Inhaled [D-Ala2]-Dynorphin 1-6 Prevents Hyperacetylation and Release of High Mobility Group Box 1 in a Mouse Model of Acute Lung Injury.

COVID-19 is a respiratory infection caused by the SARS-CoV-2 virus that can rapidly escalate to life-threatening pneumonia and acute respiratory distress syndrome (ARDS). Recently, extracellular high mobility group box 1 (HMGB1) has been identified as an essential component of cytokine storms that occur with COVID-19; HMGB1 levels correlate significantly with disease severity. Thus, the modulation of HMGB1 release may be vital for treating COVID-19. HMGB1 is a ubiquitous nuclear DNA-binding protein whose biological function depends on posttranslational modifications, its redox state, and its cellular localization. The acetylation of HMGB1 is a prerequisite for its translocation from the nucleus to the cytoplasm and then to the extracellular milieu. When released, HMGB1 acts as a proinflammatory cytokine that binds primarily to toll-like receptor 4 (TLR4) and RAGE, thereby stimulating immune cells, endothelial cells, and airway epithelial cells to produce cytokines, chemokines, and other inflammatory mediators. In this study, we demonstrate that inhaled [D-Ala2]-dynorphin 1-6 (leytragin), a peptide agonist of δ-opioid receptors, significantly inhibits HMGB1 secretion in mice with lipopolysaccharide- (LPS-) induced acute lung injury. The mechanism of action involves preventing HMGB1's hyperacetylation at critical lysine residues within nuclear localization sites, as well as promoting the expression of sirtuin 1 (SIRT1), an enzyme known to deacetylate HMGB1. Leytragin's effects are mediated by opioid receptors, since naloxone, an antagonist of opioid receptors, abrogates the leytragin effect on SIRT1 expression. Overall, our results identify leytragin as a promising therapeutic agent for the treatment of pulmonary inflammation associated with HMGB1 release. In a broader context, we demonstrate that the opioidergic system in the lungs may represent a promising target for the treatment of inflammatory lung diseases.

Molecular and behavioural abnormalities in the FUS-tg mice mimic frontotemporal lobar degeneration: Effects of old and new anti-inflammatory therapies.

Genetic mutations in FUS, a DNA/RNA-binding protein, are associated with inherited forms of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A novel transgenic FUS[1-359]-tg mouse line recapitulates core hallmarks of human ALS in the spinal cord, including neuroinflammation and neurodegeneration, ensuing muscle atrophy and paralysis, as well as brain pathomorphological signs of FTLD. However, a question whether FUS[1-359]-tg mouse displays behavioural and brain pro-inflammatory changes characteristic for the FTLD syndrome was not addressed. Here, we studied emotional, social and cognitive behaviours, brain markers of inflammation and plasticity of pre-symptomatic FUS[1-359]-tg male mice, a potential FTLD model. These animals displayed aberrant behaviours and altered brain expression of inflammatory markers and related pathways that are reminiscent to the FTLD-like syndrome. FTLD-related behavioural and molecular Journal of Cellular and Molecular Medicine features were studied in the pre-symptomatic FUS[1-359]-tg mice that received standard or new ALS treatments, which have been reported to counteract the ALS-like syndrome in the mutants. We used anti-ALS drug riluzole (8 mg/kg/d), or anti-inflammatory drug, a selective blocker of cyclooxygenase-2 (celecoxib, 30 mg/kg/d) for 3 weeks, or a single intracerebroventricular (i.c.v.) infusion of human stem cells (Neuro-Cells, 500 000-CD34+ ), which showed anti-inflammatory properties. Signs of elevated anxiety, depressive-like behaviour, cognitive deficits and abnormal social behaviour were less marked in FUS-tg-treated animals. Applied treatments have normalized protein expression of interleukin-1ß (IL-1ß) in the prefrontal cortex and the hippocampus, and of Iba-1 and GSK-3ß in the hippocampus. Thus, the pre-symptomatic FUS[1-359]-tg mice demonstrate FTLD-like abnormalities that are attenuated by standard and new ALS treatments, including Neuro-Cell preparation.

Endotoxaemia resulting from decreased serotonin tranporter (5-HTT) function: a reciprocal risk factor for depression and insulin resistance?

Depression and diabetes are serious diseases with an increasing global prevalence. Intriguingly, recent meta-analyses have highlighted an asymmetrical relationship between the two conditions as depressed patients were found to display a higher risk of developing type 2 diabetes than those individuals suffering from diabetes are to become depressed. Based on recent findings, we favor a hypothesis where by decreased peripheral serotonin (5-HT) transporter (5-HTT) function is a reciprocal risk factor for the co-morbidity of depression and diabetes, as it can trigger inflammatory pathogenetic mechanisms of both conditions. Higher intestinal levels of 5-HT and 5-HT3 receptor stimulation lead to increased intestinal permeability in 5-HTT deficient mice, which is viewed one of the most relevant animal models of depression. We hypothesize that this leakage of bacterial endotoxins can activate both central and peripheral Toll-like receptor 4 (TLR4), which inhibits insulin signaling and IRS1/PI3K/Akt and thus, contribute to the pathogenesis of diabetes and depression that are associated with this pathway. Antidepressant therapies, which also suppress intestinal 5-HTT, may have potentiating effects on the association between depression and diabetes. It is also of interest that high carbohydrate and fat intake ("cafeteria-type diet") increases intestinal 5-HT leading to TLR4 activation. Thus, endotoxaemia and inflammation owing to increased intestinal 5-HT may underpin the depression and diabetes association, where the risk of the latter pathology becomes particularly preeminent after the onset of depression and not vice versa. The evidence presented here shows the further investigation into peripheral mechanisms that linked diabetes to depression is clearly warranted.

Mitochondrial H2O2 as an enable signal for triggering autophosphorylation of insulin receptor in neurons.

BACKGROUND: Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H2O2 production, which plays a role in receptor autophosphorylation. However, the kinetic characteristics of the H2O2 signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date. RESULTS: Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H2O2 signal precedes receptor autophosphorylation and represents a single spike with a peak at 5-10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H2O2 signal. The mechanism by which insulin triggers the H2O2 signal involves modulation of succinate dehydrogenase activity. Insulin dose-response for receptor autophosphorylation is well described by hyperbolic function (Hill coefficient, nH, of 1.1±0.1; R2=0.99). N-acetylcysteine (NAC), a scavenger of H2O2, dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal (Hill coefficient, nH, of 8.0±2.3; R2=0.97), signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H2O2 signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H2O2 signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H2O2 generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H2O2-generating system in neurons during the autophosphorylation process. CONCLUSIONS: In this study, we demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial H2O2 signal exceeds a certain threshold. This finding provides novel insights into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H2O2 signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of mitochondrial H2O2 signaling may lead to the development of cerebral insulin resistance.

H2O2 Signalling Pathway: A Possible Bridge between Insulin Receptor and Mitochondria.

This review is focused on the mechanistic aspects of the insulin-induced H2O2 signalling pathway in neurons and the molecules affecting it, which act as risk factors for developing central insulin resistance. Insulin-induced H2O2 promotes insulin receptor activation and the mitochondria act as the insulin-sensitive H2O2 source, providing a direct molecular link between mitochondrial dysfunction and irregular insulin receptor activation. In this view, the accumulation of dysfunctional mitochondria during chronological ageing and Alzheimer's disease (AD) is a risk factor that may contribute to the development of dysfunctional cerebral insulin receptor signalling and insulin resistance. Due to the high significance of insulin-induced H2O2 for insulin receptor activation, oxidative stress-induced upregulation of antioxidant enzymes, e.g., in AD brains, may represent another risk factor contributing to the development of insulin resistance. As insulin-induced H2O2 signalling requires fully functional mitochondria, pharmacological strategies based on activating mitochondria biogenesis in the brain are central to the treatment of diseases associated with dysfunctional insulin receptor signalling in this organ.

Dicholine salt of succinic acid, a neuronal insulin sensitizer, ameliorates cognitive deficits in rodent models of normal aging, chronic cerebral hypoperfusion, and beta-amyloid peptide-(25-35)-induced amnesia.

BACKGROUND: Accumulated evidence suggests that insulin resistance and impairments in cerebral insulin receptor signaling may contribute to age-related cognitive deficits and Alzheimer's disease. The enhancement of insulin receptor signaling is, therefore, a promising strategy for the treatment of age-related cognitive disorders. The mitochondrial respiratory chain, being involved in insulin-stimulated H2O2 production, has been identified recently as a potential target for the enhancement of insulin signaling. The aim of the present study is to examine: (1) whether a specific respiratory substrate, dicholine salt of succinic acid (CS), can enhance insulin-stimulated insulin receptor autophosphorylation in neurons, and (2) whether CS can ameliorate cognitive deficits of various origins in animal models. RESULTS: In a primary culture of cerebellar granule neurons, CS significantly enhanced insulin-stimulated insulin receptor autophosphorylation. In animal models, CS significantly ameliorated cognitive deficits, when administered intraperitoneally for 7 days. In 16-month-old middle-aged C57Bl/6 mice (a model of normal aging), CS enhanced spatial learning in the Morris water maze, spontaneous locomotor activity, passive avoidance performance, and increased brain N-acetylaspartate/creatine levels, as compared to the age-matched control (saline). In rats with chronic cerebral hypoperfusion, CS enhanced spatial learning, passive avoidance performance, and increased brain N-acetylaspartate/creatine levels, as compared to control rats (saline). In rats with beta-amyloid peptide-(25-35)-induced amnesia, CS enhanced passive avoidance performance and increased activity of brain choline acetyltransferase, as compared to control rats (saline). In all used models, CS effects lasted beyond the seven-day treatment period and were found to be significant about two weeks following the treatment. CONCLUSION: The results of the present study suggest that dicholine salt of succinic acid, a novel neuronal insulin sensitizer, ameliorates cognitive deficits and neuronal dysfunctions in animal models relevant to age-related cognitive impairments, vascular dementia, and Alzheimer's disease.

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons.

BACKGROUND: Accumulated evidence suggests that hydrogen peroxide (H2O2) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H2O2 in neuronal insulin receptor activation and the origin of insulin-induced H2O2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H2O2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons. RESULTS: Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H2O2 release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H2O2 scavenger, inhibited both insulin-stimulated H2O2 release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H2O2 production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H2O2 release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN. CONCLUSION: Results of the present study suggest that insulin-induced H2O2 is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in the insulin receptor autophosphorylation in neurons.