Pharmacological inhibition of pleckstrin homology domain leucine-rich repeat protein phosphatase is neuroprotective: differential effects on astrocytes

Pleckstrin homology domain and leucine-rich repeat protein Phosphatase 1 (PHLPP1) inhibits protein kinase B (Akt) survival signaling in neurons. Small molecule pan-PHLPP inhibitors (selective for PHLPP1 and PHLPP2) may offer a translatable method to induce Akt neuroprotection. We tested several recently discovered PHLPP inhibitors (NSC117079 and NSC45586; benzoic acid, 5-[2-[4-[2-(2,4-diamino-5-methylphenyl)diazenyl]phenyl]diazenyl]-2-hydroxy-,sodium salt.) in rat cortical neurons and astrocytes and compared the biochemical response of these agents with short hairpin RNA (shRNA)-mediated PHLPP1 knockdown (KD). In neurons, both PHLPP1 KD and experimental PHLPP inhibitors activated Akt and ameliorated staurosporine (STS)-induced cell death. Unexpectedly, in astrocytes, both inhibitors blocked Akt activation, and NSC117079 reduced viability. Only PHLPP2 KD mimicked PHLPP inhibitors on astrocyte biochemistry. This suggests that these inhibitors could have possible detrimental effects on astrocytes by blocking novel PHLPP2-mediated prosurvival signaling mechanisms. Finally, because PHLPP1 levels are reportedly high in the hippocampus (a region prone to ischemic death), we characterized hippocampal changes in PHLPP and several Akt targeting prodeath phosphatases after cardiac arrest (CA)-induced brain injury. PHLPP1 levels increased in rat brains subjected to CA. None of the other Akt inhibitory phosphatases increased after global ischemia (i.e., PHLPP2, PTEN, PP2A, and PP1). Selective PHLPP1 inhibition (such as by shRNA KD) activates Akt survival signaling in neurons and astrocytes. Nonspecific PHLPP inhibition (by NSC117079 and NSC45586) only activates Akt in neurons. Taken together, these results suggest that selective PHLPP1 inhibitors should be developed and may yield optimal strategies to protect injured hippocampal neurons and astrocytes-namely from global brain ischemia.