Cell-permeable PEP-1-SH3GL3 protects neurons from ischemic damage by reducing oxidative stress and pro-inflammatory...

SH3-domain GRB2-like 3 (SH3GL3), also known as endophilin A3, is highly expressed in the hippocampus; however, comprehensive studies on SH3GL3 expression after ischemia and its protective roles against ischemic damage in the hippocampus are lacking. Here, we examined SH3GL3 expression in the hippocampal CA1 region. SH3GL3 immunoreactivity gradually increased, peaking 12 h after ischemia/reperfusion, and then decreased over time post-ischemia. However, SH3GL3 immunoreactivity increased again in the hippocampal CA1 region 7 days after ischemia and SH3GL3 immunoreactivity in this group was detected in neurons as well as astrocytes. To elucidate SH3GL3's role against oxidative stress and ischemic damage, we synthesized the cell-permeable PEP-1-SH3GL3 fusion protein to facilitate crossing the cell membrane and blood-brain barrier. In an in vitro study, treatment with PEP-1-SH3GL3, unlike its control protein (SH3GL3), was delivered into HT22 hippocampal cells in a concentration- and incubation time-dependent manner. Additionally, PEP-1-SH3GL3 significantly reduced hydrogen peroxide (H₂O₂)-induced cell death, reactive oxygen species formation, and DNA fragmentation in HT22 cells. In an in vivo ischemia study, treatment with PEP-1-SH3GL3 was delivered into hippocampus and the protein was peaked 3 h after ischemia and protein treatment and thereafter decreased with time after ischemia and protein treatment. Treatment with PEP-1-SH3GL3 significantly reduced traveled distance one day after ischemia and increased the number of surviving cells in the hippocampal CA1 region four days after ischemia in gerbils. PEP-1-SH3GL3 treatment decreased ischemia-induced phagocytic microglia in the stratum pyramidale of the CA1 region four days after ischemia. Furthermore, treatment with PEP-1-SH3GL3 significantly mitigated ischemia-induced oxidative stress (hydroperoxides and malondialdehyde) and pro-inflammatory cytokine release (interleukin-1β, interleukin-6, tumor necrosis factor-α) in the hippocampus 3 and 6 h after ischemia, respectively. These results suggest that PEP-1-SH3GL3 may serve as a therapeutic agent to inhibit neuronal damage caused by cerebral ischemia.