Efficacy of Intravitreal Multi-Characteristic Opsin (MCO-010) Optogenetic Gene Therapy in a Mouse Model of Leber Congenital Amaurosis.

Leber congenital amaurosis (LCA) is a sight-threatening inherited retinal disorder (IRD) caused by numerous genetic mutations. Multi-characteristic opsin (MCO)-based optogenetic therapy allows the recruitment of residual cells of the retina in LCA for alternative vision transduction while being mutation-agnostic. Using mice, we investigated the efficacy of an adeno-associated virus2 (AAV2)-transduced ambient light-activatable MCO (MCO-010) containing a metabotropic glutamate receptor-6 bipolar cell-specific promoter/enhancer.

Engineered sensor actuator modulator as aqueous humor outflow actuator for gene therapy of primary open-angle glaucoma.

Glaucoma, a blinding eye disease with optic neuropathy, is usually associated with elevated intraocular pressure (IOP). The currently available pharmacological and surgical treatments for glaucoma have significant limitations and side effects, which include systemic reactions to medications, patient non-compliance, eye infections, surgical device failure, and damage to the eye. Here, we present Sensor-Actuator-Modulator (SAM), an engineered double mutant version of the bacterial stretch-activated mechanosensitive channel of large conductance (MscL) that directly senses tension in the membrane lipid bilayer of cells and in response, transiently opens its large nonspecific pore to release cytoplasmic fluid.

Novel Small Molecules with Anti-Inflammatory and Anti-Angiogenic Activity in a Mouse Model of Oxygen-Induced Retinopathy.

Retinopathy of prematurity (ROP) has a dual-phase disease pathology; in phase 1, hyperoxia-induced vaso-obliteration occurs in the retinal vasculature due to increased oxidative stress (OS) and inflammation, followed by phase 2, where hypoxia increases the overproduction of growth factors, inducing retinal neovascularization. Toll-like receptor 2 and -4 (TLR2 and TLR4) overactivation, hyper-inflammation, macrophages, and neutrophil infiltration contribute to the developing ROP. AVR-121 and AVR-123 are novel classes of small-molecule dual inhibitors of TLR2/4 tested in a human leukemia monocytic cell line (THP-1) and cord-blood-derived mononuclear cells (CBMCs).

Response of heterologously expressed pressure sensor-actuator-modulator macromolecule to external mechanical stress.

Cells from different organs in the body experience a range of mechanical and osmotic pressures that change in various diseases, including neurological, cardiovascular, ophthalmological, and renal diseases. Here, we demonstrate the use of an engineered Sensor-Actuator-Modulator (SAM) of microbial origin derived from a mechanosensitive channel of large conductance (MscL) for sensing external mechanical stress and modulating activities of mammalian cells. SAM is reliably expressed in the mammalian cell membrane and acts as a tension-activated pressure release valve.

Neuroprotective and Anti-Inflammatory Activities of Hybrid Small-Molecule SA-10 in Ischemia/Reperfusion-Induced Retinal Neuronal Injury Models.

Unlabelled: Embolism, hyperglycemia, high intraocular pressure-induced increased reactive oxygen species (ROS) production, and microglial activation result in endothelial/retinal ganglion cell death. Here, we conducted in vitro and in vivo ischemia/reperfusion (I/R) efficacy studies of a hybrid antioxidant-nitric oxide donor small molecule, to assess its therapeutic potential for ocular stroke.

Methods: To induce I/R injury and inflammation, we subjected R28 and primary microglial cells to oxygen glucose deprivation (OGD) for 6 h in vitro or treated these cells with a cocktail of TNF-α, IL-1β and IFN-γ for 1 h, followed by the addition of (10 µM).

Novel Thiol Containing Hybrid Antioxidant-Nitric Oxide Donor Small Molecules for Treatment of Glaucoma.

Oxidative stress induced death and dysregulation of trabecular meshwork (TM) cells contribute to the increased intraocular pressure (IOP) in primary open angle (POAG) glaucoma patients. POAG is one of the major causes of irreversible vision loss worldwide. Nitric oxide (NO), a small gas molecule, has demonstrated IOP lowering activity in glaucoma by increasing aqueous humor outflow and relaxing TM.

Near-Infrared Laser-Based Spatially Targeted Nano-enhanced Optical Delivery of Therapeutic Genes to Degenerated Retina.

Non-viral delivery of therapeutic genes into targeted areas of retina is essential for re-functionalizing the retinal circuitry. While a focused ultrafast laser beam has been recently used for intra-ocular delivery of molecules, it poses the significant technical challenge of overcoming aberrations of the eye and maintaining a tightly focused spot on the retinal cell membrane. Furthermore, to minimize collateral damage and increase the throughput of gene delivery, we introduced a weakly focused near-infrared (NIR) continuous wave (CW) or pulsed laser beam on to the cells wherein the intensity is locally enhanced by gold nanorods bound to the cell membranes to permit gene insertion.

Hybrid Compound SA-2 is Neuroprotective in Animal Models of Retinal Ganglion Cell Death.

Purpose: Determine the toxicity, bioavailability in the retina, and neuroprotective effects of a hybrid antioxidant-nitric oxide donor compound SA-2 against oxidative stress-induced retinal ganglion cell (RGC) death in neurodegenerative animal models.

Methods: Optic nerve crush (ONC) and ischemia reperfusion (I/R) injury models were used in 12-week-old C57BL/6J mice to mimic conditions of glaucomatous neurodegeneration. Mice were treated intravitreally with either vehicle or SA-2.

Basics on the use of acid-sensing ion channels' inhibitors as therapeutics.

Since the discovery of acid-sensing ion channels in 1997, their importance in the health of neurons and other non-neuronal cells has gained significant importance. Acid-sensing ion channels play important roles in mediating pain sensation during diseases such as stroke, inflammation, arthritis, cancer, and recently migraine. More interestingly, acid-sensing ion channels may explain the sex differences in pain between males and females.

Neuroprotective Effects of Psalmotoxin-1, an Acid-Sensing Ion Channel (ASIC) Inhibitor, in Ischemia Reperfusion in Mouse Eyes.

Purpose: The purpose of the current study is to assess changes in the expression of Acid-Sensing Ion Channel (ASIC)1a and ASIC2 in retinal ganglion cells (RGCs) after retinal ischemia and reperfusion (I/R) injury and to test if inhibition of ASIC1a provides RGC neuroprotection.

Methods: Transient ischemia was induced in one eye of C57BL/6 mice by raising intraocular pressure to 120 mmHg for 60 min followed by retinal reperfusion by restoring normal pressure. RGC function was measured by Pattern electroretinography (PERG).

Neuroprotective effects of inhibitors of Acid-Sensing ion channels (ASICs) in optic nerve crush model in rodents.

Purpose: The purpose of the current study was to assess the potential involvement of acid-sensing ion channel 1 (ASIC1) in retinal ganglion cell (RGC) death and investigate the neuroprotective effects of inhibitors of ASICs in promoting RGC survival following optic nerve crush (ONC).

Results: ASIC1 protein was significantly increased in optic nerve extracts at day 7 following ONC in rats. Activated calpain-1 increased at 2 and 7 days following ONC as evidenced by increased degradation of α-fodrin, known substrate of calpain.

Glucocorticoid therapy and ocular hypertension.

The projected number of people who will develop age-related macular degeneration in estimated at 2020 is 196 million and is expected to reach 288 million in 2040. Also, the number of people with Diabetic retinopathy will grow from 126.6 million in 2010 to 191.

CD44 induced enhancement of phosphatase activity and calcium influx: Modifications of EGR-1 expression and cell proliferation.

The purpose of this study was to investigate how CD44 impaired Akt phosphorylation, EGR-1 expression and cell proliferation. E6.1 Jurkat cells, which lack endogenous CD44 expression, were engineered to express CD44.

Sigma-1 receptor stimulation protects retinal ganglion cells from ischemia-like insult through the activation of extracellular-signal-regulated kinases 1/2.

Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.

Changes in retinal aquaporin-9 (AQP9) expression in glaucoma.

The eye contains numerous water channel proteins and the roles of AQPs (aquaporins) in the retina are blurred, especially under disease conditions. The purpose of this study was to investigate the expression of AQP9 gene and proteins affected by elevated IOP (intraocular pressure) in a rat model of glaucoma induced by intravitreous injection of hypertonic saline into the episcleral veins. The gene and protein expressions of AQP9 were investigated by real-time PCR and Western blotting.

Fluorescent protein-labeled glucocorticoid receptor alpha isoform trafficking in cultured human trabecular meshwork cells.

Purpose: To characterize the roles of the cytoskeleton and heat shock protein 90 (HSP90) in steroid-induced glucocorticoid receptor alpha (GRα) translocation in cultured human trabecular meshwork cells.

Methods: Stably transfected red fluorescent protein (RFP)-GRα NTM5 cell lines were developed. Nuclear localization of RFP-GRα in NTM5 cells treated with vehicle (ethanol), dexamethasone (DEX), or RU486 was measured in cytosolic and nuclear fractions by western blotting and laser confocal microscopy.

Protein profiling of human nonpigmented ciliary epithelium cell secretome: the differentiation factors characterization for retinal ganglion cell line.

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), or glutamate.

Changes in ocular aquaporin expression following optic nerve crush.

Purpose: Changes in the expression of water channels (aquaporins; AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush (ONC). This study was designed to analyze changes in the expression of AQP4 (water selective channel) and AQP9 (water and lactate channel) following ONC in the rat.

Changes in ocular aquaporin-4 (AQP4) expression following retinal injury.

Purpose: Changes in the expression of water channels or aquaporins (AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury. This study was designed to analyze changes in the expression of AQP4 following elevation of intraocular pressure (IOP) and after intravitreal endothelin-1 injection and the potential involvement of the ubiquitin-dependent proteasome.

Sigma-1 receptor regulation of voltage-gated calcium channels involves a direct interaction.

Purpose: The sigma-1 receptor belongs to a recently discovered family of transmembrane proteins expressed in the central nervous system, including the eye, and mediates the regulation of ion channels. The exact function of sigma receptors remains to be elucidated. The purpose of this study was to investigate the effect of sigma-1 receptor ligands on calcium homeostasis in a retinal ganglion cell line (RGC)-5 and rat primary RGCs.

Stress-induced changes in neuronal Aquaporin-9 (AQP9) in a retinal ganglion cell-line.

The water channel, Aquaporin-9 (AQP9) is enriched in selected neuronal populations and is unique its ability to act as a lactate-glycerol channel supplying neurons with alternative fuel under ischaemic conditions. AQP9 was detected in RGC-5 cells, a retinal ganglion cell-line, primary RGCs, and retina by Western blotting, real-time PCR (RT-PCR) and immunohistochemistry. RGC-5 cells subjected to a hypotonic stress increased their cell volume that was blocked by known inhibitor of AQP9 (phloretin (40 microM)).

Parallel signaling pathways in endothelin-1-induced proliferation of U373MG astrocytoma cells.

Endothelin-1 (ET-1) is a potent mitogen for many cells, especially when its levels are elevated under pathological conditions, as seen in tumor cell progression and astroglial activation in neuropathies. While ET-1 is known to cause astroglial proliferation, in the present study, multiple signaling pathways involved in ET-1-mediated astrocyte proliferation were characterized. Treatment with PD98059 and U0126 (MEK inhibitors) inhibited not only ET-1-induced cell proliferation but also ET-1-activated phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in U373MG astrocytoma cells.

Localization of endothelin-converting enzyme in bovine optic nerve and retina.

A significant loss and remodeling of the lamina cribrosa tissue leading to the excavation of the optic nerve is seen in glaucoma. Elevated endothelin-1 (ET-1) levels are detected in the aqueous humor of patients of open-angle glaucoma and in the plasma of patients with normal- tension glaucoma. Optic nerve damage, including axonal loss, can be mimicked by ET-1 injection near the optic nerve.

Characterization of endothelin-converting enzyme activities in ARPE-19 cells, a human retinal pigmented epithelial cell line.

Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET- 1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line.