title: Effects of targeted suppression of glutaryl-CoA dehydrogenase by lentivirus-mediated shRNA and excessive intake of lysine on apoptosis in rat striatal neurons.
abstract: In glutaric aciduria type 1 (GA1), glutaryl-CoA dehydrogenase (GCDH) deficiency has been shown to be responsible for the accumulation of glutaric acid and striatal degeneration. However, the mechanisms by which GA1 induces striatal degeneration remain unclear. In this study, we aimed to establish a novel neuronal model of GA1 and to investigate the effects of GCDH deficiency and lysine-related metabolites on the viability of rat striatal neurons. Thus we constructed a lentiviral vector containing short hairpin RNA targeted against the GCDH gene expression (lentivirus-shRNA) in neurons. A virus containing a scrambled short hairpin RNA construct served as a control. Addition of lysine (5 mmol/L) was used to mimic hypermetabolism. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis was assessed using Hoechst33342 staining and Annexin V-PE/7-AAD staining. The mitochondrial membrane potential (MPP) was monitored using tetramethylrhodamine methyl ester. The expression levels of caspases 3, 8, and 9 were determined by Western blotting. We found that lentivirus-shRNA induced apoptosis and decreased MMP levels in neurons, and addition of 5 mmol/L lysine enhanced this effect markedly. Lentivirus-shRNA upregulated the protein levels of caspases 3 and 9 regardless of the presence of 5 mmol/L lysine. The expression level of caspase 8 was higher in neurons co-treated with lentivirus-shRNA and 5 mmol/L lysine than in control. Benzyloxy-carbonyl-Val-Ala-Asp(OMe)-fluoromethylketone, a pan-caspase inhibitor, blocked the apoptosis induced by lentivirus-shRNA and 5 mmol/L lysine to a great extent. These results indicate that the targeted suppression of GCDH by lentivirus-mediated shRNA and excessive intake of lysine may be a useful cell model of GA1. These also suggest that GA1-induced striatal degeneration is partially caspase-dependent.
authors: Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors: Jinzhi
authors: J
authors: Gao
authors: Cai
authors: C
authors: Zhang
authors: Xi
authors: X
authors: Fu
authors: Qin
authors: Q
authors: Yi
authors: Fengyan
authors: F
authors: Tian
authors: Qin
authors: Q
authors: Ning
authors: Xiaoping
authors: X
authors: Luo
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D001928:Brain Diseases, Metabolic / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D061945:Caspase Inhibitors / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D055785:Gene Knockdown Techniques; D050770:Glutaryl-CoA Dehydrogenase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008239:Lysine / Q000378:metabolism / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000235:genetics; D017072:Neostriatum / Q000166:cytology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D061945:Caspase Inhibitors; D034741:RNA, Small Interfering; D050770:Glutaryl-CoA Dehydrogenase; D008239:Lysine
references: Hum Mol Genet. 2002 Feb 15;11(4):347-57
references: 11854167
references: J Cereb Blood Flow Metab. 2012 Jul;32(7):1362-76
references: 22453628
references: J Neurochem. 2006 May;97(3):899-910
references: 16573641
references: Biochim Biophys Acta. 2010 Jun;1802(6):552-60
references: 20302929
references: J Vis Exp. 2011 May 23;(51):
references: 21654619
references: Mol Genet Metab. 2011 Dec;104(4):425-37
references: 21944461
references: Biochim Biophys Acta. 2008 Jun;1782(6):385-90
references: 18348873
references: Int J Dev Neurosci. 2005 Dec;23(8):695-701
references: 16213122
references: Curr Gene Ther. 2008 Dec;8(6):474-82
references: 19075630
references: Metab Brain Dis. 2010 Jun;25(2):191-8
references: 20437086
references: J Clin Invest. 2007 Nov;117(11):3258-70
references: 17932566
references: PLoS One. 2012;7(11):e48644
references: 23133649
references: Free Radic Biol Med. 2012 Aug 1;53(3):595-609
references: 22595027
references: Brain. 2006 Apr;129(Pt 4):899-910
references: 16446282
references: J Inherit Metab Dis. 2005;28(1):57-67
references: 15702406
references: J Mol Neurosci. 2006;30(1-2):105-6
references: 17192649
references: Virol J. 2010 Sep 21;7:248
references: 20858246
references: J Bioenerg Biomembr. 2011 Feb;43(1):31-8
references: 21249436
references: Vaccine. 2012 Mar 28;30(15):2499-509
references: 22342915
references: Neurotoxicology. 2007 Sep;28(5):931-7
references: 17368774
references: Methods Mol Biol. 2008;442:95-112
references: 18369781
references: Curr Opin Mol Ther. 2009 Oct;11(5):554-64
references: 19806504
references: Hum Gene Ther. 2005 Jan;16(1):17-25
references: 15703485
references: Mol Genet Metab. 2012 Sep;107(1-2):72-80
references: 22520952
references: Biochem Med. 1975 Jan;12(1):12-21
references: 1137568
references: Neuroscience. 2005;135(1):111-20
references: 16111821
references: Toxicol Sci. 2010 Apr;114(2):323-34
references: 20047973
references: J Inherit Metab Dis. 2004;27(6):813-8
references: 15505386
references: Int J Dev Neurosci. 2007 Oct;25(6):391-8
references: 17643899
references: Brain. 2009 Jul;132(Pt 7):1764-82
references: 19433437
references: Neurobiol Dis. 2012 Nov;48(2):202-11
references: 21889981
references: Neuropediatrics. 2000 Dec;31(6):303-6
references: 11508549
references: Hum Mol Genet. 2011 Jun 15;20(12):2422-34
references: 21493629
references: Am J Med Genet C Semin Med Genet. 2006 May 15;142C(2):86-94
references: 16602100
references: Front Neuroendocrinol. 2010 Jul;31(3):341-58
references: 20457175
references: Dis Model Mech. 2009 May-Jun;2(5-6):206-10
references: 19407324
references: Mol Pharmacol. 2003 Dec;64(6):1295-308
references: 14645659
references: J Huazhong Univ Sci Technolog Med Sci. 2012 Feb;32(1):107-112
references: 22282255
references: J Biol Chem. 2005 Jun 10;280(23):21830-6
references: 15840571
references: Neurochem Int. 2004 Apr;44(5):345-53
references: 14643752
references: Carcinogenesis. 2006 Aug;27(8):1538-46
references: 16513679
references: Brain. 2005 Apr;128(Pt 4):711-22
references: 15689364
references: PLoS One. 2011;6(7):e22231
references: 21779398
references: J Inherit Metab Dis. 2003;26(7):713-4
references: 14707522
references: PLoS One. 2011;6(6):e20831
references: 21698251
references: Pediatr Res. 2006 Jun;59(6):840-7
references: 16641220
references: Med Sci Monit. 2011 Jul;17(7):PH55-9
references: 21709643
references: Brain. 2006 Aug;129(Pt 8):e54
references: 16870879
references: Hum Mol Genet. 2008 Dec 15;17(24):3854-63
references: 18775954
references: Pediatr Res. 2005 Jun;57(6):771-6
references: 15774829
references: J Inherit Metab Dis. 2004;27(6):825-8
references: 15505388
references: J Hum Nutr Diet. 2012 Dec;25(6):514-9
references: 22845646
references: Biosci Rep. 1999 Feb;19(1):27-34
references: 10379904
references: Mol Genet Metab. 2010 Oct-Nov;101(2-3):178-82
references: 20732827
references: J Inherit Metab Dis. 2011 Jun;34(3):677-94
references: 21431622
references: J Neurosci Res. 1993 Aug 1;35(5):567-76
references: 8377226
references: J Inherit Metab Dis. 2004;27(6):861-8
references: 15505393
references: Methods Mol Biol. 2010;614:3-35
references: 20225033
references: Neurology. 2002 Dec 10;59(11):1802-4
references: 12473778
references: J Neurol Sci. 2012 Nov 15;322(1-2):254-62
references: 22669122
references: Ann Indian Acad Neurol. 2012 Jan;15(1):31-4
references: 22412270
references: J Biol Chem. 2011 May 20;286(20):17777-84
references: 21454630
references: Apoptosis. 2010 Nov;15(11):1403-9
references: 20127416