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The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells.

Growth/differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor beta (TGF-β) superfamily. It is widely distributed in the nervous system, where it has been shown to play an important role in neuronal maintenance. The present study investigates the role of endogenous GDF-15 in sciatic nerve (SN) lesions using wild-type (WT) and GDF-15 knock-out (KO) mice. SN of 5-6-month-old mice were crushed or transected. Dorsal root ganglia (DRG) and nerve tissue were analyzed at different time points from 6 h to 9 weeks post-lesion. Both crush and transection induced GDF-15 mRNA and protein in the distal portion of the nerve, with a peak at day 7. DRG neuron death did not significantly differ between the genotypes; similarly, remyelination of regenerating axons was not affected by the genotype. Alternative macrophage activation and macrophage recruitment were more pronounced in the KO nerve. Protrusion speed of axons was similar in the two genotypes but WT axons showed better maturation, as indicated by larger caliber at 9 weeks. Furthermore, the regenerated WT nerve showed better performance in the electromyography test, indicating better functional recovery. We conclude that endogenous GDF-15 is beneficial for axon regeneration following SN crush.

The pan-neurotrophin receptor p75NTR is expressed in the adult brain in a discrete pattern. Although numerous studies have addressed its implications for hippocampal functions, the generated sets of data are surprisingly conflicting. We have therefore set out to re-investigate the impact of a deletion of the full-length p75NTR receptor on several parameters of the dentate gyrus (DG), including neurogenesis and hippocampus-related behavior by using p75NTR(ExIII) knockout mice. Moreover, we investigated further parameters of the DG (cholinergic innervation, dendritic spines). In addition, we analyzed on the morphological level the impact of aging by comparing adult and aged p75NTR(ExIII) mice and their age-matched littermates. Adult (4-6 months old), but not aged (20 months old), p75NTR(ExIII) knockout mice display an enhanced volume of the DG. However, adult neurogenesis within the adult DG was unaffected in both adult and aged p75NTR(ExIII) knockout mice. We could further demonstrate that the change in the volume of the DG was accompanied by an increased cholinergic innervation and increased spine densities of granule cells in adult, but not aged p75NTR deficient mice. These morphological changes in the adult p75NTR deficient mice were accompanied by specific alterations in their behavior, including altered behavior in the Morris water maze test, indicating impairments in spatial memory retention.

The neurotrophin receptor p75NTRhas been implicated in mediating neuronal apoptosis after injury to the CNS. Despite its frequent induction in pathologic states, there is limited understanding of the mechanisms that regulate p75NTRexpression after injury. Here, we show that after focal cerebral ischemiain vivoor oxygen–glucose deprivation in organotypic hippocampal slices or neurons, p75NTRis rapidly induced. A concomitant induction of proNGF, a ligand for p75NTR, is also observed. Induction of this ligand/receptor system is pathologically relevant, as a decrease in apoptosis, after oxygen–glucose deprivation, is observed in hippocampal neurons or slices after delivery of function-blocking antibodies to p75NTRor proNGF and inp75NTRandngfhaploinsufficient slices. Furthermore, a significant decrease in infarct volume was noted inp75NTR−/− mice compared with the wild type. We also investigated the regulatory mechanisms that lead to post-ischemic induction of p75NTR. We demonstrate that induction of p75NTRafter ischemic injury is independent of transcription but requires active translation. Basal levels of p75NTRin neurons are maintained in part by the expression of microRNA miR-592, and an inverse correlation is seen between miR-592 and p75NTRlevels in the adult brain. After cerebral ischemia, miR-592 levels fall, with a corresponding increase in p75NTRlevels. Importantly, overexpression of miR-592 in neurons decreases the level of ischemic injury-induced p75NTRand attenuates activation of pro-apoptotic signaling and cell death. These results identify miR-592 as a key regulator of p75NTRexpression and point to a potential therapeutic candidate to limit neuronal apoptosis after ischemic injury.

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss.

It is becoming increasingly clear that members of the transforming growth factor-β (TGF-β) family have roles in the central nervous system that extend beyond their well-established roles as neurotrophic and neuroprotective factors. Recent findings have indicated that the TGF-β signaling pathways are involved in the modulation of both excitatory and inhibitory synaptic transmission in the adult mammalian brain. In this review, we discuss how TGF-β, bone morphogenetic protein and activin signaling at central synapses modulate synaptic plasticity, cognition and affective behavior. We also discuss the implications of these findings for the molecular understanding and potential treatment of neuropsychiatric diseases, such as anxiety, depression and other neurological disorders.

Extracellular signal-regulated kinase (ERK) is a versatile protein kinase that regulates many cellular functions. Growing evidence suggests that ERK1/2 plays a crucial role in promoting cell death in a variety of neuronal systems, including neurodegenerative diseases. It is believed that the magnitude and the duration of ERK1/2 activity determine its cellular function. In this review, we summarize recent evidence for a role of ERK1/2 in neuronal death. Furthermore, we discuss the mechanisms involved in ERK1/2 mediating neuronal death.

Transforming growth factor-beta (TGF-beta) and glial-cell-line-derived neurotrophic factor (GDNF) have been shown to synergize in several paradigms of neuronal survival. We have previously shown that cerebellar granule neurons (CGN) degenerate in low potassium via ERK1/2 (extra-cellular-regulated kinase)-dependent plasma membrane (PM) damage and caspase-3-dependent DNA fragmentation. Here, we have investigated the putative synergistic function of GDNF and TGF-beta in CGN degeneration. GDNF alone prevents low-potassium-induced caspase-3 activation and DNA fragmentation but does not affect either low-potassium-induced ERK activation or PM damage. TGF-beta alone does not affect low-potassium-induced DNA fragmentation but potentiates low-potassium-induced PM damage. This effect of TGF-beta is independent of ERK1/2 activation but dependent on p38-MAPK (mitogen-activated protein kinase) activation. When co-applied with TGF-beta, GDNF paradoxically antagonizes TGF-beta-induced potentiation of PM damage by inhibiting TGF-beta-induced p38-MAPK activation. In addition, PI3K (phosphatidylinositol 3-kinase) inhibitors abolish the GDNF effect. This study thus demonstrates a differential mechanism of action of GDNF and TGF-beta on CGN degeneration. GDNF inhibits caspase-3-dependent DNA fragmentation but does not affect ERK-dependent PM damage. However, GDNF can attenuate TGF-beta-induced p38-MAPK-dependent PM damage via the PI3K pathway.

GDNF is a potent neurotrophic factor for nigrostriatal dopaminergic neurons in vitro and in animal models of Parkinson's disease (PD), but has largely failed when tested in therapeutic applications in human PD. We report here that GDNF requires transforming growth factor-beta (TGF-beta) to elicit its neurotrophic activity. Lesioning the mouse nigrostriatal system with MPTP significantly upregulates striatal TGF-beta2 mRNA levels. As expected, GDNF protects against the destructive effects of MPTP, including losses of TH-ir nigral neurons, striatal dopamine and TH-ir fibers. Application of antibodies neutralizing all three TGF-beta isoforms to the MPTP-lesioned striatum abolishes the neurotrophic effect of GDNF. We show that TGF-beta antibodies are not toxic and do not interfere with retrograde transport of iodinated GDNF, suggesting that TGF-beta antibodies do not impair internalization and retrograde trafficking of GDNF. We conclude that striatal TGF-beta may be essential for permitting exogenous GDNF to act as a neuroprotective factor.

Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTR (ExIII) knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTR (ExIV) knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG), leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX) positive cells and reduced numbers of apoptotic cells in p75NTR (ExIV) knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3) positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities) as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTR (ExIV) knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone.

Growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) is a new member of the transforming growth factor beta (TGF-beta) superfamily, which has most recently been found in activated macrophages (MPhi). We have now investigated GDF-15/MIC-1 in human MPhi after exposure to oxidized low-density lipoproteins (oxLDL) related mediators in vitro and in arteriosclerotic carotid arteries. Using RT-PCR and Western blotting a pronounced induction of GDF-15/MIC-1 expression by oxLDL, C6-ceramide, tumor necrosis factor (TNFalpha) and hydrogen peroxide (H2O2) was found in cultured human MPhi. In 11 human arteriosclerotic carotid arteries, immunohistochemical analyses supported by computer-assisted morphometry and regression analyses demonstrated a significant colocalization of GDF-15/MIC-1 immunoreactivity (IR) with oxLDL IR and manganese superoxide dismutase (MnSOD) IR in CD68 immunoreactive (ir) MPhi, which were also expressing AIF-IR (apoptosis-inducing factor), caspase-3-IR (CPP32), PARP-IR, c-Jun/AP-1-IR and p53-IR. Our data suggest that GDF-15/MIC-1 is inducible in human MPhi by oxLDL and its mediators in vitro and is supposed to contribute to oxidative stress dependent consequences in arteriosclerotic plaques, e.g. modulating apoptosis and inflammatory processes in activated MPhi.

Multiple evidence suggests that fibroblast growth factors (FGFs), most prominently FGF-2, affect astroglial proliferation, maturation, and transition to a reactive phenotypein vitro, and after exogenous administration,in vivo. Whether this reflects a physiological role of endogenous FGF is unknown. Using FGF-2 and FGF-5 single- and double mutant mice we show now a region-specific reduction of glial fibrillary acidic protein (GFAP), but not of S100 in gray matter astrocytes. FGF-2 is apparently the major regulator of GFAP, because in mice deficient for FGF-2, GFAP is distinctly reduced in cortex and striatum, whereas in FGF-5-/-animals only a reduction in the midbrain tegmentum can be observed. In FGF-2-/-/FGF-5-/-double mutant animals, GFAP-immunoreactivity is reduced in all three brain regions. Cortical astrocytes cultured from FGF-2-/-/FGF-5-/-double mutant mice revealed reduced levels of GFAP, but not S100 as compared with wild-type littermates. This phenotype could be rescued by exogenous FGF-2 but not FGF-5 (10 ng/ml). Electron microscopy revealed reduced levels of intermediate filaments in perivascular astroglial endfeet. This defect was accompanied by enhanced permeability of the blood-brain barrier (BBB), as detected by albumin extravasation. Levels of the tight junction proteins Occludin and ZO-1 were reduced in blood vessels of FGF-2-/-/FGF-5-/-double mutant mice as compared with wild-type littermates. Our data support the notion that endogenous FGF-2 and FGF-5 regulate GFAP expression in a region-specific manner. The observed defect in astroglial differentiation is accompanied by a defect in BBB function arguing for an indirect or direct role of FGFs in the regulation of BBB permeabilityin vivo.

c-Ret encodes a receptor tyrosine kinase that is essential for normal development of the kidney as well as enteric and sympathetic neurons. Since sympathetic neurons and neuroendocrine chromaffin cells originate from a common progenitor cell, we have examined the relevance of c-Ret for the development of adrenal chromaffin cells by analyzing mouse mutants lacking c-Ret. Adrenal chromaffin cells express c-Ret mRNA at embryonic day (E) 12.5 and 13.5, yet levels of expression decline at later embryonic and postnatal ages. Adrenal medullae of c-Ret deficient mice show normal numbers of tyrosine hydroxylase (TH)-immunoreactive cells at E13.5 and at birth. Ultrastructurally, adrenal chromaffin cells of c-Ret−/− mice appear unaltered: chromaffin cells develop typical secretory chromaffin granules, the morphological hallmark of chromaffin cells, and synaptic terminals appear normal. However, adrenaline levels and numbers of chromaffin cells immunoreactive for the adrenaline synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) are reduced by about 30% in c-Ret-deficient mice arguing for a direct or indirect role of c-Ret in the regulation of PNMT. Thus, despite expression of c-Ret, adrenal chromaffin cells develop largely normal in mice lacking c-Ret. We therefore conclude that sympathetic neurons and neuroendocrine chromaffin cells profoundly differ in their requirement for c-Ret signaling during development.

We have previously shown that the neurotrophic effect of glial cell line-derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)beta. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFbeta promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFbeta, pretreatment with TGFbeta confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFbeta-induced recruitment of the glycosyl-phosphatidylinositol-anchored GDNF receptor (GFR)alpha1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRalpha1 can promote survival in the absence of TGFbeta. Our data suggest that TGFbeta is involved in GFRalpha1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects.

FGF-2 is a potent neurotrophic factor for several populations of CNS neurons and has been shown to protect hippocampal neurons from glutamate-induced cell death in vitro and in vivo. Mechanisms underlying the neurotrophic and protective actions of FGF-2 have been resolved only in part. Using glutamate-treated cultured hippocampal neurons we show that FGF-2 shares its neuroprotective capacity with GDNF. Hippocampal neurons express glial-cell-line-derived neurotrophic factor (GDNF), its receptors c-Ret and the lipid-anchored GDNF family receptor-alpha1 (GFRalpha-1), and the FGF receptor 1 (FGFR I). Neutralizing antibodies to GDNF abolish the neuroprotective effect of FGF-2. In support of the notion that GDNF is required to permit the protective effects of FGF-2 we find that FGF-2 up-regulates GDNF and GFRalpha-1 in hippocampal neurons. Furthermore, FGF-2-induced GDNF causes enhanced phosphorylation of c-Ret and the signaling components Akt and Erk. A putative downstream target of FGF-2 and GDNF are bcl-2 gene family members, whose mRNAs are differentially up-regulated by the two factors. Together, these data suggest that GDNF is an important protective factor for glutamate-lesioned hippocampal neurons and an essential mediator of the neuroprotective actions of FGF-2.

The activation of epidermal growth factor receptor (EGFR) affects multiple aspects of neural precursor behaviour, including proliferation and migration. Telencephalic precursors acquire EGF responsiveness and upregulate EGFR expression at late stages of development. The events regulating this process and its significance are still unclear. We here show that in the developing and postnatal hippocampus (HP), growth/differentiation factor (GDF) 15 and EGFR are co-expressed in primitive precursors as well as in more differentiated cells. We also provide evidence that GDF15 promotes responsiveness to EGF and EGFR expression in hippocampal precursors through a mechanism that requires active CXC chemokine receptor (CXCR) 4. Besides EGFR expression, GDF15 ablation also leads to decreased proliferation and migration. In particular, lack of GDF15 impairs both processes in the cornu ammonis (CA) 1 and only proliferation in the dentate gyrus (DG). Importantly, migration and proliferation in the mutant HP were altered only perinatally, when EGFR expression was also affected. These data suggest that GDF15 regulates migration and proliferation by promoting EGFR signalling in the perinatal HP and represent a first description of a functional role for GDF15 in the developing telencephalon.

Neurotrophin-4 (NT-4) is perhaps the still most enigmatic member of the neurotrophin family. We show here thatNT-4is expressed in neurons of paravertebral and prevertebral sympathetic ganglia, i.e., the superior cervical (SCG), stellate (SG), and celiac (CG) ganglion. Mice deficient forNT-4showed a significant reduction (20–30%) of preganglionic sympathetic neurons in the intermediolateral column (IML) of the thoracic spinal cord. In contrast, neuron numbers in the SCG, SG, and CG were unchanged. Numbers of axons in the thoracic sympathetic trunk (TST) connecting the SG with lower paravertebral ganglia were also reduced, whereas axon numbers in the cervical sympathetic trunk (CST) were unaltered. Axon losses in the TST were paralleled by losses of synaptic terminals on SG neurons visualized by electron microscopy. Furthermore, immunoreactivity for the synaptic vesicle antigen SV2 was clearly reduced in the SG and CG. Levels of catecholamines and tyrosine hydroxylase immunoreactivity were dramatically reduced in the SG and the CG but not in the SCG. Despite this severe phenotype in the sympathetic system, blood pressure levels were not reduced and displayed a pattern more typical of deficits in baroreceptor afferents. Numbers of IML neurons were unaltered at postnatal day 4, suggesting a postnatal requirement for their maintenance. In light of these and previous data, we hypothesize that NT-4 provided by postganglionic sympathetic neurons is required for establishing and/or maintaining synapses of IML neurons on postganglionic cells. Impairment of synaptic connectivity may consequently reduce impulse flow, causing a reduction in transmitter synthesis in postganglionic neurons.

Based on recent evidence from in vitro and gene knockout/insertion studies, this short review summarizes the molecular scenario underlying the development of adrenal chromaffin cells and their preganglionic innervation. During migration of neural crest cells from the dorsal surface of the neural tube to their destinations in the sympathetic primordia and adrenal glands, precursors of the so-called sympathoadrenal (SA) cell lineage are exposed to signals from the notochord and ventral neural tube probably including the protein, Sonic hedgehog. These, and signals in the region of the dorsal aorta (members of the family of bone morphogentic proteins), where SA progenitor cells subsequently assemble, are essential for the induction of the adrenergic phenotype. SA progenitor cells subsequently differentiate into paravertebral and prevertebral sympathetic neurones, intra- and extra-adrenal chromaffin cells and intermediate SIF (small intensely fluorescent) cells. Based on in vitro studies with isolated SA and chromaffin progenitor cells, glucocortiocids have been claimed as essential for suppressing neuronal commitment and for channelling SA cells towards the chromaffin phenotype. However, mice deficient for a functional glucocorticoid receptor possess the full complement of adrenal chromaffin cells at birth, suggesting that signals other than glucocorticoid hormones may be important in triggering chromaffin cell differentiation. The cholinergic neurones that are preganglionic to adrenal chromaffin cells have their cell bodies located in the intermediolateral column (IML) of the spinal cord. For their normal development, these neurones require signals from the adrenal medulla, which include neurotrophin-4, a major neurotrophic factor of adrenal chromaffin cells. Taken together, these data provide a more complete picture of molecular signalling in the development of one of the most important neuroendocrine tissues in vertebrates.

Free radical formation is considered to be a major cause of dopaminergic (DAergic) cell death in the substantia nigra leading to Parkinson's disease (PD). In this study we employed several radical donors including iron and sodium nitroprusside to induce toxic effects on DAergic neurons cultured from the embryonic rat midbrain floor. Overall cell survival was assessed by assaying LDH, and DAergic neuron survival was monitored by counting tyrosine hydroxylase-positive cells. Our data suggest that the DAergic neuron population is about fourfold more susceptible to free-radical-mediated damage than the total population of midbrain neurons. Application of the neurotrophic factors GDNF and NT-4, for which DAergic neurons have specific receptors, prior to toxin administration protected these neurons from toxin-mediated death, which, fully or in part, occurs under the signs of apoptosis. These findings underscore the importance of GDNF and NT-4 in designing future therapeutical concepts for PD.

Glial cell line-derived neurotrophic factor (GDNF), an important factor for developing and lesioned pre- and postganglionic sympathetic neurons, and its congeners signal through a receptor complex consisting of the tyrosine kinase c-Ret and a lipid-anchored alpha receptor (GFR alpha 1-4). Using in situ hybridization we show now that the mRNA for GFR alpha-2 is abundant in the adult rat adrenal medulla and its chromaffin cells. Coexpression of c-Ret and GFR alpha-1 mRNA's is restricted to a scarce subpopulation of medullary sympathetic neurons. Both GFR alpha-1 and GFR alpha-2 mRNA's are associated with preganglionic nerve trunks in the adrenal cortex. It is conceivable therefore that GDNF and related factors may activate chromaffin and preganglionic Schwann cells through a GFR-alpha receptor in absence of c-Ret.

The bone morphogenetic proteins have been implicated in several inductive processes throughout vertebrate development including nervous system patterning. Recently, these proteins have also emerged as candidates for regulating survival of mesencephalic dopaminergic and sympathetic neurons. Interestingly, we have found that several bone morphogenetic proteins can be detected in developing embryonic day 14 rat dorsal root ganglia by means of reverse transcription-polymerase chain reaction and immunocytochemistry. To further elucidate their potential role during the period of ontogenetic neuron death, serum-free cultures of dorsal root sensory neurons from developing chick and rat embryos were treated with distinct bone morphogenetic proteins with or without simultaneous addition of other "established" neurotrophic factors. Our results show that bone morphogenetic proteins exert survival promoting effects on their own, and that they can positively modulate the effects of neurotrophins on sensory neurons. In particular, growth/differentiation factor-5, bone morphogenetic protein-2, -4, -7 and -12 significantly increased the survival promoting effects of neurotrophin-3 and nerve growth factor on cultured dorsal root ganglion neurons. These results fit well into the current concept that neurotrophic factors may act synergistically in ensuring neuronal survival. Moreover, these data suggest potential instructive interactions of bone morphogentic proteins and neurotrophins during sensory neuron development. Finally, the documented neurotrophic capacity of bone morphogenetic protein family members may have potential relevance for the treatment of peripheral neuropathies.

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-beta 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.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP) constitute a subfamily of transforming growth factor-betas (TGF-betas) with prominent roles in the regulation of neuron survival and differentiation. Although numerous members of the TGF-beta superfamily are important regulators of glial cell functions in health and disease, it is unknown whether any member of the GDNF subfamily may have functions in normal or pathological glial cell performances. To begin to address this issue, we have studied expression and putative functions of GDNF, NTN, PSP, and their receptors in two cell lines representing models for oligodendrocyte progenitor cells (OLI-neu) and immature oligodendrocytes (OLN-93), respectively. RT-PCR analysis revealed expression of all three growth factor mRNAs in OLI-neu and OLN-93 cells. Expression was weak in OLI-neu cells, while both NTN and PSP mRNAs were strongly expressed in OLN-93 cells. Furthermore, OLI-neu and OLN-93 cells expressed transcripts encoding the GDNF receptors Ret and GFRalpha-1. The two splice variants for GFRalpha-2 were exclusively synthesized in OLI-neu cells. Similarly, primary O-2A progenitor cells and enriched mature oligodendrocytes expressed Ret, GFRalpha-1 and GFRalpha-2 mRNAs. Both GDNF and NTN stimulated DNA synthesis monitored by BrdU incorporation of OLI-neu cells in a dose-dependent fashion. Co-administration of TGF-beta significantly reduced this effect. Similarly, PDGF co-applied with GDNF or NTN down-regulated proliferation in OLI-neu cells. In contrast, OLN-93 cells did not respond to GDNF or NTN with increased incorporation of BrdU. Expression of GDNF, NTN, and their receptors and distinct effects in two model cell lines of oligodendrocyte development suggest that functions of members of the GDNF family and their receptors may not be restricted to neurons and may be implicated in oligodendrocyte development.

When fetal calf serum (FCS) alone is used as a trophic support for cultured chicken parasympathetic ciliary ganglionic (cCG) neurons, it does not show any survival-promoting effects on these neurons. When FCS is applied to heparin-affinity chromatography, however, potent survival-promoting activity is obtained in the fraction eluted with 0.5 M NaCl. Using cCG neurons as a bioassay system, this neurotrophic activity was purified by a combination of heparin-affinity chromatography, gel filtration chromatography, and Sep-Pak C18 cartridge. The 40-50-kDa fractions from the gel filtration column with strong survival-promoting activity were shown to contain insulin-like growth factor II (IGF-II) by immunoblot analysis. By acidification, the survival-promoting activity and IGF-II were translocated together from the 40-50-kDa to the 7-10-kDa fractions, and the survival-promoting activity in the 7-10-kDa fractions was blocked by an anti-IGF-II neutralizing monoclonal antibody. These results indicate that the neurotrophic substance in 0.5 M NaCl-eluate from heparin-affinity chromatography is IGF-II and that mechanisms may exist in vivo for the activation of latent IGF-II, whose biological effects may be blocked by its specific binding proteins.

Neurotrophins and their trk receptors constitute major classes of signaling molecules with important actions in the developing and adult nervous system. With regard to the sympathoadrenal cell lineage, which gives rise to sympathetic neurons and chromaffin cells, neurotrophin-3 (NT-3) and nerve growth factor (NGF) are thought to influence developing sympathetic neurons. Neurotrophin requirements of chromaffin cells of the adrenal medulla are less well understood than those for NGF. In order to provide the bases for understanding of putative functions of neurotrophins for the development and maintenance of chromaffin cells and their preganglionic innervation, in situ hybridization has been used to study the expression of brain-derived neurotrophic factor (BDNF) and NT-3, together with their cognate receptors trkB and trkC, in the adrenal gland and in the intermediolateral column (IML) of the spinal cord. BDNF is highly expressed in the embryonic adrenal cortex and later in cells of the cortical reticularis zone. Adrenal medullary chromaffin cells fail to express detectable levels of mRNAs for BDNF, NT-3, and their cognate receptors trkB and trkC. Neurons in the IML express BDNF and trkB, and low levels of NT-3 and trkC. Our data make it unlikely that BDNF and NT-3 serve as retrograde trophic factors for IML neurons but suggest roles of BDNF and NT-3 locally within the spinal cord and possibly for sensory nerves of the adrenal cortex.

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.

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.

Macrophages are widely distributed in lymphohaemopoietic and many other mammalian tissues, where they are mainly involved in host defence mechanisms, phagocytosis, wound repair, and secretion of growth factors. Increasing evidence suggests that secretory products of macrophages can influence adrenal gland functions. In the present study, we have used specific antibodies to ED1 (cytoplasmic antigen), ED2 (membrane antigen), ED8 (membrane antigen), and OX-6 (MHC class II/membrane antigen) as markers for macrophages to examine their distribution within the adult rat adrenal gland. ED2 and OX-6 recognize distinct subpopulations of adrenal gland macrophages, whereas macrophages immunoreactive (-ir) for ED1 and ED8 could not be detected. OX-6-ir macrophages were most numerous in the cortical reticularis and glomerulosa zones, while only few cells were found in the zona fasciculata and in the adrenal medulla. Macrophages immunoreactive for ED2 were restricted to the adrenal medulla. The majority of these macrophages were associated with vascular sinuses or chromaffin cells. By double-immunolabelling we found that most of ED2-ir medullary macrophages contain neurotrophin-4 (NT-4)-like ir. Attempts to clarify whether macrophages take up NT-4 from NT-4-ir chromaffin cells indicated that medullary macrophages are immunonegative for chromogranin A and neuropeptide Y, two major secretory products of chromaffin cells. In situ hybridizations and immunofluorescence showed expression of the neurotrophin receptor TrkA, but not TrkB in the adrenal medulla. In vitro studies indicated that NT-4, similar to nerve growth factor, can induce c-fos-ir in chromaffin cells. We conclude that chromaffin cells are putative targets for adrenal medullary NT-4, whose functions remain to be clarified.

Summary Based on recent evidence from in vitro and gene knock-out/knock-in studies this short review summarizes the molecular scenario underlying the development of autonomic neurons from the neural crest. The focus is on the sympathoadrenal (SA) cell lineage. While migrating ventrally precursors of this cell lineage are exposed to signals from notochord/ventral neural tube probably including the protein sonic hedgehog. These and signals in the region of the dorsal aorta (members of the family of bone morphogenetic proteins), where SA progenitor cells subsequently assemble, are essential for the induction of the adrenergic phenotype. SA progenitor cells subsequently differentiate into paravertebral and prevertebral sympathetic neurons, intra- and extra-adrenal chromaffin cells and intermediate SIF (small intensely fluorescent ) cells. Based on in vitro studies with isolated SA and chromaffin progenitor cells glucocorticoids have been claimed as essential for suppressing a neuronal commitment and channeling SA cells towards the chromaffin phenotype. Unexpectedly, mice deficient for a functional glucocorticoid receptor possess the full complement of adrenal chromaffin cells at birth. We present a hypothetical scenario consistent with these data, in which chromaffin cell development would be the default pathway in the SA cell lineage, while development into a neuronal direction requires specific growth factor signaling, which is probably distinct for paravertebral and prevertebral sympathetic neurons.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta (TGF-beta) superfamily, is a potent neurotrophic factor for several neuron populations in the central and peripheral nervous system. Members of the neurotrophin, neurokine, and TGF-beta families of growth factors can affect neurons beyond their capacity to promote survival. They can play instructive roles including the determination of a particular transmitter phenotype. Here, we show that GDNF enhances the number of calretinin (CaR)-positive neurons in serum-free cultures of striatal cells isolated from embryonic rats. The effect is dose-dependent, can be elicited with concentrations as low as 0.1 ng/ml, and is not accompanied by increased incorporation of 5-bromo-2'-desoxyuridine and appearance of glial fibrillary acidic protein-positive cells. Similar, but weaker effects can be elicited by brain-derived neurotrophic factor, neurotrophin-3 and -4, fibroblast growth factor-2. Ciliary neurotrophic factor, nerve growth factor, and TGF-beta 1 do not affect striatal CaR expression. GDNF can augment CaR-positive cells at any time point and with a minimal exposure of 18 hr, suggesting induction of the phenotype rather than increased survival. By reverse transcription polymerase chain reaction (RT-PCR), we show that GDNF is expressed in the E16 striatum and in cultures derived from this tissue. GDNF also protected striatal CaR-positive neurons against glutamate toxicity. We conclude that striatal GDNF, in addition to its retrograde trophic role for nigrostriatal dopaminergic neurons, may also act locally within the striatum (e.g., by inducing the CaR phenotype and protecting these cells against toxic insult).

The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein A beta. Proteolysis by gamma-secretase is the last processing step resulting in release of A beta. Cleavage occurs after residue 40 of A beta [A beta(1-40)], occasionally after residue 42 [A beta(1-42)]. Even slightly increased amounts of this A beta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD. Unexpectedly we have identified in neurons the endoplasmic reticulum (ER) as the site for generation of A beta(1-42) and the trans-Golgi network (TGN) as the site for A beta(1-40) generation. It is interesting that intracellular generation of A beta seemed to be unique to neurons, because we found that nonneuronal cells produced significant amounts of A beta(1-40) and A beta(1-42) only at the cell surface. The specific production of the critical A beta isoform in the ER of neurons links this compartment with the generation of A beta and explains why primarily ER localized (mutant) proteins such as the presenilins could induce AD. We suggest that the earliest event taking place in AD might be the generation of A beta(1-42) in the ER.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) 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-beta 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-beta superfamily, but act indirectly, possibly through glial cells.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote survival and differentiation of midbrain dopaminergic (DAergic) neurons in vitro and in vivo. This is consistent with their expression and that of their cognate receptors, trkB and trkC, in the nigrostriatal system. Degeneration of DAergic neurons of the substantia nigra and alpha-synuclein-positive aggregates in the remaining substantia nigra (SN) neurons are hallmarks of Parkinson's disease (PD). Reduced expression of BDNF has been reported in the SN from PD patients. Moreover, mutations in the BDNF gene have been found to play a role in the development of familial PD. We show now that haploinsufficiencies of the neurotrophin receptors trkB and/or trkC cause a reduction in numbers of SN neurons in aged (21-23 month old) mice, which is accompanied by a reduced density in striatal tyrosine hydroxylase immunoreactive (TH-ir) fibers. These aged mutant mice, in contrast to wild-type littermates, display an accumulation of alpha-synuclein in the remaining TH-positive neurons of the SN. We conclude that impairment in trkB and/or trkC signaling induces a phenotype in the aged SN, which includes two hallmarks of PD, losses of TH positive neurons and axons along with massive neuronal deposits of alpha-synuclein.

Basic fibroblast growth factor (bFGF; FGF-2) has potent trophic effects on developing and toxically impaired midbrain dopaminergic (DAergic) neurons which are crucially affected in Parkinson's disease. The trophic effects of FGF-2 are largely indirect, both in vitro and in vivo, and possibly involve intermediate actions of astrocytes and other glial cells. To further investigate the cellular and molecular mechanisms underlying the restorative actions of FGF-2, and to analyse in more detail the changes within astroglial cells in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned striatum, we have studied striatal expression and regulation of connexin-43 (cx43), the principal gap junction protein of astroglial cells, along with the expression of glial fibrillary acidic protein (GFAP), FGF-2, and functional coupling. Our results show an immediate, yet transient increase in cx43 mRNA, and a sustained increase in FGF-2 mRNA, GFAP-positive cells, and cx43-immunoreactive punctata following the MPTP lesion, without any induction of functional coupling between astrocytes and other glial cells as revealed by dye coupling of patched cells. Unilateral administration of FGF-2 in a piece of gelfoam caused a further increase in cx43-positive punctata immediately adjacent to the implant, which was more pronounced than after application of a gelfoam containing the non-trophic control protein in cytochrome C. These changes were parallelled by a small increase in cx43 protein determined by Western blot, but not by alterations in the coupling state of cells in the vicinity of the gelfoam implant. Although our data indicate that MPTP and exogenous FGF-2 may alter expression and protein levels of cx43, they do not support the notion that increases in cellular coupling may underly the trophic and widespread actions of FGF-2 in the MPTP-model of Parkinson's disease. © 1996 Wiley-Liss, Inc.

We have studied the postnatal expression of neurotrophins, their cognate high-affinity trk receptors and the low-affinity NGF receptor (p75LNGFR) in the rat adrenal gland using RT-PCR. Neurotrophin mRNAs were detectable during the whole postnatal period. Strongest signals were obtained for BDNF and NT4/5. Expression of trkA, trkB, trkC and p75LNGFR was found at all ages studied. Signals for trkA were highest in the adult adrenal medulla, whereas signals for p75LNGFR were highest in the adult adrenal cortex. Cur data suggest still largely enigmatic roles for neurotrophins in functions of the adrenal medulla and possibly also the cortex.

Glia cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-beta (TGF-beta) superfamily, has been implicated in the survival, morphological and functional differentiation of midbrain dopaminergic neurons and motoneurons in vitro and in vivo. The factor may thus have utility in the treatment of various human neurodegenerative disorders. Mechanisms regulating expression of GDNF in normal and diseased brain as a possible means to increase the local availability of GDNF are only beginning to be explored. We have established and employed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to study and compare levels of expression of GDNF mRNA in several cell types and to investigate its regulation. GDNF expression was clearly evident in primary cultured astrocytes, the glioma B49 and C6 cell, but less pronounced in the Schwannoma RN22 cell lines. Little or no signal could be observed in neuroblastoma cell lines (IMR32, LAN-1) or the pheochromocytoma cell line PC12, emphasizing the glial character of this factor. Using the C6 cell line we found that fibroblast growth factor-2 (FGF-2; bFGF) can increase GDNF mRNA levels, whereas FGF-1, platelet-derived growth factor (PDGF), and vasoactive intestinal polypeptide (VIP) are apparently ineffective. Several other factors (forskolin, kainic acid, triiodothyronine dexamethasone, GDNF, TGF-beta 1, and interleukin-6) appear to have slightly negative effects on GDNF mRNA levels at the concentrations tested. To further explore the relationship between FGF-2 and GDNF, we also addressed the question whether GDNF, like FGF-2, may have an effect on C6 cell proliferation. We conclude that (1) glial and glial tumor cells, rather than neuronal cell lines, express GDNF, (2) that FGF-2 has a prominent inductive effect on GDNF expression and (3) that GDNF stimulates C6 cell proliferation. Finally, these data suggest that neurotrophic actions of FGF-2 in mixed glial-neuronal cell cultures might be mediated in part by GDNF.

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-beta (TGF-beta) fails to maintain sensory neurons cultured from embryonic day (E) 8 chick dorsal root ganglia (DRG), although DRG neurons are immunoreactive for the TGF-beta receptor type II, which is essential for TGF-beta signaling. However, in combination with various concentrations of NT-3 and NT-4, but not NGF, TGF-beta 3 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-beta decreases neuron survival suggesting that endogenous TGF-beta in these cultures affects the efficacies of neurotrophins. Consistent with this notion and a modulatory role of TGF-beta in neurotrophin functions is the observation that TGF-beta 2 and -beta 3 immunoreactivities and TGF-beta 3 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-beta and LIF in vitro, and possibly also in vivo.

Transforming growth factors beta (TGF-beta), a family of pleiotropic cytokines, are widely distributed in the developing and adult nervous system. In order to further determine the neural functions of TGF-beta, we have localized the TGF-beta isoforms 1, 2 and 3 in the adult rat adrenal medulla and studied the neuroprotective capacity of one representative family member, TGF-beta 2, for those spinal cord neurons which innervate adrenal chromaffin cells and which die after destruction of the adrenal medulla. Unilateral electrothermal destruction of the adrenal medulla led to the disappearance of 25% of sympathetic preganglionic neurons, which are located in the intermediolateral (IML) column of thoracic spinal cord segments 7-10 and can be selectively marked by NADPH-diaphorase. The neurons which disappeared following adrenomedullectomy constitute the full set of neurons that innervate the adrenal medulla. Implantation of gelfoam soaked with 0.5 micrograms TGF-beta 2 into the adrenal wound cavity rescued all spinal cord neurons in the IML ipsilaterally to the lesioned side. Cytochrome c was not effective. Injections of [125I]TGF-beta 2 into the adrenal medulla did not result in retrograde transport and subsequent labelling of spinal cord neurons, suggesting that TGF-beta may exert its neuroprotective actions by indirect mechanisms. TGF-beta applied to cultured adrenocortical cells did not overtly increase the amount of mRNA for fibroblast growth factor-2, an established trophic molecule for sympathetic preganglionic spinal cord neurons. The mechanisms by which TGF-beta exerts its neurotrophic effect are therefore unclear. Even so, our data provide the first evidence that TGF-beta may play an important role in vivo in the control of maintenance of a population of spinal cord neurons.

Neurotrophins have multiple functions for the development of the nervous system. They can promote survival and differentiation of select neuronal populations, but have also been shown to play instructive roles in the determination of the transmitter phenotype of neurons. We have investigated the influence of neurotrophins on the expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), a histochemical marker for nitric oxide synthase, in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age we found NADPH-d reactivity becoming apparent in the spinal cord and predominantly expressed in preganglionic autonomic nuclei. Numbers of NADPH-d-positive neurons in spinal cord cultures were very low 24 h after plating. They did not change significantly until day 4 in vitro. However, treatment with the neurotrophins BDNF, NT-3 or NT-4 significantly increased their numbers. The effect became apparent after just 24 h, and was significant with concentrations as low as 1 ng/ml. Treatment with BDNF, NT-3 and NT-4 also augmented numbers of NADPH-d-positive neurons when initiated after three or five days in culture, and became consistently apparent within 24 h. This suggests that the neurotrophin-mediated increase in NADPH-d-positive neurons is unlikely to be due to promotion of neuron survival. NGF and two members of the transforming growth factor-beta superfamily, which have pronounced trophic effects on select neuron populations in vitro, TGF-beta 1 and GDNF, were not effective. Combined application of NT-4 and NT-3 had no additive effect. Our data therefore suggest that neurotrophins are involved in the developmental regulation of NADPH-d activity in neuron populations of the spinal cord. Neuron populations affected may include preganglionic autonomic neurons. NADPH-d activity may be induced in neurons expressing the enzyme constitutively, yet at undetectable levels, or may be induced de novo.

Growth/differentiation factor 5 (GDF5) is a novel member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. We show here that GDF5 is expresed in the developing CNS including the mesencephalon and acts as a neurotrophic, survival promoting molecule for rat dopaminergic midbrain neurons, which degenerate in Parkinson's disease. Recombinant human GDF5 supports dopaminergic neurons, dissected at embryonic day (E) 14 and cultured for 8 days under serum-free conditions, to almost the same extent as TGF-beta 3, and is as effective as glial cell line-derived neurotrophic factor (GDNF), two established trophic factors for midbrain dopaminergic neurons. In contrast to TGF-beta and GDNF, GDF5 augments numbers of astroglial cells in the cultures, suggesting that it may act indirectly and through pathways different from those triggered by TGF-beta and GDNF. GDF5 also protects dopaminergic neurons against the toxicity of N-methylpyridinium ion (MPP+), which selectively damages dopaminergic neurons through mechanisms currently debated in the etiology of Parkinson's disease (PD). GDF5 may therefore now be tested in animal models of PD and might become useful in the treatment of PD.

In order to evaluate the neurotrophic potential that interleukins may have for nigrostriatal dopaminergic neurons, we have applied the interleukins 1 α, 1β, and 2 through 12 to cultures of E14 rat midbrain floor cells enriched for dopaminergic neurons. IL-6 and -7 were the only interleukins that modestly (130%, as compared to controls, 100%) promoted survival of dopaminergic neurons visualized by their immunoreactivity for tyrosine hydroxylase over an 8-day culture period. The effect was not mediated by astroglial cells. We conclude that most interleukins per se may not act as neurotrophic factors for dopaminergic neurons, although several of them occur in the embryonic and adult CNS.

Using in situ hybridization and reverse transcription combined with the polymerase chain reaction (RT-PCR) we have studied the expression and developmental regulation of interleukin-6 (IL-6) and its receptor mRNAs in rat adrenal medulla. Labeling for both transcripts was much more intense over the adrenal medulla than over cortical regions at postnatal day 70. Levels of mRNAs of both genes measured by RT-PCR increased from birth to adulthood. In vitro survival and neurite growth of chromaffin cells from early postnatal rats were not affected by IL-6. Even so, the presence and developmental regulation of IL-6 and its receptor are consistent with a possible auto- or paracrine role of IL-6 in rat adrenal medulla.

The superfamily of transforming growth factors-beta (TGF-beta) comprises an expanding list of multifunctional proteins serving as regulators of cell proliferation and differentiation. Prominent members of this family include the TGF-beta s 1-5, activins, bone morphogenetic proteins and a recently discovered glial cell line-derived neurotrophic factor (GDNF). In the present study we demonstrate and compare the survival promoting and neuroprotective effects of TGF-beta 1, -2 and -3, activin A and GDNF for midbrain dopaminergic neurons in vitro. All proteins increase the survival of tyrosine hydroxylase-immunoreactive dopaminergic neurons isolated from the embryonic day (E) 14 rat mesencephalon floor to varying extents (TGF-beta s 2.5-fold, activin A and GDNF 1.6-fold). TGF-beta s, activin A and GDNF did not augment numbers of very rarely observed astroglial cells visualized by using antibodies to glial fibrillary acidic protein and had no effect on cell proliferation monitored by incorporation of BrdU. TGF-beta 1 and activin A protected dopaminergic neurons against N-methyl-4-phenylpiridinium ion toxicity. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that TGF-beta 2 mRNA, but not GDNF mRNA, is expressed in the E14 rat midbrain floor and in mesencephalic cultures. We conclude that TGF-beta s 1-3, activin A and GDNF share a neurotrophic capacity for developing dopaminergic neurons, which is not mediated by astroglial cells and not accompanied by an increase in cell proliferation.

Neurotrophic factors are operationally defined as molecules that promote the survival and differentiation of neurons. Chemically, they belong to divergent classes of molecules but most of the classic neurotrophic factors are proteins. Together with stem cells, viral vectors and genetically engineered cells, they constitute important tools in neuroprotective and regenerative neurobiology. Protein neurotrophic molecules signal through receptors located on the cell membrane. Their downstream signaling exploits pathways that are often common to chemically different factors and frequently target a relatively restricted set of transcription factors, RNA interference and diverse molecular machinery involved in the life vs. death decisions of neurons. Application of neurotrophic factors with the aim of curing or, at least, improving the outcome of neurodegenerative diseases requires (1) profound knowledge of the complex molecular pathology of the disease, (2) the development of animal models as closely as possible resembling the human disease, (3) the identification of target cells to be addressed, (4) intense efforts in chemical engineering to ensure the stability of molecules or to design carriers and small analogs with the ability to cross the blood–brain barrier and (5) scrutinity with regard to possible side effects. Last, but not least, engineering efforts to optimize administration, e.g., by designing the right canulae and infusion devices, are important for the successful translation of preclinical advances into clinical benefit. This article presents selected examples of neurotrophic factors that are currently being tested in animal models or developed for transfer to the clinic, with a major focus on factors with the potential of becoming applicable in various forms of retinal degeneration.

The transforming growth factors beta (TGF-beta), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well-defined steps of cellular actions possibly controlled by TGF-beta s, and since TGF-beta mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regeneration) or transfected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post-lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF-beta 1-3 mRNA were determined for each segment. We demonstrate that TGF-beta 1 mRNA levels are higher than those of TGF-beta 3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF-beta 2 mRNA could not be detected. For TGF-beta 1-3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF-beta immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF-beta-IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF-beta-IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF-beta-IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF-beta expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF-beta mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a "reactive" Schwann cell phenotype after peripheral nerve lesion might involve an upregulation of TGF-beta expression.

Chromaffin cells of the adrenal medulla were used to study the release of neurotrophic factors operationally defined by their capacity to promote the in vitro survival of embryonic neurons from the peripheral and central nervous system. Chromaffin cells are closely related to sympathetic neurons in terms of their transmitters and specific proteins and, like sympathetic neurons, receive preganglionic cholinergic, aminergic and peptidergic neuronal inputs. The issue of whether chromaffin cells store and secrete neurotrophic factors is therefore pertinent to the question whether trophic mechanisms may be involved in neuronal interactions and what modes of secretion are employed to liberate neurotrophic factors from neurons. Cell culture media conditioned by purified bovine chromaffin cells supported several neuron populations in vitro. Stimulation of the chromaffin cells with the eholinergic agonist carbaehol (10−4 M) increased in parallel the output of neurotrophic factor activity (assayed on chick ciliary ganglionic neurons) as well as two components specifically located in chromaffin granules, chromogranin A and catecholamines. The release of all three components was partially blocked by the Ca2+ channel blocker verapamil (10−5M), suggesting co-storage and -release of neurotrophic factors, chromogranin A and catecholamines in/from chromaffin granules. Neurotrophic factor activity for ciliary ganglionic neurons accumulating in the medium of unstimulated chromaffin cells decreased with time, and so did catecholamines. In contrast, amounts of neurotrophic factors and catecholamines released by challenging cells with carbaehol did not significantly decline up to 62 h. The neurotrophic factor activity tested on chick ciliary, sensory and spinal cord neurons as well as on rat hippocampal neurons was heat- and trypsin-labile and could not be blocked by polyclonal antibodies against bovine nerve growth factor and the chromogranin A, B, and C. Defined fragments of chromogranin A and pancreastatin were devoid of neurotrophic activity. Our results suggest the presence of one or several neurotrophic factors in chromaffin granules, which can be released by exocytosis and may be potentially relevant for the maintenance of neurons innervating the adrenal medulla.

Neuroblastoma cells have been shown to contain growth factors, which may be involved in the regulation of cell proliferation and/or differentiation. We have examined whether human IMR-32 neuroblastoma cells store factors with a capacity to promote neuron survival and differentiation. Heparin affinity chromatography, cell blotting, and isoelectric focusing of IMR-32 cell extracts revealed multiple neurotrophic activities at molecular weights of 16.8, 24.1, 39.0, 45.3, 52.3 and isoelectric points of 4.0, 5.0, 8.3, 9.0. Immunocytochemistry, immunoblotting, and radio-immunoassay with specific antibodies suggests that one neurotrophic activity is immunologically identical with basic fibroblast growth factor (bFGF). This assumption is supported by Northern blot analysis, which shows a 6.0 kb bFGF transcript.

The extracellular membrane-associated protein F-spondin has been implicated in cell–matrix and cell–cell adhesion and plays an important role in axonal pathfinding. We report here that F-spondin is expressed in non-neuronal cells in the embryonic chicken ciliary ganglion (CG) and robustly promotes survival of cultured CG neurons. Using deletion constructs of F-spondin we found that the amino-terminal Reelin/Spondin domain cooperates with thrombospondin type 1 repeat (TSR) 6, a functional TGFβ-activation domain. In ovo treatment with blocking antibodies raised against the Reelin/Spondin domain or the TSR-domains caused increased apoptosis of CG neurons during the phase of programmed cell death and loss of about 30% of the neurons compared to controls. The Reelin/Spondin domain receptor — APP and its downstream signalling molecule disabled-1 are expressed in CG neurons. F-spondin induced rapid phosphorylation of disabled-1. Moreover, both blocking the central APP domain and interference with disabled-1 signalling disrupted the survival promoting effect of F-spondin. Taken together, our data suggest that F-spondin can promote neuron survival by a mechanism involving the Reelin/Spondin and the TSR domains.

Growth factors with already established multiple effects on non-neural cells continue to be of considerable interest to researchers with regard to the nervous system, where regulation of cell maintenance and plasticity in relation to lesion and regeneration is part of their functional repertoire. Fibroblast growth factors, interleukins, and type beta transforming growth factors are prominent representatives of such proteins. Ciliary neurotrophic factor is another multifunctional neurokine. The proposed role of this molecule as a 'lesion factor', however, is still not firmly settled.

To further clarify the function of basic fibroblast growth factor (bFGF) in the nervous system, we have examined its distribution, lesion-dependent regulation, retrograde transport, and trophic roles on rat hypoglossal neurons. In adult rats, bFGF-like immunoreactivity is localized in hypoglossal motoneurons, drastically reduced 2 days after axotomy, and re-expressed by 11 days. Neuron numbers and morphology assessed by Nissl staining are not affected by the lesion. 125J bFGF is specifically retrogradely transported by hypoglossal motoneurons from their peripheral nerve terminals. Moreover, bFGF stimulates the in vitro survival of hypoglossal neurons (ED50 2 ng/ml). In vivo administration of bFGF prevents lesion-induced motoneuron death to 14% in 7 day old rats and to 60% in 18 day old rats, but not the axotomy-induced decrease of choline acetyltransferase activity in the hypoglossal nucleus of adult rats. These results are consistent with a neurotrophic role of bFGF in the hypoglossal system. © 1992 Wiley-Liss, Inc.