Neuronal cell death caused by glutamate excitotoxicity is associated with cerebral ischemia, traumatic brain injury, epilepsy and neurodegenerative disorders including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (Faden et al. 1989;Bradford 1995;Mattson et al. 1999;Coyle 2006;Lizasoain et al. 2006). Glutamate overstimulation of NMDA, a-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainic acid (KA) receptors results in an influx of extracellular calcium and sodium and the release of calcium from intracellular stores. Increased intracellular calcium initiates a range of cell damaging processes involving phospholipases, proteases, kinases, phosphatases and ROS/RNS that can trigger a host of neuronal cell death pathways. Depending on the intensity of glutamate receptor over-stimulation, the resulting neuronal death can display a variety of features ranging from mostly necrotic to mostly apoptotic (Choi et al. 1987;Lipton and Rosenberg 1994). Moreover, regardless of the neuronal cell death outcome, glutamate receptor stimulation activates the c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1) transcription Abbreviations used: BSS, balance salt solution; DIV, day in vitro; DMEM, Dulbecco's modified Eagle medium; MTS, 3-(4,5,dimethyliazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; NB, neurobasal; PBS, phosphate-buffered saline. AbstractNeuronal cell death caused by glutamate excitotoxicity is prevalent in various neurological disorders and has been associated with the transcriptional activation of activator protein-1 (AP-1). In this study, we tested 19 recently isolated AP-1 inhibitory peptides, fused to the cell penetrating peptide TAT, for their efficacy in preventing cell death in cortical neuronal cultures following glutamate excitotoxicity. Five peptides (PYC19D-TAT, PYC35D-TAT, PYC36D-TAT, PYC38D-TAT, PYC41D-TAT) displayed neuroprotective activity in concentration responses in both L-and retro-inverso D-isoforms with increasing levels of neuroprotection peaking at 83%. Interestingly, the D-TAT peptide displayed a neuroprotective effect increasing neuronal survival to 25%. Using an AP-1 luciferase reporter assay, we confirmed that the AP-1 inhibitory peptides reduce AP-1 transcriptional activation, and that c-Jun and c-Fos mRNA following glutamate exposure is reduced. In addition, following glutamate exposure the AP-1 inhibitory peptides decreased calpain-mediated a-fodrin cleavage, but not neuronal calcium influx. Finally, as neuronal death following glutamate excitotoxicity was transcriptionally independent (actinomycin D insensitive), our data indicate that activation of AP-1 proteins can induce cell death via nontranscriptional pathways. Thus, these peptides have potential application as therapeutics directly or for the rational design of small molecule inhibitors in both apoptotic and necrotic neuronal death associated with AP-1 activation.

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