June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
P1-CPP promotes Foxo1 and Creb signaling and reduces apoptosis in Neurotrophic Factor-Deprived Primary Retinal Ganglion Cells
Author Affiliations & Notes
  • Gretchen A Johnson
    Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Jennifer Hien Pham
    Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Raghu R Krishnamoorthy
    Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Ram H Nagaraj
    Ophthalmology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States
  • Dorota Luiza Stankowska
    Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Footnotes
    Commercial Relationships   Gretchen Johnson None; Jennifer Pham None; Raghu Krishnamoorthy None; Ram Nagaraj None; Dorota Stankowska None
  • Footnotes
    Support  T32 Grant AG020494
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2005. doi:
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      Gretchen A Johnson, Jennifer Hien Pham, Raghu R Krishnamoorthy, Ram H Nagaraj, Dorota Luiza Stankowska; P1-CPP promotes Foxo1 and Creb signaling and reduces apoptosis in Neurotrophic Factor-Deprived Primary Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2005.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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