Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population. More than 80% of AMD cases are of the dry-AMD classification, which characteristically features geographic atrophy resulting from the loss of retinal pigment epithelial (RPE) cells (1, 2). Dry-AMD often converts to wet-AMD, which is visionthreatening. Accumulation of undigested cellular debris (e.g., drusen), oxidative stress, and subsequent death and degeneration of RPE cells are important contributors to AMD pathology (3,4). Accumulating evidence suggests that the cellular sphingolipid mediator, ceramide (Cer), which acts as a second messenger to induce apoptosis, might play a role in RPE cell death (5-7). Here, we investigate for the first time the role of Cer in human RPE-derived ARPE19 cells under oxidative stress by removing excess cellular Cer via genetic manipulation of Cer-hydrolyzing enzymes.In addition to being a critical component of membrane structure and function, Cer serves as a vital intracellular second messenger. A steady-state level of Cer is necessary for cell proliferation and differentiation (8,9), while excessive amounts of Cer are often toxic and can result in cellular apoptosis (10). High concentrations of Cer can result in the induction of cell death, leading to tissue damage and organ failure, such as what is observed in retinal degeneration Sph) is the major source for Sph 1-phosphate production, which has an opposing role to Cer and provides cytoprotection. Here, we investigated the role of Cer in human RPE-derived ARPE19 cells under hydrogen peroxide-induced oxidative stress, and show that Cer and hexosyl-Cer levels increase in the oxidatively stressed ARPE19 cells, which can be prevented by overexpression of lysosomal ASAH1. This study demonstrates that oxidative stress generates sphingolipid death mediators in retinal cells and that induction of ASAH1 could rescue retinal cells from oxidative stress by hydrolyzing excess Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress. J. Lipid Res. 2019. 60: 30-43. Abstract Over 11 million people in the United States alone have some form of age-related macular degeneration (AMD). Oxidative stress, cell death, and the degeneration of retinal pigment epithelial (RPE) cells contribute to AMD pathology. Recent evidence suggests that ceramide (Cer), a cellular sphingolipid mediator that acts as a second messenger to induce apoptosis, might play a role in RPE cell death. The lysosomal breakdown of Cer by acid ceramidase [N-acylsphingosine amidohydrolase (ASAH)1] into sphingosine (Supplementary key words N-acylsphingosine amidohydrolase 1 • ceramide • hexosyl-ceramide • lysosome, retinal pigment epithelium • retinal degeneration • age-related macular degeneration

Sphingolipids as Emerging Mediators in Retina Degeneration

“…Oxidative stress increases Cer levels and induces apoptosis in human RPE cells and this was replicated by using C2- and C6-Cer (Barak et al, 2001). Oxidative stress increases both Cer and hexosyl-Cer levels in ARPE-19 cells, a human RPE cell line, promoting cell death; this death is prevented by over-expression of acid ceramidase, which increases Sph levels but not those of S1P (Sugano et al, 2018). Interestingly, increased serum levels of hexosyl-Cer have been reported in patients with late stage AMD (Pujol-Lereis et al, 2018).…”

“…Interestingly, increased serum levels of hexosyl-Cer have been reported in patients with late stage AMD (Pujol-Lereis et al, 2018). Noteworthy, oxidative stress leads to increased S1P levels in control RPE cells, which might reflect an attempt to counteract Cer action toward cell death (Sugano et al, 2018). Laser exposure, a frequent treatment for retina neovascularization, induces human RPE cell death concomitant with Cer overproduction (Barak et al, 2005).…”

“…These mice show age-dependent retina degeneration and RPE alterations, with an impaired autophagic flux (Wu et al, 2015); it still remains to be defined whether Sph increase leads to these changes. Puzzlingly, overexpression of acid ceramidase in ARPE19 cells increases their Sph levels, and protects these cells from oxidative stress, with no concomitant increase in S1P levels (Sugano et al, 2018). Hence, much remains to be investigated regarding Sph actions in the retina in order to design new therapies through the adequately modulation of sphingolipid levels.…”

Sphingosine Kinase 2 Phosphorylation of FTY720 is Unnecessary for Prevention of Light-Induced Retinal Damage

“…We found that Cer plays a crucial role in light-induced degeneration of photoreceptor cells and were able to conclude that FTY720 prevents excess Cer formation during light exposure 22 . We have also shown in our in vitro studies that reduction of Cer by overexpression of acid Ceramidase (ASAH1), a lysosomal enzyme which degrades excess Cer, in retinal pigment epithelial (RPE) cells provides significant protection from oxidative stress-induced cell death, further highlighting the role of Cer in retinal degeneration 24 .…”

Elusive Roles of the Different Ceramidases in Human Health, Pathophysiology, and Tissue Regeneration

“…An age-dependent inhibition of ACDase leads to ceramide accumulation, an increase in oxidative stress, and the death of retinal cells and erythrocytes [ 27 , 28 ]. By contrast, it was reported that kidney cells collected from aged mice show an elevated expression of Asah1 mRNA compared to that of young mice [ 29 ].…”