Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.

The interplay of environmental luminance and genetics in the retinal dystrophy induced by the dominant RPE65 mutation.

SignificanceIn humans, genetic mutations in the retinal pigment epithelium (RPE) 65 are associated with blinding diseases, for which there is no effective therapy alleviating progressive retinal degeneration in affected patients. Our findings uncovered that the increased free opsin caused by enhancing the ambient light intensity increased retinal activation, and when compounded with the RPE visual cycle dysfunction caused by the heterozygous D477G mutation and aggregation, led to the onset of retinal degeneration.

IMPDH1 retinal variants control filament architecture to tune allosteric regulation.

Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.

ALDH2/SIRT1 Contributes to Type 1 and Type 2 Diabetes-Induced Retinopathy through Depressing Oxidative Stress.

Clinical observations found vision-threatening diabetic retinopathy (DR) occurs in both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) patients, but T1DM may perform more progressive retinal abnormalities at the same diabetic duration with or without clinical retinopathy. In the present study, T1DM and T2DM patients without manifestations of DR were included in our preliminary clinical retrospective observation study to investigate the differentiated retinal function at the preclinical stage. Then, T1DM and T2DM rat models with 12-week diabetic duration were constructed to explore the potential mechanism of the discrepancy in retinal disorders. Our data demonstrated T1DM patients presented a poor retinal function, a higher allele frequency for ALDH2GA/AA, and a depressed aldehyde dehydrogenase 2 (ALDH2) activity and silent information regulator 1 (SIRT1) level, compared to T2DM individuals. In line with this, higher amplitudes of neurovascular function-related waves of electroretinograms were found in T2DM rats. Furthermore, the retinal outer nuclear layers were reduced in T1DM rats. The levels of retinal oxidative stress biomarkers including total reactive oxygen species, NADPH oxidase 4 and mitochondrial DNA damage, and inflammatory indicators covering inducible/endothelial nitric acid synthase ratio, interleukin-1, and interleukin-6 were obviously elevated. Notably, the level of retinal ALDH2 and SIRT1 in T1DM rats was significantly diminished, while the expression of neovascularization factors was dramatically enhanced compared to T2DM. Together, our data indicated that the ALDH2/SIRT1 deficiency resulted in prominent oxidative stress and was in association with DR progression. Moreover, a differentiating ALDH2/SIRT1 expression may be responsible for the dissimilar severity of DR pathological processes in chronic inflammatory-related T1DM and T2DM.

Glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase inhibition in the presence of pro-inflammatory cytokines contribute to the metabolic imbalance of diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1ß and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.

Doxycycline Ameliorates the Severity of Experimental Proliferative Vitreoretinopathy in Mice.

After successful surgeries for patients with rhegmatogenous retinal detachment, the most common cause of retinal redetachment is proliferative vitreoretinopathy (PVR), which causes severe vision impairment and even blindness worldwide. Until now, the major treatment for PVR is surgical removal of the epiretinal membrane, while effective treatment to prevent PVR is still unavailable. Therefore, we investigated the potential of doxycycline, an antibiotic in the tetracycline class, to treat PVR using a mouse model. We used the human retinal pigment epithelial cell line, ARPE-19, for in vitro and in vivo studies to test doxycycline for PVR treatment. We found that doxycycline suppressed the migration, proliferation, and contraction of ARPE-19 cells with reduced p38 MAPK activation and total MMP activity. Intravitreal doxycycline and topical tetracycline treatment significantly ameliorated the PVR severity induced by ARPE-19 cells in mice. PVR increased the expression of MMP-9 and IL-4 and p38 MAPK phosphorylation and modestly decreased IL-10. These effects were reversed by doxycycline and tetracycline treatment in the mouse retina. These results suggest that doxycycline will be a potential treatment for PVR in the future.

A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase.

Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 µs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.

The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis.

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12-/- (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, ß and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Expression of the SARS-CoV-2 Receptor ACE2 in Human Retina and Diabetes-Implications for Retinopathy.

Purpose: To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. Methods: Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. Results: We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. Conclusions: Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.

GD3 synthase deletion alters retinal structure and impairs visual function in mice.

Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.

Correcting QUEST Magnetic Resonance Imaging-Sensitive Free Radical Production in the Outer Retina In Vivo Does Not Correct Reduced Visual Performance in 24-Month-Old C57BL/6J Mice.

Purpose: To test the hypothesis that acutely correcting a sustained presence of outer retina free radicals measured in vivo in 24-month-old mice corrects their reduced visual performance. Methods: Male C57BL/6J mice two and 24 months old were noninvasively evaluated for unremitted production of paramagnetic free radicals based on whether 1/T1 in retinal laminae are reduced after acute antioxidant administration (QUEnch-assiSTed [QUEST] magnetic resonance imaging [MRI]). Superoxide production was measured in freshly excised retina (lucigenin assay). Combining acute antioxidant administration with optical coherence tomography (i.e., QUEST OCT) tested for excessive free radical-induced shrinkage of the subretinal space volume. Combining antioxidant administration with optokinetic tracking tested for a contribution of uncontrolled free radical production to cone-based visual performance declines. Results: At two months, antioxidants had no effect on 1/T1 in vivo in any retinal layer. At 24 months, antioxidants reduced 1/T1 only in superior outer retina. No age-related change in retinal superoxide production was measured ex vivo, suggesting that free radical species other than superoxide contributed to the positive QUEST MRI signal at 24 months. Also, subretinal space volume did not show evidence for age-related shrinkage and was unresponsive to antioxidants. Finally, visual performance declined with age and was not restored by antioxidants that were effective per QUEST MRI. Conclusions: An ongoing uncontrolled production of outer retina free radicals as measured in vivo in 24 mo C57BL/6J mice appears to be insufficient to explain reductions in visual performance.

ßA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity.

ßA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as ßA3 and ßA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that ßA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of ßA3/A1-crystallin in metabolism of retinal astrocytes. We found that ßA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but ßA3-crystallin does not. Loss of ßA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited ßA1-knockdown (KD) mice, but not in ßA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified ßA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced ßA1-crystallin and higher levels of PTP1B in the vitreous humor.

An animal model for mitochondrial tyrosyl-tRNA synthetase deficiency reveals links between oxidative phosphorylation and retinal function.

Mitochondria maintain a distinct pool of ribosomal machinery, including tRNAs and tRNAs activating enzymes, such as mitochondrial tyrosyl-tRNA synthetase (YARS2). Mutations in YARS2, which typically lead to the impairment of mitochondrial protein synthesis, have been linked to an array of human diseases including optic neuropathy. However, the lack of YARS2 mutation animal model makes us difficult to elucidate the pathophysiology underlying YARS2 deficiency. To explore this system, we generated YARS2 knockout (KO) HeLa cells and zebrafish using CRISPR/Cas9 technology. We observed the aberrant tRNATyr aminoacylation overall and reductions in the levels in mitochondrion- and nucleus-encoding subunits of oxidative phosphorylation system (OXPHOS), which were especially pronounced effects in the subunits of complex I and complex IV. These deficiencies manifested the decreased levels of intact supercomplexes overall. Immunoprecipitation assays showed that YARS2 bound to specific subunits of complex I and complex IV, suggesting the posttranslational stabilization of OXPHOS. Furthermore, YARS2 ablation caused defects in the stability and activities of OXPHOS complexes. These biochemical defects could be rescued by the overexpression of YARS2 cDNA in the YARS2KO cells. In zebrafish, the yars2KO larva conferred deficient COX activities in the retina, abnormal mitochondrial morphology, and numbers in the photoreceptor and retinal ganglion cells. The zebrafish further exhibited the retinal defects affecting both rods and cones. Vision defects in yars2KO zebrafish recapitulated the clinical phenotypes in the optic neuropathy patients carrying the YARS2 mutations. Our findings highlighted the critical role of YARS2 in the stability and activity of OXPHOS and its pathological consequence in vision impairments.

Apocynin ameliorates NADPH oxidase 4 (NOX4) induced oxidative damage in the hypoxic human retinal Müller cells and diabetic rat retina.

NADPH oxidase (NOX) is a main producers of reactive oxygen species (ROS) that may contribute to the early pathogenesis of diabetic retinopathy (DR). ROS has harmful effects on endogenous neuro-survival factors brain-derived neurotrophic factor (BDNF) and sirtuin 1 (SIRT1) are necessary for the growth and survival of the retina. The role of NOX isoforms NOX4 in triggering ROS in DR is not clear. Here we determine the protective effects of a plant-derived NOX inhibitor apocynin (APO) on NOX4-induced ROS production which may contribute to the depletion of survival factors BDNF/SIRT1 or cell death in the diabetic retinas. Human retinal Müller glial cells (MGCs) were treated with hypoxia mimetic agent cobalt chloride (CoCl2) in the absence or presence of APO. Molecular analysis demonstrates that NOX4 is upregulated in CoCl2-treated MGCs and in the diabetic retinas. Increased NOX4 was accompanied by the downregulation of BDNF/SIRT1 expression or in the activation of apoptotic marker caspase-3. Whereas, APO treatment downregulates NOX4 and subsequently upregulates BDNF/SIRT1 or alleviate caspase-3 expression. Accordingly, in the diabetic retina we found a positive correlation in NOX4 vs ROS (p = 0.025; R2 = 0.488) and caspase-3 vs ROS (p = 0.04; R2 = 0.428); whereas a negative correlation in BDNF vs ROS (p = 0.009; R2 = 0.596) and SIRT1 vs ROS (p = 0.0003; R2 = 0.817) respectively. Taken together, NOX4-derived ROS could be a main contributor in downregulating BDNF/SIRT1 expression or in the activation of caspase-3. Whereas, APO treatment may minimize the deleterious effects occurring due to hyperglycemia and/or diabetic mimic hypoxic condition in early pathogenesis of DR.

A Comprehensive Study of the Retinal Phenotype of Rpe65-Deficient Dogs.

The Rpe65-deficient dog has been important for development of translational therapies of Leber congenital amaurosis type 2 (LCA2). The purpose of this study was to provide a comprehensive report of the natural history of retinal changes in this dog model. Rpe65-deficient dogs from 2 months to 10 years of age were assessed by fundus imaging, electroretinography (ERG) and vision testing (VT). Changes in retinal layer thickness were assessed by optical coherence tomography and on plastic retinal sections. ERG showed marked loss of retinal sensitivity, with amplitudes declining with age. Retinal thinning initially developed in the area centralis, with a slower thinning of the outer retina in other areas starting with the inferior retina. VT showed that dogs of all ages performed well in bright light, while at lower light levels they were blind. Retinal pigment epithelial (RPE) inclusions developed and in younger dogs and increased in size with age. The loss of photoreceptors was mirrored by a decline in ERG amplitudes. The slow degeneration meant that sufficient photoreceptors, albeit very desensitized, remained to allow for residual bright light vision in older dogs. This study shows the natural history of the Rpe65-deficient dog model of LCA2.

Hydrogen sulfide is synthesized endogenously in both retinal artery and retina mostly via CSE.

Hydrogen sulfide (H2S) is an important gasotransmitter expressed in various tissues of the organism, including the eye. It is known that H2S is localized especially in the retina and corneal layers in bovine eye. The enzymes that mediate H2S synthesis are 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). Herein, we aimed to investigate the concentration levels and distribution profiles of these enzymes in bovine retina and retinal artery. Enzyme levels were measured by ELISA and distribution were determined by immunofluorescence microscopic analysis. Much higher concentrations of CBS and CSE have been detected in the retinal artery compared to the retina. In both tissues, particulary 3-MST was found at the lowest level while, CSE was determined to be the most abundant enzyme among the others. CBS distribution was shown in both endothelial and smooth muscle layers, while CSE was seen especially in the endothelial layer of the retinal artery. In the retina, CBS and CSE were expressed in cone-basil cells and retinal ganglion cells, while CSE was also present in bipolar cells. Our results indicated that H2S is synthesized endogenously in ocular tissues. The widespread expression of H2S synthesizing enzymes in the retina and retinal artery of the bovine eye, which has anatomical similarities with the human eye, may suggest a protective role for H2S against retinal vascular diseases as well as a regulatory role in the retinal vascular tone.

Fluorescent detection of PARP activity in unfixed tissue.

Poly-ADP-ribose-polymerase (PARP) relates to a family of enzymes that can detect DNA breaks and initiate DNA repair. While this activity is generally seen as promoting cell survival, PARP enzymes are also known to be involved in cell death in numerous pathologies, including in inherited retinal degeneration. This ambiguous role of PARP makes it attractive to have a simple and fast enzyme activity assay, that allows resolving its enzymatic activity in situ, in individual cells, within complex tissues. A previously published two-step PARP activity assay uses biotinylated NAD+ and streptavidin labelling for this purpose. Here, we used the fluorescent NAD+ analogues ε-NAD+ and 6-Fluo-10-NAD+ to assess PARP activity directly on unfixed tissue sections obtained from wild-type and retinal degeneration-1 (rd1) mutant retina. In standard UV microscopy ε-NAD+ incubation did not reveal PARP specific signal. In contrast, 6-Fluo-10-NAD+ resulted in reliable detection of in situ PARP activity in rd1 retina, especially in the degenerating photoreceptor cells. When the 6-Fluo-10-NAD+ based PARP activity assay was performed in the presence of the PARP specific inhibitor olaparib, the activity signal was completely abolished, attesting to the specificity of the assay. The incubation of live organotypic retinal explant cultures with 6-Fluo-10-NAD+, did not produce PARP specific signal, indicating that the fluorescent marker may not be sufficiently membrane-permeable to label living cells. In summary, we present a new, rapid, and simple to use fluorescence assay for the cellular resolution of PARP activity on unfixed tissue, for instance in complex neuronal tissues such as the retina.

Deficit of human ornithine aminotransferase in gyrate atrophy: Molecular, cellular, and clinical aspects.

Gyrate Atrophy (GA) of the choroid and retina (MIM# 258870) is an autosomal recessive disorder due to mutations of the OAT gene encoding ornithine-delta-aminotransferase (OAT), associated with progressive retinal deterioration and blindness. The disease has a theoretical global incidence of approximately 1:1,500,000. OAT is mainly involved in ornithine catabolism in adults, thus explaining the hyperornithinemia as hallmark of the disease. Patients are treated with an arginine-restricted diet, to limit ornithine load, or the administration of Vitamin B6, a precursor of the OAT coenzyme pyridoxal phosphate. Although the clinical and genetic aspects of GA are known for many years, the enzymatic phenotype of pathogenic variants and their response to Vitamin B6, as well as the molecular mechanisms explaining retinal damage, are poorly clarified. Herein, we provide an overview of the current knowledge on the biochemical properties of human OAT and on the molecular, cellular, and clinical aspects of GA.

Purification of a Src family tyrosine protein kinase from bovine retinas.

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

Spatio-temporal expression profile of Mettl3/Mettl14 during mouse retina development / Perfil de expresión espacio-temporal de Mettl3 / Mettl14 durante el desarrollo de la retina del ratón

SUMMARY: The Mettl3/Mettl14 methyltransferase complex installs the most ubiquitous internal mRNA modification- N6-methyladenosine (m6A). The vertebrate retina development is a multi-step process that requires fine-tuning of multiple cellular events, but very little is known about the potential function of Mettl3 and Mettl14 in this process. In this study, we demonstrated the spatio-temporal expression of Mettl3 and Mettl14 during retina development in mouse by quantitative PCR and immunofluorescence staining. We found that these two components of methyltransferase complex could be detected from the beginning of retina development; and the expression of Mettl3 and Mettl14 were gradually restricted to inner nuclear layer (INL) and ganglion cell layer (GCL); Double labeling showed that Mettl3 and Mettl14 had similar expression patterns in mature retinal INL and GCL. Overall, our spatio-temporal expression data provided the foundation for future research on the function of m6A modification in the retina development.

RESUMEN: El complejo Mettl3 / Mettl14 metiltransferasa establece la modificación interna más significativa de ARNm: N6- metiladenosina (m6A). El desarrollo de la retina de los vertebrados es un proceso de varios pasos que requiere múltiples eventos celulares; existe muy poca información sobre la función potencial de Mettl3 y Mettl14 en este proceso. En este estudio, demostramos la expresión espacio-temporal de Mettl3 y Mettl14 durante el desarrollo de la retina en ratón mediante PCR cuantitativa y tinción de inmunofluorescencia. Descubrimos que estos dos componentes del complejo de metiltransferasa podían ser detectados desde el comienzo del desarrollo de la retina; la expresión de Mettl3 y Mettl14 se restringió gradualmente a la capa nuclear interna (INL) y la capa de células ganglionares (GCL); se observó que Mettl3 y Mettl14 tenían patrones de expresión similares en INL y GCL retinianos maduros. En general, nuestros datos de expresión espacio-temporal proporcionan información para futuras investigaciones sobre la función de la modificación de m6A en el desarrollo de la retina.