Kerstin Krieglstein

Bone Morphogenetic Proteins: Neurotrophic Roles for Midbrain Dopaminergic Neurons and Implications of Astroglial Cells

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF‐β) superfamily that have been implicated in tissue growth and remodelling. Recent evidence suggests that several BMPs are expressed in the developing and adult brain. Specifically, we show that BMP 2 and BMP 6 are expressed in the developing midbrain floor of the rat. We studied potential neurotrophic effects of BMPs on the in vitro survival, transmitter uptake and protection against MPP+ toxicity of mesencephalic dopaminergic neurons cultured from the embryonic midbrain floor at embryonic day (E) 14. At 10 ng/ml and under serum‐free conditions, most BMPs promoted the survival of dopaminergic neurons visualized by tyrosine hydroxylase immunocytochemistry during an 8‐day culture period, but to varying extents (relative potencies: BMP 6 = 12 > 2, 4, 7). BMPs 6 and 12 were as effective as fibroblast growth factor‐2 (FGF‐2) and glial cell line‐derived neurotrophic factor, promoting survival 1.7‐fold compared with controls. BMPs 9 and 11 were not effective. Dose‐response curves revealed an EC50 for BMPs 2, 6 and 12 of 2 ng/ml. BMPs 2, 4, 6, 7, 9 and 12 also promoted DNA synthesis and astroglial cell differentiation, visualized by 5‐bromodeoxyuridine (BrdU) incorporation and glial fibrillary acidic protein (GFAP) immunocytochemistry respectively. Suppression of cell proliferation and subsequent maturation of GFAP‐positive cells by 5‐fluorodeoxyuridine or aminoadipic acid abolished the neuron survival‐promoting effect of BMP 2. This suggests that BMPs, like other non‐TGF‐β factors affecting dopaminergic neuron survival, act indirectly, probably by stimulating the synthesis and/or release of glial‐derived trophic factors. BMP 6 and BMP 7 also increased the uptake of [3H]dopamine without affecting the uptake of [3H]5‐hydroxytryptamine and [3H]GABA, underscoring the specificity of the trophic effect. We conclude that several BMPs share a neurotrophic capacity for dopaminergic midbrain neurons with other members of the TGF‐β superfamily, but act indirectly, possibly through glial cells.

Neural functions of the transforming growth factors β

II. The TGF-13 isoforms and their functions in the nervous system 1. TGF-I3s are widely distributed in the developing and adult nervous system 2. TGF-13s control important events in neural development (a) TGF-13s govern early steps in morphogenesis and phenotypic decisions (b) TGF-13s and radial glial cell functions (c) TGF-I3s control proliferation of neural progenitors (d) TGF-13s control neuron survival (e) GF-I3s affect axon growth 3. TGF-I3s orchestrate the responses of the nervous system to lesions (a) TGF-13s co-operate with and determine the actions of other cytokines (b) Expression of TGF-13s is altered in neural lesions and neurodegenerative diseases (c) TGF-13s regulate astroglial cell proliferation and phenotype (d) TGF-13s and the microglial response (e) TGF-13s and the Schwann cell response (f) TGF-13s are potent neuroprotective agents 4. TGF-I3s in the treatment of neurological disorders

TGF-β regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins

We investigated putative roles of transforming growth factor (TGF)-beta expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-beta2 and -beta3 as well as the TGF-beta receptor TbetaR-II, but are not ir for TGF-beta1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-beta3 mRNA and TGF-beta biological activity in cultures of E8 CG neurons. None of the TGF-beta isoforms--beta1, beta2, or beta3--has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-beta significantly promotes CG neuron survival. However, TGF-beta does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-beta released from CG neurons using an antibody to TGF-beta1/-beta2/-beta3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti-pan-TGF-beta antibody could be rescued by adding exogenous TGF-beta. Together, these data suggest that para-/autocrine TGF-beta signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons.

Expression, localization, and function of transforming growth factor-beta s in embryonic chick spinal cord, hindbrain, and dorsal root ganglia.

We have studied the localizations of transforming growth factor-beta (TGF-beta) 2 and 3 immunohistochemically using isoform-specific antibodies and TGF-beta 3 mRNA by in situ hybridization in the nervous system of the 3- to 15-day-old chick embryo with special reference to spinal cord, hindbrain, and dorsal root ganglia (DRG). At embryonic day (E) 3, TGF-beta 3 mRNA as well as TGF-beta 2 and 3 immunoreactivities (IRs) were most prominent in the notochord, wall of the aorta, and dermomyotome. At E5 and E7, strong TGF-beta 2 and 3 IR were seen in or on radial glia of spinal cord and hindbrain. Radial glia in the floor plate region and ventral commissure gave the most intense signal. In the DRG, fiber strands of intense IRs representing extracellular matrix or satellite cells were seen. Neuronal perikarya did not become IR for TGF-beta 2 and 3 until E11, but even then the moderate signals for TGF-beta 3 mRNA could not be specifically localized to the neuronal cell bodies. In E11 and older embryos, spinal cord glial or glial progenitor cells, but not neuronal cell bodies were labeled for TGF-beta 3 mRNA. Immunocytochemistry and western blot analysis indicated that E8 DRG neurons have the TGF-beta receptor type II, and treatment of these cells with NGF induces expression of TGF-beta 3 mRNA. The TGF-beta isoforms 1, 2, and 3 did not promote survival of E8 DRG neurons in dissociated cell cultures. All three TGF-beta isoforms, however, promoted neurite growth from E8 DRG explants, but were less potent than nerve growth factor. Our data suggest identical localizations of TGF-beta 2 and -beta 3 IR in the developing chick and mammalian nervous systems, underscoring the general importance of TGF-beta s in fundamental events of neural development.

TRANSFORMING GROWTH FACTOR-BETA MEDIATES THE NEUROTROPHIC EFFECT OF FIBROBLAST GROWTH FACTOR-2 ON MIDBRAIN DOPAMINERGIC NEURONS

Fibroblast growth factor (FGF)‐2 is an established neurotrophic factor for dopaminergic (DAergic) neurons in the ventral midbrain. Its survival and differentiation‐promoting effects on DAergic neurons in vitro and in vivo are crucially dependent on the presence, numerical expansion and maturation of astroglial cells. We show now that transforming growth factor (TGF)‐β, an established trophic factor for DAergic neurons and product of astroglial cells, mediates the trophic effect of FGF‐2 on DAergic neurons cultured from the embryonic rat midbrain floor. Antibodies to TGF‐β that neutralize the isoforms ‐β1, ‐β2 and ‐β3 abolish the trophic effect of FGF‐2. FGF‐2 increases TGF‐β3 mRNA and amounts of biologically active TGF‐β determined in a mink lung epithelial cell assay in a time‐dependent manner. FGF‐2 also induces levels of active TGF‐β in neonatal rat astrocytes cultured from midbrain, striatum and cortex. We conclude that TGF‐β is required for mediating the survival promoting effect of FGF‐2 on DAergic and, possibly, cortical and striatal neurons grown in the presence of glial cells.

Microencapsulated Ciliary Neurotrophic Factor: Physical Properties and Biological Activities

Controlled drug release in the CNS and PNS is still an obstacle to the treatment of neurodegenerative disorders. We have prepared a variety of microspheres containing either ciliary neurotrophic factor (CNTF) or genetically engineered cells able to synthesize and release this cytokine. CNTF is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. However, when given in therapeutically effective doses by systemic injections, it produces numerous adverse side effects. In order to minimize these effects we have microencapsulated it in biopolymers (chitosans, alginates, and copolymers in various proportions to achieve different kinetic properties). Size distribution profiles were determined by an image analysis system and surface characteristics were assessed by electron microscopy. The total content of CNTF as well as the amounts released per day were determined by ELISA and in vitro bioassays. The results from the release kinetics demonstrate that long-term secretion (up to 24 days) of CNTF is achieved by combining chitosan with copolymerized lactic and glycolic acid, whereas microspheres made of alginate provided only relatively short-term release (2-12 days). Neuron survival and neurite outgrowth in cultures of ciliary ganglia were supported by microencapsulated CNTF, indicating biological stability of CNTF. Genetically engineered human kidney cells 293 continued synthesizing CNTF within spheres and the released amounts of CNTF in the culture medium were comparable to the amounts secreted from monolayers (1 ng/ml of supernatant from confluent cultures) or even higher. These studies provide a basis for future testing of CNTF in encapsulated preparations using animal models of neurodegenerative disorders.

GDF-15/MIC-1 a novel member of the TGF-beta superfamily.

We have cloned, expressed, and raised antibodies against a novel member of the TGF-beta superfamily, growth/differentiation factor-15 (GDF-15). The predicted protein is identical to macrophage inhibitory cytokine-1 (MIC-1), which was discovered simultaneously. GDF-15 is a more distant member of the TGF-beta superfamily and does not belong to one of the known TGF-beta subfamilies. In the CNS, GDF-15/MIC-1 mRNA is abundantly expressed by the choroid plexus. In addition we have preliminary evidence that GDF-15/MIC-1 is a potent trophic factor for selected classes of neurons in vitro and in vivo. Thus, GDF-15 is a novel neurotrophic factor with prospects for the treatment of disorders of the CNS.

Differential regulation of distinct phenotypic features of serotonergic neurons by bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs), growth and differentiation factor 5 (GDF5) and glial cell line‐derived neurotrophic factor (GDNF) are members of the transforming growth factor‐β superfamily that have been implicated in tissue growth and differentiation. Several BMPs are expressed in embryonic and adult brain. We show now that BMP‐2, –6 and –7 and GDF5 are expressed in the embryonic rat hindbrain raphe. To start to define roles for BMPs in the regulation of serotonergic (5‐HT) neuron development, we have generated serum‐free cultures of 5‐HT neurons isolated from the embryonic (E14) rat raphe. Addition of saturating concentrations (10 ng/mL) of BMP‐6 and GDF5 augmented numbers of tryptophan hydroxylase (TpOH) ‐immunoreactive neurons and cells specifically taking up 5,7‐dihydroxytryptamine (5,7‐DHT) by about two‐fold. Alterations in 5‐HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of short‐term treatments. Importantly, BMP‐7 selectively induced 5,7‐DHT uptake without affecting TpOH immunoreactivity. BMP‐6 and –7 also promoted DNA synthesis and increased numbers of cells immunoreactive for vimentin and glial fibrillary acidic protein (GFAP). Pharmacological suppression of cell proliferation or glial development abolished the induction of serotonergic markers by BMP‐6 and –7, suggesting that BMPs act indirectly by stimulating synthesis or release of glial‐derived serotonergic differentiation factors. Receptor bodies for the neurotrophin receptor trkB, but not trkC, abolished the BMP‐mediated effects on serotonergic development, suggesting that the glia‐derived factor is probably brain‐derived neurotrophic factor (BDNF) or neurotrophin‐4. In support of this notion, we detected increased levels of BDNF mRNA in BMP‐treated cultures. Together, these data suggest both distinct and overlapping roles of several BMPs in regulating 5‐HT neuron development.

Distinct modulatory actions of TGF-β and LIF on neurotrophin-mediated survival of developing sensory neurons

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are important for the regulation of survival and differentiation of distinct, largely non-overlapping populations of embryonic sensory neurons. We show here that the multifunctional cytokine transforming growth factor-β (TGF-β) fails to maintain sensory neurons cultured from embryonic day (E) 8 chick dorsal root ganglia (DRG), although DRG neurons are immunoreactive for the TGF-β receptor type II, which is essential for TGF-β signaling. However, in combination with various concentrations of NT-3 and NT-4, but not NGF, TGF-β3 causes a further significant increase in neuron survival. In DRG cell cultures treated with NGF, NT-3, and NT-4, a neutralizing antibody to TGF-β decreases neuron survival suggesting that endogenous TGF-β in these cultures affects the efficacies of neurotrophins. Consistent with this notion and a modulatory role of TGF-β in neurotrophin functions is the observation that TGF-β2 and-β3 immunoreactivities and TGF-β3 mRNA are located in embryonic chick DRG in close association with neurons from E5 onwards. We also show that leukemia inhibitory factor (LIF) significantly decreases NGF-mediated DRG neuron survival. Together, these data indicate that actions and efficacies of neurotrophins are under distinct control by TGF-β and LIF in vitro, and possibly also in vivo.

Bovine Chromaffin Cells Release a Transforming Growth Factor-β-Like Molecule Contained Within Chromaffin Granules

Abstract: Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)‐β in terms of its biological activity. The assay used to assess the activity of TGF‐β is based on cells transfected with a plasminogen activator inhibitor‐1 promoter‐luciferase construct. The assay is highly specific in detecting TGF‐β1, ‐β2, and ‐β3 but does not detect several cytokines and growth factors, such as fibroblast growth factor‐2, transforming growth factor‐α, platelet‐derived growth factor‐AB, insulin‐like growth factor‐I, or neurotrophin‐3 or ‐4. Moreover, we show that this assay does not detect a wide range of TGF‐β superfamily members (activin A, bone morphogenetic protein‐2, ‐4, ‐6, and ‐7, growth/differentiation factor‐5, and glial cell line‐derived neurotrophic factor). Chromaffin granules contain ∼1 ng of TGF‐β/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF‐β1/β2/β3. TGF‐β is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10−5M). These data suggest that TGF‐β is stored in chromaffin granules and can be released by exocytosis.