去水卫矛醇对斑马鱼神经发育毒性评价

目的 探讨去水卫矛醇(dianhydrogalactitol,DAG)诱导斑马鱼胚胎及幼鱼神经发育毒性作用及其机制。方法 在一般毒性评价的基础上,对斑马鱼胚胎进行相应的分组暴露给药,采用幼鱼的自主运动反应、光照刺激反应等试验观察DAG对斑马鱼神经及行为的影响;通过脑部组织病理学检查、吖啶橙染色,观察DAG对斑马鱼脑部的组织影响;利用实时荧光定量PCR方法测定斑马鱼幼鱼体内多巴胺神经元相关基因(DAT、TH、GCH1)及神经抑/促凋亡相关基因(Bax、Bcl-2)的相对表达量。结果 DAG在20,40,75 mg·mL^-1下能明显抑制斑马鱼的自主运动,且自主运动抑制率呈现明显的浓度相关性;DAG在20,40,75 mg·mL^-1下对斑马鱼反应速度有较明显的抑制作用,反应能力下降率呈现浓度相关性;DAG各浓度组斑马鱼脑组织形态变小,但组织结构均未见显著异常;采用吖啶橙染色检测斑马鱼整体胚胎细胞凋亡情况,发现给药组头部绿色荧光比对照组明显,说明细胞凋亡增多,且细胞凋亡呈剂量依赖性增加,与表观一致;DAG在75,150,300,425,600 mg·L^-1下可导致斑马鱼幼鱼多巴胺能神经元相关基因DAT、TH、GCH1的mRNA相对表达量下调,Bax/Bcl-2的RNA相对表达量随着给药浓度增加而上升。结论 高浓度DAG对斑马鱼胚胎和幼鱼具有神经发育毒性作用,可能与多巴胺能神经元的抑制作用有关。     

基于MRI自动化定量海马体积诊断颞叶癫痫患者海马硬化

目的 评估基于MRI自动化定量海马体积诊断颞叶癫痫患者海马硬化（HS）的效能。方法 回顾性分析经术后病理检查证实的64例存在HS的颞叶癫痫患者,采用AccuBrain软件对头部3D T1WI进行自动化定量分析,检测海马体积指数（HVI）;以病理结果为诊断金标准,统计2名医师目测诊断HS结果。采用受试者工作特征（ROC）曲线获得HVI诊断HS的截断值及曲线下面积（AUC）,计算HVI诊断HS的敏感度、特异度和准确率,评价2种诊断方式所获结果的一致性。结果 64例中,38例左侧HS、26例右侧HS。医师目测诊断HS的敏感度、特异度、准确率分别为90.63%（58/64）、100%（64/64）及95.31%（122/128）。ROC曲线结果显示,HVI诊断HS的最佳截断值为0.185,AUC为0.936。HVI诊断HS的敏感度、特异度、准确率分别为87.50%（56/64）、93.75%（60/64）及90.63%（116/128）。医师目测与HVI诊断HS的准确率一致性较强（Kappa=0.684,P<0.05）。结论 MRI自动化定量海马体积可用于诊断颞叶癫痫患者HS,其诊断效能较高。     

转铁蛋白受体1对淀粉样蛋白前体/早老素1转基因小鼠神经元的保护作用

目的 探讨转铁蛋白受体1(TfR1)在淀粉样蛋白前体（APP）/早老素1（PS1）转基因小鼠脑内异常表达情况及其对阿尔茨海默病（AD）神经元的保护作用。 方法 首先,利用免疫荧光及Western blotting技术检测出生后1月（P1M）至P12M各发育时间点,APP/PS1转基因小鼠与野生型小鼠大脑TfR1的表达情况;其次,取APP/PS1转基因与野生型新生小鼠原代海马神经元培养,培养12 d后利用TfR1 shRNA质粒干扰TfR1基因的表达,利用Western blotting技术检测干扰后细胞TfR1的表达变化;ELISA技术检测TfR1干扰前后细胞β-淀粉样蛋白(Aβ)_1-42的分泌量;利用微管相关蛋白2(MAP2)标记神经元突起,观察TfR1干扰前、后神经元突起的生长变化;最后,利用FM1-43染色观察由TfR1介导的轴质运输中囊泡的运输情况。 结果 在APP/PS1转基因小鼠生长发育过程中,随着年龄的增长TfR1的表达呈现先增加后减少的趋势,在P6M之后明显降低,且与对照组相比差异有显著性;TfR1 shRNA 干扰后可以使原代神经元细胞内TfR1基因沉默,使其突起明显变细、变长并影响囊泡的运输。与对照组相比,TfR1基因在APP/PS1转基因小鼠原代神经元中表达量减少,荧光减弱。 结论 APP、PS1基因突变可导致TfR1的表达下降;APP/PS1转基因小鼠原代神经元经TfR1 shRNA干扰Aβ_1-42分泌量增多,影响神经元突起的生长,使轴质运输速率减慢,囊泡的活动减缓,加重AD病情。故TfR1的表达可以对神经元起到保护作用。     

Clinicopathological features of low-grade malignant endolymphatic sac tumors

Background

Low-grade malignant endolymphatic sac tumor (ELST) is a rare neoplasm, occurring in the inner ear and invading the temporal bone. This study aims to investigate the clinicopathological features of low-grade malignant ELSTs.

A nonstop variant in REEP1 causes peripheral neuropathy by unmasking a 3′UTR‐encoded,aggregation‐inducing motif

Single‐nucleotide variants that abolish the stop codon (“nonstop” alterations) are a unique type of substitution in genomic DNA. Whether they confer instability of the mutant mRNA or result in expression of a C‐terminally extended protein depends on the absence or presence of a downstream in‐frame stop codon, respectively. Of the predicted protein extensions, only few have been functionally characterized. In a family with autosomal dominant Charcot‐Marie‐Tooth disease type 2, that is, an axonopathy affecting sensory neurons as well as lower motor neurons, we identified a heterozygous nonstop variant in REEP1. Mutations in this gene have classically been associated with the upper motor neuron disorder hereditary spastic paraplegia (HSP). We show that the C‐terminal extension resulting from the nonstop variant triggers self‐aggregation of REEP1 and of several reporters. Our findings support the recently proposed concept of 3′UTR‐encoded “cryptic amyloidogenic elements.” Together with a previous report on an aggregation‐prone REEP1 deletion variant in distal hereditary motor neuropathy, they also suggest that toxic gain of REEP1 function, rather than loss‐of‐function as relevant for HSP, specifically affects lower motor neurons. A search for similar correlations between genotype, phenotype, and effect of mutant protein may help to explain the wide clinical spectra also in other genetically determined disorders.     

The Cellular Composition and Glia–Neuron Ratio in the Spinal Cord of a Human and a Nonhuman Primate: Comparison With Other Species and Brain Regions

The cellular composition of brains shows largely conserved, gradual evolutionary trends between species. In the primate spinal cord, however, the glia–neuron ratio was reported to be greatly increased over that in the rodent spinal cord. Here, we re‐examined the cellular composition of the spinal cord of one human and one nonhuman primate species by employing two different counting methods, the isotropic fractionator and stereology. We also determined whether segmental differences in cellular composition, possibly reflecting increased fine motor control of the upper extremities, may explain a sharply increased glia–neuron ratio in primates. In the cynomolgus monkey spinal cord, the isotropic fractionator and stereology yielded 206–275 million cells, of which 13.3–25.1% were neurons (28–69 million). Stereological estimates yielded 21.1% endothelial cells and 65.5% glial cells (glia–neuron ratio of 4.9–5.6). In human spinal cords, the isotropic fractionator and stereology generated estimates of 1.5–1.7 billion cells and 197–222 million neurons (13.4% neurons, 12.2% endothelial cells, 74.8% glial cells), and a glia–neuron ratio of 5.6–7.1, with estimates of neuron numbers in the human spinal cord based on morphological criteria. The non‐neuronal to neuron ratios in human and cynomolgus monkey spinal cords were 6.5 and 3.2, respectively, suggesting that previous reports overestimated this ratio. We did not find significant segmental differences in the cellular composition between cervical, thoracic and lumbar levels. When compared with brain regions, the spinal cord showed gradual increases of the glia–neuron ratio with increasing brain mass, similar to the cerebral cortex and the brainstem. Anat Rec, 301:697–710, 2018. © 2017 Wiley Periodicals, Inc.     

Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain

We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ⁹-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1–688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca²⁺, or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).A number of vertebrate and invertebrate transient receptor potential (TRP) ion channels have been implicated in temperature sensation (1–3), but only the rat menthol receptor TRP subtype M8 (TRPM8) and the rat capsaicin receptor TRP subtype V1 (TRPV1) have been shown to possess intrinsic thermosensitivity (4, 5). In 2003, Story et al. (6) proposed that the mouse TRPA1 is a noxious cold sensor. Story et al. (6) showed that TRPA1 was present in nociceptive primary sensory neurons and that CHO cells heterologously expressing the mouse TRPA1 displayed cold sensitivity. Most subsequent studies of cold responses in heterologous TRPA1 expression systems, isolated primary sensory neurons, and whole animals have provided evidence in support of mouse and rat TRPA1 being involved in noxious cold transduction (7). Interestingly, a familial episodic pain syndrome triggered by cold is caused by a gain-of-function mutation in the TRPA1 gene, indicating that TRPA1 may have a key role in human noxious cold sensation (8). Thus, human TRPA1 (hTRPA1) may be a relevant drug target for treatment of this condition and other pathological conditions, such as inflammation, nerve injury, and chemotherapy-induced neuropathy, that are characterized by TRPA1-dependent cold allodynia or hypersensitivity (7). However, in vitro studies of the expressed hTRPA1 have generated conflicting findings (8–15), and no study has provided evidence that mammalian TRPA1 channels are, indeed. intrinsically cold-sensitive proteins, which would require examination of the purified protein in a defined membrane environment.Heterologous expression studies of chimeric or mutated TRPA1 channels have proposed that the N-terminal region plays an important role in thermal and chemical sensitivity of both mammalian and insect TRPA1 (14, 16–19). Initial studies indicated that mammalian TRPA1 is activated by cysteine-reactive electrophilic compounds and oxidants, such as diallyl disulfide in garlic (9, 10, 20, 21). Targeted gene mutations have identified cysteines present in the N terminus of TRPA1 as important for electrophilic and oxidative TRPA1 channel activation (22, 23). Because several of these cysteines are involved in protein disulfide formation (24–26), it is not unlikely that such mutations will have pronounced effects on the overall TRPA1 channel structure and function (7). Electrophilic compounds can also covalently bind to cysteines in the transmembrane segments and the C-terminal domain of mammalian TRPA1 (23, 26), and the electrophiles p-benzoquinone, isovelleral, and polygodial robustly activate the heterologously expressed triple mutant hTRPA1-3C (27, 28) that was initially used to identify certain N-terminal cysteine residues in hTRPA1 as key targets for electrophiles (22). However, it is yet to be shown that covalent binding sites outside the N-terminal ankyrin repeat domain (ARD) contribute to the regulation of channel gating.Another key feature of mammalian TRPA1 is the responsiveness to nonelectrophilic compounds with very different chemical structures, such as menthol and the cannabinoids Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and Δ⁹-tetrahydrocannabiorcol (C16) (7). However, if nonelectrophilic compounds activate TRPA1 directly and at the same site on TRPA1 is not known. The site of action of nonelectrophilic TRPA1 activators is important to clarify, because some TRPA1 activators are antinoiceptive (29, 30), and nontissue-damaging TRPA1 activators may be used clinically for pharmacological regulation of TRPA1 channel activity (29).Here, we have purified and inserted hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) into lipid bilayers for functional studies using patch-clamp electrophysiology to explore the inherent thermo- and chemosensitivity of hTRPA1. Because of the great potential of TRPA1 as a drug target for treatment of human pain and the existence of mammalian TRPA1 species differences (7), the human variant of TRPA1 was chosen for these studies. We addressed the role of the N-terminal ARD in cold and chemical sensitivity by deleting the N-terminal ARD, something that cannot be studied in cells heterologously expressing TRPA1, because the N-terminal ARD is needed for insertion of the ion channel into the plasma membrane (31). Our findings consolidate hTRPA1 as a multimodal nocisensor responding to cold and chemicals. It is suggested that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophilic compounds are important in hTRPA1 channel gating. Targeting chemical interaction sites outside the N-terminal ARD may provide possibilities to fine tune TRPA1-based drug therapies [e.g., for treatment of pain associated with cold hypersensitivity (7) and cardiovascular disease (32)].     

GAP,an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

Genetically encoded calcium indicators allow monitoring subcellular Ca²⁺ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca²⁺ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg²⁺, tunable Ca²⁺ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca²⁺ indicators for organelles and becomes a valuable tool for in vivo Ca²⁺ imaging applications.Ca²⁺ is involved in the regulation of many intracellular processes that take place both in the cytosol and inside organelles (1–3). Therefore, accurate measurement of the calcium concentration ([Ca²⁺]) inside organelles is essential to discriminate discrete Ca²⁺ signals between the different compartments. Although synthetic Ca²⁺ indicators can be loaded into organelles, the signal has poor selectivity, as the dye is also present in the cytosol and must be carefully removed before measurements (4). The main advantage of Genetically Encoded Ca²⁺ Indicators (GECIs) is their ability to be targeted to specific intracellular locations. Both bioluminescent and fluorescent proteins have been successfully used to measure subcellular [Ca²⁺]. The photoprotein aequorin (5), purified from the jellyfish Aequorea victoria, was the first protein-based Ca²⁺ indicator, injected into cells in the early 1970s (6). After cloning of its cDNA (7), recombinant aequorin became the most frequently used probe to measure Ca²⁺ in organelles, including mitochondria (8), the endoplasmic reticulum (ER) (9), the nucleus (10), the Golgi apparatus (11), or secretory vesicles (12).Fluorescent GECIs achieve a better spatial resolution than bioluminescent sensors. They are generally composed of one or two fluorescent proteins, most of them variants of GFP, fused to a Ca²⁺-binding protein (13). Recently, a single EF-hand motif has been inserted in the GFP moiety to generate a Ca²⁺ fluorescent probe (14). Since the first cameleon based on FRET (15), the number of GECIs has exponentially increased, attempting optimization of critical features such as adequate expression, signal strength, or dynamic range. However, the in vivo use in mammals, one of the main applications of GECIs, has grown more slowly and has disclosed severe limitations (16, 17). Transgenic sensors usually showed a low expression, often resulting in its inactivation or reduced dynamic range. With the exception of troponin derivatives, most of the available GECIs, namely cameleons, camgaroos, pericams, or GCaMPs (circularly permutated EGFP-based Ca²⁺ sensors), are based on calmodulin, a highly regulated ubiquitous protein that binds a large number of targets (13). Although the interference with endogenous calmodulin has been reduced in the improved cameleons (18), the interaction with other cellular proteins cannot be ruled out. Thus, the loss of Ca²⁺ sensitivity observed in vivo may reflect the interaction of the probe with endogenous partners, which may disturb cellular functions.The jellyfish aequorin exhibits a number of advantages over mammalian EF-hand proteins. It is not toxic and appears not to interfere with other intracellular Ca²⁺-binding molecules, even when microinjected at high concentrations in mammalian cells. Moreover, the use of aequorin as a bioluminescence sensor has been extensively reported, ranging from subcellular Ca²⁺ measurements in many different cell types up to whole organisms, including transgenic animals (19–21).Here we describe a family of fluorescent Ca²⁺ sensors based on the fusion of two jellyfish proteins, GFP and apoaequorin. This Ca²⁺ probe shows a larger dynamic range compared with other GECIs and a robust photonic and thermal stability. It can be targeted to distinct compartments such as the nucleus, cytosol, or mitochondria, where it selectively and accurately monitors dynamic Ca²⁺ changes. In addition, we have generated a variant with a lower Ca²⁺ affinity suited for imaging Ca²⁺ changes in organelles with high resting [Ca²⁺] such as the ER or the Golgi apparatus. Finally, we demonstrate its in vivo applicability by generating transgenic mice where the Ca²⁺ biosensor maintained its in vitro features.     

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目的探讨实验性牙周炎治疗前后心脑血管疾病相关因子变化及其对大脑海马区的影响。方法 2011年6月至2012年6月在河北医科大学口腔医院选取纯种6月龄雄性SD大鼠30只,随机分为正常组、牙周炎组和牙周炎治疗组,每组10只,通过牙周结扎建立实验性牙周炎模型。牙周炎模型建立成功后,去除牙周结扎丝建立实验性牙周炎治疗模型。牙周炎组、牙周炎治疗组分别于成功建模后12周处死大鼠,取上颌骨标本观察牙周组织变化,检测血清中C-反应蛋白（CRP）、细胞间黏附分子-1（ICAM-1）和E-选择素变化,取大脑中海马区观察神经元细胞的变化。结果实验性牙周炎附着丧失和单位面积破骨细胞数目明显多于正常组和牙周炎治疗组,血清中CRP、ICAM-1和E-选择素在牙周炎时升高,治疗后降低,海马区神经元细胞在牙周炎与治疗后也明显不同。结论实验性牙周炎和局部治疗牙周炎不仅可以影响牙周组织,还可影响血清中的CRP、ICAM-1和E-选择素,进而对心脑血管系统产生影响。     

Multiple system atrophy variant with severe hippocampal pathology

The striatonigral and olivopontocerebellar systems are known to be vulnerable in multiple system atrophy (MSA), showing neuronal loss, astrogliosis, and alpha-synuclein-immunoreactive inclusions. MSA patients who displayed abundant neuronal cytoplasmic inclusions (NCIs) in the regions other than the striatonigral or olivopontocerebellar system have occasionally been diagnosed with variants of MSA. In this study, we report clinical and pathologic findings of MSA patients characterized by prominent pathologic involvement of the hippocampus. We assessed 146 consecutively autopsied MSA patients. Semi-quantitative analysis of anti-alpha-synuclein immunohistochemistry revealed that 12 of 146 patients (8.2%) had severe NCIs in two or more of the following areas: the hippocampal granule cells, cornu ammonis areas, parahippocampal gyrus, and amygdala. In contrast, the remaining 134 patients did not show severe NCIs in any of these regions. Patients with severe hippocampal involvement showed a higher representation of women (nine women/three men; Fisher's exact test, p = 0.0324), longer disease duration (13.1 ± 5.9 years; Mann–Whitney U-test, p = 0.000157), higher prevalence of cognitive impairment (four patients; Fisher's exact test, p = 0.0222), and lower brain weight (1070.3 ± 168.6 g; Mann–Whitney U-test, p = 0.00911) than other patients. The hippocampal granule cells and cornu ammonis area 1/subiculum almost always showed severe NCIs. The NCIs appeared to be ring-shaped or neurofibrillary tangle-like, fibrous configurations. Three of 12 patients also had dense, round-shaped NCIs that were morphologically similar to pick bodies. The patients with Pick body-like inclusions showed more severe atrophy of the medial temporal lobes and broader spreading of NCIs than those without. Immunohistochemistry for hyperphosphorylated tau and phosphorylated TDP-43 revealed minimal aggregations in the hippocampus of the hippocampal MSA patients. Our observations suggest a pathological variant of MSA that is characterized by severe involvement of hippocampal neurons. This phenotype may reinforce the importance of neuronal alpha-synucleinopathy in the pathogenesis of MSA.