Endogenous brain-derived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo.

Neuronal growth factors regulate the survival of neurons by their survival and death-promoting activity on distinct populations of neurons. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) promote neuronal survival via tyrosine kinase (Trk) receptors, whereas NGF and BDNF can also induce apoptosis in developing neurons through p75(NTR) receptors in the absence of their respective Trk receptors. Using mutant mice and inactivation of neurotrophins and their receptors with antibodies in rats, we show that endogenous NT-3 induces death of adult BDNF-dependent, axotomized corticospinal neurons (CSNs). When NT-3 is neutralized, the neurons survive even without BDNF, suggesting complete antagonism. Whereas virtually all unlesioned and axotomized CSNs express both trkB and trkC mRNA, p75 is barely detectable in unlesioned CSNs but strongly upregulated in axotomized CSNs by day 3 after lesion, the time point when cell death occurs. Blocking either cortical TrkC or p75(NTR) receptors alone prevents death, indicating that the opposing actions of NT-3 and BDNF require their respective Trk receptors, but induction of death depends on p75(NTR) cosignaling. The results show that neuronal survival can be regulated antagonistically by neurotrophins and that neurotrophins can induce neuronal death in the adult mammalian CNS. We further present evidence that signaling of tyrosine kinase receptors of the trk family can be crucially involved in the promotion of neuronal death in vivo.

Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system.

Multiple sclerosis is a demyelinating disorder of the central nervous system (CNS), in which an immune attack directed against myelin constituents causes myelin destruction and death of oligodendrocytes, the myelin-producing cells. Here, the efficacy of nerve growth factor (NGF), a growth factor for neurons and oligodendrocytes, in promoting myelin repair was evaluated using the demyelinating model of experimental allergic encephalomyelitis (EAE) in the common marmoset. Surprisingly, we found that NGF delayed the onset of clinical EAE and, pathologically, prevented the full development of EAE lesions. We demonstrate by immunocytochemistry that NGF exerts its antiinflammatory effect by downregulating the production of interferon gamma by T cells infiltrating the CNS, and upregulating the production of interleukin 10 by glial cells in both inflammatory lesions of EAE and normal-appearing CNS white matter. Thus, NGF, currently under investigation in human clinical trials as a neuronal trophic factor, may be an attractive candidate for therapy of autoimmune demyelinating disorders.

Brain-derived neurotrophic factor levels in the nervous system of wild-type and neurotrophin gene mutant mice.

Although brain-derived neurotrophic factor is the most abundant and widely distributed neurotrophin in the nervous system, reproducible determinations of its levels have been hampered by difficulties in raising suitable monoclonal antibodies. Following immunization of mice with recombinant fish and mammalian brain-derived neurotrophic factor, monoclonal antibodies were generated and used in an immunoassay based on the recognition of two different epitopes. Neither antibody crossreacts with neurotrophin homodimers other than brain-derived neurotrophic factor, although reactivity was detected with brain-derived neurotrophic factor/neurotrophin-3 heterodimers. As both nerve growth factor and neurotrophin-3 are known to affect the development of a variety of neurons expressing the brain-derived neurotrophic factor (bdnf) gene, this assay was used to determine levels in tissues isolated from newborn mice carrying a null mutation in the nerve growth factor (ngf) or the neurotrophin-3 (nt3) gene. Marked differences were observed between mutants and wild-type littermates in the PNS, but not in the CNS, suggesting that neither nerve growth factor nor neurotrophin-3 is a unique regulator of brain-derived neurotrophic factor levels in the newborn mouse CNS.

Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation?

Brain-derived neurotrophic factor (BDNF) has potent effects on neuronal survival and plasticity during development and after injury. In the nervous system, neurons are considered the major cellular source of BDNF. We demonstrate here that in addition, activated human T cells, B cells, and monocytes secrete bioactive BDNF in vitro. Notably, in T helper (Th)1- and Th2-type CD4(+) T cell lines specific for myelin autoantigens such as myelin basic protein or myelin oligodendrocyte glycoprotein, BDNF production is increased upon antigen stimulation. The BDNF secreted by immune cells is bioactive, as it supports neuronal survival in vitro. Using anti-BDNF monoclonal antibody and polyclonal antiserum, BDNF immunoreactivity is demonstrable in inflammatory infiltrates in the brain of patients with acute disseminated encephalitis and multiple sclerosis. The results raise the possibility that in the nervous system, inflammatory infiltrates have a neuroprotective effect, which may limit the success of nonselective immunotherapies.

Human nerve growth factor beta (hNGF-beta): mammary gland specific expression and production in transgenic rabbits.

Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-beta) cDNA were generated. Expression of hNGF-beta mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-beta was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-beta depicted a molecular weight of 13,261 Da per subunit. The biological activity of the hNGF-beta was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-beta demonstrated full biological activity when compared to commercial recombinant hNGF-beta.

Melanoma-inhibiting activity, a novel serum marker for progression of malignant melanoma.

Melanoma-inhibiting activity (MIA) was isolated previously as a small soluble protein secreted from malignant melanoma cell lines in vitro. In vivo, highly restricted expression patterns in melanocytic tumors were identified. We therefore quantitated serum levels of MIA protein by means of a nonradioactive ELISA and investigated whether MIA provides a clinically useful parameter in patients with malignant melanomas. Here, we report enhanced MIA serum levels in 13 and 23% of patients with stage I and II disease, respectively, and in 100% with stage III or IV disease. Compared with S-100 and soluble intercellular adhesion molecule 1 serum levels in these patients, MIA was the most sensitive marker. Response to therapy in stage IV disease correlated with changes in MIA serum levels. Measuring repeatedly sera of 350 patients with a history of stage I or II melanoma during follow-up, we detected 32 patients developing positive MIA values. At the time of serum analysis, 15 of them had developed metastases, and one presented with metastatic disease 6 months later. In contrast, none of the patients with normal MIA serum levels developed metastases during the follow-up period of 6-12 months. In conclusion, MIA represents a novel serum marker for systemic malignant melanoma revealing the highest sensitivity and specificity among currently available markers. Useful clinical applications include staging of primary melanomas, detection of progression from localized to metastatic disease during follow-up, and monitoring therapy of advanced melanomas.

Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies.

Mouse monoclonal antibodies were raised against bacterially expressed protein sequences of the NR2A, NR2B, NR2C and NR2D subunits of the rat NMDA receptor. From immunoblots of rat brain proteins, the apparent molecular weights of these subunits were 165, 170, 135 and 145 kDa, respectively. Proteins of similar masses were observed on immunoblots of specifically transfected HEK293 cells. Deglycosylation with endoglycosidase F reduced the mass of each endogenous NR2 subunit by approximately 10 kDa. In distribution studies, NR2A-immunoreactive protein (IRP) was located throughout the adult rat brain, NR2B-IRP was primarily in the forebrain, NR2C-IRP was predominantly in the cerebellum and NR2D-IRP was mainly found in the thalamus, midbrain and brainstem. Whereas NR2A- and NR2C-IRPs increased during rat brain post-natal development, NR2B- and NR2D-IRPs were abundant at birth and declined with age, especially in cerebellum. NR2-IRPs of mouse, rabbit, frog and human brain were of sizes similar to those of the corresponding rat subunits and were similarly distributed. In summary, NR2 subunits are large glycoproteins whose specific expression profiles in the brain are developmentally and regionally regulated and which are similarly expressed in a variety of species.

Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes.

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.

Effect of nerve growth factor on peptide neurons in dorsal root ganglia after taxol or cisplatin treatment and in diabetic (db/db) mice.

In our study we have used morphological and radio-immunological methods for the investigation of calcitonin gene-related peptide (CGRP) and substance P in cervical dorsal root ganglia (DRGs) in mice after administration of taxol or cisplatin and in spontaneously diabetic animals (db/db mice). The results were compared to findings in animals receiving recombinant human nerve growth factor (rhNGF). Morphometric analysis did not reveal any significant changes of cell size distribution in diabetic and taxol-treated mice, whereas cisplatin induced a significant decrease in the number of large- and medium-sized neurons, indicating neuronal atrophy. This finding correlated with a highly significant loss of neuropeptides after cisplatin-application. Measurement of peptide levels in the taxol-treated groups and in diabetic mice demonstrated a decrease predominantly for CGRP. Application of 10 mg/kg NGF caused a significant elevation in peptide-immunoreactivity in control animals and in taxol-treated mice, i.e., statistically significant increase in peptide concentrations and in the number of substance P- and CGRP-immunoreactive DRG-neurons, suggesting a recruitment of additional peptide cells. In diabetic animals a restoration in CGRP-content was observed under NGF-treatment; however, in this model the quantitative parameters did not demonstrate further elevation above control levels. Our data support the hypothesis that NGF exerts a major effect on the metabolism of transmitters associated with nociception and sensation in "healthy" controls and in various models of toxic and metabolic neuropathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral myelin protein-22 expression in Charcot-Marie-Tooth disease type 1a sural nerve biopsies.

Peripheral myelin protein-22 (PMP22) is expressed in myelinating Schwann cells and shows significant homology to murine growth arrest-specific gene gas3. Charcot-Marie-Tooth disease type 1a (CMT1a) is a common hereditary demyelinating neuropathy. Recently it was demonstrated that the gene for PMP22 is duplicated in CMT1a patients. A gene dosage mechanism has been postulated to cause CMT1a. According to this hypothesis, the increase in copy number of PMP22 gene would lead to an elevated expression of PMP22 and thereby cause the demyelinating phenotype of CMT1a. In the present communication we analyzed PMP22 mRNA and protein expression in sural nerve biopsies from CMT1a patients and normal controls. We show that PMP22 mRNA expression in CMT1a is not uniform. We found both elevated as well as normal PMP22 mRNA levels in patients. Interestingly, the highest PMP22 mRNA level was found in the least affected patient. In contrast to the mRNA levels, PMP22 was clearly reduced in all CMT1a patients as shown by immunohistochemistry. Thus the CMT1a phenotype may not be strictly correlated with increased PMP22 mRNA and protein expression. Possible roles of PMP22 in the pathogenesis of CMT1a are discussed.

A crucial role for neurotrophin-3 in oligodendrocyte development.

The neurotrophins nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 promote the survival of subpopulations of vertebrate neurons in vitro, but so far only nerve growth factor has been demonstrated to be essential for normal neuronal development; no neurotrophin has previously been shown to function in normal glial cell development. We found recently that neurotrophin-3 promotes the survival of pure oligodendrocyte precursor cells in vitro, and, although by itself it induces only a small percentage of these cells to synthesize DNA, in combination with platelet-derived growth factor it induces the majority of them to do so. Neither of these factors, however, has been shown to contribute to oligodendrocyte precursor cell proliferation in vivo or to stimulate pure populations of these cells to proliferate (as opposed to synthesize DNA) in vitro. Here we show that neurotrophin-3 and platelet-derived growth factor collaborate to promote clonal expansion of oligodendrocyte precursor cells in vitro and to drive the intrinsic clock that times oligodendrocyte development. We also show that neurotrophin-3 helps stimulate the proliferation of oligodendrocyte precursor cells in vivo and is thus required for normal oligodendrocyte development.

Modulation of p145c-kit function in cells of patients with acute myeloblastic leukemia.

The function of the steel factor receptor, p145c-kit, in patient-derived acute myeloblastic leukemia (AML) cells was investigated. Steel factor stimulation of AML cells coexpressing p145c-kit and the progenitor cell antigen CD34 resulted in complete receptor down-regulation, a marked decrease of CD34 antigen expression, and the induction of the granulocyte lineage antigen CD15. These changes in surface marker expression paralleled morphological differentiation to granulated blasts and promyelocytes. Interestingly, the same phenotype was achieved by IL-3 stimulation of AML cells. p145c-kit extracellular domain-specific antibodies had either blocking or enhancing effects on ligand binding, receptor phosphorylation and down-regulation, and induction of cell proliferation. Correlations of these phenomena with distinct effects of antibody stimulation on cell substrate phosphorylation provide clues for the dissection of the p145c-kit signal and the analysis of its relevance for AML.

Characterization of hemopoietic cell populations from human cord blood expressing c-kit.

Human cord blood or bone marrow cells expressing the CD34 surface antigen include a population of pluripotent progenitors. We identified and isolated a subpopulation of cells coexpressing CD34 and c-kit, a transmembrane receptor with tyrosine kinase activity. Novel monoclonal antibodies (16A6, 14A3, 3D6) directed against the extracellular domain of c-kit were used for immunofluorescence labeling and sorting of low-density mononuclear cells (MNCs) from umbilical cord blood and bone marrow. The frequency of c-kit-labeled MNCs from cord blood (mean 5.0% +/- 2.1%, n = 16) was similar to that from adult bone marrow (mean 3.7% +/- 1.3%, n = 4). On average, 1.4% of CD34-positive cells were recorded in cord blood and 2.1% in bone marrow MNCs. Roughly 60% of CD34-positive cells coexpressed c-kit. The ability of CD34+/c-kit+ cells to form multilineage colonies (CFU-GEMM) was assayed after sorting with an antibody that did not show any significant effect on c-kit ligand (RL) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation. For CD34+/c-kit+ cells, we found a 20- to 50-fold enrichment as against total MNCs, and a 2-fold enrichment if compared with the CD34+/c-kit-population. To study expression of c-kit in lymphocytic precursors, monoclonal anti-CD7 or anti-CD10 antibodies were used simultaneously. In contrast to CD34-expressing cells, however, no consistent double-labeled subpopulation of lymphocytic cells was detected. Furthermore, coexpression of CD38 (73% +/- 14%, n = 4) or CD33 (29% +/- 12%, n = 5) on a majority of c-kit-positive cells showed their lineage commitment to erythropoiesis and granulocytopoiesis.

Characterization of mono- and polyclonal antibodies against highly purified choline acetyltransferase: a monoclonal antibody shows reactivity in human brain.

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.