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.

Protection of cortical neurons from excitotoxicity by conjugated linoleic acid.

Conjugated linoleic acid (CLA) is an isomeric mixture of 18-carbon polyunsaturated fatty acids with conjugated double bonds derived from linoleic acid. CLA is sourced nutritionally from dairy products and ruminant animal meat and is associated with beneficial heath effects in several disorders, including cancer, atherosclerosis, diabetes, obesity, and inflammation. We investigated the potential for CLA to protect neurons from death in stroke simulated by exposing cultured mouse embryonic cortical neurons to glutamate. CLA (10 30 µM) significantly protected neuronal survival in response to glutamate (3 µM) excitotoxicity when given concurrently with glutamate. CLA (30 µM) also reduced neuron death when given up to 5 h after glutamate exposure (73.1 ± 13.3% protection, p < 0.01), suggesting potential utility as a post-injury therapeutic tool. The cis-9, trans-11 CLA isomer, which comprises about 40% of the commercial CLA mixture used, was identified as the active neuroprotective species. The other major species, trans-10, cis-12 CLA (40%) was without significant neuroprotective effect. CLA significantly increased neuronal Bcl-2 levels when given with glutamate and attenuated glutamate-induced dissipation of mitochondrial membrane potential, suggesting a stabilizing influence on mitochondrial function. These results show that CLA is capable of strong neuroprotective effects in glutamate excitotoxicity at concentrations likely achieved by consumption of CLA as a dietary supplement.