Raju V. S. Rajala

Involvement of Insulin/Phosphoinositide 3-Kinase/Akt Signal Pathway in 17β-Estradiol-mediated Neuroprotection

In the present study, we tested the hypothesis that 17β-estradiol (βE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H2O2)-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both βE2 and 17α-estradiol (αE2) significantly protected against H2O2-induced retinal neuron degeneration; however, progesterone had no effect. βE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [32γATP] were used as substrate. Phospho-Akt levels were also transiently increased by βE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of βE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of βE2, suggesting that βE2 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 βE2 and αE2, but not progesterone, identified phosphorylated insulin receptor β-subunit (IRβ) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRβ activation of PI3K. Systemic administration of βE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of βE2 activated retinal IRβ, 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.

Nonredundant Role of Akt2 for Neuroprotection of Rod Photoreceptor Cells from Light-Induced Cell Death

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.

Loss of Neuroprotective Survival Signal in Mice Lacking Insulin Receptor Gene in Rod Photoreceptor Cells

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. Rod-specific 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 light-induced 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.

Nanoparticle-assisted targeted delivery of eye-specific genes to eyes significantly improves the vision of blind mice in vivo

Application of viruses as a carrier, though not safe, to deliver genes to eye tissue was successful. However, a safer, nonviral, biocompatible lipid-based nanoparticle has never been tested to treat blinding eye diseases. We created an artificial virus using a nanoparticle, liposome-protamine-DNA complex (LPD), modified with a cell permeable peptide and a nuclear localization signaling (NLS) peptide, to deliver a functional gene for eye disease treatment. In the eye, a photochemical, 11-cis-retinal, allows the visual pigment rhodopsin to absorb light in the visible range. Without the photochemical, we lose the ability to see light. Retinal pigment epithelium protein 65 (Rpe65) is the key enzyme in regulating the availability of photochemical; deficiency of this gene results in a blinding eye disease. Here we show for the first time that LPD promotes efficient delivery in a cell specific-manner, and a long-term expression of Rpe65 gene to mice lacking Rpe65 gene, leading to in vivo correction of blindness. Thus, LPD nanoparticles could provide a promising, efficient, nonviral method of gene delivery with clinical applications in eye disease treatment.

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor.

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 α-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.

Corneal Cell Survival in Adenovirus Type 19 Infection Requires Phosphoinositide 3-Kinase/Akt Activation

ABSTRACT Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3β and nuclear translocation of the p65 subunit of NF-κB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

MicroRNA‐184 modulates canonical Wnt signaling through the regulation of frizzled‐7 expression in the retina with ischemia‐induced neovascularization

Aberrant activation of Wnt signaling contributes to ischemia‐induced retinal neovascularization in oxygen‐induced retinopathy (OIR), although the underlying mechanism is so far unclear. Here, we show that microRNA‐184 (miR‐184) is significantly down‐regulated in the retina of OIR mice, and miR‐184 negatively modulates Wnt signaling both in vivo and in vitro. Furthermore, we show that the Wnt receptor, frizzled‐7, is a downstream target of miR‐184, and delivery of miR‐184 mimic inhibits Wnt signaling in the OIR retina. These results suggest that decreased levels of miR‐184 are responsible, at least in part, for the aberrant activation of Wnt signaling in ischemia‐induced retinal neovascularization.

Insulin growth factor 1 receptor/PI3K/AKT survival pathway in outer segment membranes of rod photoreceptors.

PURPOSEnThe authors previously reported that physiological light induces the tyrosine phosphorylation of insulin receptors (IRs), which leads to the activation of the phosphoinositide 3-kinase (PI3K) and Akt (serine/threonine protein kinase B) survival pathway in rod photoreceptor cells. Tissue-specific deletion of IRs from photoreceptors resulted in stress-induced photoreceptor degeneration. Insulin growth factor 1 receptor (IGF-1R) is highly related in sequence and structure to the IR and shares 70% sequence identity overall and 84% identity within the tyrosine kinase domain. The role of IGF-1R in photoreceptor function is unknown. In this study the authors examined IGF-1R signaling in rod outer segment (ROS) membranes.nnnMETHODSnIGF-1R localization was examined in the plasma and disc membranes of ROS. Activation of the IGF-1R/PI3K/Akt pathway was analyzed using specific antibodies against phospho-tyrosine, IGF-1R, and phospho-Akt. PI3K activity was determined in the anti-phospho-tyrosine and anti-IGF-1R immunoprecipitates. Glutathione-S-transferase fusion proteins containing two Src homology 2 (SH2) domains of the p85 subunit of PI3K and their mutants were used to study the molecular interaction between IGF-1R and p85. In vivo IGF-1R signaling was studied in rats exposed to physiological light or to constant light.nnnRESULTSnIGF-1R is predominately localized to plasma membranes of ROS. These studies indicate that light stress results in an increase in tyrosine phosphorylation of IGF-1R and an increase in PI3K enzyme activity in anti-phosphotyrosine and anti-IGF-1R immunoprecipitates of ROS and retinal homogenates. The authors observed that light stress induces tyrosine phosphorylation of IGF-1R in ROS membranes, which leads to the binding of p85 through N-SH2 and C-SH2 domains. Finally, the authors observed a significant activation of Akt in light-stressed retinas, indicating activation of the Akt survival pathway downstream of IGF-1R activation.nnnCONCLUSIONSnLight stress induced the activation of PI3K through activation and binding of IGF-1R, which leads to activation of the Akt survival pathway in photoreceptors.

Deletion of the p85α Regulatory Subunit of Phosphoinositide 3-Kinase in Cone Photoreceptor Cells Results in Cone Photoreceptor Degeneration

PURPOSEnDownregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85α regulatory subunit.nnnMETHODSnMice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85α regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85α knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin-specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy.nnnRESULTSnAn age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85α cKO mice.nnnCONCLUSIONSnThe present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.

Activation and membrane binding of retinal protein kinase Bα/Akt1 is regulated through light‐dependent generation of phosphoinositides

Akt is a phospholipid‐binding protein and the downstream effector of the phosphoinositide 3‐kinase (PI3K) pathway. Akt has three isoforms: Akt1, Akt2, and Akt3. All of these isoforms are expressed in rod photoreceptor cells, but the individual functions of each isoform are not known. In this study, we found that light induces the activation of Akt1. The membrane binding of Akt1 to rod outer segments (ROS) is insulin receptor (IR)/PI3K‐dependent as demonstrated by reduced binding of Akt1 to ROS membranes of photoreceptor‐specific IR knockout mice. Membrane binding of Akt1 is mediated through its Pleckstrin homology (PH) domain. To determine whether binding of the PH domain of Akt1 to photoreceptor membranes is regulated by light, various green fluorescent protein (GFP)/Akt1‐PH domain fusion proteins were expressed in rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The R25C mutant PH domain of Akt1, which does not bind phosphoinositides, failed to associate with plasma membranes in a light‐dependent manner. This study suggests that light‐dependent generation of phosphoinositides regulates the activation and membrane binding of Akt1 in vivo. Our results also suggest that actin cytoskeletal organization may be regulated through light‐dependent generation of phosphoinositides.