Autophagy is required for maintaining of oligodendrocyte precursor cells in optic nerve

Abstract

Purpose : Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved mechanism by which cytoplasmic components are sequestered in autophagosomes and delivered to lysosomes for degradation. Apoptosis, a form of programmed cell death, is a common process by which cells are removed during development. A complex crosstalk takes place between apoptosis and autophagy which determines the life or death of cells. While the importance of this crosstalk in survival of oligodendrocyte precursor cells (OPCs) is poorly understood, the survival of OPCs is crucial for myelination of retinal ganglion cell axons in optic nerve (ON). This study was undertaken to understand the role of autophagy in the self-renewal and survival of ON OPCs.

Methods : OPCs (A2B5⁺ cells) were isolated from optic nerve based on a method developed in our lab and were transfected with green florescent protein (GFP)-LC3ll and red fluorescent protein RFP−GFP tandem fluorescent tagged LC3II. A2B5⁺ cells were treated with the endoplasmic reticulum (ER) stress inducer tunicamycin, alone or with 3-methyl-adenine (3-MA). Immunoblotting was used to determine levels of apoptosis markers; cleaved PARP (CPARP) and cleaved caspase 3 (CC3). Adeno-associated virus-BECN1-shRNA was used for down-regulation of Beclin 1 (pro-autophagy gene) expression in A2B5⁺ cells.

Results : Blocking autophagic flux with chloroquine (CHQ) led to accumulation of bright GFP-LC3 puncta in GFP-LC3 transfected A2B5⁺ cells. Consistently, cells transfected with RFP-GFP-LC3 revealed a significant increase in the number of autolysosomes relative to autophagosomes, suggesting activated autophagy flux. Inhibition of autophagy by 3-MA resulted in a significant reduction in proliferation of A2B5⁺ cells. Similarly, down-regulation of Beclin 1 expression led to a significant reduction in proliferation of A2B5⁺ cells, suggesting the existence of a role for autophagy during proliferation of these cells. The apoptotic markers, CPARP and CC3 were highly elevated in tunicamycin treated cells in which autophagy was inhibited by 3MA.

Conclusions : We conclude that a basal level of autophagy is required for self-renewal of ON OPCs. Moreover, we also found that autophagy in ON OPCs can play an important pro-survival role in response to ER stress.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.