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A study was conducted to examine the phototransduction functional significance of photoreceptor cyclic nucleotide-gated (CNG) channel associated with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The findings indicated that raft lipids play an important in modulation of the plasma membrane localization and functional activity of photoreceptor CNG channels.

The two-hybrid assay of protein-protein interaction in the yeast is used to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. The results reveal that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.

Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K+ affinity of the RPE-expressed α1-Na+/K+-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis. [ABSTRACT FROM AUTHOR]

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor β-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRβ) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate thea interaction between the p85 subunit of PI3K and the cytoplasmic region of IRβ. Under conditions where IRβ autophosphorylates, substitution of Y1322F and M1325P in IRβ resulted in the abolition of p85 binding to the IRβ, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRβ was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRβ (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRβ-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. [ABSTRACT FROM AUTHOR]

Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1−/−) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm's canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1−/− outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1−/− eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology. [ABSTRACT FROM AUTHOR]

The original concept that lipid and protein components are randomly distributed in cellular membranes has been challenged by evidence of compartmentalization of such components into discrete membrane microdomains (known as lipid rafts). The lipid microdomain hypothesis has generated significant controversy and rigorous inquiry to test the idea that such domains concentrate machinery to mediate cellular processes such as signaling, synaptic plasticity, and endocytosis. As such, a large number of studies have used biochemical, cell biological, and biophysical methodologies to define the composition of membrane microdomains in experimental contexts. Although biochemical preparation strategies are not without limitations (as discussed herein), the isolation of detergent-resistant and detergent-free membrane domains can provide important information about the segregation of lipids and proteins in membranes. In this chapter, we describe methodologies to isolate membranes from cell or tissue sources with biophysical/biochemical properties of membrane microdomains and also provide methods for subsequent classical or mass spectrometry-based lipid analytical approaches., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The immune-privileged environment and complex organization of retinal tissue support the retina's essential role in visual function, yet confound inquiries into cell-specific inflammatory effects that lead to dysfunction and degeneration. Caveolin-1 (Cav1) is an integral membrane protein expressed in several retinal cell types and is implicated in immune regulation. However, whether Cav1 promotes or inhibits inflammatory processes in the retina (as well as in other tissues) remains unclear. Previously, we showed that global-Cav1 depletion resulted in reduced retinal inflammatory cytokine production but paradoxically elevated retinal immune cell infiltration. We hypothesized that these disparate responses are the result of differential cell-specific Cav1 functions in the retina., Methods: We used Cre/lox technology to deplete Cav1 specifically in the neural retinal (NR) compartment to clarify the role NR-specific Cav1 (NR-Cav1) in the retinal immune response to intravitreal inflammatory challenge induced by activation of Toll-like receptor-4 (TLR4). We used multiplex protein suspension array and flow cytometry to evaluate innate immune activation. Additionally, we used bioinformatics assessment of differentially expressed membrane-associated proteins to infer relationships between NR-Cav1 and immune response pathways., Results: NR-Cav1 depletion, which primarily affects Müller glia Cav1 expression, significantly altered immune response pathway regulators, decreased retinal inflammatory cytokine production, and reduced retinal immune cell infiltration in response to LPS-stimulated inflammatory induction., Conclusions: Cav1 expression in the NR compartment promotes the innate TLR4-mediated retinal tissue immune response. Additionally, we have identified novel potential immune modulators differentially expressed with NR-Cav1 depletion. This study further clarifies the role of NR-Cav1 in retinal inflammation.

Purpose: Polymorphisms at the caveolin-1/2 locus are associated with glaucoma and IOP risk and deletion of caveolin-1 (Cav1) in mice elevates IOP and reduces outflow facility. However, the specific location/cell type responsible for Cav1-dependent regulation of IOP is unclear. We hypothesized that endothelial Cav1 in the conventional outflow (CO) pathway regulate IOP via endothelial nitric oxide synthase (eNOS) signaling., Methods: We created a mouse with targeted deletion of Cav1 in endothelial cells (Cav1ΔEC) and evaluated IOP, outflow facility, outflow pathway distal vascular morphology, eNOS phosphorylation, and tyrosine nitration of iridocorneal angle tissues by Western blotting., Results: Endothelial deletion of Cav1 resulted in significantly elevated IOP versus wild-type mice but not a concomitant decrease in outflow facility. Endothelial Cav1 deficiency did not alter the trabecular meshwork or Schlemm's canal morphology, suggesting that the effects observed were not due to developmental deformities. Endothelial Cav1 deletion resulted in eNOS hyperactivity, modestly increased protein nitration, and significant enlargement of the drainage vessels distal to Schlemm's canal. L-Nitro-arginine methyl ester treatment reduced outflow in Cav1ΔEC but not wild-type mice and had no effect on the size of drainage vessels. Endothelin-1 treatment decrease the outflow and drainage vessel size in both wild-type and Cav1ΔEC mice., Conclusions: Our results suggest that hyperactive eNOS signaling in the CO pathway of both Cav1ΔEC and global Cav1 knockout mice results in chronic dilation of distal CO vessels and protein nitration, but that Cav1 expression in the trabecular meshwork is sufficient to rescue CO defects reported in global Cav1 knockout mice.

The compartmentalization of cellular membranes into discrete membrane microdomains (known as lipid rafts) challenged the original definition of membranes as containing randomly distributed lipid and protein components. The lipid microdomain hypothesis has generated significant controversy and rigorous inquiry based on the attractive idea that such domains concentrate machinery to mediate cellular events such as signaling and endocytosis. As such, numerous studies have used biochemical, cell biological, and biophysical methodologies to define the composition of such domains in a variety of experimental contexts. In this chapter, we describe methodologies to isolate membranes from cell or tissue sources with biophysical/biochemical properties of membrane microdomains that are amenable to subsequent classical or mass spectrometry-based lipid analytical approaches.

Purpose: Recent evidence suggests that ceramide metabolism plays an important role in retinal photoreceptor cell survival and apoptosis. The purpose of this study was to characterize sphingolipids in the retina with special emphasis on the very-long-chain-containing saturated (VLC-FA) and polyunsaturated (VLC-PUFA) fatty acid-containing species. The VLC-FAs and VLC-PUFAs are synthesized by the ELOVL4 protein, which is involved in human Stargardt's macular dystrophy type 3 (STGD3)., Methods: Total lipids were extracted from retina and other tissues, and different sphingolipid classes were isolated and purified using various combinations of liquid- and solid-phase separation. Purified sphingolipids were analyzed by high-performance thin layer chromatography (HPTLC), gas chromatography (GC), and GC-MS (GC-mass spectrometry)., Results: Nonsialylated sphingolipids (NSLs) comprised approximately 3.5% of total retinal lipids of which 70% was sphingomyelin. Ceramide and glycosylceramides (GCs) constituted

Rod and cone photoreceptor cyclic nucleotide-gated (CNG) channels play pivotal roles in phototransduction. This work investigates the functional significance of photoreceptor CNG channel association with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The primary subunits of cone and rod CNG channels, CNGA3 and CNGA1, respectively, were heterologously expressed in HEK 293 cells, and channel activity was determined by ratiometric measurement of [Ca (2+)] i in response to cyclic guanosine monophosphate (cGMP) stimulation. CNGA3 was found to be largely insoluble following Triton X-100 extraction and cofractionationed with biochemically isolated membrane domains enriched in caveolin-1. Cofractionation of both natively expressed CNGA3 and CNGB1 (the modulatory subunit of the rod CNG channel) with the low buoyant density, caveolin-1-enriched membranes was also confirmed in mouse retinas. The functional significance of this association was established by the observed negative effects of depletion of raft lipids on the channel activity. Treatment with the cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), significantly inhibited CNGA3 and CNGA1 activation in response to cGMP stimulation. MCD treatment lowered cellular cholesterol levels by approximately 45% without altering fatty acid composition, suggesting that the inhibition of channel activity by MCD treatment is not due to perturbation of other membrane lipids. Treatment with the sphingolipid biosynthesis inhibitor myriocin resulted in impaired activation and cytosolic redistribution of CNGA3, suggesting that the integrity of the membrane domains is critical for the channel cellular processing and plasma membrane localization. This study demonstrates the association of photoreceptor CNG channels with membrane domains enriched in raft lipids and indicates, for the first time, that raft lipids modulate the plasma membrane localization and functional activity of photoreceptor CNG channels.

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 beta-subunit of the insulin receptor (IRbeta) in ROS membranes, which leads to the association of PI3K enzyme activity with IRbeta. Retinas from light- or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IRbeta, 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 IRbeta and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IRbeta 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 IRbeta 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 stress-induced apoptosis.