Kaempferol Improves Alzheimer's Disease by Inhibiting Neuronal Ferroptosis via Activating GPX4/AKR1C3 Signaling Pathway.

Anti-LINGO-1 antibody ameliorates cognitive impairment, promotes adult hippocampal neurogenesis, and increases the abundance of CB1R-rich CCK-GABAergic interneurons in AD mice

Context: Neuroligin-1 (NLGN1) is a cell adhesion protein located on the excitatory postsynaptic membrane. β-Amyloid (Aβ)-induced neuroinflammation decreases NLGN1 expression through epigenetic mechanisms. Triptolide (T10) and tripchlorolide (T4) exert protective effects on synapses in Alzheimer's disease (AD) mice, but the mechanisms remain unclear.Objective: The effects of T10 and T4 on hippocampal NLGN1 expression in AD mice and the epigenetic mechanisms were assessed using chromatin immunoprecipitation and methylated DNA immunoprecipitation.Materials and methods: Sixty APP/PS1 transgenic mice were randomly divided into an AD model group, a T10-treated group and a T4-treated group (n = 20); 20 wild-type littermates served as the control group. APP/PS1 transgenic mice were intraperitoneally injected with T10 (0.1 mg/kg) and T4 (25 μg/kg) once per day for 60 days. NLGN1 expression was examined using western blotting and quantitative PCR.Results: T10 and T4 increased the levels of the NLGN1 protein and mRNA in hippocampus of AD mice. T10 and T4 inhibited the binding of HDAC2 (p< 0.01) and MeCP2 (p< 0.01 and p< 0.05, respectively) to the NLGN1 promoter, and cytosine methylation (1.2305 ± 0.1482/1.2554 ± 0.3570 vs. 1.6578 ± 0.1818, p< 0.01) at the NLGN1 promoter in the hippocampus of AD mice. T10 and T4 increased the level of acetylated histone H3 (0.7733 ± 0.1611/0.8241 ± 0.0964 vs. 0.5587 ± 0.0925, p< 0.01) at the NLGN1 promoter in the hippocampus of AD mice.Conclusions: T10 and T4 may increase hippocampal NLGN1 expression in AD mice through epigenetic mechanisms, providing a new explanation for the mechanism underlying the protective effects of T10 and T4 on synapses.

Alzheimer's disease (AD) is a progressive degenerative condition. In order to treat AD, the use of a "drug repositioning" or "repurposing" approach with potential disease-modifying compounds has been increased. The new generation antipsychotics are commonly used in AD and other dementias for the treatment of psychosis and behavioral symptoms, and several animal models have shown the effects of these potential disease-modifying compounds. In this study, we examined whether long-term clozapine treatment could reduce amyloid beta (Aβ) deposition and cognitive impairment in transgenic mice of AD, Tg-APPswe/PS1dE9. AD mice were fed clozapine at 20mg/kg/day for 3months from 4.5months of age. Intake of clozapine improved the Aβ-induced memory impairment and suppressed Aβ levels and plaque deposition in the brain of AD mice. Clozapine upregulated Trk, brain-derived neurotrophic factor, cyclin-dependent kinase-5, and p35 in the cortex and hippocampus of AD mice and activated AMP-activated protein kinase (AMPK). As a downstream effector of AMPK, beta-secretase expression was decreased by clozapine administration. Moreover, clozapine-phosphorylated synapsin I at Ser9 and Ser549 sites in the hippocampus and cortex of AD mice, which may be involved in synaptic strength. This study suggests that as one of candidate for multi-target approach of AD treatment, clozapine is proposed as a therapeutic drug for treatment of AD patients.

Xanthoceras sorbifolium oil ameliorates neuroinflammation and learning and memory impairment in Alzheimer's disease mice by regulating gut microbiota and unsaturated fatty acid metabolism

In this study, we have shown the potential of a voxel-based analysis for imaging amyloid plaques and its utility in monitoring therapeutic response in Alzheimer’s disease (AD) mice using manganese oxide nanoparticles conjugated with an antibody of Aβ1-40 peptide (HMON-abAβ40). T1-weighted MR brain images of a drug-treated AD group (n=7), a nontreated AD group (n=7), and a wild-type group (n=7) were acquired using a 7.0 T MRI system before (D−1), 24-h (D+1) after, and 72-h (D+3) after injection with an HMON-abAβ40 contrast agent. For the treatment of AD mice, DAPT was injected intramuscularly into AD transgenic mice (50 mg/kg of body weight). For voxel-based analysis, the skull-stripped mouse brain images were spatially normalized, and these voxels’ intensities were corrected to reduce voxel intensity differences across scans in different mice. Statistical analysis showed higher normalized MR signal intensity in the frontal cortex and hippocampus of AD mice over wild-type mice on D+1 and D+3 (P<0.01, uncorrected for multiple comparisons). After the treatment of AD mice, the normalized MR signal intensity in the frontal cortex and hippocampus decreased significantly in comparison with nontreated AD mice on D+1 and D+3 (P<0.01, uncorrected for multiple comparisons). These results were confirmed by histological analysis using a thioflavin staining. This unique strategy allows us to detect brain regions that are subjected to amyloid plaque deposition and has the potential for human applications in monitoring therapeutic response for drug development in AD.

Beta-amyloid (Aβ) accumulation in the brains of Alzheimer's disease (AD) patients leads to mitochondrial dysfunction and ferroptosis in neurons. Voltage-dependent anion channel 1 (VDAC1) is a major protein in the mitochondrial outer membrane. It has been reported that VDAC1 associated with mitochondrial dysfunction and ferroptosis. However, the mechanism by which VDAC1 regulates mitochondrial dysfunction and ferroptosis of neurons in AD remains unclear. This study is aimed at investigating the mechanism of action of VDAC1 in mitochondrial dysfunction and ferroptosis in neurons of the AD model. In this study, we determined cell viability after treatment with Aβ1-42 via the MTT assay. The SOD, MDA, ROS, and MMP production was measured via the SOD kit, MDA kit, DCFDA staining, and JC-1 staining. The memory abilities of mice were detected via the Morris water maze test. The expression of AMPK/mTOR, Wnt/β-catenin, and GPX4 regulated by VDAC1 was detected via western blotting. Our present study showed that PC12 cells had decreased cell viability, increased LDH release, and decreased GPX4 expression after Aβ1-42 treatment. Meanwhile, Aβ1-42 induced MMP and SOD downregulation and increased MDA and ROS generation in PC12 cells. In addition, the expression of VDAC1 is increased in the brain tissue of AD mice and Aβ1-42-treated PC12 cells. Further investigation of the role of VDAC1 in regulating AD found that all effects induced by Aβ1-42 were reversed by inhibition of VDAC1. Additionally, inhibition of VDAC1 activates the AMPK/mTOR and Wnt/β-catenin pathways. Taken together, these findings demonstrate that inhibition of VDAC1 alleviates mitochondrial dysfunction and ferroptosis in AD neurons by activating AMPK/mTOR and Wnt/β-catenin.

Synaptic loss induced by beta-amyloid (Aβ) plays a critical role in the pathophysiology of Alzheimer’s disease (AD), but the mechanisms underlying this process remain unknown. In this study, we found that oridonin (Ori) rescued synaptic loss induced by Aβ1–42 in vivo and in vitro and attenuated the alterations in dendritic structure and spine density observed in the hippocampus of AD mice. In addition, Ori increased the expression of PSD-95 and synaptophysin and promoted mitochondrial activity in the synaptosomes of AD mice. Ori also activated the BDNF/TrkB/CREB signaling pathway in the hippocampus of AD mice. Furthermore, in the Morris water maze test, Ori reduced latency and searching distance and increased the number of platform crosses in AD mice. These data suggest that Ori might prevent synaptic loss and improve behavioral symptoms in Aβ1–42-induced AD mice.

The pathology of Alzheimer's disease (AD) includes β-amyloid (Aβ) (plaques) and neurofibrillary tangles (NFTs). This study aimed to explore the efficacy of Huatuo Zaizao pill (HTZP) in an AD mouse model induced by injecting Aβ1-42, and the neuroprotective mechanism of HTZP in AD. C57BL/6 (B6) mice were randomly divided into 4 groups (n = 10, per group): control group, AD model group, and 2 different doses of HTZP treated groups. The Morris water maze test was carried out on AD mice to assess the learning ability after treatment with HTZP for 15 day. The levels of inflammatory factors and the nuclear factor-κB (NF-κB) pathway were examined by western blot and real-time polymerase chain reaction (PCR). The content of microglia was investigated by immunofluorescence. This study revealed that a cognitive disorder could be mitigated when the AD mice were treated with HTZP, which might be associated with the decreased level of pro-inflammatory factors, and the inhibitory activities of microglia. Additionally, phosphorylation of IκB and NF-κB p65 could be reduced by prohibiting the neuroinflammation of NF-κB activation in the hippocampus of AD mice. These results showed that HTZP could mitigate a cognitive disorder, diminish the activation of microglia, and inhibit the content of inflammatory factors through the NF-κB pathway in Aβ1-42-induced AD mice. HTZP may be an appropriate agent for AD treatment in the future.

Prior research has reported beneficial effects of melatonin in rodent models of Alzheimer's disease (AD). This study evaluated the effect of ramelteon (Rozerem, a melatonin receptor agonist) on spatial learning & memory and neuropathological markers in a transgenic murine model of AD (the B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J transgenic mouse strain; hereafter 'AD mice'). Three months of daily ramelteon treatment (~3mg/kg/day), starting at 3 months of age, did not produce an improvement in the cognitive performance of AD mice (water maze). In contrast to wild-type control mice, AD mice did not show any evidence of having learned the location of the escape platform. The cortex and hippocampus of AD mice contained significant quantities of beta-amyloid plaques and PARP-positive (poly ADP ribose polymerase) cells, indicating apoptosis. Six months of ramelteon treatment, starting at 3 months of age, did not produce any change in these neuropathological markers. The ability of long term melatonin treatment to improve cognition and attenuate neuropathology in AD mice did not generalize to this dosage of ramelteon.

To explore the pharmacological mechanism of Changpu-Yizhi-Wan (CYW) in the treatment of Alzheimer's disease (AD) from the perspective of ferroptosis based on network pharmacology and experimental verification. The Encyclopedia of Traditional Chinese Medicine 2.0 (ETCM2.0) database was used to collect the active components of CYW, and the putative targets were predicted in ETCM2.0 and SwissTargetPrediction database. The AD related targets were collected from GeneCards, comparative toxicogenomics database (CTD), Online Mendelian Inheritance in Man (OMIM), DisGeNET and Therapeutic Target Database (TTD), the ferroptosis related targets were collected from FerrDb V2 database, and the common targets of CYW, AD and ferroptosis were calculated by Venny2.1 platform. Protein-protein interaction (PPI) analysis was performed by STRING database, and the active compounds-target network and the PPI network were constructed using Cytoscape software. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway enrichment analysis were performed through DAVID database. RSL3 was used to induce HT22 cells to establish a neuronal ferroptosis cell model, and the inhibitory effect of CYW on neuronal ferroptosis was evaluated by cell viability assay, intracellular iron assay and lipid peroxidation staining. The ferroptosis-associated key protein expressions of Nrf2, SLC7A11, GPX4 and FTH1 were detected by Western blot. A total of 100 candidate compounds were identified from CYW, and 1129 putative targets were obtained. 3924 AD-related targets and 564 ferroptosis-related targets were collected, respectively. There were 78 common targets between them and CYW targets, which were potential targets for CYW to regulate ferroptosis in the treatment of AD. PPI network analysis identified 10 key targets, including TP53, IL6, STAT3, HIF1A, NFE2L2, and others. GO, KEGG and Reactome enrichment analysis showed that 78 potential targets were involved in the regulation of ferroptosis and Nrf2-mediated gene transcription. Molecular docking showed that some active components of CYW had good affinity with Nrf2. In RSL3-induced HT22 cells, CYW significantly improved cell viability, reduced intracellular iron levels and inhibited lipid peroxidation, and improved the protein expression of Nrf2, SLC7A11, GPX4 and FTH1. The pharmacological mechanism of CYW in the treatment of AD may be related to the regulation of Nrf2/SLC7A11/GPX4/FTH1 axis to inhibit neuronal ferroptosis.

Depression is highly correlated with Alzheimer's disease (AD), but treatment for the comorbidity of both is lacking. Phosphodiesterase‐4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP (cAMP), has been considered as a promising target for treatment of both memory loss in AD and depression. Using mouse models of AD and potent PDE4 inhibitors and PDE4‐subtype knockout mice, we demonstrated that PDE4D plays a role in the comorbidity of memory loss and depression in AD. Treatment with the PDE4 inhibitor rolipram or roflumilast reversed memory deficits in novel object recognition and Morris water‐maze tests in APP/PS1 double transgenic and 3xTg‐AD mice, which are widely used models for AD. Similarly, in the tail‐suspension and forced‐swimming tests, the PDE4 inhibitors reversed the decreased immobility in AD mice, suggesting antidepressant‐like effects. The effects of PDE4 inhibitors were mimicked in the tests using mice deficient in PDE4D, which displayed memory‐enhancing and antidepressant‐like effects, relative to the WT mice microinfused with beta‐amyloid peptide 1–42 into the hippocampus. In addition, the PDE4 inhibitor treatment reversed the decreased ratio of Bcl‐2/BAX and inhibited the increased expression of PDE4D in the cerebral cortex and hippocampus of AD mice. Further, the PDE4 inhibitors reversed the decreased levels of cAMP and expression of phosphorylated cAMP response element‐binding protein (CREB) and brain derived neurotrophic factor (BDNF) in AD mice. These were mimicked by PDE4D deficiency and treatment with a PDE4D relatively selective inhibitor. In conclusions, these results suggest that PDE4 is an important target for the comorbidity of memory loss and depression in AD, which appears to be mediated by PDE4D‐cAMP signaling.Support or Funding InformationThis work was supported by research grants from NIH/NIA AG031687 and NIH/NIAAA HHSN275201700001C (both to HTZ) and the National Natural Science Foundation of China (81441111 to HW)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline; recent studies suggest that neuronal ferroptosis plays a key role in its pathogenesis. Danggui Shaoyao San (DSS), a traditional Chinese medicine formula, has shown demonstrated neuroprotective effects, but its precise mechanisms in AD treatment remain unclear. This study aims to investigate the mechanism of DSS in treating AD by inhibiting neuronal ferroptosis, explore whether DSS alleviates AD by suppressing neuronal ferroptosis via the AMPK/Sp1/ACSL4 pathway. Chemical composition of DSS was identified by LC-MS/MS, followed by network pharmacology to predict targets and pathways. Molecular docking assessed binding affinities between DSS compounds and key proteins (AMPK, Sp1, ACSL4). In vivo experiments on APP/PS1 mice evaluated DSS effects on cognitive function, oxidative stress markers, lipid peroxidation, and ferroptosis-related proteins. Network pharmacology analysis suggested that DSS regulates lipid metabolism and inhibits neuronal ferroptosis via the AMPK pathway. Molecular docking revealed strong binding affinities between DSS compounds and AMPK downstream proteins, Sp1 and ACSL4. In vivo experiments showed that DSS improved cognitive function, enhanced antioxidant capacity, reduced lipid peroxide accumulation, and decreased Fe2+ content in brain tissue. Furthermore, DSS increased the expression of FTH, p-AMPK, and GPX4 while decreasing Sp1 and ACSL4 levels, thereby inhibiting ferroptosis. DSS alleviates AD symptoms by suppressing neuronal ferroptosis via the AMPK/Sp1/ACSL4 axis, representing a novel lipid metabolism-targeted therapeutic strategy.

Alzheimer's disease (AD) is the most common cause of dementia. Mitochondrial dysfunction has been widely reported in AD due to its important role in cellular metabolism and energy production. Complex IV (cytochrome c oxidase, COX) of mitochondrial electron transport chain, is particularly vulnerable in AD. Defects of COX in AD have been well documented, but there is little evidence to support the genetic association of the COX-related genes with AD. In this study, we investigated the genetic association between 17 nuclear-encoded COX-related genes and AD in 1572 Han Chinese. The whole exons of these genes were also screened in 107 unrelated AD patients with a high probability of hereditarily transmitted AD. Variants in COX6B1, NDUFA4, SURF1, and COX10 were identified to be associated with AD. An integrative analysis with data of eQTL, expression and pathology revealed that most of the COX-related genes were significantly downregulated in AD patients and mouse models, and the AD-associated variants in COX6B1, SURF1, and COX10 were linked to altered mRNA levels in brain tissues. Furthermore, mRNA levels of Ndufa4, Cox5a, Cox10, Cox6b2, Cox7a2, and Lrpprc were significantly correlated with Aβ plaque burden in hippocampus of AD mice. Convergent functional genomics analysis revealed strong supportive evidence for the roles of COX6B1, COX10, NDUFA4, and SURF1 in AD. As the result of our comprehensive analysis of the COX-related genes at the genetic, expression, and pathology levels, we have been able to provide a systematic view for understanding the relationships of the COX-related genes in the pathology of AD.

Although our understanding of the molecular biology of Alzheimer's disease (AD) continues to improve, the etiology of the disease, particularly the involvement of gut microbiota disturbances, remains a challenge. Outer membrane vesicles (OMVs) play a key role in central nervous system diseases, but the impact of OMVs on AD progression remains unclear. In this study, we hypothesized that AD-derived OMVs (OMVsAD) were a risk factor in AD pathology. To test our hypothesis, young APP/PS1 mice (AD mice) were given OMVsAD by gavage. Young AD mice were euthanized 120days after gavage to assess the intestinal barrier, gut microbiota diversity, mediators of neuroinflammation, glial markers, amyloid burden, and short-chain fatty acid (SCFA) levels. Our results showed that OMVsAD accelerated cognitive dysfunction after 120days of intragastric administration. Morris water maze experiment and new object recognition test showed that OMVsAD caused significantly poorer spatial ability learning and memory of the AD mice. We observed the OMVsAD-treated APP/PS1 mice display OMVs disrupting the intestinal barrier compared with controls of normal human-derived OMVs. Compared with the OMVsHC group, claudin-5 and ZO-1 related to the intestinal barrier were significantly downregulated in the OMVsAD group. The OMVsAD activate microglia in the cerebral cortex and hippocampus of AD mice, and the levels of IL-1β, IL-6, TNF-α, and NF-Κb were upregulated. We also found that OMVsAD increased Aβ production. 16S rRNA sequencing showed that OMVsAD negatively regulated the α- and β-diversity index of intestinal flora and reduced the levels of SCFA. OMVsAD may change the intestinal flora of young AD, damage the intestinal mucosa and blood-brain barrier, and accelerate AD neuropathological damage.

Pyrrolidine dithiocarbamate (PDTC) is a clinically tolerated inhibitor of nuclear factor-kappaB (NF-kappaB), antioxidant and antiinflammatory agent, which provides protection in brain ischemia models. In neonatal hypoxia-ischemia model, PDTC activates Akt and reduces activation of glycogen synthase kinase 3beta (GSK-3beta). Because chronic inflammation, oxidative stress, and increased GSK-3beta activity are features of Alzheimer's disease (AD) pathology, we tested whether PDTC reduces brain pathology and improves cognitive function in a transgenic animal model of AD. A 7 month oral treatment with PDTC prevented the decline in cognition in AD mice without altering beta-amyloid burden or gliosis. Moreover, marked oxidative stress and activation of NF-kappaB were not part of the brain pathology. Instead, the phosphorylated form of GSK-3beta was decreased in the AD mouse brain, and PDTC treatment increased the phosphorylation of Akt and GSK-3beta. Also, PDTC treatment increased the copper concentration in the brain. In addition, PDTC rescued cultured hippocampal neurons from the toxicity of oligomeric Abeta and reduced tau phosphorylation in the hippocampus of AD mice. Finally, astrocytic glutamate transporter GLT-1, known to be regulated by Akt pathway, was decreased in the transgenic AD mice but upregulated back to the wild-type levels by PDTC treatment. Thus, PDTC may improve spatial learning in AD by interfering with Akt-GSK pathway both in neurons and astrocytes. Because PDTC is capable of transferring external Cu2+ into a cell, and, in turn, Cu2+ is able to activate Akt, we hypothesize that PDTC provides the beneficial effect in transgenic AD mice through Cu2+-activated Akt pathway.