INHIBITION OF LET-7I AS A MEANS TO ENHANCE THE NEUROPROTECTIVE EFFICACY OF PROGESTERONE IN THE ISCHEMIC BRAIN

The occurrence of ischemic stroke is relatively rare among pre-menopausal women. Strikingly, this risk doubles every 10 years after the menopausal transition; and women are likely to experience worse outcomes and higher mortality post stroke than men. Since both estrogen (E2) and progesterone (P4) levels decline precipitously following the menopause, this hormonal reduction may, at least partially, contribute to the higher risk and worse outcomes. By inference, these hormones could play a critical role in protecting women against ischemic stroke. In this dissertation project, we focus on P4, the relatively understudied of the two hormones. And while P4 has been shown to be a potent neuroprotectant in various experimental models of stroke, the underlying mechanisms remain unclear. One known mediator of P4's protective function is brain-derived-neurotrophic-factor (BDNF), which has an established role in promoting neuronal differentiation, survival, and synaptogenesis. In addition, emerging literature and data from our laboratory strongly support the indispensable role of glia in P4's neuroprotective program and thus, may also play a significant role in post-stroke recovery. We recently reported that P4 induces a significant release of BDNF from primary astrocytes, through a putative membrane-associated progesterone receptor consisting of progesterone-receptor-membrane-component-1 (Pgrmc1). This receptor is abundantly expressed in various regions of brain and mediates such effects of P4 in the central nervous system (CNS) as anti-apoptotic effects, spinogenesis, and BDNF release. What is not known, however, is how the expression of this receptor is regulated. This dissertation was aimed to elucidate what regulates the expression of Pgrmc1 and BDNF in glia and how such regulation influences the neuroprotective function of P4 in the ischemic brain. Based on the observation that Let-7i regulates the expression of Pgrmc1 in a peripheral cell type, and our in silico analysis that revealed that both Pgrmc1 and BDNF are potential targets of let-7i, we hypothesized that let-7i represses P4's neuroprotective effects by down-regulating the expression of both Pgrmc1 and BDNF in glia, leading to: 1) suppression of P4-induced BDNF release from glia, and 2) attenuation of the beneficial effects of P4 on neuronal survival and markers of synaptogenesis in the ischemic brain. Using primary cortical astrocytes as an experimental model, we found that let-7i negatively regulated the expression of Pgrmc1 and BDNF. This was correlated with a reduction in P4-induced BDNF release from these cells. Under such conditions of reduced expression of both Pgrmc1 and BDNF, P4 was unable to protect primary neurons against oxygen-glucose-deprivation (OGD) or regulate markers of synaptogenesis. In our in vivo model of transient ischemic stroke, we found that protective effects of P4 were greatly enhanced in animals that were concomitantly treated with an inhibitor (antagomir) of let-7i. The combined treatment also enhanced synaptogenesis in the peri-infarct region. Collectively, the data presented here suggested that in the ischemic brain, let-7i negatively influences P4-induced neuroprotection via regulation of the Pgrmc1/BDNF axis. As such, inhibition of let-7i maybe an effective means to enhance the efficacy of P4 in treating ischemic stroke.