Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice.

BACKGROUND: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies. METHODS: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau. RESULTS: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity. CONCLUSION: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.

Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice.

BACKGROUND: Obesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity. METHODS: Gene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry. RESULTS: Obese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1ß, IL1RN, TNF-α and TGFß-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice. CONCLUSIONS: Macrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.

Kinetic assessment and therapeutic modulation of metabolic and inflammatory profiles in mice on a high-fat and cholesterol diet.

The kinetics of metabolic and inflammatory parameters associated with obesity were evaluated in a murine diet-induced obesity (DIO) model using a diet high in fat and cholesterol. Cellular infiltration and mediator production were assessed and shown to be therapeutically modulated by the PPARgamma agonist rosiglitazone. C57BL/6 mice were maintained on a 45% fat/ 0.12% cholesterol (HF/CH) or Chow diet for 3, 6, 16, or 27 weeks. Flow cytometry was employed to monitor peripheral blood monocytes and adipose tissue macrophages (ATM). Gene expression and protein analysis methods were used to evaluate mediator production from total epididymal fat (EF), stromal vascular fraction (SVF), and sorted SVF cells. To investigate therapeutic intervention, mice were fed a HF/CH diet for 12 weeks and then a diet formulated with rosiglitazone (5 mg/kg) for an additional 6 weeks. A HF/CH diet correlated with obesity and a dramatic proinflammatory state. Therapeutic intervention with rosiglitazone attenuated the HF/CH induced inflammation. In addition, a novel population was found that expressed the highest levels of the pro-inflammatory mediators CCL2 and IL-6.

Short-chain fatty acids act as antiinflammatory mediators by regulating prostaglandin E(2) and cytokines.

AIM: To investigate the effect of short-chain fatty acids (SCFAs) on production of prostaglandin E(2) (PGE(2)), cytokines and chemokines in human monocytes. METHODS: Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative real-time polymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE(2), cytokines and chemokines in the supernatant. The effect of SCFAs in vivo was examined by intraplantar injection into rat paws. RESULTS: Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE(2) and that this effect can be enhanced in the presence of lipopolysaccharide (LPS). In addition, we demonstrate that PGE(2) production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 (MCP-1) production and LPS-induced interleukin-10 (IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE(2), MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-alpha and interferon-gamma in human PBMC. Finally, we show that SCFAs and LPS can induce PGE(2) production in vivo by intraplantar injection into rat paws (P < 0.01). CONCLUSION: SCFAs can have distinct antiinflammatory activities due to their regulation of PGE(2), cytokine and chemokine release from human immune cells.

Partial bladder outlet obstruction selectively abolishes protein kinase C induced contraction of rabbit detrusor smooth muscle.

PURPOSE: Despite the acute onset, partial bladder outlet obstruction in the rabbit induces detrusor remodeling similar to that in men with benign prostatic hyperplasia in terms of its impact on structural and functional alterations in smooth muscle. We determined if partial bladder outlet obstruction induced remodeling alters the protein kinase C signaling pathway that leads to contraction. MATERIALS AND METHODS: Smooth muscle from control animals and those subjected to 2 weeks of partial bladder outlet obstruction were mounted for isometric force recording, measurement of myosin light chain phosphorylation and levels of adducin phosphorylation. Bladder muscle strips were stimulated by phorbol dibutyrate or carbachol in the presence and absence of bisindolylmaleimide-1. RESULTS: Smooth muscle strips from animals subjected to partial bladder outlet obstruction showed little to no increase in stress in response to phorbol dibutyrate and no increase in myosin light chain phosphorylation levels. Muscle strips from control animals produced a robust contraction with concomitant increases in myosin light chain phosphorylation. Inhibition of protein kinase C by bisindolylmaleimide-1 significantly depressed carbachol induced contractions of muscle strips from control animals but it had no effect on carbachol induced contractions of muscle strips from outlet obstructed animals. Phorbol dibutyrate increased phospho-adducin levels in muscle strips from the 2 animal sources, suggesting that protein kinase C could be activated. CONCLUSIONS: We propose that partial bladder outlet obstruction does not alter protein kinase C activation, but rather abolishes or uncouples the pathway(s) downstream of protein kinase C, leading to contraction. Loss of this pathway may contribute to the loss of normal voiding behavior and the resultant decompensated state.

Partial bladder outlet obstruction abolishes the receptor- and G protein-dependent increase in calcium sensitivity in rabbit bladder smooth muscle.

Partial bladder outlet obstruction (PBOO) alters the function of the whole bladder and produces specific alterations in the contractility of the bladder smooth muscle cell. The goal of this study was to test the hypothesis that PBOO affects smooth muscle contraction at the level of the receptor- and G protein-dependent increase in myofilament Ca2+ sensitivity. To address this question, we used alpha-toxin-permeabilized strips of bladder smooth muscle from control animals and animals subjected to 2 wk of PBOO. Increasing free [Ca2+] increased force in permeabilized strips from control animals; the addition of 10 microM carbachol and 10 microM GTP increased both the Ca2+ sensitivity of the contractions and the maximal levels of force attained. In contrast, although increases in [Ca2+] increased force in permeabilized strips from PBOO animals, the addition of carbachol and GTP had no additional effects. Myosin light chain phosphorylation levels increased with [Ca2+], and although they tended to be higher in strips from PBOO animals, they did not reach statistical significance. Assessment of G protein activity from both animal models suggests this is not a site responsible for the loss of carbachol and GTP enhancement of myofilament Ca2+ sensitivity. The addition of phorbol dibutyrate increased the Ca2+ sensitivity of force development in strips from both animal models, suggesting that an alteration in PKC signaling is not involved. Our results are consistent with the hypothesis that PBOO decreases receptor-mediated myofilament calcium sensitization and that the site of action is downstream from either the G proteins or PKC.

Partial bladder outlet obstruction alters Ca2+ sensitivity of force, but not of MLC phosphorylation, in bladder smooth muscle.

Partial bladder outlet obstruction in the rabbit produces changes in bladder function similar to those seen clinically in patients with obstructive uropathies. Whole organ function is significantly altered, as are the smooth muscle cells inside the bladder wall. This study was designed to determine whether outlet obstruction alters smooth muscle function at the level of contractile filaments. Rabbit bladders were partially obstructed for 2 wk. Triton X-100 was used to provide a detergent-skinned bladder smooth muscle preparation that would allow control of the intracellular environment while the ability to shorten and develop force is maintained. Ca2+-force and Ca2+-myosin light chain (MLC) phosphorylation relations and maximal velocity of shortening were determined. The Ca2+ sensitivity of force was significantly lower in tissues from animals subjected to outlet obstruction compared with tissues from control animals. In contrast, no difference was noted in the Ca2+ sensitivity of MLC phosphorylation. Maximal levels of stress and MLC phosphorylation were similar in both animal groups. Maximal velocity of shortening was significantly slower in tissues from outlet-obstructed animals at all Ca2+ concentrations compared with tissues from control animals. Ultrastructurally, detergent skinning had little effect on structural integrity. Moreover, tissues from obstructed animals showed an increase in the number of sarcolemmal attachment plaque structures. We suggest that partial bladder outlet obstruction produces deleterious (e.g., decrease in Ca2+ sensitivity of force) and compensatory (e.g., increase in membrane attachment plaques) changes in bladder smooth muscle cells.

Effect of partial outlet obstruction on rabbit urinary bladder smooth muscle function.

Bladder outlet obstruction secondary to benign prostate hyperplasia is associated with many cellular changes. This study was designed to determine whether these changes involve the contractile apparatus. Bladder smooth muscles from rabbits subjected to partial outlet obstruction for 2 wk were mounted for isometric force, isotonic shortening velocity, and myosin light chain (MLC) phosphorylation levels. Muscle strips from obstructed bladders exhibited spontaneous phasic activity; muscle strips from control bladders did not. Muscle strips from obstructed bladders exhibited increased sensitivity and higher levels of stress in response to the cumulative addition of KCl or carbachol compared with control. During noncumulative addition of KCl or carbachol, no differences in sensitivity were noted. Muscle strips from obstructed bladders had elevated basal MLC phosphorylation levels and stimulation produced small increases in MLC phosphorylation compared with control. V(max) during KCl stimulation of muscle strips from obstructed bladders was 10-fold lower than control. Our results suggest that bladder outlet obstruction produces a muscle cell that develops higher levels of force but with greatly reduced cross bridge cycling rates.