Inhibition of microfold cells ameliorates early pathological phenotypes by modulating microglial functions in Alzheimer's disease mouse model.

BACKGROUND: The gut microbiota has recently attracted attention as a pathogenic factor in Alzheimer's disease (AD). Microfold (M) cells, which play a crucial role in the gut immune response against external antigens, are also exploited for the entry of pathogenic bacteria and proteins into the body. However, whether changes in M cells can affect the gut environments and consequently change brain pathologies in AD remains unknown. METHODS: Five familial AD (5xFAD) and 5xFAD-derived fecal microbiota transplanted (5xFAD-FMT) naïve mice were used to investigate the changes of M cells in the AD environment. Next, to establish the effect of M cell depletion on AD environments, 5xFAD mice and Spib knockout mice were bred, and behavioral and histological analyses were performed when M cell-depleted 5xFAD mice were six or nine months of age. RESULTS: In this study, we found that M cell numbers were increased in the colons of 5xFAD and 5xFAD-FMT mice compared to those of wild-type (WT) and WT-FMT mice. Moreover, the level of total bacteria infiltrating the colons increased in the AD-mimicked mice. The levels of M cell-related genes and that of infiltrating bacteria showed a significant correlation. The genetic inhibition of M cells (Spib knockout) in 5xFAD mice changed the composition of the gut microbiota, along with decreasing proinflammatory cytokine levels in the colons. M cell depletion ameliorated AD symptoms including amyloid-ß accumulation, microglial dysfunction, neuroinflammation, and memory impairment. Similarly, 5xFAD-FMT did not induce AD-like pathologies, such as memory impairment and excessive neuroinflammation in Spib-/- mice. CONCLUSION: Therefore, our findings provide evidence that the inhibiting M cells can prevent AD progression, with therapeutic implications.

Positional scanning of natural product hispidol's ring-B: discovery of highly selective human monoamine oxidase-B inhibitor analogues downregulating neuroinflammation for management of neurodegenerative diseases.

Multifunctional molecules might offer better treatment of complex multifactorial neurological diseases. Monoaminergic pathways dysregulation and neuroinflammation are common convergence points in diverse neurodegenerative and neuropsychiatric disorders. Aiming to target these diseases, polypharmacological agents modulating both monoaminergic pathways and neuroinflammatory were addressed. A library of analogues of the natural product hispidol was prepared and evaluated for inhibition of monoamine oxidases (MAOs) isoforms. Several molecules emerged as selective potential MAO B inhibitors. The most promising compounds were further evaluated in vitro for their impact on microglia viability, induced production of proinflammatory mediators and MAO-B inhibition mechanism. Amongst tested compounds, 1p was a safe potent competitive reversible MAO-B inhibitor and inhibitor of microglial production of neuroinflammatory mediators; NO and PGE2. In-silico study provided insights into molecular basis of the observed selective MAO B inhibition. This study presents compound 1p as a promising lead compound for management of neurodegenerative disease.

Transplantation of gut microbiota derived from Alzheimer's disease mouse model impairs memory function and neurogenesis in C57BL/6 mice.

Alzheimer's disease (AD) is a neurodegenerative disease that causes memory and cognitive decline. Although many studies have attempted to clarify the causes of AD occurrence, it is not clearly understood. Recently, the emerging role of the gut microbiota in neurodegenerative diseases, including AD, has received much attention. The gut microbiota composition of AD patients and AD mouse models is different from that of healthy controls, and these changes may affect the brain environment. However, the specific mechanisms by which gut microbiota that influence memory decline are currently unclear. In this study, we performed fecal microbiota transplantation (FMT) to clarify the role of 5xFAD mouse-derived microbiota in memory decline. We observed that FMT from 5xFAD mice into normal C57BL/6 mice (5xFAD-FMT) decreased adult hippocampal neurogenesis and brain-derived neurotrophic factor expression and increased p21 expression, resulting in memory impairment. Microglia in the hippocampus of the 5xFAD-FMT mice were activated, which caused the elevation of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1ß). Moreover, we observed that pro-inflammatory cytokines increased in the colon and plasma of 5xFAD-FMT mice. The gut microbiota composition of the 5xFAD-FMT mice was different from that of the control mice or wild type-FMT mice. Collectively, 5xFAD mouse-derived microbiota decreased neurogenesis by increasing colonic inflammation, thereby contributing to memory loss. Our findings provide further evidence concerning the role of gut microbial dysbiosis in AD pathogenesis and suggest that targeting the gut microbiota may be a useful therapeutic strategy for the development of novel candidates for the treatment of AD.

Identification of ortho catechol-containing isoflavone as a privileged scaffold that directly prevents the aggregation of both amyloid ß plaques and tau-mediated neurofibrillary tangles and its in vivo evaluation.

In this study, polyhydroxyisoflavones that directly prevent the aggregation of both amyloid ß (Aß) and tau were expediently synthesized via divergent Pd(0)-catalyzed Suzuki-Miyaura coupling and then biologically evaluated. By preliminary structure-activity relationship studies using thioflavin T (ThT) assays, an ortho-catechol containing isoflavone scaffold was proven to be crucial for preventing both Aß aggregation and tau-mediated neurofibrillary tangle formation. Additional TEM experiment confirmed that ortho-catechol containing isoflavone 4d significantly prevented the aggregation of both Aß and tau. To investigate the mode of action (MOA) of 4d, which possesses an ortho-catechol moiety, 1H-15N HSQC NMR analysis was thoroughly performed and the result indicated that 4d could directly inhibit both the formation of Aß42 fibrils and the formation of tau-derived neurofibrils, probably through the catechol-mediated nucleation of tau. Finally, 4d was demonstrated to alleviate cognitive impairment and pathologies related to Alzheimer's disease in a 5XFAD transgenic mouse model.

Phytohormone Abscisic Acid Improves Memory Impairment and Reduces Neuroinflammation in 5xFAD Mice by Upregulation of LanC-Like Protein 2.

Alzheimer's disease (AD), a type of dementia, is the most common neurodegenerative disease in the elderly. Neuroinflammation caused by deposition of amyloid ß (Aß) is one of the most important pathological causes in AD. The isoprenoid phytohormone abscisic acid (ABA) has recently been found in mammals and was shown to be an endogenous hormone, acting in stress conditions. Although ABA has been associated with anti-inflammatory effects and reduced cognitive impairment in several studies, the mechanisms of ABA in AD has not been ascertained clearly. To investigate the clearance of Aß and anti-inflammatory effects of ABA, we used quantitative real-time polymerase chain reaction and immunoassay. ABA treatment inhibited Aß deposition and neuroinflammation, thus resulting in improvement of memory impairment in 5xFAD mice. Interestingly, these effects were not associated with activation of peroxisome proliferator-activated receptor gamma, well known as a molecular target of ABA, but related with modulation of the LanC-like protein 2 (LANCL2), known as a receptor of ABA. Taken together, our results indicate that ABA reduced Aß deposition, neuroinflammation, and memory impairment, which is the most characteristic pathology of AD, via the upregulation of LANCL2. These data suggest that ABA might be a candidate for therapeutics for AD treatment.

Trans-Cinnamaldehyde Alleviates Amyloid-Beta Pathogenesis via the SIRT1-PGC1α-PPARγ Pathway in 5XFAD Transgenic Mice.

Abnormal amyloid-ß (Aß) accumulation is the most significant feature of Alzheimer's disease (AD). Among the several secretases involved in the generation of Aß, ß-secretase (BACE1) is the first rate-limiting enzyme in Aß production that can be utilized to prevent the development of Aß-related pathologies. Cinnamon extract, used in traditional medicine, was shown to inhibit the aggregation of tau protein and Aß aggregation. However, the effect of trans-cinnamaldehyde (TCA), the main component of cinnamon, on Aß deposition is unknown. Five-month-old 5XFAD mice were treated with TCA for eight weeks. Seven-month-old 5XFAD mice were evaluated for cognitive and spatial memory function. Brain samples collected at the conclusion of the treatment were assessed by immunofluorescence and biochemical analyses. Additional in vivo experiments were conducted to elucidate the mechanisms underlying the effect of TCA in the role of Aß deposition. TCA treatment led to improvements in cognitive impairment and reduced Aß deposition in the brains of 5XFAD mice. Interestingly, the levels of BACE1 were decreased, whereas the mRNA and protein levels of three well-known regulators of BACE1, silent information regulator 1 (SIRT1), peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC1α), and PPARγ, were increased in TCA-treated 5XFAD mice. TCA led to an improvement in AD pathology by reducing BACE1 levels through the activation of the SIRT1-PGC1α-PPARγ pathway, suggesting that TCA might be a useful therapeutic approach in AD.

A Novel and Selective p38 Mitogen-Activated Protein Kinase Inhibitor Attenuates LPS-Induced Neuroinflammation in BV2 Microglia and a Mouse Model.

Neuroinflammation is an important pathological feature in neurodegenerative diseases. Accumulating evidence has suggested that neuroinflammation is mainly aggravated by activated microglia, which are macrophage like cells in the central nervous system. Therefore, the inhibition of microglial activation may be considered for treating neuroinflammatory diseases. p38 mitogen-activated protein kinase (MAPK) has been identified as a crucial enzyme with inflammatory roles in several immune cells, and its activation also relates to neuroinflammation. Considering the proinflammatory roles of p38 MAPK, its inhibitors can be potential therapeutic agents for neurodegenerative diseases relating to neuroinflammation initiated by microglia activation. This study was designed to evaluate whether NJK14047, a recently identified novel and selective p38 MAPK inhibitor, could modulate microglia-mediated neuroinflammation by utilizing lipopolysaccharide (LPS)-stimulated BV2 cells and an LPS-injected mice model. Our results showed that NJK14047 markedly reduced the production of nitric oxide and prostaglandin E2 by downregulating the expression of various proinflammatory mediators such as nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α and interleukin-1ß in LPS-induced BV2 microglia. Moreover, NJK14047 significantly reduced microglial activation in the brains of LPS-injected mice. Overall, these results suggest that NJK14047 significantly reduces neuroinflammation in cellular/vivo model and would be a therapeutic candidate for various neuroinflammatory diseases.

Bardoxolone Methyl Suppresses Hepatitis B Virus Large Surface Protein Variant W4P-Related Carcinogenesis and Hepatocellular Carcinoma Cell Proliferation Via the Inhibition of Signal Transducer and Activator of Transcription 3 Signaling.

Bardoxolone methyl (CDDO-me) is a synthetic triterpenoid that has been shown to suppress various cancers and inflammation. It has been implicated for the suppression of signal transducer and activator of transcription 3 (STAT3)-mediated signaling, which plays crucial roles in the development and progression of hepatocellular carcinoma (HCC). Previously, we showed that hepatitis B virus (HBV) large surface protein (LHB) variant W4P promotes carcinogenesis and tumor progression through STAT3 activation. Thus, we examined the anti-cancer activity of CDDO-me against HCC using W4P-LHB-expressing NIH3T3 cells and HepG2 and Huh7 HCC cell lines. CDDO-me exerted cytotoxic activity against W4P-LHB-expressing NIH3T3 cells, HepG2 cells, and Huh7 cells, and induced apoptotic cell death in a dose-dependent manner, demonstrating its anti-cancer activity against HCC. Sublethal concentrations of CDDO-me suppressed STAT3 activation by W4P-LHB ectopic expression and interleukin-6 treatment in W4P-LHB-NIH3T3 and Huh7 cells respectively. The suppression of STAT3 activation by CDDO-me in W4P-LHB-NIH3T3 cells was further confirmed by decreased cyclin D1 protein levels and increased p21 and p53 mRNA synthesis. In addition, CDDO-me treatment resulted in decreased cell migration and colony formation in in vitro assays using W4P-LHB-NIH3T3, HepG2, or Huh7 cell lines, supporting its anti-cancer activity through STAT3 inhibition. Furthermore, -CDDO-me administration significantly suppressed tumor growth induced by W4P-LHB-expressing NIH3T3 cells in nude mice, confirming its anti-cancer activity. Collectively, our findings demonstrated that CDDO-me is capable of suppressing STAT3 activation in HCC cells and cells transformed by the natural variant of HBV protein. The results suggest that CDDO-me can be a potential therapeutic agent against HCC, especially tumors related to HBV mutations.

Survivin enhances hippocampal neurogenesis and cognitive function in Alzheimer's disease mouse model.

AIMS: Cognitive impairment is associated with reduced hippocampal neurogenesis; however, the causes of decreased hippocampal neurogenesis remain highly controversial. Here, we investigated the role of survivin in the modulation of hippocampal neurogenesis in AD. METHODS: To investigate the effect of survivin on neurogenesis in neural stem cells (NSCs), we treated mouse embryonic NSCs with a survivin inhibitor (YM155) and adeno-associated viral survivin (AAV-Survivin). To explore the potential role of survivin expression in AD, AAV9-Survivin or AAV9-GFP were injected into the dentate gyrus (DG) of hippocampus of 7-month-old wild-type and 5XFAD mice. Cognitive function was measured by the Y maze and Morris water maze. Neurogenesis was investigated by BrdU staining, immature, and mature neuron markers. RESULTS: Our results indicate that suppression of survivin expression resulted in decreased neurogenesis. Conversely, overexpression of survivin using AAV-Survivin restored neurogenesis in NSCs that had been suppressed by YM155 treatment. Furthermore, the expression level of survivin decreased in the 9-month-old 5XFAD compared with that in wild-type mice. AAV-Survivin-mediated overexpression of survivin in the DG in 5XFAD mice enhanced neurogenesis and cognitive function. CONCLUSION: Hippocampal neurogenesis can be enhanced by survivin overexpression, suggesting that survivin could serve as a promising therapeutic target for the treatment of AD.

Eugenol relieves the pathological manifestations of Alzheimer's disease in 5×FAD mice.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-ß (Aß) and excessive neuroinflammation, resulting in neuronal cell death and cognitive impairments. Eugenol, a phenylpropene, is the main component of Syzygium aromaticum L. (Myrtaceae) and has multiple therapeutic effects, including neuroprotective and anti-inflammatory effects, through multimodal mechanisms. PURPOSE: We aimed to investigate the effect of eugenol on AD pathologies using a 5× familiar AD (5×FAD) mouse model. METHODS: Eight-month-old 5×FAD and wild-type mice were administered with eugenol (10 or 30 mg/kg/day, p.o) for 2 months. Y-maze and Morris water maze tests were performed to assess the cognitive function of mice. After the behavioral test, molecular analysis was conducted to investigate the therapeutic mechanism of eugenol. RESULTS: Our findings indicate that eugenol treatment effectively mitigated cognitive impairments in 5×FAD mice. This beneficial effect was associated with a decrease in AD pathologies, including neuronal cell loss and Aß deposition. Specifically, eugenol inhibited necroptosis activation and increased microglial phagocytosis, which were the underlying mechanisms for the observed reductions in neuronal cell loss and Aß deposition, respectively. CONCLUSION: Overall, our data suggest that eugenol would be a potential therapeutic candidate for AD.

Chemical Knockdown of Phosphorylated p38 Mitogen-Activated Protein Kinase (MAPK) as a Novel Approach for the Treatment of Alzheimer's Disease.

Targeted protein degradation (TPD) provides unique advantages over gene knockdown in that it can induce selective degradation of disease-associated proteins attributed to pathological mutations or aberrant post-translational modifications (PTMs). Herein, we report a protein degrader, PRZ-18002, that selectively binds to an active form of p38 MAPK. PRZ-18002 induces degradation of phosphorylated p38 MAPK (p-p38) and a phosphomimetic mutant of p38 MAPK in a proteasome-dependent manner. Given that the activation of p38 MAPK plays pivotal roles in the pathophysiology of Alzheimer's disease (AD), selective degradation of p-p38 may provide an attractive therapeutic option for the treatment of AD. In the 5xFAD transgenic mice model of AD, intranasal treatment of PRZ-18002 reduces p-p38 levels and alleviates microglia activation and amyloid beta (Aß) deposition, leading to subsequent improvement of spatial learning and memory. Collectively, our findings suggest that PRZ-18002 ameliorates AD pathophysiology via selective degradation of p-p38, highlighting a novel therapeutic TPD modality that targets a specific PTM to induce selective degradation of neurodegenerative disease-associated protein.

Synthesis and Biological Evaluation of O6-Aminoalkyl-Hispidol Analogs as Multifunctional Monoamine Oxidase-B Inhibitors towards Management of Neurodegenerative Diseases.

Oxidative catabolism of monoamine neurotransmitters by monoamine oxidases (MAOs) produces reactive oxygen species (ROS), which contributes to neuronal cells' death and also lowers monoamine neurotransmitter levels. In addition, acetylcholinesterase activity and neuroinflammation are involved in neurodegenerative diseases. Herein, we aim to achieve a multifunctional agent that inhibits the oxidative catabolism of monoamine neurotransmitters and, hence, the detrimental production of ROS while enhancing neurotransmitter levels. Such a multifunctional agent might also inhibit acetylcholinesterase and neuroinflammation. To meet this end goal, a series of aminoalkyl derivatives of analogs of the natural product hispidol were designed, synthesized, and evaluated against both monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B). Promising MAO inhibitors were further checked for the inhibition of acetylcholinesterase and neuroinflammation. Among them, compounds 3aa and 3bc were identified as potential multifunctional molecules eliciting submicromolar selective MAO-B inhibition, low-micromolar AChE inhibition, and the inhibition of microglial PGE2 production. An evaluation of their effects on memory and cognitive impairments using a passive avoidance test confirmed the in vivo activity of compound 3bc, which showed comparable activity to donepezil. In silico molecular docking provided insights into the MAO and acetylcholinesterase inhibitory activities of compounds 3aa and 3bc. These findings suggest compound 3bc as a potential lead for the further development of agents against neurodegenerative diseases.

Hesperidin Improves Memory Function by Enhancing Neurogenesis in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by memory and cognitive impairments. Neurogenesis, which is related to memory and cognitive function, is reduced in the brains of patients with AD. Therefore, enhancing neurogenesis is a potential therapeutic strategy for neurodegenerative diseases, including AD. Hesperidin (HSP), a bioflavonoid found primarily in citrus plants, has anti-inflammatory, antioxidant, and neuroprotective effects. The objective of this study was to determine the effects of HSP on neurogenesis in neural stem cells (NSCs) isolated from the brain of mouse embryos and five familial AD (5xFAD) mice. In NSCs, HSP significantly increased the proliferation of NSCs by activating adenosine monophosphate (AMP)-activated protein kinase (AMPK)/cAMP-response element-binding protein (CREB) signaling, but did not affect NSC differentiation into neurons and astrocytes. HSP administration restored neurogenesis in the hippocampus of 5xFAD mice via AMPK/brain-derived neurotrophic factor/tropomyosin receptor kinase B/CREB signaling, thereby decreasing amyloid-beta accumulation and ameliorating memory dysfunction. Collectively, these preclinical findings suggest that HSP is a promising candidate for the prevention and treatment of AD.

Glibenclamide modulates microglial function and attenuates Aß deposition in 5XFAD mice.

Severe neuroinflammation is known as a main pathology of neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). In these diseases, excessive microglial activation is one of the main causes of inflammation in the central nervous system. Therefore, inhibition of activated microglia may be suggested as a treatment for neuroinflammatory diseases. Glibenclamide, known as a therapeutics for type 2 diabetes in clinical trials has been shown to be effective in the inhibiting inflammatory conditions of various diseases. However, studies on the effects of glibenclamide for improving AD pathologies are little known. In this study, we tested glibenclamide on microglial cell line BV2 and 5XFAD mice. We found that glibenclamide significantly inhibited nitric oxide (NO) at 10 µM and 40 µM in BV2 cells induced by lipopolysaccharide (LPS) stimulation. In addition, we confirmed that 40 µM of glibenclamide reduced pro-inflammatory cytokines and proteins in the LPS-stimulated microglial cells. The anti-inflammatory effect of glibenclamide was further tested in APP/PS1 transgenic mouse. Although further analysis would be needed to confirm whether glibenclamide affects behavioral performance, our data suggests that glibenclamide may be a therapeutic option for AD treatment.

A selective p38α/ß MAPK inhibitor alleviates neuropathology and cognitive impairment, and modulates microglia function in 5XFAD mouse.

BACKGROUND: Chronic neuroinflammation, aggressive amyloid beta (Aß) deposition, neuronal cell loss, and cognitive impairment are pathological presentations of Alzheimer's disease (AD). Therefore, resolution of neuroinflammation and inhibition of Aß-driven pathology have been suggested to be important strategies for AD therapy. Previous efforts to prevent AD progression have identified p38 mitogen-activated protein kinases (MAPKs) as a promising target for AD therapy. Recent studies showed pharmacological inhibition of p38α MAPK improved memory impairment in AD mouse models. METHODS: In this study, we used an AD mouse model, 5XFAD, to explore the therapeutic potential of NJK14047 which is a novel, selective p38α/ß MAPK inhibitor. The mice were injected with 2.5 mg/kg NJK14047 or vehicle every other day for 3 months. Morris water maze task and histological imaging analysis were performed. Protein and mRNA expression levels were measured using immunoblotting and qRT-PCR, respectively. In vitro studies were conducted to measure the cytotoxicity of microglia- and astrocyte-conditioned medium on primary neurons using the MTT assay and TUNEL assay. RESULTS: NJK14047 treatment downregulated phospho-p38 MAPK levels, decreased the amount of Aß deposits, and reduced spatial learning memory loss in 9-month-old 5XFAD mice. While the pro-inflammatory conditions were decreased, the expression of alternatively activated microglial markers and microglial phagocytic receptors was increased. Furthermore, NJK14047 treatment reduced the number of degenerating neurons labeled with Fluoro-Jade B in the brains of 5XFAD mice. The neuroprotective effect of NJK14047 was further confirmed by in vitro studies. CONCLUSION: Taken together, a selective p38α/ß MAPK inhibitor NJK14047 successfully showed therapeutic effects for AD in 5XFAD mice. Based on our data, p38 MAPK inhibition is a potential strategy for AD therapy, suggesting NJK14047 as one of the promising candidates for AD therapeutics targeting p38 MAPKs.