Here we report that cones show signs of nutritional imbalance during the period of cone degeneration in RP mice. The microarray analysis suggested that there are changes in cellular metabolism involving the insulin/mTOR pathway at the onset of cone death. We showed that inhibition of mTOR in wild type mice resulted in the same pattern of loss of red/green opsin as seen during degeneration. In accord with changes in p*-mTOR, and its sensitivity to glucose, we saw an upregulation of HIF-1α and GLUT1, suggesting that glucose uptake, and/or the intracellular levels of glucose, may be compromised in cones of RP mice. Additionally, systemic administration of insulin prolonged cone survival, whereas depletion of endogenous insulin had 7the reverse effect. The systemic treatment with insulin prevented the upregulation of HIF-1α in cones seen normally during cone degeneration, suggesting that insulin was directly acting on cones. Interestingly, a prolonged treatment of insulin during a time span of 7 weeks instead of 4 weeks did not show any significant improvement of cone survival (see Supplementary Fig. 10
online). This may reflect the feedback loop of the pathway in which S6K1 acts directly onto the insulin-receptor substrate (IRS). While the cross talk in cones between the insulin receptor, mTOR, HIF-1α, S6K1 and IRS remains to be investigated, the results strengthen the notion that nutrient availability in cones may be altered during the period of cone degeneration and that the insulin/mTOR pathway plays a crucial role. A recent report showed that constitutive expression of proinsulin in the rd10 mouse model of RP delays photoreceptor death, both of rods and cones37
. However, proinsulin seems not to act through the insulin receptor as mice treated with proinsulin did not develop hyperglycemia. Proinsulin blocks developmental cell death and thus may interfere with the apoptotic pathway in the postnatal retina. Although downstream effectors, such as PI3K/Akt, are regulated signaling by insulin and proinsulin, the relationship between the results of the studies reported here on insulin and the proinsulin effects remains to be determined.
Macroautophagy, which is controlled by mTOR through its downstream target S6K1, was not detected during cone degeneration, while CMA appeared to be activated. Increased LAMP-2A levels at the lysosomal membrane are suggestive of an activation of CMA. In addition, the observations concerning mTOR, HIF-1α, and GLUT1 are consistent with starvation and CMA. However, additional experiments are needed to prove that starvation and CMA are indeed occurring, and further, to prove that they are an important contributor to cone death. The lack of detectable macroautophagy does not rule out the possibility that macroautophagy might occur for a short period of time (e.g. 24 hours) prior to the activation of CMA. The data only show that macroautophagy is not the main form of autophagy over an extended period of time, which is consistent with the notion that macroautophagy is a short-term response. The prolonged inhibition of macroautophagy is likely due to increased S6K1 activity as seen by increased p*-S6 levels. S6K1 is positively regulated by mTOR and AMP-activated protein kinase (AMPK)28
, which reads out cellular ATP levels. Therefore, while mTOR may report metabolic problems with respect to glucose uptake, and reduce energy consuming processes and improve glycolysis through HIF-1α, AMPK may report normal cellular ATP levels and inhibit macroautophagy. This may represent a specific response to the energy requirements of cones. Most of the glucose taken up by PRs never enters the Krebs cycle38
. Thus the shortage of glucose may not cause a shortage of ATP. Lactate, provided by Muller glia, can generate ATP via the Krebs cycle39
. However, glucose is needed to generate NADPH in the pentose phosphate cycle, and NADPH is required for synthesis of phospholipids, the building blocks of cell membranes. PRs constantly shed their membranes at the tip of the OSs. Since reduced levels of glucose would result in reduction of membrane synthesis, the rate of OS phagocytosis by the RPE may be higher than the rate of membrane synthesis by cones. Consistent with this, OS shortening preceded cell death in these 4 models, as is also observed in human cases of RP17
. Additionally, changes that affect lipid metabolism were also seen by the microarray analysis.
Why does the loss of rods result in cone death in RP? The previous hypotheses mentioned in the Introduction have in common that they can’t fully explain the pathology found in humans. The rod and cone death kinetics shown here clearly argue against a toxin produced by dying rods as a cause for cone death since the onset of cone death always occurred after the major rod death period. If a rod toxin caused cone death, then the onset of cone death should have either coincided with the onset of rod death or should have started shortly thereafter, since this would be the period of peak toxin production. Interestingly, the lack of a trophic factor produced by healthy rods and required for cone survival would agree with the onset of cone death seen in all four models as one would expect the onset of cone death during the end stages of rod death. However, the progression of cone death and the end phase of rod death make this unlikely hypothesis as the sole reason for cone death. In the two PDE mutants and in the Rho-KO mutant, cones were dying for many weeks after the end phase of rod death, suggesting that they could survive quite awhile in the absence of rods. In addition, in the P23H model, rods died so slowly during the end phase of rod death, that during the entire period of cone death, rods were still present. The hypothesis that a lack of a rod trophic factor being the main cause for cone death seems unlikely given these discrepancies. However, the present investigation cannot rule out the lack of rod trophic factor given that the 4 mouse models used are in 4 different genetic backgrounds. Background differences could result in differences, such as different levels of such a factor, which might account for the observed differences in the progression of cone death.
How could our observations of nutritionally deprived cones explain the dependence of cones on rods? The OS-RPE interactions are vital since the RPE shuttles nutrition and oxygen from the choroidal vascaulture to PRs. Roughly 95% of all PRs in mouse and human are rods and approximately 20–30 OSs contact one RPE cell40,41
. Thus, only 1–2 of those RPE-OS contacts are via cones. During the collapse of the ONL, the remaining cone:RPE interactions are likely perturbed. If these interactions drop below a threshold required for the proper flow of nutrients, the loss of rods might result in a reduced flow of nutrients to cones. In all 4 mouse models, the onset of cone death occurred when the ONL reached one row of cells. This cell density could therefore represent the critical threshold. Then, while the remaining rods die due to a mutation in a rod-specific gene, cone death may begin due to nutrient deprivation. In accord with this notion, cone death progressed more slowly when the remaining rods died slowly. This mechanism would also explain why the loss of cones does not lead to rod death42,43
. Since in humans and mouse, cones are less than 5% of all PRs, the critical threshold that perturbs OS-RPE interactions would not be reached. Further support for this idea is provided by studies in zebrafish where the overall ratio of rods to cones is reversed (1:8). Additionally, the distribution of rods and cones in zebrafish is uneven such that certain regions are cone-rich whereas other regions are rod-rich. A recently isolated mutation in a cone-specific gene resulted in rod death, but only in regions of high cone density44
, leading Stearns and co-workers to conclude that cell density is the crucial determinant. We propose that once a critical threshold of cell density is breached, improper OS-RPE interactions result in reduced flow of nutrients (e.g. glucose). This results in reduced OS membrane synthesis, which in turn further contributes to a reduced uptake of nutrients from the RPE. Ultimately, prolonged starvation, as suggested by the activation of CMA, leads to cell death. Since starvation can occur slowly over extended periods of time, and because the rate may fluctuate due to fluctuations in nutrient uptake, the slow and irregular demise of cones observed in humans may be expected. Therefore, this study not only provides a new mechanism of cone death in RP that should direct future therapeutic approaches, but also consolidates the data from the literature with respect to the death kinetics of rods and cones seen in mice and patients with different RP mutations.