A Class I HDAC Inhibitor Rescues Synaptic Damage and Neuron Loss in APP-Transfected Cells and APP/PS1 Mice through the GRIP1/AMPA Pathway

As a neurodegenerative disease, Alzheimer’s disease (AD) seriously affects the health of older people. Changes in synapses occur first over the course of the disease, perhaps even before the formation of Aβ plaques. Histone deacetylase (HDAC) mediates the damage of Aβ oligomers to dendritic spines. Therefore, we examined the relationship between HDAC activity and synaptic defects using an HDAC inhibitor (HDACI), BG45, in the human neuroblastoma SH-SY5Y cell line with stable overexpression of Swedish mutant APP (APPsw) and in APP/PS1 transgenic mice during this study. The cells were treated with 15 μM BG45 and the APP/PS1 mice were treated with 30 mg/kg BG45. We detected the levels of synapse-related proteins, HDACs, tau phosphorylation, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors using Western blotting and immunohistochemistry. We also measured the expression of cytoskeletal proteins in the cell model. The mRNA levels of the glutamate ion receptor alginate subunit 2 (GRIK2), sodium voltage-gated channel beta subunit (SCN3B), synaptophysin (SYP), Grm2 (the gene encoding glutamate receptor subunit 2 (GluR2)), Grid2IP, glutamate receptor interacting protein 1 (GRIP1), and GRIP2 were detected to explore the effects of the HDACI on regulating the expression of synaptic proteins and AMPA receptors. According to our studies, the expressions of HDAC1, HDAC2, and HDAC3 were increased, which were accompanied by the downregulation of the synapse-related proteins SYP, postsynaptic dendritic protein (PSD-95), and spinophilin as early as 24 h after transfection with the APPsw gene. BG45 upregulated the expression of synapse-related proteins and repaired cytoskeletal damage. In vivo, BG45 alleviated the apoptosis-mediated loss of hippocampal neurons, upregulated synapse-related proteins, reduced Aβ deposition and phosphorylation of tau, and increased the levels of the synapse-related genes GRIK2, SCN3B, SYP, Grm2, and Grid2IP. BG45 increased the expression of the AMPA receptor subunits GluA1, GluA2, and GluA3 on APPsw-transfected cells and increased GRIP1 and GRIP2 expression and AMPA receptor phosphorylation in vivo. Based on these results, HDACs are involved in the early process of synaptic defects in AD models, and BG45 may rescue synaptic damage and the loss of hippocampal neurons by specifically inhibiting HDAC1, HDAC2, and HDAC3, thereby modulating AMPA receptor transduction, increasing synapse-related gene expression, and finally enhancing the function of excitatory synapses. BG45 may be considered a potential drug for the treatment of early AD in further studies.     

硫代多联吡啶铂(Ⅱ)配合物的合成、性质及在光催化制氢中的应用

合成了2个新配体4-(4-硫代乙酸)甲苯基-6-苯基-2,2'-联吡啶HC^N^N(PhCH₂SCOCH₃)(L3)和6-(4-硫代乙酸)甲苯基-2,2'-联吡啶 HC^N^N (CH₂SCOCH₃)(L5)及其发光的铂(Ⅱ)配合物ClPtC^N^N(PhCH₂SCOCH₃)(C3)和ClPtC^N^N(CH₂SCOCH₃)(C5). 通过¹H NMR谱和质谱对它们的结构进行了表征, 采用X射线单晶衍射分析确定了C3的晶体结构. 利用紫外-可见吸收光谱、 发射光谱及激发态寿命测定研究了它们的光物理性质和电化学性质, 以及配合物作为光敏剂在光催化制氢中的应用. 通过系列配合物产氢效率的比较, 揭示了它们的产氢效率和激发态寿命的关系.     

Substrate-dependent targeting of eukaryotic translation initiation factor 4A by pateamine A: negation of domain-linker regulation of activity

Central to cap-dependent eukaryotic translation initiation is the eIF4F complex, which is composed of the three eukaryotic initiation factors eIF4E, eIF4G, and eIF4A. eIF4A is an RNA-dependent ATPase and an ATP-dependent helicase that unwinds local secondary structure in mRNA to allow binding of the 43S ribosomal complex. The marine natural product pateamine A (PatA) has been demonstrated to inhibit cap-dependent initiation by targeting eIF4A and disrupting its protein-protein interactions while increasing its enzymatic activities. Here we demonstrate that the increased activity is caused by the induction of global conformational changes within eIF4A. Furthermore, binding of PatA is dependent on substrate (RNA and ATP) binding, and the increased activity upon PatA binding is caused by relief of a negative regulatory function of the eIF4A unique domain linker.     

mRNA transport to and translation in neuronal dendrites

Transport of mRNA is an important biological process in all cells that sets up gradients of translated protein from the site of mRNA docking and translation. Neurons are highly polarized cells where the targeted movement of RNAs and local translation at that site have been shown to be integral to the proper functioning of the neuron. Indeed, this specialized biological function for localized RNAs in particular neurons may in part confer a selective advantage on these cells such that they "out-compete" others in the race to establish synaptic connectivity. In this mini-review we highlight some of the salient features of RNA targeting and translation in neurons.     

Brownian dynamics simulations of binding mRNA cap analogues to eIF4E protein

The binding of five analogues of the 5'-end mRNA cap, differing in their electrostatic and hydrodynamic properties, to the eukaryotic initiation factor eIF4E was simulated by means of Brownian dynamics methods. Electrostatic and hydrodynamic models of eIF4E protein and the ligands were prepared using established molecular electrostatics and hydrodynamics simulation methods for predicting ionization states of titratable groups, adequate for given experimental conditions, and for computing their translational and rotational diffusion tensors, respectively. The diffusional encounter rate constants obtained from simulations are compared with bimolecular association rate constants resulting from stopped-flow spectrofluorimeter measurements. A very good agreement between simulations and experiments was achieved, which indicates that the kinetics of binding 5'-mRNA caps can be satisfactory explained by referring to the Brownian motion of the particles with the electrostatic steering of the ligands toward the eIF4E binding site and electrostatic desolvation contributions upon complex formation.     

Overexpression and unique rearrangement of VH2 transcripts in immunoglobulin variable heavy chain genes in ankylosing spondylitis patients

To identify immunoglobulin variable heavy chain (VH) gene usages in Korean ankylosing spondylitis (AS) patients, expression level of VH2 genes from peripheral blood mononuclear cells (PBMCs) of 8 AS patients and 9 healthy donors was analysed by quantitative real-time PCR (Q-PCR). Q-PCR results demonstrated VH2 genes were overexpressed in AS patients (Relative amount of mRNA of VH2 genes to a house-keeping gene, 7.13 ± 7.77 vs, 0.68 ± 0.55; P < 0.0001). The sequence analysis revealed the majority of them contained CDC42 binding protein kinase beta (CDC42 BPB) genes. The insertion of CDC42 BPB gene was confirmed by PCR with primers corresponding CDC42 BPB and CH genes. Our study revealed VH2 overexpression and unique rearrangement in Ig VH genes from peripheral blood of AS patients. This may imply aberrant immunoglobulin gene rearrangement in B cell occurs in Korean AS patients, which requires further investigation.     

Structural and thermodynamic behavior of eukaryotic initiation factor 4E in supramolecular formation with 4E-binding protein 1 and mRNA cap analogue, studied by spectroscopic methods

The structural and thermodynamic behavior of the complex formation of eIF4E with either or both mRNA cap analogue (m7GTP, m7GpppA, or m7GpppG) and 4EBP1 has been investigated by spectroscopic measurements. Although the circular dichroism (CD) spectrum of eIF4E was little affected by the association with any cap analogue, the association constant of eIF4E with m7GpppA/G, estimated from the fluorescence quenching, was about 10 times larger than that with m7GTP. The van't Hoff analyses showed that the m7GpppA/G binding is enthalpy-driven with a large negative deltaH(o), and this is in contrast with the entropy-driven binding of m7GTP, where the positive deltaS(o) is large enough to overcome an increase of deltaH(o). This different behavior obviously originates in the interaction of the second nucleotide in m7GpppA with eIF4E, suggesting the importance of the nucleotide sequence linked to the m7Gppp terminal moiety, in addition to the specific interaction with the m7G base, for the recognition of mRNA cap structure by eIF4E. On the other hand, the CD spectra indicated that the binding of 4EBP1, an endogenous eIF4E-regulatory protein without having any defined secondary structure, shifted the m7GTP- or m7GpppA/G-bound eIF4E to an irregular structure, although such a structural change was not observed for eIF4E alone. The association constant of 4EBP1 with m7GTP- or m7GpppA/G-bound eIF4E was by two orders of magnitude larger than that with eIF4E alone. These results suggest the close interrelation in the supramolecular formation of 4EBP-eIF4E-mRNA cap structure.     

Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5′,5′-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging – they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands – folic acid, biotin, glucose, and cholesterol – to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure–activity relationship (SAR) study using model fluorescent probes and cap–ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.

Ligand assisted cellular delivery of negatively charged dinucleotides, which are potential antagonists of the protooncogenic protein eIF4E.     

超声刻蚀法构建分级结构的超疏水表面

在湿法刻蚀和超声空化的基础上, 采用超声刻蚀法制备了具有微纳米分级结构的超疏水表面. 以等体积比的硝酸/乙醇(体积分数为4%)和双氧水(质量分数为30%)的混合溶液作为刻蚀剂, 在室温下对60Si2Mn钢、 60^#钢、 T10钢、 Cr06钢、 65Mn钢和硅钢表面超声刻蚀2~10 min, 构建出多种形貌的微纳米分级结构. 上述表面经氟硅烷修饰后具有超疏水性, 水的表观接触角高达157.0°, 155.8°, 157.4°, 154.9°, 157.6°和156.8°, 滚动角分别为6.5°, 19.2°, 6.1°, 7.8°, 6.7°和7.2°. 与常规刻蚀方法相比, 超声刻蚀的化学刻蚀作用因与空化作用耦合而得到强化和改变, 从而在钢表面构建出分级结构. 由于材料表面微结构形貌和固/液界面接触状态不同, 制得的超疏水表面表现出的润湿行为也不同. 超声刻蚀法简单易行, 成本低廉, 适用于其它金属表面构建微纳米分级结构和超疏水表面.     

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor ��

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor γ (PPARγ) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPARγ activity or knockdown of PPARγ expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPARγ through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPARγ siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress-induced gene expression by suppressing translation via activation of PPARγ in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPARγ.     

Neddylation is required for presynaptic clustering of mGlu7 and maturation of presynaptic terminals

Neddylation is a posttranslational modification in which NEDD8 is conjugated to a target substrate by cellular processes similar to those involved in ubiquitination. Recent studies have identified PSD-95 and cofilin as substrates for neddylation in the brain and have shown that neddylation modulates the maturation and stability of dendritic spines in developing neurons. However, the precise substrates and functional consequences of neddylation at presynaptic terminals remain elusive. Here, we provide evidence that the mGlu7 receptor is a target of neddylation in heterologous cells and rat primary cultured neurons. We found that mGlu7 neddylation is reduced by agonist treatment and is required for the clustering of mGlu7 in the presynaptic active zone. In addition, we observed that neddylation is not required for the endocytosis of mGlu7, but it facilitates the ubiquitination of mGlu7 and stabilizes mGlu7 protein expression. Finally, we demonstrate that neddylation is necessary for the maturation of excitatory presynaptic terminals, providing a key role for neddylation in synaptic function.Subject terms: Cellular neuroscience, Molecular neuroscience     

双核茂金属催化剂的合成、表征与应用

使用桥连配体锂盐与MCl₄络合, 合成了4个不同结构的双核茂金属化合物[μ,μ-(CH₂)₃]{[C(H)·(η⁵-C₅H₄)(η⁵-C₁₃H₈)](MCl₂)}₂[M=Zr or Ti](4, 5)和[μ,μ-(CH₂)₃]{[C(H)(η⁵-C₅H₄)(η⁵-C₉H₆)]·(MCl₂)}₂[M=Zr or Ti](6, 7), 配体和化合物都经过核磁氢谱(¹H NMR)、 碳谱(¹³C NMR)、 红外光谱(IR)及元素分析等表征, 确认了化学结构. 以甲基铝氧烷(MAO)为助催化剂, 化合物4~7为催化剂催化丙烯聚合, 考察了聚合温度、 乙烯压力、 铝钛或铝锆比对催化剂活性及聚合物分子量的影响. 结果表明, 多亚甲基桥连双核茂金属是高活性乙烯和丙烯聚合催化剂, 乙烯聚合活性最高达到7.5× 10⁶ g PE/(mol Zr·h)(化合物6), 丙烯聚合活性达 10 × 10⁵ g sPP/(mol Zr·h)(化合物4). 所得间规聚丙烯(sPP)的间规度指数(SI, r) 达到90%. 在同样条件下, 双核化合物的催化活性、 聚合物分子量M_w(> 100000)以及分子量分布(MWD>2.5)均比相应的单核化合物高(M_w<70000, MWD≤2), 表明该体系中存在较强的核效应.     

Fluorescent Turn-On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap-Recognizing Proteins

The m⁷G cap is a unique nucleotide structure at the 5′-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m⁷G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m⁷GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.     

Phosphorothioate Modification of mRNA Accelerates the Rate of Translation Initiation to Provide More Efficient Protein Synthesis

Messenger RNAs (mRNAs) with phosphorothioate modification (PS‐mRNA) to the phosphate site of A, G, C, and U with all 16 possible combinations were prepared, and the translation reaction was evaluated using an E. coli cell‐free translation system. Protein synthesis from PS‐mRNA increased in 12 of 15 patterns when compared with that of unmodified mRNA. The protein yield increased 22‐fold when the phosphorothioate modification at A/C sites was introduced into the region from the 5′‐end to the initiation codon. Single‐turnover analysis of PS‐mRNA translation showed that phosphorothioate modification increases the number of translating ribosomes, thus suggesting that the rate of translation initiation (rate of ribosome complex formation) is positively affected by the modification. The method provides a new strategy for improving translation by using non‐natural mRNA.