Characterization of mouse homolog of brain acyl-CoA hydrolase: molecular cloning and neuronal localization

Acyl-CoA hydrolase could provide a mechanism via its potency to modulate cellular concentrations of acyl-CoAs for the regulation of various cellular events including fatty acid metabolism and gene expression. However, only limited evidence of this is available. To better understand the physiological role of this enzyme, we characterized a mouse brain acyl-CoA hydrolase, mBACH. The cloned cDNA for mBACH encoded a 338-amino-acid polypeptide with >95% identity to the human and rat homologs, indicating that the BACH gene is highly conserved among species. This was supported by the similarity in genomic organization of the BACH gene between humans and mice. Bacterially expressed mBACH was highly active against long-chain acyl-CoAs with a relatively broad specificity for chain length. While palmitoyl-CoA hydrolase activity was widely distributed in mouse tissues, it was marked in the brain, consistent with mBACH being almost exclusively distributed in this tissue, where >80% of the enzyme activity was explained by mBACH present in the cytosol. Immunohistochemistry demonstrated a neuronal localization of mBACH in both the central and peripheral nervous systems. In neurons, mBACH was distributed throughout the cell body and neurites. Although four isoforms except mBACH itself, that may be generated by the alternative use of exons of a single mBACH gene, were cloned, their mRNA levels in the brain were estimated to be negligible. However, a 50-kDa polypeptide besides the major one of 43-kDa seemed to be translated from the mBACH mRNA with differential in-frame ATG triplets used as the initiation codon. These findings will contribute to the functional analysis of the BACH gene using mice including genetic studies.     

ARIP1,2 mRNA及其蛋白在小鼠脑组织表达和分布的比较研究

目的比较新发现的激活素受体相互作用蛋白1,2(ARIP1,2)在小鼠脑组织的表达与分布。方法采用Northern杂交检测ARIP1,2 mRNA,Western杂交及免疫组化染色检测ARIP1,2蛋白。结果Northern杂交显示ARIP1,2 mRNA在小鼠组织中的表达形式明显不同,ARIP1主要在脑组织表达,ARIP2组织表达广泛。Western杂交进一步揭示ARIP1在大脑、小脑、海马、下丘脑及垂体表达,而ARIP2在大脑、小脑、海马、下丘脑、垂体及脾脏均有不同程度表达。免疫组化染色显示,ARIP1,2成熟蛋白在大脑、小脑及垂体均有表达,ARIP1在大脑和小脑皮质主要表达在小细胞神经元,而ARIP2主要表达在大细胞神经元。ARIP1在神经垂体和腺垂体表达水平明显高于ARIP2。结论ARIP1,2均表达于脑和垂体,但其表达的细胞类型和强度明显不同,这种差异可能与其在神经细胞中介导激活素生物学活性差异有关。     

Cytoskeletal characterization of a neuroectodermal cell line and embryonic mouse brain

The cytoskeleton of a newly isolated mouse embryo brain-derived cell line and of dissected mesencephalon-rhombencephalon samples of the 10- to 11-day-old mouse embryo, constituted of a ventricular zone only, has been examined by immunocytochemistry, electrophoretic separation and Western blotting techniques. In accordance with their epithelial organization, the ventricular cells contain cytokeratins as main constituents of their cytoskeleton. Vimentin has been also revealed as early as day 10.5 of gestation. The complex cytokeratin polypeptide pattern puts this histological type of epithelia into the category of complex, rather than simple, epithelia and calls for explanations other than keratinization for the presence and functional role of the high-molecular weight, basic keratins. The cytoskeletal composition of the embryo brain-derived gliogenic cell line reflects the vimentin- and cytokeratin-positive character of the ventricular cells.     

Glucocorticoid induction of the α2,6 sialyltransferase enzyme in a mouse neural cell line

Combined sialyltransferase (ST) activities were induced in the HN9 hippocampal cell line following treatment with the synthetic glucocorticoid dexamethasone (dex) for 24 hr. Induction occurred in a dose-dependent manner, with the maximum induction of a 2-fold increase over control enzyme levels occurring at a concentration of 10^-8 M dex. A minimum of 6 hr pretreatment with drug was required before significant induction could be detected and elevated enzyme levels persisted for up to 48 hr post-treatment. The induced form of the enzyme showed an increase in reaction maximum velocity (V_max) while showing no change in affinity (K_m) for the acceptor substrate asialofetuin. The α2,6 ST enzyme was demonstrated to be the primary enzyme induced and there was no change in expression of the α2,3 ST enzyme. Lectin blot analysis demonstrated an increase in the levels of the α2,6-linked cellular sialoglycoproteins and a parallel decrease in the α2,3 sialoglycoprotein levels. J. Neurosci. Res. 51:619–626, 1998. © 1998 Wiley-Liss, Inc.     

Up-regulation of the Ire1-mediated signaling molecule, Bip, in ischemic rat brain.

The endoplasmic reticulum (ER) is thought to play important roles in various neurological diseases via multifactorial and complex mechanisms. The Ire1-mediated signal is part of one ER signaling pathways; the signal induces the expression of an ER-resident protein, Bip/GRP78, and is thought to be involved in cell death under ER stress. In this study, we examined time-dependent Bip expression after transient middle cerebral artery occlusion and characterized the Bip-positive cells. Ire1- mediated molecules, Bip, were rapidly up-regulated in the ischemic area after 3.5 h recirculation. Their immunoreactivity continued to increase until 24-48 h. Immunofluorescence staining revealed Bip up-regulation in ischemic neurons, which were TUNEL positive. Our studies suggest that the Ire1-mediated signal might be associated with ischemic neuronal damage.     

Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes

Protease-activated receptor-1 (PAR1) is a G-protein coupled receptor that is proteolytically activated by blood-derived serine proteases. Although PAR1 is best known for its role in coagulation and hemostasis, recent findings demonstrate that PAR1 activation has actions in the central nervous system (CNS) apart from its role in the vasculature. Rodent studies have demonstrated that PAR1 is expressed throughout the brain on neurons and astrocytes. PAR1 activation in vitro and in vivo appears to influence neurodegeneration and neuroprotection in animal models of stroke and brain injury. Because of increasing evidence that PAR1 has important and diverse roles in the CNS, we explored the protein localization and function of PAR1 in human brain. PAR1 is most intensely expressed in astrocytes of white and gray matter and moderately expressed in neurons. PAR1 and GFAP co-localization demonstrates that PAR1 is expressed on the cell body and on astrocytic endfeet that invest capillaries. PAR1 activation in the U178MG human glioblastoma cell line increased PI hydrolysis and intracellular Ca(2+), indicating that PAR1 is functional in human glial-derived tumor cells. Primary cultures of human astrocytes and human glioblastoma cells respond to PAR1 activation by increasing intracellular Ca(2+). Together, these results demonstrate that PAR1 is expressed in human brain and functional in glial tumors and cultures derived from it. Because serine proteases may enter brain tissue and activate PAR1 when the blood brain barrier (BBB) breaks down, pharmacological manipulation of PAR1 signaling may provide a potential therapeutic target for neuroprotection in human neurological disorders.     

Expression of mRNA encoding alpha1E and alpha1G subunit in the brain of a rat model of absence epilepsy

Low voltage-activated calcium channels are thought to play a key role in the generation of spike and waves discharges characteristic of absence epilepsy. Therefore, the expression level of mRNA encoding calcium channel alpha1E and alpha1G subunits was measured in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Using quantitative RT-PCR and in situ hybridization, no difference was found in alpha1G mRNA expression between GAERS and control animals, while a decreased expression of alpha1E was seen in the cerebellum and the brain stem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed.     

Characterization of a multipotent neural progenitor cell line cloned from an adult p53-/- mouse cerebellum

Here we report developmental characteristics of clonal cell line 2Y6f1, which was established from an adult p53(-/-) mouse cerebellum. 2Y6f1 began as a homogeneous population of small polygonal epithelial cells, but during passages it gradually became heterogeneous, containing cells of varying size and shape that expressed either neuron- or astrocyte-specific proteins. Supplements to the culture medium altered the levels of some of the cell type markers. For example, addition of insulin increased expression of neurofilaments, while cholera toxin increased that of glial fibrillary acidic protein. In a colony assay, 2Y6f1 cells gave rise to both homogeneous and heterogeneous colonies, consistent with the idea that they contained multipotent neural progenitor cells. Establishment of subclones that were exclusively neuronal or astroglial in differentiation further supported the conclusion that 2Y6f1 cells have many features that may qualify them as bona fide stem cells and make them a useful new model in neural stem cell biology.     

异体神经干细胞脑移植后免疫排斥反应的研究

目的 探讨中枢神经系统异体神经干细胞移植后免疫排斥反应的问题. 方法 选取同一批次的成年克隆山羊8只,采用随机数字表法分成2组:神经干细胞移植组和对照组.采用开颅手术的方式分别将异体神经干细胞及相同剂量的生理盐水移植到山羊脑皮层下,抽血测定移植后2组动物不同时间点(移植前1周、移植当时、移植后1周、3周及3个月)血中IL-2及IL-10的水平,以了解移植后动物全身免疫排斥反应的情况;并通过免疫组化的方法 检测移植局部病理切片CD3+细胞浸润的情况,了解移植局部免疫排斥反应的情况. 结果神经干细胞移植组在移植后1周、3周及3个月时血中IL-2水平较对照组明显升高,IL-10水平较对照组明显下降,差异均有统计学意义(P＜0.05).移植局部在急性期(细胞移植后1周)和慢性期(细胞移植后3个月)两个时间点均有大量CD3+细胞浸润,与对照组相比差异均有统计学意义(P＜0.05). 结论 中枢神经系统异体神经干细胞移植后可发生急、慢性全身和局部免疫排斥反应.     

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.     

Calcyon in the rat brain: cloning of cDNA and expression of mRNA

Calcyon is a 24 kD protein recently cloned from a human brain cDNA library and shown to interact with the dopamine receptor 1 (D1R) of D1-like receptors. This interaction shifts the effector coupling of D1R to stimulate a calcium signaling pathway, without influencing the D1R-adenylyl-cAMP pathway. To obtain more knowledge about the potential role of calcyon in the brain, we cloned rat calcyon cDNA and studied its distribution in the brain. Northern blot analysis and RT-PCR revealed that rat calcyon mRNA was expressed only in the brain. With the use of the in situ hybridization technique, we studied rat calcyon mRNA distribution in the brain and related it to the distribution of D1R and dopamine receptor 5 (D5R) mRNAs. Prominent calcyon mRNA signals were found in several brain regions, including hippocampus, hypothalamus, cerebellum, and medial prefrontal cortex. Less abundant calcyon mRNA expression was observed in the dorsal striatum region, where D1R mRNA is highly expressed and where D1R/cAMP-DARPP-32 signaling pathway is of great functional importance. The strongest expression of D5R mRNA was found in the hippocampus and cerebellum, where D1R mRNA expression was relatively low. In conclusion, rat calcyon appears to be a brain specific protein. There is a certain overlap between calcyon mRNA distribution and that of the D1R and D5 mRNAs, indicating that calcyon might be associated not only with D1R but also with D5R.     

Immunohistochemical analysis of the localization of guanine nucleotide-binding protein in the mouse brain

A guanine nucleotide-binding protein, G0, is a heterotrimer with the alpha- and beta gamma-subunits (referred to here as alpha 0 and beta gamma, respectively). We examined the distribution pattern of the anti-alpha 0 and anti-beta gamma immunoreactive products in the hippocampus, and cerebral and cerebellar cortices of the mouse brain. In the hippocampus, alpha 0- and beta gamma-immunoreactivities were localized in the neuropil of the stratum oriens, stratum radiatum and stratum lacunosum-moleculare, but were absent from the cell bodies of the pyramidal cells and their apical dendritic shafts. In the cerebral cortex, alpha 0- and beta gamma-immunoreactivities were seen in the neuropil of all 6 layers of the cerebral cortex, especially in the uppermost molecular layer (layer I), and were absent from cell bodies of neurons and their apical dendritic shafts. In the cerebellar cortex, the molecular layer was heavily stained with anti-alpha 0 and beta gamma-antibodies. The present study revealed that the distribution pattern of beta gamma-immunoreactivities in these structures of the mouse brain was strikingly similar to that of the alpha 0-immunoreactivities.     

Characterization of Gs alpha mRNA transcripts in primary cultures of rat brain astrocytes.

A cDNA clone encoding a stimulatory G-protein alpha subunit (Gs alpha) was isolated from a cDNA library derived from cultured rat astrocytes. The nucleotide sequence of the cDNA indicated that it corresponds to the Gs alpha-2 form of Gs alpha mRNA, one of four Gs alpha mRNAs known to be derived by alternative splicing from the human Gs alpha gene. A ribonuclease protection assay using cRNA from this clone allowed distinction between the Gs alpha-1 and Gs alpha-2 mRNAs, which encode the 52-kDa (Gs-L) forms of Gs alpha. Astrocytes express relatively high amounts of Gs alpha-1 mRNA, much lower amounts of the Gs alpha-2 mRNA, and no detectable amounts of the mRNAs (Gs alpha-3 and Gs alpha-4) encoding the two 45-kDa forms of Gs alpha (Gs alpha-S). Similar results were obtained with RNA samples isolated from whole brain. The 45-kDa form of Gs alpha protein was not detectable by immunoblot analysis of a membrane preparation from rat cerebral cortex (the source of the astrocyte cultures). These results indicate that the expression of Gs alpha forms in astrocytes is similar to that found in whole brain.     

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.     

HDAC inhibitors dysregulate neural stem cell activity in the postnatal mouse brain

The mammalian central nervous system (CNS) undergoes significant expansion postnatally, producing astrocytes, oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy, a condition commonly treated with valproate/valproic acid (VPA), a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs, however, may be essential for the differentiation of distinct subpopulations of neurons and glia. Here, we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7–P14, combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG, rostral migratory stream (RMS), and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed, however, that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA, OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity, we found that short term treatment with VPA in vivo reduced neurosphere numbers and size, a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor, Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively, these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered, especially in individuals whose brains are actively undergoing key postnatal time windows of development.