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C-Glycosylflavones Alleviate Tau Phosphorylation and Amyloid Neurotoxicity through GSK3? Inhibition.

ABSTRACT: Alzheimer's disease (AD) is the most common brain disorder worldwide. Aberrant tau hyperphosphorylation and accumulation play critical roles in the formation of neurofibrillary tangles highly associated with neuronal dysfunction and cognitive impairment in AD pathogenesis. Glycogen synthase kinase-3? (GSK3?) is a key kinase responsible for tau hyperphosphorylation. Selective inhibition of GSK3? is a promising strategy in AD therapy. Corn silks (CS, Zea mays L.) have been traditionally used as a medicinal herb and recently noted for their potentially cognitive benefits. However, the neuroprotective components of CS and their molecular mechanism have received little attention to date. As part of our effort screening phytochemicals against a broad panel of kinases targeting AD tauopathy, we found inhibition of GSK3? by CS extracts. Subsequent bioassay-guided fractionation led to the isolation and identification of two 6-C-glycosylflavones, isoorientin (1) and 3'-methoxymaysin (2), with selective inhibition against GSK3? in vitro. Enzyme kinetics and molecular docking studies demonstrated that 1 specifically inhibited GSK3? via an ATP noncompetitive mechanism, acting as a substrate competitive inhibitor of GSK3?. Further in vitro cellular studies demonstrated that 1 effectively attenuated tau phosphorylation mediated by GSK3? and was neuroprotective against ?-amyloid-induced tau hyperphosphorylation and neurotoxicity in SH-SY5Y cells. The C-glycosylflavones represent new lead candidates with a novel mechanism of action for the development of AD phytopharmaceuticals.

SUBMITTER: Liang Z

PROVIDER: S-EPMC7355085 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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C-Glycosylflavones Alleviate Tau Phosphorylation and Amyloid Neurotoxicity through GSK3β Inhibition.

Liang Zhibin Z Zhang Bei B Su Wei Wen WW Williams Philip G PG Li Qing X QX

ACS chemical neuroscience 20160603 7

Alzheimer's disease (AD) is the most common brain disorder worldwide. Aberrant tau hyperphosphorylation and accumulation play critical roles in the formation of neurofibrillary tangles highly associated with neuronal dysfunction and cognitive impairment in AD pathogenesis. Glycogen synthase kinase-3β (GSK3β) is a key kinase responsible for tau hyperphosphorylation. Selective inhibition of GSK3β is a promising strategy in AD therapy. Corn silks (CS, Zea mays L.) have been traditionally used as a ...[more]

PMID: 27213824

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Project description:Tau hyperphosphorylation has been linked directly to the formation of toxic neurofibrillary tangles (NFTs) in tauopathies, however, prior to NFT formation, the sequence of pathological events involving tau phosphorylation remains unclear. Here, the effect of glycogen synthase kinase 3β (GSK3β) on tau pathology was examined independently for each step of transcellular propagation; namely, tau intracellular aggregation, release, cellular uptake and seeding activity. We find that overexpression of GSK3β-induced phosphorylated 0N4R tau led to a higher level of tau oligomerization in SH-SY5Y neuroblastoma cells than wild type 0N4R, as determined by several orthogonal assays. Interestingly, the presence of GSK3β also enhanced tau release. Further, we demonstrated that cells endocytosed more monomeric tau protein when pre-phosphorylated by GSK3β. Using an extracellular vesicle (EVs)-assisted tau neuronal delivery system, we show that exosomal GSK3β-phosphorylated tau, when added to differentiated SH-SY5Y cells, induced more efficient tau transfer, showing much higher total tau levels and increased tau aggregate formation as compared to wild type exosomal tau. The role of a primary tau phosphorylation site targeted by microtubule-affinity regulating kinases (MARKs), Ser262, was tested by pseudo-phosphorylation using site-directed mutagenesis to aspartate (S262D). S262D tau overexpression significantly enhanced tau release and intracellular tau accumulation, which were concurrent with the increase of pathological states of tau, as determined by immunodetection. Importantly, phosphorylation-induced tau accumulation was augmented by co-transfecting S262D tau with GSK3β, suggesting a possible interplay between Ser262 phosphorylation and GSK3β activity in tau pathology. Lastly, we found that pre-treatment of cells with amyloid-β (Aβ) further tau phosphorylation and accumulation when Ser262 pre-phosphorylation was present, suggesting that S262 may be a primary mediator of Aβ-induced tau toxicity. These findings provide a potential therapeutic target for treating tau-related disorders by targeting specific phospho-tau isoforms and further elucidate the GSK3β-mediated pathological seeding mechanisms.

Project description:Introduction and aimsAlzheimer's disease (AD) is characterized by the abnormal accumulation of hyperphosphorylated tau proteins and amyloid-beta (Aβ) peptides. Recent studies have shown that many microRNAs (miRNAs) are dysregulated in AD, and modulation of these miRNAs can influence the development of tau and Aβ pathology. The brain-specific miRNA miR-128, encoded by MIR128-1 and MIR128-2, is important for brain development and dysregulated in AD. In this study, the role of miR-128 in tau and Aβ pathology as well as the regulatory mechanism underlying its dysregulation were investigated.MethodsThe effect of miR-128 on tau phosphorylation and Aβ accumulation was examined in AD cellular models through miR-128 overexpression and inhibition. The therapeutic potential of miR-128 in AD mouse model was assessed by comparing phenotypes of 5XFAD mice administered with miR-128-expressing AAVs with 5XFAD mice administered with control AAVs. Phenotypes examined included behavior, plaque load, and protein expression. The regulatory factor of miR-128 transcription was identified through luciferase reporter assay and validated by siRNA knockdown and ChIP analysis.ResultsBoth gain-of-function and loss-of-function studies in AD cellular models reveal that miR-128 represses tau phosphorylation and Aβ secretion. Subsequent investigations show that miR-128 directly inhibits the expression of tau phosphorylation kinase GSK3β and Aβ modulators APPBP2 and mTOR. Upregulation of miR-128 in the hippocampus of 5XFAD mice ameliorates learning and memory impairments, decreases plaque deposition, and enhances autophagic flux. We further demonstrated that C/EBPα transactivates MIR128-1 transcription, while both C/EBPα and miR-128 expression are inhibited by Aβ.ConclusionOur findings suggest that miR-128 suppresses AD pathogenesis, and could be a promising therapeutic target for AD. We also find a possible mechanism underlying the dysregulation of miR-128 in AD, in which Aβ reduces miR-128 expression by inhibiting C/EBPα.

Project description:Alzheimer's disease is a neuropathological condition with abnormal accumulation of extracellular Amyloid-β plaques and intracellular neurofibrillary tangles of Microtubule-associated protein Tau (Tau) in the brain. In pathological conditions, Tau undergoes post-translational modifications such as hyperphosphorylation by the activity of cellular kinases, which eventually leads to protein aggregation in neurons. Melatonin is a neuro-hormone that is mainly secreted from the pineal gland and functions to modulate the cellular kinases. In our study, we have checked the neuroprotective function of Melatonin by MTT and LDH assay, where Melatonin inhibited the Tau aggregates-mediated cytotoxicity and membrane leakage in Neuro2A cells. The potency of Melatonin has also been studied for the quenching of intracellular reactive oxygen species level by DCFDA assay and caspase 3 activity. Melatonin was shown to reduce the GSK3β mRNA and subsequent protein level as well as the phospho-Tau level (pThr181 and pThr212-pSer214) in okadaic acid-induced Neuro2A cells, as observed by western blot and immunofluorescence assay. Further, Melatonin has increased the cellular Nrf2 level and its nuclear translocation as an oxidative stress response in Tauopathy. The Melatonin was found to induce pro- and anti-inflammatory cytokines levels in N9 microglia. The mRNA level of cellular kinases such as as-GSK3β, MAPK were also studied by qRT-PCR assay in Tau-exposed N9 and Neuro2A cells. The immunomodulatory effect of Melatonin was evident as it induced IL-10 and TGF-β cytokine levels and activated MAP3K level in Tau-exposed microglia and neurons, respectively. Melatonin also downregulated the mRNA level of pro-inflammatory markers, IL-1β and Cyclooxygenase-2 in N9 microglia. Together, these findings suggest that Melatonin remediated the cytokine profile of Tau-exposed microglia, reduced Tau hyperphosphorylation by downregulating GSK3β level, and alleviated oxidative stress via Nrf2 nuclear translocation.

Dataset Information

C-Glycosylflavones Alleviate Tau Phosphorylation and Amyloid Neurotoxicity through GSK3? Inhibition.

Publications

C-Glycosylflavones Alleviate Tau Phosphorylation and Amyloid Neurotoxicity through GSK3β Inhibition.

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