Here, we confirmed that in the intact retina, light stress activated the lysosomal-mediated autophagic pathway and triggered the formation of AVs as measured by the increased levels of lysozyme, cathepsin S, and LAMP2 (
Fig. 1), the increased expression of LC3 mRNA (
Fig. 1), and the lipidated forms of LC3 and LC3II (
Fig. 2), respectively. However, based on these readouts, only macroautophagy, but not microautophagy and CMA, could be augmented by rapamycin treatment. To test whether, under light stress conditions, rapamycin treatment might contribute to photoreceptor stability, photoreceptor survival and function were tested after 10 days of light damage in the presence or absence of daily injections of rapamycin. Rapamycin was found to improve rod photoreceptor survival and function (
Figs. 3A,
3B) compared with vehicle-treated animals; yet rod output, as determined by b-wave analysis, was unaffected (
Fig. 3C). On the other hand, though cone survival was unaffected by either light damage or rapamycin treatment, animals treated with rapamycin had significantly reduced cone photoreceptor cell–mediated retinal responses, when tested with UV, green, and white light test flashes (
Fig. 5). Image analysis revealed that GFP-LC3–positive AVs are localized to cones, but not rods (
Figs. 6,
7). Thus, the effects of rapamycin on rods appear to be macroautophagy independent, whereas in cones, macroautophagy might play an essential role in altering the cone-mediated ERG response. Cone b-wave amplitudes might be reduced by autophagy, participating in the shortening of the cone outer segments and by removing the light-sensitive pigment (photostasis). Alternatively, autophagy-dependent or -independent changes in signaling at the cone-bipolar cell synapse might result in reduced cone b-wave amplitudes. Similar autophagy-independent pathways might be responsible for the lack of an improvement in rod b-wave amplitudes in light of the increased rod responses. On a separate note, the differential effects on structure and function (i.e., improved or no effect on rod and cone survival vs. the lack of an effect or a negative effect on rod and cone b-wave amplitudes, respectively) highlights the importance of investigating both aspects of a cell when testing therapeutic compounds. It has been suggested that rapamycin, acting through the mTOR pathway, prevents or reduces neurodegeneration in a number of neurologic diseases. Thus far, inhibition or activation of the mTOR pathway has been used in two different models of retinal degeneration. Kaushal
47 has shown that P23H rhodopsin, a misfolding mutant that is retained in the endoplasmic reticulum, can be successfully removed by increasing autophagy in a cellular expression system, suggesting that this mechanism might be useful in clearing these misfolded proteins associated with retinal degeneration in vivo. On the other hand, the induction of the insulin/mTOR pathway and, hence, the reduction of autophagy was found to delay cone cell death in a mouse model of retinitis pigmentosa, presumably by reducing starvation in the remaining cones.
48 Here, in light damage, the mTOR pathway might be involved in increasing AVs in cones, thus aiding in the removal of damaged cellular components. It is unclear why AVs could not be documented in rods, as previously shown by Reme et al.
11 in the rat, but the difference might include the experimental paradigm (a light intensity switch during the day vs. constant light) and the timing (1–3 days after the light stimulus onset vs. 10 days). Rapamycin has been shown in muscle cells to cause a metabolic shift from glucose use to fatty acid oxidation,
49 reducing glycolysis by approximately 40%. Although we found that light damage reduced the expression of 6-phosphofructokinase, the rate-limiting protein for glycolysis,
16 rapamycin did not further affect 6-PFK mRNA levels (data not shown), making it unlikely that the rapamycin effects occurred through changes in metabolism, although this possibility cannot be excluded. Finally, rapamycin is not a single-target molecule. It has been shown that rapamycin activates a number of autophagy-independent pathways. Rapamycin has been used to inhibit VEGF expression and activity,
26 to affect wound healing,
27 to act as a general immunosuppressant,
27,28 and to decrease protein synthesis by inhibiting translation.
29,30 As mentioned above, blocking VEGF activity has been shown to provide neuroprotection in light damage (Bemelmans A et al.
IOVS. 2007; 48:ARVO E-Abstract 1692), and VEGF has been shown to be proapoptotic in the presence of increased TGF-β.
31 Increased levels of VEGF and TGF-β in light damage that could be eliminated or blunted by rapamycin suggest that the effect of rapamycin on rod survival is dependent on this autophagy-independent pathway. Finally, because rapamycin is administered systemically, protective effects produced by autophagy-independent mechanisms on other cells in the eye, or even elsewhere in the body, cannot be excluded.