Small Molecule-Activated O-GlcNAcase for Spatiotemporal Removal of O-GlcNAc in Live Cells.

The nutrient sensor O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification found on thousands of nucleocytoplasmic proteins. O-GlcNAc levels in cells dynamically respond to environmental cues in a temporal and spatial manner, leading to altered signal transduction and functional effects. The spatiotemporal regulation of O-GlcNAc levels would accelerate functional interrogation of O-GlcNAc and manipulation of cell behaviors for desired outcomes. Here, we report a strategy for spatiotemporal reduction of O-GlcNAc in live cells by designing an O-GlcNAcase (OGA) fused to an intein triggered by 4-hydroxytamoxifen (4-HT). After rational protein engineering and optimization, we identified an OGA-intein variant whose deglycosidase activity can be triggered in the desired subcellular compartments by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated that 4-HT activation of the OGA-intein fusion can likewise potentiate inhibitory effects in breast cancer cells by virtue of the reduction of O-GlcNAc. The spatiotemporal control of O-GlcNAc through the chemically activatable OGA-intein fusion will facilitate the manipulation and functional understanding of O-GlcNAc in live cells.

An update on Ym1 and its immunoregulatory role in diseases.

Ym1 is a rodent-specific chitinase-like protein (CLP) lacking catalytic activity, whose cellular origins are mainly macrophages, neutrophils and other cells. Although the detailed function of Ym1 remains poorly understood, Ym1 has been generally recognized as a fundamental feature of alternative activation of macrophages in mice and hence one of the prevalent detecting targets in macrophage phenotype distinguishment. Studies have pointed out that Ym1 may have regulatory effects, which are multifaceted and even contradictory, far more than just a mere marker. Allergic lung inflammation, parasite infection, autoimmune diseases, and central nervous system diseases have been found associations with Ym1 to varying degrees. Thus, insights into Ym1's role in diseases would help us understand the pathogenesis of different diseases and clarify the genuine roles of CLPs in mammals. This review summarizes the information on Ym1 from the gene to its expression and regulation and focuses on the association between Ym1 and diseases.

Treatment of GM2 Gangliosidosis in Adult Sandhoff Mice Using an Intravenous Self-Complementary Hexosaminidase Vector.

BACKGROUND: GM2 gangliosidosis is a neurodegenerative, lysosomal storage disease caused by the deficiency of ß-hexosaminidase A enzyme (Hex A), an α/ß-subunit heterodimer. A novel variant of the human hexosaminidase α-subunit, coded by HEX M, has previously been shown to form a stable homodimer, Hex M, that hydrolyzes GM2 gangliosides (GM2) in vivo. MATERIALS & METHODS: The current study assessed the efficacy of intravenous (IV) delivery of a self-complementary adeno-associated virus serotype 9 (scAAV9) vector incorporating the HEXM transgene, scAAV9/HEXM, including the outcomes based on the dosages provided to the Sandhoff (SD) mice. Six-week-old SD mice were injected with either 2.5E+12 vector genomes (low dose, LD) or 1.0E+13 vg (high dose, HD). We hypothesized that when examining the dosage comparison for scAAV9/HEXM in adult SD mice, the HD group would have more beneficial outcomes than the LD cohort. Assessments included survival, behavioral outcomes, vector biodistribution, and enzyme activity within the central nervous system. RESULTS: Toxicity was observed in the HD cohort, with 8 of 14 mice dying within one month of the injection. As compared to untreated SD mice, which have typical survival of 16 weeks, the LD cohort and the remaining HD mice had a significant survival benefit with an average/median survival of 40.6/34.5 and 55.9/56.7 weeks, respectively. Significant behavioral, biochemical and molecular benefits were also observed. The second aim of the study was to investigate the effects of IV mannitol infusions on the biodistribution of the LD scAAV9/HEXM vector and the survival of the SD mice. Increases in both the biodistribution of the vector as well as the survival benefit (average/median of 41.6/49.3 weeks) were observed. CONCLUSION: These results demonstrate the potential benefit and critical limitations of the treatment of GM2 gangliosidosis using IV delivered AAV vectors.

Relationship Between O-GlcNAcase Expression and Prognosis of Patients With Osteosarcoma.

Several studies have demonstrated a role of O-GlcNAcylation (O-GlcNAc) in tumorigenesis of various carcinomas by modification of tumor-associated proteins. However, its implication in the pathogenesis of osteosarcoma remains unclear. This study aimed to investigate the levels of O-GlcNAc and the expressions of O-linked N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA) in human osteosarcoma tissues, by using immunohistochemistry; and to find correlations between the levels or expressions and several clinicopathologic parameters. There were 109 first diagnosed osteosarcoma patients, including Enneking stage IIB (n=70) and III (n=39). Correlations between the immunoreactive score (IRS) and clinicopathologic parameters, overall survival, and metastasis-free survival were evaluated. A positive correlation was found between the IRS of OGA and the percentage of postchemotherapeutic tumor necrosis (r=0.308; P=0.017). Univariate analysis revealed significantly lower OGA IRS in metastatic patients (P=0.020) and poor chemotherapeutic-responder patients (P=0.001). By multivariate analysis, presence of tumor metastasis (P=0.002) and lower OGA IRS (P=0.004) was significantly associated with shorter overall survival. Subgroup analysis in stage IIB osteosarcoma (n=70) demonstrated that male sex (P=0.019), presence of tumor recurrence (P=0.026), poor chemotherapeutic responder (P=0.022), and lower OGA IRS (P=0.019) were significantly correlated with short metastasis-free survival. But, lower OGA IRS was the only independent predictor for short metastasis-free survival (P=0.006). Our findings suggested that O-GlcNAc pathway, especially OGA, may involve in pathogenesis and aggressiveness of osteosarcoma. Low level of OGA expression may be used as a poor prognostic indicator.

SREBP1-Induced Glutamine Synthetase Triggers a Feedforward Loop to Upregulate SREBP1 through Sp1 O-GlcNAcylation and Augments Lipid Droplet Formation in Cancer Cells.

Glutamine and lipids are two important components of proliferating cancer cells. Studies have demonstrated that glutamine synthetase (GS) boosts glutamine-dependent anabolic processes for nucleotide and protein synthesis, but the role of GS in regulating lipogenesis remains unclear. This study identified that insulin and glutamine deprivation activated the lipogenic transcription factor sterol regulatory element-binding protein 1 (SREBP1) that bound to the GS promoter and increased its transcription. Notably, GS enhanced the O-linked N-acetylglucosaminylation (O-GlcNAcylation) of the specificity protein 1 (Sp1) that induced SREBP1/acetyl-CoA carboxylase 1 (ACC1) expression resulting in lipid droplet (LD) accumulation upon insulin treatment. Moreover, glutamine deprivation induced LD formation through GS-mediated O-GlcNAc-Sp1/SREBP1/ACC1 signaling and supported cell survival. These findings demonstrate that insulin and glutamine deprivation induces SREBP1 that transcriptionally activates GS, resulting in Sp1 O-GlcNAcylation. Subsequently, O-GlcNAc-Sp1 transcriptionally upregulates the expression of SREBP1, resulting in a feedforward loop that increases lipogenesis and LD formation in liver and breast cancer cells.

An integrated strategy for the identification and screening of anti-allergy components from natural products based on calcium fluctuations and cell extraction coupled with HPLC-Q-TOF-MS.

Allergic diseases are a significant public health problem worldwide. Traditional Chinese medicines (TCMs) with reported anti-allergy effects may be important sources for the development of new anti-allergy drugs. Thus, establishing an analytical method that can simultaneously identify and screen anti-allergic compounds in TCMs is important. The increased concentrations of intracellular calcium ions resulting in mast cell degranulation releasing active mediators play a key role in allergic diseases, which can be used as a potential index to identify anti-allergic herbs and compounds. In this study, we provide a new strategy that was applied to screening natural anti-allergic compounds based on fluorescence calcium ion (Ca2+) fluctuation integrated with cell extract and high-performance liquid chromatography-mass spectrometry (HPLC-MS). A low-cost, convenient fluorescence detection Ca2+ signaling method was established and successfully applied to identify three herbs. Then, the method was integrated with biospecific cell fishing and HPLC-MS to screen potential active components that have the effect of stabilizing the cell membrane of rat basophilic leukemia granulocytes (RBL-2H3). Seven components, namely, albiflorin and paeoniflorin from Radix Paeoniae Alba, ononin and formononetin from Radix Astragali, cimifugin, 4'-O-ß-D-glucosyl-5-O-methylvisamminol, and prim-O-glucosylcimifugin from Radix Saposhnikoviae were fished. These seven compounds have the effect of inhibiting cell Ca2+ influx. 4'-O-ß-D-Glucosyl-5-O-methylvisamminol, prim-O-glucosylcimifugin, paeoniflorin, ononin, and formononetin significantly inhibit the release of ß-hexosaminidase, which is equivalent to the positive drug. In conclusion, the integrated strategy of fluorescence detection calcium ion kinetic method binding with biospecific cell fishing was an effective mode to identify and screen natural anti-allergic compounds.

Altered Macrophage Function Associated with Crystalline Lung Inflammation in Acid Sphingomyelinase Deficiency.

Deficiency of ASM (acid sphingomyelinase) causes the lysosomal storage Niemann-Pick disease (NPD). Patients with NPD type B may develop progressive interstitial lung disease with frequent respiratory infections. Although several investigations using the ASM-deficient (ASMKO) mouse NPD model revealed inflammation and foamy macrophages, there is little insight into the pathogenesis of NPD-associated lung disease. Using ASMKO mice, we report that ASM deficiency is associated with a complex inflammatory phenotype characterized by marked accumulation of monocyte-derived CD11b+ macrophages and expansion of airspace/alveolar CD11c+ CD11b- macrophages, both with increased size, granularity, and foaminess. Both the alternative and classical pathways were activated, with decreased in situ phagocytosis of opsonized (Fc-coated) targets, preserved clearance of apoptotic cells (efferocytosis), secretion of Th2 cytokines, increased CD11c+/CD11b+ cells, and more than a twofold increase in lung and plasma proinflammatory cytokines. Macrophages, neutrophils, eosinophils, and noninflammatory lung cells of ASMKO lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, which formed large eosinophilic polygonal Charcot-Leyden-like crystals. In addition to providing insight into novel features of lung inflammation that may be associated with NPD, our report provides a novel connection between ASM and the development of crystal-associated lung inflammation with alterations in macrophage biology.

Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the ß-subunit of ß-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.

GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the ß-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.

Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme ß-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

RELMα is Induced in Airway Epithelial Cells by Oncostatin M Without Requirement of STAT6 or IL-6 in Mouse Lungs In Vivo.

Resistin-like molecule alpha (RELMα) and YM-1 are secreted proteins implicated in murine models of alternatively activated macrophage (AA/M2) accumulation and Th2-skewed inflammation. Since the gp130 cytokine Oncostatin M (OSM) induces a Th2-like cytokine and AA/M2 skewed inflammation in mouse lung, we here investigated regulation of RELMα and YM-1. Transient pulmonary overexpression of OSM by Adenovirus vector (AdOSM) markedly induced RELMα and YM-1 protein expression in total lung. In situ hybridization showed that RELMα mRNA was highly induced in airway epithelial cells (AEC) and was co-expressed with CD68 mRNA in some but not all CD68+ cells in parenchyma. IL-6 overexpression (a comparator gp130 cytokine) induced RELMα, but at significantly lower levels. IL-6 (assessing IL-6-/- mice) was not required, nor was STAT6 (IL-4/13 canonical signalling) for AdOSM-induction of RELMα in AEC. AEC responded directly to OSM in vitro as assessed by pSTAT3 activation. RELMα-deficient mice showed similar inflammatory cell infiltration and cytokine responses to wt in response to AdOSM, but showed less accumulation of CD206+ AA/M2 macrophages, reduced induction of extracellular matrix gene mRNAs for COL1A1, COL3A1, MMP13, and TIMP1, and reduced parenchymal alpha smooth muscle actin. Thus, RELMα is regulated by OSM in AEC and contributes to extracellular matrix remodelling in mouse lung.

Galectins, Eosinophiles, and Macrophages May Contribute to Schistosoma japonicum Egg-Induced Immunopathology in a Mouse Model.

Schistosomiasis is a severe public health problem, which can cause tissue fibrosis and can even be fatal. Previous studies have proven that galectins and different kinds of cells involve in the regulation of tissue fibrosis process. In this study, outbred Kunming mice were infected with Schistosoma japonicum (S. japonicum). Our results showed that compared with uninfected mice, there were severe egg granulomatous inflammation and tissue fibrosis in the livers, spleens, and large intestines of S. japonicum-infected mice at 8 weeks post-infection (p.i.), and the number of eosinophils by hematoxylin and eosin staining and CD68 macrophage-positive area by immunohistochemical staining were significantly increased. Detected by using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), at 8 weeks after S. japonicum infection, the mRNA expression levels of galectin (Gal)-1, Gal-3, CD69, eosinophil protein X (EPX), and chitinase 3-like protein 3 (Ym1) were significantly increased in liver, spleen, and large intestine; eotaxin-1 (CCL11) and eosinophil cationic protein were significantly increased in both liver and spleen; eotaxin-2 (CCL24) and Arginase1 (Arg1) were significantly increased in both spleen and large intestine; and CD200R was significantly increased in both liver and large intestine. However, interleukin (IL)-1ß and inducible nitric oxide synthase (iNOS) were only significantly increased in liver. The M2/M1 ratio of CD200R/CD86 genes was significantly increased in liver, and ratios of Ym1/IL-1ß and Ym1/iNOS were significantly increased in liver, spleen, and large intestine of S. japonicum-infected mice. Ex vivo study further confirmed that the levels of Gal-1, Gal-3, CD200R, Arg1, and Ym1 were significantly increased, and the ratios of CD200R/CD86 and Ym1/IL-1ß were significantly increased in peritoneal macrophages isolated from S. japonicum-infected mice at 8 weeks p.i. In addition, correlation analysis showed that significant positive correlations existed between mRNA levels of Gal-1/Gal-3 and EPX in liver, between Gal-3 and Ym1 in both liver and large intestine, and between Gal-3 and CD200R in peritoneal macrophages of S. japonicum-infected mice. Our data suggested that Gal-1, Gal-3, eosinophils, and macrophages are likely involved in the development of egg granulomatous response and fibrosis induced by S. japonicum infection.

Blockage of O-linked GlcNAcylation induces AMPK-dependent autophagy in bladder cancer cells.

BACKGROUND: High levels of the post-translational modification O-GlcNAcylation (O-GlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of O-GlcNAcylation on autophagy in bladder cancer remains unclear. Here, we evaluate the change in autophagic activity in response to O-GlcNAcylation and explore the potential mechanisms. METHODS: O-GlcNAcylation levels in bladder cancer cells were altered through pharmacological or genetic manipulations: treating with 6-diazo-5-oxo-norleucine (DON) or thiamet-G (TG) or up- and downregulation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA). Autophagy was determined using fluorescence microscopy and western blotting. Co-immunoprecipitation (Co-IP) assays were performed to evaluate whether the autophagy regulator AMP-activated protein kinase (AMPK) was O-GlcNAc modified. RESULTS: Cellular autophagic flux was strikingly enhanced as a result of O-GlcNAcylation suppression, whereas it decreased at high O-GlcNAcylation levels. Phosphorylation of AMPK increased after the suppression of O-GlcNAcylation. We found that O-GlcNAcylation of AMPK suppressed the activity of this regulator, thereby inhibiting ULK1 activity and autophagy. CONCLUSION: We characterized a new function of O-GlcNAcylation in the suppression of autophagy via regulation of AMPK. GRAPHICAL ABSTRACT: Blockage of O-linked GlcNAcylation induces AMPK dependent autophagy in bladder cancer cells.

TSPO Modulates IL-4-Induced Microglia/Macrophage M2 Polarization via PPAR-γ Pathway.

Microglia activation has been reported to be associated with pathogenesis of neuroinflammation, central nervous system damage, and degeneration diseases. With various damage-associated molecules released, M1 polarization of microglia emerges early after injury and followed by M2 polarization. In this study, we demonstrate using a primary microglia polarization model that, during the M2 polarization of microglia, the protein expression of translocator protein (TSPO) was decreased and peroxisome proliferator-activated receptor (PPAR-γ) activation was observed. In addition, we found TSPO antagonist PK11195 treatment enhanced PPAR-γ expression in M2-polarized microglia, while TSPO agonist FGIN-1-27 and TSPO overexpression in microglia significantly suppressed PPAR-γ expression in both the cytoplasm and nucleus. Then, real-time quantitative PCR was used to detect the expression of M2 polarization markers in microglia after TSPO ligand treatment, the data showed that PK11195 promoted the expression of CD206, Arg-1, YM-1, and FIZZ-1 induced by interleukin-4 (IL-4), and FGIN-1-27 and TSPO overexpression inhibited the expression of these molecules. Furthermore, the release of BDNF, CNTF-1, IGF-1, and NGF-1 from microglia was determined by enzyme-linked immunosorbent assay; these trophic factors showed similar trends with expression of M2 polarization markers. Levels of BDNF, CNTF-1, IGF-1, and NGF-1 were obviously upregulated by PK11195 and downregulated by FGIN-1-27 and TSPO overexpression. We propose that IL-4 in the hypoxic ischemia brain site induces the M2 polarization of microglia, and TSPO inhibits the M2 polarization and trophic factor release through PPAR-γ pathway.

Evidence of a compensatory regulation of colonic O-GlcNAc transferase and O-GlcNAcase expression in response to disruption of O-GlcNAc homeostasis.

O-GlcNAcylation is a post-translational modification of thousands of intracellular proteins that dynamically regulates many fundamental cellular processes. Cellular O-GlcNAcylation levels are regulated by a unique couple of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which adds and removes the GlcNAc residue, respectively. Maintenance of O-GlcNAc homeostasis is essential to ensure optimal cellular function and disruption of this homeostasis has been linked to the etiology of several human diseases including cancer. The mechanisms through which the cell maintains O-GlcNAc homeostasis are not fully understood but several studies have suggested that a reciprocal regulation of OGT and OGA expression could be one of them. In this study, we investigated the putative regulation of OGT and OGA expression in response to disruption in O-GlcNAc homeostasis in colon. We provide in vitro and in vivo evidences that in colon cells, modulation of O-GlcNAcylation levels leads to a compensatory regulation of OGT and OGA expression in an attempt to restore basal O-GlcNAcylation levels. Our results also suggests that the regulation of colonic OGA expression in response to changes in O-GlcNAc homeostasis occurs mostly at the transcriptional level whereas OGT regulation seems to rely mainly on post-transcriptional mechanisms.

dPGS Regulates the Phenotype of Macrophages via Metabolic Switching.

The synthetic compound dendritic polyglycerol sulfate (dPGS) is a pleiotropic acting molecule but shows a high binding affinity to immunological active molecules as L-/P-selectin or complement proteins leading to well described anti-inflammatory properties in various mouse models. In order to make a comprehensive evaluation of the direct effect on the innate immune system, macrophage polarization is analyzed in the presence of dPGS on a phenotypic but also metabolic level. dPGS administered macrophages show a significant increase of MCP1 production paralleled by a reduction of IL-10 secretion. Metabolic analysis reveals that dPGS could potently enhance the glycolysis and mitochondrial respiration in M0 macrophages as well as decrease the mitochondrial respiration of M2 macrophages. In summary the data indicate that dPGS polarizes macrophages into a pro-inflammatory phenotype in a metabolic pathway-dependent manner.

Nephropathy in Hypertensive Animals Is Linked to M2 Macrophages and Increased Expression of the YM1/Chi3l3 Protein.

Macrophages contribute to a continuous increase in blood pressure and kidney damage in hypertension, but their polarization status and the underlying mechanisms have not been clarified. This study revealed an important role for M2 macrophages and the YM1/Chi3l3 protein in hypertensive nephropathy in a mouse model of hypertension. Bone marrow cells were isolated from the femurs and tibia of male FVB/N (control) and transgenic hypertensive animals that overexpressed the rat form of angiotensinogen (TGM(rAOGEN)123, TGM123-FVB/N). The cells were treated with murine M-CSF and subsequently with LPS+IFN-γ to promote their polarization into M1 macrophages and IL-4+IL-13 to trigger the M2 phenotype. We examined the kidneys of TGM123-FVB/N animals to assess macrophage polarization and end-organ damage. mRNA expression was evaluated using real-time PCR, and protein levels were assessed through ELISA, CBA, Western blot, and immunofluorescence. Histology confirmed high levels of renal collagen. Cells stimulated with LPS+IFN-γ in vitro showed no significant difference in the expression of CD86, an M1 marker, compared to cells from the controls or the hypertensive mice. When stimulated with IL-4+IL-13, however, macrophages of the hypertensive group showed a significant increase in CD206 expression, an M2 marker. The M2/M1 ratio reached 288%. Our results indicate that when stimulated in vitro, macrophages from hypertensive mice are predisposed toward polarization to an M2 phenotype. These data support results from the kidneys where we found an increased infiltration of macrophages predominantly polarized to M2 associated with high levels of YM1/Chi3l3 (91,89%), suggesting that YM1/Chi3l3 may be a biomarker of hypertensive nephropathy.

Genetic knockout of myosin light chain kinase (MLCK210) prevents cerebral microhemorrhages and attenuates neuroinflammation in a mouse model of vascular cognitive impairment and dementia.

The blood-brain barrier (BBB) is critical in maintenance of brain homeostasis, and loss of its functional integrity is a key feature across a broad range of neurological insults. This includes both acute injuries such as traumatic brain injury and stroke, as well as more chronic pathologies associated with aging, such as vascular cognitive impairment and dementia (VCID). A specific form of myosin light chain kinase (MLCK210) is a major regulator of barrier integrity in general, including the BBB. Studies have demonstrated the potential of MLCK210 as a therapeutic target for peripheral disorders involving tissue barrier dysfunction, but less is known about its potential as a target for chronic neurologic disorders. We report here that genetic knockout (KO) of MLCK210 protects against cerebral microhemorrhages and neuroinflammation induced by chronic dietary hyperhomocysteinemia. Overall, the results are consistent with an accumulating body of evidence supporting MLCK210 as a potential therapeutic target for tissue barrier dysfunction and specifically implicate it in BBB dysfunction and neuroinflammation in a model of VCID.

Facile Formation of ß-thioGlcNAc Linkages to Thiol-Containing Sugars, Peptides, and Proteins using a Mutant GH20 Hexosaminidase.

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.

Calcitonin gene-related peptide decreases IL-1beta, IL-6 as well as Ym1, Arg1, CD163 expression in a brain tissue context-dependent manner while ameliorating experimental autoimmune encephalomyelitis.

Activation states of immune cells (among them, the so-called pro- or anti-inflammatory states) influence the pathogenesis of multiple sclerosis (MS). The neuropeptide calcitonin gene-related peptide (CGRP) can exert a pro- or anti-inflammatory role in a context-dependent manner. In mice CGRP was found to attenuate the development of experimental autoimmune encephalomyelitis (EAE, a common MS animal model). We analyzed CGRP effects on the expression of cytokines and markers of activation states, as well as its intracellular cascade, following intrathecal administration during EAE immunization. Real Time quantitative-PCR (RT-PCR) showed that IL-1beta and IL-6 (associated to a pro-inflammatory state in EAE), but also Ym1 (also known as Chil3), Arg1 and CD163 (associated to an anti-inflammatory state in EAE) were decreased in the encephalon (devoid of cerebellum). In the cerebellum itself, IL-1beta and Ym1 were decreased. TNF-alpha (associated to a pro-inflammatory state in EAE), but also IL-10 (associated to another type of anti-inflammatory state) and BDNF were unchanged in these two regions. No changes were detected in the spinal cord. Additional tendencies toward a change (as revealed by RT-PCR) were again decreases: IL-10 in the encephalon and Arg1 in the spinal cord. CGRP decreased percentage of Ym1+/CD68+ immunoreactive cells and cell density of infiltrates in the cervical spinal cord pia mater. Instead, Ym1 in the underlying parenchyma and at thoracic and lumbar levels, as well as Arg1, were unchanged. In cultured microglia the neuropeptide decreased Ym1, but not Arg1, immunoreactivity. Inducible NOS (iNOS) was unchanged in spinal cord microglia and astrocytes. The neuropeptide increased the activation of ERK1/2 in the astrocytes of the spinal cord and in culture, but did not influence the activation of ERK1/2 or p38 in the spinal cord microglia. Finally, in areas adjacent to infiltration sites CGRP-treated microglia showed a larger ramification radius. In conclusion, CGRP-induced EAE amelioration was associated to a concomitant, context-dependent decrease in the expression of markers belonging to both pro- or anti-inflammatory activation states of immune cells. It can be hypothesized that CGRP-induced EAE attenuation is obtained through a novel mechanism that promotes down-regulation of immune cell activation that facilitates the establishment of a beneficial environment in EAE provided possibly also by other factors.