Transient intraneuronal Aβ rather than extracellular plaque pathology correlates with neuron loss in the frontal cortex of APP/PS1KI mice

The accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles consisting of hyperphosphorylated tau protein are pathological features of Alzheimer’s disease (AD) commonly modeled in mice using known human familial mutations; however, the loss of neurons also found to occur in AD is rarely observed in such models. The mechanism of neuron degeneration remains unclear but is of great interest as it is very likely an important factor for the onset of adverse memory deficits occurring in individuals with AD. The role of Aβ in the neuronal degeneration is a matter of controversial debates. In the present study we investigated the impact of extracellular plaque Aβ versus intraneuronal Aβ on neuronal cell death. The thalamus and the frontal cortex of the APP/PS1KI mouse model were chosen for stereological quantification representing regions with plaques only (thalamus) or plaques as well as intraneuronal Aβ (frontal cortex). A loss of neurons was found in the frontal cortex at the age of 6 months coinciding with the decrease of intraneuronal immunoreactivity, suggesting that the neurons with early intraneuronal Aβ accumulation were lost. Strikingly, no neuron loss was observed in the thalamus despite the development of abundant plaque pathology with levels comparable to the frontal cortex. This study suggests that plaques have no effect on neuron death whereas accumulation of intraneuronal Aβ may be an early transient pathological event leading to neuron loss in AD. O. Wirths and T. A. Bayer have equally contributed to this work.     

Japanese Encephalitis Virus infection induces IL-18 and IL-1beta in microglia and astrocytes: correlation with in vitro cytokine responsiveness of glial cells and subsequent neuronal death

IL-1β and IL-18 are members of the IL-1 family of ligands, and their receptors are members of the IL-1 receptor family. Although several biological properties overlap for these cytokines, differences exist. In order to assess functional importance of these two cytokines in viral encephalitis, we have exploited an experimental model of Japanese Encephalitis (JE) and subsequent in vitro cell culture system. We report for the first time that in Japanese Encephalitis, microglia and astrocytes both produce IL-18 and IL-1β. In vitro, these two cytokines differentially activate microglia and astrocyte, and also alter the by stander neuronal survival following treatment with these two cytokines.     

Neuronal COX-2 expression and phosphorylation of pRb precede p38 MAPK activation and neurofibrillary changes in AD temporal cortex

In Alzheimer's disease (AD) brain, increased levels of cyclooxygenase-2 (COX-2), cell cycle markers, and p38 MAP kinase (MAPK) can be detected in neuronal cells. Besides mediating COX-2 expression, p38 MAPK is suggested to mediate cell cycle progression through phosphorylation of the retinoblastoma protein (pRb). In this study, we show that neuronal immunoreactivity for phosphorylated p38 MAPK does not correlate with COX-2 or phosphorylated pRb (ppRb) in control and AD temporal cortex. Immunoreactivity for activated p38 MAPK co-localizes with AT8 immunoreactivity and increases with the occurrence of neurofibrillary tangles and plaques. On the other hand, COX-2 immunoreactivity co-localizes and correlates with ppRb immunoreactivity in pyramidal neurons. COX-2 and ppRb do not co-localize with AT8 and decrease with increasing pathology. These results suggest that p38 MAPK does not mediate COX-2 expression and pRb inactivation, which are involved in cellular changes in pyramidal neurons early in AD pathogenesis.     

丹参诱导大鼠骨髓间充质干细胞分化为神经元样细胞中的钙离子检测

目的 检测大鼠骨髓间充质干细胞经丹参注射液诱导分化的神经元样细胞内钙离子浓度,以期为骨髓间充质干细胞应用于神经系统疾病的治疗提供理论依据.方法 从成年大鼠骨髓中获取骨髓间充质干细胞,体外扩增培养,经碱性成纤维生长因子预诱导后施加10mL/L丹参注射液于骨髓间充质干细胞培养液中.运用免疫荧光检测神经元特异性核蛋白(NeuN)在诱导后细胞与经新生大鼠海马获取的体外培养海马神经元中的表达.激光共聚焦技术检测诱导后的细胞内钙离子浓度.并与原代培养海马神经元内的钙离子浓度进行比较.结果 大鼠骨髓间充质干细胞经碱性成纤维生长因子和丹参注射液处理后,可表达NeuN,并具有神经元样的表型.诱导分化的神经元样细胞内钙离子浓度为984.75±79.51,原代培养海马神经元内钙离子浓度为769.42±60.93,两者比较差异无统计学意义(P>0.05).结论 丹参注射液诱导骨髓间充质干细胞分化的神经元样细胞具有神经元的某些特征.     

Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases

Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367–382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-l-Leucyl-l-Leucyl-l-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.     

Spinal cord tau pathology in cervical spondylotic myelopathy

We conducted an immunohistochemical and ultrastructural examination of the spinal cords from 11 cases of cervical spondylotic myelopathy (CSM), together with those from 11 age- and sex-matched control subjects. Immunostaining with AT8 antibody revealed various numbers of tau-positive neuropil thread-like structures (NTSs), often demonstrating a conspicuous astrocytic foot-like perivascular or subpial arrangement, and glial cells with short and thick processes, so-called thorn-shaped astrocytes (TSAs), in the affected cervical cords in 8 of the 11 CSM cases (73%). A number of tau-positive neuronal cytoplasmic pretangles/tangles were also found in the gray matter in all the CSM cases (100%). No such astrocytic or neuronal tau lesions were found in the control subjects. The tau deposited in the NTSs and TSAs was predominantly 4-repeat tau, whereas the neuronal cytoplasmic pretangles/tangles contained both 3-repeat and 4-repeat tau. Ultrastructurally, paired helical filaments about 20 nm wide, together with glial filaments, were detected occasionally in the astrocytic processes. In conclusion, the present findings indicate that astrocytic and neuronal tau lesions appear in the affected cervical cord during the disease process of CSM.     

Effect of Retinoic Acid on Ferritin H Expression During Brain Development and Neuronal Differentiation

Abstract

We have previously shown that brain ferritin H expression, which has been associated with iron utilization, is developmentally regulated. Because retinoic acid (RA) regulates gene expression and is involved in cellular differentiation, we tested the hypothesis that RA regulates ferritin H during brain development and neuronal differentiation. RA, administered to rats on postnatal day 1, produced a 4-fold increase in brain ferritin H mRNA (p<0.01) after 24 h. To examine whether RA-stimulated neuronal differentiation contributed to this up-regulation, ferritin and ferritin H mRNA were measured in human neuronal precursor cells (NTera-2, NT2) before and after 4-weeks of RA-stimulated differentiation into post-mitotic neurons. Differentiation resulted in a 2-fold increase in both ferritin and ferritin H mRNA (p<0.05). Immunocytochemistry and Northern analysis showed significant elevations in ferritin expression that began as early as 24 h after RA treatment. While there was also a significant increase in the labile iron pool after RA treatment, this did not occur until 72 h. These data show that RA regulates ferritin H expression during rat brain development and neuronal differentiation and suggests a new role for RA in brain iron metabolism.     

aFGF基因在大鼠脑皮质损伤神经元的表达

目的：为aFGF对成年大鼠脑损伤后脑内神经元的作用提供理论依据。方法：应用原位杂交和免疫组织化学方法观察了大鼠脑损伤后aFGF基因mRNA和蛋白在皮质神经元的表达变化。结果：脑损伤后15min及30min伤侧大脑皮质神经元aFGF mRNA和蛋白表达增加，aFGF mRNA表达于伤手6h达高峰，持续至伤后24h，以后表达逐步降低，于伤后72h恢复至正常水平。神经元aFGF蛋白表达于伤后1h达高峰，持续至伤后3h，伤后24h表达恢复正常。结论：脑损伤后皮质神经元aFGF基因表达增加与神经元抗损伤修复机制有关。     

盐酸多奈哌齐对D-半乳糖致原代培养神经元损伤的保护作用及其机制

目的:探讨盐酸多奈哌齐(安理申)防治D-半乳糖致原代培养神经元损伤的作用机制。方法:在原代培养第6d,以50mM D-半乳糖(D-gal)作用神经元72h造成自由基损伤模型,用10％盐酸多奈哌齐药物血清防治损伤。倒置显微镜下观察神经元的生长发育形态,MTT法在酶标仪上检测神经元代谢率,PI染色在流式细胞仪上检测神经元凋亡构成比,HE染色观察神经元形态、形态计量神经突起密度,RT-PCR检测糖醛还原酶信使RNA(AR-mRNA)的含量。t检验,比较正常对照神经元、受D-gal攻击神经元和受D-gal攻击同时得到盐酸多奈哌齐防治神经元之间的差别。结果与讨论:受D-gal攻击而受损伤的神经元,生长发育显著迟缓,盐酸多奈哌齐防治使其明显恢复;代谢率从0.762±0.030(n=33)降低到0.543±0.064(n=11,P<0.01),盐酸多奈哌齐防治使其回升到0.652±0.028(n=11,P<0.01);凋亡构成比从0.060±0.029(n=19)增高到0.356±0.215(n=19,P<0.01),盐酸多奈哌齐防治使其回降到0.154±0.130(n=19,P<0.01);出现变性坏死的形态变化,盐酸多奈哌齐防治使其好转,但神经突起浆质中出现均匀、细小的颗粒;神经突起密度从0.557±0 0422(n=10)降低到0.468±0.0330(n=10,P<0.01),盐酸多奈哌齐防治使其回升到0.481±0.0387(η=10,P>0.05);无AR-mRNA的表达。表明盐酸多奈哌     

Laminar distribution of neuron degeneration in posterior cingulate cortex in Alzheimer's disease

Summary The laminar distribution of neuron losses in posterior cingulate cortex were evaluated in 25 clinically and neuropathologically diagnosed cases of dementia of the Alzheimer type (DAT). The layer of maximal neuron loss in area 23a for each DAT case was determined by comparison with mean neuron densities for each layer of 17 neurologically intact control cases. The DAT cases were separated into five classes: class 1, 12% of all DAT cases, no or less than 40% neuron loss in any layer; class 2, 24%, maximal neuron losses in layers II or III; class 3, 28%, losses mainly in layer IV; class 4, 12%, losses mainly in layers V or VI; class 5, 24%, severe losses in all layers. An analysis of large and small neurons showed that in class 2 there was an equal loss of both in layer IIIa–b, in class 3 mostly small neurons were lost in layer IV, in class 4 mostly large neurons were lost in layers III, IV and V, while in class 5 there was no selectivity. The age of disease onset and length of the disease were the same for all classes, although classes 4 and 5 tended to have an earlier onset. No measures of thioflavin S-stained neuritic plaque (NP) or neurofibrillary tangle (NFT) density discriminated among these classes. In 64% of all DAT cases there was a progressive shift in NFT from ventral area 30 where most were in layer II to areas 23a–b where there was a balance between those in superficial and deep layers to dorsal area 23c where most were in layers V and VI. There were four cases with massive numbers of NFT in area 23a (1802±477 versus 261±44 for all other cases). Since one of these cases was in each of classes 1, 2, 3 and 5, it is unlikely that this form of amyloid deposition is related to laminar specificities in neuron degeneration. Finally, neuron and NFT densities in the hippocampal formation were the same for each class. In conclusion, differential laminar changes in neuron density provide a basis for neuropathological subtyping of DAT which is more sensitive than measures of thioflavin S-stained NP and NFT densities either in neocortex or the hippocampus.Supported by NIH-NINDS grants, NS18745, NS14944 and PONS19632