Preservation of vision after CaMKII-mediated protection of retinal ganglion cells.

Retinal ganglion cells (RGCs) are the sole output neurons that transmit visual information from the retina to the brain. Diverse insults and pathological states cause degeneration of RGC somas and axons leading to irreversible vision loss. A fundamental question is whether manipulation of a key regulator of RGC survival can protect RGCs from diverse insults and pathological states, and ultimately preserve vision. Here, we report that CaMKII-CREB signaling is compromised after excitotoxic injury to RGC somas or optic nerve injury to RGC axons, and reactivation of this pathway robustly protects RGCs from both injuries. CaMKII activity also promotes RGC survival in the normal retina. Further, reactivation of CaMKII protects RGCs in two glaucoma models where RGCs degenerate from elevated intraocular pressure or genetic deficiency. Last, CaMKII reactivation protects long-distance RGC axon projections in vivo and preserves visual function, from the retina to the visual cortex, and visually guided behavior.

Comparative proteomic study of retinal ganglion cells undergoing various types of cellular stressors.

Retinal ganglion cell (RGC) damage serves as a key indicator of various retinal degenerative diseases, including diabetic retinopathy (DR), glaucoma, retinal arterial and retinal vein occlusions, as well as inflammatory and traumatic optic neuropathies. Despite the growing body of data on the RGC proteomics associated with these conditions, there has been no dedicated study conducted to compare the molecular signaling pathways involved in the mechanism of neuronal cell death. Therefore, we launched the study using two different insults leading to RGC death: glutamate excitotoxicity and optic nerve crush (ONC). C57BL/6 mice were used for the study and underwent NMDA- and ONC-induced damage. Twenty-four hours after ONC and 1 h after NMDA injection, we collected RGCs using CD90.2 coupled magnetic beads, prepared protein extracts, and employed LC-MS for the global proteomic analysis of RGCs. Statistically significant changes in proteins were analyzed to identify changes to cellular signaling resulting from the treatment. We identified unique and common alterations in protein profiles in RGCs undergoing different types of cellular stresses. Our study not only identified both unique and shared proteomic changes but also laid the groundwork for the future development of a therapeutic platform for testing gene candidates for DR and glaucoma.

Development of TRIB3-Based Therapy as a Gene-Independent Approach to Treat Retinal Degenerative Disorders.

Inherited retinal degeneration (RD) constitutes a heterogeneous group of genetic retinal degenerative disorders. The molecular mechanisms underlying RD encompass a diverse spectrum of cellular signaling, with the unfolded protein response (UPR) identified as a common signaling pathway chronically activated in degenerating retinas. TRIB3 has been recognized as a key mediator of the PERK UPR arm, influencing various metabolic pathways, such as insulin signaling, lipid metabolism, and glucose homeostasis, by acting as an AKT pseudokinase that prevents the activation of the AKT → mTOR axis. This study aimed to develop a gene-independent approach targeting the UPR TRIB3 mediator previously tested by our group using a genetic approach in mice with RD. The goal was to validate a therapeutic approach targeting TRIB3 interactomes through the pharmacological targeting of EGFR-TRIB3 and delivering cell-penetrating peptides targeting TRIB3 → AKT. The study employed rd10 and P23H RHO mice, with afatinib treatment conducted in p15 rd10 mice through daily intraperitoneal injections. P15 P23H RHO mice received intraocular injections of cell-penetrating peptides twice at a 2-week interval. Our study revealed that both strategies successfully targeted TRIB3 interactomes, leading to an improvement in scotopic A- and B-wave ERG recordings. Additionally, the afatinib-treated mice manifested enhanced photopic ERG amplitudes accompanied by a delay in photoreceptor cell loss. The treated rd10 retinas also showed increased PDE6ß and RHO staining, along with an elevation in total PDE activity in the retinas. Consequently, our study demonstrated the feasibility of a gene-independent strategy to target common signaling in degenerating retinas by employing a TRIB3-based therapeutic approach that delays retinal function and photoreceptor cell loss in two RD models.

Chronic canaliculitis with canaliculoliths due to Providencia stuartii infection.

Canaliculitis, inflammation of the lacrimal canaliculi, can be caused by numerous pathogens, most commonly bacteria from the genera Actinomyces, Streptococcus, and Staphylococcus. Primary canaliculitis often requires surgical canaliculolith removal and appropriate antibiotic coverage. The authors report a case of a 77-year-old woman with a history of punctal plugs who presented with chronic canaliculitis with canaliculoliths that grew Providencia stuartii. P. stuartii has not previously been described as a cause of primary canaliculitis. This case highlights a new organism that causes canaliculitis with canaliculoliths and stresses the importance of speciation and antibiotic sensitivity testing following canaliculotomy and curettage. P. stuartii should be considered in the differential for bacterial canaliculitis with canaliculoliths, especially in patients with persistent symptoms on topical antibiotic therapy without canaliculotomy.

The endoplasmic reticulum: Homeostasis and crosstalk in retinal health and disease.

The endoplasmic reticulum (ER) is the largest intracellular organelle carrying out a broad range of important cellular functions including protein biosynthesis, folding, and trafficking, lipid and sterol biosynthesis, carbohydrate metabolism, and calcium storage and gated release. In addition, the ER makes close contact with multiple intracellular organelles such as mitochondria and the plasma membrane to actively regulate the biogenesis, remodeling, and function of these organelles. Therefore, maintaining a homeostatic and functional ER is critical for the survival and function of cells. This vital process is implemented through well-orchestrated signaling pathways of the unfolded protein response (UPR). The UPR is activated when misfolded or unfolded proteins accumulate in the ER, a condition known as ER stress, and functions to restore ER homeostasis thus promoting cell survival. However, prolonged activation or dysregulation of the UPR can lead to cell death and other detrimental events such as inflammation and oxidative stress; these processes are implicated in the pathogenesis of many human diseases including retinal disorders. In this review manuscript, we discuss the unique features of the ER and ER stress signaling in the retina and retinal neurons and describe recent advances in the research to uncover the role of ER stress signaling in neurodegenerative retinal diseases including age-related macular degeneration, inherited retinal degeneration, achromatopsia and cone diseases, and diabetic retinopathy. In some chapters, we highlight the complex interactions between the ER and other intracellular organelles focusing on mitochondria and illustrate how ER stress signaling regulates common cellular stress pathways such as autophagy. We also touch upon the integrated stress response in retinal degeneration and diabetic retinopathy. Finally, we provide an update on the current development of pharmacological agents targeting the UPR response and discuss some unresolved questions and knowledge gaps to be addressed by future research.

Adeno-associated virus mediated gene therapy for neuroprotection of retinal ganglion cells in glaucoma.

Glaucoma is a group of diseases typically characterized by the degeneration of the optic nerve and is one of the world's leading causes of blindness. Although there is no cure for glaucoma, reducing intraocular pressure is an approved treatment to delay optic nerve degeneration and retinal ganglion cell (RGC) death in most patients. Recent clinical trials have evaluated the safety and efficacy of gene therapy vectors for the treatment of inherited retinal degenerations (IRDs), and the results are promising, generating enthusiasm for the treatment of other retinal diseases. While there have been no reports on successful clinical trials for gene therapy-based neuroprotective treatment of glaucoma, and only a few studies assessing the efficacy of gene therapy vectors for the treatment of Leber hereditary optic neuropathy (LHON), the potential for neuroprotective treatment of glaucoma and other diseases affecting RGCs is still widely recognized. Here, we review recent progress and cover current limitations pertaining to targeting RGCs with adeno-associated virus-based gene therapy for the treatment of glaucoma.

Comparative Proteomic Study of Retinal Ganglion Cells Undergoing Various Types of Cellular Stressors.

Retinal ganglion cell (RGC) damage serves as a key indicator of various retinal degenerative diseases, including diabetic retinopathy (DR), glaucoma, retinal arterial and retinal vein occlusions, as well as inflammatory and traumatic optic neuropathies. Despite the growing body of data on the RGC proteomics associated with these conditions, there has been no dedicated study conducted to compare the molecular signaling pathways involved in the mechanism of neuronal cell death. Therefore, we launched the study using two different insults leading to RGC death: glutamate excitotoxicity and optic nerve crush (ONC). C57BL/6 mice were used for the study and underwent NMDA- and ONC-induced damages. Twenty-four hours after ONC and 1 hour after NMDA injection, we collected RGCs using CD90.2 coupled magnetic beads, prepared protein extracts, and employed LC-MS for the global proteomic analysis of RGCs. Statistically significant changes in proteins were analyzed using the Shiny Go program to identify GO biological processes and molecular functions resulting from the treatment. We identified unique and common alterations in protein profiles in RGCs undergoing different types of cellular stressors. Additionally, we observed the absence of certain proteins in treated RGCs compared to the control group. Our study not only identified both unique and shared proteomic changes but also laid the groundwork for the future development of a therapeutic platform for testing gene candidates for DR and glaucoma.

Posttranslational modifications of proteins in diseased retina.

Posttranslational modifications (PTMs) are known to constitute a key step in protein biosynthesis and in the regulation of protein functions. Recent breakthroughs in protein purification strategies and current proteome technologies make it possible to identify the proteomics of healthy and diseased retinas. Despite these advantages, the research field identifying sets of posttranslationally modified proteins (PTMomes) related to diseased retinas is significantly lagging, despite knowledge of the major retina PTMome being critical to drug development. In this review, we highlight current updates regarding the PTMomes in three retinal degenerative diseases-namely, diabetic retinopathy (DR), glaucoma, and retinitis pigmentosa (RP). A literature search reveals the necessity to expedite investigations into essential PTMomes in the diseased retina and validate their physiological roles. This knowledge would accelerate the development of treatments for retinal degenerative disorders and the prevention of blindness in affected populations.

Proteomic analysis of diabetic retinas.

Introduction: As a metabolic disease, diabetes often leads to health complications such as heart failure, nephropathy, neurological disorders, and vision loss. Diabetic retinopathy (DR) affects as many as 100 million people worldwide. The mechanism of DR is complex and known to impact both neural and vascular components in the retina. While recent advances in the field have identified major cellular signaling contributing to DR pathogenesis, little has been reported on the protein post-translational modifications (PTM) - known to define protein localization, function, and activity - in the diabetic retina overall. Protein glycosylation is the enzymatic addition of carbohydrates to proteins, which can influence many protein attributes including folding, stability, function, and subcellular localization. O-linked glycosylation is the addition of sugars to an oxygen atom in amino acids with a free oxygen atom in their side chain (i.e., threonine, serine). To date, more than 100 congenital disorders of glycosylation have been described. However, no studies have identified the retinal O-linked glycoproteome in health or disease. With a critical need to expedite the discovery of PTMomics in diabetic retinas, we identified both global changes in protein levels and the retinal O-glycoproteome of control and diabetic mice. Methods: We used liquid chromatography/mass spectrometry-based proteomics and high throughput screening to identify proteins differentially expressed and proteins differentially O-glycosylated in the retinas of wildtype and diabetic mice. Results: Changes in both global expression levels of proteins and proteins differentially glycosylated in the retinas of wild-type and diabetic mice have been identified. We provide evidence that diabetes shifts both global expression levels and O-glycosylation of metabolic and synaptic proteins in the retina. Discussion: Here we report changes in the retinal proteome of diabetic mice. We highlight alterations in global proteins involved in metabolic processes, maintaining cellular structure, trafficking, and neuronal processes. We then showed changes in O-linked glycosylation of individual proteins in the diabetic retina.

The Phase 3 INVIGORATE Trial of Reproxalap in Patients with Seasonal Allergic Conjunctivitis.

Purpose: There is an unmet need for new treatments for allergic conjunctivitis. Objective: To assess the activity of reproxalap, a novel reactive aldehyde species modulator, in a real-world model of seasonal allergen exposure. Methods: The INVIGORATE Trial, a prospective, quadruple-masked, vehicle-controlled, crossover, sequence-randomized Phase 3 trial, tested the efficacy of reproxalap in adults with a history of moderate to severe allergic conjunctivitis, ragweed pollen allergy, and allergen chamber-induced ocular itching and redness. Patients were randomly assigned (1:1) to receive 0.25% reproxalap ophthalmic solution or vehicle, followed by a 2-week washout period before crossing over to the other test article. The primary endpoint was ocular itching from 110 to 210 minutes after chamber entry; the key secondary endpoint was ocular redness over the chamber duration (0-4 scales for both endpoints). Results: Of the 95 randomly assigned patients, 89 completed all visits (reproxalap to vehicle: n = 46; vehicle to reproxalap: n = 43). Primary and key secondary endpoints were met: reproxalap significantly reduced ocular itching (mean [SE]: -0.50 [0.03], p < 0.001) and redness (-0.14 [0.01], p < 0.001) relative to vehicle. Responder analyses confirmed the clinical relevance of both end points. Reproxalap was safe and well tolerated. No clinically significant changes in safety assessments were observed. No serious or severe treatment-emergent adverse events (TEAEs) were reported. The most commonly reported TEAE was mild and transient installation site irritation after reproxalap versus vehicle administration. Conclusion: In this well-controlled allergen chamber trial, reproxalap was statistically superior to vehicle across typical symptoms and signs of allergic conjunctivitis. Trial Registration: NCT04207736.

Delphi Panel Consensus Regarding Current Clinical Practice Management Options for Demodex blepharitis.

Purpose: To obtain consensus on Demodex blepharitis (DB) treatment using a modified Delphi panel process. Methods: Literature search identified gaps in knowledge surrounding treatment of DB. Twelve ocular surface disease experts comprised the Demodex Expert Panel on Treatment and Eyelid Health (DEPTH). They completed a live roundtable discussion in addition to 3 surveys consisting of scaled, open-ended, true/false, and multiple-choice questions pertaining to the treatment of DB. Consensus for scaled questions using a 1 to 9 Likert scale was predefined as median scores of 7-9 and 1-3. For other question types, consensus was achieved when 8 of 12 panelists agreed. Results: The experts agreed that an effective therapeutic agent for treatment of DB would likely decrease the necessity of mechanical intervention, such as lid scrubs or blepharoexfoliation (Median = 8.5; Range 2-9). When treating DB, panelists believed that collarettes serve as a surrogate for mites, and that eliminating or reducing collarettes should be the main clinical goal of treatment (Median = 8; Range 7-9). The panelists would treat patients with at least 10 collarettes, regardless of other signs or symptoms and agreed that DB can be cured, but there is always the possibility for a reinfestation (n = 12). There was also consensus that collarettes, and therefore mites, are the primary treatment target and the way by which clinicians can monitor patient response to therapy (Median = 8; Range 7-9). Conclusion: Expert panelists achieved consensus on key facets of DB treatment. Specifically, there was consensus that collarettes are pathognomonic for DB, that DB patients with >10 collarettes should be treated even in the absence of symptoms, and that treatment efficacy can be tracked by collarette resolution. By increasing awareness about DB, understanding the goals of and monitoring treatment efficacy, patients will receive better care and, ultimately, better clinical outcomes.

Clinical diagnosis and management of Demodex blepharitis: the Demodex Expert Panel on Treatment and Eyelid Health (DEPTH).

BACKGROUND: Twelve ocular surface disease experts convened to achieve consensus about Demodex blepharitis (DB) using a modified Delphi panel process. METHODS: Online surveys were administered using scaled, open-ended, true/false, and multiple-choice questions. Consensus for questions using a 1 to 9 Likert scale was predefined as median scores of 7-9 and 1-3. For other question types, consensus was achieved when 8 of 12 panellists agreed. Questions were randomized, and results of each survey informed the following survey. RESULTS: Twelve practitioners comprised the Demodex Expert Panel on Treatment and Eyelid Health (DEPTH). Following 3 surveys, experts agreed that DB is chronic (n = 11) and recurrent (n = 12) and is often misdiagnosed. Consensus was achieved regarding inflammation driving symptoms (median = 7; range 7-9), collarettes as the most common sign (n = 10) and pathognomonic for DB (median = 9; range 8-9), and itching as the most common symptom (n = 12). Panellists agreed that DB may be diagnosed based on collarettes, mites, and/or patient symptoms (n = 10) and felt that patients unresponsive to typical therapies should be evaluated for DB (n = 12). Consensus about the most effective currently available OTC treatment was not reached. CONCLUSIONS: The Delphi methodology proved effective in establishing consensus about DB, including signs, symptoms, and diagnosis. Consensus was not reached about the best treatment or how to grade severity. With increased awareness, eyecare practitioners can offer DB patients better clinical outcomes. A follow-up Delphi panel is planned to obtain further consensus surrounding DB treatment.

TFOS Lifestyle Report Executive Summary: A Lifestyle Epidemic - Ocular Surface Disease.

The Tear Film & Ocular Surface Society (TFOS) Workshop entitled 'A Lifestyle Epidemic: Ocular Surface Disease' was a global initiative undertaken to establish the direct and indirect impacts of everyday lifestyle choices and challenges on ocular surface health. This article presents an executive summary of the evidence-based conclusions and recommendations of the 10-part TFOS Lifestyle Workshop report. Lifestyle factors described within the report include contact lenses, cosmetics, digital environment, elective medications and procedures, environmental conditions, lifestyle challenges, nutrition, and societal challenges. For each topic area, the current literature was summarized and appraised in a narrative-style review and the answer to a key topic-specific question was sought using systematic review methodology. The TFOS Lifestyle Workshop report was published in its entirety in the April 2023 and July 2023 issues of The Ocular Surface journal. Links to downloadable versions of the document and supplementary material, including report translations, are available on the TFOS website: http://www.TearFilm.org.

The contribution of the putamen to sensory aspects of pain: insights from structural connectivity and brain lesions.

Cerebral cortical activity is heavily influenced by interactions with the basal ganglia. These interactions occur via cortico-basal ganglia-thalamo-cortical loops. The putamen is one of the major sites of cortical input into basal ganglia loops and is frequently activated during pain. This activity has been typically associated with the processing of pain-related motor responses. However, the potential contribution of putamen to the processing of sensory aspects of pain remains poorly characterized. In order to more directly determine if the putamen can contribute to sensory aspects of pain, nine individuals with lesions involving the putamen underwent both psychophysical and functional imaging assessment of perceived pain and pain-related brain activation. These individuals exhibited intact tactile thresholds, but reduced heat pain sensitivity and widespread reductions in pain-related cortical activity in comparison with 14 age-matched healthy subjects. Using magnetic resonance imaging to assess structural connectivity in healthy subjects, we show that portions of the putamen activated during pain are connected not only with cortical regions involved in sensory-motor processing, but also regions involved in attention, memory and affect. Such a framework may allow cognitive information to flow from these brain areas to the putamen where it may be used to influence how nociceptive information is processed. Taken together, these findings indicate that the putamen and the basal ganglia may contribute importantly to the shaping of an individual subjective sensory experience by utilizing internal cognitive information to influence activity of large areas of the cerebral cortex.

Bilateral posterior capsule rupture and anterior vitreous prolapse from vigorous eye rubbing.

PURPOSE: We present the case of a 71-year-old male who developed decreased visual acuity eleven years after uncomplicated bilateral cataract extraction and in-the-bag IOL implantation following recent bouts of vigorous eye rubbing. OBSERVATIONS: On examination, the posterior capsules were ruptured centrally in both eyes, and there was anterior vitreous prolapse bilaterally. While both IOLs remained in the visual axis, there was mild bilateral decentration and resultant decreased vision. CONCLUSION AND IMPORTANCE: To the authors' knowledge, this is the first reported case of bilateral simultaneous posterior capsule rupture with anterior vitreous prolapse due to eye rubbing. Contrasting to previous case reports, this scenario demonstrates that severe eye rubbing can have variable severity and complications. Frequent and vigorous eye rubbing can be a precipitating cause of late postoperative posterior capsule rupture and should be avoided in pseudophakic patients, especially those with older IOL models.

Reproxalap Activity and Estimation of Clinically Relevant Thresholds for Ocular Itching and Redness in a Randomized Allergic Conjunctivitis Field Trial.

INTRODUCTION: This clinical trial assessed the activity of reproxalap, a novel reactive aldehyde species modulator, and estimated clinically relevant thresholds for changes in ocular itching and redness in an allergic conjunctivitis field trial. METHODS: This was a randomized, double-masked, vehicle-controlled phase 2 trial. Patients with ragweed-associated allergic conjunctivitis were assessed over 28 days in an environmental setting with approximately four doses per day of either 0.25% reproxalap, 0.5% reproxalap, or vehicle. Patients recorded ocular itching, redness, tearing, and eyelid swelling scores (each with a 0-4 scale, except for a 0-3 scale for swelling), and completed the Allergic Conjunctivitis Quality of Life Questionnaire at the beginning and end of the trial. RESULTS: Mixed model of repeated measures analysis demonstrated statistically lower itching and tearing scores (pooled P = 0.026 and P < 0.001, respectively) and numerically lower redness and eyelid swelling scores than vehicle on days when pollen exceeded the 95th percentile value. Using three anchor-based and three distribution-based approaches, the meaningful within-patient change and the between-group meaningful difference for patient-reported ocular itching and redness was estimated to be approximately 0.5. The most common treatment-emergent adverse event associated with reproxalap was transient irritation upon instillation. CONCLUSION: In a field clinical trial, reproxalap was well tolerated and superior to vehicle in reducing ocular itching on high-pollen days. The clinical meaningfulness threshold estimates of 0.5 units are among the first such calculations generated for the standard ocular itching and redness scores, providing important context for the clinical interpretation of clinical trials in allergic conjunctivitis.

While allergic conjunctivitis affects millions of patients worldwide, treatments with new mechanisms have not been introduced in decades. Reproxalap, a medicine being investigated as a treatment for allergic conjunctivitis, works by regulating reactive aldehyde species­molecules that are increased in a variety of inflammatory diseases. This clinical trial assessed the activity of reproxalap and estimated what amount of change in ocular itching and redness should be considered clinically important. Patients with ragweed-associated allergic conjunctivitis were assessed over 28 days and were given one of three possible eye drops at approximately four doses per day: 0.25% reproxalap; 0.5% reproxalap; or vehicle, which was composed of the same ingredients but does not contain reproxalap. Patients recorded ocular itching, redness, tearing, and eyelid swelling (all scales ranged from 0 [none] to 4 [severe] except for eyelid swelling, which ranged from 0 to 3), and completed a quality-of-life questionnaire on allergic conjunctivitis at the beginning and end of the trial. The results indicated that reproxalap was significantly better than vehicle in reducing itching and tearing scores and was better than vehicle in reducing redness and eyelid swelling scores on days when pollen counts were high. The trial also suggested that a reduction in ocular itching and redness scores of approximately 0.5 or more (scale 0­4) is likely to be clinically important. Overall, reproxalap was well tolerated and no safety concerns were noted. The most common side effect was transient ocular discomfort after eye drop administration.

What is enantiotoky, and why doesn't it exist?

Arrhenotoky, in which females arise from fertilized eggs and males from unfertilized eggs, has evolved multiple times in animals, most prominently in the insect order Hymenoptera. An alternative form of haplodiploidy, in which females are haploid and males diploid-here named enantiotoky-is not known to exist. An illustrated thought experiment shows that if it does evolve, it will be very unstable and is expected to disappear very quickly.

Comparative Morphology of the Stinger in Social Wasps (Hymenoptera: Vespidae).

The physical features of the stinger are compared in 51 species of vespid wasps: 4 eumenines and zethines, 2 stenogastrines, 16 independent-founding polistines, 13 swarm-founding New World polistines, and 16 vespines. The overall structure of the stinger is remarkably uniform within the family. Although the wasps show a broad range in body size and social habits, the central part of the venom-delivery apparatus-the sting shaft-varies only to a modest extent in length relative to overall body size. What variation there is shows no apparent correlation with social habits. This is consistent with the hypothesis that stinger size is constrained by the demands of a flight-worthy body. The sting lancets bear distinct, acute barbs in all examined species except in members of the Stenogastrinae. Barbs vary considerably among species in number, their summed lengths, and the relative degree of serration (summed length relative to lancet width). Where they are numerous and strong, it increases the likelihood of the stinger remaining fatally embedded in the skin of a vertebrate adversary (sting autotomy). Although an index that combines the number and strength of barbs is a more natural measure of overall serration, the number of barbs alone is almost as good a predictor of the likelihood of sting autotomy. Across the family as a whole, the tendency to sting autotomy is concentrated in the swarm-founding New World polistines.

Protocol for evaluating the role of a gene in protecting mouse retinal ganglion cells.

The degeneration of retinal ganglion cells (RGCs) leads to irreversible vision loss in a variety of pathological states. Here, we describe a protocol to evaluate the role of a gene in protecting mouse RGCs when they sustain injuries from excitotoxicity or axonal damage. This protocol includes the procedures for gene transfer through AAV intravitreal injection, induction of RGC injuries by NMDA-induced excitotoxicity or optic nerve crush, and retina immunohistochemistry to assess RGC survival. For complete details on the use and execution of this protocol, please refer to Guo et al. (2021).