Purchase this article with an account.
Sandrine Joly, Caroline Bachem, Helena Cayzac, Vincent Pernet; The role of Sphingosine Kinase 1 in N-Methyl-D-Aspartate-induced excitotoxicity of mouse retinal neurons. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):36.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Sphingosine 1-phosphate (S1P) is a bioactive lipid involved in many pathological events. In the current project, we investigated the role of S1P-synthesizing enzymes, Sphingosine Kinases (SphK), in N-Methyl-D-Aspartate (NMDA)-mediated excitotoxicity in C57BL/6 adult mice, an injury model mimicking the effects of ischemia-reperfusion.
The expression of SphK1 and SphK2 were studied by Western blotting and immunohistochemistry. Expression changes of SphK1, SphK2, and those of S1P-degrading enzymes Sphingosine 1-phosphate lyase 1 (SGPL1), Sphingosine 1-phosphate Phosphatase 1 (SGPP1) were assessed by qRT-PCR at 3, 6, 12, 24 and 48 hours after injecting 2 μL of 20 mM of NMDA in the vitreous space. In intact mouse groups, SphK1 overexpression in retinal ganglion cells (RGCs) was induced with an adeno-associated virus serotype 2 (AAV2) containing SphK1 cDNA. Four and 8 weeks after AAV2 injections, the effects of SphK1 overexpression on retinal histology were observed by labeling RGCs and amacrine cells with antibodies directed against β3Tubulin, choline-acetyl transferase and calretinin.
By Western blotting, the expression of SphK1 appeared higher than that of SphK2 in retinal lysates, suggesting that SphK1 is the main enzyme producing S1P in the retinal tissue. By immunohistochemistry, SphK1 was detected throughout retinal layers, including those containing RGCs and amacrine cells. Six hours after NMDA administration in the eyeball, the mRNA level of SphK1 was up-regulated by 15 fold and sharply decreased at 12 hrs. By contrast, the level of SphK2 mRNA was weakly affected by NMDA. Sgpl1 mRNA steadily rose after NMDA injection and peaked at 24 hrs while the levels of Sgpp1 mRNA only showed a trend toward a decrease. In eyes treated with AAV2.SphK1, the immunohistochemical signal of SphK1 increased in RGC cell bodies, axons and in vesicular-like structures in the innermost region of the inner plexiform layer (IPL). Retinal cell layer measurements did not show structural changes due to SphK1 overexpression 4 weeks after AAV2 injection. However, the IPL thickness was reduced by ~50% after 8 weeks, indicating that sustained increase of SphK1, alone, can alter synapse maintenance in the retina.
Our results suggest that SphK1 up-regulation may contribute to retinal synapse degradation in pathological conditions such as those caused by NMDA-induced excitotoxicity.
This PDF is available to Subscribers Only