mTORC1 regulates high levels of protein synthesis in retinal ganglion cells of adult mice.

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.

Signaling roles of phosphoinositides in the retina.

The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (also known as phosphatidylinositol phosphates or PIPs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of phosphoinositide kinases and phosphoinositide phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule, phosphatidylinositol. PIP signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane budding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIPs in general, in this review, we discuss recent studies and advances in PIP lipid signaling in the retina. We specifically focus on PIP lipids from vertebrate (e.g., bovine, rat, mouse, toad, and zebrafish) and invertebrate (e.g., Drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIPs revealed from animal models and human diseases, and methods to study PIP levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PIP-modifying enzymes/phosphatases and further unravel PIP regulation and function in the different cell types of the retina.

mTORC1 and mTORC2 expression in inner retinal neurons and glial cells.

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.

A non-canonical rhodopsin-mediated insulin receptor signaling pathway in retinal photoreceptor neurons.

We previously reported a ligand-independent and rhodopsin-dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein-protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep™ density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep™ is a non-ionic iodixanol-based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.

Loss of human disease protein retinitis pigmentosa GTPase regulator (RPGR) differentially affects rod or cone-enriched retina.

It is unclear how genes, such as RPGR (retinitis pigmentosa guanine triphosphatase regulator) that are expressed in both rods and cones, cause variable disease pathogenesis. Using transcriptomic analysis, we show that loss of RPGR in a rod-dominant mouse retina (Rpgr(ko)) results in predominant alterations in genes involved in actin cytoskeletal dynamics, prior to onset of degeneration. We validated these findings and found an increase in activated RhoA-GTP levels and polymerized F-actin in the Rpgr(ko) mouse retina. To assess the effect of the loss of RPGR in the all-cone region of the human retina, we used Nrl(-/-) (neural retina leucine zipper) mice, to generate Rpgr(ko)::Nrl(-/-) double-knock-out (Rpgr-DKO) mice. These mice exhibited supranormal cone response to light and substantially retained retinal architecture. Transcriptomic analysis revealed predominant up-regulation of retinal pigmented epithelium (RPE)-specific genes associated with visual cycle, whereas fatty acid analysis showed mild decrease in docosahexaenoic acid in the retina of the Rpgr-DKO mice when compared with the Nrl(-/-) mice. Our data reveal new insights into distinct intracellular pathways that are involved in RPGR-associated rod and cone dysfunction and provide a platform to design new treatment modalities.

Redundant and Nonredundant Functions of Akt Isoforms in the Retina.

Serine/threonine kinase Akt is a downstream effector of the phosphoinositide 3-kinase pathway that is involved in many processes, including providing neuroprotection to stressed photoreceptor cells. Akt exists in three isoforms designated as Akt1, Akt2, and Akt3. All of these isoforms are expressed in the retina. We previously reported that Akt2 knockout mice were susceptible to light stress-induced photoreceptor degeneration, whereas Akt1 deletion had no effect on the retina. We hypothesized that the phenotype of Akt2 knockout mice may be due to the inactivation of specific substrate(s) in the retina. Yeast two-hybrid screening of a bovine retinal cDNA library with Akt2 identified a multidomain protein, POSH (plenty of SH3s), that acts as a scaffold for the JNK pathway of neuronal death. Our results suggest a stable interaction between Akt2 and POSH. Previous studies show that overexpression of POSH leads to cell death. The cell death that we observed in Akt2 knockout mice could be due to the absence of inactivation of POSH-mediated JNK signaling in the retina.

Nanoparticles as Delivery Vehicles for the Treatment of Retinal Degenerative Diseases.

Over the last few years, huge progress has been made in the understanding of molecular mechanisms underlying the pathogenesis of retinal degenerative diseases. Such knowledge has led to the development of gene therapy approaches to treat these devastating disorders. Non-viral gene delivery has been recognized as a prospective treatment for retinal degenerative diseases. In this review, we will summarize the constituent characteristics and recent applications of three representative nanoparticles (NPs) in ocular therapy.

Constitutive Activation Mutant mTOR Promote Cone Survival in Retinitis Pigmentosa Mice.

Studies form our laboratory and others show that the oncogenic tyrosine kinase and serine threonine kinase signaling pathways are essential for cone photoreceptor survival. These pathways are downregulated in mouse models of retinal degenerative diseases. In the present study, we found that activation mutants of mTOR delayed the death of cones in a mouse model of retinal degeneration. These studies suggest that oncogenic protein kinases may be useful as therapeutic agents to treat retinal degenerations that affect cones.

Impaired association of retinal degeneration-3 with guanylate cyclase-1 and guanylate cyclase-activating protein-1 leads to leber congenital amaurosis-1.

One-fifth of all cases of Leber congenital amaurosis are type 1 (LCA1). LCA1 is a severe form of retinal dystrophy caused by loss-of-function mutations in guanylate cyclase 1 (GC1), a key member of the phototransduction cascade involved in modulating the photocurrents. Although GC1 has been studied for some time, the mechanisms responsible for its regulation and membrane targeting are not fully understood. We reported earlier that retinal degeneration 3 (RD3) protein interacts with GC1 and promotes its targeting to the photoreceptor outer segments (POS). Here, we extend our studies to show a direct association between RD3 and guanylate cyclase activating protein 1 (GCAP1). Furthermore, we demonstrate that this functional interaction is important for GC1 targeting to POS. We also show that most LCA1-causing mutations in GC1 result in lost GC1 interaction with RD3 or GC1 being targeted to the plasma membrane. Our data suggest that GC1, GCAP1, and RD3 form a complex in the endoplasmic reticulum that targets GC1 to POS. Interruption of this assembly is likely the underlying mechanism for a subset of LCA1. This study offers insights for the development of therapeutic strategies to treat this severe form of blindness.

Photoreceptor Neuroprotection: Regulation of Akt Activation Through Serine/Threonine Phosphatases, PHLPP and PHLPPL.

Serine/threonine kinase Akt is a downstream effector of insulin receptor/PI3K pathway that is involved in many processes, including providing neuroprotection to stressed rod photoreceptor cells. Akt signaling is known to be regulated by the serine/threonine phosphatases, PHLPP (PH domain and leucine rich repeat protein phosphatase) and PHLPPL (PH domain and leucine rich repeat protein phosphatase-like). We previously reported that both phosphatases are expressed in the retina, as well as in photoreceptor cells. In this study, we examined the PHLPP and PHLPPL phosphatase activities towards non-physiological and physiological substrates. Our results suggest that PHLPP was more active than PHLPPL towards non-physiological substrates, whereas both PHLPP and PHLPP dephosphorylated the physiological substrates of Akt1 and Akt3 with similar efficiencies. Our results also suggest that knockdown of PHLPPL alone does not increase Akt phosphorylation, due to a compensatory increase of PHLPP, which results in the dephosphorylation of Akt. Therefore, PHLPP and PHLPPL regulate Akt activation together when both phosphatases are expressed.

Class I Phosphoinositide 3-Kinase Exerts a Differential Role on Cell Survival and Cell Trafficking in Retina.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides. They are responsible for coordinating a diverse range of cell functions including proliferation, cell survival, degranulation, vesicular trafficking, and cell migration. The PI 3-kinases are grouped into three distinct classes: I, II, and III. Class III PI3K has been shown to be involved in intracellular protein trafficking, whereas class I PI3K is known to regulate cell survival following activation of cell surface receptors. However, studies from our laboratory and others have shown that class I PI3K may also be involved in photoreceptor protein trafficking. Therefore, to learn more about the role of class I and class III P13K in trafficking and to understand the impact of the lipid content of trafficking cargo vesicles, we developed a methodology to isolate trafficking vesicles from retinal tissue. PI3K class I and III proteins were enriched in our extracted trafficking vesicle fraction. Moreover, levels of ether phosphatidylethanolamine (PE) and ether phosphatidylcholine (PC) were significantly higher in the trafficking vesicle fraction than in total retina. These two lipid classes have been suggested to be involved with fusion/targeting of trafficking vesicles.

Modulation of mouse rod photoreceptor responses by Grb14 protein.

Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14(-/-) mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τREC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14(-/-) mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τD, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized.

Non-canonical regulation of phosphatidylinositol 3-kinase gamma isoform activity in retinal rod photoreceptor cells.

BACKGROUND: Phosphatidylinositol 3-Kinases (PI3Ks) are a family of lipid kinases that phosphorylate the D3-hydroxyls of the inositol ring of phosphoinositides, and are responsible for coordinating a diverse range of cellular functions. A canonical pathway of activation of PI3Ks through the interaction of RA-domain with Ras proteins has been well established. In retinal photoreceptors, we have identified a non-canonical pathway of PI3Kγ activation through the interaction of its RA-domain with a putative Ras-like domain (RLD) in alpha subunit of cyclic nucleotide-gated channel (CNGA1) in retinal rod photoreceptors. RESULTS: The interaction between PI3Kγ and CNGA1 does not appear to play a role in regulation of CNG channel activity, but PI3Kγ uses CNGA1 as an anchoring module to achieve close proximity to its substrate to generate D3-phosphoinositides. CONCLUSIONS: Our studies suggest a functional non-canonical PI3Kγ activation in retinal rod photoreceptor cells.

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.

Insulin receptor signaling in cones.

In humans, age-related macular degeneration and diabetic retinopathy are the most common disorders affecting cones. In retinitis pigmentosa (RP), cone cell death precedes rod cell death. Systemic administration of insulin delays the death of cones in RP mouse models lacking rods. To date there are no studies on the insulin receptor signaling in cones; however, mRNA levels of IR signaling proteins are significantly higher in cone-dominant neural retina leucine zipper (Nrl) knock-out mouse retinas compared with wild type rod-dominant retinas. We previously reported that conditional deletion of the p85α subunit of phosphoinositide 3-kinase (PI3K) in cones resulted in age-related cone degeneration, and the phenotype was not rescued by healthy rods, raising the question of why cones are not protected by the rod-derived cone survival factors. Interestingly, systemic administration of insulin has been shown to delay the death of cones in mouse models of RP lacking rods. These observations led to the hypothesis that cones may have their own endogenous neuroprotective pathway, or rod-derived cone survival factors may be signaled through cone PI3K. To test this hypothesis we generated p85α(-/-)/Nrl(-/-) double knock-out mice and also rhodopsin mutant mice lacking p85α and examined the effect of the p85α subunit of PI3K on cone survival. We found that the rate of cone degeneration is significantly faster in both of these models compared with respective mice with competent p85α. These studies suggest that cones may have their own endogenous PI3K-mediated neuroprotective pathway in addition to the cone viability survival signals derived from rods.

Age-Related Changes in the Glycolytic Enzymes of M2-Isoform of Pyruvate Kinase and Fructose-1,6-Bisphosphate Aldolase: Implications to Age-Related Macular Degeneration.

Prior studies have emphasized a bioenergetic crisis in the retinal pigment epithelium (RPE) as a critical factor in the development of age-related macular degeneration (AMD). The isoforms Fructose-1,6-bisphosphate aldolase C (ALDOC) and pyruvate kinase M2 (PKM2) have been proposed to play a role in AMD pathogenesis. While PKM2 and ALDOC are crucial for aerobic glycolysis in the neural retina, they are not as essential for the RPE. In this study, we examined the expression and activity of PKM2 and ALDOC in both young and aged RPE cells, as well as in the retina and RPE tissue of mice, including an experimentally induced AMD mouse model. Our findings reveal an upregulation in PKM2 and ALDOC expression, accompanied by increased pyruvate kinase activity, in the aged and AMD mouse RPE. Conversely, there is a decrease in ALDOC expression but an increase in PKM2 expression and pyruvate kinase activity in the aged and AMD retina. Overall, our study indicates that aged and AMD RPE cells tend to favor aerobic glycolysis, while this tendency is diminished in the aged and AMD retina. These results underscore the significance of targeting PKM2 and ALDOC in the RPE as a promising therapeutic approach to address the bioenergetic crisis and prevent vision loss in AMD.

From Insight to Eyesight: Unveiling the Secrets of the Insulin-Like Growth Factor Axis in Retinal Health.

Insulin-like growth factor-1 (IGF-1) plays a diverse role in the retina, exerting its effects in both normal and diseased conditions. Deficiency of IGF-1 in humans leads to issues such as microcephaly, mental retardation, deafness, and postnatal growth failure. IGF-1 is produced in the retinal pigment epithelium (RPE) and activates the IGF-1 receptor (IGF-1R) in photoreceptor cells. When IGF-1R is absent in rod cells, it results in the degeneration of photoreceptors, emphasizing the neuroprotective function of IGF signaling in these cells. Contrastingly, in neovascular age-related macular degeneration (AMD), there is an overexpression of both IGF-1 and IGF-1R in RPE. The mechanisms behind this altered regulation of IGF-1 in diseased states are currently unknown. This comprehensive review provides recent insights into the role of IGF-1 in the health and disease of the retina, raising several unanswered questions that still need further investigation.

Age-related retinal degeneration resulting from the deletion of Shp2 tyrosine phosphatase in photoreceptor neurons.

Shp2, a critical SH2-domain-containing tyrosine phosphatase, is essential for cellular regulation and implicated in metabolic disruptions, obesity, diabetes, Noonan syndrome, LEOPARD syndrome, and cancers. This study focuses on Shp2 in rod photoreceptor cells, revealing its enrichment, particularly in rods. Deletion of Shp2 in rods leads to age-dependent photoreceptor degeneration. Shp2 targets occludin (OCLN), a tight junction protein, and its deletion reduces OCLN expression in the retina and retinal pigment epithelium (RPE). The isolation of actively translating mRNAs from rods lacking Shp2, followed by RNA sequencing, reveals alterations in cell cycle regulation. Additionally, altered retinal metabolism is observed in retinal cells lacking Shp2. Our studies indicate that Shp2 is crucial for maintaining the structure and function of photoreceptors.

Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation.

BACKGROUND: Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14? RESULTS: Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line. CONCLUSIONS: Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling.

Unlocking the role of lactate: metabolic pathways, signaling, and gene regulation in postmitotic retinal cells.

The Warburg effect, which was first described a century ago, asserts that mitotic tumor cells generate higher quantities of lactate. Intriguingly, even in typical physiological circumstances, postmitotic retinal photoreceptor cells also produce elevated levels of lactate. Initially classified as metabolic waste, lactate has since gained recognition as a significant intracellular signaling mediator and extracellular ligand. This current review endeavors to provide a concise overview and discourse on the following topics: the localization of lactate-producing enzymes, the functional significance of these enzymes, the signaling functions of lactate, and its impact on the gene expression of photoreceptors in retinal cells.