The p75 neurotrophin receptor and neuronal apoptosis

Although evidence continues to accumulate for the apoptosis-inducing role of the p75 neurotrophin receptor, several outstanding questions remain. One of these concerns the signal transduction pathway of p75, which continues to be elusive. The evidence for the roles of ceramide, c-jun kinase and NF-kappaB is discussed: none of these are able to account satisfactorily for p75 death signalling. Negative modulation of Trk signalling by p75 could account for part of the pro-apoptotic effect, but is unlikely to be a major component. Although recent evidence indicates that the juxtamembrane region is critical for causing cell death, p75 has a well-conserved death domain. This may be important for functions other than killing. In glial cells and some neurons that express p75 but not TrkA, p75 causes cell death in response to nerve growth factor (NGF) binding. In sensory neurons and PC12 cells, p75 appears to signal constitutively. In cholinergic forebrain neurons, p75 expression leads to atrophy and downregulation of cholinergic markers, rather than cell death. The major challenges in p75 research are to define its signalling pathways, and particularly the intracellular proteins with which it interacts. Another major challenge is to develop a model that reconciles the different facets of p75, such as its ability in some situations to assist TrkA to rescue NGF-dependent neurons, but to stimulate apoptosis in others.     

Diverse functions of the p75 neurotrophin receptor

The pan-neurotrophin receptor p75NTR belongs to a large family of receptors, which includes tumor necrosis factor receptors, Fas and approximately 25 other members. The p75NTR is the first receptor to be cloned molecularly. Recent years have seen the emergence of a consensus regarding the signaling pathways activated by p75NTR and its potential biological function, although receptor characterization had not been targeted for some years. We now know that p75NTR has surprisingly diverse effects, ranging from cell death to regulation of axon elongation. This diversity can be explained by the complex formation of p75NTR with other receptors and multiple signaling molecules that interact with the intracellular domain of p75NTR.     

Identification of human oral keratinocyte stem/progenitor cells by neurotrophin receptor p75 and the role of neurotrophin/p75 signaling

This study was undertaken to determine whether human oral keratinocyte stem cells characteristically express higher levels of the low-affinity neurotrophin receptor p75 and to elucidate the function of p75 in oral keratinocytes. Examination of their expression patterns and cell-cycling status in vivo showed that p75 was exclusively expressed in the basal cell layer of both the tips of the papillae and the deep rete ridges. These immunostaining patterns suggest a cluster organization; most p75(+) cells did not actively cycle in vivo. Cell sorting showed that cells in the p75(+) subset were smaller and possessed higher in vitro proliferative capacity and clonal growth potential than the p75(-) subset. Clonal analysis revealed that holoclone-type (stem cell compartment), meroclone-type (intermediate compartment), and paraclone-type (transient amplifying cell compartment) cells, previously identified in skin and the ocular surface, were present in human oral mucosal epithelium. Holoclone-type cells showed stronger p75 expression at both the mRNA and protein level than did meroclone- and paraclone-type cells. Among the several neurotrophins, nerve growth factor (NGF) and neurotrophin-3 stimulated p75(+) oral keratinocyte cell proliferation, and only NGF protected them from apoptosis. Our in vivo and in vitro findings indicate that p75 is a potential marker of oral keratinocyte stem/progenitor cells and that some neurotrophin/p75 signaling affects cell growth and survival.     

siRNA沉默p75神经营养因子受体逆转胶质瘤恶性表型

目的 利用小干扰RNA(siRNA)技术沉默与胶质瘤细胞凋亡和侵袭密切相关的p75神经营养因子受体(p75NTR)基因,观察其逆转胶质瘤恶性表型的治疗效果.方法 设计靶向p75NTR基因的siRNA片段,脂质体转染人U251胶质瘤细胞系,逆转录聚合酶链反应(RT-PCR)和免疫细胞化学方法 检测p75NTR的mRNA和蛋白表达;Transwell细胞侵袭实验检测U251细胞的侵袭力;软琼脂集落形成实验检测细胞集落形成能力;建立裸鼠颅内U251胶质瘤荷瘤模型,原位重复注射siRNA-p75NTR/脂质体复合物3次,MRI检测颅内瘤体积,免疫细胞化学SP法检测p75NTR、神经生长因子(NGF)和细胞周期蛋白(cyclin)D2表达,用TUNEL法做移植瘤细胞原位细胞凋亡检测.结果 转染siRNA基因片段的U251细胞p75NTR mRNA和蛋白质表达水平显著下降(P<0.05),细胞侵袭能力及软琼脂集落形成能力均显著降低(P<0.05);体内实验显示p75NTR的表达程度与NGF的表达呈正相关,与cyclin D2表达及原位细胞凋亡数量呈负相关,MRI示瘤体积增长缓慢,边界轮廓清晰,动物生存期明显延长(P<0.05).结论 靶向p75NTR的siRNA技术可有效降低胶质瘤细胞的侵袭及增殖能力,诱导肿瘤细胞凋亡.     

siRNA沉默p75神经营养因子受体逆转胶质瘤恶性表型

目的 利用小干扰RNA(siRNA)技术沉默与胶质瘤细胞凋亡和侵袭密切相关的p75神经营养因子受体(p75NTR)基因,观察其逆转胶质瘤恶性表型的治疗效果.方法 设计靶向p75NTR基因的siRNA片段,脂质体转染人U251胶质瘤细胞系,逆转录聚合酶链反应(RT-PCR)和免疫细胞化学方法 检测p75NTR的mRNA和蛋白表达;Transwell细胞侵袭实验检测U251细胞的侵袭力;软琼脂集落形成实验检测细胞集落形成能力;建立裸鼠颅内U251胶质瘤荷瘤模型,原位重复注射siRNA-p75NTR/脂质体复合物3次,MRI检测颅内瘤体积,免疫细胞化学SP法检测p75NTR、神经生长因子(NGF)和细胞周期蛋白(cyclin)D2表达,用TUNEL法做移植瘤细胞原位细胞凋亡检测.结果 转染siRNA基因片段的U251细胞p75NTR mRNA和蛋白质表达水平显著下降(P<0.05),细胞侵袭能力及软琼脂集落形成能力均显著降低(P<0.05);体内实验显示p75NTR的表达程度与NGF的表达呈正相关,与cyclin D2表达及原位细胞凋亡数量呈负相关,MRI示瘤体积增长缓慢,边界轮廓清晰,动物生存期明显延长(P<0.05).结论 靶向p75NTR的siRNA技术可有效降低胶质瘤细胞的侵袭及增殖能力,诱导肿瘤细胞凋亡.     

siRNA沉默p75神经营养因子受体逆转胶质瘤恶性表型

目的 利用小干扰RNA(siRNA)技术沉默与胶质瘤细胞凋亡和侵袭密切相关的p75神经营养因子受体(p75NTR)基因,观察其逆转胶质瘤恶性表型的治疗效果.方法 设计靶向p75NTR基因的siRNA片段,脂质体转染人U251胶质瘤细胞系,逆转录聚合酶链反应(RT-PCR)和免疫细胞化学方法 检测p75NTR的mRNA和蛋白表达;Transwell细胞侵袭实验检测U251细胞的侵袭力;软琼脂集落形成实验检测细胞集落形成能力;建立裸鼠颅内U251胶质瘤荷瘤模型,原位重复注射siRNA-p75NTR/脂质体复合物3次,MRI检测颅内瘤体积,免疫细胞化学SP法检测p75NTR、神经生长因子(NGF)和细胞周期蛋白(cyclin)D2表达,用TUNEL法做移植瘤细胞原位细胞凋亡检测.结果 转染siRNA基因片段的U251细胞p75NTR mRNA和蛋白质表达水平显著下降(P<0.05),细胞侵袭能力及软琼脂集落形成能力均显著降低(P<0.05);体内实验显示p75NTR的表达程度与NGF的表达呈正相关,与cyclin D2表达及原位细胞凋亡数量呈负相关,MRI示瘤体积增长缓慢,边界轮廓清晰,动物生存期明显延长(P<0.05).结论 靶向p75NTR的siRNA技术可有效降低胶质瘤细胞的侵袭及增殖能力,诱导肿瘤细胞凋亡.     

Status epilepticus induces a TrkB to p75 neurotrophin receptor switch and increases brain-derived neurotrophic factor interaction with p75 neurotrophin receptor: an initial event in neuronal injury induction

In neuronal cultures it has been demonstrated that neurotrophins can elicit neuronal death through the p75 neurotrophic receptor (p75ntr) in the absence of concomitant Trk signaling. However, it was suggested that p75ntr induces neuronal death after status epilepticus (SE) in neuronal populations that express relatively high quantities of tropomyosin receptor kinase B (TrkB). Here, using Western blot and immunohistochemistry analyses in the hippocampus, we found that 3-h SE caused a remarkable decrease in TrkB expression and phosphorylation, and a significant increase in p75ntr. TrkB modification occurs before the overexpression of the tumor suppressor protein p53, accompanies the cell damage taking place in the dentate gyrus, and precedes the CA1 neuronal injury as assessed by Fluoro-Jade B and Nissl staining. Co-immunoprecipitation of brain-derived neurotrophic factor (BDNF) or its immature form proBDNF showed increased interaction with p75ntr after its binding to TrkB was reduced. Interestingly, proBDNF also increases its binding with p75ntr after seizures that do not cause neuronal death (animals injected with pilocarpine that fail to enter SE). However, in those animals, TrkB protein levels remained unchanged and its phosphorylation increased. Our results indicate an intrinsic capacity of neurons in vivo to modify final neurotrophin output by changing the proportion of their receptors' expression and the receptors' interaction with their ligands. These early events support the idea that neurotrophins may be involved in the induction of neuronal death in vivo under pathological conditions.     

p75 neurotrophin receptor regulates basal and fluoxetine-stimulated hippocampal neurogenesis

It is widely acknowledged that neurogenesis occurs in the adult hippocampus under normal conditions and that the rate can be regulated by environmental factors, including antidepressant drugs, with concomitant effects on behaviour. Using a quick and sensitive flow cytometry method that can assess changes in the number of bromodeoxyuridine (BrdU)-positive cells in hippocampus, in combination with traditional histological cell counts in the dentate gyrus, we report that mice lacking the p75 neurotrophin receptor gene (p75^NTR−/−) have significantly reduced hippocampal neurogenesis. Chronic treatment with the antidepressant fluoxetine stimulated hippocampal cell proliferation in p75^NTR−/− animals, but it did not result in an increase above basal levels of the number of newly born neurons in the dentate gyrus. These results indicate that p75^NTR acts as a regulator of fluoxetine-stimulated as well as basal adult hippocampal neurogenesis.     

p75 neurotrophin receptor distribution and transport in cultured neurons

In this work, we define a GFP-tagged version of the p75 neurotrophin receptor (p75GFP) as a useful molecular tool for studying its distribution and cellular dynamics. Expression and subcellular localization of p75GFP have been characterized in non-neuronal (HEK 293) and in neuronal (cortical and hippocampal) cells. By monitoring movements of intracellular p75GFP in living cultured hippocampal neurons, we found that the chimeric protein was transported by tubulo-vesicular structures both anterogradely (0.1-0.5microm/s) and retrogradely (0.1-1.1microm/s), with a faster component in retrogradely moving structures. Movements of the p75GFP-containing structures were inhibited by treatment with the microtubule-disrupting agent nocodazole. Our data indicate that p75GFP is a reliable tool for studying spatial and cellular properties of p75 in CNS neurons and that p75 transport inside neurons is mediated by microtubule-associated motors.     

The p75 neurotrophin receptor in human development and disease

The functional effects of nerve growth factor (NGF) and its precursor, pro-NGF, are thought to be mediated through binding of these ligands to one or both of their receptors, TrkA and p75NTR. While the signaling pathways and downstream effects of NGF binding to TrkA are reasonably well known, those related to the binding of NGF and pro-NGF to p75NTR are less well understood. Furthermore, p75NTR appears to play functional roles that are unrelated to its ability to bind NGF and pro-NGF, some of which are ligand-independent and others of which are dependent upon binding to other neurotrophins. As these functional roles and their biochemical mechanisms become better known, the importance of p75NTR, related receptors, and both extracellular ligands and intracellular interactors and effectors for human development and health has become increasingly apparent. A complete understanding of p75NTR and its cellular partners is best served by approaching the remaining questions from both sides, with studies of function in normal states and studies of dysfunction in aberrant states mutually informing one another.     

Diagnostic utility of p75 neurotrophin receptor (p75NTR) as a marker of breast myoepithelial cells.

We evaluated the low affinity neurotrophin receptor (p75NTR) as a marker of breast myoepithelial cells. Immunohistochemical staining for p75NTR was performed on paraffin sections of 122 malignant breast lesions, 28 benign lesions and the adjacent normal breast tissue. The staining pattern was compared to those of myosin heavy chain and p63. p75NTR immunostain was consistently positive and compatible with p63 and myosin immunoreactivity in the myoepithelial cells of the normal mammary gland, benign breast lesions (six usual ductal hyperplasias, six specimens with sclerosing adenosis, eight intraductal papillomas, six fibroadenomas), and carcinoma in situ (18 ductal carcinomas in situ, two noninvasive papillary carcinomas, two lobular carcinomas in situ). The luminal cells were negative for p75NTR, but rare positive cells were noticed in the solid areas of some of the usual ductal hyperplasias. Four of 64 invasive ductal carcinomas (6%) and all metaplastic carcinomas (n = 3, 100%) showed a variable degree of p75(NTR) positivity. No p75NTR expression was found in the malignant cells in all in situ carcinomas, invasive lobular carcinomas (n = 11), tubular carcinomas (n = 10), invasive papillary carcinomas (n = 6), mucinous carcinomas (n = 4), and medullary carcinomas (n = 2). No myosin immunoreactivity was seen in the luminal/tumor cells, but p63 pattern of staining in the luminal/tumor cells was quite similar to that of p75NTR. Although significant p75NTR immunoreactivity was noticed in the vessels, nerves, and stromal component of fibroadenomas, no difficulties in the evaluation of the immunostain of myoepithelial cells were encountered. Our study shows that p75NTR is a useful marker for breast myoepithelial cells and can be used to rule out invasive disease as well as to evaluate difficult for diagnosis sclerosing lesions. Our data suggest a role of neurotrophins in the development of fibroepithelial breast tumors and some of the breast carcinomas.     

人参皂苷诱导大鼠C6脑胶质瘤细胞的分化

目的:观察人参皂苷对胶质瘤细胞的诱导分化作用,探讨人参皂苷抗胶质瘤作用机制.方法:采用MTT、软琼脂克隆形成率、形态学与神经胶质纤维酸性蛋白(GFAP)免疫荧光等方法鉴定分化程度.结果:10μg/ml人参皂苷即可明显诱导大鼠C6胶质瘤细胞株的细胞分化,表现为增殖抑制,克隆形成能力丧失,细胞突起增长,GFAP表达量升高.结论:一定剂量的人参皂苷可以诱导大鼠脑胶质瘤细胞分化达到抗肿瘤的效果.     

miR-124对C6胶质瘤细胞增殖和迁移能力的影响

目的:研究miR-124对C6胶质瘤细胞增殖和迁移能力的影响.方法:体外培养C6胶质瘤细胞,依据转染不同分为空白对照组、阴性对照组和miR-124拟似物组.实时荧光PCR检测转染效率,绘制生长曲线,MTT实验检测miR-124对C6细胞增殖能力影响,计算抑制率.划痕实验检测miR-124对C6细胞迁移能力影响,计算迁移率.结果:生长曲线显示miR-124拟似物组C6细胞生长能力受到明显抑制,转染后第3天,miR-124拟似物组C6细胞数目显著低于阴性对照组(5.410±0.463Vs.6.917±0.385;P＜0.01);miR-124拟似物组mRNA表达量显著高于阴性对照组和空白对照组(5.92±0.56 vs.0.93±0.13和1.00±0.12;P＜0.01);MTT增殖实验显示miR-124抑制C6细胞增殖能力,miR-124拟似物组抑制率显著高于阴性对照物组(42.90±5.169 vs.10.24±3.351;P＜0.01);划痕实验显示miR-124明显抑制C6细胞迁移能力,阴性对照组迁移率显著高于miR-124拟似物组(98.79±1.210vs.81.72±5.972;P＜0.05).结论:miR-124能够显著抑制C6胶质瘤细胞的增殖和迁移能力.     

Attenuation of opioid analgesic tolerance in p75 neurotrophin receptor null mutant mice

Repeated exposure to opioid drugs can lead to the development of tolerance, which manifests as a reduction in analgesic potency, and physical dependence, a response indicated by a withdrawal syndrome. Accumulating evidence suggests that the nerve growth factor (NGF) family of neurotrophins may have an important modulatory role in the induction of opioid analgesia and opioid addiction. Because neurotrophins universally bind the p75 neurotrophin receptor (p75NTR), we investigated whether the activity of this receptor is involved in the development of opioid analgesic tolerance and physical dependence. We found that in both the wild-type and p75NTR−/− mice an acute systemic (i.p.) injection of morphine produced a maximal analgesic response as measured by the thermal tail-immersion test. Repeated injection of morphine over 5 days in wild-type mice resulted in a progressive decline of the analgesic effect and a concomitant loss of the agonist potency, reflecting development of morphine tolerance. However, the loss of morphine analgesia was not observed in p75NTR−/− mice. In the second part of this study, mice were given escalating doses of systemic (i.p.) morphine over 5 days and subsequently challenged with the opioid receptor antagonist naloxone. This challenge precipitated a robust withdrawal syndrome that was comparable in wild-type mice and p75NTR−/− mice. The findings suggest that p75NTR activity plays a critical role in the development of opioid analgesic tolerance but not in the induction or the expression of opioid physical dependence.     

Knockdown of DSPP inhibits the migration and invasion of glioma cells

Dentin sialophosphoprotein (DSPP) is a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins and has been proved to contribute to the migration of a variety of solid tumor cells. However, whether DSPP participates in the pathogenic process of glioma remains unknown. In this study, we aimed to investigate the expression and biological function of DSPP in human glioma cells. We demonstrated through Western blot that DSPP is overexpressed in glioma tissues comparing to normal brain tissues. To investigate the role of DSPP in glioma carcinogenesis, we reduced the DSPP expression by small interfering RNA (siRNA) and found that DSPP silencing significantly inhibited the migration and invasion of glioma cells, the critical characteristics of glioma. Furthermore, we showed that DSPP down-regulation significantly decreased the activation of the AKT/mTOR/p70S6K pathway in glioma cells. Taken together, these findings indicate that knockdown of DSPP inhibits glioma cells migration and invasion, suggesting that targeting DSPP might be a potentially effective therapeutic strategy for treating glioma.     

Knockdown of TFPI-2 promotes migration and invasion of glioma cells

Glioblastoma is the most malignant primary brain tumor. Due to its highly promigratory and proinvasive properties, standard therapy including surgery, chemotherapy and radiation fails in eradicating this highly aggressive type of cancer. Here, we evaluated the role of TFPI-2, a Kunitz-type serine protease inhibitor, which has been previously described as a tumor suppressor gene in several types of cancer, including glioma. TFPI-2 expression was absent in five of nine investigated high-grade glioma cell lines. Lentiviral knockdown of TFPI-2 in two of the TFPI-2-expressing cell lines (MZ-18 and Hs 638) was associated with pronounced changes in the cellular behavior: glioma cell proliferation, migration and invasion were significantly increased in TFPI-2 knockdown cells in comparison to empty vector-transfected control cells. Since TFPI-2 might exert its tumor suppressor function by inhibiting MMPs, we subsequently analyzed the effects of specific MMP inhibitors on cell invasion of TFPI-2 KD cells vs. control cells. The data obtained from these experiments suggest that the anti-invasive properties of TFPI-2 are associated with inhibition of MMP-1 and MMP-2, while inhibition of MMP-9 seems to play a minor role in this context. Our findings underscore the important role of TFPI-2 as a tumor suppressor gene and indicate that TFPI-2 may be a useful diagnostic marker for the aggressive phenotype of glial tumors.     

Expression and p75 neurotrophin receptor dependence of cholesterol synthetic enzymes in adult mouse brain

Normal brain function depends critically on cholesterol. Although cholesterol is synthesized locally in the adult brain, the precise anatomical localization of cholesterogenic enzymes is not known. Here we show that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAred) and 7-dehydrocholesterol reductase (7dhcred), the first and last enzymes, respectively, in the cholesterol biosynthesis pathway, are co-expressed in neurons throughout adult murine brain. Co-localization is most prominent in cortical, hippocampal, and cholinergic neurons. Since adult hippocampal and cholinergic neurons express p75 neurotrophin receptors (p75NTR) we hypothesized that p75NTR regulates expression of cholesterogenic enzymes. Treatment of Neuro2a neuroblastoma cells or primary cerebellar cultures with siRNA downregulates p75NTR and decreases the expression level of HMG-CoAred and 7dhcred. Native neuroblastoma cell lines with differential expression of p75NTR differentially express 7dhcred; 7dhcred expression correlates with p75NTR expression. This suggests that, in p75NTR-expressing cells, p75NTR regulates cholesterol synthesis through regulation of HMG-CoAred and 7dhcred expression. The unexpected localization of cholesterogenic enzymes in adult neurons suggests that at least some adult neurons retain the ability to synthesize cholesterol.     

双氢青蒿素抑制胶质瘤干细胞侵袭迁移的机制

BACKGROUND: Dihydroartmisinin can promote apoptosis of glioma cells GL261, but its effect on glioma stem cells is still unknown.OBJECTIVE: To investigate the preliminary mechanism that dihydroartemisinin inhibits migration and invasion of glioma stem cells.METHODS:Glioma stem cells were isolated from mouse malignant glioma cell lines GL261. Immunofluorescence analysis was conducted to identify the characteristics of glioma stem cells. Migration and invasion abilities of glioma stem cells were analyzed by Transwell assay. The mRNA expressions of Toll-like receptor 2, matrix metalloproteinase-2 and matrix metalloproteinase-9 were examined by real-time fluorescence quantitative PCR.RESULTS AND CONCLUSION: The characteristics of glioma stem cells were identified by CD133 and Nestin staining. The migration and invasion of glioma stem cells were attenuated by dihydroartemisinin dose-dependently. Moreover, the mRNA expression of Toll-like receptor 2, matrix metalloproteinase-2 and matrix metalloproteinase-9 was also decreased by dihydroartemisinin in a dose dependent manner. These results suggest that dihydroartemisinin inhibits the migration and invasion of glioma stem cells probably through attenuation of Toll-like receptor signaling pathway.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

The p75 neurotrophin receptor influences NT-3 responsiveness of sympathetic neurons in vivo.

To determine the role of the p75 neurotrophin receptor (p75NTR) in sympathetic neuron development, we crossed transgenic mice with mutations in p75NTR, nerve growth factor (NGF) and neurotrophin-3 (NT-3). Neuron number is normal in sympathetic ganglia of adult p75NTR-/- mice. Mice heterozygous for a NGF deletion (NGF+/-) have 50% fewer sympathetic neurons. In the absence of p75NTR (p75NTR-/- NGF+/-), however, neuron number is restored to wild-type levels. When NT-3 levels are reduced (p75NTR-/- NGF+/- NT3 +/-), neuron number decreases compared to p75NTR-/- NGF+/- NT3+/+. Thus, without p75NTR, NT3 substitutes for NGF, suggesting that p75 alters the neurotrophin specificity of TrkA in vivo.