Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110 -3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the ␤-subunit of the insulin receptor (IR␤) in ROS membranes, which leads to the association of PI3K enzyme activity with IR␤. Retinas from light-or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IR␤, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P 2 as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IR␤ and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IR␤ immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IR␤ in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P 3 . We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stressinduced apoptosis.Many cell proliferation and cell survival pathways are initiated upon activation of tyrosine kinase receptors, which transduce their signals by recruiting a variety of cytoplasmic signaling proteins (1, 2). Many of the signaling proteins contain phosphotyrosine binding domains, Src homology 2 (SH2) 1 domains, and Src homology 3 (SH3) domains, which are involved in mediating protein-protein interactions (4, 5).The phosphotyrosine-dependent interaction between different phosphotyrosine binding and SH2 domain-containing proteins with activated receptors initiates cellular changes that take place in response to a wide range of extracellular signals (2).One of the SH2 domain-containing proteins, phosphoinositide 3-kinase (PI3K), consists of an ϳ85-kDa regulatory subunit (p85) and a ϳ110-kDa catalytic subunit (p110), the latter being responsible for the phosphorylation of phosphoinositides at the D3 position and of serine residues in proteins (7, 8). The p85 subunit contains an SH3 domain capable of binding to proline-rich sequences, a p110 binding domain, and two SH2 domains. PI3K was initially found to be associated with middle-T/pp60c-Src (9), pp60v-Src, and platelet-derived growth factor receptors in both normal and transformed NIH3T3 fibroblast cells (10, 11). PI3K activity increases in response to platelet-derived growth factor binding to its receptor, in large part because the p85-p110 complex is translocated from the cytosol to the plasma membrane, by the direct binding of the p85 SH2 domain to tyro...

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, but the role of IR activity in vivo is unknown. We previously reported that light causes increased tyrosine phosphorylation of the IR in vivo, which leads to the downstream activation of the phosphoinositide 3-kinase and Akt pathway in rod photoreceptor cells. The functional role of IR in rod photoreceptor cells is not known. We observed that light stress induced tyrosine phosphorylation of the IR in rod photoreceptor cells, and we hypothesized that IR activation is neuroprotective. To determine whether IR has a neuroprotective role on rod photoreceptor cells, we used the Cre/lox system to specifically inactivate the IR gene in rod photoreceptors. Rodspecific IR knock-out mice have reduced the phosphoinositide 3-kinase and Akt survival signal in rod photoreceptors. The resultant mice exhibited no detectable phenotype when they were raised in dim cyclic light. However, reduced IR expression in rod photoreceptors significantly decreased retinal function and caused the loss of photoreceptors in mice exposed to bright light stress. These results indicate that reduced expression of IR in rod photoreceptor cells increases their susceptibility to lightinduced photoreceptor degeneration. These data suggest that the IR pathway is important for photoreceptor survival and that activation of the IR may be an essential element of photoreceptor neuroprotection. Insulin receptor (IR)2 signaling provides a trophic signal for transformed retinal neurons in culture (1), but the role of the IR in vivo is unknown. IR activation has been shown to rescue retinal neurons from apoptosis through a phosphoinositide 3-kinase (PI3K) cascade (1). We previously reported that light induces tyrosine phosphorylation of the retinal IR and that this activation leads to the binding of PI3K to rod outer segment (ROS) membranes (2). More recently, we demonstrated that IR activation is mediated through the G-protein-coupled receptor rhodopsin (3). IR signaling is also involved in 17␤-estradiolmediated neuroprotection in the retina (4). Recent evidence suggests a down-regulation of IR kinase activity in diabetic retinopathy that is associated with the deregulation of downstream signaling molecules (5). Deletion of several downstream effector molecules of the IR signaling pathway, such as IRS-2 (6), Akt2 (7), and Bcl-xl (8), in the retina resulted in a photoreceptor degeneration phenotype. These studies clearly indicate the importance of the IR signaling pathway in the retina.The IR is highly conserved, and the high degree of IR signaling homology between Caenorhabditis elegans, Drosophila, and humans suggests functional conservation in mammalian retina. The IR regulates neuronal survival in C. elegans (9). In Drosophila, the IR serves an important function to guide retinal photoreceptor axons from the retina to the brain during development (10), and the IR influences the size and number of photoreceptors (11). The lack of IR activation leads to neurod...

In the present study, we tested the hypothesis that 17␤-estradiol (␤E2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H 2 O 2 )-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both ␤E2 and 17␣-estradiol (␣E2) significantly protected against H 2 O 2 -induced retinal neuron degeneration; however, progesterone had no effect. ␤E2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [32 ␥ATP] were used as substrate. Phospho-Akt levels were also transiently increased by ␤E2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of ␤E2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of ␤E2, suggesting that ␤E2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that ␤E2 and ␣E2, but not progesterone, identified phosphorylated insulin receptor ␤-subunit (IR␤) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IR␤ activation of PI3K. Systemic administration of ␤E2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of ␤E2 activated retinal IR␤, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.

The Akt kinases mediate cell survival through phosphorylation and inactivation of apoptotic machinery components. Akt signaling provides a trophic signal for transformed retinal neurons in culture, but the in vivo role of Akt activity is unknown. In this study, we found that all three Akt isoforms were expressed in rod photoreceptor cells. We investigated the functional roles of Akt1 and Akt2, two of the isoforms of Akt, and their biological significance in light-induced retinal degeneration. Consistent with the hypothesis that Akt activity is important to circumvent stress-induced apoptosis, herein we report the novel finding that rod photoreceptor cells in Akt2 knock-out mice exhibited a significantly greater sensitivity to stress-induced cell death than rods in heterozygous or wild-type mice. Under similar conditions, Akt1 deletion had no effect on the retina. The presence of three Akt isoforms in the retina is suggestive of a functional redundancy; however, our studies clearly demonstrate that, under stress, Akt1 and Akt3 cannot complement the specific survival signals driven by Akt2. Furthermore, we show that Akt2 is specially activated is response to light stress. The results presented in this study provide the first direct evidence that Akt2 has a nonredundant neuroprotective role in photoreceptor survival and maintenance.

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319 -43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific ␣-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65 ؊/؊ mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction. Insulin receptor (IR)2 activation has been shown to rescue retinal neurons from apoptosis through a phosphoinositide 3-kinase (PI3K) cascade (1). Particular insights have come from observations that IR substrate-2 (principle substrate of the IR) knock-out mice lose up to 50% of their photoreceptors by 2 weeks of age, due to increased apoptosis (2). Very recently, we have shown that Ak2, another downstream target of the IR, is essential for photoreceptor survival and maintenance, since ablation of this protein in the retina resulted in stress-induced retinal degeneration (3). Specific deletion of bcl-xl (downstream effector of Akt) from rod photoreceptor cells also results in stress-induced retinal degeneration (4). These studies clearly suggest that the IR pathway is important for photoreceptor survival and maintenance.Many retinal degenerative diseases show an early loss of rod cells that is followed by loss of cone cells, and the pathological phenotype for this loss is apoptosis (5-7). The induction of cell death is a highly regulated process and can be suppressed by a variety of extracellular signals (8, 9). The ability of trophic factors to promote survival is mediated, at least in part, by PI3K (1, 10 -12). Activation of PI3K is a critical step in signal transdu...

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that is expressed in cancer cells. Its role in tumor metabolism is not definitively established, but investigators have suggested that regulation of PKM2 activity can cause accumulation of glycolytic intermediates and increase flux through the pentose phosphate pathway. Recent evidence suggests that PKM2 also may have non-metabolic functions, including as a transcriptional co-activator in gene regulation. We reported previously that PKM2 is abundant in photoreceptor cells in mouse retinas. In the present study, we conditionally deleted PKM2 (rod-cre PKM2-KO) in rod photoreceptors and found that the absence of PKM2 causes increased expression of PKM1 in rods. Analysis of metabolic flux from U-13C glucose shows that rod-cre PKM2-KO retinas accumulate glycolytic intermediates, consistent with an overall reduction in the amount of pyruvate kinase activity. Rod-cre PKM2-KO mice also have an increased NADPH availability could favor lipid synthesis, but we found no difference in phospholipid synthesis between rod-cre PKM2 KO and PKM2-positive controls. As rod-cre PKM2-KO mice aged, we observed a significant loss of rod function, reduced thickness of the photoreceptor outer segment layer, and reduced expression of photoreceptor proteins, including PDE6β. The rod-cre PKM2-KO retinas showed greater TUNEL staining than wild-type retinas, indicating a slow retinal degeneration. In vitro analysis showed that PKM2 can regulate transcriptional activity from the PDE6β promoter in vitro. Our findings indicate that both the metabolic and transcriptional regulatory functions of PKM2 may contribute to photoreceptor structure, function, and viability.

Fenofibrate, a specific agonist of peroxisome proliferator–activated receptor-α (PPARα), displays robust therapeutic effects on diabetic retinopathy (DR) in patients with type 2 diabetes. Our recent studies have shown that PPARα is downregulated in the diabetic retina, which contributes to the pathogenesis of DR. However, the mechanism for diabetes-induced downregulation of PPARα remains unknown. We investigated the role of microRNA-21 (miR-21) in regulating PPARα in DR. miR-21 was overexpressed, while PPARα levels were decreased in the retina of db/db mice, a model of type 2 diabetes. Such alterations were also observed in palmitate-treated retinal endothelial cells. miR-21 targeted PPARα by inhibiting its mRNA translation. Knockout of miR-21 prevented the decrease of PPARα, alleviated microvascular damage, ameliorated inflammation, and reduced cell apoptosis in the retina of db/db mice. Intravitreal injection of miR-21 inhibitor attenuated PPARα downregulation and ameliorated retinal inflammation in db/db mice. Further, retinal miR-21 levels were increased, while PPARα levels were decreased in oxygen-induced retinopathy (OIR). Knockout of miR-21 prevented PPARα downregulation and ameliorated retinal neovascularization and inflammation in OIR retinas. In conclusion, diabetes-induced overexpression of miR-21 in the retina is at least partly responsible for PPARα downregulation in DR. Targeting miR-21 may represent a novel therapeutic strategy for DR.

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.