Astrocytic poly(ADP-ribose) polymerase-1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP-1-induced depletion of cellular NAD(+), glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP-dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP-1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP-1 activation by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC(50) = 78.2 +/- 2.7 microM). HPLC revealed MNNG-induced time-dependent reductions in NAD(+) (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [(3)H]glutamate uptake rate (V(max)) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 +/- 2.8 in control cells to 2.1 +/- 0.8 nmol/min/mg protein 4 h post-MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP-1, directly indicating PARP-1 involvement, and by adding exogenous NAD(+) (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co-culture with PARP-1(-/-) astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP-1 can raise glutamate levels sufficiently to have receptor-mediated effects at neighboring neurons. Taken together, these experiments showed that PARP-1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity.
Tang
Kim San
KS
Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada.
Suh
Sang Won
SW
Alano
Conrad C
CC
Shao
Zongjun
Z
Hunt
Waylon T
WT
Swanson
Raymond A
RA
Anderson
Christopher M
CM
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D018920:Coculture Techniques; D018698:Glutamic Acid / Q000378:metabolism*; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D009243:NAD / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D000477:Alkylating Agents; D009243:NAD; D008769:Methylnitronitrosoguanidine; D018698:Glutamic Acid; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases