Neuroprotective role of sphingolipid rheostat in excitotoxic retinal ganglion cell death

Abstract

The glutamate excitotoxicity has been suggested as a factor involved in the loss of retinal neuronal cells, including retinal ganglion cell (RGC), in various retinal degenerative diseases including ischemia-reperfusion injury, diabetic retinopathy, and glaucoma. Excitotoxic RGC death is caused not only by direct damage to RGCs but also by indirect damage due to the inflammation of retinal glial cells. Sphingosine 1-phosphate (S1P) and ceramides are bioactive sphingolipids which have been shown to possess important physiological roles in cellular survival and apoptosis, and the balance between S1P and ceramide, sphingolipid rheostat, has been suggested to be important for determining cellular fate. Therefore, we conducted the present study to clarify the neuroprotective role of sphingolipid rheostat in excitotoxic RGC death in vivo and in vitro. Acute RGC death was induced by intravitreal N-methyl-d-aspartate (NMDA) injection in the mouse. The mRNA expression of sphingosine kinase (SphK1/SphK2) was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of SphK1/2, S1P, S1P-receptor (S1PR), glial fibrillary acidic protein (GFAP), Iba1, and CD31 were examined by immunostaining. Retinal sphingolipids and ceramides were quantified by liquid chromatography with tandem mass spectrometry. The neuroprotective effect of the sphingosine kinase inhibitor (SKI) on RGC death was assessed by RGC count and Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Further, the in vitro effect of SKI was investigated using rat primary cultured RGCs and glial cells. In addition, MG5 cells and A1 cells, which were mouse microglia and astrocyte cell-line, were also used. The expression of cleaved-caspase-3, GFAP, and Iba1 in RGCs, primary glial cells, MG5 cells, and A1 cells was assessed by immunostaining. NMDA injection resulted in mRNA upregulation of SphK1; however, SphK2 was reduced in the mouse retina. SphKs, S1P, S1PR1, S1PR2, and GFAP expression increased in the early-stage NMDA group, whereas S1P and GFAP were higher in the late-stage NMDA + SKI group. In the NMDA group, S1P expression was lower whereas sphingosine, C20, C22, and C24 ceramides showed higher levels. The proportion of very-long-chain ceramide was elevated in the NMDA group but reduced in the NMDA + SKI group. SKI treatment significantly increased RGC survival in retinal wholemount analysis and decreased apoptosis in the ganglion cell layer and inner nuclear layer. In vitro, SKI suppressed excitotoxic RGC death, cleaved-caspase-3 expression, and activated glial cells. The findings in the present study provide the first evidence demonstrating the involvement of sphingolipid rheostat in the neuroprotection against excitotoxic RGC death. Therefore, regulation of sphingolipid rheostat might serve as a potential therapy for retinal degenerative disease.

Keywords: Ceramide; Glutamate excitotoxicity; RGC death; S1P; Sphingolipid rheostat; Sphingosine kinase inhibitor.