Differential Developmental Deficits in Retinal Function in the Absence of either Protein Tyrosine Sulfotransferase-1 or -2

To investigate the role(s) of protein-tyrosine sulfation in the retina and to determine the differential role(s) of tyrosylprotein sulfotransferases (TPST) 1 and 2 in vision, retinal function and structure were examined in mice lacking TPST-1 or TPST-2. Despite the normal histologic retinal appearance in both Tpst1−/− and Tpst2−/− mice, retinal function was compromised during early development. However, Tpst1−/− retinas became electrophysiologically normal by postnatal day 90 while Tpst2−/− mice did not functionally normalize with age. Ultrastructurally, the absence of TPST-1 or TPST-2 caused minor reductions in neuronal plexus. These results demonstrate the functional importance of protein-tyrosine sulfation for proper development of the retina and suggest that the different phenotypes resulting from elimination of either TPST-1 or -2 may reflect differential expression patterns or levels of the enzymes. Furthermore, single knock-out mice of either TPST-1 or -2 did not phenocopy mice with double-knockout of both TPSTs, suggesting that the functions of the TPSTs are at least partially redundant, which points to the functional importance of these enzymes in the retina

Flavin Homeostasis in the Mouse Retina During Aging and Diabetes as a Marker for Retina Health

Nutritive and therapeutic intervention to restore the retina energy metabolism can lead to delayed onset and progression of blindness because of aging and type I and II diabetes.Biomedical Engineering, Department o

Modulation of SOD3 Levels Is Detrimental to Retinal Homeostasis

Retinal oxidative stress is a common secondary feature of many retinal diseases. Though it may not be the initial insult, it is a major contributor to the pathogenesis of highly prevalent retinal dystrophic diseases like macular degeneration, diabetic retinopathy, and retinitis pigmentosa. We explored the role of superoxide dismutase 3 (SOD3) in retinal homeostasis since SOD3 protects the extracellular matrix (ECM) from oxidative injury. We show that SOD3 is mainly extracellularly localized and is upregulated as a result of environmental and pathogenic stress. Ablation of SOD3 resulted in reduced functional electroretinographic responses and number of photoreceptors, which is exacerbated with age. By contrast, overexpression showed increased electroretinographic responses and increased number of photoreceptors at young ages, but appears deleterious as the animal ages, as determined from the associated functional decline. Our exploration shows that SOD3 is vital to retinal homeostasis but its levels are tightly regulated. This suggests that SOD3 augmentation to combat oxidative stress during retinal degenerative changes may only be effective in the short-term

Complement factor H, vitronectin, and opticin are tyrosine-sulfated proteins of the retinal pigment epithelium.

Lack of tyrosine sulfation of ocular proteins results in disorganized photoreceptor structure and drastically reduced visual function, demonstrating the importance of this post-translational modification to vision. To understand the role that tyrosine sulfation plays in the function of ocular proteins, we identified some tyrosine-sulfated proteins in the retinal pigment epithelium using two independent methods, immuno-affinity column purification with an anti-sulfotyrosine specific antibody and computer-based sequence analysis of retinal pigment epithelium secretome by means of the prediction program Sulfinator. Radioactive labeling followed by thin layer electrophoresis revealed that three proteins, vitronectin, opticin, and complement factor H (CFH), were post-translationally modified by tyrosine sulfation. The identification of vitronectin and CFH as tyrosine-sulfated proteins is significant, since both are deposited in drusen in the eyes of patients with age-related macular degeneration (AMD). Furthermore, mutations in CFH have been determined to be a major risk factor in the development of AMD. Future studies that seek to understand the role of CFH in the development of AMD should take into account the role that tyrosine sulfation plays in the interaction of this protein with its partners, and examine whether modulating sulfation provides a potential therapeutic target

The Intersection of Serine Metabolism and Cellular Dysfunction in Retinal Degeneration

In the past, the importance of serine to pathologic or physiologic anomalies was inadequately addressed. Omics research has significantly advanced in the last two decades, and metabolomic data of various tissues has finally brought serine metabolism to the forefront of metabolic research, primarily for its varied role throughout the central nervous system. The retina is one of the most complex neuronal tissues with a multitude of functions. Although recent studies have highlighted the importance of free serine and its derivatives to retinal homeostasis, currently few reviews exist that comprehensively analyze the topic. Here, we address this gap by emphasizing how and why the de novo production and demand for serine is exceptionally elevated in the retina. Many basic physiological functions of the retina require serine. Serine-derived sphingolipids and phosphatidylserine for phagocytosis by the retinal pigment epithelium (RPE) and neuronal crosstalk of the inner retina via D-serine require proper serine metabolism. Moreover, serine is involved in sphingolipid–ceramide balance for both the outer retina and the RPE and the reductive currency generation for the RPE via serine biosynthesis. Finally and perhaps the most vital part of serine metabolism is free radical scavenging in the entire retina via serine-derived scavengers like glycine and GSH. It is hard to imagine that a single tissue could have such a broad and extensive dependency on serine homeostasis. Any dysregulation in serine mechanisms can result in a wide spectrum of retinopathies. Therefore, most critically, this review provides a strong argument for the exploration of serine-based clinical interventions for retinal pathologies

Expression of the human usherin c.2299delG mutation leads to early-onset auditory loss and stereocilia disorganization

Abstract Usher syndrome (USH) is the leading cause of combined deafness and blindness, with USH2A being the most prevalent form. The mechanisms responsible for this debilitating sensory impairment remain unclear. This study focuses on characterizing the auditory phenotype in a mouse model expressing the c.2290delG mutation in usherin equivalent to human frameshift mutation c.2299delG. Previously we described how this model reproduces patient’s retinal phenotypes. Here, we present the cochlear phenotype, showing that the mutant usherin, is expressed during early postnatal stages. The c.2290delG mutation results in a truncated protein that is mislocalized within the cell body of the hair cells. The knock-in model also exhibits congenital hearing loss that remains consistent throughout the animal’s lifespan. Structurally, the stereocilia bundles, particularly in regions associated with functional hearing loss, are disorganized. Our findings shed light on the role of usherin in maintaining structural support, specifically in longer inner hair cell stereocilia, during development, which is crucial for proper bundle organization and hair cell function. Overall, we present a genetic mouse model with cochlear defects associated with the c.2290delG mutation, providing insights into the etiology of hearing loss and offering potential avenues for the development of effective therapeutic treatments for USH2A patients

PSG2-immunoaffinity column purification of tyrosine-sulfated proteins from cow RPE.

<p>(A). The elution profile was monitored by following absorbance at 280 nm. Following loading, the column was washed with buffers W1, W2, and W3. Elution was performed in buffer W3 containing 4 mM sulfated pentapeptide (EB). (B). Twenty-six microliter aliquots from input (IN), flow-through (FT), wash 1 (W1), and wash 2 (W2) were fractionated by SDS-PAGE, and proteins were visualized by staining with Coomassie blue dye. (C). Left, SDS-PAGE of 26 µL of wash 3 (W3) and eluted samples (EB) from the immunoaffinity column stained with Coomassie blue (CB) and right, immunoblotted with PSG2. Asterisks indicate the bands that were prominent on Coomassie blue-stained gel (CB) and were also recognized by PSG2 as tyrosine-sulfated.</p

Native human RPE vitronectin is tyrosine-sulfated.

<p>Vitronectin was immunoprecipitated from 500 µg human RPE lysates using anti-VTN antibody (lane 2) or mouse IgG (lane 3). Immunoprecipitants were fractionated by SDS-PAGE, transferred, and immunoblotted using anti-VTN antibody or anti-sulfotyrosine PSG2 antibody. Immunoprecipitation and western blots were repeated 3 independent times using biologically different human RPE samples. (B). Ectopically-expressed vitronectin is tyrosine-sulfated. Recombinant VTN or empty vector (pcDNA3.1) were transfected into HEK 293T cells and immunoprecipitated from conditioned media using anti-VTN antibody (lane 4) or mouse IgG (lane 3). Immunoprecipitants were electrophoresed and immunoblotted using anti-VTN antibody or anti-sulfotyrosine PSG2 antibody. Immunoprecipitation and western blots were repeated 3 independent times after independent VTN transfections. (C). ³⁵S-metabolic labeling of recombinant vitronectin <i>in vitro</i>. Vitronectin-transfectants were radiolabelled with ³⁵Sulfate. Following radiolabeling, vitronectin was immunoprecipitated and blots were either subjected to autoradiography (AR) or immunoblotted with anti-VTN antibody. (D). Radiolabeled vitronectin bands were excised from the membrane along with equivalent areas from mouse IgG immunoprecipitants, and alkaline hydrolysis was performed. The samples were then spiked with sulfo-amino standards tyrosine sulfate, threonine sulfate, and serine sulfate, and subjected to thin layer electrophoresis (TLE) on cellulose plates. Following TLE analysis, sulfo-amino standards (NHD) were visualized either by spraying with Ninhydrin or autoradiography (AR). TLE experiments were repeated at least three independent times.</p