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Growth/differentiation factor−15 (Gdf-15) is a member of the transforming growth factor-β (Tgf-β) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson’s disease (PD) by comparing Gdf-15 +/+ and Gdf-15 −/− mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 +/+ and Gdf-15 −/− mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 +/+ and Gdf-15 −/− mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.

Neuroendocrine chromaffin cells exist in both intra- and extra-adrenal locations; the organ of Zuckerkandl (OZ) constitutes the largest accumulation of extra-adrenal chromaffin tissue in mammals. The OZ disappears postnatally by modes that are still enigmatic but can be maintained by treatment with glucocorticoids (GC). Whether the response to GC reflects a pharmacological or a physiological role of GC has not been clarified. Using mice with a conditional deletion of the GC-receptor (GR) gene restricted to cells expressing the dopamine β-hydroxylase (DBH) gene [GR(fl/fl) ; DBHCre abbreviated (GR(DBHCre) )], we now present the first evidence for a physiological role of GC signalling in the postnatal maintenance of the OZ: postnatal losses of OZ chromaffin cells in GR(DBHCre) mice are doubled compared to wild-type littermates. We find that postnatal cell loss in the OZ starts at birth and is accompanied by autophagy. Electron microscopy reveals autophagic vacuoles and autophagolysosomes in chromaffin cells. Autophagy in OZ extra-adrenal chromaffin cells is confirmed by showing accumulation of p62 protein, which occurs, when autophagy is blocked by deleting the Atg5 gene (Atg5(DBHCre) mice). Cathepsin-D, a lysosomal marker, is expressed in cells that surround chromaffin cells and are positive for the macrophage marker BM8. Macrophages are relatively more abundant in mice lacking the GR, indicating more robust elimination of degenerating chromaffin cells in GR(DBHCre) mice than in wild-type littermates. In summary, our results indicate that extra-adrenal chromaffin cells in the OZ show signs of autophagy, which accompany their postnatal numerical decline, a process that is controlled by GR signalling.

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.

Purpose Several members of the fibroblast growth factor (FGF) family have been shown to be dysregulated in individuals with major depression, and treatment with antidepressants has been reported to increase FGF-2 mRNA levels in the forebrain. Methods We have used the tail suspension test (TST), and olfactory bulbectomy (OBX), and FGF-2 deficient mice to investigate putative roles of FGF-2 as an antidepressant and mediator of antidepressive drug actions. Results FGF-2 applied intraventricularly generated antidepressant-like effects in the TST. FGF-2, similar to the antidepressant amitriptyline, attenuated neuron demise in the piriform cortex and posterolateral cortical nucleus of the amygdala following OBX. Moreover, OBX induced reduction in hippocampal neurogenesis could be ameliorated by subsequent treatment with either amitriptyline or FGF-2. Furthermore, FGF-2 was effective in reversing depressive-like behavior induced by OBX, monitored in the locomotor activity and the passive avoidance test. In bulbectomized FGF-2 deficient mice, treatment with amitriptyline protected neurons, but failed to reverse behavioral alterations. Conclusions Together, these results suggest that FGF-2 constitutes both a potential target for antidepressive treatments and an important growth factor in the cytokine network underlying the actions of antidepressive drugs. The results further suggest a requirement of endogenous FGF-2 for mediating behavioral, but not neuroprotective actions of amitriptyline.

Fibroblast growth factor 2 (FGF-2) was the first growth factor discovered that exerted prominent protective and regenerative effects in an animal model of Parkinson's disease, the MPTP-lesioned dopaminergic nigrostriatal system. To address the putative physiological relevance of endogenous FGF-2 for midbrain dopaminergic neurons, we have analysed densities of tyrosine hydroxylase (TH)positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum and amygdala of adult FGF-2-deficient mice. We found that densities of TH-immunoreactive (ir) cells in the SN as well as densities of TH-ir fibers in the striatum and amygdala were unaltered as compared with wild-type littermates. There is evidence to suggest that growth factor deficits do not become apparent unless a system is challenged in a lesioning paradigm. We therefore tested the ability of the nigrostriatal system with respect to its ability to cope with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) intoxication. Treatment with 20 mg/kg MPTP on three consecutive days reduced dopamine levels in the striatum by about 80%. Densities of TH-positive neurons in the SN were reduced by 71%. However, both parameters did not significantly differ between FGF-2 (-/-) mice and wild-type littermates. Our results therefore suggest that FGF-2, despite its prominent pharmacological potency as a neurotrophic factor for the dopaminergic nigrostriatal system, is not crucial for maintaining its structural integrity and ability to cope with MPTP intoxication.

The diversification of neural-crest-derived sympathoadrenal (SA) progenitor cells into sympathetic neurons and neuroendocrine adrenal chromaffin cells was thought to be largely understood. In-vitro studies with isolated SA progenitor cells had suggested that chromaffin cell differentiation depends crucially on glucocorticoids provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor gene had revealed that adrenal chromaffin cells develop mostly normally in these mice. Alternative cues from the adrenal cortex that may promote chromaffin cell determination and differentiation have not been identified. We therefore investigated whether the chromaffin cell phenotype can develop in the absence of an adrenal cortex, using mice deficient for the nuclear orphan receptor steroidogenic factor-1 (SF1), which lack adrenal cortical cells and gonads. We show that in Sf1–/– mice typical chromaffin cells assemble correctly in the suprarenal region adjacent to the suprarenal sympathetic ganglion. The cells display most features of chromaffin cells, including the typical large chromaffin granules. Sf1–/–chromaffin cells are numerically reduced by about 50% compared with the wild type at embryonic day (E) 13.5 and E17.5. This phenotype is not accounted for by reduced survival or cell proliferation beyond E12.5. However, already at E12.5 the `adrenal' region in Sf1–/– mice is occupied by fewer PHOX2B+ and TH+ SA cells as well as SOX10+ neural crest cells. Our results suggest that cortical cues are not essential for determining chromaffin cell fate, but may be required for proper migration of SA progenitors to and/or colonization of the adrenal anlage.

Evidence is presented that the seminal vesicle of the bull is an important source of a protein which is very similar to nerve growth factor from mouse submaxillary gland. Its chemical and biological properties in vitro are compared with those of nerve growth factor from other sources.

Phox2B, a homeodomain transcription factor closely related to Phox2A, is expressed in peripheral and central noradrenergic neurons. In neural crest (NC) derivatives Phox2B is restricted to sympathetic and parasympathetic ganglia, enteric neurons, and adrenal and extraadrenal chromaffin cells. Similar to MASH-1, Phox2B has been implicated in synchronizing pan-neuronal and catecholaminergic phenotype-specific aspects of neurogenesis. The role of Phox2B for the differentiation of the neuroendocrine NC derivatives, the adrenal medullary chromaffin cells, has not been explored. We have previously reported that in MASH-1-deficient mice most chromaffin cells are arrested at the early neuroblast stage and lack catecholaminergic differentiation. We show now that in Phox2B knockout/lacZ knockin mice the maturation of presumptive chromaffin cells is arrested at an even earlier stage of development. The cells lack the catecholaminergic marker enzyme TH and fail to form a centrally located medulla. In contrast to MASH-1 (−/−) mice they do not express dHand, Phox2A, c-ret, neurofilament, neuron-specific tubulin, and NCAM and appear ultrastructurally more immature. Many of these cells die by apoptosis. Despite the complete lack of differentiation, few lacZ-positive adrenal cells can still be found at E16.5. We conclude that Phox2B regulates very early events in the differentiation of adrenal chromaffin cells distinct to steps, which essentially require MASH-1.

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.

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 investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.

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)-beta in terms of its biological activity. The assay used to assess the activity of TGF-beta is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-beta 1, -beta 2, and -beta 3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-alpha, platelet-derived growth factor-AB, insulin-like growth factor-1, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-beta 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 approximately 1 ng of TGF-beta/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-beta 1/beta 2/beta 3. TGF-beta is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10(-5) M). These data suggest that TGF-beta is stored in chromaffin granules and can be released by exocytosis.

The developmental diversification of neural crest-derived sympathoadrenal (SA) progenitor cells into neuroendocrine adrenal chromaffin cells and sympathetic neurons has been thought to be largely understood. Based on two decades of in vitro studies with isolated SA progenitor and chromaffin cells, it was widely assumed that chromaffin cell development crucially depends on glucocorticoid hormones provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor has revealed that the chromaffin cell phenotype develops largely normally in these mice, except for the induction of the adrenaline synthesizing enzyme phenylethylamine N-methyl transferase. In a search for novel candidate genes that might be involved in triggering the sympathetic neuron/chromaffin cell decision, we have studied putative contributions of transforming growth factor (TGF)-alpha, BMP-4, and the transcription factor MASH-1, molecules with distinct expressions in SA progenitor cells, in their migratory pathways and final destinations. TGF-beta2 and -beta3 and BMP-4 are highly expressed in the wall of the dorsal aorta and in the adrenal anlagen during and after immigration of SA progenitors but expressed at much lower levels in sympathetic ganglia. We found that neutralizing antibodies against all three TGF-beta isoforms applied to the chorionic-allantoic membrane (CAM) of quail embryos interfere with proliferation of immigrated adrenal chromaffin cells but do not affect their specific neuroendocrine ultrastructural phenotype. Grafting of noggin-producing cells to the CAM, which scavenges BMPs, interferes with visceral arch and limb development but does not overtly affect the chromaffin phenotype. The transcription factor MASH-1 promotes early differentiation of SA progenitors. Mice deficient for MASH-1 lack sympathetic ganglia, whereas the adrenal medulla previously has been reported to be present. We show here that most adrenal medullary cells in MASH-1(-/-) mice identified by Phox2b immunoreactivity lack the catecholaminergic marker tyrosine hydroxylase. More surprisingly, most cells do not contain chromaffin granules and display a neuroblast-like ultrastructure and show strongly enhanced expression of c-RET comparable to that observed in sympathetic ganglia. Together, our data suggest that TGF-betas and BMP-4 do not seem to be essential for chromaffin cell differentiation. In contrast with previous reports, however, MASH-1 apparently plays a crucial role in chromaffin cell development.

The development of sympathetic neurons and chromaffin cells is differentially controlled at distinct stages by various extrinsic and intrinsic signals. Here we use conditional deletion of Dicer1 in neural crest cells and noradrenergic neuroblasts to identify stage specific functions in sympathoadrenal lineages. Conditional Dicer1 knockout in neural crest cells of Dicer1Wnt1Cre mice results in a rapid reduction in the size of developing sympathetic ganglia and adrenal medulla. In contrast, Dicer1 elimination in noradrenergic neuroblasts of Dicer1DbhiCre animals affects sympathetic neuron survival starting at late embryonic stages and chromaffin cells persist at least until postnatal week 1. A differential function of Dicer1 signaling for the development of embryonic noradrenergic and cholinergic sympathetic neurons is demonstrated by the selective increase in the expression of Tlx3 and the cholinergic marker genes VAChT and ChAT at E16.5. The number of Dbh, Th and TrkA expressing noradrenergic neurons is strongly decreased in Dicer1-deficient sympathetic ganglia at birth, whereas Tlx3+/ Ret+ cholinergic neurons cells are spared from cell death. The postnatal death of chromaffin cells is preceded by the loss of Ascl1, mir-375 and Pnmt and an increase in the markers Ret and NF-M, which suggests that Dicer1 is required for the maintenance of chromaffin cell differentiation and survival. Taken together, these findings demonstrate distinct stage and lineage specific functions of Dicer1 signaling in differentiation and survival of sympathetic neurons and adrenal chromaffin cells.

Connexins (cx) constitute a family of transmembrane proteins that form gap junction channels allowing metabolic and electrical coupling of cellular networks. Initial studies on the expression of cx in the developing brain have suggested that cx may undergo dynamic changes and may possibly be implicated in synchronizing development and differentiation of neural progenitor cells and young neurons. We have investigated expression of cx26, cx32, cx43, and cx45 in the midbrain floor, where nigrostriatal dopaminergic neurons originate and differentiate. This neuron population is of major importance in regulating motor-functions. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed low levels of cx26-mRNA in the midbrain floor at E12, which gradually increased during pre- and postnatal development, reaching a maximum in the adult. Cx32-mRNA-levels reached a first peak at E16, and showed highest levels in adulthood. Cx43 was highly expressed at E12, decreased until E18, and subsequently increased again until adulthood. Cx45 mRNA was prominent at all developmental ages, but slightly decreased after the first postnatal week. Double-labeling for the dopaminergic neuronal marker tyrosine hydroxylase (TH), and cx-immunoreactivities (ir) evaluated by quantitative confocal laser microscopy revealed both distinct and similar developmental patterns for the individual cx investigated. Cx26 was highest at E14, decreased towards birth, and subsequently increased again reaching about 50% of the E14 level in the adult. Cx32-ir peaked at E16 and dropped to low levels after birth. Cx43-ir was highest at E12, decreased sharply at E14, reached its lowest levels at birth, but modestly increased again afterwards. Cx45-ir showed a biphasic pattern, with two prominent peaks at E12 and E18, followed by a massive postnatal decrease. Taken together, our results reveal that expression and ir of cx in the midbrain floor and dopaminergic neurons, respectively, follow cx-type specific patterns that temporally coincide with important steps of midbrain morphogenesis, as e.g. progenitor cell formation and migration (E12), early differentiation (E14-16), target encounter (E16-18) and postnatal functional maturation of the nigrostriatal system.

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.

Transforming growth factor-βs (TGF-βs) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, differentiation, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-βs, growth/differentiation factor-15 (GDF-15). GDF-15 is identical to macrophage inhibitory cytokine-1 (MIC-1). GDF-15/MIC-1 mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-15/MIC-1 is a potent survival promoting and protective factor for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the embryonic rat midbrain floor. The trophic effect of GDF-15/MIC-1 was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-15/MIC-1 probably acts directly on neurons. GDF-15/MIC-1 also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neuronsin vivo. Unilateral injections of GDF-15/MIC-1 into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 μg each) immediately before a 6-OHDA injection (8 μg) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least 1 month. Administration of 5 μg of GDF-15/MIC-1 in the same paradigm also provided significant neuroprotection. GDF-15/MIC-1 also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-15/MIC-1 is a novel neurotrophic factor with prominent effects on DAergic and serotonergic neurons. GDF-15/MIC-1 may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.

Fibroblast growth factor (FGF)-2 is an abundant astroglial cytokine. We have previously shown that FGF-2 downregulates gap junctions in primary astroglial cultures (B. Reuss et al., 1998, Glia 22, 19-30). We demonstrate now that FGF-2 induces astroglial dopamine (DA) sensitivity and D1 dopamine-receptor (D1DR) antigen and message in cortical and striatal astroglial cultures. On the functional level 10 micromol/L DA triggered transient increases in astroglial [Ca(2+)](i). In gap-junction-coupled cells, no FGF-2-dependent changes in proportions of DA-responsive cells were observable. However, uncoupling with octanol or 18alpha-glycirrhetinic acid isolated the smaller population of astrocytes intrinsically sensitive to DA which was significantly increased by FGF-2 in cortical and striatal cultures. Administration of DR-specific substances revealed that FGF-2 upregulated D1DR. These results indicate that downregulation of astroglial gap junctions by FGF-2 is accompanied by an upregulation of D1DR and DA sensitivity, adding a new aspect to the role of FGF-2 in the regulation of brain functions.

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.

Chromaffin cells and sympathetic neurons are derivatives of the sympathoadrenal cell lineage of the neural crest. Although they are similar with respect to many cell biological aspects, chromaffin cells, in contrast to sympathetic neurons, continue to synthesize DNA and proliferate through their whole life span. Large numbers of neural and hormonal signals have been implicated in the regulation of chromaffin cell proliferation based on in vitro studies. We have previously shown that chromaffin cells synthesize and release transforming growth factor-beta (TGF-beta) and that exogenously applied TGF-beta suppresses chromaffin cell proliferation in vitro. We show now that TGF-beta is also a physiologically relevant factor in the control of cell division in developing chromaffin cells. We have neutralized endogenous TGF-beta in quail embryos using a monoclonal antibody recognizing all three TGF-beta isoforms, TGF-beta1, -beta2, and -beta3. Embryos deprived of TGF-beta show a prominent increase in numbers of tyrosine hydroxylase (TH)-immunoreactive adrenal chromaffin cells and TH-positive cells incorporating 5'-bromo-2'deoxyuridine. This is the first documentation of the physiological significance of a factor that has been suggested to play a role in the regulation of chromaffin cell mitosis based on in vitro experiments.

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.

Serotonergic neurons in the central nervous system are crucial in the control of autonomic functions and behavior. Mechanisms by which development and maintenance of the serotonergic transmitter phenotype is regulated include activation of protein kinase A (PKA). Using cultures established from the E14 rat raphe we show here that forskolin (10 microM) increases numbers of neurons expressing tryptophan hydroxylase (TpOH), the key enzyme of serotonin synthesis, and uptake of the false serotonergic transmitter 5, 7-dihydroxytryptamine (5,7-DHT). As shown by short-term treatments the effect is due to phenotype induction rather than survival. To begin to understand downstream or parallel signaling pathways required for the PKA-mediated induction of serotonergic markers, we have studied the putative implication of brain-derived neurotrophic factor (BDNF) and its receptor trkB. Treatment of raphe neurons with forskolin induced BDNF mRNA assayed by competitive RT-PCR. Moreover, trkB-IgG receptor bodies fully prevented the forskolin-induced numerical increase in TpOH- and 5,7-DHT-positive cells suggesting an implication of a TrkB-activated pathway. TrkC-IgG had no effect. K252b, a specific inhibitor of trk kinase activity likewise abolished the induction of serotonergic markers by forskolin. In turn, the inductive effect of BDNF on serotonergic markers was blocked by KT5720, a specific inhibitor of PKA. Taken together, these data suggest that co-activation of cAMP- and trkB-dependent signaling pathways plays a crucial role in the regulation of the serotonergic neuronal phenotype.

Molecular mechanisms underlying the generation of distinct cell phenotypes is a key issue in developmental biology. A major paradigm of determination of neural cell fate concerns the development of sympathetic neurones and neuroendocrine chromaffin cells from a common sympathoadrenal (SA) progenitor cell. Two decades of in vitro experiments have suggested an essential role of glucocorticoid receptor (GR)-mediated signalling in generating chromaffin cells. Targeted mutation of the GR should consequently abolish chromaffin cells. The present analysis of mice lacking GR gene product demonstrates that animals have normal numbers of adrenal chromaffin cells. Moreover, there are no differences in terms of apoptosis and proliferation or in expression of several markers (e.g. GAP43, acetylcholinesterase, adhesion molecule L1) of chromaffin cells in GR-deficient and wild-type mice. However, GR mutant mice lack the adrenaline-synthesizing enzyme PNMT and secretogranin II. Chromaffin cells of GR-deficient mice exhibit the typical ultrastructural features of this cell phenotype, including the large chromaffin granules that distinguish them from sympathetic neurones. Peripherin, an intermediate filament of sympathetic neurones, is undetectable in chromaffin cells of GR mutants. Finally, when stimulated with nerve growth factor in vitro, identical proportions of chromaffin cells from GR-deficient and wild-type mice extend neuritic processes. We conclude that important phenotypic features of chromaffin cells that distinguish them from sympathetic neurones develop normally in the absence of GR-mediated signalling. Most importantly, chromaffin cells in GR-deficient mice do not convert to a neuronal phenotype. These data strongly suggest that the dogma of an essential role of glucocorticoid signalling for the development of chromaffin cells must be abandoned.

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.

We have cloned a novel member of the transforming growth factor-beta (TGF-beta) superfamily from a human placental cDNA library. The sequence is identical to five very recently published sequences, of which only one (macrophage inhibitory cytokine-1, MIC-1) has been characterized in terms of function. In light of the present data demonstrating the wide distribution of the mRNA and putative multifunctionality, we propose to name this molecule growth/differentiation factor-15/MIC-1 (GDF-15/MIC-1). The deduced amino acid sequence reveals typical features of a secreted molecule. The epithelium of the choroid plexus is the only site in the adult brain expressing detectable levels of GDF-15/MIC-1 mRNA. Many epithelia of non-neural tissues including those of the prostate and intestinal mucosa, bronchi and bronchioli, secretory tubuli of the submandibular gland, and lactating mammary gland are prominent sites of GDF-15/MIC-1 synthesis. GDF-15/MIC-1 is also strongly expressed by macrophages in the adrenal gland. Thus, GDF-15/MIC-1, like many other members of the TGF-beta superfamily, is widely distributed in adult tissues, being most strongly expressed in epithelial cells and macrophages.

Serotonergic (5-HT) neurons of the CNS develop as two separate clusters, a rostral and a caudal group, within the brain stem raphe. We show here that the transforming growth factors -β2 and -β3 (TGF-β) and the TGF-β type II receptor are expressed in the embryonic rat raphe, when 5-HT neurons develop and differentiate. To investigate putative roles of TGF-βs in the regulation of 5-HT neuron development we have generated serum-free cultures isolated either from the rostral or the caudal embryonic rat raphe, respectively. In cultures from the caudal E14 raphe saturating concentrations (5 ng/ml) of TGF-β2 and -β3 augmented numbers of tryptophan hydroxylase (TpOH) -immunoreactive neurons and cells specifically taking up 5,7-dihydroxytryptamine (5,7-DHT) by about 1.7-fold over a period of 4 days. Treatment with TGF-βs also increased uptake of 3H- 5HT uptake about 1.7-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 delayed short-term treatments. Comparing rostral and caudal raphe cultures from different embryonic ages suggests that distinct effects of TGF-βs reflect the responsiveness of 5-HT neurons at different ages rather than of different origins. J. Neurosci Res. 56:531–538, 1999. © 1999 Wiley-Liss, Inc.

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.

Embryonic day 14 rat midbrain cultures were kept for 7 days in vitro and then intoxicated with radical donors iron and sodium-nitroprusside for 24 h. Tyrosine-hydroxylase positive neurons in cultures which were additionally treated with growth/differentiation factor-5 (GDF-5) survived to a significantly higher percentage as compared to sister cultures without factor supplementation. Since the degeneration of TH positive cells is a key feature in Parkinson's disease, GDF-5 might be a putative therapeutical agent for this disorder.

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.

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).

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.

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.

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 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.

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.

Ten years of research within the DFG-funded Collaborative Research Grant SFB 488 at the University of Heidelberg have added many new facets to our understanding of chromaffin cell development. Glucocorticoid signaling is no longer the key for understanding the determination of the chromaffin phenotype, yet a novel role has been attributed to glucocorticoids: they are essential for the postnatal maintenance of adrenal and extra-adrenal chromaffin cells. Transcription factors, as, e.g. MASH1 and Phox2B, have similar, but also distinct functions in chromaffin and sympathetic neuronal development, and BMP-4 not only induces sympathoadrenal (SA) cells at the dorsal aorta and within the adrenal gland, but also promotes chromaffin cell maturation. Chromaffin cells and sympathetic neurons share a common progenitor in the dorsal neural tube (NT) in vivo, as revealed by single cell electroporations into the dorsal NT. Thus, specification of chromaffin cells is likely to occur after cell emigration either during migration or close to colonization of the target regions. Mechanisms underlying the specification of chromaffin cells vs. sympathetic neurons are currently being explored.

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.

Following a previous study in which we showed ameliorative effects of basic fibroblast growth factor (FGF-2) locally applied to the nigrostriatal system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice, we investigated FGF-2 actions at different time intervals after the lesion and effects on non-dopaminergic striatal transmitter systems. A triple intraperitoneal injection of 30 mg/kg MPTP at 24 h intervals caused a reduction of striatal dopamine to 23% of control levels that lasted for at least 4 weeks. Four micrograms FGF-2 soaked into gel foam and placed onto the right striatum partially and bilaterally restored dopamine levels and tyrosine hydroxylase activity after 2 weeks, when the treatment started simultaneously or 1 day after the toxin lesion. FGF-2 was ineffective, if administration commenced with a delay of 7 days. Striatal neurotransmitters that are known to be linked to the dopaminergic system were also altered by the MPTP treatment. GABA was significantly increased, while somatostatin levels were reduced. Upon FGF-2 administration both GABA and somatostatin levels were partially normalized. Our data are consistent with the notion that FGF-2 protects and rescues acutely and subacutely MPTP-lesioned nigrostriatal neurons and that its effects must be mainly indirect. Likewise, positive effects of FGF-2 on non-dopaminergic neurons may be due to the partial restoration of striatal dopamine.

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.

We have raised a monoclonal antibody (MAB-1E10) reactive with the intact forms but not the processing products of the chromaffin cell vesicle protein chromogranin B (CgB). The antibody recognizes rat and human, but not bovine and chick adrenal chromaffin cells. In addition, MAB-1E10 immunoreactivity was detected in rat PC 12 pheochromocytoma cells and in pituitaries. Several other tissues, including pancreas, small intestine and superior cervical ganglia, which are known to contain CgB in endocrine cells or neurons, respectively, were found not to be reactive with MAB-1E10. Using short-term cultures of dissociated adrenal chromaffin cells from Hannover-Wistar rats, we found that the expression of intact CgB is developmentally regulated. Between embryonic day 19 and postnatal day 40, about 80% of adrenal chromaffin cells--identified by their reactivity with an antibody against the enzyme dopamine-beta-hydroxylase--were found to be reactive with MAB-1E10. The proportion of positive cells subsequently decreased to about 5% at postnatal day 90. In the presence of glucocorticoids, this decrease was reduced to about 45% CgB-positive cells at postnatal day 90. In another rat strain, Sprague-Dawley rats, the proportion of MAB-1E10-immunoreactive chromaffin cells (about 50%) remained constant from birth to adulthood. Our results indicate that CgB is differentially expressed and/or processed in different rat tissues, strains and during development, and furthermore, that expression or processing in rat chromaffin cells might be regulated by glucocorticoids. Intact CgB appears to be a marker for a subpopulation of chromaffin cells, but its function(s) remains to be clarified.

Chromaffin granules, the secretory organelles of the neuron-like adrenal medullary chromaffin cells, have previously been shown to store and liberate neurotrophic activities that support in vitro survival of several neuron populations including those innervating the adrenal medulla. Molecules resembling fibroblast growth factor and ciliary neurotrophic factor have been identified among these activities. Since chromaffin granules store a variety of neuropeptides and many neuropeptides can have pleiotropic effects on neuronal growth and maintenance we have tested 24 different neuropeptides for their capacities to promote survival of embryonic chick ciliary, dorsal root and sympathetic ganglionic neurons. Peptides tested included several derivatives of proenkephalin (Leu- and met-enkephalin, fragments BAM 22, B, F and E), somatostatin, substance P, neuropeptide Y, neurotensin, VIP, bombesin, secretin, pancreastatin, dynorphin B, dynorphin 1–13, β-endorphin, alpha-, beta-, and gamma-MSH. Control cultures received saturating concentrations of ciliary neurotrophic or nerve growth factor (CNTF; NGF), or no trophic supplements. At 1 × 10 −5 M leu- and met-enkephalin as well as somatostatin supported sympathetic neurons to the same extent as NGF. At the same concentrations, leuenkephalin, the proenkephalin fragments BAM 22 and E, and somatostatin maintained about half of the dorsal root ganglionic neurons supported by NGF, but were not effective on ciliary neurons. VIP promoted the survival of approximately 50% of the ciliary and embryonic day 10 dorsal root ganglionic neurons as compared to saturating amounts of CNTF, but required the presence of non-neuronal cells in the cultures to be effective. Neurotensin (1 × 10 −5 M ) had a small effect on ciliary neurons. All other peptides had no effect on any neuron population tested. Our results suggest that neuropeptides that occur in the adrenal medulla may play a role for the maintenance of cholinergic, sensory, and sympathetic neurons innervating the adrenal medulla.

Fibroblast growth factor (FGF)-2 is known to have important pleiotropic effects in neuronal and glial cells during various physiological and pathological events. To investigate the role of endogenous FGF-2 in the differentiation of astrocytes, we studied the expression of glial fibrillary acidic protein (GFAP) in the hindbrain of the FGF-2 null mouse. GFAP was drastically decreased in a region-specific manner in the hindbrain of the adult and developing FGF-2 null mouse without an associated change in the expression of alternate markers for astrocytes. The deficit was evident in the astrocytes of pontine and medullary gray matter but not in the white matter. The astrocytes of the gray and white matter were seen to express FGF-2 and FGF receptors in a distinct pattern. The methylation of histone H3 at lysine 4 residue (H3K4me2) associated with the STAT (signal transducer and activator of transcription)-binding site of the GFAP promoter was significantly decreased in the gray matter of the FGF-2 null mouse, suggesting a role for FGF-2 in the epigenetic regulation of astrocyte differentiation in vivo. These observations underscore the importance of FGF-2 in astroglial differentiation in the hindbrain and the heterogeneity of astrocytes in their requirement for FGF-2 as a differentiation inducing signal.© 2011 Wiley-Liss, Inc.