Neurotrophin expression by spinal motoneurons in adult and developing rats.

Expression of the neurotrophins NT-4, brain-derived neurotrophic factor (BDNF), and NT-3 in adult rat lumbosacral spinal cord motoneurons is reported. A sensitive in situ hybridization procedure demonstrates localization of the mRNA for each of these neurotrophins within spinal motoneurons of the adult and in early postnatal development. A majority of adult rat spinal cord lumbar motoneurons (approximately 63%) express NT-4 mRNA as assessed by counting motoneurons in the L4 and L5 segments of two adult rat spinal cords on adjacent cresyl violet-stained and in situ hybridization sections. Similarly, a majority of lumbar motoneurons (approximately 73%) express BDNF mRNA. Further analyses of adjacent lumbar spinal cord sections revealed that many, although not all motoneurons coexpress both NT-4 and BDNF mRNAs. At birth, the mRNA encoding NT-3 is expressed in motoneurons, but BDNF mRNA is not apparent until postnatal day 5 (P5) and NT-4 mRNA first appears at P9. The potential biological significance of neurotrophin mRNA expression in spinal motoneurons is supported by immunohistochemical localization of each neurotrophin protein in adult motoneurons. We discuss the potential role of spinal cord neurotrophins as autocrine or paracrine factors involved in modulating motoneuron synaptic function.

The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.

Chondroitin sulfate proteoglycans (CS-PGs) expressed by reactive astrocytes may contribute to the axon growth-inhibitory environment of the injured CNS. The specific potentially inhibitory CS-PGs present in areas of reactive gliosis, however, have yet to be thoroughly examined. In this study, we used immunohistochemistry, combined immunohistochemistry-in situ hybridization, immunoblot analysis, and reverse transcription-PCR to examine the expression of specific CS-PGs by reactive astrocytes in an in vivo model of reactive gliosis: that is, the glial scar, after cortical injury. Neurocan and phosphacan can be localized to reactive astrocytes 30 d after CNS injury, whereas brevican and versican are not expressed in the chronic glial scar. Neurocan is also expressed by astrocytes in primary cell culture. Relative to the amount present in cultured astrocytes or uninjured cortex, neurocan expression increases significantly in the glial scar resulting from cortical injury, including the re-expression of the neonatal isoform of neurocan. In contrast, phosphacan protein levels are decreased in the glial scar compared with the uninjured brain. Because these CS-PGs are capable of inhibiting neurite outgrowth in vitro, our data suggest that phosphacan and neurocan in areas of reactive gliosis may contribute to axonal regenerative failure after CNS injury.

Neurogenesis and neuronal migration in the neonatal rat forebrain anterior subventricular zone do not require GFAP-positive astrocytes.

A prolific neuronal progenitor cell population in the anterior portion of the neonatal rat forebrain subventricular zone, the SVZa, is specialized for the production of olfactory bulb interneurons. At all ages, SVZa-derived cells traverse a tangential migratory pathway, the rostral migratory stream (RMS), while en route to the olfactory bulb. Unlike other neuronal progenitor cells of the forebrain, migrating progeny of SVZa progenitors express neuronal-specific proteins and continue to divide into adulthood. Recent studies indicate that in the adult, migrating SVZa-derived cells are ensheathed by astrocytes, although the function of these astrocytes has not been determined. To explore the possible role(s) of astrocytes in the rat SVZa and RMS, we examined the expression of astroglial-specific genes in the postnatal SVZa and RMS using RT-PCR, in situ hybridization, and immunohistochemistry during (Postnatal Days 1-10) and after the period of peak olfactory bulb interneuron generation. We also examined the expression of neuronal-specific genes throughout the rostral-caudal extent of the postnatal subventricular zone to determine if differential cell type-specific gene expression could distinguish the neurogenic SVZa as a region distinct from the remainder of the SVZ. We found little to no astrocyte-specific gene expression in the P0-P7 SVZa, although the neuron-specific isoforms of tubulin (T alpha 1 and beta-III tubulin) were expressed abundantly in the SVZa and RMS. In contrast, astrocyte-specific genes were strongly expressed in the SVZ posterior to the SVZa. GFAP expressions begins to appear in some restricted areas of the rostral migratory stream after the first postnatal week. These data suggest that astroglia are not involved in the generation or migration of most olfactory bulb interneurons. Moreover, the scarcity of glial markers in the neonatal SVZa indicates that the forebrain subventricular zone includes a distinct neurogenic anterior region containing predominantly committed neuronal progenitor cells.

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NeuN, a neuronal specific nuclear protein in vertebrates.

A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.

Expression of NGF receptor in the rat forebrain detected with in situ hybridization and immunohistochemistry.

The expression of nerve growth factor (NGF) receptor mRNA and NGF receptor protein was examined in the adult rat basal forebrain using in situ hybridization and immunohistochemical techniques. NGF receptor mRNA and protein were detected within cells in the medial septum, diagonal band of Broca, and nucleus basalis of Meynert. Controls showed that the hybridization signal was not due to nonspecific binding of the probe to heterologous RNAs or other molecules. As expected, the distribution of NGF receptor mRNA-containing cells correlated nicely with the distribution of NGF receptor immunoreactive cells in each of these areas. These data extend previous work which suggests that neurons in these areas express the NGF receptor mRNA and manufacture functional NGF receptors. NGF receptor immunoreactivity was also detected in the arcuate nucleus of the hypothalamus, in the leptomeninges at the base of the brain and overlying the tectum, and within ependymal regions along the lateral walls of the cerebral ventricles. A few weakly stained neurons in the lateral hypothalamus and ventrolateral striatum were also consistently observed. In contrast, NGF receptor mRNA was not detected within any meningial, ependymal, or hypothalamic tissues using in situ hybridization. A cross-linking/immunoprecipitation assay demonstrated normal, membrane-bound NGF receptors within extracts of dorsal superior colliculus, ventromedial hypothalamic, and overlying meningial tissues, proving that the staining observed in these areas was not a non-specific artifact associated with the immunohistochemistry. The lack of hybridization in these areas may reflect levels of NGF receptor mRNA which are too low to be detected by the in situ hybridization methods being used. Alternatively, the staining may represent innervation of these areas by afferents whose cell bodies are located elsewhere, and whose terminals contain the NGF receptor protein.

Expression of NGF and NGF receptor mRNAs in the developing brain: evidence for local delivery and action of NGF.

Nerve growth factor (NGF) is a well-documented target-derived trophic factor in the peripheral nervous system. Recently, proteins as well as mRNAs for both NGF and its receptor (NGF-R) have been detected in diverse areas in the central nervous system (CNS). Considerable evidence suggests that NGF also functions in the target synthesis/retrograde transport mode in the brain. For example, NGF is synthesized in the target hippocampus, as indicated by the presence of NGF message, and interacts with the receptors on terminals projecting from basal forebrain, where receptor mRNA is detectable. Spatial separation of NGF and receptor gene expression is consistent with the target mechanism of action. To ascertain whether local action may also occur in the CNS, we used sensitive nuclease protection assays to study the relationship of NGF and NGF-R expression in the developing brain. Our results indicate that in some brain areas, such as diencephalon, postnatal hippocampus, and olfactory bulb, NGF message was highly expressed, while receptor mRNA was virtually undetectable, suggesting that these areas serve as target sources of NGF for distant afferent neurons. By contrast, in other brain areas, such as cerebellum, striatum, perinatal olfactory bulb, and prenatal hippocampus, NGF and NGF-R mRNAs were coexpressed and coregulated developmentally. Consequently, local delivery and action of the trophic molecule may occur in these areas during these periods. We tentatively conclude that NGF may act through both distant and local modes in the developing CNS.

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.

Developmentally regulated expression of the nerve growth factor receptor gene in the periphery and brain.

Nerve growth factor (NGF) regulates development and maintenance of function of peripheral sympathetic and sensory neurons. A potential role for the trophic factor in brain has been detected only recently. The ability of a cell to respond to NGF is due, in part, to expression of specific receptors on the cell surface. To study tissue-specific expression of the NGF receptor gene, we have used sensitive cRNA probes for detection of NGF receptor mRNA. Our studies indicate that the receptor gene is selectively and specifically expressed in sympathetic (superior cervical) and sensory (dorsal root) ganglia in the periphery, and by the septum-basal forebrain centrally, in the neonatal rat in vivo. Moreover, examination of tissues from neonatal and adult rats reveals a marked reduction in steady-state NGF receptor mRNA levels in sensory ganglia. In contrast, a 2- to 4-fold increase was observed in the basal forebrain and in the sympathetic ganglia over the same time period. Our observations suggest that NGF receptor mRNA expression is developmentally regulated in specific areas of the nervous system in a differential fashion.

Expression and structure of the human NGF receptor.

The nucleotide sequence for the human nerve growth factor (NGF) receptor has been determined. The 3.8 kb receptor mRNA encodes a 427 amino acid protein containing a 28 amino acid signal peptide, an extracellular domain containing four 40 amino acid repeats with six cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155 amino acid cytoplasmic domain. The sequence of the extracellular domain of the NGF receptor predicts a highly ordered structure containing a negatively charged region that may serve as the ligand-binding site. This domain is conserved through evolution. Transfection of a full-length cDNA in mouse fibroblasts results in stable expression of NGF receptors that are recognized by monoclonal antibodies to the human NGF receptor and that bind [125I]NGF.

Gene transfer and molecular cloning of the human NGF receptor.

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

The nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias.

Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12-q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differentiated acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21.