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 (EC₅₀ = 78.2 ± 2.7 μM). 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 [³H]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.