Forkhead box P3 (Foxp3)+ regulatory T (Treg) cells maintain the immune tolerance and prevent inflammatory responses in the periphery. However, the presence of Treg cells in the central nervous system under steady state has not been studied. Here, for the first time, we show a substantial TCRαβ+CD4+Foxp3+ T-cell population (cerebral Treg cells) in the normal rat cerebrum, constituting more than 15% of the cerebral CD4+ T-cell compartment. Cerebral Treg cells showed an activated/memory phenotype and expressed many Treg-cell signature genes at higher levels than peripheral Treg cells. Consistent with their activated/memory phenotype, cerebral Treg cells robustly restrained the LPS-induced inflammatory responses of brain microglia/macrophages, suggesting a role in maintaining the cerebral homeostasis by inhibiting the neuroinflammation. In addition, brain astrocytes were the helper cells that sustained Foxp3 expression in Treg cells through IL-2/STAT5 signaling, showing that the interaction between astrocytes and Treg cells contributes to the maintenance of Treg-cell identity in the brain. Taken together, our work represents the first study to characterize the phenotypic and functional features of Treg cells in the normal rat cerebrum. Our data have provided a novel insight for the contribution of Treg cells to the immunosurveillance and immunomodulation in the cerebrum under steady state.

Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection in vitro and in rodent models of Parkinson’s disease and stroke. In the present study, we investigated the structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB’s 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.

Background: Glioblastoma is the most prevalent brain tumor with the poorest prognosis. Results: Methylene blue enhances oxygen consumption, reduces lactate production, and inhibits glioblastoma cell proliferation. Conclusion: Reversal of the Warburg effect could inhibit glioblastoma cell proliferation. Significance: Modulation of cancer cell bioenergetics and reversal of Warburg effect might provide a novel therapy for glioblastoma.

Background: Somatic IDH1 mutations are common in several types of cancer. Results: IDH1 mutation increases the proportion of palmitate derived from [13 C]glutamine under hypoxic conditions. Conclusion: IDH1 mutation can stimulate tumor cell reductive glutamine metabolism. Significance: IDH1 mutation may cause the unique glutamine-dependent metabolic phenotype observed in tumors.

Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca 2؉ entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP-and angiotensin IIinduced whole cell currents and Ca 2؉ entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose-and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca 2؉ entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Canonical transient receptor potential 6 (TRPC6) is a nonselective cation channel and participates in a diverse array of cellular functions by regulating intracellular Ca 2ϩ signaling (1). In particular, TRPC6 channels are highly expressed in vascular smooth muscle cells (VSMCs) 2 and play a key role in regulating myogenic tone in vascular tissues (2-4). Multiple mechanisms are involved in TRPC6 channel activation and regulation. These include membrane receptor activation (5), Ca 2ϩ store depletion (6), stretch (7,8), membrane lipids (9), and trafficking (10, 11). The distinct activation/regulation mechanisms may be tissue/cell type-specific and thus render TRPC6 a specific function in a particular site. For instance, mechanosensitive TRPC6 residing in glomerular podocytes (7, 12) and mesangial cells (13, 14) may regulate hydrostatic pressure-driven ultrafiltration in response to changes in glomerular capillary pressure. Most likely, different mechanisms may exist in the same cell and work together in a synergistic way to regulate the cell function more precisely and efficiently (8). We recently demonstrated that TRPC6 also was a redox-sensitive channel that was activated by H 2 O 2 in a TRPC6-expressing cell line (11). However, the physiological relevance of activation of the channel by reactive oxygen species (ROS) is completely unknown.ROS are produced in G protein-coupled receptor-signaling pathway (15, 16), a pathway also linked to TRPC6 channel activation. ROS not only function as an intracellular signaling molecule in a variety of cells but are also associated with many diseases, such as hypertension (15,17). In blood vessels, all types of vascular cells can produce ROS that modulate vasoactive agent-indu...