Altered trafficking of miRNAs at mitochondria modulates mitochondrial functions and cell death in brain ischemia.

Stroke is one of the major causes of death and disabilities worldwide. The rapid induction of cell death by necrosis and apoptosis is observed at the ischemic core, while long lasting apoptosis and brain inflammation continue in the penumbra. The emerging evidence suggests a critical role of mitochondria in acute and chronic inflammation and cell death. Mitochondrial dysfunction may result in the release of mitokines and/or mitochondrial DNA into the cytoplasm and activate multiple cytosolic pathways which in turn triggers inflammation. The role of miRNA, specifically mitochondria-associated miRNAs (mitomiRs) in the regulation of mitochondrial functions is emerging. In the current study, we hypothesized that ischemia-induced mitomiRs may modulate the mitochondrial functions and such alterations under stress conditions may lead to mitochondrial dysfunction and cell death. We have demonstrated the specific pattern of miRNAs associated with mitochondria that is altered under ischemic condition induced by transient middle artery occlusion (tMCAo) in rats. The putative targets of altered miRNAs include several mitochondrial proteins which signifies their involvement in maintaining mitochondrial homeostasis. The alteration of selected miRNAs in mitochondria was further detected in a cellular models when hypoxia was induced using a chemical agent CoCl2, in three cell lines. Two candidate mitomiRs, hsa-miR-149-3p and hsa-miR-204-5p were further analyzed for their functional role during in vitro hypoxia by transfecting mitomiR mimics into cells and determining critical mitochondrial functions and cell viability. The results here emphasize the role of certain mitomiRs as an important modulator of mitochondrial function under the ischemic condition.

Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas.

Glioblastoma (GBM) is the most aggressive primary brain tumor, with ineffective anti-tumor responses and a poor prognosis despite aggressive treatments. GBM immune microenvironment is heterogenous  and activation of specific immune populations in GBM is not fully characterized. Reliable animal models are critical for defining mechanisms of anti-tumor immunity. First we analyzed the immune subpopulations present in rat C6 gliomas. Using flow cytometry we determined kinetics of infiltration of myeloid cells and T lymphocytes into glioma-bearing brains. We found significant increases of the amoeboid, pro-tumorigenic microglia/macrophages, T helper (Th) and T regulatory (Treg) cells in tumor-bearing brains, and rare infiltrating T cytotoxic (Tc) cells. Transcriptomic analyses of glioma-bearing hemispheres revealed overexpression of invasion and immunosuppression-related genes, reflecting the immunosuppressive microenvironment. Microglia, sorted as CD11b+CD45low cells from gliomas, displayed the pro-invasive and immunosuppressive type of activation. Accumulation of Th and Treg cells combined with the reduced presence of Tc lymphocytes in rat gliomas may result in the lack of effective anti-tumor responses. Transcriptional profiles of CD11b+ cells and composition of immune infiltrates in C6 gliomas indicate that rat C6 gliomas employ similar immune system evasion strategies as human GBMs.

Copious urinary excretion of a male Syrian hamster (Mesocricetus auratus) salivary gland protein after its endocrine-like release upon ß-adrenergic stimulation.

Salivary glands, although widely considered as typically exocrine, may also release specific proteins in an endocrine manner. However, endocrine release of salivary gland proteins is not generally acknowledged since the evidences are not easily demonstrable. Submandibular salivary glands (SMG) of male Syrian hamsters express male-specific secretory proteins (MSP; which are lipocalins) visible in SDS-PAGE of SMG extracts, as major bands and also detectable in immunoblots of whole-saliva and urine as low MSP crossreactions. We report here that MSP is localized in acinar cells of SMG and acute treatment with isoproterenol (IPR; non-specific ß1/ß2-adrenergic agonist) results in considerable release of MSP in SMG-saliva. Moreover, acute IPR treatment markedly depletes SMG-MSP in a dose- and time-dependent manner. However, MSP depleted from SMG, far exceeds that recovered in SMG-saliva. Blood, submandibular lymph nodes and kidney of IPR-treated males showed MSP crossreactions and SDS-PAGE of their urine revealed profuse MSP excretion; this was undetectable in IPR-treated-SMG-ablated males, confirming that a substantial amount of MSP depleted from SMG after IPR treatment enters circulation and is excreted in urine. Treatments with specific ß1- or ß2-adrenergic agonists also reduced SMG-MSP levels and resulted in copious urinary excretion of MSP. Co-treatments with specific ß1/ß2-blockers indicated that above effects of IPR, ß1- and even ß2-agonists are very likely mediated by ß1-adrenoceptors. MSP's detection by SDS-PAGE in urine after ß-agonist treatment is a compelling and easily demonstrable evidence of release into circulation of a salivary gland protein. The possible means (endocrine-like or otherwise) of MSP's release into circulation and significance of its presence in saliva, blood and urine of male hamsters are discussed.