Abstract
MicroRNAs have gained popularity as a potential treatment for many diseases, including stroke. This study identifies and characterizes a specific member of the miR-17–92 cluster, miR-20a-3p, as a possible stroke therapeutic. A comprehensive microRNA screening showed that miR-20a-3p was significantly upregulated in astrocytes of adult female rats, which typically have better stroke outcomes, while it was profoundly downregulated in astrocytes of middle-aged females and adult and middle-aged males, groups that typically have more severe stroke outcomes. Assays using primary human astrocytes and neurons show that miR-20a-3p treatment alters mitochondrial dynamics in both cell types. To assess whether stroke outcomes could be improved by elevating astrocytic miR-20a-3p, we created a tetracycline (Tet)-induced recombinant adeno-associated virus (rAAV) construct where miR-20a-3p was located downstream a glial fibrillary acidic protein promoter. Treatment with doxycycline induced miR-20-3p expression in astrocytes, reducing mortality and modestly improving sensory motor behavior. A second Tet-induced rAAV construct was created in which miR-20a-3p was located downstream of a neuron-specific enolase (NSE) promoter. These experiments demonstrate that neuronal expression of miR-20a-3p is vastly more neuroprotective than astrocytic expression, with animals receiving the miR-20a-3p vector showing reduced infarction and sensory motor improvement. Intravenous injections, which are a therapeutically tractable treatment route, with miR-20a-3p mimic 4 h after middle cerebral artery occlusion (MCAo) significantly improved stroke outcomes including infarct volume and sensory motor performance. Improvement was not observed when miR-20a-3p was given immediately or 24 h after MCAo, identifying a unique delayed therapeutic window. Overall, this study identifies a novel neuroprotective microRNA and characterizes several key pathways by which it can improve stroke outcomes.
Highlights
Stroke remains the leading cause of disability and the fifth leading cause of mortality in the USA
Ischemic stroke accounts for 80% of all strokes, and there is currently only one FDA-approved drug therapy, tissue plasminogen activator. tPA must be administered within a short time window of 3–4.5 h to be effective, which limits the proportion of patients eligible to receive this treatment (5.9–7.0%) [1, 2]
Astrocytes were extracted at 48 h post stroke from adult and middle-aged females and males using positive selection for GLAST
Summary
Since stroke risk and stroke severity are modified by the age and biological sex of the patient, it is imperative for preclinical studies of potential stroke therapies to address both variables. Epigenetic modifiers, such as small non-coding RNAs, have emerged as powerful candidates for several diseases. In the case of ischemic stroke, several studies have shown that miRNA mimics or antagomirs can regulate acute and chronic stroke outcomes [8,9,10,11,12]. MiR363-3p, identified by miRNA profiling of serum, modifies stroke outcomes only in females (adult and middle-aged) but not age-matched males [14]. In order to identify a more universally effective microRNA treatment, the present study used a novel approach by focusing on astrocytes
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