Abstract
Purpose: :
Establishing the molecular cues controlling proliferation, survival and development of retina photoreceptors (PhRs) is essential for treating retina neurodegeneration. We have shown that sphingosine-1-phosphate (S1P), which regulates survival in several cell systems as an extracellular and intracellular messenger, promoted PhR survival (ARVO 2007). We now investigated whether S1P stimulates PhR differentiation and proliferation and if docosahexaenoic acid (DHA), which promotes PhR survival and differentiation, and glial derived neurotrophic factor (GDNF), which stimulates proliferation, require S1P synthesis for their effects.
Methods: :
pure neuronal cultures from rat retina were treated with S1P, [3H]sphingosine, DHA or GDNF, with the sphingosine kinase (SphK) inhibitor DHS to inhibit S1P synthesis, with Brefeldin A (BFA) and with BML-241, a S1P membrane receptor antagonist. Opsin, peripherin and SphK levels were quantitated by immunocytochemistry and Western blot; bromodeoxyuridine (BrdU) uptake and the amount of mitotic figures were determined to quantitate proliferation; apoptosis was analyzed by TUNEL labeling. S1P synthesis was determined by measuring the amounts of [3H]S1P.
Results: :
S1P prevented apoptosis and promoted differentiation of PhRs, stimulating opsin and peripherin expression and development of apical processes; this development was inhibited with BFA, which blocks trafficking. S1P also increased proliferation in PhR progenitors. DHA and GDNF increased SphK levels in PhRs, which led to DHA stimulation of [3H]S1P synthesis in PhRs. Blocking this synthesis with DHS inhibited DHA or GDNF effects on PhR survival and differentiation or proliferation, respectively. The S1P receptor antagonist blocked S1P protection but not that of DHA.
Conclusions: :
S1P promotes survival, proliferation and differentiation of PhRs by acting on membrane receptors. DHA and GDNF upregulate SphK levels to synthesize S1P and this synthesis is essential for their stimulatory effects. We propose that at different times during development GDNF and DHA promote synthesis of S1P, which then acts as a crucial mediator controlling the amount and appropriate development of PhRs.
Keywords: photoreceptors • cell survival • retinal development