Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Expression of the Oligodendrocyte-Myelin Glycoprotein by Neurons in the Mouse Central Nervous System

Abstract: The oligodendrocyte-myelin glycoprotein (OMgp) is a 110-kDa glycosylphosphatidylinositol-linked protein that was initially identified as a myelin-specific protein but whose precise function remains unknown. In this study, immunohistochemistry, western blots, in situ hybridization, and northern blots were used to determine the distribution of OMgp in the mouse brain. OMgp is present in a concentration detectable on western blots in the brains of newborn mice, and its concentration gradually increases until day 24 of life. OMgp mRNA is also present in amounts detectable on northern blots in the brains of newborn mice, and its concentration gradually increases until day 21 of life, after which the concentration diminishes a little. Most of the OMgp in the mouse brain appears to be expressed in diverse groups of neurons, but it is particularly prominent in large projection neurons such as the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, motoneurons in the brainstem, and anterior horn cells of the spinal cord. However, OMgp is not confined to these cells and is expressed in cells in the white matter as well. The OMgp gene is placed within an intron of the neurofibromatosis type I gene and on the opposite strand. This organization raises the possibility that there may be a relationship between the functions of the products of the two genes. In support of this possibility, we show that within the mouse CNS OMgp and neurofibromin are expressed in the same cell types.     

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.     

Glial and neuronal isoforms of Neurofascin have distinct roles in the assembly of nodes of Ranvier in the central nervous system

Rapid nerve impulse conduction in myelinated axons requires the concentration of voltage-gated sodium channels at nodes of Ranvier. Myelin-forming oligodendrocytes in the central nervous system (CNS) induce the clustering of sodium channels into nodal complexes flanked by paranodal axoglial junctions. However, the molecular mechanisms for nodal complex assembly in the CNS are unknown. Two isoforms of Neurofascin, neuronal Nfasc186 and glial Nfasc155, are components of the nodal and paranodal complexes, respectively. Neurofascin-null mice have disrupted nodal and paranodal complexes. We show that transgenic Nfasc186 can rescue the nodal complex when expressed in Nfasc(-/-) mice in the absence of the Nfasc155-Caspr-Contactin adhesion complex. Reconstitution of the axoglial adhesion complex by expressing transgenic Nfasc155 in oligodendrocytes also rescues the nodal complex independently of Nfasc186. Furthermore, the Nfasc155 adhesion complex has an additional function in promoting the migration of myelinating processes along CNS axons. We propose that glial and neuronal Neurofascins have distinct functions in the assembly of the CNS node of Ranvier.     

NGI重组DNA疫苗对转基因AD小鼠的治疗作用

目的研究NGI对转基因阿尔茨海默病小鼠的治疗作用。方法构建包含小鼠OMgp细胞外8LRP重复序列,人Tenascin-R的EGF-L结构域,以及人Nogo-A的氨基端和66个氨基酸细胞外域的重组质粒,肌肉注射AD小鼠,空载体组和空白对照组为对照组。水迷宫检测小鼠行为学差别,免疫组化检测其病理差别。结果4.5月龄AD小鼠经过水迷宫隐蔽平台实验,2d后疫苗组小鼠潜伏期比对照组缩短30%（P〈0.05）。探索实验显示3月龄疫苗组比空载体组和空白对照组小鼠穿越目标象限的次数分别增加48%和44%（P〈0.05）。结论NGI重组DNA疫苗能够提高转基因AD小鼠的学习与记忆能力。     

Disrupted axo-glial junctions result in accumulation of abnormal mitochondria at nodes of ranvier

Mitochondria and other membranous organelles are frequently enriched in the nodes and paranodes of peripheral myelinated axons, particularly those of large caliber. The physiologic role(s) of this organelle enrichment and the rheologic factors that regulate it are not well understood. Previous studies suggest that axonal transport of organelles across the nodal/paranodal region is locally regulated. In this study, we have examined the ultrastructure of myelinated axons in the sciatic nerves of mice deficient in the contactin-associated protein (Caspr), an integral junctional component. These mice, which lack the normal septate-like junctions that promote attachment of the glial (paranodal) loops to the axon, contain aberrant mitochondria in their nodal/paranodal regions. These mitochondria are typically large and swollen and occupy prominent varicosities of the nodal axolemma. In contrast, mitochondria located outside the nodal/paranodal regions of the myelinated axons appear normal. These findings suggest that paranodal junctions regulate mitochondrial transport and function in the axoplasm of the nodal/paranodal region of myelinated axons of peripheral nerves. They further implicate the paranodal junctions in playing a role, either directly or indirectly, in the local regulation of energy metabolism in the nodal region.     

Essential role of oligodendrocytes in the formation and maintenance of central nervous system nodal regions.

The membrane of myelinated axons is divided into functionally distinct domains characterized by the enrichment of specific proteins. The mechanisms responsible for this organization have not been fully identified. To further address the role of oligodendrocytes in the functional segmentation of the axolemma in vivo, the distribution of nodal (Na(+) channels, ankyrin G), paranodal (paranodin/contactin-associated-protein) and juxtaparanodal (Kv1.1 K(+) channels) axonal markers, was studied in the brain of MBP-TK and jimpy mice. In MBP-TK transgenic mice, oligodendrocyte ablation was selectively induced by FIAU treatment before and during the onset of myelination. In jimpy mice, oligodendrocytes degenerate spontaneously within the first postnatal weeks after the onset of myelination. Interestingly, in MBP-TK mice treated for 1-20 days with FIAU, despite the ablation of more than 95% of oligodendrocytes, the protein levels of all tested nodal markers was unaltered. Nevertheless, these proteins failed to cluster in the nodal regions. By contrast, in jimpy mice, despite a diffused localization of paranodin, the formation of nodal clusters of Na(+) channels and ankyrin G was observed. Furthermore, K(+) channels clusters were transiently visible, but were in direct contact with nodal markers. These results demonstrate that the organization of functional domains in myelinated axons is oligodendrocyte dependent. They also show that the presence of these cells is a requirement for the maintenance of nodal and paranodal regions.     

Targeted overexpression of osteoactivin in cells of osteoclastic lineage promotes osteoclastic resorption and bone loss in mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ～2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

The oligodendrocyte-myelin glycoprotein belongs to a distinct family of proteins and contains the HNK-1 carbohydrate

The complete primary structure of the human oligodendrocyte-myelin glycoprotein (OMgp), a glycophospholipid-linked membrane protein of oligodendrocytes and central nervous system myelin, has been determined. The deduced amino acid sequence predicts a polypeptide of 433 amino acids which includes a 17-amino acid leader sequence. OMgp consists of four domains: (a) a short cysteine-rich motif at the NH2 terminus; (b) a series of tandem leucine-rich repeats (LRs) present in several other proteins where they may play roles in adhesion; (c) a serine/threonine-rich region that contains probable attachment sites for O-linked carbohydrates; and (d) a hydrophobic COOH-terminal segment that is likely to be cleaved concomitant with the attachment of lipid during biosynthesis of OMgp. OMgp shares the first three of its four domains with the platelet glycoprotein Ib, which is responsible for the initial adhesion of platelets to the exposed subendothelium during hemostasis. Together with glycoprotein Ib and several other proteins, OMgp belongs to a family of proteins that contain both an NH2-terminal cysteine-rich motif and an adjacent series of LRs. In addition, we report that a subpopulation of OMgp molecules contains the HNK-1 carbohydrate, which has been shown to mediate interactions among cells in the central nervous system.     

Oligodendrocyte myelin glycoprotein growth inhibition function requires its conserved leucine-rich repeat domain,not its glycosylphosphatidyl-inositol anchor

The oligodendrocyte myelin glycoprotein (OMgp) inhibits neurite outgrowth and axonal regeneration after brain injury, but its normal function remains unknown. Several observations suggest its implication in cell growth regulation. Here we report an analysis of the domain requirement in OMgp proliferation inhibitory function. We first studied the OMgp protein sequence in 14 mammal species and observed a high conservation of its leucine-rich repeat (LRR) domain. The deletion of this LRR domain is responsible for a total loss of function in an in vitro expression system. The possible three-dimensional structure of the LRR domain of OMgp was modelled using the structure of Yersinia pestis YopM cytotoxin as a template. The predicted arrangement of the LRR segments is compatible with a function of OMgp as a binding protein. The OMgp is a glycosylphosphatidyl-inositol-linked protein anchored in the plasma membrane of oligodendrocytes and neurones. Using deletion mutagenesis, we demonstrated the dispensability of the glycosylphosphatidyl-inositol anchor for OMgp proliferation inhibition function. Our results suggest that OMgp is part of a receptor complex, either as a coreceptor or as a membrane-bound or soluble ligand, involved in the transmission of a growth suppressive signal.     

Bax and Bcl-2 interaction in a transgenic mouse model of familial amyotrophic lateral sclerosis

It has been proposed that mutations in copper/zinc-superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl-2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS-linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl-2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl-2, Bcl-XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl-2 and Bcl-XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl-2 in transgenic mSOD1 mice, overexpression of Bcl-2 increases the formation of Bcl-2:Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl-2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl-2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl-2:Bax heterodimers. Based on these findings, it may be concluded that Bcl-2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.     

Down-regulation of p27Kip1 expression is required for development and function of T cells

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27(Kip1) is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4(-)CD8(-) thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27(Kip1) expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27(Kip1) expression. The development of thymocytes in the transgenic strain in which p27(Kip1) is most abundant (p27-Tg(high) mice) appeared to be blocked at the CD4(-)CD8(-)CD25(+)CD44(low) stage. Peripheral T cells from p27-Tg(high) mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg(high) mice. These results suggest that down-regulation of p27(Kip1) expression is required for the development, proliferation, and immunoresponsiveness of T cells.     

Constitutive overexpression of Id-1 in mammary glands of transgenic mice results in precocious and increased formation of terminal end buds, enhanced alveologenesis, delayed involution

Inhibitor of differentiation-1 (Id-1) has been shown to play an essential role in cell proliferation, invasion, migration, and anti-apoptosis. However, the effect of Id-1 in mammary gland development remains unknown. Here, we generated MMTV-Id-1 transgenic mice to study the role of Id-1 in mammary gland development. In virgin mice, Id-1 overexpression led to precocious development and delayed regression of terminal end buds (TEBs) compared with wild-type mice. The number of BrdU-positive cells and the expression of Wnt signaling molecules, β-catenin and cyclin D1, which regulate ductal extension and TEB formation in virgin, were statistically higher in Id-1 transgenic mice than in wild-type mice. Id-1 also had an effect on the formation and proliferation of lobuloalveolar structures during early and mid-pregnancy. Id-1 transgenic mice had more lobulated and prominent alveolar budding than wild-type mice and had significantly greater counts of lobuloalveolar structures in early pregnancy. The expression of BrdU, β-catenin, and cyclin D1 was also predominantly increased in Id-1 transgenic mice. Moreover, Id-1 transgenic mice showed delayed involution. Id-1 regulated the expression levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax, and resulted in delay of apoptotic peak during postlactational involution. We also found that Id-1 was able to modulate expression of the regulators of Wnt/β-catenin signaling such as phospho-Akt, BMP2, FGF3, and RAR-β in tubuloalveolar development of mammary glands. Taken together, our results suggest that Id-1 plays a pivotal role in mammary gland development through Wnt signaling-mediated acceleration of precocity and alveologenesis and Bcl-2 family members-mediated delay of involution.     

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.     

High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.