Lysyl oxidase like-1 deficiency in optic nerve head astrocytes elicits reactive astrocytosis and alters functional effects of astrocyte derived exosomes.

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro, positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis.

CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity.

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.

Glioprotection of Retinal Astrocytes After Intravitreal Administration of Memantine in the Mouse Optic Nerve Crush Model.

BACKGROUND In glaucoma, non-intraocular pressure (IOP)-related risk factors can result in increased levels of extracellular glutamate, which triggers a cascade of neurodegeneration characterized by the excessive activation of N-methyl-D-aspartate (NMDA). The purpose of our study was to evaluate the glioprotective effects of memantine as a prototypic uncompetitive NMDA blocker on retinal astrocytes in the optic nerve crush (ONC) mouse model for glaucoma. MATERIAL AND METHODS Optic nerve crush was performed on all of the right eyes (n=8), whereas left eyes served as contralateral healthy controls (n=8) in Balb/c/Sca mice. Four randomly assigned mice received 2-µl intravitreal injections of memantine (1 mg/ml) after ONC in the experimental eye. One week after the experiment, optic nerves were dissec-ted and stained with methylene blue. Retinae were detached from the sclera. The tissue was immunostained. Whole-mount retinae were investigated by fluorescent microscopy. Astrocyte counts for each image were performed manually. RESULTS Histological sections of crushed optic nerves showed consistently moderate tissue damage in experimental groups. The mean number of astrocytes per image in the ONC group was significantly lower than in the healthy control group (7.13±1.5 and 10.47±1.9, respectively). Loss of astrocytes in the memantine-treated group was significantly lower (8.83±2.2) than in the ONC group. Assessment of inter-observer reliability showed excellent agreement among observations in control, ONC, and memantine groups. CONCLUSIONS The ONC is an effective method for investigation of astrocytic changes in mouse retina. Intravitreally administered memantine shows a promising glioprotective effect on mouse retinal astrocytes by preserving astrocyte count after ONC.

Caspase-3-Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging.

Mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain.

Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes.

Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute critically to extracellular matrix synthesis during development and throughout life. In glaucoma, and in related disorders affecting the optic nerve and the optic nerve head, pathological changes include altered astrocyte gene and protein expression resulting in their activation and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA in order to slow glaucoma and related pathologies are of high clinical relevance. Herein, we describe the development of standardized methods that will allow for the systematic advancement of such glioprotective strategies. These include isolation, purification and culture of primary adult rat ONHAs, optimized immunocytochemical protocols for cell type validation, as well as plate reader-based assays determining cellular viability, proliferation and the intracellular redox state. We validated and standardized our protocols by performing a glioprotection study using primary ONHAs. Specifically, we measured protection against exogenously-applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress in the retina and optic nerve head by the prototypic antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Levels of oxidative stress were increased in the response to exogenously applied tBHP and were assessed by 6-carboxy-2', 7' dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase; the half-maximal effect (EC50) for tBHP was 212 ± 25 µM. This was paralleled very effectively in the assays measuring cell death and cell viability with half-maximal effects of 241 ± 20 µM and 194 ± 5 µM for tBHP in the lactate dehydrogenase (LDH) release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays, respectively. Pre-treatment with 100 µM Trolox decreased the sensitivity of ONHAs to tBHP. Half-maximal effects increased to 396 ± 12 µM tBHP in the LDH release assay and to 383 ± 3 µM tBHP in the MTT assay. Vehicle treatment (0.1% v/v ethanol) did not significantly affect cellular responses to tBHP. Antioxidant treatment increases ONHA viability and reduces the deleterious effects of oxidative stress. Our experiments provide important feasibility data for utilizing primary rat ONHAs in plate reader-based assays assessing novel therapeutics for glioprotection of the optic nerve and the optic nerve head in glaucoma and related disorders. Furthermore, our novel, standardized protocols have the potential to be readily adapted to high-throughput and high-content testing strategies.

Antioxidant drug therapy approaches for neuroprotection in chronic diseases of the retina.

The molecular pathways contributing to visual signal transduction in the retina generate a high energy demand that has functional and structural consequences such as vascularization and high metabolic rates contributing to oxidative stress. Multiple signaling cascades are involved to actively regulate the redox state of the retina. Age-related processes increase the oxidative load, resulting in chronically elevated levels of oxidative stress and reactive oxygen species, which in the retina ultimately result in pathologies such as glaucoma or age-related macular degeneration, as well as the neuropathic complications of diabetes in the eye. Specifically, oxidative stress results in deleterious changes to the retina through dysregulation of its intracellular physiology, ultimately leading to neurodegenerative and potentially also vascular dysfunction. Herein we will review the evidence for oxidative stress-induced contributions to each of the three major ocular pathologies, glaucoma, age-related macular degeneration, and diabetic retinopathy. The premise for neuroprotective strategies for these ocular disorders will be discussed in the context of recent clinical and preclinical research pursuing novel therapy development approaches.

Resveratrol and calcium signaling: molecular mechanisms and clinical relevance.

Resveratrol is a naturally occurring compound contributing to cellular defense mechanisms in plants. Its use as a nutritional component and/or supplement in a number of diseases, disorders, and syndromes such as chronic diseases of the central nervous system, cancer, inflammatory diseases, diabetes, and cardiovascular diseases has prompted great interest in the underlying molecular mechanisms of action. The present review focuses on resveratrol, specifically its isomer trans-resveratrol, and its effects on intracellular calcium signaling mechanisms. As resveratrol's mechanisms of action are likely pleiotropic, its effects and interactions with key signaling proteins controlling cellular calcium homeostasis are reviewed and discussed. The clinical relevance of resveratrol's actions on excitable cells, transformed or cancer cells, immune cells and retinal pigment epithelial cells are contrasted with a review of the molecular mechanisms affecting calcium signaling proteins on the plasma membrane, cytoplasm, endoplasmic reticulum, and mitochondria. The present review emphasizes the correlation between molecular mechanisms of action that have recently been identified for resveratrol and their clinical implications.

PLL-g-PEG Polymer Inhibits Antibody-Drug Conjugate Uptake into Human Corneal Epithelial Cells In Vitro.

Purpose: Antibody-drug conjugates (ADCs) are a relatively recent advance in the delivery of chemotherapeutics that improve targeting of cytotoxic agents. However, despite their antitumor activity, severe ocular adverse effects, including vision loss, have been reported for several ADCs. The nonspecific uptake of ADCs into human corneal epithelial cells (HCECs) and their precursors via macropinocytosis has been proposed to be the primary mechanism of ocular toxicity. In this study, we evaluated the ability of a novel polymer, poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), to decrease the ADC rituximab-mc monomethylauristatin F (MMAF) (RIX) uptake into human corneal epithelial (HCE-T) cells. Methods: HCE-T cells were exposed to increasing concentrations of RIX to determine inhibition of cell proliferation. HCE-T cells were treated with PLL-g-PEG, the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA), or vehicle. After 30 min of incubation, RIX was added. ADC was detected by fluorescent anti-human immunoglobulin G and fluorescently conjugated dextran as viewed by microscopy. Results: RIX caused dose-dependent inhibition of HCE-T cell proliferation. EIPA significantly reduced RIX uptake and decreased macropinocytosis as assessed by direct quantification of RIX using a fluorescently conjugated anti-human antibody as well as quantification of macropinocytosis using fluorescently conjugated dextran. PLL-g-PEG resulted in a dose-dependent inhibition of RIX uptake with half-maximal inhibitory concentrations of 0.022%-0.023% PLL-g-PEG. Conclusion: The data show PLL-g-PEG to be a potent inhibitor of RIX uptake by corneal epithelial cells and support its use as a novel therapeutic approach for the prevention of ocular adverse events associated with ADC therapy.

Prospects and limitations of cumate-inducible lentivirus as a tool for investigating VEGF-A-mediated pathology in diabetic retinopathy.

Diabetic retinopathy (DR) is a multifactorial disease displaying vascular-associated pathologies, including vascular leakage and neovascularization, ultimately leading to visual impairment. However, animal models accurately reflecting these pathologies are lacking. Vascular endothelial growth factor A (VEGF-A) is an important factor in the development of micro- and macro-vascular pathology in DR. In this study, we evaluated the feasibility of using a cumate-inducible lentivirus (LV) mediated expression of vegf-a to understand DR pathology in vitro and in vivo. Retinal pigment epithelial cells (ARPE-19) were transduced with cumate-inducible LV expressing vegf-a, with subsequent analysis of vegf-a expression and its impact on cell proliferation, viability, motility, and permeability. Cumate tolerability in adult Wistar rat eyes was assessed as an initial step towards a potential DR animal model development, by administering cumate via intravitreal injections (IVT) and evaluating consequent effects by spectral domain optical coherence tomography (SD-OCT), flash electroretinography (fERG), ophthalmic examination (OE), and immunohistochemistry. Transduction of ARPE-19 cells with cumate-inducible LV resulted in ~ 2.5-fold increase in vegf-a mRNA and ~ threefold increase in VEGF-A protein secretion. Transduced cells displayed enhanced cell proliferation, viability, permeability, and migration in tube-like structures. However, IVT cumate injections led to apparent retinal toxicity, manifesting as retinal layer abnormalities, haemorrhage, vitreous opacities, and significant reductions in a- and b-wave amplitudes, along with increased microglial activation and reactive gliosis. In summary, while cumate-inducible LV-mediated vegf-a expression is valuable for in vitro mechanistic studies in cellular drug discovery, its use is not a feasible approach to model DR in in vivo studies due to cumate-induced retinal toxicity.

A case for neuroprotection in ophthalmology: developments in translational research.

Cellular aging occurs by the lifelong accumulation of oxidative damage leading to neuronal apoptosis, termed 'neurodegeneration', and the functional deficits of aging. Loss of visual function is one of the most important quality of life measures for older adults. We discuss recent clinical and laboratory advances in the neuroprotective treatment of the aging eye with particular emphasis on the three major ocular neurodegenerative conditions: glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy (DR).

Immune System, Inflammation and Autoantigens in Wet Age-Related Macular Degeneration: Pathological Significance and Therapeutic Importance.

Wet age-related macular degeneration (wAMD) is a chronic inflammation-associated neurodegenerative disease affecting the posterior part of the eye in the aging population. Aging results in the reduced functionality of cells and tissues, including the cells of the retina. Initiators of a chronic inflammatory and pathologic state in wAMD may be a result of the accumulation of inevitable metabolic injuries associated with the maintenance of tissue homeostasis from a young age to over 50. Apart from this, risk factors like smoking, genetic predisposition, and failure to repair the injuries that occur, alongside attempts to rescue the hypoxic outer retina may also contribute to the pathogenesis. Aging of the immune system (immunosenescence) and a compromised outer blood retinal barrier (BRB) result in the exposure of the privileged milieu of the retina to the systemic immune system, further increasing the severity of the disease. When immune-privileged sites like the retina are under pathological stress, certain age- and disease-related conditions may necessitate assistance from cells distant from the resident ones to help restore the functionality of the tissue. As a necessary part of tissue repair, inflammation is a major response to disease and recruits immune cells to the site of damage. We suspect that the specific reparative inflammatory responses are controlled by an autoantigen-T cell-mediated mechanism, a process that may be hindered in wAMD.

Dietary Alcohol Consumption Elicits Corneal Toxicity Through the Generation of Cellular Oxidative Stress.

Purpose: Clinical data suggest that alcohol use is associated with the development of signs and symptoms of dry eye disease. However, preclinical data investigating ocular toxicity after dietary alcohol consumption are lacking. In this study, we investigated the effects of alcohol on the ocular surface, in human corneal epithelial cells (HCE-T) in vitro and in C57BL/6JRj mice in vivo. Methods: HCE-T were exposed to clinically relevant doses of ethanol. To determine the effects of dietary alcohol consumption in vivo, wild-type mice were administered the Lieber-DeCarli liquid diet (5% vol/vol ethanol or isocaloric control) for 10 days ad libitum. Corneal fluorescein staining was performed to assess ocular surface damage. Histopathological and gene expression studies were performed on cornea and lacrimal gland tissue. Results: Sublethal doses of ethanol (0.01%-0.5%) resulted in a dose-dependent increase of cellular oxidative stress in corneal epithelial cells and a significant increase in NFE2L2 and downstream antioxidant gene expression, as well as an increase in NFκB signaling; short-term exposure (0.5%, 4 h) triggered significant corneal epithelial cell barrier breakdown. Exposure to the alcohol-containing diet caused a 3-fold increase in corneal fluorescein staining, with no effect on tear volumes. Corneal thickness was significantly reduced in the alcohol diet group, and corneal tissue revealed dysregulated antioxidant and NFκB signaling. Our data provide the first published evidence that alcohol exposure causes ocular toxicity in mice. Conclusions: Our results are consistent with clinical studies linking past alcohol consumption to signs of ocular surface disease.

Structure-Function Relationships in the Rodent Streptozotocin-Induced Model for Diabetic Retinopathy: A Systematic Review.

The streptozotocin (STZ)-induced rodent model is one of the most commonly employed models in preclinical drug discovery for diabetic retinopathy (DR). However, standardization and validation of experimental readouts are largely lacking. The aim of this systematic review was to identify and compare the most useful readouts of STZ-induced DR and provide recommendations for future study design based on our findings. We performed a systematic search using 2 major databases, PubMed and EMBASE. Only articles describing STZ-induced DR describing both functional and structural readouts were selected. We also assessed the risk of bias and analyzed qualitative data in the selected studies. We identified 21 studies that met our inclusion/exclusion criteria, using either rats or mice and study periods of 2 to 24 weeks. Glucose level thresholds used to define hyperglycemia were inconsistent between studies, however, most studies used either 250 or 300.6 mg/dL as a defining criterion for hyperglycemia. All included studies performed electroretinography (ERG) and reported a reduction in a-, b-, or c-wave and/or oscillatory potential amplitudes. Spectral-domain optical coherence tomography and fluorescein angiography, as well as immunohistochemical and histopathological analyses showed reductions in retinal thickness, vascular changes, and presence of inflammation. Risk of bias assessment showed that all studies had a high risk of bias due to lack of reporting or correctly following procedures. Our systematic review highlights that ERG represents the most consistent functional readout in the STZ model. However, due to the high risk of bias, caution must be used when interpreting these studies.

Topical Porphyrin Antioxidant Protects Against Ocular Surface Pathology in a Novel Rabbit Model for Particulate Matter-Induced Dry Eye Disease.

Purpose: Particulate matter (PM) is a primary cause for the development of acute and chronic dry eye disease, especially irritant-induced conjunctivitis. The purpose of the present study was to determine the effects of fine atmospheric PM on the rabbit ocular surface, and determine the protective effects of a synthetic antioxidant, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), in vitro and in vivo. Methods: Rabbit corneal epithelial cells (SIRC) were exposed to increasing concentrations of PM to determine the effects on cell motility and viability. The in vivo effects of topically instilled PM were tested in New Zealand White rabbits. Comprehensive ophthalmic exams and corneal fluorescein staining were performed. Results: Exposure to PM resulted in dose-dependent cell death and impaired cellular motility; Mn-TM-2-PyP protected against PM-induced cytotoxicity and significantly increased SIRC cell motility. In vivo, exposure to PM (5 mg/ml, topical, 3 times daily for 7 days) resulted in signs of dry eye, notably hyperemia, increased corneal fluorescein staining, and decreased tear volumes. Mn-TM-2-PyP significantly improved hyperemia and corneal fluorescein readouts but had no effect on tear production. Lifitegrast (Xiidra®) showed similar pharmacologic efficacy to Mn-TM-2-PyP. Conclusion: Overall, these data provide evidence that PM induces phenotypes of ocular surface disease responsive to antioxidant and immunosuppressant therapy. To our knowledge this is the first report of a large animal model to study PM-induced ocular surface disease. The present work provides standardized experimental paradigms for the comprehensive in vitro and in vivo testing of novel therapeutic approaches targeting PM-induced conjunctivitis and dry-eye.

High cortical spreading depression susceptibility and migraine-associated symptoms in Ca(v)2.1 S218L mice.

OBJECTIVE: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. METHODS: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. RESULTS: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. INTERPRETATION: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema.

Preclinical Efficacy of Pro- and Anti-Angiogenic Peptide Hydrogels to Treat Age-Related Macular Degeneration.

Pro-angiogenic and anti-angiogenic peptide hydrogels were evaluated against the standard of care wet age-related macular degeneration (AMD) therapy, Aflibercept (Eylea®). AMD was modeled in rats (laser-induced choroidal neovascularization (CNV) model), where the contralateral eye served as the control. After administration of therapeutics, vasculature was monitored for 14 days to evaluate leakiness. Rats were treated with either a low or high concentration of anti-angiogenic peptide hydrogel (0.02 wt% 8 rats, 0.2 wt% 6 rats), or a pro-angiogenic peptide hydrogel (1.0 wt% 7 rats). As controls, six rats were treated with commercially available Aflibercept and six with sucrose solution (vehicle control). Post lasering, efficacy was determined over 14 days via fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT). Before and after treatment, the average areas of vascular leak per lesion were evaluated as well as the overall vessel leakiness. Unexpectedly, treatment with pro-angiogenic peptide hydrogel showed significant, immediate improvement in reducing vascular leak; in the short term, the pro-angiogenic peptide performed better than anti-angiogenic peptide hydrogel and was comparable to Aflibercept. After 14 days, both the pro-angiogenic and anti-angiogenic peptide hydrogels show a trend of improvement, comparable to Aflibercept. Based on our results, both anti-angiogenic and pro-angiogenic peptide hydrogels may prove good therapeutics in the future to treat wet AMD over a longer-term treatment period.

Poly(lactic-co-glycolic acid) Nanoparticles Encapsulating the Prenylated Flavonoid, Xanthohumol, Protect Corneal Epithelial Cells from Dry Eye Disease-Associated Oxidative Stress.

Oxidative stress is a known contributor to the progression of dry eye disease pathophysiology, and previous studies have shown that antioxidant intervention is a promising therapeutic approach to reduce the disease burden and slow disease progression. In this study, we evaluated the pharmacological efficacy of the naturally occurring prenylated chalconoid, xanthohumol, in preclinical models for dry eye disease. Xanthohumol acts by promoting the transcription of phase II antioxidant enzymes. In this study, xanthohumol prevented tert-butyl hydroperoxide-induced loss of cell viability in human corneal epithelial (HCE-T) cells in a dose-dependent manner and resulted in a significant increase in expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of phase II endogenous antioxidant enzymes. Xanthohumol-encapsulating poly(lactic-co-glycolic acid) nanoparticles (PLGA NP) were cytoprotective against oxidative stress in vitro, and significantly reduced ocular surface damage and oxidative stress-associated DNA damage in corneal epithelial cells in the mouse desiccating stress/scopolamine model for dry eye disease in vivo. PLGA NP represent a safe and efficacious drug delivery vehicle for hydrophobic small molecules to the ocular surface. Optimization of NP-based antioxidant formulations with the goal to minimize instillation frequency may represent future therapeutic options for dry eye disease and related ocular surface disease.

Severe and progressive neurotransmitter release aberrations in familial hemiplegic migraine type 1 Cacna1a S218L knock-in mice.

Familial hemiplegic migraine type 1 (FHM1) is caused by mutations in the CACNA1A gene, encoding neuronal presynaptic Ca(V)2.1 (P/Q-type) Ca(2+) channels. These channels mediate neurotransmitter release at many central synapses and at the neuromuscular junction (NMJ). Mutation S218L causes a severe neurological phenotype of FHM and, additionally, ataxia and susceptibility to seizures, delayed brain edema, and fatal coma after minor head trauma. Recently, we generated a Cacna1a S218L knock-in mutant mouse, displaying these features and reduced survival. A first electrophysiological study showed high susceptibility for cortical spreading depression, enhanced neuronal soma Ca(2+) influx, and at diaphragm NMJs, a considerable increase of neurotransmitter release. We here assessed the function of S218L knock-in NMJs at several muscle types in great detail. Pharmacological analyses using specific Ca(V) subtype-blocking toxins excluded compensatory contribution of non-Ca(V)2.1 channels. Endplate potentials were considerably broadened at many NMJs. High rate (40 Hz)-evoked acetylcholine release was slightly reduced; however, it was not associated with block of neurotransmission causing weakness, as assessed with grip strength measurements and in vitro muscle contraction experiments. The synaptopathy clearly progressed with age, including development of an increased acetylcholine release at low-rate nerve stimulation at physiological extracellular Ca(2+) concentration and further endplate potential broadening. Our results suggest enhanced Ca(2+) influx into motor nerve terminals through S218L-mutated presynaptic Ca(V)2.1 channels, likely because of the earlier reported negative shift of activation potential and reduced inactivation. Similar severe aberrations at central synapses of S218L mutant mice and humans may underlie or contribute to the drastic neurological phenotype.

Control of intracellular calcium signaling as a neuroprotective strategy.

Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed.

Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants.

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.