Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.     

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation [published erratum appears in J Cell Biol 1998 Dec 28;143(7):following 2090]

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.     

The differential distribution of beta tubulin mRNAs in individual mammalian brain cells

It has been shown by in vitro translation of polyadenylated messenger RNAs (poly(A)+ mRNAs) that the mRNAs encoding both alpha and beta tubulin isotypes are present at much higher relative levels in the developing rat brain than they are in the adult, suggesting that the requirements for tubulin subunits vary with cell type and/or with the developmental stages of a particular cell type. The postnatally developing rat cerebellum, with its readily identifiable cell populations that perform the gamut of developmental tasks, is a suitable model for analyzing specific cellular mRNA distributions during development. In this report, by in situ hybridization techniques it is shown that, by comparison to total cellular poly(A)+ mRNA levels, there is relatively more of the total beta tubulin mRNAs in mitotically active external granule layer cells than in those in the internal granule layer. These results show that migration and differentiation of these granule cells is accompanied by a decrease in their beta tubulin mRNA levels relative to the levels in granule cells of the external granule cell layer. Furthermore, the relative levels of beta tubulin mRNA both in the prenatally formed Purkinje cells and the postnatally formed stellate cells are two to fourfold less than in the granule cells of the internal granule cell layer.     

Expression and functional interaction of hepatocyte growth factor- scatter factor and its receptor c-met in mammalian brain

Hepatocyte growth factor-scatter factor (HGF-SF) is a pleiotropic cytokine with mito-, morpho-, and motogenic effects on a variety of epithelial and endothelial cells. HGF-SF activity is mediated by the c- met protooncogene, a membrane-bound tyrosine kinase. Here, we demonstrate that both genes are expressed in developing and adult mammalian brains. HGF-SF mRNA is localized in neurons, primarily in the hippocampus, the cortex, and the granule cell layer of the cerebellum, and it is also present at high levels in ependymal cells, the chorioid plexus, and the pineal body. c-met is expressed in neurons, preferentially in the CA-1 area of the hippocampus, the cortex, and the septum, as well as in the pons. In the embryonic mouse, brain HGF-SF and c-met are expressed as early as days 12 and 13, respectively. Neuronal expression of HGF-SF is evolutionary highly conserved and detectable beyond the mammalian class. Incubation of septal neurons in culture with HGF-SF leads to a rapid increase of c-fos mRNA levels. The results demonstrate the presence of a novel growth factor-tyrosine kinase signaling system in the brain, and they suggest that HGF-SF induces a functional response in a neuronal subpopulation of developing and adult CNS.     

Localization of immunoreactive transthyretin (prealbumin) and of transthyretin mRNA in fetal and adult rat brain

We used a combination of immunohistochemical and molecular-biological techniques to investigate the localization of transthyretin (TTR) in the brains of adult and fetal rats. The immunohistochemical studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of TTR using the unlabeled peroxidase-antiperoxidase method. TTR mRNA levels were measured by Northern-blot analysis of poly (A+) RNA, followed by hybridization to 32P-labeled TTR cDNA; TTR mRNA was localized in brain tissue sections by in situ hybridization. Immunoreactive TTR was found to be specifically localized in the choroid plexus epithelial cells of adult rat brain. High levels of TTR mRNA were found in poly (A+) RNA samples obtained from the choroid plexus. In addition, the specific localization of TTR mRNA in the epithelial cells of the choroid plexus was demonstrated by in situ hybridization. Neither immunoreactive TTR nor TTR mRNA were found in other regions of adult rat brains. The levels of TTR mRNA in the choroid plexus were at least 30 times higher than those observed in the adult liver. Immunoreactive TTR was observed in the brains of fetal rats on as early as the 11th day of gestation. This immunoreactive TTR was localized in the tela choroidea, the developmental forerunner of the choroid plexus. Immunoreactive TTR was also observed in the fetal choroid plexus as it began to form (14th day of gestation) as well as in the more completely developed choroid plexus (18th day of gestation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular expression of MAP 2 kinase in rat brain

The cellular localization of microtubule-associated protein (MAP) 2 kinase mRNA in rat brain was examined by in situ hybridization histochemistry using a synthetic oligonucleotide probe. MAP 2 kinase was expressed in both neuronal and non-neuronal cells. ‘Areas of high density of mRNA label by the MAP 2 kinase probe appeared to be associated with high cellular packing density. Thus, MAP 2 kinase expression was particularly high in regions such as the locus coeruleus, the piriform cortex, the dentate gyrus granule cell layer, pyramidal cells of the hippocampus, the mitral cells of the olfactory bulb, and the large motor neurons of the V and VII nerves. This apparent ubiquitous distribution suggests an important role of MAP 2 kinase in the cellular functions in most cells of the adult brain.     

Expression of ribosomal protein L4 (rpL4) during neurogenesis and 5-azacytidine (5AzC)-induced apoptotic process in the rat

5-Azacytidine (5AzC) induces neuronal apoptosis in rat and mouse fetuses. 5AzC also induces apoptosis in undifferentiated PC12 cells, and ribosomal protein L4 (rpL4) mRNA expression increases prior to apoptosis. To clarify the roles of rpL4 during neurogenesis, we first examined the distribution of rpL4 mRNA in the developing rat brain by in situ hybridization and RT-PCR, and compared the results to the distribution of TUNEL- or PCNA-positive cells. rpL4 mRNA expression was strong in the ventricular zone (VZ), subventricular zone (SVZ), cortical plate (CP), cerebral cortex, granule cell layer (GCL), pyramidal cell layer (Py) and external granular layer (EGL) during embryonic and early postnatal days, and it was remarkably weakened thereafter. A lot of PCNA-positive cells were observed in VZ, SVZ, and EGL during embryonic and early postnatal days, and such distribution of PCNA-positive cells was almost identical to rpL4 mRNA distribution. Only few TUNEL-positive cells were observed in VZ, SVZ, cerebral cortex, EGL, and hippocampus during embryonic and early postnatal days, and the regions with TUNEL-positive cells were not identical to rpL4 mRNA distribution. Next, the changes of rpL4 mRNA expression in the brain of 5AzC-treated rat fetuses were examined by in situ hybridization and RT-PCR. Apoptotic cells appeared at 9 to 24 hours after treatment (HAT). However, the rpL4 mRNA expression was unchanged during the apoptotic process. From the results, it is suggested that rpL4 would have certain roles in cell proliferation and differentiation during neurogenesis, but have no roles in 5AzC-induced apoptosis in the fetal brain.     

Neuronal expression of myeloperoxidase is increased in Alzheimer's disease

Myeloperoxidase, a heme protein expressed by professional phagocytic cells, generates an array of oxidants which are proposed to contribute to tissue damage during inflammation. We now report that enzymatically active myeloperoxidase and its characteristic amino acid oxidation products are present in human brain. Further, expression of myeloperoxidase is increased in brain tissue showing Alzheimer's neuropathology. Consistent with expression in phagocytic cells, myeloperoxidase immunoreactivity was present in some activated microglia in Alzheimer brains. However, the majority of immunoreactive material in brain localized with amyloid plaques and, surprisingly, neurons including granule and pyramidal neurons of the hippocampus. Confirming neuronal localization of the enzyme, several neuronal cell lines as well as primary neuronal cultures expressed myeloperoxidase protein. Myeloperoxidase mRNA was also detected in neuronal cell lines. These results reveal the unexpected presence of myeloperoxidase in neurons. The increase in neuronal myeloperoxidase expression we observed in Alzheimer disease brains raises the possibility that the enzyme contributes to the oxidative stress implicated in the pathogenesis of the neurodegenerative disorder.     

Temporal and spatial patterns of expression of p35, a regulatory subunit of cyclin-dependent kinase 5, in the nervous system of the mouse

The protein p35 is a regulatory subunit of cyclin-dependent kinase 5. It has no recognized homology to cyclins but binds to and activates cyclin-dependent kinase 5 directly in the absence of other protein molecules. Cyclin-dependent kinase 5 was initially isolated by homology to the key cell cycle regulator cdc2 kinase and later identified as a neuronal kinase that phosphorylates histone H1, tau or neurofilaments. This kinase is localized in axons of the developing and mature nervous system. To understand the role of p35 as a regulator of cyclin-dependent kinase 5 activity in the CNS, we examined the pattern of expression of p35 mRNA in the nervous system of embryonic, early postnatal and adult mice. In separate experiments, we also examined the spatial distribution of cyclin-dependent kinase 5 mRNA and the activity of cyclin-dependent kinase 5/p35 kinase complex. Postmitotic cells express p35 mRNA immediately after they leave the zones of cell proliferation. It is also expressed in developing axonal tracts in the brain. Cyclin-dependent kinase 5 mRNA is present in postmitotic and in proliferative cells throughout the embryonic central nervous system. During early postnatal period signal for p35 mRNA declines while that for cyclin-dependent kinase 5 mRNA increases throughout the brain. In the adult brain although both p35 and cyclin-dependent kinase 5 mRNAs are expressed at relatively high levels in certain structures associated with the limbic system, considerable differences exist in the patterns of their distribution in other parts of the brain. These data suggest that the p35/cyclin-dependent kinase 5 complex may be associated with early events of neuronal development such as neuronal migration and axonal growth while in the limbic system of the mature brain it may be associated with the maintenance of neuronal plasticity.     

Localization of type I insulin-like growth factor receptor messenger RNA in the adult rat brain by in situ hybridization.

Using multiple 35S-labeled oligonucleotide probes concurrently, the type I insulin-like growth factor receptor (IGF-I-R) mRNA was demonstrated by Northern blot hybridization in newborn and adult rat brain as a single species of approximately 11 kilobases. The probes were used to localize IGF-I-R mRNA by in situ hybridization in slices of adult rat brain. The highest levels of IGF-I-R mRNA expression were found in the glomerular and mitral cell body layers of the olfactory bulb, the granule cell body layers of the dentate gyrus and cerebellum, the pyramidal cell body layers of the piriform cortex and Ammon's horn, and the choroid plexus. The lowest levels of IGF-I-R mRNA expression were found in white matter. At the cellular level, IGF-I-R mRNA was expressed by a variety of neurons, by epithelial cells of the choroid plexus, and by ependymal cells of the third ventricle. Of the neuron types studied, the highest levels of IGF-I-R mRNA were consistently found in perikarya of mitral and tufted cells in the olfactory bulb, in pyramidal cells of the piriform cortex and Ammon's horn, and in granule cells of the dentate gyrus. There was a close congruency between the distribution of IGF-I binding and IGF-I-R mRNA at the regional level. Neuropil layers in the cerebral cortex, olfactory bulb, hippocampus, and cerebellum contained a high level of IGF-I binding, whereas the adjacent cell body layers contained a high level of the IGF-I-R mRNA. We conclude that in these regions, IGF-I-R mRNA is synthesized in neuronal cell bodies, and the receptors are transported to axons and dendrites in adjacent synapse-rich layers, where appropriate IGF effects are achieved.     

Regional and cellular codistribution of interleukin 1 beta and nerve growth factor mRNA in the adult rat brain: possible relationship to the regulation of nerve growth factor synthesis

We have found a regional distribution of IL 1 beta mRNA and IL 1 activity in the normal adult rat brain, which reveals at least partially a colocalization with nerve growth factor (NGF). The predominantly neuronal signal patterns were found over the granule cells of the dentate gyrus, the pyramidal cells of the hippocampus, the granule cells of the cerebellum, the granule and periglomerular cells of the olfactory bulb, and over dispersed cells of the ventromedial hypothalamus and of the frontal cortex. In these areas also the highest levels of IL 1 activity were observed. In the striatum and septum much lower levels of IL 1 beta mRNA and IL 1 activity (shown for the striatum), most likely synthesized by glial cells, could be determined. IL 1 beta-expressing cells were mainly found in brain regions that also synthesize NGF mRNA as shown by in situ hybridization. NGF mRNA could be demonstrated over pyramidal cells of the hippocampus, granule cells of the dentate gyrus, periglomerular cells of the olfactory bulb and over prefrontal cortex neurons. These data indicate that IL 1 beta, among other factors, might also play a regulatory role in the synthesis of NGF in the CNS, as has been demonstrated in the peripheral nervous system (Lindholm, D., R. Heumann, M. Meyer, and H. Thoenen. 1987. Nature (Lond.). 330:658-659).     

Localization of insulin-like growth factor-I mRNA in rat brain by in situ hybridization--relationship to IGF-I receptors

Recent evidence has demonstrated regional synthesis of insulin-like growth factor I (IGF-I) in rat brain, which is also known to contain widespread specific type I IGF receptors. In order to precisely define sites of IGF-I mRNA synthesis, and their relationship to IGF-I receptor sites, we have applied the techniques of in situ hybridization and in vitro receptor autoradiography in rat brain. Frozen sections of adult rat brain and liver were hybridized with 32P-labeled cDNA inserts for human IGF-I (780 base pairs) or a positive control transthyretin cDNA (1430 base pairs) probe, or a series of negative probes, followed by film or emulsion autoradiography. Receptor autoradiography was performed on similar sections using 125I-IGF-I in buffer, some chambers containing excess unlabeled IGF-I. Hybridization of IGF-I probe was clearly seen only in three major brain regions: the olfactory bulb, hippocampus and cerebellum, whereas transthyretin only hybridized to choroid plexus as expected, and other probes showed no hybridization. In olfactory bulb, hybridization was greatest in the internal granular and mitral cell layers, with lower levels in the glomerular layer, where IGF-I receptors were concentrated. In hippocampus, hybridization was to pyramidal cells of Ammon's horn in CA1 and CA2 layers and dentate gyrus, with some labeling in CA3. IGF-I receptors were most dense in CA2, CA3, CA4, and dentate gyrus. In cerebellum, hybridization was to the granule cell layer, with IGF-I receptors primarily in the adjacent molecular layer. We have clearly demonstrated precise sites of local IGF-I synthesis in adult rat brain, adjacent to, and sometimes overlapping sites of high density IGF-I receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer''s disease.

Clones for the amyloid beta protein precursor gene were isolated from a cDNA library prepared from the frontal cortex of a patient who had died with a histologically confirmed diagnosis of Alzheimer's disease; they were used to investigate the tissue and cellular distribution of amyloid beta protein precursor mRNA in brain tissues from control patients and from Alzheimer's disease patients. Amyloid beta protein precursor mRNA was expressed in similar amounts in all control human brain regions examined, but a reduction of the mRNA level was observed in the frontal cortex from patients with Alzheimer's disease. By in situ hybridization amyloid beta protein precursor mRNA was present in granule and pyramidal cell bodies in the hippocampal formation and in pyramidal cell bodies in the cerebral cortex. No specific labelling of glial cells or endothelial cells was found. The same qualitative distribution was observed in tissues from control patients and from patients with Alzheimer's disease. Senile plaque amyloid thus probably derives from neurones. The tissue distribution of amyloid beta protein precursor mRNA and its cellular localization demonstrate that its expression is not confined to the brain regions and cells that exhibit the selective neuronal death characteristic of Alzheimer's disease.     

Expression mapping of cytotoxic T-lymphocyte antigen-2α gene transcripts in mouse brain

Cytotoxic T-lymphocyte antigen-2alpha (CTLA-2alpha), an inhibitor peptide homologous to the proregion of mouse cathepsin L, was originally discovered and expressed in mouse-activated T-cells and mast cells. Expressed recombinant CTLA-2alpha is shown to exhibit selective inhibition to cathepsin L-like cysteine proteinases. However, its in vivo targets in mammalian tissues are yet to be identified. We carried out in situ hybridization studies to examine the expression pattern of CTLA-2alpha mRNA and determine the specific cell types synthesizing CTLA-2alpha in the mouse brain. CTLA-2alpha mRNA was detected in various neuronal populations within the telencephalon in cerebral cortices, olfactory system, septum, basal ganglia, amygdala and highest levels were observed in the hippocampus. Within the diencephalon high density of positive cells was found in mediodorsal and lateral posterior thalamic nuclei and medial habenular nucleus (MHb). In the hypothalamus, high density of CTLA-2alpha mRNA labeling was seen in the suprachiasmatic nucleus (Sch), optic tract, arcuate nucleus, and median eminence. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus were also densely labeled. Other mesencephalic expression sites were the superior colliculus, periaqueductal gray, paramedian raphe nucleus, and inferior colliculus. In the rhombencephalon, strong labeling was detected in the pontine, vestibular, and reticular nuclei. Intense expression was also noted within cerebellar cortex in Purkinje neurons and at a moderate level in granule cell layer, stellate, and basket cells. A possible function of this novel inhibitor peptide in relation to learning, memory, and diseases is discussed.     

Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system.

We have localized dihydropyridine (DHP-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel alpha 1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel alpha 1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B alpha 1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and alpha 1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.