. Chandra A. Lehtonen H.J. Launonen V. Aaltonen L.A. Pugh C.W. Mihai R. Trudgian D. Kessler B. Baynes J.W. Ratcliffe P.J. Tomlinson I.P. Pollard P.J. Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status.J. Pathol. 2011; 225: 4-11Crossref PubMed Scopus (186) Google Scholar, 7.Chen Y.B. Brannon A.R. Toubaji A. Dudas M.E. Won H.H. Al-Ahmadie H.A. Fine S.W. Gopalan A. Frizzell N. Voss M.H. Russo P. Berger M.F. Tickoo S.K. Reuter V.E. Hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cancer: recognition of the syndrome by pathologic features and the utility of detecting aberrant succination by immunohistochemistry.Am. J. Surg. Pathol. 2014; 38: 627-637Crossref PubMed Scopus (175) Google Scholar). In both cases, intracellular fumarate concentrations are elevated; in fumarate hydratase deficient cells, the fumarate concentration is about 5 mm (8.Ternette N. Yang M. Laroyia M. Kitagawa M. O'Flaherty L. Wolhulter. K. Igarashi K. Saito K. Kato K. Fischer R. Berquand A. Kessler B.M. Lappin T. Frizzell N. Soga T. Adam J. Pollard P.J. Inhibition of mitochondrial aconitase by succination in fumarate hydratase deficiency.Cell Rep. 2013; 3: 689-700Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), whereas fumarate levels increase up to fivefold in adipocytes grown in the presence of high (30 mm) versus normal (5 mm) glucose concentrations (2.Nagai R. Brock J.W. Blatnik M. Baatz J.E. Bethard J. Walla M.D. Thorpe S.R. Baynes J.W. Frizzell N. Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress.J. Biol. Chem. 2007; 282: 34219-34228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In the adipocyte the increase in fumarate and succinated proteins develops as a direct result of mitochondrial stress induced by nutrient excess. Mechanistically, excess glucose without increased ATP demand inhibits the electron transport chain resulting in an elevated NADH/NAD+ ratio. This inhibits NAD+-dependent Krebs cycle enzymes and leads to an increase in fumarate and protein succination (9.Frizzell N. Thomas S.A. Carson J.A. Baynes J.W. Mitochondrial stress causes increased succination of proteins in adipocytes in response to glucotoxicity.Biochem. J. 2012; 445: 247-254Crossref PubMed Scopus (40) Google Scholar). In support of this we have also shown that low concentrations of chemical uncouplers of oxidative phosphorylation (OXPHOS) can decrease fumarate concentrations and protein succination (9.Frizzell N. Thomas S.A. Carson J.A. Baynes J.W. Mitochondrial stress causes increased succination of proteins in adipocytes in response to glucotoxicity.Biochem. J. 2012; 445: 247-254Crossref PubMed Scopus (40) Google Scholar). The physiological consequences of protein succination include a decrease in the functionality of the target protein (8.Ternette N. Yang M. Laroyia M. Kitagawa M. O'Flaherty L. Wolhulter. K. Igarashi K. Saito K. Kato K. Fischer R. Berquand A. Kessler B.M. Lappin T. Frizzell N. Soga T. Adam J. Pollard P.J. Inhibition of mitochondrial aconitase by succination in fumarate hydratase deficiency.Cell Rep. 2013; 3: 689-700Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 10.Blatnik M. Frizzell N. Thorpe S.R. Baynes J.W. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by fumarate in diabetes: formation of S-(2-succinyl)cysteine, a novel chemical modification of protein and possible biomarker of mitochondrial stress.Diabetes. 2008; 57: 41-49Crossref PubMed Scopus (104) Google Scholar, 11.Adam J. Hatipoglu E. O'Flaherty L. Ternette N. Sahgal N. Lockstone H. Baban D. Nye E. Stamp G.W. Wolhuter K. Stevens M. Fischer R. Carmeliet P. Maxwell P.H. Pugh C.W. Frizzell N. Soga T. Kessler B.M. El-Bahrawy M. Ratcliffe P.J. Pollard. P.J. Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling.Cancer Cell. 2011; 20: 524-537Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 12.Piroli G.G. Manuel A.M. Walla M.D. Jepson M.J. Brock J.W. Rajesh M.P. Tanis R.M. Cotham W.E. Frizzell N. Identification of protein succination as a novel modification of tubulin.Biochem. J. 2014; 462: 231-245Crossref PubMed Scopus (25) Google Scholar), for example succination of adiponectin prevents the formation of multimeric complexes and reduces plasma adiponectin levels in diabetes (4.Frizzell N. Rajesh M. Jepson M.J. Nagai R. Carson J.A. Thorpe S.R. Baynes J.W. Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin.J. Biol. Chem. 2009; 284: 25772-25781Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Considering the impact of glucotoxicity driven mitochondrial stress in the adipocyte, we predicted that deficiencies in OXPHOS associated with NADH accumulation would also result in increased protein succination. S-(2-succino)cysteine two-dimensional brainstem cerebellum cysteine pyridylethylation crus 1 ansiform lobule cysteine succination by fumarate cortex endoplasmic reticulum fastigial nucleus gas chromatography gigantocellular reticular nucleus glutathione hereditary leiomyomatosis and renal cell carcinoma 4-hydroxy-2-nonenal inferior olive mitochondrial diseases methionine oxidation microtubular pellet olfactory bulb oxidative phosphorylation polyvinylidene difluoride radioimmuno precipitation assay supernatant after tubulin polymerization sciatic nerve striatum cleared supernatant voltage-dependent anion channel vestibular nuclei. Mitochondrial respiratory chain disorders encompass a broad range of encephalopathies and myopathies associated with the defective assembly, activity or maintenance of the OXPHOS machinery (13.Tucker E.J. Compton A.G. Thorburn D.R. Recent advances in the genetics of mitochondrial encephalopathies.Curr. Neurol. Neurosci. Rep. 2010; 10: 277-285Crossref PubMed Scopus (39) Google Scholar), and are estimated to occur in about 1 in 5,000 live births (14.Skladal D. Halliday J. Thorburn D.R. Minimum birth prevalence of mitochondrial respiratory chain disorders in children.Brain. 2003; 126: 1905-1912Crossref PubMed Scopus (363) Google Scholar). A common feature in most mitochondrial diseases (MD) is a failure to thrive because of reduced mitochondrial energy production; both the brain and muscle are usually affected because of their high dependence on oxidative metabolism (13.Tucker E.J. Compton A.G. Thorburn D.R. Recent advances in the genetics of mitochondrial encephalopathies.Curr. Neurol. Neurosci. Rep. 2010; 10: 277-285Crossref PubMed Scopus (39) Google Scholar). Leigh syndrome is one of the most common manifestations of MD and is characterized by progressive neurodegeneration with bilateral necrotizing lesions of the brainstem and basal ganglia, resulting in lactic acidosis, ataxia, seizures, dystonia, and respiratory failure (15.Finsterer J. Leigh and Leigh-like syndrome in children and adults.Pediatr. Neurol. 2008; 39: 223-235Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 16.Rahman S. Blok R.B. Dahl H.H. Danks D.M. Kirby D.M. Chow C.W. Christodoulou J. Thorburn D.R. Leigh syndrome: clinical features and biochemical and DNA abnormalities.Ann. Neurol. 1996; 39: 343-351Crossref PubMed Scopus (612) Google Scholar). Mutations in genes encoding the five complexes of the OXPHOS machinery can lead to Leigh syndrome; however, the majority of these mutations affect subunits of complexes I and IV (17.Ruhoy I.S. Saneto R.P. The genetics of Leigh syndrome and its implications for clinical practice and risk management.Appl. Clin. Genet. 2014; 7: 221-234PubMed Google Scholar), and both mitochondrial and nuclear encoded proteins may be affected (17.Ruhoy I.S. Saneto R.P. The genetics of Leigh syndrome and its implications for clinical practice and risk management.Appl. Clin. Genet. 2014; 7: 221-234PubMed Google Scholar, 18.Pagniez-Mammeri H. Loublier S. Legrand A. Bénit P. Rustin P. Slama A. Mitochondrial complex I deficiency of nuclear origin I. Structural genes.Mol. Genet. Metab. 2012; 105: 163-172Crossref PubMed Scopus (42) Google Scholar, 19.Pagniez-Mammeri H. Rak M. Legrand A. Bénit P. Rustin P. Slama A. Mitochondrial complex I deficiency of nuclear origin II. Non-structural genes.Mol. Genet. Metab. 2012; 105: 173-179Crossref PubMed Scopus (26) Google Scholar). Complex I is a large (980 kDa) l-shaped protein assembly consisting of 45 peptides, with one flavin mononucleotide and eight iron–sulfur clusters (20.Mimaki M. Wang X. McKenzie M. Thorburn D.R. Ryan M.T. Understanding mitochondrial complex I assembly in health and disease.Biochim. Biophys. Acta. 2012; 1817: 851-862Crossref PubMed Scopus (279) Google Scholar). One of the first identified mutations of complex I encoded Ndufs4, a small (18 kDa) assembly protein (21.van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Demonstration of a new pathogenic mutation in human complex I deficiency: A 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit.Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 22.Loeffen J.L. Smeitink J.A. Trijbels J.M. Janssen A.J. Triepels R.H. Sengers R.C. van den Heuvel L.P. Isolated complex I deficiency in children: clinical, biochemical and genetic aspects.Hum. Mutat. 2000; 15: 123-134Crossref PubMed Scopus (255) Google Scholar, 23.Budde S.M. van den Heuvel L.P. Janssen A.J. Smeets R.J. Buskens C.A. DeMeirleir L. Van Coster R. Baethmann M. Voit T. Trijbels J.M. Smeitink J.A. Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene.Biochem. Biophys. Res. Commun. 2000; 275: 63-68Crossref PubMed Scopus (163) Google Scholar). Ndufs4 assists in the final stages of complex I assembly, and its absence results in the formation of a smaller ∼830 kDa subcomplex that lacks the NADH dehydrogenase module and has significantly less electron shuttling activity than the intact holoenzyme (24.Calvaruso M.A. Willems P. van den Brand M. Valsecchi F. Kruse S. Palmiter R. Smeitink J. Nijtmans L. Mitochondrial complex III stabilizes complex I in the absence of NDUFS4 to provide partial activity.Hum. Mol. Genet. 2012; 21: 115-120Crossref PubMed Scopus (81) Google Scholar, 25.Lazarou M. McKenzie M. Ohtake A. Thorburn D.R. Ryan M.T. Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I.Mol. Cell. Biol. 2007; 27: 4228-4237Crossref PubMed Scopus (201) Google Scholar). Ndufs4 mutations are associated with brainstem deterioration in humans (26.Leshinsky-Silver E. Lebre A.S. Minai L. Saada A. Steffann J. Cohen S. Rötig A. Munnich A. Lev D. Lerman-Sagie T. NDUFS4 mutations cause Leigh syndrome with predominant brainstem involvement.Mol. Genet. Metab. 2009; 97: 185-189Crossref PubMed Scopus (47) Google Scholar), and a recently described Ndufs4 knockout mouse (Ndufs4 KO) exhibits many of the clinical and neurological symptoms observed in human Leigh syndrome (27.Kruse S.E. Watt W.C. Marcinek D.J. Kapur R.P. Schenkman K.A. Palmiter R.D. Mice with mitochondrial complex I deficiency develop a fatal encephalomyopathy.Cell Metab. 2008; 7: 312-320Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 28.Quintana A. Kruse S.E. Kapur R.P. Sanz E. Palmiter R.D. Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 10996-11001Crossref PubMed Scopus (168) Google Scholar). One of the most common clinical features of MD is lactic acidosis, derived from the accumulation of pyruvate and elevated NADH. Increased lactate or lactate:pyruvate ratios have been measured in the blood, urine, and cerebrospinal fluid of a large number of Leigh syndrome patients (15.Finsterer J. Leigh and Leigh-like syndrome in children and adults.Pediatr. Neurol. 2008; 39: 223-235Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 16.Rahman S. Blok R.B. Dahl H.H. Danks D.M. Kirby D.M. Chow C.W. Christodoulou J. Thorburn D.R. Leigh syndrome: clinical features and biochemical and DNA abnormalities.Ann. Neurol. 1996; 39: 343-351Crossref PubMed Scopus (612) Google Scholar). Increases in other organic acids in urine have also been reported (16.Rahman S. Blok R.B. Dahl H.H. Danks D.M. Kirby D.M. Chow C.W. Christodoulou J. Thorburn D.R. Leigh syndrome: clinical features and biochemical and DNA abnormalities.Ann. Neurol. 1996; 39: 343-351Crossref PubMed Scopus (612) Google Scholar), indicating that metabolic acidosis is a prominent clinical feature. Interestingly, a study designed to find new diagnostic metabolites in MD demonstrated that within certain age ranges the measurement of urinary fumarate and malate was a more useful discriminator of MD than lactate or other organic acids (29.Barshop B.A. Metabolomic approaches to mitochondrial disease: correlation of urine organic acids.Mitochondrion. 2004; 4: 521-527Crossref PubMed Scopus (59) Google Scholar). Barshop's findings support the hypothesis that MD derived from OXPHOS deficiencies may exhibit increased protein succination because of the accumulation of NADH and subsequently fumarate. In this study we report for the first time that protein succination is present in the brain in an animal model of Leigh syndrome, the Ndufs4 KO mouse, suggesting that this modification may be an important biochemical link between the genetic defect and the onset of neuropathology observed in Leigh syndrome. Unless otherwise noted, all chemicals were purchased from Sigma/Aldrich Chemical Co (St. Louis, MO). Criterion polyacrylamide gels, nitrocellulose membranes and Precision Plus protein ladder were purchased from BioRad Laboratories (Richmond, CA). Polyvinylidene difluoride (PVDF) membranes and ECL Plus chemiluminescent substrate were from GE Healthcare (Piscataway, NJ). The preparation of the polyclonal anti-2SC antibody has been described previously (2.Nagai R. Brock J.W. Blatnik M. Baatz J.E. Bethard J. Walla M.D. Thorpe S.R. Baynes J.W. Frizzell N. Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress.J. Biol. Chem. 2007; 282: 34219-34228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The following commercial antibodies were used: α-tubulin DM1A, VDAC2 9412 from Cell Signaling Technology, Inc. (Danvers, MA); β-tubulin TUB2.1 from Santa Cruz Biotechnology (Dallas, TX); HNE (HNE-11-S) from Alpha Diagnostics International, Inc. (San Antonio, TX); Ndufs4 2C7CD4AG3 and DJ1 ab4150 from Abcam (Cambridge, MA); VDAC1 clone N152B/23 from Antibodies Inc. (Davis, CA), and glutathione from Virogen Corporation (Watertown, MA). Animal care and use procedures were carried out in accordance with protocols written under the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by The University of South Carolina Animal Care and Use Committee. Ndufs4 heterozygous mice were obtained from Drs. Richard Palmiter and Albert Quintana (Seattle Children's Research institute, Seattle, WA). After 2 weeks of acclimatization to the University of South Carolina School of Medicine Animal Facility, breeding pairs were mated, and the litters were weaned at 21 days of age. Genotyping was performed as previously described (28.Quintana A. Kruse S.E. Kapur R.P. Sanz E. Palmiter R.D. Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 10996-11001Crossref PubMed Scopus (168) Google Scholar). KO and WT mice were sacrificed by CO2 asphyxiation at early (3 weeks), middle (6 weeks), and late (9 weeks) stages, and brain, sciatic nerves and skeletal muscle were removed immediately; tissues with the exception of the brain (see below) were snap frozen in liquid nitrogen and stored at −80 °C until further use. Some brains were subjected to macro dissection before freezing; isolated regions included the brainstem (BS), cerebellum (CB), cerebral cortex (Ctx), striatum (Str), and olfactory bulb (OB). For the microdissection of CB and BS areas, the posterior half of the brain was frozen and sliced at 400 μm in a cryostat (Microm HM 560) between Bregma −5.40 and −7.00 mm, according to a mouse brain atlas (30.Franklin K.B.J Paxinos G. The mouse brain in stereotaxic coordinates. 3rd Ed. Academic Press, Elsevier, New York, NY2008Google Scholar). Brain slices were mounted on microscope glass slides, and the vestibular Nuclei (VN), inferior olive/gigantocellular reticular nuclei (IO/Gi), fastigial nuclei (FN), and crus 1 ansiform lobule (Crus 1) were punched out of the slices using a Harvard Apparatus puncher (1.0 mm diameter); all punches from the same region were collected in one tube per mouse. Radioimmunoprecipitation assay (RIPA) lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 0.1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, pH 7.4), with the addition of 2 mm diethylenetriaminepentaacetic acid and a protease inhibitor mixture (P8340, Sigma/Aldrich) was added to tissues that were used for gel separation procedures. Homogenization was performed by pulse sonication at 2 watts using a Model 100 sonic dismembrator (Fisher Scientific, Fair Lawn, NJ) for 30 s prior to resting on ice for 30 min in lysis buffer. The homogenate was clarified from nuclei and unbroken cells by centrifugation at 900 × g for 10 min at 4 °C. Protein in the supernatants was precipitated with 9 volumes of cold acetone for 10 min on ice. After centrifugation at 3000 × g for 10 min and removal of the acetone, the protein pellet was resuspended in 500 μl RIPA buffer. For the identification of succination sites in VDAC2 (see ”Identification of Succinated Cysteines in Tubulin and VDAC2 by LC-MS/MS” below), subcellular fractions were prepared from WT Ctx and from WT and Ndufs4 KO BS, according to Frezza et al. (31.Frezza C. Cipolat S. Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.Nat. Protoc. 2007; 2: 287-295Crossref PubMed Scopus (857) Google Scholar). Briefly, tissues were homogenized in 10 mm Tris-HCl (pH 7.4), 1 mm EGTA, 200 mm sucrose, and a protease inhibitor mixture (P8340, Sigma/Aldrich), using a Teflon pestle operated homogenizer. The homogenate was cleared of cell nuclei and unbroken cells by centrifugation at 600 × g for 10 min at 4 °C; the supernatant was further centrifuged at 7000 × g for 10 min at 4 °C. The second supernatant was considered the cytosolic fraction and the pellet containing the mitochondria was resuspended in homogenization buffer. Analysis by Western blotting of the initial homogenate, cytosol and mitochondrial fractions showed an enrichment of mitochondrial markers in the mitochondrial fraction (citrate synthase); tubulin was found concentrated in the cytosolic fraction (data not shown). The protein content in the different samples was determined by the Lowry assay (32.Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Vector Preparation: The lentiviral vectors were prepared by the University of South Carolina Viral Vector Facility. Briefly, TRC2 Fh1 shRNA, clone- TRCN0000246831 or SHC202 MISSION TRC2 pLKO.5-puro Non-Mammalian shRNA control plasmids (Sigma/Aldrich) were used to generate the lentiviral vectors. The vectors also contained a puromycin resistance gene. 15 μg vector plasmid, 10 μg psPAX2 packaging plasmid (Addgene 12260, Cambridge, MA), 5 μg pMD2.G envelope plasmid (Addgene 12259) and 2.5 μg pRSV-Rev plasmid (Addgene 12253) were transfected into 293T cells. The filtered conditioned medium was collected and stored at −80 °C until use. N1E-115 cells (subclone N1E-115–1 neuroblastoma cells) were obtained from Sigma (08062511). The cells were grown in non-differentiation medium (NDM, 90% DMEM (Gibco, Grand Island, NY) with 25 mm glucose, no pyruvate, 25 mm HEPES, 4 mm Glutamine) and 10% Fetal Bovine Serum (Atlanta Biologicals, Atlanta, GA) in 25 cm2 flasks. At 80% confluence, the cells in 25 cm2 were washed with Dulbecco's PBS and 1.5 ml NDM was added prior to addition of 300 μl of lentiviral vector conditioned medium for cell transduction. The cells were incubated for 18 h before returning the volume to 4.5 ml with NDM. After 48 h selection of transduced cells with puromycin (1.75 μg/ml, determined by a dose response curve, Sigma/Aldrich) was initiated for 1 week. At the end of this period, the remaining cells were expanded and differentiated into neurons in the presence of 2% FBS, 1.25% DMSO in DMEM in addition to 1 μg/ml puromycin for 5 days. The cells were harvested in RIPA as described previously (12.Piroli G.G. Manuel A.M. Walla M.D. Jepson M.J. Brock J.W. Rajesh M.P. Tanis R.M. Cotham W.E. Frizzell N. Identification of protein succination as a novel modification of tubulin.Biochem. J. 2014; 462: 231-245Crossref PubMed Scopus (25) Google Scholar) and the levels of fumarase (to assess efficiency of shRNA knockdown) and protein succination were determined by immunoblotting (described below). In a separate experiment the levels of fumarate were determined by GC-MS (described below). The quantification of fumarate was performed by GC-MS at the David H. Murdock Research Institute (DHMRI, Kannapolis, NC). Metabolite extraction was performed in an adaptation of previous methods (9.Frizzell N. Thomas S.A. Carson J.A. Baynes J.W. Mitochondrial stress causes increased succination of proteins in adipocytes in response to glucotoxicity.Biochem. J. 2012; 445: 247-254Crossref PubMed Scopus (40) Google Scholar). Briefly, N1E-115 cells grown in 25 cm2 flasks were washed three times with ice-cold PBS followed by the immediate addition of 20 volumes ice-cold chloroform/methanol (2:1). The samples were vortexed and allowed to stand on ice for 10 min with intermittent vortexing prior to addition of 0.2 volumes H2O. The samples were vortexed and allowed to stand on ice for an additional 2 min, followed by centrifugation at 3220 × g for 20 min. The aqueous supernatant was transferred into a clean tube and dried under air. The extraction was repeated an additional time by adding equal parts of methanol and deionized water, centrifuging, and transferring the aqueous layer into the respective tube to dry. The protein interface for each sample was removed for quantification of protein by Lowry assay (32.Lowry O.H. Rosenbrough N.J. Far" @default.
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• W2300917170 date "2016-02-01" @default.
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• W2300917170 title "Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome" @default.
• W2300917170 cites W1775749144 @default.
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• W2300917170 doi "https://doi.org/10.1074/mcp.m115.051516" @default.
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