Retinal Metabolic Profile on IMPG2 Deficiency Mice with Subretinal Lesions.

The interphotoreceptor matrix (IPM) is the extracellular matrix between the photoreceptors and the retinal pigment epithelium (RPE). The IPM has two proteoglycans: the IPM proteoglycans 1 and 2 (IMPG1 and IMPG2, respectively). Patients with mutations on IMPG2 develop subretinal vitelliform lesions that affect vision. We previously created an IMPG2 knockout (KO) mice model that generates subretinal lesions similar to those found in humans. These subretinal lesions in IMPG2 KO mice retinas are, in part, composed of mislocalized IMPG1. In addition, IMPG2 KO mice show microscopic IMPG1 material accumulation between the RPE and the photoreceptor outer segments. In this work we discuss the possibility that material accumulation on IMPG2 KO mice retinas affects photoreceptor metabolism. To further investigate this idea, we used targeted metabolomics to profile retinal metabolome on IMPG2 KO mice. The metabolite set enrichment analysis showed reduced glutamate metabolism, urea cycle, and galactose metabolism suggesting affected energy metabolism in mice retinas of IMPG2 KO mice with subretinal lesion.

HSP90α is needed for the survival of rod photoreceptors and regulates the expression of rod PDE6 subunits.

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90ß. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2 months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6 months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90ß paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.

Interphotoreceptor matrix proteoglycans IMPG1 and IMPG2 proteolyze in the SEA domain and reveal localization mutual dependency.

The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of photoreceptor neurons. Interphotoreceptor matrix proteoglycan 1 and 2 (IMPG1 and IMPG2) are major components of the IPM. Both proteoglycans possess SEA (sperm protein, enterokinase and agrin) domains, which may support proteolysis. Interestingly, mutations in the SEA domains of IMPG1 and IMPG2 are associated with vision disease in humans. However, if SEA domains in IMPG molecules undergo proteolysis, and how this contributes to vision pathology is unknown. Therefore, we investigated SEA-mediated proteolysis of IMPG1 and IMPG2 and its significance to IPM physiology. Immunoblot analysis confirmed proteolysis of IMPG1 and IMPG2 in the retinas of wildtype mice. Point mutations mimicking human mutations in the SEA domain of IMPG1 that are associated with vision disease inhibited proteolysis. These findings demonstrate that proteolysis is part of the maturation of IMPG1 and IMPG2, in which deficits are associated with vision diseases. Further, immunohistochemical assays showed that proteolysis of IMPG2 generated two subunits, a membrane-attached peptide and an extracellular peptide. Notably, the extracellular portion of IMPG2 trafficked from the IPM around the inner segment toward the outer segment IPM by an IMPG1-dependent mechanism. This result provides the first evidence of a trafficking system that shuttles IMPG1 and IMPG2 from the inner to outer IPM in a co-dependent manner. In addition, these results suggest an interaction between IMPG1-IMPG2 and propose that mutations affecting one IMPG could affect the localization of the normal IMPG partner, contributing to the disease mechanism of vision diseases associated with defective IMPG molecules.

Proteoglycan IMPG2 Shapes the Interphotoreceptor Matrix and Modulates Vision.

Photoreceptor neurons are surrounded by an extracellular matrix, called the interphotoreceptor matrix (IPM). Activities crucial to vision occur within the IPM, including trafficking of nutrients and metabolites, retinal attachment, and interactions needed for normal outer segment phagocytosis. The IPM includes the following two unique proteoglycans: IPM proteoglycan 1 (IMPG1) and IMPG2. Patients with mutations in IMPG1/IMPG2 develop visual deficits with subretinal material accumulation, highlighting the critical role of the IPM in vision. To determine the role of these proteoglycans in retinal physiology and the pathologic mechanisms that lead to vision loss, we generated mouse models lacking IMPG1/IMPG2. In normal retina, IMPG1 and IMPG2 occupy distinct IPM compartments, represent the main source of chondroitin sulfate and are fundamental for the constitution of the cone-specific glycocalyx stained by the PNA (peanut agglutinin) lectin marker. No evident morphologic or functional deficits were found in mice lacking IMPG1. In the absence of IMPG2, IMPG1 abnormally accumulated at the subretinal space need, likely leading to the formation of subretinal lesions and reduced visual function. Interestingly, mice lacking both IMPG1 and IMPG2, regardless of sex, showed normal retinal structure and function, demonstrating that the aberrant IMPG1 distribution is the main cause of the visual alterations observed in the absence of IMPG2. In conclusion, our results show the dependence of secreted proteoglycans such as IMPG1 on the extracellular environment to properly integrate into the matrix, demonstrate the role of IMPG2 in shaping the IPM, and shed light on the potential mechanisms leading to the development of subretinal lesions and vision loss.SIGNIFICANCE STATEMENT The photoreceptors are specialized neurons that drive phototransduction in the mammalian retina. These cells are organized and surrounded by an extracellular matrix, the interphotoreceptor matrix (IPM). Mutations in IPM proteoglycans are associated with blindness in humans. Our studies show that two specific proteoglycans of the IPM, IPM proteoglycan 1 (IMPG1) and IMPG2, form a dynamic structure with distinct localization and dependency. When IMPG2 is absent, IMPG1 cannot integrate into the IPM, leading to abnormal proteoglycan accumulation and visual deficits. This work adds a new layer of understanding to IPM physiology and describes the pathologic events following deficits in proteoglycans, providing novel possibilities for visual restoration in patients with IMPG-related pathologies.

ARL13B, a Joubert Syndrome-Associated Protein, Is Critical for Retinogenesis and Elaboration of Mouse Photoreceptor Outer Segments.

Mutations in the Joubert syndrome-associated small GTPase ARL13B are linked to photoreceptor impairment and vision loss. To determine the role of ARL13B in the development, function, and maintenance of ciliated photoreceptors, we generated a pan-retina knock-out (Six3-Cre) and a rod photoreceptor-specific inducible conditional knock-out (Pde6g-CreERT2) of ARL13B using murine models. Embryonic deletion of ARL13B led to defects in retinal development with reduced cell proliferation. In the absence of ARL13B, photoreceptors failed to develop outer segment (OS) membranous discs and axonemes, resulting in loss of function and rapid degeneration. Additionally, the majority of photoreceptor basal bodies did not dock properly at the apical edge of the inner segments. The removal of ARL13B in adult rod photoreceptor cells after maturation of OS resulted in loss of photoresponse and vesiculation in the OS. Before changes in photoresponse, removal of ARL13B led to mislocalization of rhodopsin, prenylated phosphodiesterase-6 (PDE6), and intraflagellar transport protein-88 (IFT88). Our findings show that ARL13B is required at multiple stages of retinogenesis, including early postnatal proliferation of retinal progenitor cells, development of photoreceptor cilia, and morphogenesis of photoreceptor OS discs regardless of sex. Last, our results establish a need for ARL13B in photoreceptor maintenance and protein trafficking.SIGNIFICANCE STATEMENT The normal development of photoreceptor cilia is essential to create functional, organized outer segments with stacked membrane discs that house the phototransduction proteins necessary for sight. Our study identifies a complex role for ARL13B, a small GTPase linked to Joubert syndrome and visual impairment, at various stages of photoreceptor development. Loss of ARL13B led to defects in retinal proliferation, altered placement of basal bodies crucial for components of the cilium (transition zone) to emanate, and absence of photoreceptor-stacked discs. These defects led to extinguished visual response and dysregulated protein trafficking. Our findings show the complex role ARL13B plays in photoreceptor development, viability, and function. Our study accounts for the severe retinal impairment observed in ARL13B-linked Joubert syndrome patients.

Phototransduction early steps model based on Beer-Lambert optical law.

The amount of available rhodopsin on the photoreceptor outer segment and its change over time is not considered in classic models of phototransduction. Thus, those models do not take into account the absorptance variation of the outer segment under different brightness conditions. The relationship between the light absorbed by a medium and its absorptance is well described by the Beer-Lambert law. This newly proposed model implements the absorptance variation phenomenon in a set of equations that admit photons per second as input and results in active rhodopsins per second as output. This study compares the classic model of phototransduction developed by Forti et al. (1989) to this new model by using different light stimuli to measure active rhodopsin and photocurrent. The results show a linear relationship between light stimulus and active rhodopsin in the Forti model and an exponential saturation in the new model. Further, photocurrent values have shown that the new model behaves equivalently to the experimental and theoretical data as published by Forti in dark-adapted rods, but fits significantly better under light-adapted conditions. The new model successfully introduced a physics optical law to the standard model of phototransduction adding a new processing layer that had not been mathematically implemented before. In addition, it describes the physiological concept of saturation and delivers outputs in concordance to input magnitudes.

Global and ocular hypothermic preconditioning protect the rat retina from ischemic damage.

Retinal ischemia could provoke blindness. At present, there is no effective treatment against retinal ischemic damage. Strong evidence supports that glutamate is implicated in retinal ischemic damage. We investigated whether a brief period of global or ocular hypothermia applied 24 h before ischemia (i.e. hypothermic preconditioning, HPC) protects the retina from ischemia/reperfusion damage, and the involvement of glutamate in the retinal protection induced by HPC. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. One day before ischemia, animals were submitted to global or ocular hypothermia (33°C and 32°C for 20 min, respectively) and fourteen days after ischemia, animals were subjected to electroretinography and histological analysis. Global or ocular HPC afforded significant functional (electroretinographic) protection in eyes exposed to ischemia/reperfusion injury. A marked alteration of the retinal structure and a decrease in retinal ganglion cell number were observed in ischemic retinas, whereas global or ocular HPC significantly preserved retinal structure and ganglion cell count. Three days after ischemia, a significant decrease in retinal glutamate uptake and glutamine synthetase activity was observed, whereas ocular HPC prevented the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate induced alterations in retinal function and histology which were significantly prevented by ocular HPC. These results support that global or ocular HPC significantly protected retinal function and histology from ischemia/reperfusion injury, probably through a glutamate-dependent mechanism.

Therapeutic efficacy of melatonin in reducing retinal damage in an experimental model of early type 2 diabetes in rats.

Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages and provokes significant retinal alterations. The aim of this work was to analyze the effect of melatonin on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water. Three weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25 mg/kg). One day or 3 wk after vehicle or STZ injection, animals were subcutaneously implanted with a pellet of melatonin. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12 wk of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Melatonin, which did not affect glucose metabolism in control or diabetic rats, prevented the decrease in the electroretinogram a-wave, b-wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels. In addition, melatonin prevented the decrease in retinal catalase activity. These results indicate that melatonin protected the retina from the alterations observed in an experimental model of DR associated with type 2 diabetes.

Ischemic conditioning protects the rat retina in an experimental model of early type 2 diabetes.

Diabetic retinopathy is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages, and provokes significant retinal alterations. We investigated the effect of ischemic conditioning on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water, and 3 weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25mg/kg). Retinal ischemia was induced by increasing intraocular pressure to 120 mm Hg for 5 min; this maneuver started 3 weeks after vehicle or STZ injection and was weekly repeated in one eye, while control eyes were submitted to a sham procedure. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12 weeks of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Brief ischemia pulses in one eye and a sham procedure in the contralateral eye did not affect glucose metabolism in control or diabetic rats. Ischemic pulses reduced the decrease in the electroretinogram a-wave, b-wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels observed in diabetic animals. In addition, ischemic conditioning prevented the decrease in retinal catalase activity induced by T2DM. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies to treat diabetic retinopathy associated with T2DM.

Retinal changes in an experimental model of early type 2 diabetes in rats characterized by non-fasting hyperglycemia.

Diabetic retinopathy is a leading cause of acquired blindness in young, but also in elder adults, mostly affected by type 2 diabetes mellitus (T2DM). The aim of this work was to develop an experimental model of early human T2DM in adult rats, and to analyze retinal functional, morphological, and biochemical changes arising during the early stages of the moderate metabolic derangement. For this purpose, animals were divided in four groups: adult male Wistar rats receiving: tap water and citrate buffer i.p. (group 1), tap water with 30% sucrose and citrate buffer i.p. (group 2), tap water and 25mg/kg i.p streptozotocin (STZ, group 3), or 30% sucrose and STZ (group 4). Fasting and postprandial glycemia, fructosamine and serum insulin levels were assessed. In addition, i.p. glucose and insulin tolerance tests were performed. Retinal function (electroretinogram, ERG) and morphology (optical microscopy), retinal nitric oxide synthase (NOS) activity (using (3)H-arginine), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), and TNFα levels (ELISA) were evaluated. At 6 and 12 weeks of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in most metabolic tests, as compared with the other groups. At 12 weeks of treatment, a significant decrease in the ERG a- and b- wave and oscillatory potential amplitudes, and a significant increase in retinal NOS activity, TBARS, TNFα, glial fibrillary acidic protein in Müller cells, and vascular endothelial growth factor levels were observed. These results indicate that the combination of diet-induced insulin resistance and a slight secretory impairment resulting from a low-dose STZ treatment mimics some features of human T2DM at its initial stages, and provokes significant retinal alterations.

Effect of experimental glaucoma on the non-image forming visual system.

Glaucoma is a leading cause of blindness worldwide, characterized by retinal ganglion cell degeneration and damage to the optic nerve. We investigated the non-image forming visual system in an experimental model of glaucoma in rats induced by weekly injections of chondroitin sulphate (CS) in the eye anterior chamber. Animals were unilaterally or bilaterally injected with CS or vehicle for 6 or 10 weeks. In the retinas from eyes injected with CS, a similar decrease in melanopsin and Thy-1 levels was observed. CS injections induced a similar decrease in the number of melanopsin-containing cells and superior collicular retinal ganglion cells. Experimental glaucoma induced a significant decrease in the afferent pupil light reflex. White light significantly decreased nocturnal pineal melatonin content in control and glaucomatous animals, whereas blue light decreased this parameter in vehicle- but not in CS-injected animals. A significant decrease in light-induced c-Fos expression in the suprachiasmatic nuclei was observed in glaucomatous animals. General rhythmicity and gross entrainment appear to be conserved, but glaucomatous animals exhibited a delayed phase angle with respect to lights off and a significant increase in the percentage of diurnal activity. These results indicate the glaucoma induced significant alterations in the non-image forming visual system.

Effect of nitroxyl on the hamster retinal nitridergic pathway.

There is a growing body of evidence on the role of nitric oxide (NO) in retinal physiology. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO(-)), because it is formed by a number of diverse biochemical reactions. The aim of the present report was to comparatively analyze the effect of HNO and NO on the retinal nitridergic pathway in the golden hamster. For this purpose, sodium trioxodinitrate (Angeli's salt) and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) were used as HNO and NO releasers, respectively. Angeli's salt and DEA/NO significantly decreased nitric oxide synthase activity. In addition, Angeli's salt (but not DEA/NO) significantly decreased l-arginine uptake. DEA/NO significantly increased cGMP accumulation at low micromolar concentrations, while Angeli's salt affected this parameter with a threshold concentration of 200muM. Although Angeli's salt and DEA/NO significantly diminished reduced glutathione and protein thiol levels in a similar way, DEA/NO was significantly more effective than AS in increasing S-nitrosothiol levels. None of these compounds increased retinal lipid peroxidation. These results suggest that HNO could regulate the hamster retinal nitridergic pathway by acting through a mechanism that only partly overlaps with that involved in NO response.

Effect of neurosteroids on the retinal gabaergic system and electroretinographic activity in the golden hamster.

It has been established that neurosteroids can either inhibit or enhance GABA(A) receptor activity. Although GABA is the main inhibitory neurotransmitter in the mammalian retina, the effects of neurosteroids on retinal GABAergic activity have not been investigated. The aim of this work was to study the neurochemical and electroretinographic effects of neurosteroids in the golden hamster. On one hand, pregnenolone sulfate inhibited and allotetrahydrodeoxycorticosterone increased GABA-induced [36Cl]- uptake in neurosynaptosomes. On the other hand, in whole retinas, pregnenolone sulfate increased, whereas allotetrahydrodeoxycorticosterone decreased high potassium-induced [3H]GABA release. The effect of both neurosteroids on GABA release was Ca2+-dependent, as in its absence release was not altered. The intravitreal injection of pregnenolone sulfate or vigabatrin (an irreversible inhibitor of GABA degradation) significantly decreased scotopic b-wave amplitude, whereas the opposite effect was evident when bicuculline or allotetrahydrodeoxycorticosterone were injected. A protein with a molecular weight close to that of hamster adrenal cytochrome P450 side-chain cleavage (P450scc) was detected in the hamster retina. P450scc-like immunoreactivity was localized in the inner nuclear and the ganglion cell layers. These results indicate that neurosteroids significantly modulate retinal GABAergic neurotransmission and electroretinographic activity. In addition, the selective localization of P450scc suggests that neurosteroid biosynthesis might occur only in some layers of the hamster retina.