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Growth/differentiation factor-15 (GDF-15) is a widely expressed distant member of the TGF-β superfamily with prominent neurotrophic effects on midbrain dopaminergic neurons. We show here that GDF-15-deficient mice exhibit progressive postnatal losses of spinal, facial, and trigeminal motoneurons. This deficit reaches a ∼20% maximum at 6 months and is accompanied by losses of motor axons and significant impairment of rotarod skills. Similarly, sensory neurons in dorsal root ganglia (L4, L5) are reduced by 20%, whereas sympathetic neurons are not affected. GDF-15 is expressed and secreted by Schwann cells, retrogradely transported along adult sciatic nerve axons, and promotes survival of axotomized facial neurons as well as cultured motor, sensory, and sympathetic neurons. Despite striking similarities in the GDF-15 and CNTF knock-out phenotypes, expression levels of CNTF and other neurotrophic factors in the sciatic nerve were unaltered suggesting that GDF-15 is a genuine novel trophic factor for motor and sensory neurons.

Extracellular signal-regulated kinase (ERK) activation has been shown to promote neuronal death in various paradigms. We demonstrated previously that the late and sustained ERK activation in cerebellar granule neurons (CGNs) cultured in low potassium predominantly promotes plasma membrane (PM) damage. Here, we examined the effects of a well established neuronal survival factor, insulin-like growth factor 1 (IGF-1), on the ERK cell death pathway. Stimulation of CGNs with IGF-1 induced an early and transient ERK activation but abrogated the appearance of late and sustained ERK. Withdrawal or readdition of IGF-1 after 4 h in low potassium failed to prevent sustained ERK activation and cell death. IGF-1 activated the protein kinase A (PKA) to mediate ERK inhibition via c-Raf phosphorylation at an inhibitory site (Ser259). Phosphatidylinositol 3-kinase (PI3K) or PKA inhibitors, but not a specific Akt inhibitor, abrogated PKA signaling. This suggests that the PI3K/PKA/c-Raf-Ser259 pathway mediates ERK inhibition by IGF-1 independent of Akt. In addition, adenoviral-mediated expression of constitutively active MEK (mitogen-activated protein kinase kinase) or Sindbis viral-mediated expression of mutant Raf Ser259Ala both attenuated IGF-1-mediated prevention of PM damage. Activation of caspase-3 promoted DNA damage. Its inhibition by IGF-1 was both PI3K and Akt dependent but PKA independent. 8-Br-cAMP, an activator of PKA, induced phosphorylation of c-Raf-Ser259 and inhibited ERK activation without affecting caspase-3. This indicates a selective role for PKA in ERK inhibition through c-Raf-Ser259 phosphorylation. Together, these data demonstrate that IGF-1 can positively and negatively regulate the ERK pathway in the same neuronal cell, and provide new insights into the PI3K/Akt/PKA signaling pathways in IGF-1-mediated neuronal survival.

Development of midbrain dopaminergic neurons is known to depend on inductive signals derived from the ventral midline, including Sonic hedgehog (Shh) as one of the identified molecules. Here we show that in addition to Shh, transforming growth factor (TGF)-β is required for both induction and survival of ventrally located midbrain dopaminergic neurons. Like Shh, TGF-β is expressed in early embryonic structures such as notochord and floor plate, as well as in the area where mibrain dopaminergic neurons are developing. Treatment of cells dissociated from the rat embryonic day (E) 12 mibrain floor with TGF-β significantly increases the number of tyrosine hydroxylase (TH)-positive dopaminergic neurons within 24 hr. Neutralization of TGF-βin vitrocompletely abolishes the induction of dopaminergic neurons. In the absence of TGF-β, Shh cannot induce TH-positive neurons, and vice versa, neutralizing endogenous Shh abolishes the capacity of TGF-β to induce dopaminergic neuronsin vitro. Furthermore, neutralization of TGF-βin vivoduring chick E2–7 but not E4–7 resulted in a significant reduction in TH-positive neurons in the ventral midbrain floor but not in the locus coeruleus or diencephalon, which suggests that the TGF-β is required for the induction of mesencephalic dopaminergic neurons with a critical time period at E2/E3. Furthermore, neutralization of TGF-β between E6 and 10, a time period during maturation of mesencephalic dopaminergic neurons when no further inductive cues are required, also resulted in a significant loss of dopaminergic neurons, suggesting that TGF-β is required for the promotion of survival of ventral midbrain dopaminergic neurons as well. Together, our results identify TGF-β as an essential mediator for the induction and maintenance of midbrain dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for several populations of CNS and peripheral neurons. Synthesis and storage of GDNF by the neuron-like adrenal medullary cells suggest roles in adrenal functions and/or in the maintenance of spinal cord neurons that innervate the adrenal medulla. We show that unilateral adrenomedullectomy causes degeneration of all sympathetic preganglionic neurons within the intermediolateral column (IML) of spinal cord segments T7–T10 that project to the adrenal medulla.In situhybridization revealed that IML neurons express the glycosylphosphatidylinositol-linked α receptor 1 and c-Ret receptors, which are essential for GDNF signaling. IML neurons also display immunoreactivity for transforming growth factor-β (TGF-β) receptor II. Administration of GDNF (recombinant human, 1 μg) in Gelfoam implanted into the medullectomized adrenal gland rescued all Fluoro-Gold-labeled preganglionic neurons projecting to the adrenal medulla after four weeks. Cytochromecapplied as a control protein was not effective. The protective effect of GDNF was prevented by co-administration to the Gelfoam of neutralizing antibodies recognizing all three TGF-β isoforms but not GDNF. This suggests that the presence of endogenous TGF-β was essential for permitting a neurotrophic effect of GDNF. Our data indicate that GDNF has a capacity to protect a population of autonomic spinal cord neurons from target-deprived cell death. Furthermore, our results demonstrate for the first time that the previously reported requirement of TGF-β for permitting trophic actions of GDNFin vitro(Krieglstein et al., 1998) also applies to thein vivosituation.

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7–T10, disappear. Administration of NT-4 in gelfoams (6 μg) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7–T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla.

TrkA high-affinity receptors are essential for the normal development of sympathetic paravertebral neurons and subpopulations of sensory neurons. Paravertebral sympathetic neurons and chromaffin cells of the adrenal medulla share an ontogenetic origin, responsiveness to NGF, and expression of TrkA. Which aspects of development of the adrenal medulla might be regulated via TrkA are unknown. In the present study we demonstrate that mice deficient for TrkA, but not the neurotrophin receptor TrkB, show an early postnatal progressive reduction of acetylcholinesterase (AChE) enzymatic activity in the adrenal medulla and in preganglionic sympathetic neurons within the thoracic spinal cord, which are also significantly reduced in number. Quantitative determinations of specific AChE activity revealed a massive decrease (−62%) in the adrenal gland and a lesser, but still pronounced, reduction in the thoracic spinal cord (−40%). Other markers of the adrenal medulla and its innervation, including various neuropeptides, chromogranin B, secretogranin II, amine transporters, the catecholamine-synthesizing enzymes tyrosine hydroxylase and PNMT, synaptophysin, and L1, essentially were unchanged. Interestingly, AChE immunoreactivity appeared unaltered, too. Preganglionic sympathetic neurons, in contrast to adrenal medullary cells, do not express TrkA. They must, therefore, be affected indirectly by the TrkA knock-out, possibly via a retrograde signal from chromaffin cells. Our results suggest that signaling via TrkA, but not TrkB, may be involved in the postnatal regulation of AChE activity in the adrenal medulla and its preganglionic nerves.

The specific mechanisms underlying the restorative effects of adrenal chromaffin grafts in experimental parkinsonism are still obscure. Recent findings indicated an involvement of graft-induced trophic interactions in the course of recovery-related events. Evidence that basic fibroblast growth factor (bFGF), a potent trophic protein for neurons, (1) is present in chromaffin cells (Blottner et al., 1989) and (2) exerts trophic activities on embryonic mesencephalic neurons in vitro (Ferrari et al., 1989) provided the rationale for administering bFGF in gel foam implants unilaterally to the striatum of 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned mice. Simultaneous bFGF/MPTP treatment diminished bilaterally the reduction of striatal dopamine (DA) levels observed in cytochrome c/MPTP-treated mice and led to an ipsilateral reappearance of tyrosine hydroxylase (TH)-like immunoreactive fibers, most notably adjacent to the implant, 2 weeks after the surgery. Determinations of TH activities and TH immunoblotting demonstrated that bFGF almost fully reversed the loss of TH activity on either side but restored TH protein more on the ipsilateral than on the contralateral side. Furthermore, differences in dihydroxyphenylacetic acid levels, which were about twice as high on the contralateral side yet still reduced with respect to untreated mice, supported our assumption that the molar TH activity was increased on the untreated side, possibly due to an intrinsic compensatory up- regulation. Delayed administration of bFGF starting 8 d after the MPTP treatment was equally effective with regard to morphological parameters. Our results suggest that bFGF partially prevents the deleterious chemical and morphological consequences of an MPTP-mediated nigrostriatal lesion. Thus, bFGF mimics at least the morphological effects of chromaffin cell grafts to the MPTP-lesioned brain.