Excitotoxicity‐induced endocytosis mediates neuroprotection by TAT‐peptide‐linked JNK inhibitor

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07535.x Excitotoxicity and cerebral ischemia induce strong endocytosis in neurons, and we here investigate its functional role in neuroprotection by a functional transactivator of transcription (TAT)‐peptide, the c‐Jun N‐terminal kinase (JNK) inhibitor D‐JNKI1, against NMDA‐excitotoxicity in vitro and neonatal ischemic stroke in P12 Sprague–Dawley rats. In both situations, the neuroprotective efficacy of D‐JNKI1 was confirmed, but excessively high doses were counterproductive. Importantly, the induced endocytosis was necessary for neuroprotection, which required that the TAT‐peptide be administered at a time when induced endocytosis was occurring. Uptake by other routes failed to protect, and even promoted cell death at high doses. Blocking the induced endocytosis of D‐JNKI1 with heparin or with an excess of D‐TAT‐peptide eliminated the neuroprotection. We conclude that excitotoxicity‐induced endocytosis is a basic property of stressed neurons that can target neuroprotective TAT‐peptides into the neurons that need protection. Furthermore, it is the main mediator of neuroprotection by D‐JNKI1. This may explain promising reports of strong neuroprotection by TAT‐peptides without apparent side effects, and warns that the timing of peptide administration is crucial. Excitotoxicity‐induced endocytosis provides specific drug‐targeting for neuroprotection. There is currently no neuroprotective pharmacotherapy for stroke. This is partly because of drug side‐effects, but these might be reduced if delivery specificity could be increased. We recently showed that TAT peptides can be targeted to the cells that need them by excitotoxicity‐induced endocytosis, which is clathrin/dynamin‐dependent. Our present study shows that excitotoxicity‐induced endocytosis of a TAT‐linked JNK‐inhibitory peptide gives neuroprotection, whereas constitutive endocytosis does not.