脑血管内皮细胞敲除Prmt5导致脑血管损伤及星形胶质细胞活化*

目的: 研究蛋白质精氨酸甲基转移酶5(protein arginine methyltransferase 5,Prmt5)在小鼠脑血管发育、稳态维持中的功能,并考察脑血管内皮细胞特异性敲除Prmt5后对中枢神经系统的影响。方法: 利用脑血管内皮细胞特异性表达SP-A-Cre转基因小鼠和Prmt5条件基因打靶小鼠交配,构建脑血管内皮细胞特异性Prmt5敲除小鼠。利用H-E染色、免疫荧光染色、激光散斑成像、Sulfo-NHS-Biotin染料灌注等方法评价脑血管内皮细胞特异性Prmt5敲除小鼠脑血管结构、脑血流量、血脑屏障渗透性等;利用实时定量PCR进一步检测补体C1q(complement C1q,C1q)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin 1β,IL-1β)等细胞因子的表达水平。通过免疫荧光、Western blot等检测胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、S100钙结合蛋白β(S100 calcium-binding protein β protein,S100β)和补体C3(complement C3,C3)的表达,检测小鼠皮层、丘脑和小脑中星形胶质细胞活化水平。结果: 脑血管内皮细胞特异性敲除Prmt5导致血管损伤, C1q、TNF-α和IL-1β等炎症因子表达水平上调,活化星形胶质细胞比例明显增加。结论: 脑血管内皮细胞中Prmt5在小鼠脑血管稳态维持中发挥了重要功能。     

综述与专论: 运动调节星形胶质细胞改善阿尔茨海默病机制研究

阿尔茨海默病（Alzheimer’s disease， AD） 是老年人中最常见的中枢神经系统退行性疾病之一， AD的病理性改变与星形胶质细胞（astrocytes）密切相关。星形胶质细胞是血脑屏障和三方突触的重要组成部分。越来越多的证据表明，在AD早期阶段星形胶质细胞已出现分子和细胞水平的改变。星形胶质细胞的功能会影响AD的发生发展。多项研究表明，运动可以通过调节星形胶质细胞的激活、营养因子的释放、能量代谢、炎症及氧化应激反应，延缓AD的病理变化及改善AD认知功能障碍。本文对运动通过星形胶质细胞改善AD认知功能障碍的作用及机制进行综述和展望，旨在为AD的预防和治疗提供新策略。     

综述:星形胶质细胞介导的β-淀粉样蛋白代谢与阿尔茨海默病早期的关系

星形胶质细胞是中枢神经系统中含量最丰富的细胞,研究表明,星形胶质细胞与阿尔茨海默病(Alzheimer's disease,AD)病程有关,尤其是对AD主要致病蛋白β-淀粉样蛋白(β-amyloid protein,Aβ)的产生、内化和降解过程起着重要的调节作用．本文讨论星形胶质细胞中Aβ的产生,星形胶质细胞对Aβ的内化、降解和清除的机制,并阐释星形胶质细胞在Aβ代谢中的作用与AD早期发病机制的关系．     

星形胶质细胞在阿尔茨海默病中的功能变化及分子机制

星形胶质细胞在脑内数量最多,分布最广,对神经元有营养支持的作用,并且能够调控神经元的活性。越来越多的证据表明星形胶质细胞激活参与阿尔茨海默病(Alzheimer's disease,AD)的发生和发展。在AD病理情况下,星形胶质细胞在多种因子如β淀粉样蛋白(beta-amyloid,Aβ)和促炎细胞因子的作用下被激活,激活的星形胶质细胞进一步释放一氧化氮(Nitric oxide,NO)和多种炎性因子增强炎症级联反应。功能失常的星形胶质细胞会促进Aβ的产生,减弱对Aβ的摄取和清除,导致Aβ聚集沉积形成老年斑。激活的星形胶质细胞释放的炎症因子还能显著增加神经元内tau蛋白的异常过度磷酸化,产生神经纤维缠结。本文对星形胶质细胞在AD中参与神经变性的功能变化和分子机制进行总结,为星形胶质细胞作为靶点预防及治疗AD提供一定的理论依据。     

星形胶质细胞在神经系统疾病中的作用

激活的星形胶质细胞会产生和释放的神经递质、神经营养因子和促炎因子等,对神经元既有保护作用,也有毒性作用,在阿尔茨海默病、帕金森症、癫痫、缺血性脑损伤等多种神经系统疾病的发生发展过程中有着重要作用。对近年来国内外有关星形胶质细胞参与神经系统疾病进程的最新研究进展作了综述,并对今后研究工作进行了展望。     

LPS诱导海马星形胶质细胞活化与活化的星形胶质细胞对海马神经元的影响

探讨脂多糖(Lipopolysaccharide,LPS)对长时间存活大鼠海马内星形胶质细胞的反应以及对神经元的影响。方法:本实验用10只健康成年雄性SD大鼠,海马CA3区注射LPS 10μ1．7和14d后,尼氏染色观察神经元的变化,免疫组织化学染色结合图像分析方法观察海马CA3区注射部位胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)、的表达变化。结果:脂多糖可促进海马星形胶质细胞的活化,但并不能引起海马区神经元的损伤。结论:星形胶质细胞在脑损伤后的脑内炎症反应起了一定的作用,但并不能引起神经元的损伤。     

原代培养星形胶质细胞和小胶质细胞的特性与鉴定

本研究旨在明确原代培养的星形胶质细胞和小胶质细胞不同代次的生长特性,优化高效获取状态一致细胞的技术方法。将新生乳鼠的脑组织进行原代分离培养胶质细胞,通过细胞增殖检测试剂盒（cell counting kit-8,CCK-8）测定混合胶质细胞增殖曲线,使用流式细胞术检测两类细胞比例,并通过免疫荧光染色鉴定两类胶质细胞分型情况。生长曲线显示P0和P1代混合胶质细胞增殖活力最好;通过170 r/min机械振摇30 min可获得97.3%的高纯度小胶质细胞,该纯化方法得到的P0、P1、P2代离子钙接头蛋白-1（ionized calcium-binding adapter molecule 1,Iba-1）阳性小胶质细胞的形态及其M1、M2表型比例无代次差别;通过星形胶质细胞表面抗原-2（astrocyte cell surface antigen-2,ACSA-2）磁珠抗体分选的方法可获得纯度达到95.7%的星形胶质细胞,该纯化方法得到的P0、P1、P2代胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）阳性星形胶质细胞的形态及其A1、A2表型比例无代次差别。本研究详述了原代分离培养的小胶质细胞和星形胶质细胞的生长特点,证明了获取两类胶质细胞的最佳代次,优化了获取两类胶质细胞的技术方法,验证了连续培养两代不会影响其功能表型。本结果为研究神经系统炎症相关疾病的分子机制提供了技术支撑。     

星形胶质细胞的生物学功能

人类大脑由两类细胞组成:一类是神经元,另一类是神经胶质细胞。神经胶质细胞的数量约为神经元的10倍,但其作用长期以来一直被认为仅限于在神经元之间充当填充物,填满大脑中的剩余空间,同时为神经元提供营养。但近年来认识到神经胶质细胞的主要成员星形胶质细胞能够感知外界刺激,它的反应选择性甚至高于相邻神经元。神经元的反应活动很多都要经过星形胶质细胞的介导才能完成。本文介绍了星形胶质细胞在神经调制、突触调节和神经血管系统偶联方面的一些新进展,以期在不久的将来对星形胶质细胞的功能有更深入的了解,并能应用于临床实践。     

脊髓背角浅层Hes5+星形胶质细胞在机械痛敏中发挥“门控”作用

近年来,人们对星形胶质细胞的功能有了更多更深入的认识。2020年11月,日本九州大学Makoto Tsuda教授实验室发表了题为脊髓背角浅层星形胶质细胞在机械痛敏中发挥"门控"作用的研究论文,揭示星形胶质细胞在机械痛传递过程中发挥关键作用(Nat Neurosci,2020,23:1376~1387)。     

NDRG2与GFAP在不同脑区星形胶质细胞的表达与分布

目的:观察NDRG2(N-myc下游调节基因2)与GFAP(胶质纤维酸性蛋白)在不同脑区星形胶质细胞的表达与分布。方法:利用免疫荧光NDRG2与GFAP双标技术以及Western Blot技术观察皮层、海马及纹状体等不同脑区星形胶质细胞NDRG2和GFAP的表达与分布。结果:免疫荧光结果显示NDRG2阳性细胞广泛而均匀地分布于不同脑区,并与GFAP存在较好的共定位;NDRG2与GFAP标记的星形胶质细胞形态不尽相同。Western Blot结果显示NDRG2在皮层中表达比海马和纹状体多,而GFAP在海马中表达比皮层和纹状体多。结论:NDRG2广泛表达于不同脑区星形胶质细胞,并于GFAP存在较好的共定位。     

Effects of DISC1 on Alzheimer’s disease cell models assessed by iTRAQ proteomics analysis

Alzheimer’s disease (AD) is a form of neurodegenerative disease in the elderly with no cure at present. In a previous study, we found that the scaffold protein, disrupted in Schizophrenia 1 (DISC1) is down-regulated in the AD brains, and ectopic expression of DISC1 can delay the progression of AD by protecting synaptic plasticity and down-regulating BACE1. However, the underlying mechanisms remain not to be elucidated. In the present study, we compared the proteomes of normal and DISC1^high AD cells expressing the amyloid precursor protein (APP) using isobaric tag for relative and absolute quantitation (iTRAQ) and mass spectrometry (MS). The differentially expressed proteins (DEPs) were identified, and the protein–protein interaction (PPI) network was constructed to identify the interacting partners of DISC1. Based on the interaction scores, NDE1, GRM3, PTGER3 and KATNA1 were identified as functionally or physically related to DISC1, and may therefore regulate AD development. The DEPs were functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases with the DAVID software, and the Non-supervised Orthologous Groups (eggNOG) database was used to determine their evolutionary relationships. The DEPs were significantly enriched in microtubules and mitochondria-related pathways. Gene set enrichment analysis (GSEA) was performed to identify genes and pathways that are activated when DISC1 is overexpressed. Our findings provide novel insights into the regulatory mechanisms underlying DISC1 function in AD.     

Alterations and Mechanism of Gut Microbiota in Graves’ Disease and Hashimoto’s Thyroiditis

To explore the role of gut microbiota in Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). Seventy fecal samples were collected, including 27 patients with GD, 27 with HT, and 16 samples from healthy volunteers. Chemiluminescence was used to detect thyroid function and autoantibodies (FT3, FT4, TSH, TRAb, TGAb, and TPOAb); thyroid ultrasound and 16S sequencing were used to analyze the bacteria in fecal samples; KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous Groups) were used to analyze the functional prediction and pathogenesis. The overall structure of gut microbiota in the GD and HT groups was significantly different from the healthy control group. Proteobacteria and Actinobacteria contents were the highest in the HT group. Compared to the control group, the GD and HT groups had a higher abundance of Erysipelotrichia, Cyanobacteria, and Ruminococcus_2 and lower levels of Bacillaceae and Megamonas. Further analysis of KEGG found that the “ABC transporter” metabolic pathway was highly correlated with the occurrence of GD and HT. COG analysis showed that the GD and HT groups were enriched in carbohydrate transport and metabolism compared to the healthy control group but not in amino acid transport and metabolism. Our data suggested that Bacillus, Blautia, and Ornithinimicrobium could be used as potential markers to distinguish GD and HT from the healthy population and that “ABC transporter” metabolic pathway may be involved in the pathogenesis of GD and HT.     

Tau蛋白寡聚体与阿尔茨海默病

阿尔茨海默病（AD）是严重影响老年人健康的一种神经退行性疾病。AD主要两个病理特征是tau蛋白组成的神经原纤维缠结和β淀粉样蛋白组成的Aβ斑块。Tau蛋白是目前研究AD机制和防治药物的一个重要靶点。Tau蛋白的寡聚体形式被认为是最具神经毒性的,并且其能在神经元之间传播,诱导胞内的正常tau蛋白聚集。本综述结合近年来的文献报道,对tau寡聚体的制备手段、形成机理、神经毒性、传播机制以及治疗前景等方面做了系统总结和讨论,为人们深入认识tau寡聚体提供参考。     

In Silico Design of Dual-Binding Site Anti-Cholinesterase Phytochemical Heterodimers as Treatment Options for Alzheimer’s Disease

The number of patients with neurodegenerative diseases, particularly Alzheimer’s disease (AD), continues to grow yearly. Cholinesterase inhibitors (ChEIs) represent the first-line symptomatic drug treatment for mild-to-moderate AD; however, there is an unmet need to produce ChEIs with improved efficacy and reduced side effects. Herein, phytochemicals with reported anti-acetylcholinesterase (AChE) activity were ranked in silico for their anti-AChE potential. Ligands with a similar or higher binding affinity to AChE than galantamine were then selected for the design of novel dual-binding site heterodimeric drugs. In silico molecular docking of heterodimers with the target enzymes, AChE and butyrylcholinesterase (BuChE), were performed, and anti-cholinesterase binding affinities were compared with donepezil. Drug-likeliness properties and toxicity of the heterodimers were assessed using the SwissADME and ProTox-II webservers. Nine phytochemicals displayed similar or higher binding affinities to AChE than galantamine: sanguinarine > huperzine A > chelerythrine > yohimbine > berberine > berberastine > naringenin > akuammicine > carvone. Eleven heterodimeric ligands were designed with phytochemicals separated by four- or five-carbon alkyl-linkers. All heterodimers were theoretically potent AChE and BuChE dual-binding site inhibitors, with the highest affinity achieved with huperzine-4C-naringenin, which displayed 34% and 26% improved affinity to AChE and BuChE, respectively, then the potent ChEI drug, donepezil. Computational pharmacokinetic and pharmacodynamic screening suggested that phytochemical heterodimers would display useful gastrointestinal absorption and with relatively low predicted toxicity. Collectively, the present study suggests that phytochemicals could be garnered for the provision of novel ChEIs with enhanced drug efficacy and low toxicity.     

Development of naringenin-O-alkylamine derivatives as multifunctional agents for the treatment of Alzheimer’s disease

In this study, a series of naringenin-O-alkylamine derivatives were designed and obtained by introducing an alkylamine fragment into the naringenin skeleton. The in vitro biological activity results revealed that compounds 5f and 7k showed good antioxidant activity with ORAC values of 2.3eq and 1.2eq, respectively. Compounds 5f and 7k were reversible and excellent huAChE inhibitors with IC₅₀ values of 0.91 μM and 0.57 μM, respectively. Moreover, compounds 5f and 7k could inhibit self-induced Aβ_1–42 aggregation with 62.1% and 43.8% inhibition rate, respectively, and significantly inhibited huAChE-Aβ_1–40 aggregation with 51.7% and 43.4% inhibition rate, respectively. In addition, compounds 5f and 7k were selective metal chelators and remarkably inhibited Cu²⁺-induced Aβ_1–42 aggregation with 73.5% and 68.7% inhibition rates, respectively. Furthermore, compounds 5f and 7k could cross the blood-brain barrier in vitro and displayed good neuroprotective effects and anti-inflammatory properties. Further investigation showed that compound 5f did not show obvious hepatotoxicity and displayed a good hepatoprotective effect by its antioxidant activity. The in vivo study displayed that compound 5f significantly improved scopolamine-induced mice memory impairment. Therefore, compound 5f was a potential multifunctional candidate for the treatment of AD.     

Olfactory neuropathology in Alzheimer’s disease: a sign of ongoing neurodegeneration

Olfactory neuropathology is a cause of olfactory loss in Alzheimer’s disease (AD). Olfactory dysfunction is also associated with memory and cognitive dysfunction and is an incidental finding of AD dementia. Here we review neuropathological research on the olfactory system in AD, considering both structural and functional evidence. Experimental and clinical findings identify olfactory dysfunction as an early indicator of AD. In keeping with this, amyloid-β production and neuroinflammation are related to underlying causes of impaired olfaction. Notably, physiological features of the spatial map in the olfactory system suggest the evidence of ongoing neurodegeneration. Our aim in this review is to examine olfactory pathology findings essential to identifying mechanisms of olfactory dysfunction in the development of AD in hopes of supporting investigations leading towards revealing potential diagnostic methods and causes of early pathogenesis in the olfactory system.     

Design,synthesis, and evaluation of 3,7-substituted coumarin derivatives as multifunctional Alzheimer’s disease agents

Multitarget directed ligands (MTDLs) are emerging as promising treatment options for Alzheimer’s disease (AD). Coumarin derivatives serve as a good starting point for designing MTDLs due to their inherent inhibition of monoamine oxidase (MAO) and cholinesterase enzymes, which are complicit in AD’s complex pathophysiology. A preliminary series of 3,7-substituted coumarin derivatives were synthesised and evaluated for enzyme inhibitory activity, cytotoxicity as well as neuroprotective ability. The results indicated that the compounds are weak cholinesterase inhibitors with five compounds demonstrating relatively potent inhibition and selectivity towards MAO-B with IC₅₀ values between 0.014 and 0.498 hx00B5;µM. Significant neuroprotective effects towards MPP⁺-compromised SH-SY5Y neuroblastoma cells were also observed, with no inherent cytotoxicity at 10 µM for all compounds. The overall results demonstrated that substitution of the phenylethyloxy moiety at the 7-position imparted superior general activity to the derivatives, with the propargylamine substitution at the 3-position, in particular, displaying the best MAO-B selectivity and neuroprotection.     

Lysine 63-linked ubiquitination of tau oligomers contributes to the pathogenesis of Alzheimer’s disease

Ubiquitin-modified tau aggregates are abundantly found in human brains diagnosed with Alzheimer’s disease (AD) and other tauopathies. Soluble tau oligomers (TauO) are the most neurotoxic tau species that propagate pathology and elicit cognitive deficits, but whether ubiquitination contributes to tau formation and spreading is not fully understood. Here, we observed that K63-linked, but not K48-linked, ubiquitinated TauO accumulated at higher levels in AD brains compared with age-matched controls. Using mass spectrometry analyses, we identified 11 ubiquitinated sites on AD brain-derived TauO (AD TauO). We found that K63-linked TauO are associated with enhanced seeding activity and propagation in human tau-expressing primary neuronal and tau biosensor cells. Additionally, exposure of tau-inducible HEK cells to AD TauO with different ubiquitin linkages (wild type, K48, and K63) resulted in enhanced formation and secretion of K63-linked TauO, which was associated with impaired proteasome and lysosome functions. Multipathway analysis also revealed the involvement of K63-linked TauO in cell survival pathways, which are impaired in AD. Collectively, our study highlights the significance of selective TauO ubiquitination, which could influence tau aggregation, accumulation, and subsequent pathological propagation. The insights gained from this study hold great promise for targeted therapeutic intervention in AD and related tauopathies.     

Design,synthesis, and evaluation of chalcone-Vitamin E-donepezil hybrids as multi-target-directed ligands for the treatment of Alzheimer’s disease

A novel series of chalcone-Vitamin E-donepezil hybrids was designed and developed based on multitarget-directed ligands (MTDLs) strategy for treating Alzheimer’s disease (AD). The biological results revealed that compound 17f showed good AChE inhibitory potency (ratAChE IC₅₀ = 0.41 µM; eeAChE IC₅₀ = 1.88 µM). Both the kinetic analysis and docking study revealed that 17f was a mixed type AChE inhibitor. 17f was also a good antioxidant (ORAC = 3.3 eq), selective metal chelator and huMAO-B inhibitor (IC₅₀ = 8.8 µM). Moreover, it showed remarkable inhibition of self- and Cu²⁺-induced Aβ_1–42 aggregation with a 78.0 and 93.5% percentage rate at 25 µM, respectively, and disassembled self-induced and Cu²⁺-induced aggregation of the accumulated Aβ_1–42 fibrils with 72.3 and 84.5% disaggregation rate, respectively. More importantly, 17f exhibited a good neuroprotective effect on H₂O₂-induced PC12 cell injury and presented good blood-brain barrier permeability in vitro. Thus, 17f was a promising multi-target-directed ligand for treating AD.