Expression of trkB in human neuroblastoma in relation to MYCN expression and retinoic acid treatment

Expression of full-length trkB can be found in some highly malignant neuroblastoma tumors with an amplified MYCN gene. This contrasts sympathetic neuroblasts, from which neuroblastomas are thought to arise, which neither express trkB nor are dependent on the p145(trkB) ligands, brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5, for their normal development. In this study we show that trkB was expressed in two out of five neuroblastoma tumors with amplified MYCN, while no trkB expression was observed when the MYCN gene was overexpressed in a non-MYCN-amplified neuroblastoma cell line. This shows that MYCN overexpression per se is not sufficient to induce trkB expression. trkB expression and BDNF responsiveness in neuroblastoma cells can be induced by all-trans-retinoic acid (RA). When SH-SY5Y cells were stimulated with a combination of RA and BDNF, norepinephrine and tyrosine hydroxylase levels were unaltered, showing that the cells did not change toward a more catecholaminergic sympathetic phenotype. However, expression of growth-associated protein 43, indicative of a neuronal phenotype, was elevated. Vesicular acetylcholine transporter, choline acetyl transferase, and neuropeptide tyrosine mRNA levels also increased in RA-BDNF-treated cells, which could suggest that these cells develop into a sympathetic cholinergic phenotype. In addition, treatment with RA-induced expression of the platelet-derived growth factor receptor-alpha. As previously shown for BDNF, platelet-derived growth factor stimulated growth of the RA-treated cells, findings that could have clinical relevance. If these receptors mediate a mitogenic signal in vivo also, this might limit the effect of RA treatment on neuroblastoma patients.     

脑源性神经营养因子在体外诱导SH-SY5Y细胞分化过程中的作用

目的 探讨不同浓度的脑源性神经营养因子(BDNF)在体外诱导SH-SY5Y细胞分化时,对G2期及其前后细胞周期相关蛋白mRNA表达的影响.方法 采用全反式维甲酸(RA)和低(1μg/L)、中(10μg/L)、高(100μg/L)3种不同浓度BDNF在无血清培养液中相继诱导SH-SY5Y细胞分化,形成均一的具有神经元形态的细胞.以一步法实时定量RT-PCR检测其G2期细胞周期相关蛋白mRNA的表达,荧光激活细胞分类术(FACS)检测各组细胞时相.结果 在各浓度BDNF组中周期蛋白B1(cyclin B1)的mRNA含量与对照组及RA组比较均明显降低(P<0.05);仅高浓度组cyclin B2和周期蛋白依赖性激酶1(Cdk1)的mRNA含量显著降低(P<0.05),;低浓度和中浓度BDNF组Cdk5 mRNA含量均高于RA组(P<0.05).BDNF各浓度组处于G1期的细胞百分率均显著高于RA和对照组(P<0.01),而处于S期的细胞百分率均显著低于对照组(P<0.01),且低浓度及高浓度组同时低于对照组(P<0.01).结论 提示BDNF在有效促进SH-SY5Y细胞进一步分化的同时,没有引起G2期及其前后细胞周期相关蛋白mRNA表达的异常升高,BDNF无诱导细胞凋亡的危险,可能有减缓AD进程的作用,并且呈剂量依赖性.     

BDNF对诱导分化的SH-SY5Y细胞G_1期相关蛋白表达的影响

研究不同浓度的脑源性神经营养因子(BDNF)在体外诱导SH-SY5Y细胞分化时对G1期细胞周期相关蛋白表达的影响。采用全反式维甲酸(RA)和低(1ng/ml)、中(10ng/ml)、高(100ng/ml)三种不同浓度BDNF在无血清培养液中相继诱导SH-SY5Y细胞分化,形成均一的具有神经元形态的细胞。以实时定量RT-PCR法检测G1期细胞周期相关蛋白(包括cdk4,cdk6,cyclin D1,Cyclin E,p16和p27)的mRNA水平的表达变化。结果显示:不同浓度的BDNF处理后,Cdk4mRNA的表达水平均低于未分化细胞组(P<0.05);Cdk6和Cyclin D1的mRNA表达水平与RA组比较显著降低(P<0.05),Cyclin E和p16的mRNA表达水平仅在高浓度BDNF处理后明显降低,与未分化组比较P<0.05,p27的mRNA表达水平无显著变化。以上结果提示BD-NF在诱导SH-SY5Y细胞分化的同时无意促使其再进入细胞周期。同时,用RA预处理5d后改换BDNF培养SH-SY5Y细胞,可使细胞展现出成熟神经细胞的形态,是体外研究某些脑疾病的理想细胞模型。     

Amyloid precursor protein is a biomarker for transformed human pluripotent stem cells

Increasing evidence suggests an important function of the β-amyloid precursor protein (APP) in malignant disease in humans; however, the biological basis for this evidence is not well understood at present. To understand the role of APP in transformed pluripotent stem cells, we studied its expression levels in human testicular germ cell tumors using patient tissues, model cell lines, and an established xenograft mouse model. In the present study, we demonstrate the cooperative expression of APP with prominent pluripotency-related genes such as Sox2, NANOG, and POU5F1 (Oct3/4). The closest homologue family member, APLP2, showed no correlation to these stem cell factors. In addition, treatment with histone deacetylase (HDAC) inhibitors suppressed the levels of APP and stem cell markers. Loss of pluripotency, either spontaneously or as a consequence of treatment with an HDAC inhibitor, was accompanied by decreased APP protein levels both in vitro and in vivo. These observations suggest that APP represents a novel and specific biomarker in human transformed pluripotent stem cells that can be selectively modulated by HDAC inhibitors.     

NMDA receptor/amyloid precursor protein interactions: a comparison between wild-type and amyloid precursor protein mutations associated with familial Alzheimer's disease

Two recent reports showed that amyloid precursor protein (APP) may contribute to postsynaptic mechanisms via the regulation of the surface trafficking of excitatory N-methyl-D-aspartate (NMDA) receptors. Here we have investigated the interactions and surface trafficking of NR1-1a/NR2A and NR1-1a/NR2B NMDA receptor subtypes with three APP mutations linked to familial Alzheimer's disease, APP695(Indiana), APP695(London) and APP695(Swedish). Flag-tagged mutated APP695s were generated and shown to be expressed at equivalent levels to wild-type APP695 in mammalian cells. Each APP mutant co-precipitated with NR1-1a/NR2A and NR1-1a/NR2B receptors following co-expression in mammalian cells. Further, as found for wild-type APP695, each enhanced NMDA receptor surface expression with no concomitant increase in total NR1-1a, NR2A or NR2B subunit expression. Thus these three familial APP mutations behave as wild-type APP695 with respect to their association with assembled NMDA receptors and their APP695-enhanced receptor cell surface trafficking.     

ADAM10 expression and promoter haplotype in Alzheimer's disease

Alzheimer's disease is confirmed at autopsy according to the accumulation of brain neuritic plaques and neurofibrillary tangles in the brain. Neuritic plaques contain amyloid-β (Aβ) and lower levels of Aβ correspond to an increase in ADAM10 α-secretase activity. ADAM10 α-secretase activity produces a soluble amyloid precursor protein (APP) alpha (sAPPα) product and negates the pathological production of Aβ. In this investigation, it was hypothesized that genetic variation with the ADAM10 promoter is associated with ADAM10 expression levels as well as cerebrospinal fluid sAPPα levels. Results from this investigation suggest that the ADAM10 rs514049-rs653765 C-A promoter haplotype is associated with: (1) higher CSF sAPPα levels in cognitively normal controls compared with Alzheimer's disease (AD) patients, (2) higher postmortem brain hippocampus, but not cerebellum, ADAM10 protein levels in subjects with low plaque scores compared with those with high plaque scores, and (3) higher promoter activity for promoter-only reporter constructs compared with promoter 3' untranslated region (3'UTR) constructs in the human neuroblastoma SHSY5Y cell line, but not in HepG2 or U118 cell lines. Taken together, these findings suggest that ADAM10 expression is modulated according to a promoter haplotype that is influenced in a brain region- and cell type-specific manner.     

Lamotrigine attenuates deficits in synaptic plasticity and accumulation of amyloid plaques in APP/PS1 transgenic mice

Hyperactivity and its compensatory mechanisms may causally contribute to synaptic and cognitive deficits in Alzheimer's disease (AD). Blocking the overexcitation of the neural network, with levetiracetam (LEV), a sodium channel blocker applied in the treatment of epilepsy, prevented synaptic and cognitive deficits in human amyloid precursor protein (APP) transgenic mice. This study has brought the potential use of antiepileptic drugs (AEDs) in AD therapy. We showed that the chronic treatment with lamotrigine (LTG), a broad-spectrum AED, suppressed abnormal spike activity, prevented the loss of spines, synaptophysin immunoreactivity, and neurons, and thus attenuated the deficits in synaptic plasticity and learning and memory in APP and presenilin 1 (PS1) mice, which express human mutant APP and PS1. In contrast with LEV, which failed to reduce the generation of amyloid β, the chronic LTG treatment reduced the cleavage of APP by β-secretase and thus the numbers and the size of amyloid plaques in the brains of APP and PS1 mice. Moreover, the levels of brain-derived neurotrophic growth factor (BDNF) and nerve growth factor (NGF) were enhanced in the brains of APP and PS1 mice by the chronic LTG treatment. Therefore, these observations demonstrate that LTG attenuates AD pathology through multiple mechanisms, including modulation of abnormal network activity, reduction of the generation of amyloid beta and upregulation of BDNF and NGF.     

骨髓基质细胞体外诱导分化成神经元和胶质细胞

目的　探索骨髓基质细胞 (bonemarrowstromalcells,BMSC)体外诱导分化为神经元和神经胶质细胞的可行性 ,为BMSC在神经科学领域内的应用奠定基础。方法　以成年犬BMSC为实验对象 ,利用碱性成纤维细胞生长因子 (bFGF)、表皮生长因子 (EGF)、维甲酸 (RA)、脑源性神经营养因子 (BDNF)、胶质细胞系来源神经营养因子 (GDNF)等作为增殖及分化诱导因子 ,进行增殖培养、分化诱导 ;免疫组化法进行细胞性质鉴定。结果　加入bFGF、EGF增殖培养 72h可见细胞分裂相 (成纤维细胞样细胞 )。加入RA、BDNF、GDNF诱导 3d ,部分细胞有NSE、GFAP成分表达 ;第 10d可见有神经元、神经胶质形态样细胞形成 ,经细胞成分 (NSE、GFAP)鉴定证实为神经元、神经胶质细胞。结论　BMSC在体外培养条件下 ,经过bFGF、EGF、RA、BDNF、GDNF等因子的“程序性”作用 ,可以分化成神经元和神经胶质细胞     

Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells

The mechanisms that underlie the altered neurotransmitter system in Alzheimer's disease (AD) are not well understood. Amyloid precursor protein (APP) is a precursor protein for beta-amyloid, an important trigger protein in the pathogenesis of AD. Duplication of the APP gene as well as APP genes that contain certain mutations has been reported to be associated with familial AD (FAD), and a role of APP in neurotransmission has been suggested recently. This study examines the role of APP in exocytosis in PC12 cells using transfected human growth hormone (hGH) as a reporter for secretion. It was found that overexpression of APP or expression of the Swedish FAD mutation (APPsw) in PC12 cells significantly increased the basal secretion and constitutive secretion of hGH. Expression of an APP phosphorylation-deficient mutant decreased both basal and constitutive secretion relative to the APP wild-type, suggesting a role for APP-Thr668 phosphorylation in secretion in PC12 cells. Overexpression of X11alpha, a protein that stabilizes cellular APP, also increased the basal secretion of hGH but, contrary to APP, decreased the constitutive secretion of hGH, suggesting that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion of hGH may result from APP-X11alpha interaction. These results demonstrate an unknown role for APP in secretion, and suggest that elevated levels of APP or APP mutation in FAD brains contribute to the altered neurotransmitter pathology of AD through stimulation of basal and constitutive secretion.     

Circulating levels of IL-11 and leukaemia inhibitory factor (LIF) do not significantly participate in the production of acute-phase proteins by the liver

To investigate the contribution of IL-11 and LIF to acute-phase protein (APP) production, we first analysed the effects of IL-11 and LIF on production of C-reactive protein (CRP), fibrinogen, and haptoglobin by human primary hepatocytes. We also measured the serum levels of IL-11, LIF, and CRP in serum from patients with inflammatory rheumatic diseases to assess the role of these cytokines in the APP response in vivo. We included patients with conditions associated with a high APP response such as rheumatoid arthritis (RA) or spondylarthropathy (SpA), and others usually associated with a weak APP response such as systemic lupus erythematosus (SLE), in order to investigate whether these cytokines could account for the differences in APP responses. Our results showed that IL-11 and LIF induced only minimal stimulation on production of APP by human primary hepatocytes compared with IL-6, known as the major inducer. Serum levels of CRP were elevated in RA and SpA, and significantly higher than in SLE patients. Despite the presence of a high APP response in some of our patients and despite the fact that we used sensitive assays to measure IL-11 and LIF, serum levels of both cytokines were not detected in any of the tested sera. In conclusion, our results show that circulating levels of IL-11 or LIF do not contribute significantly to the production of APP in vivo, and that they do not account for the difference in APP response between SLE and other inflammatory rheumatic diseases.