Does GDNF exert its neuroprotective effects on photoreceptors in the rd1 retina through the glial glutamate transporter GLAST?
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Delyfer
Marie-Noëlle
MN
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. marie-noelle.delyfer@libertysurf.fr
Simonutti
Manuel
M
Neveux
Nathalie
N
Léveillard
Thierry
T
Sahel
José-Alain
JA
D000818:Animals; D004195:Disease Models, Animal; D027341:Excitatory Amino Acid Transporter 1 / Q000378:metabolism*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology*; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009924:Organ Culture Techniques; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012162:Retinal Degeneration / Q000378:metabolism / Q000517:prevention & control*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D027341:Excitatory Amino Acid Transporter 1; C495040:Gdnf protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D018696:Neuroprotective Agents; D012333:RNA, Messenger; C493393:Slc1a3 protein, mouse; D005973:Glutamine; D018698:Glutamic Acid