Inactivation of fibroblast growth factor receptor signaling in myelinating glial cells results in significant loss of adult spiral ganglion neurons accompanied by age-related hearing impairment.

Hearing loss has been attributed to many factors, including degeneration of sensory neurons in the auditory pathway and demyelination along the cochlear nerve. Fibroblast growth factors (FGFs), which signal through four receptors (Fgfrs), are produced by auditory neurons and play a key role in embryonic development of the cochlea and in neuroprotection against sound-induced injury. However, the role of FGF signaling in the maintenance of normal auditory function in adult and aging mice remains to be elucidated. Furthermore, the contribution of glial cells, which myelinate the cochlear nerves, is poorly understood. To address these questions, we generated transgenic mice in which Fgfr1 and Fgfr2 were specifically inactivated in Schwann cells and oligodendrocytes but not in neurons. Adult mutant mice exhibited late onset of hearing impairment, which progressed markedly with age. The hearing impairment was accompanied by significant loss of myelinated spiral ganglion neurons. The pathology extended into the cochlear nucleus, without apparent loss of myelin or of the deletion-bearing glial cells themselves. This suggests that perturbation of FGF receptor-mediated glial function leads to the attenuation of glial support of neurons, leading to their loss and impairment of auditory functions. Thus, FGF/FGF receptor signaling provides a potentially novel mechanism of maintaining reciprocal interactions between neurons and glia in adult and aging animals. Dysfunction of glial cells and FGF receptor signaling may therefore be implicated in neurodegenerative hearing loss associated with normal aging.

p53 deficiency fails to prevent increased programmed cell death in the Bcl-X(L)-deficient nervous system.

Bcl-X(L) mice display a similar neurodevelopmental phenotype as rb, DNA ligase IV, and XRCC4 mutant embryos, suggesting that endogenous Bcl-X(L) expression may protect immature neurons from death caused by DNA damage and/or cell cycle dysregulation. To test this hypothesis, we generated bcl-x/p53 double mutants and examined neuronal cell death in vivo and in vitro. Bcl-X(L)-deficient primary telencephalic neuron cultures were highly susceptible to the apoptotic effects of cytosine arabinoside (AraC), a known genotoxic agent. In contrast, neurons lacking p53, or both Bcl-X(L) and p53, were markedly, and equivalently, resistant to AraC-induced caspase-3 activation and death in vitro indicating that Bcl-X(L) lies downstream of p53 in DNA damage-induced neuronal death. Despite the ability of p53 deficiency to protect Bcl-X(L)-deficient neurons from DNA damage-induced apoptosis in vitro, p53 deficiency had no effect on the increased caspase-3 activation and neuronal cell death observed in the developing Bcl-X(L)-deficient nervous system. These findings suggest that Bcl-X(L) expression in the developing nervous system critically regulates neuronal responsiveness to an apoptotic stimulus other than inadequate DNA repair or cell cycle abnormalities.

Bid regulation of neuronal apoptosis.

Bid is a BH3 domain only pro-apoptotic member of the Bcl-2 family which interacts with Bax to regulate apoptosis. Bax-deficient embryos show decreased neuronal programmed cell death in vivo and resistance to cytosine arabinoside (AraC)-induced neuronal apoptosis in vitro. In this report, we demonstrate that Bid-deficient embryos show no neurodevelopmental abnormalities, and Bid-deficiency has no effect on the in vitro apoptotic response of either telencephalic neural precursor cells or neurons to AraC-induced death. We conclude that bid does not play an essential role in either naturally occurring or genotoxin-induced neuronal cell death.

Apoptosis and brain development.

Neuronal cell death in the embryonic brain was first recognized almost a century ago. Its significance for normal nervous system development and function has been a major focus of neuroscientific investigation ever since. Remarkable progress has been made in defining the cellular processes controlling neuronal cell death and studies performed over the last ten years have revealed extensive homology between the molecules regulating programmed cell death in Caenorhabditis elegans and apoptosis in mammalian cells. Targeted gene disruptions of members of the bcl-2 and caspase gene families have demonstrated particularly significant roles for bcl-x, bax, caspase-9 and caspase-3 in mammalian brain development. As expected from previous studies of synapse-bearing neurons and neurotrophic factors, reduced neuronal cell death in mice bearing mutations in key pro-apoptotic molecules resulted in increased numbers of neurons in a variety of neuronal subpopulations. However, targeted gene disruptions also demonstrated a heretofore underappreciated significance of neural precursor cell death and immature neuron death in nervous system development. Pathological activation of apoptotic death pathways may lead to neuroanatomic abnormalities and possibly to developmental disabilities.

Neural precursor cell apoptosis and glial tumorigenesis following transplacental ethyl-nitrosourea exposure.

Neural precursor cells (NPCs) populate the embryonic ventricular zone and persist in the subependymal zone of the adult brain. We hypothesized that hereditary and/or acquired mutations in apoptosis-associated genes, such as p53 and caspases, may protect NPCs from DNA damage-induced death and predispose them to subsequent neoplastic transformation. To test this hypothesis, we exposed NPCs from wild-type and targeted gene-disrupted mouse embryos (p53, caspase-9, caspase-3, and bax mutants) to ethyl-nitrosourea (ENU), a known DNA mutagen and neural carcinogen, and measured NPC viability. We found that ENU produced caspase-3 activation and apoptotic NPC death 6-24 h after administration both in vivo and in vitro. This effect was critically dependent on p53 and caspase-9 expression. The long-term effect of intrauterine ENU exposure was examined in control and p53-deficient mice. High grade glial tumors were found in 60% of p53(-/-) young adult mice exposed to ENU on gestational day 12.5 but not in p53(+/-) or p53(+/+) littermates or in untreated p53-deficient mice. All the tumors were located supratentorially and possessed strong immunoreactivity for glial fibrillary acidic protein and the anti-apoptotic molecule Bcl-X(L). These results suggest that intrauterine exposure of NPCs to certain DNA damaging agents may synergistically interact with specific genetic abnormalities (e.g. p53 deficiency) to produce glial neoplasms in the adult brain.

Neuronal plasticity and survival in mood disorders.

Studies at the basic and clinical levels demonstrate that neuronal atrophy and cell death occur in response to stress and in the brains of depressed patients. Although the mechanisms have yet to be fully elucidated, progress has been made in characterizing the signal transduction cascades that control neuronal atrophy and programmed cell death and that may be involved in the action of antidepressant treatment. These pathways include the cyclic adenosine monophosphate and neurotrophic factor signal transduction cascades. It is notable that these same pathways have been demonstrated to play a pivotal role in cellular models of neural plasticity. This overlap of plasticity and cell survival pathways, together with studies demonstrating that neuronal activity enhances cell survival, suggests that neuronal atrophy and death could result from a disruption of the mechanisms underlying neural plasticity. The role of these pathways and failure of neuronal plasticity in stress-related mood disorders are discussed.

Tryptophan hydroxylase: purification by affinity chromatography on calmodulin-sepharose.

Tryptophan hydroxylase (EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)) from rat mesencephalic tegmentum has been purified by sequential chromatography on Blue-Sepharose, DE-52, and calmodulin-Sepharose. The hydroxylase is excluded from Blue-Sepharose and is eluted from DE-52 with a step-wise NaCl gradient. Tryptophan hydroxylase binds to calmodulin-Sepharose in the presence of calcium and is eluted with either EGTA or calmodulin itself, but not with tryptophan. The purification scheme is rapid (5-6 h) and yields an enzyme with a specific activity of 225 nmol 5-HTP/mg min, representing a 400-fold purification with 7% recovery. The tryptophan hydroxylase preparation was judged to be > 95% pure using the present isolation procedure.

Tetrahydrobiopterin biosynthesis in C6 glioma cells: induction of GTP cyclohydrolase I gene expression by lipopolysaccharide and cytokine treatment.

The possibility that 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis is stimulated in glial cells by treatment with lipopolysaccharide (LPS) and tumor necrosis factor (TNF-alpha) was examined in the astrocyte-derived C6 glioma cell line. Under basal culture conditions BH4 levels were found to be at the limit of detection. Concurrent treatment with 10 micrograms/ml LPS and 50 ng/ml TNF-alpha caused a time-dependent 13-fold increase in the levels of BH4. This treatment paradigm also induced nitric oxide synthase activity, as evidenced by increased levels of nitrite, an oxidized metabolite of NO, in the culture medium. LPS and TNF-alpha treatment led to a 25-fold increase in GTPCH enzyme activity, the first and rate-limiting enzyme in BH4 synthesis, and a corresponding 23-fold increase in GTPCH protein levels. Northern blot analysis showed that increased levels of GTPCH mRNA preceded changes in GTPCH protein, GTPCH enzyme activity and BH4 levels and reached a maximal of 44-fold that was sustained for at least 48 h. These results demonstrate that LPS and TNF-alpha stimulate de-novo BH4 biosynthesis and suggest that C6 cells offer a model system for studying the molecular events that control the induction of GTPCH gene expression and BH4 synthesis in glial cells.

Tryptophan hydroxylase: cloning and expression of the rat brain enzyme in mammalian cells.

A cDNA encoding full-length tryptophan hydroxylase was produced by reverse transcriptase-PCR from rat brain mRNA and expressed transiently in a human fibroblast cell line. Catalytic activity was low unless transfected cells were grown in the presence of FeSO4. Recombinant tryptophan hydroxylase was found almost exclusively within the soluble compartment of the cell and was dependent on tryptophan and tetrahydrobiopterin for activity. The catalytic activity of recombinant tryptophan hydroxylase was stimulated > 25-fold by Fe(II) and to a somewhat lesser extent by the polyanions heparin and phosphatidylserine. The enzyme was inhibited by desferrioxamine and dopamine, both of which complex iron. When extracts from transfected cells were subjected to sucrose gradient centrifugation and analytical gel filtration, the recombinant enzyme behaved the same as the native enzyme from brain. A monoclonal antibody against phenylalanine hydroxylase that cross-reacts with brain tryptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant tryptophan hydroxylase expressed in mammalian cells is assembled into tetramers of approximately 220,000 daltons. Its catalytic and physical properties appear to be very similar to those of the native enzyme from brain.

Expression and deletion mutagenesis of tryptophan hydroxylase fusion proteins: delineation of the enzyme catalytic core.

cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1-98) or catalytic (amino acids 99-444) domains of the enzyme, were cloned and expressed as glutathione S-transferase fusion proteins in E. coli. The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S-transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99-444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.

Neurokinin-3 receptors modulate dopamine cell function and alter the effects of 6-hydroxydopamine.

Neurokinin-3 receptor expression within rat midbrain dopamine neurons was demonstrated using a combination of in situ hybridization and receptor autoradiographic techniques. Continuous intranigral infusion of the neurokinin-3 receptor agonist senktide selectively increased striatal dopamine metabolism over a period of several days, followed by apparent development of tolerance. In contrast, in animals with moderate unilateral 6-hydroxydopamine-induced lesions of nigrostriatal dopamine cells, intranigral senktide infusion increased dopamine turnover in the surviving dopamine neurons and reduced functional dopamine asymmetry (reflected by spontaneous rotations) over the 2-week period tested. Thus nigral neurokinin receptors can modulate normal dopamine cell activity and may provide a therapeutic target in the treatment of Parkinson's disease.

Human fascioliasis in Shropshire.

Five patients with fascioliasis presented with malaise, pruritus, and pain in the right hypochondrium. Eosinophilia, increase in serum gamma globulin, and pyrexia were other features. Four patients treated with oral bithionol recovered completely and it is suggested that this is an effective form of therapy. The double diffusion precipitin test, being speedy and simple, was used as a screening procedure for diagnosis. The source of infection was wild watercress growing in a farm ditch.