The role of miRNA in regulating Progesterone's neuroprotective function in the ischemic brain

Abstract: Stroke has been reported as the fourth leading cause of death for Americans and it is a leading cause of adult disability. The risk of ischemic stroke increases significantly with aging. Gender appears to play a profound role, with the incidence being higher in women. A large body of studies has suggested that women in postmenopausal state are at greater risk of ischemic stroke and are likely to experience much more severe impacts. A considerable amount of research has supported that progesterone (P4) is a potent neuroprotectant that may exert beneficial effects in various neurodegenerative diseases and stroke. Our laboratory has reported that Brain-derived neurotrophic factor (BDNF) is a critical mediator for P4 neuroprotective actions. BDNF has well-defined roles in synaptogenesis and neuronal survival. We recently reported that P4 enhances BDNF release from glia, but not from neurons, by acting via a novel membrane-associated progesterone receptor, Pgrmc1. Here, we identified a member of the Let-7 microRNA (miRNA) family as a potential negative regulator of Pgrmc1. Our data demonstrated an inverse association between the expression levels of Let-7 and the transcripts of Pgrmc1 and BDNF in post-ischemic mouse cortex. Literature supports the antagomir (synthetic inhibitor) of Let-7 miRNA significantly reduced infarct volume and improved neurological deficits in a rodent ischemic stroke model. When combined, these lines of evidence have strongly supported our hypothesis that in the stroked brain, Let-7 negatively regulates Pgrmc1 gene expression, which disrupts P4-induced BDNF release from glia and ultimately leads to the attenuation of P4’s positive effect on synaptogenesis. Methods:

1. Cell culture: Mouse primary cortical neurons and primary cortical astrocytes were derived from postnatal day 1 male pups. Primary astrocytes were maintained in DMEM with sodium pyruvate/10% FBS + 1% penicillin/streptomycin. Primary neurons were maintained in Neurobasal A media with 10% FBS + 1% penicillin/streptomycin. 2.Treatment of primary astrocytes: Primary astrocytes were transfected with either Let-7 mimics or inhibitors. qPCR was used to confirm the overexpression and knock down of Let-7, as well as expression of their potential targets. Western blotting was used the determine the protein levels of the miRNA potential targets. 3.Treatment of neuronal cultures with conditioned media (CM) from glia: Primary astrocytes were transfected with miRNA mimics/inhibitors. Then cultures were treated with vehicle control (0.1% DMSO ) or 10 nM P4 for 18 hrs. The CM was concentrated by Protein Concentrators with 9 KDa molecular-weight cutoff (Pierce) to remove P4. Concentrated CM was then applied to neuronal cultures.
2. Animal treatment: Female C57BL/6J mice were bilaterally ovariectomized at 4.5 months old to eliminate endogenous ovarian production of P4. One week afterward, P4 pellets or cholesterol pellets were implanted in these animal subcutaneously. 2 days after pellets implantation, ischemic stroke was induced in these mice using the middle cerebral arteries occlusion method. 24hrs after stroke induction, the antagomir of let-7or scrambled control were delivered to the penumbra via intracerebral ventricular (ICV) injection.