Dysregulation of microRNAs (miRNAs) has been tied to several neurological disorders, including ischemic stroke. It has also been established that social environments can modulate miRNA profiles. We have previously shown that post-stroke social isolation (SI) is linked to poor stroke outcomes and that miR-181c-5p emerged as one of few lead miRNAs that was downregulated in both stroke and SI. Therefore, in this study we examined the potential role of miR-181c-5p mimic in reversing the detrimental effects of post-stroke SI. Two to three-month-old C57BL/6 male mice were pair-housed (PH) for at least two weeks. After two weeks, mice underwent stroke survival surgery using middle cerebral artery occlusion (MCAO) and were randomly assigned to one of two housing conditions: stroke isolation (ST-ISO) or stroke pair-housing with a healthy partner (ST-PH). ST-ISO mice were randomized to receive either miR-181c-5p mimic or a scrambled RNA (7 mg/kg i.v./day x drug) control at 24 h and 48 h after stroke. The effects of miR-181c-5p mimic treatment were evaluated at 1, 3, and 7 days after stroke at histological, behavioral, and biochemical levels. Target genes of miR-181c-5p were then analyzed by qPCR using an RT 2 Profiler qPCR Array of pre-coated miR-181c gene targets. Temporal profile expression data suggested that miR-181c-5p was significantly downregulated (p<0.05 vs ST-PH) up to 7 days after post-stroke SI. MiR-181c-5p mimic treatment significantly increased miR-181c-5p expression in brain tissue and showed partial swift recovery in sensorimotor deficit. Target gene analysis identified downregulation of several calcium signaling-related genes, e.g., Cpne2 and Gria 1 & 2 after miR-181c-5p mimic treatment. In summary, present data suggests that miR-181c-5p is a potential target for post-stroke SI. Data also suggests that genes related to calcium and glutamate signaling might be involved in the beneficial effect of the miR-181c-5p mimic.
Determining a molecule’s mechanism of action is
paramount
during chemical probe development and drug discovery. The cellular
thermal shift assay (CETSA) is a valuable tool to confirm target engagement
in cells for a small molecule that demonstrates a pharmacological
effect. CETSA directly detects biophysical interactions between ligands
and protein targets, which can alter a protein’s unfolding
and aggregation properties in response to thermal challenge. In traditional
CETSA experiments, each temperature requires an individual sample,
which restricts throughput and requires substantial optimization.
To capture the full aggregation profile of a protein from a single
sample, we developed a prototype real-time CETSA (RT-CETSA) platform
by coupling a real-time PCR instrument with a CCD camera to detect
luminescence. A thermally stable Nanoluciferase variant (ThermLuc)
was bioengineered to withstand unfolding at temperatures greater than
90 °C and was compatible with monitoring target engagement events
when fused to diverse targets. Utilizing well-characterized inhibitors
of lactate dehydrogenase alpha, RT-CETSA showed significant correlation
with enzymatic, biophysical, and other cell-based assays. A data analysis
pipeline was developed to enhance the sensitivity of RT-CETSA to detect
on-target binding. RT-CETSA technology advances capabilities of the
CETSA method and facilitates the identification of ligand-target engagement
in cells, a critical step in assessing the mechanism of action of
a small molecule.
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