Lack of neuroprotection of inhibitory peptides targeting Jun/JNK after transient focal cerebral ischemia in spontaneously hypertensive rats.

In this study, we have assessed the ability of two TAT-fused peptides PYC36D-TAT and JNKI-1D-TAT (JNKI-1 or XG-102), which respectively inhibit jun proto-oncogene (c-Jun) and c-Jun N-terminal kinase (JNK) activation, to reduce infarct volume and improve functional outcome (adhesive tape removal) after transient focal cerebral ischemia in Spontaneously Hypertensive (SH) rats. PYC36D-TAT and JNKI-1D-TAT peptide batches used for experiments were tested in vitro and protected cortical neurons against glutamate excitotoxicity. Rats were treated intravenously with three different doses of PYC36D-TAT (7.7, 76, or 255 nmol/kg), JNKI-1D-TAT (255 nmol/kg), D-TAT peptide (255 nmol/kg), or saline (vehicle control), 10 minutes after reperfusion after 90 minutes of middle cerebral artery occlusion (MCAO). Contrary to other stroke models, no treatment significantly reduced infarct volume or improved functional score measurements compared with vehicle-treated animals when assessed 48 hours after MCAO. Additionally, assessment of the JNKI-1D-TAT peptide, when administered 1 or 2 hours after reperfusion after 90 minutes of MCAO, also did not improve histological or functional outcomes at 48 hours after occlusion. This study is the first to evaluate the efficacy of PYC36D-TAT and JNKI-1D-TAT using the SH rat, which has recently been shown to be more sensitive to AMPA receptor activation rather than to NMDA receptor activation after cerebral ischemia, and which may have contributed to the negative findings.

Characterisation of neuronal cell death in acute and delayed in vitro ischemia (oxygen-glucose deprivation) models.

Using 96 well microtitre plate sized glass wells we have established and characterised two in vitro ischemia (oxygen-glucose deprivation) models that induce acute or delayed neuronal cell death. In vitro ischemia was induced by washing cortical neuronal cultures with a balanced salt solution either with (acute model) or without (delayed model) 25mM 2-deoxy-d-glucose, and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and adding glucose containing media (delayed model) or removing the balanced salt solution/2-deoxy-d-glucose medium (acute model) and adding glucose containing media. The models were characterised with respect to in vitro ischemia dose duration, cell death time course and for necrosis, apoptosis, autophagy and necroptosis biomarkers. To this end, biomarkers for all four modes of cell death were detected in both in vitro ischemia models, although the time of onset and relative proportion of each cell death mode differed between models. While it is likely that different modes of cell death were activated in the same cell, autophagy appeared to be a prominent cell death mode, especially in the delayed model. Together these models will provide valuable tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput screening system to evaluate potential neuroprotective agents.

AP-1 inhibitory peptides attenuate in vitro cortical neuronal cell death induced by kainic acid.

This study has assessed the neuroprotective efficacy of five AP-1 inhibitory peptides in an in vitro excitotoxicity model. The five AP-1 inhibitory peptides and controls of the JNK inhibitor peptide (JNKI-1D-TAT) and TAT cell-penetrating-peptide were administered to primary cortical neuronal cultures prior to kainic acid exposure. All five AP-1 inhibitory peptides and JNKI-1D-TAT provided significant neuroprotection from kainic acid induced neuronal cell death. Kainic acid exposure induced caspase and calpain activation in neuronal cultures, with caspase-induced cleavage of α-fodrin reduced by administration of the AP-1 inhibitory peptides. Sequence analysis of the AP-1 inhibitory peptides did not reveal the presence of any secondary structures; however two peptides shared 66% amino-acid sequence homology. As a result, truncated sequences were designed and synthesised to identify the active region of the peptides. All truncated peptides were significantly neuroprotective following kainic acid and glutamate exposure. We have shown for the first time the neuroprotective efficacy of full-length and truncated AP-1 inhibitory peptides in kainic acid and glutamate neuronal excitotoxicity models. The identification of therapeutic targets, such as the AP-1 complex, is an important step for the development of pharmaceuticals to reduce neuronal loss in disorders with a prevalence of excitotoxic cell death such as epilepsy, cerebral ischaemia, and traumatic brain injury.

AP-1 inhibitory peptides are neuroprotective following acute glutamate excitotoxicity in primary cortical neuronal cultures.

Neuronal 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 alpha-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 non-transcriptional 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.