Mechanisms contributing to inhibition of retinal ganglion cell death by cell permeable peptain-1 under glaucomatous stress.

This study assesses the neuroprotective potential of CPP-P1, a conjugate of an anti-apoptotic peptain-1 (P1) and a cell-penetrating peptide (CPP) in in vitro, in vivo, and ex vivo glaucoma models. Primary retinal ganglion cells (RGCs) were subjected to either neurotrophic factor (NF) deprivation for 48 h or endothelin-3 (ET-3) treatment for 24 h and received either CPP-P1 or vehicle. RGC survival was analyzed using a Live/Dead assay. Axotomized human retinal explants were treated with CPP-P1 or vehicle for seven days, stained with RGC marker RBPMS, and RGC survival was analyzed. Brown Norway (BN) rats with elevated intraocular pressure (IOP) received weekly intravitreal injections of CPP-P1 or vehicle for six weeks. RGC function was evaluated using a pattern electroretinogram (PERG). RGC and axonal damage were also assessed. RGCs from ocular hypertensive rats treated with CPP-P1 or vehicle for seven days were isolated for transcriptomic analysis. RGCs subjected to 48 h of NF deprivation were used for qPCR target confirmation. NF deprivation led to a significant loss of RGCs, which was markedly reduced by CPP-P1 treatment. CPP-P1 also decreased ET-3-mediated RGC death. In ex vivo human retinal explants, CPP-P1 decreased RGC loss. IOP elevation resulted in significant RGC loss in mid-peripheral and peripheral retinas compared to that in naive rats, which was significantly reduced by CPP-P1 treatment. PERG amplitude decline in IOP-elevated rats was mitigated by CPP-P1 treatment. Following IOP elevation in BN rats, the transcriptomic analysis showed over 6,000 differentially expressed genes in the CPP-P1 group compared to the vehicle-treated group. Upregulated pathways included CREB signaling and synaptogenesis. A significant increase in Creb1 mRNA and elevated phosphorylated Creb were observed in CPP-P1-treated RGCs. Our study showed that CPP-P1 is neuroprotective through CREB signaling enhancement in several settings that mimic glaucomatous conditions. The findings from this study are significant as they address the pressing need for the development of efficacious therapeutic strategies to maintain RGC viability and functionality associated with glaucoma.

Brain-derived neuerotrophic factor and related mechanisms that mediate and influence progesterone-induced neuroprotection.

Historically, progesterone has been studied significantly within the context of reproductive biology. However, there is now an abundance of evidence for its role in regions of the central nervous system (CNS) associated with such non-reproductive functions that include cognition and affect. Here, we describe mechanisms of progesterone action that support its brain-protective effects, and focus particularly on the role of neurotrophins (such as brain-derived neurotrophic factor, BDNF), the receptors that are critical for their regulation, and the role of certain microRNA in influencing the brain-protective effects of progesterone. In addition, we describe evidence to support the particular importance of glia in mediating the neuroprotective effects of progesterone. Through this review of these mechanisms and our own prior published work, we offer insight into why the effects of a progestin on brain protection may be dependent on the type of progestin (e.g., progesterone versus the synthetic, medroxyprogesterone acetate) used, and age, and as such, we offer insight into the future clinical implication of progesterone treatment for such disorders that include Alzheimer's disease, stroke, and traumatic brain injury.

Estradiol and 3ß-diol protect female cortical astrocytes by regulating connexin 43 Gap Junctions.

While estrogens have been described to protect or preserve neuronal function in the face of insults such as oxidative stress, the prevailing mechanistic model would suggest that these steroids exert direct effects on the neurons. However, there is growing evidence that glial cells, such as astrocytes, are key cellular mediators of protection. Noting that connexin 43 (Cx43), a protein highly expressed in astrocytes, plays a key role in mediating inter-cellular communication, we hypothesized that Cx43 is a target of estradiol (E2), and the estrogenic metabolite of DHT, 3ß-diol. Additionally, we sought to determine if either or both of these hormones attenuate oxidative stress-induced cytotoxicity by eliciting a reduction in Cx43 expression or inhibition of Cx43 channel permeability. Using primary cortical astrocytes, we found that E2 and 3ß-diol were each protective against the mixed metabolic/oxidative insult, iodoacetic acid (IAA). Moreover, these effects were blocked by estrogen receptor antagonists. However, E2 and 3ß-diol did not alter Cx43 mRNA levels in astrocytes but did inhibit IAA-induced Cx43 gap junction opening/permeability. Taken together, these data implicate astrocyte Cx43 gap junction as an understudied mediator of the cytoprotective effects of estrogens in the brain. Given the wide breadth of disease states associated with Cx43 function/dysfunction, further understanding the relationship between gonadal steroids and Cx43 channels may contribute to a better understanding of the biological basis for sex differences in various diseases.

The endothelin receptor antagonist macitentan ameliorates endothelin-mediated vasoconstriction and promotes the survival of retinal ganglion cells in rats.

Glaucoma is a chronic and progressive eye disease, commonly associated with elevated intraocular pressure (IOP) and characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The pathological changes in glaucoma are triggered by multiple mechanisms and both mechanical effects and vascular factors are thought to contribute to the etiology of glaucoma. Various studies have shown that endothelin-1 (ET-1), a vasoactive peptide, acting through its G protein coupled receptors, ETA and ETB, plays a pathophysiologic role in glaucoma. However, the mechanisms by which ET-1 contribute to neurodegeneration remain to be completely understood. Our laboratory and others demonstrated that macitentan (MAC), a pan endothelin receptor antagonist, has neuroprotective effects in rodent models of IOP elevation. The current study aimed to determine if oral administration of a dual endothelin antagonist, macitentan, could promote neuroprotection in an acute model of intravitreal administration of ET-1. We demonstrate that vasoconstriction following the intravitreal administration of ET-1 was attenuated by dietary administration of the ETA/ETB dual receptor antagonist, macitentan (5 mg/kg body weight) in retired breeder Brown Norway rats. ET-1 intravitreal injection produced a 40% loss of RGCs, which was significantly lower in macitentan-treated rats. We also evaluated the expression levels of glial fibrillary acidic protein (GFAP) at 24 h and 7 days post intravitreal administration of ET-1 in Brown Norway rats as well as following ET-1 treatment in cultured human optic nerve head astrocytes. We observed that at the 24 h time point the expression levels of GFAP was upregulated (indicative of glial activation) following intravitreal ET-1 administration in both retina and optic nerve head regions. However, following macitentan administration for 7 days after intravitreal ET-1 administration, we observed an upregulation of GFAP expression, compared to untreated rats injected intravitreally with ET-1 alone. Macitentan treatment in ET-1 administered rats showed protection of RGC somas but was not able to preserve axonal integrity and functionality. The endothelin receptor antagonist, macitentan, has neuroprotective effects in the retinas of Brown Norway rats acting through different mechanisms, including enhancement of RGC survival and reduction of ET-1 mediated vasoconstriction.

Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells.

Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.

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. Retinal thickness was measured by spectral-domain optical coherence tomography (SD-OCT). The electroretinogram and pattern ERG (PERG) were used to assess retinal function. RGC survival was determined by counting RBPMS-positive RGCs and immunohistochemical analysis of superoxide dismutase 1 (SOD1) levels was carried out in the retina sections. Concentrations of SA-2 in the retina and choroid were determined using HPLC and MS. In addition, the direct effect of SA-2 treatment on RGC survival was assessed in ex vivo rat retinal explants under hypoxic (0.5% O2) conditions. Results: Compound SA-2 did not induce any appreciable change in retinal thickness, or in a- or b-wave amplitude in naive animals. SA-2 was found to be bioavailable in both the retina and choroid after a single intravitreal injection (2% wt/vol). An increase in SOD1 levels in the retina of mice subjected to ONC and SA-2 treatment, suggests an enhancement in antioxidant activity. SA-2 provided significant (P < 0.05) RGC protection in all three of the tested RGC injury models in rodents. PERG amplitudes were significantly higher in both I/R and ONC mouse eyes following SA-2 treatment (P ≤ 0.001) in comparison with the vehicle and control groups. Conclusions: Compound SA-2 was effective in preventing RGC death and loss of function in three different rodent models of acute RGC injury: ONC, I/R, and hypoxia.

Neuroprotective effects of curcumin on endothelin-1 mediated cell death in hippocampal neurons.

OBJECTIVES: Alzheimer's disease is a progressive neurodegenerative disease characterized by loss of hippocampal neurons leading to memory deficits and cognitive decline. Studies suggest that levels of the vasoactive peptide endothelin-1 (ET-1) are increased in the brain tissue of Alzheimer's patients. Curcumin, the main ingredient of the spice turmeric, has been shown to have anti-inflammatory, anti-cancer, and neuroprotective effects. However, the mechanisms underlying some of these beneficial effects are not completely understood. The objective of this study was to determine if curcumin could protect hippocampal neurons from ET-1 mediated cell death and examine the involvement of c-Jun in this pathway. METHODS: Primary hippocampal neurons from rat pups were isolated using a previously published protocol. Viability of the cells was measured by the live/dead assay. Immunoblot and immunohistochemical analyses were performed to analyze c-Jun levels in hippocampal neurons treated with either ET-1 or a combination of ET-1 and curcumin. Apoptotic changes were evaluated by immunoblot detection of cleaved caspase-3, cleaved fodrin, and a caspase 3/7 activation assay. RESULTS: ET-1 treatment produced a 2-fold increase in the levels of c-Jun as determined by an immunoblot analysis in hippocampal neurons. Co-treatment with curcumin significantly attenuated the ET-1 mediated increase in c-Jun levels. ET-1 caused increased neuronal cell death of hippocampal neurons indicated by elevation of cleaved caspase-3, cleaved fodrin and an increased activity of caspases 3 and 7 which was attenuated by co-treatment with curcumin. Blockade of JNK, an upstream effector of c-Jun by specific inhibitor SP600125 did not fully protect from ET-1 mediated activation of pro-apoptotic enzymes in primary hippocampal cells. DISCUSSION: Our data suggests that one mechanism by which curcumin protects against ET-1-mediated cell death is through blocking an increase in c-Jun levels. Other possible mechanisms include decreasing pro-apoptotic signaling activated by ET-1 in primary hippocampal neurons.