Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.

TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function.

Urethral obstruction produces increased voiding frequency (0.7 +/- 0.06 to 1.1 +/- 0.08 h-1) and hypertrophy of the urinary bladder (89 +/- 1.7 to 708 +/- 40 mg) with profound increments in the dimensions of afferent (4, 6) and efferent neurons (299 +/- 4.7 to 573 +/- 8.6 microns2) supplying this organ in the rat. We discovered that hypertrophied bladders of rat and human contain significantly more nerve growth factor (NGF) per milligram wet weight, protein, and DNA than normal bladders. The temporal correlation between NGF content, neuronal hypertrophy, and bladder weight was consistent with a role for this growth factor in the neurotrophic effects associated with obstruction. Autoimmunity to NGF abolished the hypertrophy of NGF-sensitive bladder neurons in the pelvic ganglion after obstruction. Relief of urethral obstruction reduced bladder size (349 +/- 78 mg), but neuronal hypertrophy (460.2 +/- 10.2 microns2) and elevated NGF levels were only partially reversed. Bladder hypertrophy (133 +/- 4.3 mg) induced by osmotic diuresis slightly increased ganglion cell area (365.2 +/- 6.1 microns2) and only doubled NGF content of the bladder. These findings provide important new evidence that parenchymal cells in the hypertrophied bladder can synthesize NGF and possibly other molecular messengers that act to alter the size and function of neurons in adult animals and man.

Nerve growth factor synthesis in vascular smooth muscle.

Details of the interdependent, trophic relation between smooth muscle and its neural innervation are not well known despite suggestions that neural influences may contribute significantly to hypertensive and other cardiovascular disease. Vascular smooth muscle is a major target of innervation by neurons of the sympathetic nervous system. Sympathetic neurons depend on a constant supply of the potent neurotrophic peptide nerve growth factor. Nerve growth factor regulates an impressive list of neuronal and perhaps muscle properties, yet its source in vessels and the determinants of its synthesis are not known. We have taken advantage of the cytoarchitecture of the aorta to demonstrate that vascular smooth muscle cells synthesize nerve growth factor. The survival of cultured sympathetic neurons is supported in a nerve growth factor-dependent manner by co-culture with pure rat aortic vascular smooth muscle cells. Furthermore, pure smooth muscle cell cultures contain nerve growth factor-specific messenger RNA. Levels of messenger nucleic acid coding for nerve growth factor in smooth muscle are regulated by contractile agonists (angiotensin II, arginine vasopressin) and the adrenergic agonist phenylephrine. This suggests a link between muscle activity and growth factor production. Secretion of nerve growth factor protein by vascular smooth muscle was measured using a sensitive two-site immunoassay. Secretion is highest during muscle growth. Secretion is elevated by angiotensin II and arginine vasopressin but slightly inhibited by phenylephrine. These results suggest that cultured vascular smooth muscle can serve as a useful model in which to study the cellular regulation of trophic factor synthesis in health and disease.

Soluble and membrane-bound factors together account for target dependence of cultured parasympathetic neurons.

The survival of avian ciliary ganglion (CG) neurons in culture depends upon an exogenous supply of trophic factor(s). Skeletal muscle, a normal ganglionic target tissue, is a well documented provider of survival-promoting activity, although the molecular basis for this ability to foster neuronal survival has not been thoroughly investigated. To identify the source of skeletal muscle support, dissociated neurons were plated into microwells containing either: a basal, trophically deficient medium; live pectoral muscle myotubes; medium conditioned by myotubes; membrane remnants of osmotically lysed myotubes; or, membrane remnants and conditioned medium. Neurons remaining in culture were counted after 1, 2, 5, and 7 days. The results reveal that neuronal survival is supported by both muscle conditioned medium and the membrane remnants of cultured myotubes. Each of these alone provides for only partial survival, while both combine to equal the activity of live myotubes. Treatment of the lysed membranes with either 1.5 M NaCl and/or 15 U heparin removed only 50-60% of the activity, suggesting that multiple factors are involved in the neuronal support obtained from lysed myotubes. This is in contrast to fibroblast remnants, which support some neuronal survival, but whose activity is wholly removed by NaCl. Conditioned medium also contains a heparin binding component which accounts for approximately 60% of its activity. These results indicate that full trophic support from the cultured target tissue requires at least two distinct active agents. The experiments further suggest that the target-derived factors responsible for neuronal survival in culture, and perhaps in vivo, are both soluble and membrane-associated molecules.

Cultured smooth muscle targets lack survival activity for ciliary ganglion neurons.

Cultured neurons require specific trophic agents in order to survive. This dependence is thought to resemble the neuron-target interdependence that develops in vivo during synaptogenesis and neuronal cell death. The notion that neurons in general derive trophic support from their synaptic targets is based primarily on studies of peripheral neurons and motor neurons. To assess the general applicability of this nerve-target relationship, we tested the ability of vascular smooth muscle (VSM) to support dissociated neurons from the chick ciliary ganglion. The ciliary ganglion contains 2 distinct neuronal populations, one of which innervates striated muscle, the other VSM. Striated muscle cocultures are known to support all of the neurons in the ganglion for extended periods. Dissociated neurons were therefore cocultured in microwells containing VSM derived from the rat or chick aorta and from the choroid coat of the chick eye. Surviving neurons were counted after 1, 2, 5, and 7 d. Striated muscle is able to support full neuronal survival in the same assay. However, in no case was VSM capable of contributing to neuronal survival in vitro. The neurons in the VSM cocultures were able to form neurites and make contacts with their putative targets, as confirmed by scanning electron and light microscopy. The presence of viable and differentiated smooth muscle cells was demonstrated in the cultures by transmission electron microscopy and analysis of smooth muscle alpha-actin. The failure of VSM and even the choroid target tissue to support the survival of their innervating neurons suggests that novel mechanisms may operate to provide trophic support for neurons innervating VSM targets.

Synergistic increase in nerve growth factor secretion by cultured vascular smooth muscle cells treated with injury-related growth factors.

Vascular smooth muscle (VSM) cells comprise one of the primary targets of the sympathetic nervous system and have been shown to secrete nerve growth factor (NGF). There is increasing evidence that changes in the levels of NGF in the adult may underlie certain pathological conditions. To investigate the potential role of altered NGF production in vascular disease, VSM cell cultures were treated with injury-related growth factors and the culture medium was assayed for NGF using a two-site enzyme-linked immunosorbent assay (ELISA). Platelet-derived growth factor (PDGF), a potent VSM mitogen, caused a dose-dependent increase in NGF secretion. After 4 hr, PDGF-treated cultures contained 10 times more NGF than control cultures. NGF release remained elevated for 48 hr, but the peak secretion occurred in the first 12 hr after treatment. Transforming growth factor beta (TGF-beta) caused a fivefold increase in NGF at 4 hr when added alone, but synergized with PDGF yielding approximately 50 times more NGF than control cultures. TGF-beta and epidermal growth factor (EGF) also displayed synergism. In contrast, basic fibroblast growth factor (bFGF), which had a modest effect alone, appeared to be additive with TGF-beta. Similarly, interleukin 1-beta (IL-1 beta), which mediates increased NGF synthesis in sciatic nerve lesions (Lindholm et al.: Nature 330:658-659, 1987), showed no synergism with TGF-beta.

Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy.

PURPOSE: The goal of this investigation was to examine the effect of immunity to nerve growth factor (NGF) on alterations in sensory nerves from the urinary bladder in the dorsal root ganglia (DRG) and their projections to the L6/S1 spinal cord following urethral obstruction in the rat. MATERIALS AND METHODS: Female Wistar rats were immunized to murine 2.5S NGF, then obstructed by partial urethral ligation for 6 weeks. Retrograde axonal tracing with FluoroGold and WGA-HRP was used to measure areas of bladder DRG cells and afferent projections in the sacral spinal cord. Multiunit activity on bladder nerves allowed recording of micturition reflexes. Immunohistochemical staining for growth associated protein (GAP)-43 in the sacral parasympathetic nucleus (SPN) was used to assess potential growth or activity of axons in the spinal cord. Voiding frequencies were then measured in awake obstructed and NGF immune-obstructed rats. RESULTS: Immunity to NGF prevented obstruction-induced hypertrophy of DRG neurons, reduced retrograde axonal labeling of sacral afferent projections, eliminated enhancement of a spinal micturition reflex and abolished the increased GAP-43 expression in the SPN. Immunity to NGF prevented the urinary frequency that accompanies obstruction. CONCLUSIONS: Our results demonstrate that obstruction of the bladder elicits structural and functional plasticity in afferents as a result of ongoing neurotrophic interactions. Neurotrophic interactions offer a potential mechanism whereby symptoms and bladder hyperactivity develop with obstruction associated with benign prostatic hyperplasia.

Mitogen-activated protein kinase-independent pathways mediate the effects of nerve growth factor and cAMP on neuronal survival.

Components of the mitogen-activated protein kinase (MAP kinase) signaling pathway, including Ras, Raf, and MAP kinase, are necessary for nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. We have investigated the role of this pathway in promoting survival of primary sympathetic neurons that die when deprived of NGF. NGF caused rapid and sustained increases (approximately 4-fold) in the activities of the ERK-1 and ERK-2 isoforms of MAP kinase. PD 098059, an inhibitor of MAP kinase kinase activation, blocked the effects of NGF on both kinase isoforms. However, PD 098059 did not attenuate the effects of NGF on neuronal survival. In addition, MAP kinase activity was not increased by chlorophenylthio-cAMP, a cell-permeable analog of cAMP that supports neuronal survival in the absence of NGF. These findings indicate that activation of MAP kinase is not required for the actions of either cAMP or NGF on neuronal survival.

Receptor-mediated stimulation and inhibition of nerve growth factor secretion by vascular smooth muscle.

Nerve growth factor (NGF) is a potent neurotrophin signaling protein, the best-known member of a family of similar neurotrophins. Specific neuronal populations depend upon the neurotrophins for normal function and disturbances in NGF and neurotrophin supply have been implicated in neurodegenerative disease, diabetes, and hypertension. This report details experiments in which the hourly pattern of NGF secretion by cultured vascular smooth muscle cells is examined. Vascular smooth muscle cells are major innervation targets of the neuronal population first discovered to be NGF-dependent: the sympathetic principal neurons. The results show that arginine vasopressin (AVP), angiotensin II (AngII), and alpha-adrenergic receptor activation, all contractile stimuli, elevate NGF secretion. However, AVP dependably does so alone while AngII requires coactivation of adenosine receptors. Adenosine alone inhibits secretion and the alpha-adrenergic increase in NGF output can be antagonized by activation of beta-adrenergic receptors. A change to fresh culture medium is also a potent stimulus to increased NGF output.

Analysis of events leading to neuronal death after infection with E1-deficient adenoviral vectors.

Although recombinant adenoviral vectors are being widely used to target genes to the nervous system, the cellular and genetic effects of recombinant adenoviral infection on neuronal function have not been well characterized. Using sympathetic neuronal cultures, we analyzed the effect of adenoviral infection on viral and neuronal gene expression and on neuronal function and viability. While a delayed cytotoxicity occurred 5 days after infection, numerous biochemical and genetic perturbations occurred within the infected cell prior to this time. This study demonstrates that numerous cellular alterations were produced by recombinant adenoviral vectors and, therefore, emphasizes the need for an analysis of the effects of these viral vectors on neuronal function in the interpretation of data regarding transgene expression induced by these vectors in neurons. It also suggests that continued improvements made to the viral vectors themselves might decrease this direct cytotoxicity and lead to improved safety and function of recombinant adenovirus in vivo.

Inhibition of phosphatidylinositol 3-kinase activity blocks depolarization- and insulin-like growth factor I-mediated survival of cerebellar granule cells.

Depolarizing concentrations of potassium promote the survival of many neuronal cell types including cerebellar granule cells. To begin to understand the intracellular mediators of neuronal survival, we have tested whether the survival-promoting effect of potassium depolarization on cerebellar granule cells is dependent on either mitogen-activated protein (MAP) kinase or phosphatidylinositol 3-kinase (PI-3-K) activity. In 7-day cerebellar granule cell cultures, potassium depolarization activated both MAP kinase and PI-3-K. Preventing the activation of MAP kinase with the MEK1 inhibitor PD98059 did not affect potassium saving. In contrast, the survival-promoting effect of 25 mM potassium was negated by the addition of 30 microM LY 294002 or 1 microM wortmannin, two distinct inhibitors of PI-3-K. The cell death induced by PI-3-K inhibition was indistinguishable from the cell death caused by potassium deprivation; LY 294002-induced death included nuclear condensation, was blocked by cycloheximide, and had the same time course as potassium deprivation-induced cell death. Cerebellar granule cells can also be maintained in serum-free medium containing either 100 ng/ml insulin-like growth factor I (IGF-I) or 800 microM cAMP. PI-3-K inhibition completely blocked the survival-promoting activity of IGF-I, but had no effect on cAMP-mediated survival. These data indicate that the survival-promoting effects of depolarization and IGF-I, but not cAMP, require PI-3-K activity.

Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway.

Neurotrophins such as nerve growth factor (NGF) regulate neuronal survival during development and are neuroprotective in certain models of injury to both the peripheral and the central nervous system. Although many effects of neurotrophins involve long-term changes in gene expression, several recent reports have focused on rapid effects of neurotrophins that do not involve synthesis of new gene products. Because enhanced formation of reactive oxygen species (ROS) represents one consequence of many insults that produce neuronal death, we hypothesized that neurotrophins might influence neuronal function and survival through acute alterations in the production of ROS. Using an oxidation-sensitive compound, dihydrorhodamine, we measured ROS formation in a central nervous system-derived neuronal cell line (GT1-1 trk) and in superior cervical ganglion neurons, both of which express the transmembrane NGF receptor tyrosine kinase, trkA. There was enhanced production of ROS in both cell types in the absence of NGF that was rapidly inhibited by application of NGF; complete inhibition of ROS generation in GT1-1 trk cells occurred within 10 min. NGF suppression of ROS formation was prevented by PD 098059, a specific inhibitor of MEK (mitogen/extracellular receptor kinase, which phosphorylates mitogen-activated protein kinase). The observation that NGF acutely blocks ROS formation in neurons through activation of the mitogen-activated protein kinase pathway suggests a novel mechanism for rapid neurotrophin signaling, and has implications for understanding neuroprotective and other effects of neurotrophins.

Neurturin, a relative of glial-cell-line-derived neurotrophic factor.

The normal development of the vertebrate nervous system entails the death of 30-70% of the neurons originally generated in most neuronal populations. This naturally occurring cell death is regulated by specific neurotrophic factors that promote neuronal survival and which are produced in limiting quantities by target cells, glial cells and neurons. These factors are also of potential utility as therapeutic agents for neurodegenerative diseases. Here we describe the purification and cloning of a new neurotrophic factor, identified on the basis of its ability to support the survival of sympathetic neurons in culture. This factor, neurturin, is structurally related to glial-cell-line-derived neurotrophic factor (GDNF). These factors can each activate the MAP kinase signalling pathway in cultured sympathetic neurons and support the survival of sympathetic neurons, as well as of sensory neurons of the nodose and dorsal root ganglia. Thus, neurturin and GDNF together now define a new family of neurotrophic factors.

Neurturin shares receptors and signal transduction pathways with glial cell line-derived neurotrophic factor in sympathetic neurons.

Neurturin (NTN) is a neurotrophic factor that shares homology with glial cell line-derived neurotrophic factor (GDNF). Recently, a receptor complex has been identified for GDNF that includes the Ret tyrosine kinase receptor and a glycosylphosphatidylinositol-linked protein termed "GDNFRalpha." However, differences in the phenotype of Ret and GDNF knockout animals suggest that Ret has at least one additional ligand. In this report, we demonstrate that NTN induces Ret phosphorylation in primary cultures of rat superior cervical ganglion (SCG) neurons. NTN also caused Ret phosphorylation in fibroblasts that were transfected stably with Ret and GDNFRalpha but not in cells expressing Ret alone. A glycosylphosphatidylinositol-linked protein also was important for NTN and GDNF signaling in SCG neurons; phosphatidylinositol-specific phospholipase C treatment of SCG cultures reduced the ability of NTN to phosphorylate Ret and the ability of NTN or GDNF to activate the mitogen-activated protein kinase pathway. NTN and GDNF also caused sustained activation of Ret and the mitogen-activated protein kinase pathway in SCG neurons. Finally, both NTN and GDNF activated the phosphatidylinositol 3-kinase pathway in SCG neurons, which may be important for the ability of NTN and GDNF to promote neuronal survival. These data indicate that NTN is a physiologically relevant ligand for the Ret receptor and suggest that NTN may have a critical role in the development of many neuronal populations.