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.

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.

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.

Differential distribution of exogenous BDNF, NGF, and NT-3 in the brain corresponds to the relative abundance and distribution of high-affinity and low-affinity neurotrophin receptors.

To evaluate effective means for delivering exogenous neurotrophins to neuron populations in the brain, we compared the distribution and transport of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) following intracerebral delivery. Rats received an injection of radioiodinated or unlabeled neurotrophin into the lateral ventricle and were killed humanely after 1.5-24 hours. Other rats received continuous infusion of unlabeled neurotrophin into the lateral ventricle, the striatum, or the hippocampus for 3-14 days. The neurotrophins were detected by autoradiography or immunohistochemistry. There were striking differences between BDNF, NGF, and NT-3 in their penetration through brain tissue. These differences occurred regardless of the site or method of delivery, but were most pronounced following a bolus intracerebroventricular (ICV) injection. After ICV injection, NGF was widely distributed in tissues around the ventricles and at the surface of the brain, whereas the penetration of BDNF into brain tissue was distinctly less than that of NGF, and the penetration of NT-3 was intermediate. An ICV injection of NGF produced prominent but transient labeling of cells that contain the low-affinity NGF receptor, whereas an injection of BDNF prominently labeled the ventricular ependyma. During ICV infusion (12 micrograms/day), the distribution of BDNF was no greater than that observed after a 12-micrograms bolus injection. A sixfold increase in the amount of BDNF infused (72 micrograms/day) produced a more widespread distribution in the brain and doubled the depth of penetration into periventricular tissues near the cannula. Corresponding to their differences in penetration, NGF was retrogradely transported by basal forebrain cholinergic neurons after ICV or intrastriatal delivery, whereas NT-3 was transported by a few basal forebrain neurons after ICV delivery, and BDNF was rarely detected in neurons after ICV delivery. Delivery of BDNF directly to the striatum or the hippocampus labeled numerous neurons in nuclei afferent to these structures. In situ hybridization studies confirmed that the high-affinity BDNF receptor (TrkB) was much more widely expressed in neurons than was the high-affinity NGF receptor (TrkA). Moreover, mRNA for truncated forms of TrkB was expressed at high levels in the ependyma, the choroid epithelium, and the gray matter. It is likely that binding of BDNF to TrkB, which appears to be more abundant and ubiquitous than TrkA, restricts the diffusion of BDNF relative to that of NGF.

The neurotrophin family of NGF-related neurotrophic factors.

The recent molecular cloning of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) has established the existence of an NGF-related family of neurotrophic factors - the neurotrophins. Purification and recombinant production of BDNF and NT-3 has allowed the initiation or extension of in vitro studies of the neuronal specificity of each of these factors. We have found that NT-3, like NGF and BDNF, promotes survival and neurite outgrowth from certain populations of sensory neurons. There appear to be both distinct and overlapping specificities of the 3 neurotrophins towards peripheral neurons - sympathetic neurons and subpopulations of neural crest and neural placode-derived sensory neurons. Using cultures of central nervous system neurons, we have recently established that BDNF: (i) promotes the survival and phenotypic differentiation of rat septal cholinergic neurons, a property consistent with the discovery of high levels of BDNF mRNA expression within the hippocampus; (ii) promotes the survival of rat nigral dopaminergic neurons and furthermore protects these neurons from two dopaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and MPTP. Thus the neurotrophic effects of these factors towards peripheral neurons and neuronal populations known to degenerate in two of the major human neurodegenerative diseases - Alzheimer's and Parkinson's disease - provokes the question of whether neurotrophic factors may have therapeutic potential in halting the progression and ameliorating the symptoms of devastating neurological disorders of the CNS or PNS, or improving regeneration of neurons of CNS or PNS after traumatic injury.

ATP binding to brain l-glutamate decarboxylase: A study by affinity chromatography.

The binding of ATP to brain l-glutamate decarboxylase (GAD) was studied by means of ATP-agarose chromatography, utilizing partially purified GAD from mouse brain after DEAE-cellulose chromatography and ammonium sulfate fractional precipitation. GAD was found to bind with a high affinity to the ATP-agarose with the ATP molecule linked to the beaded agarose through the N(6)-amino group. Agarose with ATP attached through the ribosyl hydroxyls was totally ineffective to bind the enzyme. GAD bound to the immobilized ATP could be dissociated by free ATP (10-50 mM), but not by ADP at a concentration as high as 100 mM. Mg(2+) was not a required factor for the binding. The enzyme binding to the ATP-agarose occurred under a saturating concentration (50 ?M) of pyridoxal 5?-phosphate (PLP). Moreover, GAD bound to the ATP-agarose was not dissociated by PLP even at 1.0 mM, indicating no competition of PLP with ATP for the same binding site on the enzyme. Kinetic characterization showed that binding of ATP raised the K(m) of the enzyme for PLP. Our approach provides direct evidence that there is a specific binding site on GAD for ATP, which is distinct from the binding site for PLP.

Two forms of striatal tyrosine hydroxylase from DEAE-cellulose chromatography.

Tyrosine hydroxylase (TH) in freshly prepared 45,000 g supernatant from rat striatum was fractionated by DEAE-cellulose chromatography. The elution was made with 2 vols. of buffer (50 mM Tris, pH 7.4; 2 mM dithiothreitol) followed by 4 vols. of a linear NaCl gradient (0 0.3 M) in the same buffer. TH activity was eluted in two distinct peaks: one at about 0.1 M salt (I), and the other at 0.2 M salt (II). The relationship between the two enzymes peaks was examined as follows. (1) Incubation of the supernatant in the presence of cAMP-dependent protein kinase, 1 mM ATP, 10 mM Mg2+, and 0.1 mM cAMP resulted in the elimination of peak I, with a concomitant increase of peak II. This shift of TH peaks was prevented when the protein kinase was blocked by the addition of its inhibitory modulator. (2) Incubation of the supernatant with alkaline phosphatase, an enzyme known to dephosphorylate a variety of phosphoproteins, resulted in the elimination of peak II, with a concomitant increase of peak I. (3) Only freshly prepared supernatants showed two distinct TH peaks from DEAE-cellulose. From supernatants held at 0 degrees C for 24 h. peak II was markedly reduced and peak I concomitantly increased. Since peak II appears to be readily convertible to peak I, no further fractionation was attempted. From the data obtained here, we believe that peaks I and II are respectively the nonphosphorylated and phosphorylated forms of TH. Furthermore, the endogenous distribution of the two TH forms in striatum was altered by the administration of haloperidol (2 mg/kg. i.p.), a neuroleptic drug known to activate the enzyme via a cAMP-dependent mechanism. At 90 min after the treatment, there was a marked increase of peak II, with a concomitant decrease of peak I. Thus, this procedure provides a simple means for estimating the degree of phosphorylation of TH in vivo in catecholaminergic neurons under various physiological and pharmacological conditions.

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.

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.

Pretreatment with intravenous FGF-13 reduces infarct volume and ameliorates neurological deficits following focal cerebral ischemia in rats.

Fibroblast growth factor-13 (FGF-13), novel member of FGF family has recently been molecularly cloned as a result of high throughput sequencing of a ovarian cancer cell, hippocampal, and kidney cDNA libraries. The human gene encodes for a protein with a molecular weight of 22 kDa that is most homologous to FGF-8 (70% similarity). In the current study, we tested the effects of intravenously administered FGF-13 in a model of permanent focal cerebral ischemia in Sprague-Dawley rats. FGF-13 or the vehicle was administered systematically via the tail vein 30 min prior, and 30 min and 24 h after the occlusion of the left middle cerebral artery (MCAo). Animals were weighed and evaluated behaviorally prior to and at 24 and 48 h after MCAo. The volume of cerebral infarct and swelling were determined using an image analysis system (BioQuant) and cresyl violet stained sequential sections from the forebrain region. Histopathology was evaluated to compare the therapeutic effects. We found a 63% reduction in infarct volume in FGF-13- vs. vehicle-treated animals (infarct volume was 21.9+/-3.8% in vehicle- and 8.1+/-1.6% in FGF-13-treated rats, p=0.0016) and a moderate inhibition of brain swelling by FGF-13. The reduction in infarct volume and brain swelling were associated with improvement of clinical deficits in FGF-13 treated animals (p<0.001). Histopathological examination determined that nervous tissue was better preserved in FGF-13 treated rats than those of controls. These data show that pretreatment with intravenous FGF-13 reduces infarct size and ameliorates neurological deficits following permanent focal cerebral ischemia in rats.

Modulation of neuronal choline acetyltransferase activity by factors derived from cultures of non-neuronal cells from the CNS.

We have found that cholinergic neurons in spinal cord-dorsal root ganglion cultures derived from E12-E13 mouse embryos are sensitive, as measured by changes in choline acetyltransferase activity, to factors secreted by non-neuronal cells derived from the same tissue at an identical developmental stage. Conditioned medium was produced by incubating non-neuronal cultures for 4 days in defined medium. The cholinotrophic activity present in the conditioned medium had a molecular weight of greater than 50,000 as determined by ultrafiltration and bound wheat germ lectin and heparin sepharose. Total RNA isolated from the non-neuronal cells, used to produce the conditioned medium, was translated in frog oocytes. Conditioned medium from the injected oocytes was also found to contain cholinotrophic activity. In contrast, the conditioned medium from water-injected oocytes was inactive. The interaction between the cholinotrophic activity in conditioned medium from frog oocytes and known second messengers was also examined. Dibutyryl cyclic AMP produced a concentration-dependent increase in choline acetyltransferase activity. If a maximal effective dose of dibutyryl cyclic AMP was added in conjunction with a maximal effective dose of conditioned medium from oocytes injected with total RNA a nearly additive response was noted. In contrast, the phorbol ester, phorbol 12-myristate 13-acetate, produced a biphasic change in the level of choline acetyltransferase activity; with lower doses stimulating and higher doses inhibiting the enzyme activity. When conditioned medium from oocytes injected with non-neuronal cell RNA was added in conjunction with the phorbol ester a decrease in the physiological response was noted.

Nerve growth factor and phorbol esters increase the number of choline acetyltransferase-positive cells in two morphologically distinct classes of basal forebrain neurons in primary cultures.

Nerve growth factor (NGF) has been shown to be active in the CNS as a neurotrophic agent. Cholinergic neurons of the basal forebrain are one cell type in the CNS which have been identified as a target for NGF. When dissociated cell cultures from the basal forebrain were treated for 7 days with NGF (20 ng/100 microliters), the number of choline acetyltransferase (ChAT)-immunopositive cells was increased from 30 +/- 6 to 58 +/- 3. Cholinergic cells taken from the basal forebrain exhibit 3 different morphologies: stellate, pyramidal, and bipolar. The NGF treatment was found to increase the number of stellate cells from 7 +/- 2 to 23 +/- 2 and the number of pyramidal cells from 14 +/- 2 to 26 +/- 2, but had no effect on the number of bipolar cells. The activation of protein kinase C by phorbol 12-myristate, 13-acetate (TPA) also increased the number of ChAT-positive cells in a dose-dependent manner. A maximal increase was observed with 10 ng/ml of TPA which increased the number of positive cells from a basal level of 21 +/- 4 to 42 +/- 4. As was the case with NGF, only the stellate and pyramidal cells were affected by the phorbol ester treatment. In co-addition experiments, the cultures were treated with 10 ng/100 of NGF and 10 ng/ml of TPA, with the result that there was no further increase in the number of immunopositive cells over the NGF controls. These results suggest that the mechanisms by which NGF and TPA increase the number of ChAT-positive cells are interactive at some point. The effect of TPA at the higher doses of NGF was distinctly different. When cells were treated with 20 ng/100 microliters of NGF and 0.05-50 ng/ml of TPA, the NGF response was down-regulated to the level of the vehicle-treated controls.

Purification and characterization of a soluble cyclic nucleotide-independent Ca2+-calmodulin-sensitive protein kinase from rat brain.

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.

Characterization of the responses of Purkinje cells to neurotrophin treatment.

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-immunopositve 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, approximately 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 NT4/5 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 approximately 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.

Activation of midbrain tryptophan hydroxylase by MgATP. Absence during early postnatal development.

The brain tryptophan hydroxylase is known to be activated by magnesium adenosine triphosphate (MgATP). This activation has been suspected to be a case of enzyme phosphorylation, although convincing evidence is still lacking. In supernatants (100,000 g) from adult mouse midbrains, the addition of 1 mM ATP and 10 mM MgCl2 could increase the tryptophan activity by 70-90%, when the enzyme activity was determined at a subsaturating concentration of 6-MPH4 (0.2 mM). The present study has revealed that the enzyme activation by MgATP could only be achieved from mice after 12 days of postnatal age. No activation was found in midbrain preparations from younger animals, although a substantial level of tryptophan hydroxylase activity was already present. The possibility that some required component(s) for the enzyme activation may be lacking during early development was tested by mixing a dialyzed adult preparation with the neonatal midbrain supernatant. Under these conditions, the tryptophan hydroxylase in the neonatal supernatant was activated by MgATP. Furthermore, the addition of a crude protein kinase fraction from adult midbrain cytosol was also capable of restoring the enzyme activatability in the neonatal preparation. It appears that the lack of activatability by MgATP alone during early development was due to absence of one or more biochemical factors required for the activation.

Soluble protein kinase fractions from DEAE-cellulose chromatography. A comparison between brain and heart from the rat.

The cytosol fraction from rat midbrain was chromatographed on DEAE-cellulose with a linear NaCl gradient (0-0.3 M). Two peaks of protein kinase activity were obtained when assayed with either histone or casein. A similar elution profile of the kinase activity was obtained from rat heart. The first peaks from midbrain and heart were compared in terms of their dependency upon cAMP and sensitivity to the endogenous protein kinase inhibitor. Neither of the two substances had an effect on the activity of the brain kinase. Furthermore, the dissociability of the midbrain and heart enzymes in the presence of cAMP or histone was compared by DEAE-cellulose chromatography. The heart enzyme was dissociated into a catalytic subunit characteristic of a cAMP-dependent protein kinase, whereas the brain kinase was totally unaffected by the cAMP or histone. The results of these tests indicate that although the elution profiles from DEAE-cellulose are similar between midbrain and heart, the first peak from brain contains a protein kinase that appears to be cAMP independent.

Vascular endothelial cell growth factors promote the in vitro development of rat photoreceptor cells.

We have identified and characterized a novel trophic effect of vascular endothelial cell growth factor (VEGF) on photoreceptor cells. Treatment of retinal cultures, derived from postnatal day 1 (P1) rats, with VEGF-2 resulted in a dose- and time-dependent increase in the level of rhodopsin protein, as determined by ELISA assay. After 7-9 d of treatment the VEGF-1 or VEGF-2, at a concentration of 10 ng/ml, induced a 200-300% increase in rhodopsin protein and a 220% increase in the number of rhodopsin-immunopositive cells. Treatment with VEGF-2 induced a 250% increase in the number of syntaxin-immunopositive cells and a 67% increase in high-affinity GABA uptake, both markers for amacrine cells. In contrast, there was no increase in the non-neuronal cell populations. VEGF-2 induced an approximately 300% increase in the number of bromodeoxyuridine-labeled (BrdU) retinal cells within 48 hr of treatment. After 3 d in culture both the basal and stimulated levels of BrdU incorporation were reduced, suggesting that the proliferative effect of VEGF was restricted developmentally. Furthermore, there was a developmentally dependent increase in the mitogenic response to VEGF-2, with retinal cultures derived from E15, E20, or P1 animals demonstrating a 50, 100, and 300% increase in thymidine incorporation, respectively. However, VEGF treatment resulted in an increase in the number of rhodopsin-immunopositive cells only when the cultures were derived from P1 animals. Therefore, retinal progenitor cells appear to be targets for VEGF, and thus VEGF may be involved in the regulation of the early developmental program of retinal neurogenesis.

Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures.

First described as a survival factor for chick ciliary ganglion neurons, ciliary neurotrophic factor (CNTF) has recently been shown to promote survival of chick embryo motor neurons. We now report neurotrophic effects of CNTF toward three populations of rat hippocampal neurons, the first demonstration of effects of CNTF upon rodent CNS neurons in culture. CNTF elicited an increase in the neurofilament content of hippocampal cultures prepared from embryonic day 18 (E18) rat brain. This was accompanied by increases of 2-, 28-, and 3-fold in the number of GABAergic, cholinergic, and calbindin-immunopositive cells, respectively. CNTF totally prevented the 67% loss of GABAergic neurons that occurred in control cultures over 8 d. CNTF also increased high-affinity GABA uptake and glutamic acid decarboxylase activity. Effects of CNTF were in all cases dose dependent, with maximal stimulation at approximately 100 pg/ml. When addition was delayed for 3 d, CNTF failed to elicit increases either in the number of cholinergic neurons or in GABA uptake.

Computational EST database analysis identifies a novel member of the neuropoietic cytokine family.

A novel member of the neuropoietic cytokine family has been cloned and the protein expressed and characterized. In an effort to identify novel secreted proteins, an algorithm incorporating neural network algorithms was applied to a large EST database. A full-length clone was identified that is 1710 bp in length and has a single open reading frame of 225 amino acids. This new cytokine is most homologous to cardiotrophin-1, having a similarity and an identity of 46 and 29%, respectively, and therefore we have named it cardiotrophin-like cytokine (CLC). Northern hybridization analysis identified a 1.4-kb messenger RNA that is highly expressed in spleen and peripheral leukocytes. Purified recombinant CLC induced the activation of NFkappaB and SRE reporter constructs in the TF-1, U937, and M1 cell lines. Furthermore, the signal transduction pathway for CLC was characterized in the neuroblastoma cell line SK-N-MC and found to involve tyrosine phosphorylation of gp130 and STAT-1.