Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons.
Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process.
Baptista
Márcio S
MS
Center for Neuroscience and Cell Biology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal.
Melo
Carlos V
CV
Armelão
Mário
M
Herrmann
Dennis
D
Pimentel
Diogo O
DO
Leal
Graciano
G
Caldeira
Margarida V
MV
Bahr
Ben A
BA
Bengtson
Mário
M
Almeida
Ramiro D
RD
Duarte
Carlos B
CB
D000818:Animals; D002478:Cells, Cultured; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology*; D006868:Hydrolysis / Q000187:drug effects; D009474:Neurons / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D054875:Ubiquitination
D018698:Glutamic Acid; D046988:Proteasome Endopeptidase Complex; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2
Brain Res. 2002 Jan 18;925(1):89-99
11755903
Stroke. 2001 Dec 1;32(12):2926-31
11739997
EMBO J. 1991 May;10(5):1275-84
2022191
Trends Pharmacol Sci. 1998 Dec;19(12):500-5
9871412
Nat Struct Mol Biol. 2007 Apr;14(4):280-6
17384644
Circ Res. 2007 May 11;100(9):1276-91
17495234
Neurochem Res. 2008 May;33(5):962-9
17940883
Neurochem Res. 2008 Aug;33(8):1459-65
18270816
J Neurochem. 1991 Feb;56(2):720-3
1988566
Arch Biochem Biophys. 1997 Jun 15;342(2):383-8
9186501
Neurochem Res. 2005 Aug;30(8):989-1001
16258848
Curr Opin Investig Drugs. 2005 Jul;6(7):686-99
16044664
Exp Neurol. 2008 Sep;213(1):145-53
18599042
N Engl J Med. 1994 Mar 3;330(9):613-22
7905600
J Cell Biol. 1994 Jul;126(2):331-41
8034738
Mol Cell Neurosci. 2005 Oct;30(2):279-89
16112871
J Intern Med. 1996 Nov;240(5):259-77
8946809
J Biol Chem. 1999 Aug 20;274(34):24366-71
10446215
Toxicol Appl Pharmacol. 2002 Dec 1;185(2):111-8
12490135
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):4293-8
12634427
Int J Exp Pathol. 2000 Oct;81(5):323-39
11168679
J Cereb Blood Flow Metab. 2004 Nov;24(11):1219-25
15545915
Brain Res Mol Brain Res. 1994 Feb;21(3-4):293-302
8170353
J Neurosci Res. 1995 Jan 1;40(1):134-7
7714921
J Cell Biol. 2002 Sep 30;158(7):1229-38
12356867
J Neurosci Res. 2000 Mar 15;59(6):827-32
10700020
J Neurochem. 1993 Feb;60(2):395-407
8419527
Curr Med Chem. 2006;13(28):3425-40
17168715
Brain Res Mol Brain Res. 1996 Mar;36(2):261-7
8965646
Brain Res. 2007 Jun 18;1154:80-3
17482148
Mol Cell Neurosci. 2002 Oct;21(2):223-38
12401444
Br J Clin Pharmacol. 2002 Sep;54(3):269-76
12236847
Chem Biol. 2001 Aug;8(8):739-58
11514224
Neurochem Int. 2009 Sep;55(5):288-94
19576516
Int J Biochem Cell Biol. 2005 Oct;37(10):2115-33
16125114
Cancer Res. 2007 Oct 1;67(19):9472-81
17909057
Biochem Biophys Res Commun. 1996 Dec 24;229(3):891-5
8954991
Neuron. 1991 Jul;7(1):91-100
2069816
Ann N Y Acad Sci. 1995 May 10;757:439-56
7611702
J Neurochem. 2001 Apr;77(2):353-71
11299298
Med Res Rev. 2001 Jul;21(4):245-73
11410931
Am J Physiol Cell Physiol. 2004 Oct;287(4):C834-43
15355854
J Comp Neurol. 2004 Oct 25;478(4):405-17
15384067
Cell. 1994 Sep 9;78(5):761-71
8087844
J Neurochem. 2006 Sep;98(5):1688-95
16879709
Exp Neurol. 2007 Jun;205(2):367-82
17433299
EMBO J. 2005 Oct 19;24(20):3602-12
16193061
Neurochem Int. 2005 Jul;47(1-2):136-42
15936116
Brain Res. 1996 May 6;719(1-2):161-71
8782876
Brain Res. 1993 May 14;611(1):130-8
8100173
Neurochem Int. 1999 Jul;35(1):73-80
10403432
Eur J Pharmacol. 2006 Sep 1;545(1):2-10
16831414
Mol Cell. 2001 Feb;7(2):411-20
11239469
Brain Res. 2008 May 1;1207:9-18
18377878
J Cereb Blood Flow Metab. 2003 Jan;23(1):75-87
12500093
J Cereb Blood Flow Metab. 2002 Jun;22(6):705-10
12045669
J Neurosci Res. 2008 Dec;86(16):3684-92
18655199
Cell Mol Neurobiol. 1991 Oct;11(5):485-96
1742770
J Cereb Blood Flow Metab. 1998 Apr;18(4):376-85
9538902
J Cereb Blood Flow Metab. 2008 Apr;28(4):655-73
18073773
Mol Cell. 2004 Nov 19;16(4):575-86
15546617
Cell Death Differ. 2005 Oct;12(10):1329-43
15905876
J Neurosci Res. 2002 Mar 15;67(6):787-94
11891793
Neurochem Int. 2003 Jun;42(7):549-54
12590937
Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11259-63
7479976
Stroke. 2007 Dec;38(12):3230-6
17975104
Stroke. 2000 Jul;31(7):1686-93
10884474
J Neurosci. 1994 Mar;14(3 Pt 2):1834-55
8126575
Neuron. 1988 Oct;1(8):623-34
2908446
Neurobiol Aging. 2002 Jan-Feb;23(1):145-58
11755029
Exp Neurol. 1993 Feb;119(2):291-301
8432368
J Biol Chem. 2000 Jul 7;275(27):20822-8
10781586
J Cell Sci. 2008 Feb 15;121(Pt 4):437-49
18230651
Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10599-603
7479848
J Biomed Sci. 2003;10(6 Pt 1):617-24
14576464
Annu Rev Biochem. 1998;67:425-79
9759494