The role of the Toll like receptor 4 signaling in sex-specific persistency of depression-like behavior in response to chronic stress.

Chronic stress is a major risk factor for Major Depressive Disorder (MDD), and it has been shown to impact the immune system and cause microglia activation in the medial prefrontal cortex (mPFC) involved in the pathogenesis of depression. The aim of this study is to further investigate cellular and molecular mechanisms underlying persistent depression behavior in sex specific manner, which is observed clinically. Here, we report that both male and female mice exhibited depression-like behavior following exposure to chronic stress. However, only female mice showed persistent depression-like behavior, which was associated with microglia activation in mPFC, characterized by distinctive alterations in the phenotype of microglia. Given these findings, to further investigate the underlying molecular mechanisms associated with persistent depression-like behavior and microglia activation in female mice, we used translating-ribosome affinity purification (TRAP). We find that Toll like receptor 4 (TLR4) signaling is casually related to persistent depression-like behavior in female mice. This is supported by the evidence that the fact that genetic ablation of TLR4 expression in microglia significantly reduced the persistent depression-like behavior to baseline levels in female mice. This study tentatively supports the hypothesis that the TLR4 signaling in microglia may be responsible for the sex differences in persistent depression-like behavior in female.

The Bioactive Dietary Polyphenol Preparation Alleviates Depression and Anxiety-Like Behaviors by Modulating the Regional Heterogeneity of Microglia Morphology.

SCOPE: The goal of this study is to investigate the effects of a bioactive dietary polyphenol preparation (BDPP), which is made up of grape-derived polyphenols, on microglial responses, as well as the underlying molecular mechanisms in depression and anxiety-like behaviors. METHODS AND RESULTS: The study finds that treatment with BDPP significantly decreases depression-like and anxiety-like behaviors induced by chronic stress in mice, while leaving their locomotor activity unaffected. The study also finds that BDPP treatment reverses microglia activation in the amygdala and hippocampal formation, regions of the brain involved in emotional regulation, from an amoeboid shape to ramified shape. Additionally, BDPP treatment modulates the release of pro-inflammatory cytokines such as interleukin-6 via high mobility box 1 protein and the receptor for advanced glycation end products (HMGB1-RAGE) signaling pathway in activated microglia induced by chronic stress. CONCLUSION: The findings suggest regional heterogeneity in microglial responses following chronic stress in subregions of the corticolimbic circuit. Specifically, activation of the immune-inflammatory HMGB1-RAGE pathway may provide a new avenue for preventing the manifestation of psychiatric impairments including stress-induced anxiety- and depression-like behavior, using bioactive and bioavailable polyphenols.

The bioactive dietary polyphenol preparation alleviates depression and anxiety-like behaviors by modulating the regional heterogeneity of microglia morphology.

Scope: The goal of this study is to investigate the effects of a bioactive dietary polyphenol preparation (BDPP), which is made up of grape-derived polyphenols, on microglial responses, as well as the underlying molecular mechanisms in depression and anxiety-like behaviors. Methods and results: We find that treatment with BDPP significantly decreased depression-like and anxiety-like behaviors induced by chronic stress in mice, while leaving their locomotor activity unaffected. We also find that BDPP treatment reversed microglia activation in the amygdala and hippocampal formation, regions of the brain involved in emotional regulation, from an amoeboid shape to ramified shape. Additionally, BDPP treatment modulates the release of pro-inflammatory cytokines such as interleukin-6 via high mobility box 1 protein and the receptor for advanced glycation end products (HMGB1-RAGE) signaling pathway in activated microglia induced by chronic stress. Conclusion: Our findings suggest regional heterogeneity in microglial responses following chronic stress in subregions of the corticolimbic circuit. Specifically, activation of the immune-inflammatory HMGB1-RAGE pathway might provide a new avenue for therapeutic intervention in stress-induced anxiety- and depression-like behavior, using bioactive and bioavailable polyphenols.

Dissolution Study on Grape Polyphenol Hard Gelatin Capsule Dietary Supplements.

Methods for a dissolution study by ultra-high performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC-QqQ/MS) analysis of grape polyphenol dietary supplements, namely, grape seed extract (GSE) and resveratrol (RSV) capsules, were developed following the guidance of United States Pharmacopeia (USP) <2040>. Two dissolution media, 0.1 N hydrochloric acid (pH 1.2) and 0.05 M acetate buffer (pH 4.6), were evaluated with dissolution apparatus (USP 1), 100 rpm rotation speed, and 900 ml dissolution medium volume. Dissolution profiling was performed over 120 min. Major phenolic compounds of gallic acid, catechin, epicatechin, and procyanidin B2 were quantitated to obtain the dissolution profile of GSE capsules, and trans-RSV was used for RSV capsules. Results indicated that the released trans-RSV for RSV capsules in both of the dissolution media meets the USP standards, and that for the GSE capsules, all the four marker compounds passed the dissolution test in the HCl medium but did not reach a 75% release within 60 min in the acetate buffer. These promising results suggest that the general USP dissolution protocols are adequate for the successful release of RSV capsules in HCl medium and acetate buffer and GSE capsules (in HCl medium), but may be inadequate for GSE capsules in acetate buffer. These results showed that under a low pH of 1.2 (simulated stomach environment), bioactive compounds were released on time from the GSE capsules and met the USP guidelines; however, under a higher pH of 4.6 (simulated duodenum environment), the same biomarkers failed, suggesting the need to further improve the dissolution of GSE over a wider range of pH environments to enhance bioavailability and efficacy.

Chronic Stress-Induced Depression and Anxiety Priming Modulated by Gut-Brain-Axis Immunity.

Chronic stress manifests as depressive- and anxiety-like behavior while recurrent stress elicits disproportionate behavioral impairments linked to stress-induced immunological priming. The gut-brain-microbiota-axis is a promising therapeutic target for stress-induced behavioral impairments as it simultaneously modulates peripheral and brain immunological landscapes. In this study, a combination of probiotics and prebiotics, known as a synbiotic, promoted behavioral resilience to chronic and recurrent stress by normalizing gut microbiota populations and promoting regulatory T cell (Treg) expansion through modulation of ileal innate lymphoid cell (ILC)3 activity, an impact reflecting behavioral responses better than limbic brain region neuroinflammation. Supporting this conclusion, a multivariate machine learning model correlatively predicted a cross-tissue immunological signature of stress-induced behavioral impairment where the ileal Treg/T helper17 cell ratio associated to hippocampal chemotactic chemokine and prefrontal cortex IL-1ß production in the context of stress-induced behavioral deficits. In conclusion, stress-induced behavioral impairments depend on the gut-brain-microbiota-axis and through ileal immune regulation, synbiotics attenuate the associated depressive- and anxiety-like behavior.

Neuronal Pentraxin 1 Promotes Hypoxic-Ischemic Neuronal Injury by Impairing Mitochondrial Biogenesis via Interactions With Active Bax[6A7] and Mitochondrial Hexokinase II.

Mitochondrial dysfunction is a key mechanism of cell death in hypoxic-ischemic brain injury. Neuronal pentraxin 1 (NP1) has been shown to play crucial roles in mitochondria-mediated neuronal death. However, the underlying mechanism(s) of NP1-induced mitochondrial dysfunction in hypoxia-ischemia (HI) remains obscure. Here, we report that NP1 induction following HI and its subsequent localization to mitochondria, leads to disruption of key regulatory proteins for mitochondrial biogenesis. Brain mitochondrial DNA (mtDNA) content and mtDNA-encoded subunit I of complex IV (mtCOX-1) expression was increased post-HI, but not the nuclear DNA-encoded subunit of complex II (nSDH-A). Up-regulation of mitochondrial proteins COXIV and HSP60 further supported enhanced mtDNA function. NP1 interaction with active Bax (Bax6A7) was increased in the brain after HI and in oxygen-glucose deprivation (OGD)-induced neuronal cultures. Importantly, NP1 colocalized with mitochondrial hexokinase II (mtHKII) following OGD leading to HKII dissociation from mitochondria. Knockdown of NP1 or SB216763, a GSK-3 inhibitor, prevented OGD-induced mtHKII dissociation and cellular ATP decrease. NP1 also modulated the expression of mitochondrial transcription factor A (Tfam) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), regulators of mitochondrial biogenesis, following HI. Together, we reveal crucial roles of NP1 in mitochondrial biogenesis involving interactions with Bax[6A7] and mtHKII in HI brain injury.

UGT84F9 is the major flavonoid UDP-glucuronosyltransferase in Medicago truncatula.

Mammalian phase II metabolism of dietary plant flavonoid compounds generally involves substitution with glucuronic acid. In contrast, flavonoids mainly exist as glucose conjugates in plants, and few plant UDP-glucuronosyltransferase enzymes have been identified to date. In the model legume Medicago truncatula, the major flavonoid compounds in the aerial parts of the plant are glucuronides of the flavones apigenin and luteolin. Here we show that the M. truncatula glycosyltransferase UGT84F9 is a bi-functional glucosyl/glucuronosyl transferase in vitro, with activity against a wide range of flavonoid acceptor molecules including flavones. However, analysis of metabolite profiles in leaves and roots of M. truncatula ugt84f9 loss of function mutants revealed that the enzyme is essential for formation of flavonoid glucuronides, but not most flavonoid glucosides, in planta. We discuss the use of plant UGATs for the semi-synthesis of flavonoid phase II metabolites for clinical studies.

Chemical, Manufacturing, and Standardization Controls of Grape Polyphenol Dietary Supplements in Support of a Clinical Study: Mass Uniformity, Polyphenol Dosage, and Profiles.

Bioactive dietary polyphenols in grape (Vitis vinifera) have been used in Dietary Supplements (DSs) with the aim to prevent numerous diseases, including cardiovascular and neurodegenerative diseases, and to reduce depression and anxiety. Given prior recognition that DSs can be quality challenged from the purity, authentication, adulteration, and actual concentration of targeted bioactives, to ensure consumer health protection as well as the quality and safety of grape polyphenol-based DSs, the present investigation was aimed at establishing a comprehensive quality control (QC) approach for grape polyphenol-based DSs in support of a human clinical study. In this study, the manufactured grape seed polyphenol extract (GSPE) and trans-resveratrol (RSV) capsules and Concord Grape Juice (CGJ) along with the corresponding original drug materials were analyzed using the developed different liquid chromatography/UV-visible spectroscopy/mass spectrometry (LC/UV-Vis/MS) methods. The weight variation of GSPE and RSV capsules was also evaluated according to the US Pharmacopeia (USP) tests. The results indicate that the total identified polyphenol content in each grape seed extract (GSE) capsule/CGJ is very similar and all GSE/RSV capsules pass the content/weight uniformity test. Given the complexity of these and many botanical products from the issues of purity, quality, adulteration, consistency, and their coupling to the complex chemistry in each grape-derived botanical, quality assurance and the steps needed to ensure grape-derived DSs being well homogeneous and stable and containing the known and expected bioactives at specific concentration ranges are fundamental to any research study and in particular to a clinical trial. Each of these issues is essential to provide a solid foundation upon which clinical trials with botanicals can be conducted with the goal of realizing measurable mental health outcomes such as reducing depression and anxiety as well as understanding of their underlying biological mechanisms.

Glucuronidation of Methylated Quercetin Derivatives: Chemical and Biochemical Approaches.

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'-O-methyl-quercetin-3-O-ß-d-glucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4'-O-methyl-quercetin-7-O-ß-d-glucuronide (4), 3-O-methyl-quercetin-3'-O-ß-d-glucuronide (5), and 4'-O-methyl-quercetin-3'-O-ß-d-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; 3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. A mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.

Efficient Chemical Synthesis of (Epi)catechin Glucuronides: Brain-Targeted Metabolites for Treatment of Alzheimer's Disease and Other Neurological Disorders.

Grape seed extract (GSE) is rich in flavonoids and has been recognized to possess human health benefits. Our group and others have demonstrated that GSE is able to attenuate the development of Alzheimer's disease (AD). Moreover, our results have disclosed that the anti-Alzheimer's benefits are not directly/solely related to the dietary flavonoids themselves, but rather to their metabolites, particularly to the glucuronidated ones. To facilitate the understanding of regioisomer/stereoisomer-specific biological effects of (epi)catechin glucuronides, we here describe a concise chemical synthesis of authentic standards of catechin and epicatechin metabolites 3-12. The synthesis of glucuronides 9 and 12 is described here for the first time. The key reactions employed in the synthesis of the novel glucuronides 9 and 12 include the regioselective methylation of the 4'-hydroxyl group of (epi)catechin (≤1.0/99.0%; 3'-OMe/4'-OMe) and the regioselective deprotection of the tert-butyldimethylsilyl (TBS) group at position 5 (yielding up to 79%) over the others (3, 7 and 3' or 4').

The Viral Hypothesis in Alzheimer's Disease: Novel Insights and Pathogen-Based Biomarkers.

Early diagnosis of Alzheimer's disease (AD) and the identification of significant risk factors are necessary to better understand disease progression, and to develop intervention-based therapies prior to significant neurodegeneration. There is thus a critical need to establish biomarkers which can predict the risk of developing AD before the onset of cognitive decline. A number of studies have indicated that exposure to various microbial pathogens can accelerate AD pathology. Additionally, several studies have indicated that amyloid-ß possess antimicrobial properties and may act in response to infection as a part of the innate immune system. These findings have led some to speculate that certain types of infections may play a significant role in AD pathogenesis. In this review, we will provide an overview of studies which suggest pathogen involvement in AD. Additionally, we will discuss a number of pathogen-associated biomarkers which may be effective in establishing AD risk. Infections that increase the risk of AD represent a modifiable risk factor which can be treated with therapeutic intervention. Pathogen-based biomarkers may thus be a valuable tool for evaluating and decreasing AD risk across the population.

The NLRP3 Inflammasome as a Critical Actor in the Inflammaging Process.

As a consequence of the considerable increase in the human lifespan over the last century, we are experiencing the appearance and impact of new age-related diseases. The causal relationships between aging and an enhanced susceptibility of suffering from a broad spectrum of diseases need to be better understood. However, one specific shared feature seems to be of capital relevance for most of these conditions: the low-grade chronic inflammatory state inherently associated with aging, i.e., inflammaging. Here, we review the molecular and cellular mechanisms that link aging and inflammaging, focusing on the role of the innate immunity and more concretely on the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, as well as how the chronic activation of this inflammasome has a detrimental effect on different age-related disorders.

The Use of Antimicrobial and Antiviral Drugs in Alzheimer's Disease.

The aggregation and accumulation of amyloid-ß plaques and tau proteins in the brain have been central characteristics in the pathophysiology of Alzheimer's disease (AD), making them the focus of most of the research exploring potential therapeutics for this neurodegenerative disease. With success in interventions aimed at depleting amyloid-ß peptides being limited at best, a greater understanding of the physiological role of amyloid-ß peptides is needed. The development of amyloid-ß plaques has been determined to occur 10-20 years prior to AD symptom manifestation, hence earlier interventions might be necessary to address presymptomatic AD. Furthermore, recent studies have suggested that amyloid-ß peptides may play a role in innate immunity as an antimicrobial peptide. These findings, coupled with the evidence of pathogens such as viruses and bacteria in AD brains, suggests that the buildup of amyloid-ß plaques could be a response to the presence of viruses and bacteria. This has led to the foundation of the antimicrobial hypothesis for AD. The present review will highlight the current understanding of amyloid-ß, and the role of bacteria and viruses in AD, and will also explore the therapeutic potential of antimicrobial and antiviral drugs in Alzheimer's disease.

Prophylactic effect of flavanol rich preparation metabolites in promoting resilience to a mouse model of social stress.

Major depressive disorder (MDD) is a leading cause of disability, and there is an urgent need for new therapeutics. Stress-mediated induction of pro-inflammation in the periphery contributes to depression-like behaviors both in humans and in experimental models. Inflammatory cytokine interleukin-6 (IL-6) has emerged as a potential therapeutic target. Our studies demonstrated that metabolism of flavanol rich cocoa preparation (FRP) led to the accumulation of select phenolic acids that may contribute to its anti-inflammatory activity. Using a repeated social defeat stress (RSDS) model of depression, we showed that oral administration of FRP attenuates susceptibility to RSDS-mediated depression, supporting the further development of FRP as a novel therapeutic for the treatment of stress disorders and anxiety in humans.

Potential Novel Role of COVID-19 in Alzheimer's Disease and Preventative Mitigation Strategies.

There are a number of potential implications for the field of Alzheimer's disease (AD) stemming from the global spread of SARS-CoV-2. Neuroinflammation is known to be a prominent feature of neurodegeneration and plays a major role in AD pathology. Immune response and excessive inflammation in COVID-19 may also accelerate the progression of brain inflammatory neurodegeneration, and elderly individuals are more susceptible to severe outcomes after SARS-CoV-2 infection. Individuals with type 2 diabetes (T2D) are at an increased risk for AD as well as severe outcomes after SARS-CoV-2 infection. Genetic and socioeconomic factors influencing the rates of T2D, AD, and COVID-19 severity may create an exceptionally high-risk profile for certain demographics such as African Americans and Hispanic Americans. Type I interferon response plays an important role in both host response to viral infection, as well as AD pathology and may be a sensible therapeutic target in both AD and COVID-19.

Gut Microbiome-Modified Polyphenolic Compounds Inhibit α-Synuclein Seeding and Spreading in α-Synucleinopathies.

Misfolding, aggregation and deposition of α-synuclein (α-syn) are major pathologic characteristics of Parkinson's disease (PD) and the related synucleinopathy, multiple system atrophy (MSA). The spread of α-syn pathology across brain regions is thought to play a key role in the onset and progression of clinical phenotypes. Thus, there is increasing interest in developing strategies that target and attenuate α-syn aggregation and spread. Recent studies of brain-penetrating polyphenolic acids, namely, 3-hydroxybenzoic acid (3-HBA), 3,4-dihydroxybenzoic acid (3,4-diHBA), and 3-(3-hydroxyphenyl)propionic acid (3-HPPA) that are derived from gut microbiota metabolism of dietary polyphenols, show in vitro ability to effectively modulate α-syn misfolding, oligomerization, and mediate aggregated α-syn neurotoxicity. Here we investigate whether 3-HBA, 4-hydroxybenzoic acid (4-HBA), 3,4-diHBA, or 3-HPPA interfere with α-syn spreading in a cell-based system. Using HEK293 cells overexpressing α-syn-A53T-CFP/YFP, we assessed α-syn seeding activity using Fluorescence Resonance Energy Transfer (FRET) to detect and quantify α-syn aggregation. We demonstrated that 3-HPPA, 3,4-diHBA, 3-HBA, and 4-HBA significantly attenuated intracellular α-syn seeding aggregation. To determine whether our compounds could inhibit brain-derived seeding activity, we utilized insoluble α-syn extracted from post-mortem MSA or PD brain specimens. We found that 3-HPPA effectively attenuated MSA-induced aggregation of monomer into high molecular weight aggregates capable of inducing intracellular aggregation. Outcomes from our studies suggest interactions between gut microbiome and certain dietary factors may form the basis for effective therapies that modulate pathologic α-syn propagation. Collectively, our findings provide the basis for future developments of probiotic, prebiotic, or synbiotic approaches for modulating the onset and/or progression of α-synucleinopathies.

Peroxisome Proliferator Activator Receptor Gamma Coactivator-1α Overexpression in Amyotrophic Lateral Sclerosis: A Tale of Two Transgenics.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder manifesting with upper and lower neuron loss, leading to impairments in voluntary muscle function and atrophy. Mitochondrial dysfunction in metabolism and morphology have been implicated in the pathogenesis of ALS, including atypical oxidative metabolism, reduced mitochondrial respiration in muscle, and protein aggregates in the mitochondrial outer membrane. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays an essential role in the regulation of mitochondrial biogenesis, the process by which existing mitochondria grow and divide. PGC-1α has been previously reported to be downregulated in the spinal cord of individuals with ALS. Towards targeting PGC-1α as a therapeutic mechanism, we have previously reported improved motor function and survival in the SOD1G93A mouse model of ALS by neuron-specific over-expression of PGC-1α under a neuron-specific enolase (NSE) promoter. As pharmacological intervention targeting PGC-1α would result in whole-body upregulation of this transcriptional co-activator, in the current study we investigated whether global expression of PGC-1α is beneficial in a SOD1G93A mouse model, by generating transgenic mice with PGC-1α transgene expression driven by an actin promoter. Actin-PGC-1α expression levels were assayed and confirmed in spinal cord, brain, muscle, liver, kidney, and spleen. To determine the therapeutic effects of global expression of PGC-1α, wild-type, actin-PGC-1α, SOD1G93A, and actin-PGC-1α/SOD1G93A animals were monitored for weight loss, motor performance by accelerating rotarod test, and survival. Overexpression of actin-PGC-1α did not confer significant improvement in these assessed outcomes. A potential explanation for this difference is that the actin promoter may not induce levels of PGC-1α relevant to disease pathophysiology in the cells that are specifically relevant to the pathogenesis of ALS. This evidence strongly supports future therapeutic approaches that target PGC-1α primarily in neurons.

Anti-aggregation Effects of Phenolic Compounds on α-synuclein.

The aggregation and deposition of α-synuclein (αS) are major pathologic features of Parkinson's disease, dementia with Lewy bodies, and other α-synucleinopathies. The propagation of αS pathology in the brain plays a key role in the onset and progression of clinical phenotypes. Thus, there is increasing interest in developing strategies that attenuate αS aggregation and propagation. Based on cumulative evidence that αS oligomers are neurotoxic and critical species in the pathogenesis of α-synucleinopathies, we and other groups reported that phenolic compounds inhibit αS aggregation including oligomerization, thereby ameliorating αS oligomer-induced cellular and synaptic toxicities. Heterogeneity in gut microbiota may influence the efficacy of dietary polyphenol metabolism. Our recent studies on the brain-penetrating polyphenolic acids 3-hydroxybenzoic acid (3-HBA), 3,4-dihydroxybenzoic acid (3,4-diHBA), and 3-hydroxyphenylacetic acid (3-HPPA), which are derived from gut microbiota-based metabolism of dietary polyphenols, demonstrated an in vitro ability to inhibit αS oligomerization and mediate aggregated αS-induced neurotoxicity. Additionally, 3-HPPA, 3,4-diHBA, 3-HBA, and 4-hydroxybenzoic acid significantly attenuated intracellular αS seeding aggregation in a cell-based system. This review focuses on recent research developments regarding neuroprotective properties, especially anti-αS aggregation effects, of phenolic compounds and their metabolites by the gut microbiome, including our findings in the pathogenesis of α-synucleinopathies.