Acute Inhibition Research Articles

Cannabimimetic effects of abused indazole-carboxamide synthetic cannabinoid receptor agonists AB-PINACA, 5F-AB-PINACA and 5F-ADB-PINACA in mice: Tolerance, dependence and withdrawal

BackgroundChronic abuse of synthetic cannabinoid receptor agonists (SCRAs), known as “K2″ or “Spice”, threatens public health and safety. Recently, SCRAs of the indazole-carboxamide structural class have become more prevalent. Preclinical studies investigating the tolerance and dependence potentially involved in chronic SCRA abuse is limited. The present study determined the in vivo effects of chronic exposure to indazole-carboxamide SCRAs, AB-PINACA, 5F-AB-PINACA and 5F-ADB-PINACA compared to the first-generation SCRA, JWH-018. MethodsAdult male C57Bl/6 mice were used for dose-effect determinations of hypothermic effects. Adult male NIH Swiss mice were used in biotelemetry studies to assess tolerance to hypothermic effects following repeated SCRA administration over 5 consecutive days, and to determine the role of Phase I drug metabolism via acute CYP450 inhibition in the presence of 1-ABT, a nonspecific CYP450 inhibitor. SCRA dependence was determined in adult male NIH Swiss mice via assessment of rimonabant-precipitated observable sign of withdrawal (i.e., front paw tremors). ResultsAll SCRAs elicited dose-dependent hypothermia mediated through cannabinoid CB1 receptors (CB1Rs). 1-ABT increased duration of hypothermia for all SCRAs tested, and increased the magnitude of hypothermia for all SCRAs except 5F-ADB-PINACA. Upon repeated administration, tolerance to hypothermic effects of AB-PINACA, 5F-AB-PINACA and 5F-ADB-PINACA was much less than that of JWH-018. Similarly, rimonabant-precipitated front paw tremors were much less frequent in mice treated with 5F-AB-PINACA and 5F-ADB-PINACA than in mice treated with JWH-018. ConclusionsThese findings suggest a decreased potential for tolerance and withdrawal among indazole-carboxamide SCRAs, and may imply structural class-dependent profiles of in vivo effects among SCRAs.

Dopamine Transporter, PhosphoSerine129 α-Synuclein and α-Synuclein Levels in Aged LRRK2 G2019S Knock-In and Knock-Out Mice.

The G2019S mutation in leucine rich-repeat kinase 2 (LRRK2) is a major cause of familial Parkinson’s disease. We previously reported that G2019S knock-in mice manifest dopamine transporter dysfunction and phosphoSerine129 α-synuclein (pSer129 α-syn) immunoreactivity elevation at 12 months of age, which might represent pathological events leading to neuronal degeneration. Here, the time-dependence of these changes was monitored in the striatum of 6, 9, 12, 18 and 23-month-old G2019S KI mice and wild-type controls using DA uptake assay, Western analysis and immunohistochemistry. Western analysis showed elevation of membrane dopamine transporter (DAT) levels at 9 and 12 months of age, along with a reduction of vesicular monoamine transporter 2 (VMAT2) levels at 12 months. DAT uptake was abnormally elevated from 9 to up to 18 months. DAT and VMAT2 level changes were specific to the G2019S mutation since they were not observed in LRRK2 kinase-dead or knock-out mice. Nonetheless, dysfunctional DAT uptake was not normalized by acute pharmacological inhibition of LRRK2 kinase activity with MLi-2. Immunoblot analysis showed elevation of pSer129 α-syn levels in the striatum of 12-month-old G2019S KI mice, which, however, was not confirmed by immunohistochemical analysis. Instead, total α-syn immunoreactivity was found elevated in the striatum of 23-month-old LRRK2 knock-out mice. These data indicate mild changes in DA transporters and α-syn metabolism in the striatum of 12-month-old G2019S KI mice whose pathological relevance remains to be established.

Acute systemic knockdown of Atg7 is lethal and causes pancreatic destruction in shRNA transgenic mice

ABSTRACT The notion that macroautophagy/autophagy is a potentially attractive therapeutic target for a variety of diseases, including cancer, largely stems from pre-clinical mouse studies. Most of these examine the effects of irreversible and organ confined autophagy deletion using site specific Cre-loxP recombination of the essential autophagy regulating genes Atg7 or Atg5. Model systems with the ability to impair autophagy systemically and reversibly at all disease stages would allow a more realistic approach to evaluate the consequences of authophagy inhibition as a therapeutic concept and its potential side effects. Here, we present shRNA transgenic mice that via doxycycline (DOX) regulable expression of a highly efficient miR30-E-based shRNA enabled knockdown of Atg7 simultaneously in the majority of organs, with the brain and spleen being noteable exceptions. Induced animals deteriorated rapidly and experienced profound destruction of the exocrine pancreas, severe hypoglycemia and depletion of hepatic glycogen storages. Cessation of DOX application restored apparent health, glucose homeostasis and pancreatic integrity. In a similar Atg5 knockdown model we neither observed loss of pancreatic integrity nor diminished survival after DOX treatment, but identified histological changes consistent with steatohepatitis and hepatic fibrosis in the recovery period after termination of DOX. Regulable Atg7-shRNA mice are valuable tools that will enable further studies on the role of autophagy impairment at various disease stages and thereby help to evaluate the consequences of acute autophagy inhibition as a therapeutic concept.

Opposing roles of the entero-pancreatic hormone urocortin-3 in glucose metabolism in rats

Aim/hypothesisUrocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved.MethodsWe studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis.ResultsIn rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3’s cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion.Conclusions/interpretationOur combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.Graphical abstract

Circulating corticosterone levels mediate the relationship between acute ethanol intoxication and markers of NF-κB activation in male rats

Binge drinking is a harmful pattern of alcohol use that is associated with a number of serious health problems. Of particular interest are the rapid alterations in neuroimmune gene expression and the concurrent activation of the hypothalamic-pituitary-adrenal (HPA) axis activation associated with high intensity drinking. Using a rat model of acute binge-like ethanol exposure, the present studies were designed to assess the role of corticosterone (CORT) in ethanol-induced neuroimmune gene expression changes, particularly those associated with the NFκB signaling pathway, including rapid induction of IL-6 and IκBα, and suppression of IL-1β and TNFα gene expression evident after administration of moderate to high doses of ethanol (1.5–3.5 g/kg ip) during intoxication (3 h post-injection). Experiment 1 tested whether inhibition of CORT synthesis with metyrapone and aminoglutethimide (100 mg/kg each, sc) would block ethanol-induced changes in neuroimmune gene expression. Results indicated that rapid alterations in IκBα, IL-1β, and TNFα expression were completely blocked by pretreatment with the glucocorticoid synthesis inhibitors, an effect that was reinstated by co-administration of exogenous CORT (3.75 mg/kg) in Experiment 2. Experiment 3 assessed whether these rapid alterations in neuroimmune gene expression would be evident when rats were challenged with a subthreshold dose of ethanol (1.5 g/kg) in combination with 2.5 mg/kg CORT, which showed limited evidence for additive effects of low-dose CORT combined with a moderate dose of ethanol. Acute inhibition of mineralocorticoid (spironolactone) or glucocorticoid (mifepristone) receptors, alone (Experiment 4) or combined (Experiment 5) had no effect on ethanol-induced changes in neuroimmune gene expression, presumably due to poor CNS penetrance of these drugs. Finally, Experiments 6 and 7 showed that dexamethasone (subcutaneous; a GR agonist) recapitulated effects of ethanol. Overall, we conclude that ethanol-induced CORT synthesis and release is responsible for suppression of IL-1β, TNFα, and induction of IκBα in the hippocampus through GR signaling. Interventions designed to curb these changes may reduce drinking, and subdue detrimental neuroimmune activation induced by ethanol.

Neflamapimod induces vasodilation in resistance mesenteric arteries by inhibiting p38 MAPK\u03b1 and downstream Hsp27 phosphorylation

Neflamapimod, a selective inhibitor of p38 mitogen activated protein kinase alpha (MAPKα), is under clinical investigation for its efficacy in Alzheimer’s disease (AD) and dementia with Lewy Bodies (DLB). Here, we investigated if neflamapimod-mediated acute inhibition of p38 MAPKα could induce vasodilation in resistance-size rat mesenteric arteries. Our pressure myography data demonstrated that neflamapimod produced a dose-dependent vasodilation in mesenteric arteries. Our Western blotting data revealed that acute neflamapimod treatment significantly reduced the phosphorylation of p38 MAPKα and its downstream target heat-shock protein 27 (Hsp27) involved in cytoskeletal reorganization and smooth muscle contraction. Likewise, non-selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation, and induced vasodilation. Endothelium denudation or pharmacological inhibition of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) had no effect on such vasodilation. Neflamapimod-evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell K+ channels. Altogether, our data for the first time demonstrates that in resistance mesenteric arteries, neflamapimod inhibits p38 MAPKα and phosphorylation of its downstream actin-associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which neflamapimod is being investigated.

Bruton's TK regulates myeloid cell recruitment during acute inflammation.

Background and PurposeBruton's TK (BTK) is a non‐receptor kinase best known for its role in B lymphocyte development that is critical for proliferation and survival of leukaemic cells in B‐cell malignancies. However, BTK is expressed in myeloid cells, particularly neutrophils, monocytes and macrophages where its inhibition has been reported to cause anti‐inflammatory properties.Experimental ApproachWe explored the role of BTK on migration of myeloid cells (neutrophils, monocytes and macrophages), in vitro using chemotaxis assays and in vivo using zymosan‐induced peritonitis as model systems.Key ResultsUsing the zymosan‐induced peritonitis model of sterile inflammation, we demonstrated that acute inhibition of BTK prior to zymosan challenge reduced phosphorylation of BTK in circulating neutrophils and monocytes. Moreover, pharmacological inhibition of BTK with ibrutinib specifically inhibited neutrophil and Ly6Chi monocytes, but not Ly6Clo monocyte recruitment to the peritoneum. X‐linked immunodeficient (XID) mice, which have a point mutation in the Btk gene, had reduced neutrophil and monocyte recruitment to the peritoneum following zymosan challenge. Pharmacological or genetic inhibition of BTK signalling substantially reduced human monocyte and murine macrophage chemotaxis, to a range of clinically relevant chemoattractants (C5a and CCL2). We also demonstrated that inhibition of BTK in tissue resident macrophages significantly decreases chemokine secretion by reducing NF‐κB activity and Akt signalling.Conclusion and ImplicationsOur work has identified a new role of BTK in regulating myeloid cell recruitment via two mechanisms, reducing monocyte/macrophages' ability to undergo chemotaxis and reducing chemokine secretion, via reduced NF‐κB and Akt activity in tissue resident macrophages.

The Statin Target Hmgcr Regulates Energy Metabolism and Food Intake through Central Mechanisms.

The statin drug target, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), is strongly linked to body mass index (BMI), yet how HMGCR influences BMI is not understood. In mammals, studies of peripheral HMGCR have not clearly identified a role in BMI maintenance and, despite considerable central nervous system expression, a function for central HMGCR has not been determined. Similar to mammals, Hmgcr is highly expressed in the Drosophila melanogaster brain. Therefore, genetic and pharmacological studies were performed to identify how central Hmgcr regulates Drosophila energy metabolism and feeding behavior. We found that inhibiting Hmgcr, in insulin-producing cells of the Drosophila pars intercerebralis (PI), the fly hypothalamic equivalent, significantly reduces the expression of insulin-like peptides, severely decreasing insulin signaling. In fact, reducing Hmgcr expression throughout development causes decreased body size, increased lipid storage, hyperglycemia, and hyperphagia. Furthermore, the Hmgcr induced hyperphagia phenotype requires a conserved insulin-regulated α-glucosidase, target of brain insulin (tobi). In rats and mice, acute inhibition of hypothalamic Hmgcr activity stimulates food intake. This study presents evidence of how central Hmgcr regulation of metabolism and food intake could influence BMI.

Unraveling the Effects of GSK-3β Isoform Modulation against Limbic Seizures and in the 6 Hz Electrical Kindling Model for Epileptogenesis.

Two closely related glycogen synthase kinase-3 (GSK-3) isoforms have been identified in mammals: GSK-3α and GSK-3β. GSK-3β is the most prominent in the central nervous system and was previously shown to control neuronal excitability. We previously demonstrated that indirubin and its structural analogue and the nonselective GSK-3 inhibitor BIO-acetoxime exerted anticonvulsant effects in acute seizure models in zebrafish, mice, and rats. We here examined for the first time the anticonvulsant effect of TCS2002, a specific and potent inhibitor of GSK-3β, in two models for limbic seizures: the pilocarpine rat model for focal seizures and the acute 6 Hz corneal mouse model for refractory seizures. Next, we additionally used the 6 Hz kindling model to establish differences in seizure susceptibility and seizure progression in mice that either overexpress human GSK-3β (GSK-3β OE) or lack GSK-3β (GSK-3β-/-) in neurons. We demonstrate that TCS2002 exerts anticonvulsant actions against pilocarpine- and 6 Hz-evoked seizures. Compared to wild-type littermates, GSK-3β OE mice are less susceptible to seizures but are more rapidly kindled. Interestingly, compared to GSK-3β+/+ mice, neuronal GSK-3β-/- mice show increased susceptibility to 6 Hz-induced seizures. These contrasting observations suggest compensatory neurodevelopmental mechanisms that alter seizure susceptibility in GSK-3β OE and GSK-3β-/- mice. Although the pronounced anticonvulsant effects of selective and acute GSK-3β inhibition in the 6 Hz model identify GSK-3β as a potential drug target for pharmacoresistant seizures, our data on the sustained disruption of GSK-3β activity in the transgenic mice suggest a role for GSK-3 in kindling and warrants further research into the long-term effects of selective pharmacological GSK-3β inhibition.

Clinical and Preclinical Evidence for Roles of Soluble Epoxide Hydrolase in Osteoarthritis Knee Pain.

Chronic pain due to osteoarthritis (OA) is a major clinical problem, and existing analgesics often have limited beneficial effects and/or adverse effects, necessitating the development of novel therapies. Epoxyeicosatrienoic acids (EETs) are endogenous antiinflammatory mediators, rapidly metabolized by soluble epoxide hydrolase (EH) to dihydroxyeicosatrienoic acids (DHETs). We undertook this study to assess whether soluble EH-driven metabolism of EETs to DHETs plays a critical role in chronic joint pain associated with OA and provides a new target for treatment. Potential associations of chronic knee pain with single-nucleotide polymorphisms (SNPs) in the gene-encoding soluble EH and with circulating levels of EETs and DHETs were investigated in human subjects. A surgically induced murine model of OA was used to determine the effects of both acute and chronic selective inhibition of soluble EH by N-[1-(1-oxopropy)-4-piperidinyl]-N'-(trifluoromethoxy)phenyl]-urea (TPPU) on weight-bearing asymmetry, hind paw withdrawal thresholds, joint histology, and circulating concentrations of EETs and DHETs. In human subjects with chronic knee pain, 3 pain measures were associated with SNPs of the soluble EH gene EPHX2, and in 2 separate cohorts of subjects, circulating levels of EETs and DHETs were also associated with 3 pain measures. In the murine OA model, systemic administration of TPPU both acutely and chronically reversed established pain behaviors and decreased circulating levels of 8,9-DHET and 14,15-DHET. EET levels were unchanged by TPPU administration. Our novel findings support a role of soluble EH in OA pain and suggest that inhibition of soluble EH and protection of endogenous EETs from catabolism represents a potential new therapeutic target for OA pain.

Acute inhibition of OGA sex-dependently alters the networks associated with bioenergetics, autophagy, and neurodegeneration

The accumulation of neurotoxic proteins characteristic of age-related neurodegenerative pathologies such as Alzheimer’s and Parkinson’s diseases is associated with the perturbation of metabolism, bioenergetics, and mitochondrial quality control. One approach to exploit these interactions therapeutically is to target the pathways that regulate metabolism. In this respect, the nutrient-sensing hexosamine biosynthesis pathway is of particular interest since it introduces a protein post-translational modification known as O-GlcNAcylation, which modifies different proteins in control versus neurodegenerative disease postmortem brains. A potent inhibitor of the O-GlcNAcase enzyme that removes the modification from proteins, Thiamet G (TG), has been proposed to have potential benefits in Alzheimer’s disease. We tested whether key factors in the O-GlcNAcylation are correlated with mitochondrial electron transport and proteins related to the autophagy/lysosomal pathways in the cortex of male and female mice with and without exposure to TG (10 mg/kg i.p.). Mitochondrial complex activities were measured in the protein homogenates, and a panel of metabolic, autophagy/lysosomal proteins and O-GlcNAcylation enzymes were assessed by either enzyme activity assay or by western blot analysis. We found that the networks associated with O-GlcNAcylation enzymes and activities with mitochondrial parameters, autophagy-related proteins as well as neurodegenerative disease-related proteins exhibited sex and TG dependent differences. Taken together, these studies provide a framework of interconnectivity for multiple O-GlcNAc-dependent pathways in mouse brain of relevance to aging and sex/age-dependent neurodegenerative pathogenesis and response to potential therapies.

Acute systemic inhibition of inflammation augments endothelium-dependent dilation in women with a history of preeclamptic pregnancy.

Women who have had preeclampsia demonstrate microvascular endothelial-dysfunction, mediated in part by reduced nitric oxide (NO)-dependent dilation. Preeclamptic pregnancies are associated with elevated inflammation, and inhibition of inflammation attenuates endothelial damage in animal models of preeclampsia. However, it is unclear if inhibition of vascular inflammation improves endothelial function in women after a preeclamptic pregnancy. Using the cutaneous microcirculation as a model, we hypothesized that acute systemic inhibition of vascular inflammation (oral salsalate; 1500mg/twice daily, 4days) would improve endothelium- and NO-dependent vasodilation in women with a history of preeclampsia (PE) but not in women with a history of uncomplicated pregnancy (HC). Twelve HC (30±1yrs, 10±2months postpartum) and 10 PE (30±2yrs, 8±2months postpartum) participated in a double-blind placebo-controlled study. Following each treatment, 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusion of acetylcholine (Ach, 10-7-102mM) or Ach+15mM L-NAME (NO synthase antagonist). Red blood cell flux was measured over each site by laser-Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP+local heat 43°C). ACh-induced (77±3 vs. 92±3%CVCmax; p=0.01) and NO-dependent (20±6 vs. 33±4%; p=0.02) vasodilation were attenuated in PE compared to HC. Salsalate augmented ACh-induced (95±2%CVCmax; p=0.002) and NO-dependent (39±3%; p=0.009) dilation in PE compared to placebo but had no effect in HC (all p>0.05). Salsalate treatment augmented endothelium-dependent vasodilation via NO-mediated pathways in women who have had preeclampsia, suggesting that inflammatory signaling mediates persistent endothelial dysfunction following preeclampsia.

Activity-dependent PI4P synthesis by PI4KIIIα regulates long-term synaptic potentiation

Phosphatidylinositol 4-phosphate (PI4P) is a low abundant phospholipid with important roles in lipid transport and membrane trafficking. However, little is known of its metabolism and function in neurons. Here, we investigate its subcellular distribution and functional roles in dendrites of rodent hippocampal neurons during resting state and long-term synaptic potentiation (LTP). We show that neural activity causes dynamic reversible changes in PI4P metabolism in dendrites. Upon LTP induction, PI4KIIIα, a type III phosphatidylinositol 4-kinase, localizes to the dendritic plasma membrane (PM) in a calcium-dependent manner and causes substantial increase in the levels of PI4P. Acute inhibition of PI4KIIIα activity abolishes trafficking of the AMPA-type glutamate receptor to the PM during LTP induction, and silencing of PI4KIIIα expression in the hippocampal CA1 region causes severe impairment of LTP and long-term memory. Collectively, our results identify an essential role for PI4KIIIα-dependent PI4P synthesis in synaptic plasticity of central nervous system neurons.

Increased fMRI connectivity upon chemogenetic inhibition of the mouse prefrontal cortex

While shaped and constrained by axonal connections, fMRI-based functional connectivity reorganizes in response to varying interareal input or pathological perturbations. However, the causal contribution of regional brain activity to whole-brain fMRI network organization remains unclear. Here we combine neural manipulations, resting-state fMRI and in vivo electrophysiology to probe how inactivation of a cortical node causally affects brain-wide fMRI coupling in the mouse. We find that chronic inhibition of the medial prefrontal cortex (PFC) via overexpression of a potassium channel increases fMRI connectivity between the inhibited area and its direct thalamo-cortical targets. Acute chemogenetic inhibition of the PFC produces analogous patterns of fMRI overconnectivity. Using in vivo electrophysiology, we find that chemogenetic inhibition of the PFC enhances low frequency (0.1–4 Hz) oscillatory power via suppression of neural firing not phase-locked to slow rhythms, resulting in increased slow and δ band coherence between areas that exhibit fMRI overconnectivity. These results provide causal evidence that cortical inactivation can counterintuitively increase fMRI connectivity via enhanced, less-localized slow oscillatory processes.

Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during Drosophila mesoderm invagination.

Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, we find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. We propose that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.

Age-dependent attenuation of spatial memory deficits by the histone acetyltransferase p300/CBP-associated factor (PCAF) in 3xTG Alzheimer's disease mice.

Histone acetylation, catalyzed by histone acetyltransferases, has emerged as a promising therapeutic strategy in Alzheimer's disease (AD). By longitudinally characterizing spatial memory at 3, 6, and 9 mo of age, we show that acute activation and inhibition of the histone acetyltransferase PCAF remediated memory impairments in 3xTG-AD mice in an age-related bidirectional manner. At 3 and 6 mo of age, PCAF activation ameliorated memory deficits. At 9 mo of age, PCAF activation had no effect on spatial memory, whereas PCAF inhibition improved memory deficits in females. This work reveals a complex potential therapeutic role for PCAF in AD, initially benefitting memory but becoming detrimental as the disease progresses.

SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice

Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide‑binding oligomerization domain, leucine rich repeat, and pyrin domain‑containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.

The effect of the long-term inhibition of hydrogen sulfide production on the reactivity of the cardiovascular system in Wistar rats.

In this study, we investigated the blood pressure responses of the peripheral bed invivo after chronic hydrogen sulfide (H2S) inhibition combined with acute nitric oxide (NO) deficiency. We also evaluated the role of endogenously produced H2S in the vasoactive responses of large- and medium-sized arteries invitro. Changes in integrated blood pressure responses were measured after chronic inhibition of cystathionine-γ-lyase, an enzyme involved in H2S synthesis, with DL-propargylglycine (PPG), and acute inhibition of NO-synthase with nonspecific L-NG-nitro arginine methyl ester (L-NAME), and vasoactive responses of the thoracic aorta (TA) and mesenteric artery (MA) were investigated after acute incubation with PPG. We confirmed that chronic H2S deficiency had no effect on blood pressure, heart trophycity, noradrenaline, and H2S donor vasoactive responses but induced renal hypertrophy and a decrease in acetylcholine-induced hypotensive and L-NAME-induced hypertensive responses. Acute H2S deficiency led to an increase in basal tone (MA) or active tone (TA), whereas endothelium-dependent vasorelaxation remained unaffected. Long-term administration of PPG revealed a role of endogenous H2S in the bioavailability of endothelial NO in peripheral arteries. When both H2S and NO were lacking, the activation of H2S-independent compensatory mechanisms plays an important role in maintaining the vasodilator responses of the cardiovascular system.

PTEN Regulates Dendritic Arborization by Decreasing Microtubule Polymerization Rate.

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway. Loss-of-function mutations in PTEN have been found in a subset of patients with macrocephaly and autism spectrum disorder (ASD). PTEN loss in neurons leads to somal hypertrophy, aberrant migration, dendritic overgrowth, increased spine density, and hyperactivity of neuronal circuits. These neuronal overgrowth phenotypes are present on Pten knock-out (KO) and reconstitution with autism-associated point mutations. The mechanism underlying dendritic overgrowth in Pten deficient neurons is unclear. In this study, we examined how Pten loss impacts microtubule (MT) dynamics in both sexes using retroviral infection and transfection strategies to manipulate PTEN expression and tag the plus-end MT binding protein, end-binding protein 3 (EB3). We found Pten KO neurons sprout more new processes over time compared with wild-type (WT) neurons. We also found an increase in MT polymerization rate in Pten KO dendritic growth cones. Reducing MT polymerization rate to the WT level was sufficient to reduce dendritic overgrowth in Pten KO neurons in vitro and in vivo Finally, we found that rescue of dendritic overgrowth via inhibition of MT polymerization was sufficient to improve the performance of Pten KO mice in a spatial memory task. Taken together, our data suggests that one factor underlying PTEN loss dependent dendritic overgrowth is increased MT polymerization. This opens the possibility for an intersectional approach targeting MT polymerization and mTOR with low doses of inhibitors to achieve therapeutic gains with minimal side effects in pathologies associated with loss of neuronal PTEN function.SIGNIFICANCE STATEMENT Loss of Pten function because of genetic deletion or expression of mutations associated with autism spectrum disorder (ASD), results in overgrowth of neurons including increased total dendritic length and branching. We have discovered that this overgrowth is accompanied by increased rate of microtubule (MT) polymerization. The increased polymerization rate is insensitive to acute inhibition of mechanistic target of rapamycin (mTOR)C1 or protein synthesis. Direct pharmacological inhibition of MT polymerization can slow the polymerization rate in Pten knock-out (KO) neurons to rates seen in wild-type (WT) neurons. Correction of the MT polymerization rate rescues increased total dendritic arborization and spatial memory. Our studies suggest that phosphatase and tensin homolog (PTEN) inhibits dendritic growth through parallel regulation of protein synthesis and cytoskeletal polymerization.

Orexin 1 Receptor Antagonism in the Basolateral Amygdala Shifts the Balance From Pro- to Antistress Signaling and Behavior

BackgroundStress produces differential behavioral responses through select molecular modifications to specific neurocircuitry elements. The orexin (Orx) system targets key components of this neurocircuitry in the basolateral amygdala (BLA). MethodsWe assessed the contribution of intra-BLA Orx1 receptors (Orx1Rs) in the expression of stress-induced phenotypes of mice. Using the Stress Alternatives Model, a social stress paradigm that produces two behavioral phenotypes, we characterized the role of intra-BLA Orx1R using acute pharmacological inhibition (SB-674042) and genetic knockdown (AAV-U6-Orx1R-shRNA) strategies. ResultsIn the BLA, we observed that Orx1R (Hcrtr1) messenger RNA is predominantly expressed in CamKIIα+ glutamatergic neurons and rarely in GABAergic (gamma-aminobutyric acidergic) cells. While there is a slight overlap in Hcrtr1 and Orx2 receptor (Hcrtr2) messenger RNA expression in the BLA, we find that these receptors are most often expressed in separate cells. Antagonism of intra-BLA Orx1R after phenotype formation shifted behavioral expression from stress-sensitive (Stay) to stress-resilient (Escape) responses, an effect that was mimicked by genetic knockdown. Acute inhibition of Orx1R in the BLA also reduced contextual and cued fear freezing responses in Stay animals. This phenotype-specific behavioral change was accompanied by biased molecular transcription favoring Hcrtr2 over Hcrtr1 and Mapk3 over Plcb1 cell signaling cascades and enhanced Bdnf messenger RNA. ConclusionsFunctional reorganization of intra-BLA gene expression is produced by antagonism of Orx1R, which promotes elevated Hcrtr2, greater Mapk3, and increased Bdnf expression. Together, these results provide evidence for a receptor-driven mechanism that balances pro- and antistress responses within the BLA.