Pharmacological Inhibition of Pleckstrin Homology Domain Leucine-Rich Repeat Protein Phosphatase Is Neuroprotective: Differential Effects on Astrocytes

Abstract

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,-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 astrocytesnamely from global brain ischemia.

Cold stress protein RBM3 responds to temperature change in an ultra-sensitive manner in young neurons

“…Neuron cultures were performed as previously described by our group (Jackson et al, 2013). Cortices were dissected in ice-cold Hanks balanced salt solution (HBSS; Life Technologies, Grand Island, NY, USA) containing sodium bicarbonate (SIGMA), penicillin-streptomycin (Life technologies), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Life Technologies).…”

“…Pure rat astrocyte cultures were prepared as previously reported by our group (Jackson et al, 2013). In brief, brains were collected from postnatal day 2 SD rat pups.…”

Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

“…(25) has been shown to regulate the phosphorylation state of Ser473 in numerous studies (e.g., refs. [26][27][28][29][30]. The family comprises two genes: PHLPP1, which is alternatively spliced to yield PHLPP1α and PHLPP1β, and PHLPP2 (24,31).…”

PHLPPing through history: a decade in the life of PHLPP phosphatases

“…Two compounds were identified to selectively inhibit activity of PHLPP1 and PHLPP2 with in vitro IC 50 values in the 5μM range, compared to in vitro IC 50 values in the 100μM range for PP2Cα and PP1. Treatment of cells with these inhibitors has been shown to increase Akt phosphorylation and suppress apoptosis of cells [12], promote chondrocyte proliferation [13], decrease chaperone-mediated autophagy [14], and raise levels of Akt activity in rat cortical neurons resulting in a neuroprotective phenotype [15]. The identification of such inhibitors not only provides a pharmacological breakthrough to studying PHLPP activity in vitro and in cells, but also provides the first steps to creating a potential therapeutic drug to inhibit PHLPP activity.…”