Expression of members of the proteolipid protein gene family in the developing murine central nervous system

Two homologous cDNAs were previously isolated by expression cloning with a monoclonal antibody that recognized a CNS neuronal membrane protein. Both cDNAs, M6a and M6b, bore significant homology with the major myelin proteolipid protein, PLP/DM20. Our initial studies of M6 gene expression in the adult mouse brain showed that M6a was present in neurons, PLP/DM20 in oligodendrocytes, and M6b in both neurons and glia. This led to the recognition of a novel gene family that included the oligodendrocyte-specific PLP/DM20 gene and the neuronal M6 genes. These observations supported the idea that PLP/DM20 may have functions other than myelination. In this report, we describe the spatial and temporal patterns of expression of M6a, M6b, and PLP/DM20 in the developing nervous system. PLP expression was limited to the white matter. M6a appeared in post-mitotic neurons of the brain and spinal cord as early as E10, and later in the hippocampus, cerebral cortex, and the granule cells of the cerebellum. In contrast, M6b was expressed at early embryonic stages in the ventricular zone of the spinal cord, and later during development in both neurons and glia. The early appearance of M6a and M6b mRNAs in the murine CNS suggested that these molecules might play an important role in the development of a variety of neural cell types. © 1996 Wiley-Liss, Inc.     

Differentially expressed olfactomedin-related glycoproteins (Pancortins) in the brain

Messenger RNA differential display is conducted to search for genes that are expressed in a region-specific pattern in the rodent brain. Eleven novel gene fragments are isolated. One of these genes which we call pancortin, based on its predominant mRNA expression in the cerebral cortex of the adult, is studied. These pancortin cDNA clones are grouped into four different types of cDNA, designated as pancortin-1 to -4. All pancortin cDNAs share a common sequence in the middle of their structure, having two alternative sequences at both 5′- and 3′-ends, respectively. Deduced amino acid sequence shows that all pancortins have sequences of hydrophobic amino acids at N-terminus and no obvious membrane spanning regions. In situ hybridization histochemistry using oligonucleotide probes specific for 5′- and 3′-end variable parts has revealed that these four pancortin mRNAs are expressed differentially in the adult rodent brain. Robust expression of pancortin-1 and -2 mRNA is observed in the cerebral cortex (including the hippocampus and the olfactory bulb). However, little of pancortin-3 and -4 mRNA is observed there. In the cortex, some neurons are stained by an antibody raised against Pancortin. Immuno-electron microscopic study has revealed that Pancortin-like immunoreactive products are localized mainly in the endoplasmic reticulum and not in the Golgi apparatus indicating that Pancortins are the endoplasmic reticulum-anchored proteins. Our results suggest that each Pancortin is differentially regulated and may perform different functions in the brain.     

Isolation of a mouse brain cDNA expressed in developing neuroblasts and mature neurons

A previously uncharacterized 4.5-kb mouse cDNA clone, designated mc7, was isolated and found to be predominantly expressed in brain. This cDNA predicts a 1035-bp open reading frame that encodes for a 345-amino acid polypeptide especially rich in glutamic acid residues located in the region from residues 80 to 174. Computational analysis revealed among other features, putative zinc-finger motifs and coiled-coil regions. The corresponding mc7 gene is detected in mouse, rat, pig and human genomes. In mouse the mc7 mRNA is expressed predominantly in brain and to a much lesser extent in kidney, lung and spleen. In brain it is detectable as early as embryonic day 14 while it is retained in the adult. In situ hybridization studies revealed that mc7 mRNA is widely, albeit unevenly, expressed in neurons throughout the adult brain. Developmental in situ hybridization studies in the cerebellar cortex demonstrated that at postnatal day 5 mc7 mRNA is mainly expressed in neuroblasts of the external granular layer and in developing neurons of the internal granular layer. Some staining is also present in purkinje cells becoming particularly pronounced at postnatal day 10, the time of arborarization of their dendritic tree. In the adult cerebellar cortex expression is mainly confined in purkinje cells and to a lesser extent in granule neurons. The early expression of mc7 in neuroblasts and developing neurons as well as its retention in a wide variety of mature neurons suggest that it may play a role in the process of differentiation and maturation of these cells in the brain.     

Identification and characterization of batk,a predominantly brain-specific non-receptor protein tyrosine kinase related to Csk

A novel cDNA, brain-associated tyrosine kinase (Batk), was isolated from a rat hippocampal library and appears to encode a new member of the Csk subfamily of non-receptor protein tyrosine kinases, with 52% overall amino acid identity to rat Csk. Batk resembles kinases of the Src family in that it contains a Src homology 2 (SH2) domain and an SH3 domain, followed by a tyrosine kinase domain. Analysis of incompletely spliced Batk cDNAs suggests that the genomic structure of Batk is similar to that of Csk with identical exon/intron boundaries. Batk also shows significant homology (86% overall amino acid identity) to the recently described human megakaryocyte-specific Matk. Although Batk is 41 amino acids shorter than Matk, Southern blot analysis suggests that Batk might be a rat homolog of Matk. Batk is predominantly expressed in the brain, with lower expression in the spleen and undetectable expression in other tissues. In situ hybridization and Northern blot analysis show that Batk is widely distributed throughout the adult brain, being primarily expressed in neurons, including those of the hippocampus and cortex. In contrast, embryos appear to have markedly decreased expression levels. Analysis of postnatal day 1 brain suggests that Batk may be upregulated at birth throughout the brain except in the cerebellum. In view of its homology to Csk, a negative regulator of Src family tyrosine kinases, and its generalized expression in the adult brain, we suggest that Batk may function as a brain-specific regulator of kinases involved in the development and maintenance of the nervous system. © 1994 Wiley-Liss, Inc.     

Localization of urokinase-type plasminogen activator mRNA in the adult mouse brain

Urokinase-type plasminogen activator (uPA) is an inducible serine protease, secreted by a variety of cell types, that functions in fibrinolysis and has been implicated also in events such as cell migration and tissue remodeling and repair. To explore the role of uPA in the adult brain, we have now screened the whole mouse brain for cells expressing the uPA gene through in situ hybridization using ³⁵S-complementary RNA. uPA mRNA was visualized predominantly in three regions: (1) the subicular complex, (2) the entorhinal cortex, (3) the parietal cortex, where the signal was somewhat lower and confined to layers IV and VI. Weaker signals were seen in the basolateral nucleus of the amygdala and in the anterodorsal thalamic nucleus, and also in the hilus of the dentate gyrus where labeling was slightly over background. Cells exhibiting uPA mRNA signaling were large neurons according to morphological criteria. These results support the view of uPA being involved in neuronal functions of the adult brain, specifically in the hippocampal formation and the parietal cortex.     

Rapid increase of Nurr1 mRNA expression in limbic and cortical brain structures related to coping with depression‐like behavior in mice

The immediate‐early gene Nurr1 is a member of the inducible orphan nuclear receptor family. Nurr1 is essential to the differentiation, maturation, and maintenance of midbrain dopaminergic neurons and is expressed in different brain regions. We have reported that adult mice with reduced Nurr1 expression displayed an increase in immobility response to acute stress. These mice were also deficient in the retention of emotional memory. Thus, Nurr1 expression seems to be relevant to normal cognitive processes. To investigate the response of Nurr1 to a stress stimulus, Nurr1 mRNA expression was examined by in situ hybridization in adult mice using a depression‐like behavior paradigm, the forced swim test. The Nurr1 gene was rapidly and widely up‐regulated throughout the brain, including cortical areas (i.e., prefrontal cortex, primary and secondary visual cortex, primary auditory cortex, and secondary somatosensory cortex), hippocampus (dentate gyrus, CA1, CA2, and CA3), and midbrain (substantia nigra pars compacta and ventral tegmental area) at 30 min and 3 hr after the forced swim test. Dopamine content was reduced in prefrontal cortex and midbrain following swim stress. These results suggest that the increase in Nurr1 expression might be a compensatory mechanism to counteract the changes in forebrain dopamine transmission in coping with acute stress. © 2010 Wiley‐Liss, Inc.     

Differential expression of the nerve growth factor receptor gene in multiple brain areas

Recent work has indicated that the trophic protein, nerve growth factor (NGF), is detectable in several brain regions, in addition to its well-known localization to the periphery. In addition, a number of cholinergic populations in the brain respond to NGF by increasing enzymes involved in acetylcholine metabolism. It is well recognized that responsiveness to NGF is dependent on expression of specific receptors; we have recently detected expression by the responsive rat basal forebrain/septal, cholinergic neurons, suggesting that NGF plays a physiologic role in the development of this brain pathway. To define a potential role for NGF in other rat brain regions, we isolated a rat receptor cDNA clone to use as a probe to detect receptor message by sensitive Sl nuclease protection experiments. Our studies indicate that the NGF receptor (NGF-R) gene is expressed by anatomically, functionally and biochemically diverse populations, widely distributed in the rat brain, and is not restricted to the basal forebrain/septal region. We detect NGF receptor message in frontal cortex, hippocampus, caudate, cerebellum and olfactory bulb. Moreover, developmental profiles of steady-state quantities varied differently for each area. Our observations support the contention that NGF regulates multiple brain systems, in addition to forebrain cholinergic pathways.     

Localization of mRNA for Ca/calmodulin-dependent protein kinase I in the brain of developing and mature rats

The gene expression of Ca²⁺/calmodulin-dependent protein kinase I (CaM kinase I) in the brain of developing and adult rats was examined by in situ hybridization histochemistry. During the development, CaM kinase I showed two chronological expression patterns; the persistent and relative high expression as observed in the olfactory bulb and cerebellar cortex, and the gradual decrease in the expression during the postnatal development as observed in most other brain regions. The gene expression was not detected in the germinal ventricular zone and cerebellar external granular layer. In the mature brain, CaM kinase I mRNA was expressed widely, though weakly in general, in almost all neurons, except for the olfactory bulb, cerebellum and hippocampus expressing at high intensity. These findings suggest that CaM kinase I may play a variety of neuronal Ca²⁺/calmodulin-mediated signaling processes in the developing and mature brains.     

Altered NGF protein levels in different brain areas after immunolesion

Nerve growth factor (NGF) provides critical trophic support to the cholinergic basal forebrain neurons that express high levels of the low-affinity NGF receptor (p75^NGFR) in the adult rat brain. Intraventricular injection of 192 IgG-saporin, made by coupling the monoclonal antibody to p75^NGFR 192 IgG to the cytotoxin saporin, selectively destroys the p75^NGFR-bearing neurons in the basal forebrain and was used here to examine the effects of selective cholinergic lesions on brain NGF protein levels. We showed that 192 IgG-saporin produced significant long-lasting elevation of NGF protein levels in the hippocampus, cortex, and olfactory bulb, with profound reductions of ChAT activities representing complete cholinergic deafferentations of these areas. NGF level was maintained in the basal forebrain, even though there was almost complete loss of p75^NGFR-immunoreactive cells and significant decrease of ChAT activity. In addition, a mild glial response was observed in the basal forebrain, and most of the activated astroglia expressed NGF-like immunoreactivity there. The increases in NGF protein levels in the target areas of the basal forebrain were most likely due to loss of cholinergic basal forebrain neurons and retrograde transport of NGF from these areas. Glial-derived NGF is partially responsible for the maintained level of NGF in the basal forebrain after the loss of cholinergic neurons. The accumulation of NGF protein in the target areas may have some effects on synaptic rearrangement in denervated tissues. © 1996 Wiley-Liss, Inc.     

Single-Nuclei RNA Sequencing of 5 Regions of the Human Prenatal Brain Implicates Developing Neuron Populations in Genetic Risk for Schizophrenia

BackgroundWhile a variety of evidence supports a prenatal component in schizophrenia, there are few data regarding the cell populations involved. We sought to identify cells of the human prenatal brain mediating genetic risk for schizophrenia by integrating cell-specific gene expression measures generated through single-nuclei RNA sequencing with recent large-scale genome-wide association study (GWAS) and exome sequencing data for the condition.MethodsSingle-nuclei RNA sequencing was performed on 5 brain regions (frontal cortex, ganglionic eminence, hippocampus, thalamus, and cerebellum) from 3 fetuses from the second trimester of gestation. Enrichment of schizophrenia common variant genetic liability and rare damaging coding variation was assessed in relation to gene expression specificity within each identified cell population.ResultsCommon risk variants were prominently enriched within genes with high expression specificity for developing neuron populations within the frontal cortex, ganglionic eminence, and hippocampus. Enrichments were largely independent of genes expressed in neuronal populations of the adult brain that have been implicated in schizophrenia through the same methods. Genes containing an excess of rare damaging variants in schizophrenia had higher expression specificity for developing glutamatergic neurons of the frontal cortex and hippocampus that were also enriched for common variant liability.ConclusionsWe found evidence for a distinct contribution of prenatal neuronal development to genetic risk for schizophrenia, involving specific populations of developing neurons within the second-trimester fetal brain. Our study significantly advances the understanding of the neurodevelopmental origins of schizophrenia and provides a resource with which to investigate the prenatal antecedents of other psychiatric and neurologic disorders.     

Amphiphysin I but not dynamin I nor synaptojanin mRNA expression increased after repeated methamphetamine administration in the rat cerebrum and cerebellum

Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3′ region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3′ UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²⁺ and Mg²⁺ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.     

原位杂交检测Sema4C基因在胚胎和成年小鼠组织中的表达

目的研究Sema4C基因在小鼠不同发育阶段的表达情况，为探讨其生物学功能提供相关线索。方法用地高辛进行标记并制备Sema4C特异性RNA探针。对于不同胎龄的胎鼠，用胚胎整体原位杂交(Wholemount)的方法检测Sema4C在不同发育阶段的表达，用组织芯片结合原位杂交的方法对Sema4C在成年小鼠不同组织中的表达进行检测。结果Sema4C基因在E11．5小鼠胚胎的前脑，眼原基，背根神经节，动脉弓中均有表达，E13．5小鼠胚胎除以上部位以外在上肢肢芽中也有部分表达。在成年小鼠的大脑皮层，小脑蒲肯野纤维层，视网膜视神经节细胞层和肾小管上皮细胞层中均有较高表达，而在肌肉，心脏，肺，脾脏，肝脏等组织中表达量比较低或未检测到表达。结论小鼠Sema4C基因除了在神经系统发育过程中持续高表达外，还在成年小鼠肾小管上皮中高表达，提示其可能在神经系统和肾脏生理功能方面发挥某种作用。     

Protease nexin I (PNI) in mouse brain is expressed from the same gene as in seminal vesicle

Protease nexin I (PNI), a serine protease inhibitor (serpin), is the most potent tissue inhibitor of thrombin. In the nervous system, PNI has been shown to participate in processes related to synaptic plasticity and neuronal survival. We assigned the human gene for PNI (PI7) to chromosome 2q33–35, and to syntenic regions in mouse chromosome 1. Others showed that a similar serpin was expressed in mouse seminal vesicle, which presented the possibility of a “duplicate” gene. The data also raised controversy over the quantity of PNI mRNA expressed in the brain vs peripheral tissues, such as seminal vesicle. In order to further our investigations of PNI regulation and its influence on neuronal survival and neuroprotection, it was necessary to confirm whether the nexin observed in mouse brain samples was identical to the published protease nexin I sequences. To accomplish this, we performed DNA sequence analysis of cDNAs made from RNAs isolated from mouse forebrain and hindbrain as well as from seminal vesicle. These confirmed the identity of the mouse PNI gene (SPI4) in brain and peripheral tissues. Furthermore, Northern hybridization studies indicated that the PNI message is present at lower levels in the adult brain compared to the adult seminal vesicle. Western immunoblotting showed no differences between brain and seminal vesicle PNI proteins. The PNI cDNAs generated will serve as useful probes for the continued characterization of the serpin:protease balance as it relates to nerve cell function.     

Developmental profile of GABAA-receptors in the marmoset monkey: Expression of distinct subtypes in pre- and postnatal brain

Gamma aminobutyric acid (GABA)_A-receptors are expressed in fetal mammalian brain before the onset of synaptic inhibition, suggesting their involvement in brain development. In this study, we have analyzed the maturation of the GABA_A-receptor in the marmoset monkey forebrain to determine whether distinct receptor subtypes are expressed at particular stages of pre- and postnatal ontogeny. The distribution of the subunits α1, α2, and β2,3 was investigated immunohistochemically between embryonic day 100 (6 weeks before birth) and adulthood. Prenatally, the α2- and β2,3-subunit-immunoreactivity (-IR) was prominent throughout the forebrain, whereas the α1-subunit-IR appeared in selected regions shortly before birth. The α2-subunit-IR disappeared gradually to become restricted to a few regions in adult forebrain. By contrast, the α1-subunit-IR increased dramatically after birth and replaced the α2-subunit in the basal forebrain, pallidum, thalamus, and most of the cerebral cortex. Staining for the β2,3-subunits was ubiquitous at every age examined, indicating their association with either the α1- or the α2-subunit in distinct receptor subtypes. In neocortex, the α1-subunit-IR was first located selectively to layers IV and VI of primary somatosensory and visual areas. Postnatally, it increased throughout the cortex, with the adult pattern being established only during the second year. The switch in expression of the α1- and α2-subunits indicates that the subunit composition of major GABA_A-receptor subtypes changes during ontogeny. This change coincides with synaptogenesis, suggesting that the emergence of α1-GABA_A-receptors parallels the formation of inhibitory circuits. A similar pattern has been reported in rat, indicating that the developmental regulation of GABA_A-receptors is conserved across species, possibly including man. However, the marmoset brain is more mature than the rat brain at the onset of α1-subunit expression, suggesting that α1-GABA_A-receptors are largely dispensable in utero, but may be required for information processing after birth. © 1996 Wiley-Liss, Inc.     

Localization and ontogeny of the orphan receptor OR-1 in the rat brain

This study describes the expression of the OR-1 orphan receptor in embryonic, postnatal, and adult brain tissue studied byin situ hybridization. This newly characterized member of the nuclear receptor superfamily functions as a modulator of retinoic acid and thyroid hormone signalling by influencing gene activation by these hormones from a distinct promoter region. In the fetal brain OR-1 mRNA was observed from E13–E16 in the developing pons, tegmentum, pontine flexure, medulla, inferior and superior colliculi, cerebellum, hippocampus, thalamus, striatum, and cortical plate. At E18, OR-1 was expressed in the hippocampus, cerebellum, ventricular layer of the developing cortex and cortical plate, striatum, and olfactory bulb. In the E21 to early postnatal brain the highest expression of OR-1 mRNA was seen in the hippocampus, cerebellum, striatum, and olfactory bulb. The expression of OR-1 in the cerebellum increased during postnatal development and by d P21 OR-1 mRNA had reached the levels present in the adult in the cerebellar cortex. In the adult brain the highest expression of OR-1 mRNA was observed in the Ca1 area of the hippocampus and the cerebellar cortex. We conclude that OR-1 is widely expressed in the fetal brain, whereas in the postnatal and adult brains OR-1 mRNA is more discretely localized, and that the amount of OR-1 mRNA increases in the cerebellum during postnatal development. The results of this study suggest that, in the fetal brain, OR-1 has a spatially widespread role in modulating gene activation by retinoids and thyroid hormone, whereas in the adult brain this modulation occurs only in distinct neuronal populations.