Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.视网膜色素变性（Retinitis pigmentosa, RP）是一种可导致感光细胞丢失的退行性疾病。视网膜色素变性的小鼠模型，如rd10小鼠（B6.CXBl-Pde6brd10/J），加深了我们对该疾病的认知，并为潜在治疗手段的开发提供了支撑。2011年，本团队首次证实合成孕激素类似物炔诺孕酮（Norgestrel）在两种视网膜退行性变小鼠模型（包括rd10小鼠）中具有神经保护作用。此后我们阐明了炔诺孕酮保护受胁迫感光细胞的多种机制，例如上调生长因子的表达。因此本研究旨在进一步解析炔诺孕酮的神经保护作用，具体而言，我们旨在探究小胶质细胞（microglia）可能发挥的作用——因为已有研究表明，小胶质细胞介导的炎症反应会加剧神经退行性病变。本研究向携带出生后第10天（P10）rd10幼崽的母鼠投喂添加炔诺孕酮的饲料（剂量为80mg/kg），幼鼠断奶后仍持续摄入炔诺孕酮。随后分别从喂食对照饲料或添加炔诺孕酮饲料的P15至P50的rd10小鼠体内采集组织样本。结果显示，在rd10小鼠中，投喂含炔诺孕酮的饲料可在P40前为视网膜提供显著的保护作用。小胶质细胞活性的改变与显著的保护作用相契合，表明小胶质细胞的变化参与了炔诺孕酮介导的神经保护过程。通过原代视网膜小胶质细胞与661W感光细胞样细胞的体外培养实验，我们证实rd10小鼠的小胶质细胞可诱导神经元细胞死亡。我们揭示了炔诺孕酮的全新作用机制：其可直接作用于小胶质细胞，减少促炎活化并抑制神经元细胞死亡。炔诺孕酮可有效抑制rd10小鼠视网膜中细胞因子、趋化因子以及危险相关分子模式分子（DAMP）的表达。值得注意的是，在rd10小鼠体内，炔诺孕酮可在RNA层面将fractalkine-CX3CR1信号通路的表达上调1000倍。已有研究证实，fractalkine-CX3CR1信号通路可通过调控视网膜小胶质细胞的活化与迁移发挥神经元保护作用。综上，本研究结果表明炔诺孕酮有望成为治疗视网膜色素变性的潜在手段，其在视网膜中兼具神经保护与抗炎双重功效。