Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain.     

Expression of the calmodulin-dependent protein phosphatase, calcineurin, in rat brain: developmental patterns and the role of nigrostriatal innervation.

The distribution of neurons expressing the calmodulin-dependent protein phosphatase, calcineurin (CN) was characterized in developing and adult rat brain using a combination of immunocytochemical, immunoblot and in situ hybridization approaches. Immunoblot analysis revealed a strong increase postnatally in CN protein expression. Four differently-charged isoforms of CN were observed in adult brain with apparent regional differences in isoform expression. Immunocytochemistry showed highest levels of CN in hippocampus, striatum, substantia nigra, amygdala and septal nuclei with immunoreactivity first appearing in striatum and septal nuclei, followed by hippocampus, neocortex and limbic structures. In situ hybridization demonstrated that mRNA for the catalytic subunit of CN was seen as early as postnatal day (PND) 1 in striatum, cortex and hippocampus. Since immunoreactivity was not detectable until day 4, this suggests that mRNA expression may precede that of protein by several days in these regions. Lesioning of developing and adult nigrostriatal dopamine neurons either with 6-hydroxydopamine or by surgical hemitransection had little effect on expression of CN, suggesting that CN expression is not influenced transsynaptically by dopamine. Collectively, these findings demonstrate that CN protein and mRNA expression are subject to regional and temporal control during brain development suggesting that specific synaptic connections may influence CN gene expression. However, in striatum, dopaminergic innervation does not appear to affect CN levels.     

Ontogeny of somatostatin mRNA-containing perikarya in the rat central nervous system.

The distribution of neuronal perikarya containing somatostatin mRNA in the developing rat brain was investigated with in situ hybridization histochemistry. This study describes the expression of somatostatin mRNA during selected perinatal stages and demonstrates regional changes in somatostatin mRNA expression at the single cell level. The mRNA expression closely parallels previously reported developmental localization of the peptide (Inagaki et al., 1982; Shiosaka et al., 1982). As early as embryonic day 13 (E13), somatostatin mRNA was observed in discrete spinal cord and brainstem regions. At birth, densely hybridized somata could be seen primarily in ventral and caudal brain areas with small scattered neurons in the hippocampus and dorsal neocortex. After birth, somatostatin mRNA increased in forebrain regions, such as the hippocampus, dorsal neocortex, and caudate. By postnatal day 14 (P14), the distribution in the telencephalic and diencephalic regions approached that of the adult brain. Several brain regions manifested large changes in the density of somatostatin mRNA hybridization during development. For example, the cerebellar vermis and brainstem contained somatostatin mRNA perikarya during early postnatal development but decreased in these regions in the adult. During perinatal development, increases in somatostatin mRNA content were the results of increases in both the number of neurons containing somatostatin mRNA as well as in the amount of this mRNA expressed in each cell. As the brain differentiates, the apparent numbers of somatostatin mRNA containing neurons in certain brain regions are reduced. These data provide evidence for transient somatostatinergic neurons during early development in discrete areas of the occipital cortex, pyriform cortex, cerebellum, and brainstem and suggest that this peptide may play a role in the development of these regions.     

Differential expression of gamma-aminobutyric acid type B receptor-1a and -1b mRNA variants in GABA and non-GABAergic neurons of the rat brain

To understand the heterogeneity of gamma-aminobutyric acid type B receptor (GABABR)-mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA and non-GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive in situ hybridization histochemistry and, in combination with GABA immunocytochemistry, double labeling. In situ hybridization with a pan probe, which recognizes a common sequence of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did not display significant levels of either GABABR1a or GABABR1b mRNAs. These data suggested widespread availability of GABABR-mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain structures may contribute to the heterogeneity of GABABR-mediated inhibition. Some GABA neurons possessed, but others might lack the molecular machinery for GABABR-mediated disinhibition, autoinhibition, or both.     

Increased Cyclin G1 Immunoreactivity During Alzheimer's Disease

Numerous proteins are alternatively expressed in neurons and glia during Alzheimer's disease (AD) and may contribute to the regulation of neuronal cell death or function in regenerative responses to neuronal injury. A recently described member of the cyclin gene family, cyclin G1, is expressed in post-mitotic neurons in the adult rat brain and is expressed at high levels after brain injury. In the current study we examined the expression and subcellular distribution of cyclin G1 in non-demented adult and AD brain. While low levels of cyclin G1 protein were observed in pyramidal neurons in control brain, abundant cyclin G1 immunoreactivity was present in the cytoplasm of pyramidal neurons in the neocortex and hippocampus of AD brain. Cyclin G1 immunoreactivity was not present in cells containing neurofibrillary pathology. Our results indicate that cyclin G1 is expressed in human adult brain and exhibits increased immunoreactivity in the cytoplasm of pyramidal neurons in AD. In addition, cyclin G1 immunoreactivity was not evident in cells containing cytoskeletal pathology.     

Localization of neuregulin-1alpha (heregulin-alpha) and one of its receptors, ErbB-4 tyrosine kinase, in developing and adult human brain

Using immunohistochemistry, Western blot analysis, and RT-polymerase chain reaction, we studied the distribution of neuregulin-1 splice variant alpha (NRG-1alpha) and one of its putative receptors, ErbB-4 tyrosine kinase, in human brain. In the pre- and perinatal human brain immunoreactivity was confined to numerous neurons, with the highest cell density found in cortical gray matter, hypothalamus and cerebellum. In the adult brain, single cortical gray and white matter neurons showed NRG-1alpha immunoreactivity. Occasionally, immunoreactive oligodendrocytes were observed. NRG-1alpha-expressing neurons were also found in the hypothalamus, hippocampus, basal ganglia and brain stem. Application of two antibodies recognizing alpha and beta isoforms revealed a different distribution pattern in that many cortical and hippocampal pyramidal neurons were labeled. ErbB-4 immunoreactivity was expressed in both neurons and oligodendrocytes. Our data show that NRG-1alpha expression is lower in the adult human brain than in the developing brain, and, therefore, support a role for NRG-1alpha in brain development.     

Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain

Cytosolic tyrosine kinases play a critical role both in neural development and in adult brain function and plasticity. Here we isolated a cDNA with high homology to human Ack1 and mouse Tnk2. This cDNA directs the expression of a 125-kD protein that can be autophosphorylated in tyrosines. Initially, this clone was named Pyk1 for proline-rich tyrosine kinase (Lev et al., 1995); however, since it corresponds to the mouse homolog of Ack1, here we called it Ack1/Pyk1. In this study we show that Ack1/Pyk1 mRNA and protein is highly expressed in the developing and adult brain. The highest levels of Ack1/Pyk1 expression were detected in the hippocampus, neocortex, and cerebellum. Electron microscopy studies showed that Ack1/Pyk1 protein is expressed in these regions both at dendritic spines and presynaptic axon terminals, indicating a role in synaptic function. Furthermore, we demonstrate that Ack1/Pyk1 mRNA levels are strongly upregulated by increased neural activity, produced by intraperitoneal kainate injections. During development, Ack1/Pyk1 was also expressed in the proliferative ventricular zones and in postmitotic maturing neurons. In neuronal cultures, Ack1/Pyk1 was detected in developing dendrites and axons, including dendritic tips and growth cones. Moreover, Ack1/Pyk1 colocalized with Cdc42 GTPase in neuronal cultures and coimmunoprecipitated with Cdc42 in HEK 293T cells. Altogether, our findings indicate that Ack1/Pyk1 tyrosine kinase may be involved both in adult synaptic function and plasticity and in brain development.     

Selective decrease in NR1 subunit splice variant mRNA in the hippocampus of Pb2+-exposed rats: implications for synaptic targeting and cell surface expression of NMDAR complexes

We have previously shown that exposure to environmentally relevant levels of Pb(2+) during brain development decreases the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) and NR2A genes in the hippocampus of young adult rats and was associated with deficits in hippocampal LTP and spatial learning [Neuroscience 99 (2000) 233-242]. In the present study, we demonstrate that the lower levels of NR1 subunit mRNA expressed in the Pb(2+)-exposed hippocampus are principally due to decreased levels of the NR1-4 and NR1-2 splice variants. These changes were present in the absence of changes in GluR1, PSD-95 and alphaCaMKII gene expression. A unique characteristic of these splice variants is that they lack the C1 cassette. Further, these splice variants have been shown to impart the highest cell surface expression, PKC potentiation and calcium kinetics to NMDAR complexes. Our present findings indicate that Pb(2+)-induced changes in NR1 subunit splice variant mRNA expression in the hippocampus may provide a mechanism by which Pb(2+)-exposure can modify NMDAR-mediated calcium signaling and influence the degree of synaptic plasticity.     

Identification of PSF, the polypyrimidine tract-binding protein-associated splicing factor, as a developmentally regulated neuronal protein.

The polypyrimidine tract-binding protein-associated splicing factor (PSF), which plays an essential role in mammalian spliceosomes, has been found to be expressed by differentiating neurons in developing mouse brain. The sequence of a fragment of mouse PSF was found to be remarkably similar to that of human PSF. Both the expression of PSF mRNA in cortex and cerebellum and PSF immunoreactivity in all brain areas were high during embryonic and early postnatal life and almost disappeared in adult tissue, except in the hippocampus and olfactory bulb where various neuronal populations remained PSF-immunopositive. Double-labeling experiments with anti-PSF antibody and anti-neurofilaments or anti-glial fibrillary acidic protein antibodies on sections of cortex, hippocampus, and cerebellum indicate that PSF is expressed by differentiating neurons but not by astrocytic cells. In vitro, mouse PSF was found to be expressed by differentiating cortical and cerebellar neurons. Radial glia or astrocyte nuclei were not immunopositive; however, oligodendrocytes differentiating in vitro were found to express PSF. The restricted expression of PSF suggests that this splicing factor could be involved in the control of neuronal-specific splicing events occurring at particular stages of neuronal differentiation and maturation.     

Intracortical Regionality Represented by Specific Transcription for a Novel Protein, Latexin

The monoclonal antibody (mAb) PC3.1 recognizes a subset of neurons distributed in the infragranular layers of the lateral neocortex of the rat. Immunoaffinity chromatography with mAb PC3.1 showed that this antibody specifically binds a peptide epitope on a 29 kDa protein named latexin. To study the molecular details of the protein, we isolated four independent cDNA clones for latexin from cDNA libraries of the rat cerebral cortex and whole brain using the amino acid sequences of latexin fragments. Analysis of these cDNA clones showed that the predicted primary structure of latexin consists of 223 amino acids, and has no strict homology to any sequences so far known. Western and Northern blots demonstrated that the latexin and its mRNA were expressed predominantly in neural tissues with some expression in non-neural tissues. The gene that encodes latexin in the rat appeared to have homologues in other mammalian species and in the chick. In situ hybridization showed that latexin mRNA is synthesized in a subset of neurons in the lateral but not the dorsal neocortex, and that the distribution profile of these neurons is quite similar to that of neurons expressing latexin. These results indicate that latexin is a novel class of neuronal protein which represents intracortical regionality, and suggest that the regional specification of the neocortex involves selective parcellation of neurons which express a particular gene.     

Identification of the novel developmentally regulated gene, Bdm2, which is highly expressed in fetal rat brain

Most of the neurogenesis take place during the embryonic stage; the genes expressed predominantly in this stage may play important roles in the control of development of the central nervous system. Using a differential display method, we identified the novel rat gene, brain development-related molecule 2 (Bdm2), that is expressed more abundantly in the embryonic brain than in the adult brain. Full-length Bdm2 cDNA consists of 1842 base pairs (bp) and contains an open reading frame of 1260 bp. Northern blot analysis demonstrated that Bdm2 was strongly expressed in the late embryonic brain and was still detected at lower levels in an early postnatal period; in adults, Bdm2 mRNA was decreased to an undetectable level in brain, though the expression of this mRNA was revealed in other tissues. Level of Bdm2 mRNA was maintained during neuronal differentiation of mouse embryonal carcinoma cell P19, but decreased during the differentiation to glial and unidentified non-neuronal cells. In situ hybridization study demonstrated the wide distribution of Bdm2 mRNA in the embryonic brain; in the adult brain, the hybridization signals became more restricted to the hippocampus, olfactory bulb, cerebellum, and neocortex, almost coinciding with the regions where nascent and immature neurons are present. Thus, it appears likely that Bdm2 encodes a protein that is involved in both the regulation of growth of undifferentiated neural cells and the terminal differentiation of neuronal cells.     

Functional characterization of two splice variants of rat bad and their interaction with Bcl-w in sympathetic neurons

Neuronal cell death is in many cases regulated by competitive interactions between pro- and antiapoptotic proteins of the Bcl-2 family. In this study we have identified two splice variants of the rat proapoptotic molecule Bad, which differ in their carboxy-terminal regions. Both splice variants of Bad interacted with the antiapoptotic molecule Bcl-w as shown by yeast two-hybrid assay and by co-immunoprecipitation experiments from transfected cells. mRNA expression for the two variants of bad were detected in all neonatal and adult rat tissues tested. Overexpression of either of the two isoforms of Bad in nerve growth factor (NGF)-maintained sympathetic neurons by microinjection induced the cell death of these neurons, which was neutralized by co-expression of Bcl-w. Overexpression of Bcl-w in sympathetic neurons also counteracted death induced by NGF deprivation, which was not reduced by co-expression of either of the two Bad variants. The results suggest that Bcl-w, Bad-alpha, and Bad-beta may participate in the regulation of apoptosis in the sympathetic nervous system.     

Tenascin mRNA isoforms in the developing mouse brain

The extracellular matrix glycoprotein tenascin is expressed in the developing mouse cerebellum as a group of four protein species of different molecular weights. The difference is most likely due to alternative splicing which is known to occurr in tenascin mRNA isoforms that would account for this heterogeneity, tenascin splice variants were isolated from mouse brain by the polymerase chain reaction (PCR). In agreement with Northern blot analysis, amplification by PCR revealed a general decrease in tenascin mRNA expression during development from embryonic and early postnatal to adult stages. This decrease was more pronounced for isoforms of high molecular weight compared to those of low molecular weight. In accord with the observations at the protein level, four splice variants were found to be predominantly expressed, containing insertions of either six, five, or one fibronection type III repeat, or comprising no insertion. In addition, a minor splice variant with an insertion of four fibronectin type III repeats was isolated. Three of the isolated mRNA splice variants have not yet been described for mouse tenascin. Among them, an isoform containing six alternatively spliced repeats was found to include a novel fibronectin type III repeat. The sequence of this repeat displays 96.7% similarity to a corresponding type III repeat in human tenascin, revealing a strict evolutionary conservation between tenascin molecules from different species in the region of alternative splicing. Southern blot analysis of the amplified mRNA isoforms showed that the novel mouse type III repeat is confined to splice variants with an insertion of six fibronectin type III repeats. Furthermore, in situ hybridization on sections from mouse embryos indicated that tenascin-specific mRNAs containing the novel type III repeat are predominantly expressed in the central nervous system. © 1994 Wiley-Liss, Inc.     

GABA receptor rho subunit expression in the developing rat brain

Ionotropic GABA(C) receptors are composed of rho1, rho2 and rho3 subunits. Although the distribution of rho subunit mRNAs in the adult brain has been studied, information on the developmental regulation of different rho subunits in the brain is scattered and incomplete. Here, GABA(C) receptor rho subunit expression was studied in the developing rat brain. In situ hybridization on postnatal brain slices showed rho2 mRNA expression from newborn in superficial gray layer (SGL) of superior colliculus (SuC), and from the first postnatal week in the hippocampal CA1 region and pretectal nucleus of the optic tract. rho2 mRNA was also expressed in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three rho subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, rho2 mRNA expression clearly dominated over rho1 and rho3, whereas in the superior colliculus, rho1 mRNA expression levels were similar to rho2. In both areas, a clear up-modulation of rho2 and rho3 mRNA during the first postnatal week was detected. GABA(C) receptor protein expression was confirmed in adult hippocampus, superior colliculus and dorsal lateral geniculate nucleus by immunohistochemistry. Our results demonstrate for the first time the expression of all three rho subunit mRNAs in several regions of the developing and adult rat brain. Our quantitative data allows assessment of putative subunit combinations in the superior colliculus and hippocampus. From the selective distribution of rho subunits, it may be hypothesized that GABA(C) receptors are specifically involved in aspects of visual image motion processing in the rat brain.