Abstract
Purpose :
To determine 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 retinal ganglion cells (RGCs) in a rodent model of glaucoma and human retinal explants.
Methods :
Ex vivo human retinal explants (n=3 donors) were obtained within 12h postmortem and were incubated with 12.5 µg/ml P1-CPP or vehicle for 7 days. Explants were stained with RGC marker, RBPMS, and surviving RGCs were counted using ImageJ. In a separate set of experiments, intraocular pressure (IOP) was elevated in Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once a week for a period of 2 weeks. Rats were euthanized, primary adult RGCs were isolated by the immunopanning method. Total RNA was isolated using the Trizol/column method. RNA-sequencing was performed using an Illumina platform. The resulting FASTQ files were uploaded into Galaxy for analysis with FASTQC, RNASTAR, featureCounts, and finally DESeq2. The results from DESeq2 were then assessed with Qiagen’s Ingenuity Pathway Analysis (IPA) to identify significantly upregulated pathways.
Results :
P1-CPP treatment, at 7 days significantly (p<0.005) prevented RGC loss by 35% compared to the vehicle. RNA-seq analysis from rat RGCs isolated following 2 weeks of IOP-elevation revealed that P1-CPP treated groups had several differentially expressed (DEGs), compared to vehicle-treated groups, including 6343 significantly upregulated and 5960 significantly downregulated. Some significantly upregulated pathways following P1-CPP treatment include phagosome formation, synaptic long-term depression, and CREB signaling in neurons. The IOP and vehicle-treated groups, when compared to the naïve group, demonstrated a decreased expression of members of the CREB signaling pathway (Creb-1, c-RAF, MEK1/2, ERK1/2, and p90RSK). This decline was prevented by P1-CPP treatment. Quantitative PCR further confirmed the RNA-seq findings of the increased expression of Creb-1 in P1-CPP treated rats compared to that of vehicle-treated group.
Conclusions :
P1-CPP was neuroprotective against neurotrophic factor deprivation-mediated RGC loss in human retinal explants. Mechanism of action of P1-CPP in a rodent model of glaucoma includes the activation of the pro-survival CREB signaling pathway, phagosome formation, and long-term synaptic depression to prevent cell death and vision loss.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.