GlyNAC (Glycine and N-Acetylcysteine) Supplementation in Old Mice Improves Brain Glutathione Deficiency, Oxidative Stress, Glucose Uptake, Mitochondrial Dysfunction, Genomic Damage, Inflammation and Neurotrophic Factors to Reverse Age-Associated Cognitive Decline: Implications for Improving Brain Health in Aging.

Cognitive decline frequently occurs with increasing age, but mechanisms contributing to age-associated cognitive decline (ACD) are not well understood and solutions are lacking. Understanding and reversing mechanisms contributing to ACD are important because increased age is identified as the single most important risk factor for dementia. We reported earlier that ACD in older humans is associated with glutathione (GSH) deficiency, oxidative stress (OxS), mitochondrial dysfunction, glucose dysmetabolism and inflammation, and that supplementing GlyNAC (glycine and N-acetylcysteine) improved these defects. To test whether these defects occur in the brain in association with ACD, and could be improved/reversed with GlyNAC supplementation, we studied young (20-week) and old (90-week) C57BL/6J mice. Old mice received either regular or GlyNAC supplemented diets for 8 weeks, while young mice received the regular diet. Cognition and brain outcomes (GSH, OxS, mitochondrial energetics, autophagy/mitophagy, glucose transporters, inflammation, genomic damage and neurotrophic factors) were measured. Compared to young mice, the old-control mice had significant cognitive impairment and multiple brain defects. GlyNAC supplementation improved/corrected the brain defects and reversed ACD. This study finds that naturally-occurring ACD is associated with multiple abnormalities in the brain, and provides proof-of-concept that GlyNAC supplementation corrects these defects and improves cognitive function in aging.

Supplementing Glycine and N-Acetylcysteine (GlyNAC) in Older Adults Improves Glutathione Deficiency, Oxidative Stress, Mitochondrial Dysfunction, Inflammation, Physical Function, and Aging Hallmarks: A Randomized Clinical Trial.

BACKGROUND: Elevated oxidative stress (OxS), mitochondrial dysfunction, and hallmarks of aging are identified as key contributors to aging, but improving/reversing these defects in older adults (OA) is challenging. In prior studies, we identified that deficiency of the intracellular antioxidant glutathione (GSH) could play a role and reported that supplementing GlyNAC (combination of glycine and N-acetylcysteine [NAC]) in aged mice improved GSH deficiency, OxS, mitochondrial fatty-acid oxidation (MFO), and insulin resistance (IR). To test whether GlyNAC supplementation in OA could improve GSH deficiency, OxS, mitochondrial dysfunction, IR, physical function, and aging hallmarks, we conducted a placebo-controlled randomized clinical trial. METHODS: Twenty-four OA and 12 young adults (YA) were studied. OA was randomized to receive either GlyNAC (N = 12) or isonitrogenous alanine placebo (N = 12) for 16-weeks; YA (N = 12) received GlyNAC for 2-weeks. Participants were studied before, after 2-weeks, and after 16-weeks of supplementation to assess GSH concentrations, OxS, MFO, molecular regulators of energy metabolism, inflammation, endothelial function, IR, aging hallmarks, gait speed, muscle strength, 6-minute walk test, body composition, and blood pressure. RESULTS: Compared to YA, OA had GSH deficiency, OxS, mitochondrial dysfunction (with defective molecular regulation), inflammation, endothelial dysfunction, IR, multiple aging hallmarks, impaired physical function, increased waist circumference, and systolic blood pressure. GlyNAC (and not placebo) supplementation in OA improved/corrected these defects. CONCLUSION: GlyNAC supplementation in OA for 16-weeks was safe and well-tolerated. By combining the benefits of glycine, NAC and GSH, GlyNAC is an effective nutritional supplement that improves and reverses multiple age-associated abnormalities to promote health in aging humans. Clinical Trials Registration Number: NCT01870193.

Mechanistic Role of Jak3 in Obesity-Associated Cognitive Impairments.

BACKGROUND AND AIMS: A compromise in intestinal mucosal functions is associated with several chronic inflammatory diseases. Previously, we reported that obese humans have a reduced expression of intestinal Janus kinase-3 (Jak3), a non-receptor tyrosine kinase, and a deficiency of Jak3 in mice led to predisposition to obesity-associated metabolic syndrome. Since meta-analyses show cognitive impairment as co-morbidity of obesity, the present study demonstrates the mechanistic role of Jak3 in obesity associated cognitive impairment. Our data show that high-fat diet (HFD) suppresses Jak3 expression both in intestinal mucosa and in the brain of wild-type mice. METHODOLOGY: Recapitulating these conditions using global (Jak3-KO) and intestinal epithelial cell-specific conditional (IEC-Jak3-KO) mice and using cognitive testing, western analysis, flow cytometry, immunofluorescence microscopy and 16s rRNA sequencing, we demonstrate that HFD-induced Jak3 deficiency is responsible for cognitive impairments in mice, and these are, in part, specifically due to intestinal epithelial deficiency of Jak3. RESULTS: We reveal that Jak3 deficiency leads to gut dysbiosis, compromised TREM-2-functions-mediated activation of microglial cells, increased TLR-4 expression and HIF1-α-mediated inflammation in the brain. Together, these lead to compromised microglial-functions-mediated increased deposition of ß-amyloid (Aß) and hyperphosphorylated Tau (pTau), which are responsible for cognitive impairments. Collectively, these data illustrate how the drivers of obesity promote cognitive impairment and demonstrate the underlying mechanism where HFD-mediated impact on IEC-Jak3 deficiency is responsible for Jak3 deficiency in the brain, reduced microglial TREM2 expression, microglial activation and compromised clearance of Aß and pTau as the mechanism during obesity-associated cognitive impairments. CONCLUSION: Thus, we not only demonstrate the mechanism of obesity-associated cognitive impairments but also characterize the tissue-specific role of Jak3 in such conditions through mucosal tolerance, gut-brain axis and regulation of microglial functions.

GlyNAC (Glycine and N-Acetylcysteine) Supplementation in Mice Increases Length of Life by Correcting Glutathione Deficiency, Oxidative Stress, Mitochondrial Dysfunction, Abnormalities in Mitophagy and Nutrient Sensing, and Genomic Damage.

Determinants of length of life are not well understood, and therefore increasing lifespan is a challenge. Cardinal theories of aging suggest that oxidative stress (OxS) and mitochondrial dysfunction contribute to the aging process, but it is unclear if they could also impact lifespan. Glutathione (GSH), the most abundant intracellular antioxidant, protects cells from OxS and is necessary for maintaining mitochondrial health, but GSH levels decline with aging. Based on published human studies where we found that supplementing glycine and N-acetylcysteine (GlyNAC) improved/corrected GSH deficiency, OxS and mitochondrial dysfunction, we hypothesized that GlyNAC supplementation could increase longevity. We tested our hypothesis by evaluating the effect of supplementing GlyNAC vs. placebo in C57BL/6J mice on (a) length of life; and (b) age-associated GSH deficiency, OxS, mitochondrial dysfunction, abnormal mitophagy and nutrient-sensing, and genomic-damage in the heart, liver and kidneys. Results showed that mice receiving GlyNAC supplementation (1) lived 24% longer than control mice; (2) improved/corrected impaired GSH synthesis, GSH deficiency, OxS, mitochondrial dysfunction, abnormal mitophagy and nutrient-sensing, and genomic-damage. These studies provide proof-of-concept that GlyNAC supplementation can increase lifespan and improve multiple age-associated defects. GlyNAC could be a novel and simple nutritional supplement to improve lifespan and healthspan, and warrants additional investigation.

Glycine and N-acetylcysteine (GlyNAC) supplementation in older adults improves glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, genotoxicity, muscle strength, and cognition: Results of a pilot clinical trial.

BACKGROUND: Oxidative stress (OxS) and mitochondrial dysfunction are implicated as causative factors for aging. Older adults (OAs) have an increased prevalence of elevated OxS, impaired mitochondrial fuel-oxidation (MFO), elevated inflammation, endothelial dysfunction, insulin resistance, cognitive decline, muscle weakness, and sarcopenia, but contributing mechanisms are unknown, and interventions are limited/lacking. We previously reported that inducing deficiency of the antioxidant tripeptide glutathione (GSH) in young mice results in mitochondrial dysfunction, and that supplementing GlyNAC (combination of glycine and N-acetylcysteine [NAC]) in aged mice improves naturally-occurring GSH deficiency, mitochondrial impairment, OxS, and insulin resistance. This pilot trial in OA was conducted to test the effect of GlyNAC supplementation and withdrawal on intracellular GSH concentrations, OxS, MFO, inflammation, endothelial function, genotoxicity, muscle and glucose metabolism, body composition, strength, and cognition. METHODS: A 36-week open-label clinical trial was conducted in eight OAs and eight young adults (YAs). After all the participants underwent an initial (pre-supplementation) study, the YAs were released from the study. OAs were studied again after GlyNAC supplementation for 24 weeks, and GlyNAC withdrawal for 12 weeks. Measurements included red-blood cell (RBC) GSH, MFO; plasma biomarkers of OxS, inflammation, endothelial function, glucose, and insulin; gait-speed, grip-strength, 6-min walk test; cognitive tests; genomic-damage; glucose-production and muscle-protein breakdown rates; and body-composition. RESULTS: GlyNAC supplementation for 24 weeks in OA corrected RBC-GSH deficiency, OxS, and mitochondrial dysfunction; and improved inflammation, endothelial dysfunction, insulin-resistance, genomic-damage, cognition, strength, gait-speed, and exercise capacity; and lowered body-fat and waist-circumference. However, benefits declined after stopping GlyNAC supplementation for 12 weeks. CONCLUSIONS: GlyNAC supplementation for 24-weeks in OA was well tolerated and lowered OxS, corrected intracellular GSH deficiency and mitochondrial dysfunction, decreased inflammation, insulin-resistance and endothelial dysfunction, and genomic-damage, and improved strength, gait-speed, cognition, and body composition. Supplementing GlyNAC in aging humans could be a simple and viable method to promote health and warrants additional investigation.

Severe Glutathione Deficiency, Oxidative Stress and Oxidant Damage in Adults Hospitalized with COVID-19: Implications for GlyNAC (Glycine and N-Acetylcysteine) Supplementation.

Humanity is battling a respiratory pandemic pneumonia named COVID-19 which has resulted in millions of hospitalizations and deaths. COVID-19 exacerbations occur in waves that continually challenge healthcare systems globally. Therefore, there is an urgent need to understand all mechanisms by which COVID-19 results in health deterioration to facilitate the development of protective strategies. Oxidative stress (OxS) is a harmful condition caused by excess reactive-oxygen species (ROS) and is normally neutralized by antioxidants among which Glutathione (GSH) is the most abundant. GSH deficiency results in amplified OxS due to compromised antioxidant defenses. Because little is known about GSH or OxS in COVID-19 infection, we measured GSH, TBARS (a marker of OxS) and F2-isoprostane (marker of oxidant damage) concentrations in 60 adult patients hospitalized with COVID-19. Compared to uninfected controls, COVID-19 patients of all age groups had severe GSH deficiency, increased OxS and elevated oxidant damage which worsened with advancing age. These defects were also present in younger age groups, where they do not normally occur. Because GlyNAC (combination of glycine and N-acetylcysteine) supplementation has been shown in clinical trials to rapidly improve GSH deficiency, OxS and oxidant damage, GlyNAC supplementation has implications for combating these defects in COVID-19 infected patients and warrants urgent investigation.

Supplementing Glycine and N-acetylcysteine (GlyNAC) in Aging HIV Patients Improves Oxidative Stress, Mitochondrial Dysfunction, Inflammation, Endothelial Dysfunction, Insulin Resistance, Genotoxicity, Strength, and Cognition: Results of an Open-Label Clinical Trial.

Background: Patients with HIV (PWH) develop geriatric comorbidities, including functional and cognitive decline at a younger age. However, contributing mechanisms are unclear and interventions are lacking. We hypothesized that deficiency of the antioxidant protein glutathione (GSH) contributes to multiple defects representing premature aging in PWH, and that these defects could be improved by supplementing the GSH precursors glycine and N-acetylcysteine (GlyNAC). Methods: We conducted an open label clinical trial where eight PWH and eight matched uninfected-controls were studied at baseline. PWH were studied again 12-weeks after receiving GlyNAC, and 8-weeks after stopping GlyNAC. Controls did not receive supplementation. Outcome measures included red-blood cell and muscle GSH concentrations, mitochondrial function, mitophagy and autophagy, oxidative stress, inflammation, endothelial function, genomic damage, insulin resistance, glucose production, muscle-protein breakdown rates, body composition, physical function and cognition. Results: PWH had significant defects in measured outcomes, which improved with GlyNAC supplementation. However, benefits receded after stopping GlyNAC. Conclusions: This open label trial finds that PWH have premature aging based on multiple biological and functional defects, and identifies novel mechanistic explanations for cognitive and physical decline. Nutritional supplementation with GlyNAC improves comorbidities suggestive of premature aging in PWH including functional and cognitive decline, and warrants additional investigation.

Emerging Prevention and Treatment Strategies to Control COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has now become a serious global threat after inflicting more than 8 million infections and 425,000 deaths in less than 6 months. Currently, no definitive treatment or prevention therapy exists for COVID-19. The unprecedented rise of this pandemic has rapidly fueled research efforts to discover and develop new vaccines and treatment strategies against this novel coronavirus. While hundreds of vaccines/therapeutics are still in the preclinical or early stage of clinical development, a few of them have shown promising results in controlling the infection. Here, in this review, we discuss the promising vaccines and treatment options for COVID-19, their challenges, and potential alternative strategies.

Hyperglycemia-Induced Oxidative-Nitrosative Stress Induces Inflammation and Neurodegeneration via Augmented Tuberous Sclerosis Complex-2 (TSC-2) Activation in Neuronal Cells.

Diabetes is a systemic disease mainly characterized by chronic hyperglycemia and with extensive and long-lasting spiteful complications in central nervous systems (CNS). Astrocytes play an important role in the defense mechanism of CNS, with great ability of withstanding accumulation of toxic substances. Apart from functional disorders, hyperglycemia leads to slow progressive structural abnormalities in the CNS through oxidative stress pathways. However, the molecular mechanism by which neurons die under oxidative stress induced by high glucose (HG) remains largely unclear. Here, we report that HG-induced inflammation and neurodegeneration in brain tissues, brain astrocytes (C6), and pheochromocytoma (PC-12) cells are cultured in HG conditions. Our results show that the increases in phosphorylation of Akt and ERK1/2MAPK are associated with increased accumulations of reactive oxygen species (ROS) in neuronal cells, which simultaneously enhanced phosphorylations of tuberous sclerosis complex-2 (TSC-2) and mammalian target of rapamycin (mTOR) in the diabetic brain and in HG-exposed neuronal cells. Pharmacologic inhibition of Akt or ERK1/2 or siRNA-mediated gene silencing of TSC-2 suppressed the strong downregulation of TSC-2-mTOR activation. Findings of this study also demonstrate that HG resulted in phosphorylation of NF-κB, coinciding with the increased production of inflammatory mediators and activation of neurodegenerative markers. Pretreatment of cells with antioxidants, phosphoinositide3-kinase (PI3-K)/Akt, and ERK1/2 inhibitors significantly reduced HG-induced TSC-2 phosphorylation and restored NF-κB protein expression leading to decreased production of inflammatory mediators and neurodegenerative markers. These results illustrate that ROS functions as a key signaling component in the regulatory pathway induced by elevated glucose in neuronal cell activation leading to inflammation and neurodegeneration.

Lactobacillus acidophilus attenuates Aeromonas hydrophila induced cytotoxicity in catla thymus macrophages by modulating oxidative stress and inflammation.

The pathogenesis of Aeromonas hydrophila, a potent fish pathogen, is attributed to its ability to cause motile aeromonad septicaemia leading to apoptosis in a myriad of fish species, including freshwater carp Catla catla. However, the underlying mechanism of antagonistic activity of probiotics against A. hydrophila induced apoptosis is not elucidated due to lack of appropriate in-vitro models. This study reported that the exposure of catla thymus macrophages (CTM) to A. hydrophila markedly induced cellular injuries as evidenced by elevated levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), increased apoptosis, DNA damage and decreased cellular viability. Flow cytometry analysis and Annexin-V/propidium iodide assay further confirmed increased ROS positive cells leading to cell death after infection. The quantitative real-time PCR analysis, also revealed upregulation of inducible nitric-oxide synthase (iNOS), pro-inflammatory cytokine (TNFα), cyclooxygenase2 (COX-2) and downregulation of anti-inflammatory cytokine (IL-10). Pretreatment of cells with probiotic, Lactobacillus acidophilus attenuated A. hydrophila induced apoptosis as evident from the decrease in the levels of ROS, RNS and DNA damage. Significant increase (P≤0.05) in expression of TNFα and IL-10 and decrease in iNOS and COX-2 was observed on probiotic stimulation. In-vivo study using catla fingerlings confirmed similar pattern of ROS, iNOS, NO production and cytokine expression in thymus. This study provides a comprehensive insight into the mechanistic basis of L. acidophilus induced macrophage mediated inflammatory response against A. hydrophila in CTM cells. Further, it speculates the possibility of using cost-effective in-vitro models for screening probiotic candidates of therapeutic potential in aquaculture industry.

Hyperglycemia-induced inflammation caused down-regulation of 8-oxoG-DNA glycosylase levels in murine macrophages is mediated by oxidative-nitrosative stress-dependent pathways.

High glucose-induced increase in production of reactive oxygen/nitrogen species (ROS/RNS) is recognized as a major cause of the clinical complications associated with diabetes. ROS/RNS apart from being redox agents, cause an unwanted severe physiological load to cells, also act as cellular messengers, and play a key role in activation of circulating macrophages. However, the molecular mechanisms of activation of macrophages by hyperglycemic conditions are currently unclear. In the present study, we report that high glucose (HG) causes a dramatic increase in the production of inflammatory cytokines and chemokines, at least in part through enhanced mRNA transcription. The increase in levels of inflammatory cytokines/chemokines corresponds to increased levels of ROS/RNS, which is accompanied by increased activities of Akt, ERK1/2, tuberin, down regulation of 8-oxoG-DNA glycosylase (OGG1), and increase in 8-hydroxydeoxyguanosine (8-OHdG) accumulation in DNA. Elevated levels of ROS/RNS are triggering alteration in antioxidants level, biomolecules damage, cell cycle dysregulation, and apoptosis in macrophage cells. Pretreatment of antioxidants caused decrease in the levels of ROS/RNS leads to an increase in the levels of antioxidants, decrease in biomolecules damage, alterations in Akt, ERK1/2, tuberin, upregulation of OGG1, and decrease in 8-OHdG accumulations in DNA. Further, antioxidants treatments inhibit the effects of HG on the transcriptional activity of cytokines and chemokines. Our results demonstrate that intracellular signaling pathways mediated by ROS/RNS are linked to each other by elevated glucose in macrophages activation leading to inflammation. These findings provide a mechanistic explanation of how ROS/RNS cooperate to conduct inflammatory intracellular signals in macrophages related complications in hyperglycemic conditions.

Effects of CEES and LPS synergistically stimulate oxidative stress inactivates OGG1 signaling in macrophage cells.

2-chloroethyl ethyl sulphide (CEES), a monofunctional analogue of sulfur mustard, is a strong vesicant and an alkylating chemical warfare agent. We studied the molecular mechanism of oxidative stress triggered signaling cascades in murine macrophages exposed to CEES with lipopolysaccharide (LPS). Exposure of CEES with specific dose of LPS stimulates oxidative stress caused increasing level of intracellular ROS and RNS, decreased antioxidant enzymes, increasing bimolecular damage, reduced cell viability, and cell cycle arrest. Synergistic exposure of CEES and LPS provoked significant increase in phosphorylation of MAPKs, Akt, tuberin, that down regulate OGG1 expression and 8-OHdG accumulations. Treatment with Akt and ERK1/2 inhibitors, the cells with constitutively active inhibiting activity of Akt and ERK1/2MAPK significant reduce CEES and LPS challenge tuberin but not the OGG1. In addition, the N-acetylcysteine inhibited ROS/RNS generation, elevation of antioxidants level, expression of ERK1/2, Akt, tuberin phosphorylation, resulted in deceased 8-OHdG accumulation and upregulation of OGG1 protein expression suggesting no involvement of Akt and ERK1/2MAPK pathways after CEES and LPS challenge. Collectively, our results indicate that exposure of CEES and LPS induces oxidative stress and the activation of tuberin, and 8-OHdG accumulation via upstream signaling pathways including Akt and ERK1/2MAPK pathway in macrophages but not the down regulation of OGG1.

Hyperglycemia-induced oxidative stress induces apoptosis by inhibiting PI3-kinase/Akt and ERK1/2 MAPK mediated signaling pathway causing downregulation of 8-oxoG-DNA glycosylase levels in glial cells.

Glial cells are very important for normal brain function and alterations in their activity due to hyperglycemia, could contribute to diabetes-related cognitive dysfunction. Oxidative insults often cause rapid changes in almost all cells including glial cells. However, pathophysiologic mechanisms that lead to diabetic complications are not completely elucidated. Therefore, we examined whether elevated glucose levels directly or indirectly disrupt antioxidant defense mechanisms causing alterations in signaling pathways, cell cycle dysregulation, and reactive oxygen/nitrogen species-mediated apoptosis in glial cells. Findings of this study demonstrated that exposure of glial cells to high glucose markedly induces cellular and molecular injuries, as evidenced by elevated levels of reactive oxygen/nitrogen species, biomolecules damage, cell cycle dysregulation, decrease in antioxidant enzymes, and decrease in cell viability. Pretreatment of cells with N-acetyl-L-cysteine reduced high glucose-induced cytotoxicity by increasing the levels of antioxidant enzymes, and decreasing the number of apoptotic cells. Further, at molecular level high glucose treatment resulted in a significant increase in phosphorylation of Akt, MAPKs, tuberin, down regulation of 8-oxoG-DNA glycosylase and increase in 8-hydroxydeoxyguanosine accumulations. Pretreatment of cells with N-acetyl-L-cysteine, phosphatidylinositol3-kinase/Akt and ERK1/2 inhibitors completely abolished the apoptotic effects of high glucose. Moreover, N-acetyl-L-cysteine significantly inhibited reactive oxygen/nitrogen species generation, elevated antioxidants levels, inhibited Akt, ERK1/2, tuberin phosphorylation, decreased 8-hydroxydeoxyguanosine accumulation and upregulated 8-oxoG-DNA glycosylase expression. Our results demonstrate that high glucose induces apoptosis and inhibits proliferation of glial cells, which may be mediated by the phosphorylation of tuberin, down regulation of 8-oxoG-DNA glycosylase and 8-hydroxydeoxyguanosine accumulation via activation of Akt and ERK1/2MAPK pathways.

Incidence of cystoid macular oedema in diabetic patients after phacoemulsification and free radical link to its pathogenesis.

PURPOSE: Postoperative onset of cystoid macular oedema (CME) in diabetic patients after cataract surgery is a frequent problem in working-age adults worldwide. Here, we investigate the postoperative development of CME in diabetic patients after undergoing phacoemulsification with other ailing factors associated with CME. METHODS: This prospective study included 65 Type 2 diabetic patients with no diabetic retinopathy (DR), mild to moderate DR, moderate to severe DR and proliferative DR who underwent phacoemulsification surgery. Indirect ophthalmoscopy, fluorescein angiograms and optical coherence tomography were taken for a period of 8 weeks postoperatively to determine visual outcome and development of CME. Serum samples were collected for the measurement of antioxidants and reactive oxygen species (ROS) levels. RESULTS: Our data showed that CME occurred postoperatively in 47% without pre-existing DR and 55% of eyes with pre-existing DR (p<005). Positive association was noticed between morbid conditions, like hypertension (p<0.01) and diabetic nephropathy (p<0.05), and postoperative incidence of CME. The activity of antioxidant enzymes in patients with DR was found to be lower as compared with diabetic (D) patients, but catalase activity was recorded the maximum in these patients. The ROS activity was recorded highest in the serum samples of DR becoming CME positive. CONCLUSIONS: The present results suggest that after phacoemulsification, the chance of development of CME in DR is more as compared to D. Moreover, the development of CME is significantly associated with decrease in antioxidant levels, increased ROS activities, hypertension, diabetic nephropathy, and hyperlipidaemia.