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Aims: The manner in which hydrogen sulfide (H 2 S) suppresses neuroinflammation is poorly understood. We investigated whether H 2 S polarized microglia to an anti-inflammatory (M2) phenotype by activating AMPactivated protein kinase (AMPK). Results: Three structurally unrelated H 2 S donors (5-(4-hydroxyphenyl)-3H-1,2-dithiocyclopentene-3-thione [ADT-OH], (p-methoxyphenyl) morpholino-phosphinodithioic acid [GYY4137], and sodium hydrosulfide [NaHS]) enhanced AMPK activation in BV2 microglial cells in the presence and absence of lipopolysaccharide (LPS). The overexpression of the H 2 S synthase cystathionine bsynthase (CBS) in BV2 cells enhanced endogenous H 2 S production and AMPK activation regardless of LPS stimulation. On LPS stimulation, overexpression of both ADT-OH and CBS promoted M2 polarization of BV2 cells, as evidenced by suppressed M1 and elevated M2 signature gene expression. The promoting effects of ADT-OH on M2 polarization were attenuated by an AMPK inhibitor or AMPK knockdown. Liver kinase B1 (LKB1) and calmodulin-dependent protein kinase kinase b (CaMKKb) are upstream kinases that activate AMPK. ADT-OH activated AMPK in Hela cells lacking LKB1. In contrast, both the CaMKKb inhibitor and siRNA abolished ADT-OH activation of AMPK in LPS-stimulated BV2 cells. Moreover, the CaMKKb inhibitor and siRNA blunted ADT-OH suppression on M1 gene expression and enhancement of M2 gene expression in LPS-stimulated BV2 cells. Moreover, ADT-OH promoted M2 polarization of primary microglia in an AMPK activation-and CaMKKb-dependent manner. Finally, in an LPS-induced in vivo neuroinflammation model, both ADT-OH and NaHS enhanced AMPK activation in the brain area where microglia were overactivated on LPS stimulation. Furthermore, ADT-OH suppressed M1 and promoted M2 gene expression in this in vivo model. Innovation and Conclusion: CaMKKb-dependent AMPK activation is an unrecognized mechanism underlying H 2 S suppression on neuroinflammation.

PTEN inhibitors administered prior to or immediately after experimental stroke confer acute neuroprotection. However, it remains unclear if delayed treatment with a PTEN inhibitord improves long-term functional recovery after stroke. We addressed the issue in this study. Adult male mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by treatment with a well-established PTEN inhibitor bpv or saline daily for 14 days, starting at 24 hours after MCAO. Functional recovery was assessed with behavioral tests and acute infarct volumes were analyzed histologically. Delayed bpv treatment did not reduce infarction during the acute phase, but significantly improved long-term functional recovery after MCAO. Since PTEN is a critical intrinsic inhibitory factor in axonal regeneration, we further examined bpv effects on axonal densities following MCAO using bielschowsky silver staining and immunohistochemistry with antibodies against myelin basic protein. Delayed bpv treatment significantly increased axon densities in the ischemic brain at 14 days after MCAO. Moreover, PTEN expression persistently remained high in the ischemic brain over 14 days after MCAO, and bpv treatment increased post-ischemic activation of Akt and mTOR in the ischemic brain. Akt and mTOR activation are the well-established cascades downstream to PTEN inhibition and have been shown to contribute to post-injury axonal regrowth in response to PTEN inhibition. Consistently, in an in vitro neuronal ischemia model, bpv enhanced axonal outgrowth of primary cortical neurons after oxygen-glucose deprivation and the enhancing effects were abolished by Akt/mTOR inhibition. In conclusion, delayed bpv treatment improved functional recovery from experimental stroke possibly via enhancing axonal growth and Akt/mTOR activation contributed to bpv-enhanced post-stroke axon growth.

To characterize the structures of N-glycans derived from human serum, we report a strategy that combines microchip electrophoresis, standard addition, enzymatic digestion, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We compared: (i) electrophoretic mobilities of known N-glycans from well-characterized (standard) glycoproteins through standard addition, (ii) the electrophoretic mobilities of N-glycans with their molecular weights determined by MALDI-MS, and (iii) electrophoretic profiles of N-glycans enzymatically treated with fucosidase. The key step to identify the sialylated N-glycans was to quantitatively neutralize the negative charge on both α2,3- and α2,6-linked sialic acids by covalent derivatization with methylamine. Both neutralized and nonsialylated N-glycans from these samples were then reacted with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) to provide a fluorescent label and a triple-negative charge, separated by microchip electrophoresis, and detected by laser-induced fluorescence. The methylamidation step leads to a 24% increase in the peak capacity of the separation and direct correlation of electrophoretic and MALDI-MS results. In total, 37 unique N-glycan structures were assigned to 52 different peaks recorded in the electropherograms of the serum samples. This strategy ensures the needed separation efficiency and detectability, easily resolves linkage and positional glycan isomers, and is highly reproducible.