Retinal Neuroprotective Effects of Flibanserin, an FDA-Approved Dual Serotonin Receptor Agonist-Antagonist

Purpose: To assess the neuroprotective effects of flibanserin (formerly BIMT-17), a dual 5-HT1A agonist and 5-HT2A antagonist, in a light-induced retinopathy model. Methods: Albino BALB/c mice were injected intraperitoneally with either vehicle or increasing doses of flibanserin ranging from 0.75 to 15 mg/kg flibanserin. To assess 5-HT1A-mediated effects, BALB/c mice were injected with 10 mg/kg WAY 100635, a 5-HT1A antagonist, prior to 6 mg/kg flibanserin and 5-HT1A knockout mice were injected with 6 mg/kg flibanserin. Injections were administered once immediately prior to light exposure or over the course of five days. Light exposure lasted for one hour at an intensity of 10,000 lux. Retinal structure was assessed using spectral domain optical coherence tomography and retinal function was assessed using electroretinography. To investigate the mechanisms of flibanserin-mediated neuroprotection, gene expression, measured by RT-qPCR, was assessed following five days of daily 15 mg/kg flibanserin injections. Results: A five-day treatment regimen of 3 to 15 mg/kg of flibanserin significantly preserved outer retinal structure and function in a dose-dependent manner. Additionally, a single-day treatment regimen of 6 to 15 mg/kg of flibanserin still provided significant protection. The action of flibanserin was hindered by the 5-HT1A antagonist, WAY 100635, and was not effective in 5-HT1A knockout mice. Creb, c-Jun, c-Fos, Bcl-2, Cast1, Nqo1, Sod1, and Cat were significantly increased in flibanserin-injected mice versus vehicle-injected mice. Conclusions: Intraperitoneal delivery of flibanserin in a light-induced retinopathy mouse model provides retinal neuroprotection. Mechanistic data suggests that this effect is mediated through 5-HT1A receptors and that flibanserin augments the expression of genes capable of reducing mitochondrial dysfunction and oxidative stress. Since flibanserin is already FDA-approved for other indications, the potential to repurpose this drug for treating retinal degenerations merits further investigation

Spontaneous allelic variant in deafness–blindness gene \u3ci\u3eUsh1g\u3c/i\u3e resulting in an expanded phenotype

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bwnull mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell\u27s kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw/+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene

The effects of PEGylation on LNP based mRNA delivery to the eye.

Gene therapy is now an effective approach to treat many forms of retinal degeneration. Delivery agents that are cell-specific, allow for multiple dosing regimens, and have low immunogenicity are needed to expand the utility of gene therapy for the retina. We generated eight novel lipid nanoparticles (LNPs) ranging in size from 50 nm to 150 nm by changing the PEG content from 5% to 0.5%, respectively. Subretinal injections of LNP-mRNA encoding luciferase revealed that 0.5% PEG content within nanoparticles elicits the highest expression. Similar injections of LNP delivered cre mRNA into Ai9 mice revealed cell-specific protein expression in the retinal pigment epithelium (RPE), confirmed by fundus photography and immunohistochemistry of whole globe cross-sections. To investigate mechanisms of LNP delivery to the eye, we injected mCherry mRNA using the subretinal approach in apoE-/- and Mertk-/- mice. RPE transfection was observed in both mouse models suggesting that LNP intracellular delivery is not solely dependent on apolipoprotein adsorption or phagocytosis. To investigate LNP penetration, particles were delivered to the vitreous chamber via an intravitreal injection. The 0.5% PEG particles mediated the highest luciferase activity and expression was observed in the Müller glia, the optic nerve head and the trabecular meshwork, but failed to reach the RPE. Overall, particles containing less PEG (~150 nm in size) mediated the highest expression in the eye. Thus far, these particles successfully transfect RPE, Müller cells, the optic nerve head and the trabecular meshwork based on route of administration which can expand the utility of LNP-mediated gene therapies for the eye

Increasing the Efficacy of Gold Nanorod Uptake in Stem Cell-Derived Therapeutic Cells: Implications for Stem Cell Labeling and Optical Coherence Tomography Imaging

The advancement of safe nanomaterials for use as optical coherence tomography (OCT) imaging and stem cell-labeling agents to longitudinally visually track therapeutic derived retinal stem cells to study their migration, survival rate, and efficacy is challenged by instability, intracellular aggregation, low uptake, and cytotoxicity. Here, we describe a series of hybrid lipid-coated gold nanorods (AuNRs) that could solve these issues. These nanomaterials were made via a layer-by-layer assembly approach, and their stability in biological media, mechanism, efficiency of uptake, and toxicity were compared with a commercially available set of AuNRs with a 5 nm mesoporous silica (mSiO2)-polymer coating. These nanomaterials can serve as stem cell labeling and OCT imaging agents because they absorb in the near-infrared (NIR) region away from biological tissues. Although both subtypes of AuNRs were taken up by retinal pigment epithelial, neural progenitor, and baby hamster kidney cells, slightly negatively charged hybrid lipid-coated AuNRs had minimal aggregation in biological media and within the cytoplasm of cells ( ∼3000 AuNRs / cell ) as well as minimal impact on cell health. Hybrid lipid-coated AuNRs modified with cell-penetrating peptides had the least toxicological impact, with \u3e92% cell viability. In contrast, the more “sticky” AuNRs with a 5 nm mSiO2-polymer coating showed significant aggregation in biological media and within the cytoplasm with lower-than-expected uptake of AuNRs ( ∼5400 of AuNRs/cell ) given their highly positive surface charge ( 35+ mV ). Collectively, we have demonstrated that hybrid lipid-coated AuNRs, which absorb in the NIR-II region away from biological tissues, with tuned surface chemistry can label therapeutic derived stem cells with minimal aggregation and impact on cell health as well as enhance uptake for OCT imaging applications

y-secretase inhibition of murine choroidal neovascularization is associated with reduction of superoxide and proinflammatory cytokines

PURPOSE. This study aimed to determine whether upregulation of γ-secretase could inhibit laser-induced choroidal neovascularization (CNV) and if this was associated with a reduction in both oxidative stress and proinflammatory cytokines. METHODS. γ-Secretase, or its catalytic subunit presenilin 1 (PS1), were upregulated by exposure to either pigment epithelial derived factor (PEDF) or an AAV2 vector containing a PS1 gene driven by a vascular endothelial-cadherin promoter. Retinal endothelial cells were infected with AAV2 or exposed to PEDF in the presence or absence of VEGF and in vitro angiogenesis determined. Mouse eyes either received intravitreal injection of PEDF, DAPT (a γ-secretase inhibitor) or PEDF + DAPT at the time of laser injury, or AAV2 infection 3 weeks before receiving laser burns. Lesion volume was determined 14 days post laser injury. Superoxide generation, antioxidant activity and the production of proinflammatory mediators were assessed. Knockdown of γ-secretase was achieved using siRNA. RESULTS. γ-Secretase upregulation and PS1 overexpression suppressed VEGF-induced in vitro angiogenesis and in vivo laser-induced CNV. This was associated with a reduction in the expression of VEGF and angiogenin 1 together with reduced superoxide anion generation and an increase in MnSOD compared with untreated CNV eyes. PS1 overexpression reduced proinflammatory factors and microglial activation in eyes with CNV compared with control. siRNA inhibition of γ-secretase resulted in increased angiogenesis. CONCLUSIONS. γ-Secretase, and in particular PS1 alone, are potent regulators of angiogenesis and this is due in part to stabilizing endogenous superoxide generation and reducing proinflammatory cytokine expression during CNV

Flibanserin’s neuroprotective effects are 5-HT_1A receptor-mediated.

<p>(<b>A i</b>) A spider graph representing average right-eye receptor plus values demonstrates that pre-treatment with WAY 100635 prior to 6 mg/kg flibanserin (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) decreased average receptor plus thickness as compared to flibanserin-treatment without WAY 100635 (6 mg/kg Flibanserin, <i>blue</i>). A 6 mg/kg dose of flibanserin in 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>) resulted in average receptor plus thickness that was not significantly different from vehicle-injected and naïve mice. The gray area indicates ± 2 SD of the naïve averaged data. (<b>A ii, A iii</b>) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that both pre-treatment of BALB/c mice with WAY 100635 prior to a flibanserin injection (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) and a flibanserin injection in 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>) significantly reduces the observed neuroprotective effects provided by a single 6 mg/kg dose of flibanserin (6 mg/kg Flibanserin, <i>blue</i>) in the <b>(A ii)</b> temporal quadrant and the <b>(A iii)</b> nasal quadrant. (<b>B</b>) Pre-treatment with WAY 100635 (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) mitigated the improvements to ERG a- and b-wave amplitudes provided by a single 6 mg/kg dose of flibanserin (<i>blue</i>). ERG a- and b-wave amplitudes were not improved by a single dose of 6 mg/kg flibanserin when administered to 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>). “†” indicates the flash intensity providing the b_(max,rod) value at which statistical analyses was also performed and presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159776#pone.0159776.s001" target="_blank">S1C Fig</a>. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05.</p

A one-day time course of flibanserin can preserve retinal morphology and function.

<p><b>(A i)</b> A spider graph representing average right-eye receptor plus values demonstrates that a single dose of 6 mg/kg flibanserin or greater can significantly preserve retinal morphology to thicknesses comparable to naïve mice. The gray area indicates ± 2 SD of the naïve averaged data. (<b>A ii, iii</b>) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that 6 mg/kg flibanserin provides morphological protection in the <b>(A ii)</b> temporal quadrant and the <b>(A iii)</b> nasal quadrant. Group averages are represented as mean ± standard error bar. <b>(B)</b> Mice treated with doses of 6 mg/kg and greater of flibanserin showed significantly higher ERG b-wave and a-wave responses at the highest flash intensity (3.55 log cd•s/m²) as compared to vehicle-treated mice. “†” indicates the greatest flash intensity that elicits a rod-only response (b_(max,rod)). Group differences at the “†” flash intensity were statistically evaluated and are represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159776#pone.0159776.s001" target="_blank">S1B Fig</a>. Individual ERGs were averaged with ERGs for mice within group and are represented as mean ± standard error. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05.</p