Stroke is the leading cause of disability in developed countries. However, no treatment is available beyond 3 h post-ictus. Here, we report that nuclear translocation of PTEN (phosphatase and tensin homolog deleted on chromosome TEN) is a delayed step causatively leading to excitotoxic (in vitro) and ischemic (in vivo) neuronal injuries. We found that excitotoxic stimulation of N-methyl-D-aspartate (NMDA) resulted in PTEN nuclear translocation in cultured neurons, a process requiring mono-ubiquitination at the lysine 13 residue (K13), as the translocation was prevented by mutation of K13 or a short interfering peptide (Tat-K13) that flanks the K13 residue. More importantly, using a rat model of focal ischemia, we demonstrated that systemic application of Tat-K13, even 6 h after stroke, not only reduced ischemia-induced PTEN nuclear translocation, but also strongly protected against ischemic brain damage. Our study suggests that inhibition of PTEN nuclear translocation may represent a novel after stroke therapy.

Modification of proteins by 4-hydroxy-2-nonenal (4-HNE) has been proposed to cause neurotoxicity in a number of neurodegenerative diseases, including distal axonopathy in diabetic sensory neuropathy. We tested the hypothesis that exposure of cultured adult rat sensory neurons to 4-HNE would result in the formation of amino acid adducts on mitochondrial proteins and that this process would be associated with impaired mitochondrial function and axonal regeneration. In addition, we compared 4-HNE-induced axon pathology with that exhibited by neurons isolated from diabetic rats. Cultured adult rat dorsal root ganglion (DRG) sensory neurons were incubated with varying concentrations of 4-HNE. Cell survival, axonal morphology, and level of axon outgrowth were assessed. In addition, video microscopy of live cells, western blot, and immunofluorescent staining were utilized to detect protein adduct formation by 4-HNE and to localize actively respiring mitochondria. 4-HNE induced formation of protein adducts on cytoskeletal and mitochondrial proteins, and impaired axon regeneration by approximately 50% at 3 microM while having no effect on neuronal survival. 4-HNE initiated formation of aberrant axonal structures and caused the accumulation of mitochondria in these dystrophic structures. Neurons treated with 4-HNE exhibited a distal loss of active mitochondria. Finally, the distal axonopathy and the associated aberrant axonal structures generated by 4-HNE treatment mimicked axon pathology observed in DRG sensory neurons isolated from diabetic rats and replicated aspects of neurodegeneration observed in human diabetic sensory neuropathy.

Excitotoxicity, due to overstimulation of N-methyl D-aspartate receptors (NMDARs), has a pivotal role in many neurological disorders. However, NMDAR antagonists often cause side effects, and identifying more druggable therapeutic targets for NMDAR excitotoxicity is an important goal. Activation of caspases is a downstream effect of excitotoxicity that may be critically involved in NMDAR-mediated cell death. Caspase-6 (casp6) in particular has been shown to play a key role in the pathogenesis of stroke, Huntington disease, and Alzheimer disease. Using N-methyl D-aspartate (NMDA)-induced excitotoxic injuries of primary rat neurons, we demonstrate that there is an early increase in caspase profiles after an excitotoxic event at the level of mRNA, protein, and activity. Casp6 is elevated and activated first, followed by caspase-8 and caspase-3. Similarly, known casp6 substrates huntingtin, as well as novel casp6 substrates serine/threonine kinase 3 and death domain-associated protein, are cleaved in similar temporal patterns post NMDA. On the basis of these data, we propose that casp6 may be an initiator caspase in apoptotic cascades leading to neuronal death after an excitotoxic event and suggest casp6 as a potential therapeutic target for neurological disorders where NMDAR-mediated excitotoxicity has been shown to play a role.