Fractone-heparan sulfates mediate BMP-7 inhibition of cell proliferation in the adult subventricular zone

Bone morphogenetic protein-7 (BMP-7) is a heparin-binding growth factor that inhibits cell proliferation in the subventricular zone (SVZ) of the lateral ventricle, the primary neurogenic niche in the adult brain. However, the physiological mechanisms regulating the activity of BMP-7 in the SVZ are unknown. Here, we report the inhibitory effect of BMP-7 on cell proliferation through the anterior SVZ after intracerebroventricular injection in the adult mouse. To determine whether the inhibition of cell proliferation induced by BMP-7 is dependant on heparin-binding, heparitinase-1 was intracerebroventricularly injected to N-desulfate heparan sulfate proteoglycans before BMP-7 was injected. Heparatinase-1 drastically reduced the inhibitory effect of BMP-7 on cell proliferation in the SVZ. To determine where BMP-7 binds within the niche, we visualized biotinylated-BMP-7 after intracerebroventricular injection, using streptavidin Texas red on frozen brain sections. BMP-7 binding was seen as puncta in the SVZ at the location of fractones, the particulate specialized extracellular matrix of the SVZ, which have been identified primarily by N-sulfated heparan sulfate immunoreactivity (NS-HS+). BMP binding was also seen in NS-HS+ blood vessels of the SVZ. Injection of heparitinase-1 prior to biotinylated BMP-7 resulted in the absence of signal for biotinylated-BMP-7 in the fractones and blood vessels, indicating that the binding is heparan sulfate dependant. These results indicate that BMP-7 requires heparan sulfates to bind and inhibit cell proliferation in the SVZ neurogenic niche. Heparan sulfates concentrated in fractones and SVZ blood vessels emerge as a functional stem cell niche component involved in growth factor activity.     

Fractone aging in the subventricular zone of the lateral ventricle

In adulthood, the subventricular zone (SVZ) is one of the restricted places where neurogenesis persists. In this neurogenic niche, specialized extracellular matrix (ECM) structures termed fractones contact neural stem cells and their immediate progeny. Fractones are composed of ubiquitous ECM components including heparan sulfate proteoglycans such as perlecan and agrin. We have previously shown that fractones can capture growth factors and promote growth factor activity through a heparin binding mechanism in order to regulate neurogenesis. With aging, neurogenesis is known to decrease. However, the effect of aging on fractones structure and composition remains unknown. Here, we report that, while fractone number decreased, fractone size dramatically increased with aging. Despite the changes in fractones morphology, niche cells expressing glial fibrillary acidic protein kept direct contact with fractones. Furthermore, we have observed that heparan sulfate chains contained in fractones were modified with aging. However, FGF-2 was still captured by fractones via heparan sulfates. Together, our results suggest that the changes observed in fractones structure and composition are critically related to aging of the SVZ neurogenic niche.     

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone

The tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. We show here that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo.     

体外骨形态发生蛋白4抑制乳腺癌细胞的增殖和迁移

目的:研究骨形态发生蛋白4(BMP4)对乳腺癌细胞MCF-7增殖、迁移和侵袭能力的影响.方法:将重组慢病毒Lv-BMP4及干扰慢病毒Lv-shBMP4感染MCF-7细胞,构建MCF-7/Lv-BMP4过表达组及MCF-7/Lv-shBMP4干扰实验组,同时设空白组(Blank)、慢病毒空载对照组(Lv-NC)、 过表达...     

Anatomically discrete sex differences and enhancement by testosterone of cell proliferation in the telencephalic ventricle zone of the adult canary brain

Previous work in songbirds has suggested that testosterone increases neuronal recruitment and survival in HVC but does not affect neuronal proliferation in the ventricular zone and that males and females have similar rates of proliferation except at discrete locations. Many of these conclusions are however based on limited data or were inferred indirectly. Here we specifically tested the effects of testosterone on cellular proliferation in the ventricular zone of both male and female adult canaries. We implanted adult birds of both sexes with testosterone or empty implants for 1 week and injected them with BrdU. One day later, we collected their brains and quantified BrdU-positive cells in the ventricular zone (VZ) at different rostro-caudal levels of the brain, ranging from the level where the song nucleus Area X occurs through the caudal extent of HVC. Proliferation in the dorsal part of the VZ was low and unaffected by sex or testosterone treatment. In the ventral part of the VZ, females had more proliferating cells than males, but only at rostral levels, near Area X. Also in the ventral part of the VZ, testosterone increased proliferation in birds of both sexes, but only in the mid- to caudal-VZ, caudal to the level of Area X, around the septum and HVC. We thus demonstrate here that there is both an effect of testosterone and possibly a more subtle effect of sex on cellular proliferation in the adult songbird brain, and that these effects are specific to different levels of the brain.     

Hippocampus/amygdala alterations, loss of heparan sulfates, fractones and ventricle wall reduction in adult BTBR T+ tf/J mice, animal model for autism

Multiple studies converge to implicate alterations of the hippocampus and amygdala in the pathology of autism. We have previously reported anatomical alterations of the meninges, vasculature and fractones, the specialized extracellular matrix (ECM) of the subventricular zone, in the forebrain of adult BTBR T+ tf/J mice, animal model for autism. Here, we used bisbenzidine cell nucleus staining and dual immunofluorescence histochemistry for laminin and N-sulfated heparan sulfate proteoglycans (NS-HSPG) to examine a series of brain sections containing the amygdala and hippocampus in the adult BTBR T+ tf/j mouse. We observed an excessive separation of the two hippocampi, a modified trajectory of the meninges leading to a shrunken choroid plexus in the lateral ventricle, a shorter granular layer of the dentate gyrus, and a reduced size of the amygdala nuclei. The lateral ventricle near the amygdala, and the third ventricle were shrunken. The number and size of fractones, and their immunoreactivity for NS-HSPG, were reduced throughout the third and lateral ventricles walls. Enlarged blood vessels were found at the endopiriform cortex/amygdala interface. These results show anatomical alterations of the hippocampal/amygdala that are associated with defects of the choroid plexus/ventricular system and the ECM in the BTBR T+ TF/J mouse. Similar alterations of the hippocampus/amygdala axis in humans with autism to these observed in BTBR T+ tf/J mice make this animal model highly valuable for the study of autism. Moreover, the meningo/vascular and ECM alterations in BTBR T+ Tf/J mice suggest a possible role of the brain connective tissue in autism.     

Fluctuations in cellular proliferation across the light/dark cycle in the subgranular zone of the dentate gyrus in the adult male Syrian hamster

A lifelong persistent neurogenesis occurs in the dentate gyrus of the mammalian hippocampus. Research in peripheral cell tissue has shown that the timing of cellular division of these cells coincide with the light/dark cycle, however it remains unclear as to whether there is an association between the time of day and cellular proliferation in the brain. The timing of cellular division can be studied through the use of a cellular proliferation marker, such as 5-bromo-2-deoxyuridine (BrdU), which is taken up by the DNA of dividing cells during replication. The goal of this study was to determine whether the time of day affects the number of BrdU labeled cells in the subgranular zone of the dentate gyrus of adult male Syrian hamsters. Adult males received a single systemic injection of BrdU (300 mg/kg) at either the end of the light (ZT-13) or dark phase (ZT-23) of a 14:10 LD cycle and were sacrificed 24 h or 3 days later. Sections through the hippocampus were immunolabeled for BrdU. Cellular proliferation fluctuated across the light/dark cycle during the expansion phase rather than during initial cellular proliferation. A twofold increase in number was expected between 24 and 72 h following a single BrdU injection, but this increase was only seen in the population of cells injected at the end of the light phase.     

MicroRNA-137 inhibits cell migration and invasion by targeting bone morphogenetic protein-7 (BMP7) in non-small cell lung cancer cells

MicroRNA-137 (miR-137) was reported to be dysregulated in several human cancers. However, the function and mechanism of miR-137 in non-small cell lung cancer (NSCLC) is still unclear. In the current study, we explored the role of miR-137 in NSCLC progression. Using qRT-PCR, our data showed that miR-137 was significantly down-regulated in NSCLC tissues and cell lines. In vitro functional assay, we found that over-expression of miR-137 suppressed NSCLC cells proliferation, migration and invasion, indicating that miR-137 could act as a tumor suppressor in NSCLC progression. In addition, bone morphogenetic protein-7 (BMP7) was identified as a target of miR-137 in NSCLC cells, Luciferase reporter assay suggested that miR-137 directly targeted 3’-UTR of BMP7, and correlation analysis revealed that BMP7 inversely correlated with miR-137 in NSCLC tissues. Furthermore, Restoration of BMP7 remarkably reversed the tumor suppressive effects of miR-137 on NSCLC cell proliferation, migration, and invasion. Taken together, our findings suggested that miR-137/BMP7 axis could contribute to the progression of NSCLC, suggesting miR-137 as a potential therapeutic target for the treatment of NSCLC.     

Bone morphogenetic protein 4 is induced in hepatocellular carcinoma by hypoxia and promotes tumour progression

Striking similarities exist between molecular mechanisms driving embryonic liver development and progression of hepatocellular carcinoma (HCC). Bone morphogenetic proteins (BMPs), particularly BMP4, have been proposed to regulate embryonic hepatic development. BMP expression has been observed in neoplasia but the expression and biological role of BMP4 in human HCC are unknown. We found increased BMP4 mRNA and protein in HCC cell lines and tissue samples compared to primary human hepatocytes and corresponding non‐tumourous tissue. Hypoxia further induced BMP4 expression in HCC cells, which was abolished by transfection of a dominant negative form of HIF‐1alpha (dnHIF‐1alpha). However, gel shift assays revealed only minor binding activity in nuclear extracts from (hypoxic) HCC cells to a putative hypoxia‐response element in the BMP4 promoter. Sequence analysis of the BMP4 promoter revealed two Ets‐1 binding sites, and Ets‐1 activity was increased in HCC cells under hypoxic conditions. Transfection of dnHIF‐1alpha completely abrogated hypoxia‐induced Ets‐1 activity as well as BMP4 expression. Overexpression of Ets‐1 markedly enhanced BMP4 promoter activity, while antisense Ets‐1 almost completely abolished basal as well as hypoxia‐induced BMP4 expression. These data demonstrate that Ets‐1 activity contributes to baseline expression of the BMP4 gene and is the predominant mediator of the HIF‐dependent BMP4 induction under hypoxic conditions. To determine the functional relevance of BMP4 expression, HCC cell lines were treated with antisense BMP4 constructs or siRNA against BMP4. BMP4 suppression resulted in a strong reduction of the migratory and invasive potential and anchorage‐independent growth. Furthermore, tube formation assays indicated that BMP4 expressed by HCC cells promotes vasculogenesis. Our findings demonstrate that BMP4 is increased in HCC and promotes HCC progression. Therefore, BMP4 expression may have clinical relevance, and interfering with BMP4 signalling appears as an attractive therapeutic target for this highly aggressive tumour. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

BMP4参与海人酸损伤后成年大鼠海马齿状回颗粒细胞增殖及胶质增生

目的探讨海人酸（KA）侧脑室注射致大鼠海马损伤后骨形成蛋白-4（BMP4）的表达变化及其与颗粒细胞增殖和胶质细胞增生的关系。方法将成年大鼠分为对照组与实验组。侧脑室注射KA7d后,用尼氏染色检测海马神经元丢失,用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA阳性细胞与BrdU标记细胞、GFAP阳性细胞数的变化。结果正常成年大鼠BMP4mRNA阳性细胞主要分布于海马齿状回的门区、颗粒下层、CA3、CAI区。BrdU标记细胞主要分布在齿状回颗粒下层。GFAP阳性细胞主要分布在齿状回、CA3区。在KA侧脑室注射致海马损伤后7d,海马CA3、CA4区神经元丢失明显,BMP4mRNA阳性细胞与BrdU、GFAP阳性细胞均明显增加。结论KA侧脑室注射致海马损伤后,成年大鼠海马齿状回颗粒细胞增殖增强和胶质增生可能与BMP4表达增加有关。     

Cilostazol attenuates ischemic brain injury and enhances neurogenesis in the subventricular zone of adult mice after transient focal cerebral ischemia

Evidence suggests that neurogenesis occurs in the adult mammalian brain, and that various stimuli, for example, ischemia/hypoxia, enhance the generation of neural progenitor cells in the subventricular zone (SVZ) and their migration into the olfactory bulb. In a mouse stroke model, focal ischemia results in activation of neural progenitor cells followed by their migration into the ischemic lesion. The present study assessed the in vivo effects of cilostazol, a type 3 phosphodiesterase inhibitor known to activate the cAMP-responsive element binding protein (CREB) signaling, on neurogenesis in the ipsilateral SVZ and peri-infarct area in a mouse model of transient middle cerebral artery occlusion. Mice were divided into sham operated (n=12), vehicle- (n=18) and cilostazol-treated (n=18) groups. Sections stained for 5-bromodeoxyuridine (BrdU) and several neuronal and a glial markers were analyzed at post-ischemia days 1, 3 and 7. Cilostazol reduced brain ischemic volume (P<0.05) and induced earlier recovery of neurologic deficit (P<0.05). Cilostazol significantly increased the density of BrdU-positive newly-formed cells in the SVZ compared with the vehicle group without ischemia. Increased density of doublecortin (DCX)-positive and BrdU/DCX-double positive neural progenitor cells was noted in the ipsilateral SVZ and peri-infarct area at 3 and 7 days after focal ischemia compared with the vehicle group (P<0.05). Cilostazol increased DCX-positive phosphorylated CREB (pCREB)-expressing neural progenitor cells, and increased brain derived neurotrophic factor (BDNF)-expressing astrocytes in the ipsilateral SVZ and peri-infarct area. The results indicated that cilostazol enhanced neural progenitor cell generation in both ipsilateral SVZ and peri-infarct area through CREB-mediated signaling pathway after focal ischemia.     

Meningeal/vascular alterations and loss of extracellular matrix in the neurogenic zone of adult BTBR T+ tf/J mice,animal model for autism

Autism spectrum disorders are characterized by impaired social and communication skills and seem to result from altered neural development. We sought to determine whether the anatomy of the meninges and extracellular matrix (ECM) is altered in an animal model of autism, the BTBR T+ tf/J mouse. This mouse displays white matter tract anatomical defects and exhibits several symptoms of autism. Immunofluorescence cytochemistry for laminin, a major ECM marker, was performed on series of coronal sections of the adult BTBR T+ tf/J brain and the anatomy was analyzed in comparison to B6 wild type mice. Laminin immunoreactivity visualized meninges, blood vessels and the subventricular zone (SVZ) stem cell-associated ECM structures, which I have named fractones. All BTBR T+ tf/J mice observed showed the same forebrain defects. The lateral ventricle volume was severely reduced, the falx cerebri elongated, the arteries enlarged and the choroid plexus atrophied. Compared to B6 mice, fractone numbers in BTBR T+ tf/J mice were reduced by a factor three in the SVZ of the anterior portion of the lateral ventricle. This represents the primary neurogenic zone during adulthood. Fractones were reduced by a factor 1.5 in posterior portions of the lateral ventricle. Moreover, fractone size was reduced throughout the lateral ventricle SVZ. These results show hitherto unsuspected alterations in connective tissue/vasculature and associated ECM in the adult BTBR T+ tf/J mouse. The drastic changes of the connective tissue and ECM in the neurogenic zone of the lateral ventricle may contribute to incorrect neurogenesis during developmental and adult stages.     

去松果体后成年大鼠学习记忆及侧脑室室管膜下区神经干细胞增殖的变化

目的观察松果体切除对学习记忆及侧脑室室管膜下区(SVZ)神经干细胞增殖的影响。方法将30只成年健康雄性Sprague-Dawley大鼠随机分为非手术、假手术及去松果体组,每组大鼠10只。在建立动物模型16d后,连续5d测定大鼠在Morris水迷宫的学习记忆能力,继之用免疫组织化学方法观察SVZ的增殖细胞核抗原(PCNA)阳性细胞的变化。结果去松果体组大鼠在Morris水迷宫泳游的逃避潜伏期及在原平台象限游泳距离的百分比均较非手术组或假手术组大鼠的明显延长或减少(P<0.01)。去松果体大鼠SVZ的PCNA阳性细胞数也明显低于非手术组或假手术组大鼠(P<0.01)。结论本研究首次观察到,去松果体使体内褪黑素减少,可导致学习记忆功能及SVZ神经干细胞增殖能力出现相似的明显下降趋势,说明褪黑素是确保学习记忆及神经发生得以正常进行的重要激素之一;提示褪黑素可能直接通过作用于神经干细胞上的相应受体以及间接通过提高基底前脑胆碱能系统功能来促进神经干细胞增殖,进而提高嗅觉记忆功能。     

Chronic treatment of exendin-4 affects cell proliferation and neuroblast differentiation in the adult mouse hippocampal dentate gyrus

Exendin-4 isolated from Heloderma suspectum venom acts via glucagon-like peptide 1 (GLP-1) receptor and has clinically been used in the type 2 diabetes. In this study, we investigated the effects of exendin-4 on cell proliferation and neuroblast differentiation in the subgranular zone (SGZ) of the dentate gyrus in mice. Exendin-4 was treated intraperitoneally to male ICR mice twice a day for 21 days. The exendin-4-treated group showed a significantly higher number of Ki67- (1.51-fold), doublecortin (DCX)- (2.5-fold) and 5-bromo-2′-deoxyuridine (BrdU) + DCX- (2.46-fold) immunoreactive cells in the SGZ of the dentate gyrus compared to the control group. The results of this study showed that treatment with exendin-4 increased cell proliferation neuroblast differentiation in the SGZ of the dentate gyrus, suggesting that exendin-4 promotes structural plasticity in the dentate gyrus.     

Bone morphogenetic protein-2/4 signalling pathway components are expressed in the human thymus and inhibit early T-cell development

T-cell differentiation is driven by a complex network of signals mainly derived from the thymic epithelium. In this study we demonstrate in the human thymus that cortical epithelial cells produce bone morphogenetic protein 2 (BMP2) and BMP4 and that both thymocytes and thymic epithelium express all the molecular machinery required for a response to these proteins. BMP receptors, BMPRIA and BMPRII, are mainly expressed by cortical thymocytes while BMPRIB is expressed in the majority of the human thymocytes. Some thymic epithelial cells from cortical and medullary areas express BMP receptors, being also cell targets for in vivo BMP2/4 signalling. The treatment with BMP4 of chimeric human-mouse fetal thymic organ cultures seeded with CD34+ human thymic progenitors results in reduced cell recovery and inhibition of the differentiation of human thymocytes from CD4- CD8- to CD4+ CD8+ cell stages. These results support a role for BMP2/4 signalling in human T-cell differentiation.     

Extracellular K+ accumulations and synchronous GABA-mediated potentials evoked by 4-aminopyridine in the adult rat hippocampus

Transient changes in extracellular potassium concentration ([K⁺]₀) and field potentials were evoked by 4-aminopyridine (4-AP; 50–100 M) and recorded with ion-selective microelectrodes in CA1b, CA3b and dentate sectors of adult rat hippocampal slices. Long-lasting field potentials recurred at a frequency of 1/60 s (0.016±0.003 Hz) in association with increases in [K⁺]₀ which were largest and most sustained in the dendritic regions where afferent fibers terminate (dentate>CAl>CA3) and in the hilus. In stratum radiatum of CA1 or stratum moleculare of the dentate these fields had a peak amplitude of 1.4±0.29 mV, duration 8.3±1.6 s, and were accompanied by increases in [K⁺]₀ of 1.8±0.22 mM that lasted 32±5.5 s (n = 17 slices). Interictal epileptiform potentials, which were brief (<0.2 s) and more frequent at 1/3 s (0.30±0.02 Hz) were also present in CA1, CA3 and the hilus and associated with small increases in [K⁺]₀ (0.5 mM, duration 2 s). Interictal activity was blocked by 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX; 5–20 M); the slow, less frequent potentials were resistant to both CNQX and dl-2amino-5-phosphonovaleric acid (APV; 50 M) and reversibly blocked (or attenuated by 80%) by bicuculline methiodide (BMI) (25–100 M). The BMI-sensitive potentials were also abolished by baclofen (100 M), an effect which was reversed by 2-OH-saclofen (100 M). Focal application of KCl or GABA in the absence of 4-AP evoked long-lasting field and [K⁺]₀ potentials which were similar to those evoked by 4-AP but more sustained. The proportional relationship between the amplitudes of field and K⁺ potentials with GABA closely resembled that observed for 4-AP; in contrast the slope of KCl-evoked responses was lower. Our results demonstrate that in the adult rat hippocampus 4-AP induces in many different regions accumulations of [K⁺]₀ in synchrony with the long-lasting field potentials, which are known to correspond to an intracellular long-lasting depolarization of the pyramidal cells. These changes are smaller than those which occur in the immature rat hippocampus — which may be related to differences in Na-K-ATPase and susceptibility to seizures. These events involve the activation of GABA_A receptors, are under the modulatory control of GABA_B receptors, and likely arise from the activity of GABAergic interneurons and/or afferent terminals. The long-lasting field potentials appear to reflect mainly the direct depolarizing actions of GABA and to a much more limited extent the associated accumulation of [K⁺]₀.