7836 Targeting of Mineralocorticoid Receptor Expression by miR-124-3p-Loaded Extracellular Vesicles as Potential Therapy for Vascular Cognitive Impairment
Authors: Birch Lara Anne , Fullerton Josie L , MacKenzie Scott Mackay , Work Lorraine M , Davies Eleanor 
Published: October 2024

Disclosure: L.A. Birch: None. J.L. Fullerton: None. S.M. MacKenzie: None. L.M. Work: None. E. Davies: None.

Vascular cognitive impairment (VCI) encompasses all disorders of vascular origin that result in cognitive deficits. Stroke is a leading cause of death, and doubles risk of VCI, in turn those with VCI have an increased risk of stroke. Hypertension is the most significant modifiable risk factor for both stroke and VCI. The mineralocorticoid aldosterone is an essential regulator of blood pressure, acting via the mineralocorticoid receptor (MR), and hyperaldosteronism increases risk of cerebrovascular disease including stroke and VCI. In addition to its epithelial role, MR is distributed throughout the vasculature and brain and has potential as a twofold target for VCI given that MR antagonism has proved beneficial in both stroke and VCI. Expression of the MR gene, NR3C2, can be repressed by microRNAs post-transcriptionally binding NR3C2 mRNA at its 3’ untranslated region (3’UTR). MicroRNAs circulate within extracellular vesicles (EVs), which have shown promise in therapeutic delivery and can cross the blood-brain barrier. This project aims to target MR expression by EV-mediated delivery of miRNAs targeting the NR3C2-3’UTR In silico analysis identified 74 microRNAs as potentially binding the NR3C2-3’UTR. Several were also highlighted in a recent review of circulating microRNAs implicated in stroke, including the brain-specific microRNA-124-3p (miR-124-3p). Pathway analysis also revealed additional mRNA targets of miR-124-3p relevant to VCI and aldosterone signalling, including serum/glucocorticoid regulated kinase 1 (SGK1), a protein kinase downstream of MR that mediates aldosterone action. We confirmed binding of miR-124-3p to the NR3C2-3’UTR in vitro by dual luciferase assay, with significantly decreased reporter gene expression observed after treatment with miR-124-3p precursor. Following induction of ischaemic stroke in stroke-prone spontaneously hypertensive rats by the transient middle cerebral artery occlusion model, brain miR-124-3p levels were significantly altered, with reduced expression confined to the infarct region. EVs were then isolated from rat plasma using size exclusion chromatography and loaded with miR-124-3p mimic by electroporation. When incubated with B50 rat neuronal cells, these EVs resulted in a significant 2-fold increase in miR-124-3p level, indicating successful delivery. No change in NR3C2 or SGK1 mRNA level was observed. Similarly, following oxygen-glucose deprivation, loaded EV treatment increased miR-124-3p levels but NR3C2 and SGK1 mRNA levels were unchanged. MR protein levels are still to be quantified. In conclusion, miRNA-124-3p is a promising therapeutic mediator of NR3C2 expression. Ongoing studies will include further in vitro characterization of its effects involving additional cell types of the neurovascular unit and exploration of neuroprotective effects in an in vivo bilateral carotid artery stenosis model of VCI.

Presentation: 6/3/2024