P1-CPP promotes Foxo1 and Creb signaling and reduces apoptosis in Neurotrophic Factor-Deprived Primary Retinal Ganglion Cells

Purpose: To elucidate the intracellular mechanisms underlying neuroprotective effects of the core peptide of a-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in primary retinal ganglion cells (RGCs). Targets of the investigation were limited to Creb1, Bak1/Bad, and Foxo1, based upon RNA sequencing data obtained from RGCs of IOP-elevated rats treated with P1-CPP in comparison with the vehicle.

Methods: Primary RGCs isolated from Sprague Dawley rat pups were deprived of neurotrophic factors (NT) namely, BDNF, CNTF, and Forskolin for 48 hours, either in the presence or absence of P1-CPP (4µM). After the treatments, RNA isolation was carried out using Trizol reagent. Subsequently, cDNA synthesis and qPCR analysis of the target genes expression, including Creb1 (n=2), Foxo1 (n=3), and Bak1 (n=3), was performed. Another set of RGCs subjected to the same treatments was fixed with 4% paraformaldehyde for 20 minutes and used for immunocytochemical analyses of p-CREB (n=3), FOXO1 (n=3), and BAD (n=3) protein expression. Immunostaining with an RBPMS antibody was used as an RGC marker. N indicates experimental repeats.

Results: Following NT deprivation, there was an increase in mRNA expression of Creb1 (2-fold) in RGCs treated with P1-CPP, compared to the vehicle-treated RGCs. Moreover, the phosphorylated (active) form, p-CREB, was increased (by 102%; p=0.04) in primary RGCs treated with P1-CPP, compared to the vehicle-treated group. Pro-apoptotic Bak1 mRNA expression was not changed in the P1-CPP-treated RGCs compared to the vehicle-treated group. Primary RGCs stained for BAD protein showed a decrease (by 62%; p=0.08) in the P1-CPP treated group compared to the vehicle-treated RGCs. Foxo1 mRNA levels were increased by more than 2-fold in the P1-CPP treated RGCs, compared to the vehicle-treated RGCs. FOXO1 protein was also elevated in primary RGCs treated with P1-CPP compared to the vehicle group (by 59%).

Conclusions: P1-CPP is neuroprotective against neurotrophic factor deprivation through multiple mechanisms, including early changes in the expression of mitochondrial homeostasis regulator Foxo1, activation of the pro-survival CREB pathway, and inhibition of pro-apoptotic members of the BCL-2 family of proteins.