The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D ¹⁵N-¹H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.
Humans ; Amyloid beta-Protein Precursor/chemistry* ; Micelles ; Amyloid Precursor Protein Secretases/metabolism* ; Magnetic Resonance Spectroscopy ; Recombinant Proteins

Myloid beta(Aβ) is produced by cleavage of amyloid precursor protein(APP), which is a main reason for Alzheimer's disease(AD) occurrence and development. This study preliminarily investigated the mechanism of Atractylodes macrocephala(AM) against AD based on LKB1-AMPK-TFEB pathway. The effect of AM on memory ability of AD transgenic Caenorhabditis elegans CL2241 was detected, and then the APP plasmid was transiently transferred to mouse neuroblastoma(N2 a) cells in vitro. The mice were divided into the blank control group, APP group(model group), positive control group(100 μmol·L~(-1) rapamycin), and AM low-, medium-and high-dose groups(100, 200 and 300 μg·mL~(-1)). The content of Aβ_(1-42) in cell medium, the protein level of APP, the fluorescence intensity of APP, the transcriptional activity of transcription factor EB(TFEB), the activity of lysosomes in autophagy, and autophagy flux were determined by enzyme-linked immunosorbent assay(ELISA), Western blot, fluorescence microscope, luciferase reporter gene assay, RLuc-LC3 wt/RLuc-LC3 G120 A, and mRFP-GFP-LC3, respectively. The protein expression of TFEB, LC3Ⅱ, LC3Ⅰ, LAMP2, Beclin1, LKB1, p-AMPK and p-ACC was detected by Western blot. Immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the fluorescence intensity of TFEB and the mRNA expression of TFEB and downstream target genes, respectively. The results showed that AM reduced the chemotactic index of transgenic C. elegans CL2241, and decreased the content of Aβ in the supernatant of cell culture medium at different concentrations. In addition, AM lowered the protein level of APP and the fluorescence intensity of APP in a dose-dependent manner. Transcriptional activity of TFEB and fluorescence intensity of mRFP-GFP-LC3 plasmid were enhanced after AM treatment, and the value of RLuc-LC3 wt/RLuc-LC3 G120 A was reduced. AM promoted the protein levels of TFEB, LAMP2 and Beclin1 at different concentrations, and increased the protein expression ratio of LC3Ⅱ/LC3Ⅰ in a dose-dependent manner. Immunofluorescence results revealed that AM improved the fluorescence intensity and nuclear expression of TFEB, and RT-PCR results indicated that AM of various concentrations elevated the mRNA expression of TFEB in APP transfected N2 a cells and promoted the transcription level of LAMP2 in a dose-dependent manner, and high-concentration AM also increased the mRNA levels of LC3 and P62. The protein levels of LKB1, p-AMPK and p-ACC were elevated by AM of different concentrations. In summary, AM regulating lysophagy and degrading APP are related to the activation of LKB1-AMPK-TFEB pathway.
AMP-Activated Protein Kinases/metabolism* ; Alzheimer Disease/drug therapy* ; Amyloid beta-Peptides/metabolism* ; Amyloid beta-Protein Precursor/metabolism* ; Animals ; Atractylodes/chemistry* ; Autophagy/drug effects* ; Beclin-1/pharmacology* ; Caenorhabditis elegans/metabolism* ; Macroautophagy ; Mice ; RNA, Messenger ; Sirolimus/pharmacology*

OBJECTIVETo research Angelica tenuissima Nakai (ATN) for use in novel Alzheimer's disease (AD) therapeutics.

METHODSThe effect of a 30% ethanol extract of ATN (KH032) on AD-like cognitive impairment and neuropathological and neuroinflammatory changes induced by bilateral intracerebroventricular injections of β-amyloid (Aβ) peptide (Aβ) was investigated. Male C57Bl/6 mice were randomly divided into 4 groups, 10 in each group. KH032-treated groups were administrated with a low or high dose of KH032 (50 and 200 mg/kg, respectively), intragastrically for 16 days; distilled water was applied in the sham and negative groups. Open fifield test, Y maze and Morris water maze test were used for behavior test and cognitive ability. In addition, the neuroprotective effects of KH032 in Aβ-infused mice on the histopathological markers [neuronspecific nuclear protein (NeuN), Aβ] of neurodegeneration were examined. The levels of glial fibrillary acidic protein (GFAP), NeuN, phosphorylation extracellular signal-regulated kinase (ERK)/ERK, brain-derived neurotrophic factor (BDNF), phosphorylation cAMP response element-binding (CREB)/CREB protein expression were measured by Western blot.

RESULTSKH032 treatment ameliorated cognitive impairments, reduced the overexpression of Aβ, and inhibited neuronal loss and neuroinflammatory response in the Aβ-infused mice. Moreover, KH032 treatment enhanced BDNF expression levels in the hippocampus. Finally, KH032 treatment increased phosphorylation of ERK1/2 and CREB, vital for ERK-CREB signaling.

CONCLUSIONSKH032 attenuated cognitive defificits in the Aβ-infused mice by increasing BDNF expression and ERK1/2 and CREB phosphorylation and inhibiting neuronal loss and neuroinflflammatory response, suggesting that KH032 has therapeutic potential in neurodegenerative disorders such as AD.

Alzheimer Disease ; drug therapy ; pathology ; physiopathology ; Amyloid beta-Peptides ; Angelica ; chemistry ; Animals ; Brain ; pathology ; Brain-Derived Neurotrophic Factor ; metabolism ; Cognitive Dysfunction ; complications ; drug therapy ; physiopathology ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Disease Models, Animal ; Male ; Maze Learning ; drug effects ; Memory, Short-Term ; drug effects ; Mice, Inbred C57BL ; Neurogenesis ; drug effects ; Neuroglia ; drug effects ; metabolism ; pathology ; Neurons ; drug effects ; metabolism ; pathology ; Neuroprotective Agents ; pharmacology ; therapeutic use ; Phosphorylation ; drug effects ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plaque, Amyloid ; drug therapy ; pathology ; physiopathology ; Signal Transduction ; drug effects

Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.
Alzheimer Disease ; metabolism ; pathology ; Amyloid beta-Peptides ; metabolism ; Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; chemistry ; genetics ; metabolism ; Brain ; metabolism ; Cells, Cultured ; Chloride Channels ; genetics ; metabolism ; Disease Models, Animal ; HEK293 Cells ; Humans ; Lysosomes ; genetics ; metabolism ; Mice ; Mice, Transgenic ; Microglia ; cytology ; metabolism ; Mutagenesis, Site-Directed ; Peptides ; analysis ; chemistry ; Protein Binding ; RNA Interference ; Sirtuin 1 ; antagonists & inhibitors ; genetics ; metabolism

OBJECTIVETo investigate the effects of exogenous hydrogen sulfide (H2S) on β-site APP cleaving enzyme 1 (BACE-1) and β-amyloid peptide (Aβ) metabolism in primary culture of neurons under high-glucose condition.

METHODSThe cortical neurons in primary culture under normal and high glucose (60 mmol/L) conditions for 24 h were exposed to 25, 50 and 100 µmol/L NaHS. Aβ1-42 concentration in the cell culture was measured by ELISA, and BACE-1 mRNA and protein levels were detected by fluorescent quantitative real-time PCR and Western blotting, respectively.

RESULTSCompared with the neurons cultured in normal glucose, the neurons exposed to high glucose showed significantly increased A&beta;1-42 concentration and BACE-1 mRNA and protein expressions (P<0.05). Exposure to 25, 50 and 100 µmol/L NaHS significantly decreased A&beta;1-42 concentration and BACE-1 mRNA and protein expressions in the high-glucose cell culture (P<0.05).

CONCLUSIONNeurons exposed to high glucose exhibit increased A&beta;1-42 levels and BACE-1 mRNA and protein expressions, which can be concentration-dependently decreased by NaHS.

Amyloid Precursor Protein Secretases ; metabolism ; Amyloid beta-Peptides ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; metabolism ; Cells, Cultured ; Culture Media ; chemistry ; Glucose ; chemistry ; Hydrogen Sulfide ; pharmacology ; Neurons ; drug effects ; metabolism ; Peptide Fragments ; metabolism ; Rats ; Rats, Sprague-Dawley

Aggregation and accumulation of beta-amyloid peptide (Abeta) in brain tissues contribute to the pathogenesis of Alzheimer's disease. Therefore, the promotion of Abeta clearance is one of the key targets for preventing and treatment Alzheimer's disease. Studies proved that some traditional Chinese medicine (TCM) compounds and extracts could impact the activity of degrading enzyme in amyloid peptide, the transport of hemato encephalic barrier and the phagocytosis of microglial cells, promote Abeta clearance, and improve learning and memory of animal models with Alzheimer's disease. In this review, we made an summary for the relations between Abeta and Alzheimer's disease, the Abeta clearance mechanism and the clearance effect of traditional Chinese medicines.
Alzheimer Disease ; drug therapy ; metabolism ; pathology ; Amyloid beta-Peptides ; chemistry ; metabolism ; Animals ; Blood-Brain Barrier ; drug effects ; metabolism ; Humans ; Medicine, Chinese Traditional ; methods ; Microglia ; drug effects ; metabolism ; Protein Multimerization ; drug effects

Alzheimer's disease (AD) is a complex neurodegenerative disorder which seriously causes the dementia in elderly people and afflicts millions of people worldwide. Drug discovery for Alzheimer's disease therapy has been a hot research area and a big challenge, in which development of acetylcholinesterase (AChE) inhibitors design was the most active and some AChE inhibitors are commercially available for AD medication already. However, practical using of commercial AChE inhibitors showed their limited usefulness and related adverse effects. Thus, it is extremely urgent to find novel AChE inhibitors with higher potency and less adverse effects. Based on the accurate crystallographic studies about AChE, strategies for multi-binding site AChE inhibitors have been formed, followed by design of the multi-target directed ligands. In this review, the structures and binding modes of commercial AChE inhibitors were briefly discussed, together with the development of AChE inhibitor design for AD therapy: from multi-binding site inhibitors to multi-target directed ligands.
Acetylcholinesterase ; chemistry ; metabolism ; Alzheimer Disease ; drug therapy ; Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Amyloid beta-Peptides ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; antagonists & inhibitors ; Binding Sites ; Butyrylcholinesterase ; chemistry ; metabolism ; Cholinesterase Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Drug Design ; Humans ; Ligands ; Monoamine Oxidase Inhibitors ; chemical synthesis ; chemistry ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; Structure-Activity Relationship

OBJECTIVETo observe the effects of hippocampal Abeta42 deposition on the expression of inflammatory cytokines and phosphorylated MAPK signal molecules as well as the intervention of AD by total glucosides of paeony (TGP).

METHOD12 week-old female SD rats were stereotactic injected one-time with a fibrillar Abeta42 positioning hippocampus to replicate AD pathology model and interfered with TGP. The expression of inflammatory cytokines and phosphorylated MAPK pathway signaling molecules were observed by immunohistochemistry (SABC), and SABC images were analyzed by image analysis software.

RESULTCompared with the control group, the IL-1beta, IL-6 and p-p38, p-JNK, p-MEK3/6 positive stained areas of AD pathology model group increased and their staining intensity decreased (the protein expression quantity inversely proportional to the staining intensity), while the IL-1beta, IL-6 and p-p38, p-JNK, p-MEK3/6 positive stained areas of the treatment groups decreased and their staining intensity increased compared with AD pathology model group.

CONCLUSIONAbeta42 deposition in hippocampus can induce the brain inflammation and the over-expression of IL-1beta, IL-6 and p-p38, p-JNK, p-MEK3/6. Inhibiting the over-expression of inflammatory cytokines and phosphorylated MAPK signaling molecules may be a major antagonistic mechanism of TGP against AD.

Alzheimer Disease ; drug therapy ; Amyloid beta-Peptides ; metabolism ; toxicity ; Animals ; Cytokines ; analysis ; Female ; Glucosides ; pharmacology ; therapeutic use ; Hippocampus ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinases ; metabolism ; Paeonia ; chemistry ; Peptide Fragments ; metabolism ; toxicity ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; metabolism

Silibinin, from milk thistle (Silybum marianum), is a flavonolignan with anti-oxidative and anti-inflammatory properties. It has been therapeutically used for the treatment of hepatic diseases in China, Germany and Japan. Recently, increasing evidences prove that silibinin is also a potent antitumor agent, and the major anti-tumor mechanism for silibinin is the prominent inhibition of the activities of receptor tyrosine kinases (RTKs) and their downstream signal molecules in a variety of tumor cell lines, such as epidermal growth factor receptor 1 (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) signaling pathways. Meanwhile, the evidences that silibinin selectively scavenges hydroxyl free radical (*OH) and specifically inhibits the action of nuclear factor kappaB (NF-kappaB) provide more complicated explanations for its antioxidant and anti-inflammatory effects. Some new findings such as that silibinin attenuating the cognitive deficits induced by amyloid beta protein (Abeta) peptide through its antioxidative and anti-inflammatory properties is valuable to broad the medical prospect of silibinin. In this review, we discuss the molecular pharmacological mechanisms of silibinin, focusing on its inhibition of tyrosine kinases, actions of antioxidation, free radical scavenging, immunoregulation and anti-inflammation.
Amyloid beta-Peptides ; metabolism ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Antioxidants ; pharmacology ; Enzyme Activation ; Free Radical Scavengers ; pharmacology ; Humans ; Milk Thistle ; chemistry ; Molecular Structure ; NF-kappa B ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Reactive Oxygen Species ; metabolism ; Receptor Protein-Tyrosine Kinases ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, IGF Type 1 ; metabolism ; Signal Transduction ; drug effects ; Silymarin ; chemistry ; isolation & purification ; pharmacology

Alzheimer Disease ; metabolism ; Amyloid ; chemistry ; metabolism ; Amyloid beta-Protein Precursor ; chemistry ; metabolism ; Dimerization ; Humans ; Hydrogen Bonding ; Protein Binding ; Protein Folding ; Protein Structure, Quaternary ; Protein Structure, Secondary ; Protein Structure, Tertiary