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

• DOI: 10.1074/mcp.m115.051516
• Publication Date: 2015-10-09T02:44:30Z
• Authors: Gerardo G. Piroli; Allison M. Manuel; Anna C. Clapper; Michael D. Walla; John E. Baatz; Richard D. Palmiter; Albert Quintana; Norma Frizzell
• Keywords: Knockout mouse

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when reacts with cysteine residues generate S-(2-succino)cysteine (2SC). Metabolic events reduce NADH re-oxidation can block activity; therefore we hypothesized oxidative phosphorylation deficiencies, such those observed some mitochondrial diseases, would also increased succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, model Leigh syndrome, demonstrate for first time succination is brainstem (BS), particularly vestibular nucleus. Importantly, most affected region exhibiting neurodegeneration and astrocyte microglial proliferation, these mice typically die respiratory failure attributed nucleus pathology. In contrast, no were skeletal muscle, corresponding lack muscle pathology this model. 2D SDS-PAGE followed by immunoblotting succinated proteins MS/MS analysis BS allowed us identify voltage-dependent anion channels 1 2 specific targets knockout. targeted mass spectrometry, Cys77 Cys48 identified endogenous sites 2. Given important role isoforms exchange ADP/ATP between cytosol mitochondria, already decreased capacity ATP synthesis KO mice, propose animals further decrease compromised function. These data suggest novel biochemical link may contribute progression neuropathology disease We previously formation (2SC) 1The abbreviations used are:2SCS-(2-succino)cysteine2Dtwo-dimensionalBSbrainstemCBcerebellumCPEcysteine pyridylethylationCrus 1crus ansiform lobuleC2SCcysteine fumarateCtxcortexERendoplasmic reticulumFNfastigial nucleusGCgas chromatographyGigigantocellular reticular nucleusGSHglutathioneHLRCChereditary leiomyomatosis renal cell carcinomaHNE4-hydroxy-2-nonenalIOinferior oliveMDmitochondrial diseasesMOXmethionine oxidationMtmicrotubular pelletOBolfactory bulbOXPHOSoxidative phosphorylationPVDFpolyvinylidene difluorideRIPAradioimmuno precipitation assaySMtsupernatant after tubulin polymerizationSNsciatic nerveStrstriatumSupcleared supernatantVDACvoltage-dependent channelVNvestibular nuclei. (protein succination) reaction reactive thiols (1.Alderson N.L. Wang Y. Blatnik M. Frizzell N. Walla M.D. Lyons T.J. Alt Carson J.A. Nagai R. Thorpe S.R. Baynes J.W. 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Polyvinylidene (PVDF) ECL chemiluminescent substrate GE Healthcare (Piscataway, NJ). preparation polyclonal anti-2SC antibody following commercial antibodies used: α-tubulin DM1A, VDAC2 9412 Cell Signaling Technology, Inc. (Danvers, MA); β-tubulin TUB2.1 Santa Cruz Biotechnology (Dallas, TX); HNE (HNE-11-S) Alpha Diagnostics International, (San Antonio, 2C7CD4AG3 DJ1 ab4150 Abcam (Cambridge, VDAC1 clone N152B/23 Antibodies (Davis, CA), Virogen Corporation (Watertown, MA). Animal care use procedures carried out accordance protocols written under guidelines National Institutes Health Guide Care Use Laboratory Animals approved University South Carolina Committee. heterozygous obtained Drs. Richard Albert Quintana (Seattle Children's Research institute, Seattle, WA). After weeks acclimatization School Medicine Facility, breeding pairs mated, litters weaned at 21 days age. Genotyping performed (28.Quintana WT sacrificed CO2 asphyxiation early (3 weeks), middle (6 late (9 weeks) stages, brain, nerves removed immediately; tissues exception (see below) snap frozen liquid nitrogen stored −80 °C until use. Some brains subjected macro dissection before freezing; isolated regions included (CB), cerebral (Ctx), (Str), (OB). For microdissection CB areas, posterior half sliced 400 μm cryostat (Microm HM 560) Bregma −5.40 −7.00 mm, according atlas (30.Franklin K.B.J Paxinos stereotaxic coordinates. 3rd Ed. Academic Press, Elsevier, New York, NY2008Google Brain slices mounted microscope glass slides, Nuclei (VN), olive/gigantocellular nuclei (IO/Gi), (FN), (Crus 1) punched using Harvard Apparatus puncher (1.0 diameter); punches same collected tube per mouse. 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TRC2 Fh1 shRNA, clone- TRCN0000246831 SHC202 MISSION pLKO.5-puro Non-Mammalian shRNA control plasmids (Sigma/Aldrich) vectors. contained puromycin resistance gene. 15 μg vector plasmid, psPAX2 packaging plasmid (Addgene 12260, Cambridge, MA), pMD2.G envelope 12259) 2.5 pRSV-Rev 12253) transfected 293T cells. filtered conditioned medium N1E-115 (subclone N1E-115–1 neuroblastoma cells) Sigma (08062511). non-differentiation (NDM, 90% DMEM (Gibco, Grand Island, NY) 25 glucose, pyruvate, HEPES, Glutamine) 10% Fetal Bovine Serum (Atlanta Biologicals, Atlanta, GA) cm2 flasks. At 80% confluence, washed Dulbecco's PBS 1.5 ml NDM 300 transduction. incubated 18 h returning volume 4.5 NDM. 48 selection transduced (1.75 μg/ml, dose curve, initiated week. end period, remaining expanded differentiated neurons 2% FBS, 1.25% DMSO μg/ml days. harvested (12.Piroli Scholar) fumarase (to assess efficiency knockdown) (described below). separate experiment GC-MS quantification David Murdock Institute (DHMRI, Kannapolis, NC). Metabolite extraction adaptation previous methods flasks three times ice-cold immediate 20 chloroform/methanol (2:1). vortexed stand intermittent vortexing 0.2 H2O. additional min, 3220 min. aqueous transferred clean dried air. repeated adding equal parts methanol deionized water, centrifuging, transferring layer respective dry. interface each sample Far