Ralph F. Alderson

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.

Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture.

Brain-derived neurotrophic factor (BDNF) was found to promote the survival of E17 rat embryo septal cholinergic neurons in culture, as assessed by a histochemical stain for acetylcholinesterase (AChE). A 2.4-fold increase in neuronal survival was achieved with 10 ng/ml BDNF. After initial deprivation of growth factor for 7 days, BDNF failed to bring about this increase, strongly suggesting that BDNF promotes cell survival and not just induction of AChE. BDNF was also found to increase the levels of cholinergic enzymes; choline acetyltransferase (ChAT) and AChE activities were increased by approximately 2-fold in the presence of 50 ng/ml BDNF. BDNF produced a 3-fold increase in the number of cells bearing the NGF receptor, as detected by the monoclonal antibody IgG-192. Although NGF had no additive effect with BDNF in terms of neuronal survival, suggesting that both act on a similar neuronal population, the combination of both produced an additive response, approximately a 6-fold increase, in ChAT activity.

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: Inhibition by antibodies to NGF

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.

Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes.

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors – ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) – in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors – CNTF, NGF and NT3 – at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte‐derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF‐like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte‐conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti‐NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.

Truncated TrkB mediates the endocytosis and release of BDNF and neurotrophin-4/5 by rat astrocytes and Schwann cells in vitro

Binding and cross-linking studies with radiolabeled neurotrophins demonstrate that cultured rat hippocampal astrocytes lack full-length TrkB, but do express high levels of truncated TrkB (tTrkB). In astrocytes and Schwann cells, tTrkB appears to have the novel function of mediating the endocytosis of neurotrophins into an acid-stable, Triton X-100 resistant intracellular pool that is released back into the medium in a temperature-dependent manner. Chloroquine treatment, trichloroacetic acid solubility, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that when incubated with astrocytes or Schwann cells for at least 48 h neither the intracellular nor the released neurotrophins were significantly degraded. The endocytosis and release of neurotrophins may represent a novel mechanism whereby neuroglia can regulate the local concentration of these neurotrophic factors for extended periods of time.

Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4/5 (NT‐4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron‐specific enolase‐immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT‐4/5, or NT‐3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT‐4/5 (46%), whereas NT‐3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT‐3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT‐4/5, and NT‐3, but not NGF, induced the rapid expression of the immediate early gene c‐fos. Immunocytochemical double‐labeling with antibodies to c‐fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c‐fos. After 4 days in vitro, both BDNF and NT‐3 induced the formation of c‐fos protein in calbindin‐immunopositive neurons, whereas NT‐4/5 did not. The latter results suggest that although BDNF and NT‐4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT‐4/5 are not identical.

Ciliary neurotrophic factor enhances the survival of Purkinje cells in vitro.

We have examined the effects of ciliary neurotrophic factor (CNTF) on the development of rat Purkinje cells in vitro. Cerebellar cells, derived from embryonic day 16 rat fetuses, were found to respond rapidly to CNTF treatment by induction of c‐Fos protein, such that 40% of the cells were immunopositive after 60 min. Treatment with low doses of CNTF (10‐100 pg/ml) for 8 days resulted in an ∼ 1.6‐fold increase in the number of Purkinje cells, identified by immunohistochemical staining for calbindin. Immunohistochemical staining for other Purkinje cell markers‐cyclic‐GMP‐dependent protein kinase and the low‐affinity nerve growth factor receptor‐verified increased Purkinje cell survival following CNTF treatment. In addition, CNTF increased specific high‐affinity GABA uptake by 45%, and the number of GABAergic neurons by 70%. A maximal increase in the number of Purkinje cells and GABA‐uptake was only achieved if CNTF was added within 48 h of plating the cells, further suggesting that CNTF enhances Purkinje cell survival in vitro. These results taken together strongly support a direct effect of CNTF in promoting the survival of Purkinje cells and possibly other GABAergic cerebellar neurons.

Neurotrophin-4/5 Maintains the Cholinergic Phenotype of Axotomized Septal Neurons

We examined the effect of intraseptal or intracerebroventricular (i.c.v.) infusions of NT‐4/5 or intraseptal infusions of NGF on the level of immunohistochemical staining of choline acetyltransferase (ChAT) and the low‐affinity nerve growth factor receptor (LNGFR) in the rat medial septum following unilateral transection of the fimbria. The extent of cell loss in the septum ipsilateral to the lesion, determined by cell counts of ChAT‐immunopositive neurons and expressed as a ratio comparing the lesioned to the intact sides, was 0.28 in animals that received an infusion of phosphate‐buffered saline (PBS). The ratios were 0.97 and 1.07 in animals that received an infusion of NT‐4/5 into the ipsilateral ventricle and septum respectively. Septal infusions of NGF produced a ratio of ChAT‐immunopositive cells of 1.03. The ratios of LNGFR‐immunopositive neurons increased from 0.50 in PBS‐infused animals to 0.79 and 0.83 in animals infused with NT‐4/5 via the i.c.v. and septal routes respectively, and to 0.89 with NGF via the septal route. Transection of the fimbria also reduced the expression of TrkA in the ipsilateral medial septum and vertical limb of the diagonal band of Broca in PBS‐infused animals. The loss of TrkA was ameliorated by either i.c.v. infusion of NT‐4/5 or septal infusion of NT‐4/5 or NGF. As determined by immunohistochemical staining, NT‐4/5 infused into the lateral ventricle was detected in the periventricular portions of the forebrain ipsilateral to the infusion, while NT‐4/5 or NGF infused intraseptally was detected in much of the septum, bilaterally. Furthermore, exogenous NT‐4/5 or NGF was detected in numerous neuronal perikarya in the medial septal and diagonal band nuclei. These data demonstrate that, as with NGF, i.c.v. as well as septal infusions of NT‐4/5 can maintain the phenotype of basal forebrain cholinergic neurons following axotomy.

Characterization of receptors for ciliary neurotrophic factor on rat hippocampal astrocytes

We have identified by Scatchard analysis both high (124 pM, 14.4 x106 sites/micrograms protein, 7600 sites/cell) and low (1.6 nM, 7.7x106 sites/micrograms protein, 4100 sites/cell) affinity receptors for [125I]-rat ciliary neurotrophic factor (rCNTF) on astrocytes. Ligand competition studies showed that the binding of [125I]-rCNTF was effectively competed by rCNTF and human CNTF, but not by hLIF, mIL-6 or mIL-1B. Three proteins specifically crossed-linked to [125I]-rCNTF, with the molecular weights of 190, 100, and 43 kDa, were immunoprecipitated by anti-rCNTF antibodies. Anti-LIFR or anti-gp130 antibodies immunoprecipitated the 100 and the 190 kDa proteins. CNTF induced the tyrosine phosphorylation of LIFR and gp130, as well as of proteins with the molecular weights of 88/91 and 42 kDa. The phosphorylation of the 88/91 kDa protein(s) was inhibited by pretreating the cells with staurosporine, 12-myristate 13-acetate phorbol (PMA), W7, chlorpromazine, or the intracellular Ca+2 chelator BAPTA/AM. In contrast, CNTF and PMA acted synergistically to induce the phosphorylation of two proteins with the molecular weights of 42 and 44 kDa. At later time points following CNTF treatment, c-fos messenger RNA and protein levels were increased. Collectively, these data indicate that hippocampal astrocytes express high-affinity, biologically functional receptor complexes for CNTF.

Potential Therapeutic Use of BDNF or NT-4/5 in Parkinson’s and Alzheimer’s Diseases

A growing body of evidence has revealed that the neuropathologies of Parkinson’s and Alzheimer’s diseases reflect numerous system atrophies, rather than singular deficits of ldopaminergic or cholinergic neurons, respectively (see Agid et al, 1987 and Reisberg, 1983 for reviews). Both diseases can eventually result in losses of catecholaminergic, indoleaminergic, cholinergic, and peptidergic neurons but vary in the degree to which these cell groups are affected, particularly at early stages. Research efforts have focused on the identification of common mechanisms contributing to these conditions, as well as determinations of growth factor responsiveness of the affected neuronal populations. The present report summarizes in vitro and in vivo evidence that two members of the neurotrophin family of growth factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), support the survival or maturation of phenotypic markers in rat neuronal populations that correspond to those severely affected in Parkinson’s and Alzheimer’s diseases. BDNF also promotes the expression of at least several of the neuropeptides that are also decreased in Alzheimer’s disease (Nawa et al, 1993; Croll et al, 1993). The present review, however, will survey the more extensive information currently available for the in vivo and in vitro actions of BDNF or NT-4/5 on monoaminergic, cholinergic, and GABAergic neurotransmitters.