Delyfer Marie-Noëlle, Simonutti Manuel, Neveux Nathalie, Léveillard Thierry, Sahel José-Alain
Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, Institut National de la Santé et de la Recherche Médicale (Inserm U592), Université Pierre et Marie Curie, Paris, France.
Mol Vis. 2005 Sep 1;11:677-87.
We previously demonstrated that exogenous glial cell line-derived neurotrophic factor (GDNF) induces histological and functional protection of photoreceptors in the retinal degeneration (rd1) mouse model. The mechanisms underlying such neuroprotection remain elusive. In parallel to this work, we provided evidence for the occurrence of glutamate-mediated excitotoxic phenomena contributing to rod photoreceptor death in the rd1 retina in the companion paper. In the present study, we investigated whether, as demonstrated in other models, GDNF could exert its neuroprotective effect on photoreceptors through Müller glial cells (MGC) by promoting the expression of the glial L-glutamate/L-aspartate transporter (GLAST), an endogenous neuroprotective mechanism against glutamate-mediated excitotoxicity.
Reverse transcription-polymerase chain reaction (RT-PCR) was used to compare the mRNA expression levels of GDNF receptors between rd1 and wild-type mouse retinas as well as between MGC and mixed retinal cell cultures. Recombinant GDNF was applied to pure MGC cultures, to rd1 retinal organ cultures and injected subretinally into rd1 mouse eyes. GLAST expression following GDNF treatment was measured by RT-PCR, immunoblotting and immunohistochemistry. Free glutamate and glutamine levels were quantified in rd1 retinas after GDNF or control treatment using an amino acid analyzer.
mRNA expression studies of GDNF receptors, GFRalpha-1 and Ret, demonstrated that GDNF receptors were not exclusively expressed by the degenerating photoreceptor cells but mainly by MGC. Exogenous GDNF application to MGC cultures, rd1 mouse retinal explants and in vivo rd1 mouse retinas increased the expression of GLAST by 48% in retinal explants (p<0.005) and by 25% in vivo (p<0.0005). GLAST protein expression in MGC was particularly increased around degenerative photoreceptors. Free glutamate and glutamine levels in the rd1 retina were not significantly modified by exogenous GDNF.
Our data suggest that, in the rd1 mouse retina, GDNF neuroprotective effect on photoreceptors can be mediated indirectly through the activation of MGC. We demonstrate that injection of recombinant GDNF enhances the expression of GLAST and more particularly around the degenerating photoreceptors. Since we failed to demonstrate that GDNF decreases free glutamate levels, we could not ascertain whether GDNF promoted photoreceptor-survival via an increase of glutamate uptake and, therefore, a change in glutamate distribution.
我们之前证明，外源性胶质细胞源性神经营养因子（GDNF）可诱导视网膜变性（rd1）小鼠模型中光感受器的组织学和功能保护。这种神经保护作用的潜在机制仍不清楚。在进行这项研究的同时，我们在同期发表的论文中提供了证据，证明谷氨酸介导的兴奋性毒性现象参与了rd1视网膜中视杆光感受器的死亡。在本研究中，我们调查了，如在其他模型中所证明的那样，GDNF是否可以通过促进胶质细胞L-谷氨酸/L-天冬氨酸转运体（GLAST）的表达，通过 Müller 胶质细胞（MGC）对光感受器发挥神经保护作用，GLAST是一种针对谷氨酸介导的兴奋性毒性的内源性神经保护机制。
采用逆转录-聚合酶链反应（RT-PCR）比较rd1和野生型小鼠视网膜之间以及MGC和混合视网膜细胞培养物之间GDNF受体的mRNA表达水平。将重组GDNF应用于纯MGC培养物、rd1视网膜器官培养物，并视网膜下注射到rd1小鼠眼中。通过RT-PCR、免疫印迹和免疫组织化学检测GDNF处理后GLAST的表达。使用氨基酸分析仪对GDNF或对照处理后的rd1视网膜中的游离谷氨酸和谷氨酰胺水平进行定量。
对GDNF受体GFRalpha-1和Ret的mRNA表达研究表明，GDNF受体并非仅由退化的光感受器细胞表达，而是主要由MGC表达。将外源性GDNF应用于MGC培养物、rd1小鼠视网膜外植体和体内rd1小鼠视网膜，可使视网膜外植体中GLAST的表达增加48%（p<0.005），体内增加25%（p<0.0005）。MGC中GLAST蛋白表达在退化的光感受器周围尤其增加。外源性GDNF对rd1视网膜中的游离谷氨酸和谷氨酰胺水平没有显著影响。
我们的数据表明,在rd1小鼠视网膜中，GDNF对光感受器的神经保护作用可通过激活MGC间接介导。我们证明，注射重组GDNF可增强GLAST的表达，尤其是在退化的光感受器周围。由于我们未能证明GDNF可降低游离谷氨酸水平，因此无法确定GDNF是否通过增加谷氨酸摄取从而改变谷氨酸分布来促进光感受器存活。
