CNTF protection of oligodendrocytes against natural and tumor necrosis factor-induced death.

A proportion of developing oligodendrocytes undergo natural cell death by apoptosis, and mature oligodendrocytes die, either by apoptosis or necrosis, in response to injurious signals such as cytotoxic cytokines and complement. Ciliary neurotrophic factor (CNTF), a trophic factor found in astrocytes in the central nervous system (CNS), promoted the survival and maturation of cultured oligodendrocytes. This trophic factor also protected oligodendrocytes from death induced by tumor necrosis factors (apoptosis) but not against complement (necrosis). These results suggest that CNTF functions in the survival of oligodendrocytes during development and may lead to therapeutic approaches for degenerative diseases of the CNS that involve oligodendrocyte destruction.

Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.

Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.

Selective RNA editing and subunit assembly of native glutamate receptors.

RNA editing and subunit assembly of ionotropic glutamate receptors (GluRs) were examined in an oligodendrocyte progenitor cell line, CG4, which expresses GluR2-GluR4, GluR6, GluR7, KA1, and KA2. AMPA-evoked currents rapidly desensitize, whereas kainate-evoked currents contain a steady-state component with a nearly linear current-voltage relation and a fast desensitizing component that is inwardly rectifying. The Q/R site is edited > 95% to the arginine codon in GluR2(Q607) mRNA, and < 5% in GluR6(Q621) mRNA. Immunoprecipitation experiments demonstrate that GluR6 and/or GluR7 subunits assemble with KA2, but not with GluR2-GluR4. These results indicate that oligodendrocyte progenitor cells selectively edit and assemble glutamate receptors into at least two functionally and structurally distinct heteromeric channels.

Stimulation of glycolysis by corticotropin and phorbol ester in cultured neurons.

Incubation of cultured neurons from chick embryo forebrain with corticotropin (ACTH) or the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) stimulates the production of lactate. The stimulation is seen after 2 h of treatment and is maximal after 12 h. Both ACTH (1-24) and TPA increase the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), a metabolic activator of 6-phosphofructo-1-kinase (PFK-1). This effect is concentration-dependent and is maximal after 4 h of treatment. PFK-1 activity is increased in a dose-dependent manner by ACTH (1-24) or TPA. This increase is not visible during the first 6 h and reaches its maximum after 18 h of treatment. The stimulation of PFK-1 activity is not due the increase of Fru-2,6-P2 by ACTH (1-24) or TPA, since saturating concentrations of Fru-2,6-P2 are present in the PFK-1 assay medium. Thus, it appears that ACTH (1-24) and TPA regulate glycolysis through two modes with different time responses: increase in Fru-2,6-P2 is the main mechanism operating during the first 6 h following the treatments and increase in the amount, or stable increase in activity of PFK-1, takes place during the later phase. It is suggested that the action of corticotropin on glycolysis is part of the mechanism of the neurotrophic activity of this hormone.

Activation of soluble guanylate cyclase through phosphorylation by protein kinase C in intact PC12 cells.

Soluble guanylate cyclase was found to be phosphorylated by protein kinase C in intact PC12 pheochromocytoma cells. The phosphate incorporation into guanylate cyclase upon addition of phorbol 12-O-myristate 13-acetate (PMA) to PC12 cells in culture coincided with an increased intracellular cGMP level. A strong correlation between phosphate incorporation into guanylate cyclase and increased cGMP level was also observed by time-course and dose-response studies of the PMA effect, as well as when cells were treated with various phorbol esters and diacylglycerols or with various protein kinase C inhibitors. The cAMP system and the presence of extracellular Ca2+ were found not to be involved in guanylate cyclase phosphorylation. The phosphorylation and activation of guanylate cyclase by protein kinase C represent a possible mechanism whereby agonist-stimulation of receptors coupled to phosphoinositide hydrolysis induces cGMP synthesis.

Big gene banks: nuggets for drug discovery or fool's gold?

Gene-mapping studies that look for complex traits among human populations have deepened our understanding of disease causes, but do they hold promise for identifying drug targets?

Separation from protein kinase C--a calcium-independent TPA-activated phosphorylating system.

A calcium-independent but 12-O-tetradecanoylphorbol-13-acetate (TPA)- or diacylglycerol-activated phospholipid-dependent phosphorylating activity has been separated from protein kinase C. This has been made possible by employing calcium-dependent hydrophobic interaction chromatography. The material bound to phenyl-Sepharose in the presence of calcium at low ionic strength was eluted with EGTA and was protein kinase C. While the unbound material passing through the phenyl-Sepharose column showed no appreciable protein kinase C activity, instead it had a high phosphorylating activity manifested in the absence of calcium and in the presence of TPA plus phospholipid. The identification of this phosphorylating activity, distinct from protein kinase C, leads to important clues to cellular responses monitored by TPA in the absence of calcium.

Inhibition of p185c-erbB-2 proto-oncogene expression by antisense oligodeoxynucleotides down-regulates p185-associated tyrosine-kinase activity and strongly inhibits mammary tumor-cell proliferation.

The c-erbB-2 proto-oncogene codes for a 185-kd putative growth factor receptor that is highly homologous to but distinct from the epidermal growth factor (EGF) receptor. Amplification and overexpression of c-erbB-2 occurs in a number of human tumors, in some of which it is a negative prognostic factor. This study investigates the possibility of inhibiting tumor-cell proliferation by blocking c-erbB-2 expression in the human mammary carcinoma cell line SK-Br-3 using chemically modified antisense oligodeoxynucleotides. Expression of the p185c-erbB-2 protein product was selectively reduced within 48 hours and resulted in a growth arrest of SK-Br-3 cells. Biochemical studies of tyrosine-kinase and S6-kinase activities after antisense inhibition of c-erbB-2 show that p185c-erbB-2 activates the S6-kinase signalling pathway in a nonlinear, dose-dependent manner. This may be relevant for the design of therapeutic strategies involving the inhibition of c-erbB-2 (proto)- oncogene expression.

Ciliary neurotrophic factor (CNTF) promotes low-affinity nerve growth factor receptor and CD4 expression by rat CNS microglia.

Ramified parenchymal microglia may provide immune surveillance in the nervous system and become activated in response to injury, showing increases in antigens found on macrophages, e.g. CD4 and MHCs. We investigated in adult rats the effects of a 2-week intraventricular infusion with ciliary neurotrophic factor (CNTF), a nervous system-associated cytokine, on microglia of the normal and injured corpus callosum. CNTF caused morphological changes, induced the expression of low-affinity nerve growth factor receptor and CD4 and increased the expression of complement receptor 3. Such changes were also observed after treatment of pure cultures of neonatal rat microglial cells with highly purified CNTF, suggesting a direct responsiveness to CNTF. Thus, endogenous astroglial and Schwann cell-derived CNTF may be an important component of the immune responses of the nervous system.

Convergent regulation by ciliary neurotrophic factor and dopamine of tyrosine hydroxylase expression in cultures of rat substantia nigra.

Ciliary neurotrophic factor and dopamine were found to enhance the expression of tyrosine hydroxylase immunoreactivity in cultured neurons from the substantia nigra of 16-day-old rat fetuses. The number of tyrosine hydroxylase-positive cells decreased progressively to approximately 30% by 96 h. Treatment with 5 microM dopamine maintained the tyrosine hydroxylase-positive neurons at 60% for 48 h, but not for longer. Concurrent treatment with 5 microM dopamine and 20 trophic units/ml ciliary neurotrophic factor had a greater impact on tyrosine hydroxylase-positive cells, resulting in the maintenance of 70% of the initial number for up to 72 h, but not beyond that time. When dopamine or dopamine/ciliary neurotrophic factor treatments were applied for 24 h after a 48-h delay, the number of tyrosine hydroxylase-positive cells was restored to 60 and 80%, respectively, but not restoration was observed with 96-h delayed treatments. These results suggest that dopamine and ciliary neurotrophic factor, alone or in combination, are not able to support the survival of tyrosine hydroxylase-positive neurons, but reduce their apparent numerical loss by enhancing the expression of tyrosine hydroxylase. The effects of dopamine, alone or in combination with ciliary neurotrophic factor, were predominantly mediated by D2 receptors, since they were blocked by selective D2 receptor antagonists and since the D2 receptor agonist quinpirole was able to substitute for dopamine. The effects of dopamine and ciliary neurotrophic factor were similar in astroblast-rich and in astroblast-depleted cultures, suggesting that they were not mediated through glial cells. These results extend our previous observations on locus coeruleus cultures, in which the concurrent treatment with ciliary neurotrophic factor and norepinephrine was shown to enhance tyrosine hydroxylase expression (but not survival) of noradrenergic neurons. They also consolidate the view that ciliary neurotrophic factor and the neuron's own transmitter act in convergence and in an autocrine/paracrine mode as regulators of the corresponding neurotransmitter phenotype.

Cyclic GMP alterations in fetal rat cerebrum after global intrauterine ischemia: role of guanylate cyclase phosphorylation.

Changes in the levels of cyclic AMP (cAMP) and cyclic GMP (cGMP) have been measured in brains of 20-day-old rat fetuses exposed to global intrauterine ischemia. Ischemia of different duration (0.5-30 minutes) did not alter the level of cAMP. In contrast, cGMP levels increased as a result of ischemia. This increase was seen even after a short period of ischemia (less than 5 minutes) and was maximal after 5 minutes, where a threefold increase could be observed. This stimulation was transient: after 30 min of ischemia, cGMP returned to the control level. Accumulation of cGMP can be related to the activation of guanylate cyclase, the activity of which is doubled after 15 minutes of ischemia. Immunoprecipitation of guanylate cyclase after in vivo labeling of the fetal brain with 32Pi revealed a threefold increase in the phosphorylation of the enzyme after 15 minutes of ischemia. The possible role of these modifications in cGMP metabolism during the course of ischemia is discussed.

Opposite effects of arachidonic acid and of its hydroperoxides on brain soluble guanylate cyclase activity.

Purified soluble guanylate cyclase from rat brain was found to be directly activated by arachidonic acid. Arachidonic acid hydroperoxides, obtained by the action of lipoxygenase, were inhibitory. An inhibition of soluble guanylate cyclase and a decrease of cyclic GMP content were also observed in pure neuronal cultures treated with lipoxygenase. The results suggest that lipoxygenase products are not involved in the production of cyclic GMP in neurons. This is in contrast with other tissues for which it is established that hormone-induced guanylate cyclase activation is regulated by lipoxygenase-catalysed metabolism of polyunsaturated fatty acids.

Glial cell line-derived neurotrophic factor. Potential for otoprotection.

Sensorineural hearing loss results from the degeneration of hair cells and/or auditory neurons in the cochlea of the inner ear. BDNF and NT-3 were shown to support survival of auditory neurons both in vitro and in vivo. Cochlea from P3-P4 rats were cultured as floating explants and hair cells in the organ of Corti were identified by phalloidin-FITC immunostaining. Treatment with cisplatin (35 micrograms/mL) or neomycin (0.6 mM) resulted in 21.2 +/- 6.0% and 7.4 +/- 4.7% surviving hair cells, respectively, after 3 days in culture. GDNF, added together with the ototoxins, increased their number to 46.7% and 37.4%, respectively. In cultures of dissociated cochlea from 4-week-old rat, cisplatin (5 mg/mL) added 24 h after seeding resulted in only 6.1 +/- 1.2% surviving neurons. However, when cisplatin was added together with GDNF (10 ng/mL), 32.8 +/- 1.0% of the neurons survived. The efficacy of GDNF in animal models of ototoxicity was tested next. Guinea pigs were pretreated with GDNF in one ear, delivered either by infusion into the inner ear (scala tympani) with Alzet minipumps (50 ng/mL at a 0.5 microL/h), or injected into the middle ear (120 microL at 1 mg/mL) through the tympanic membrane. The ear that did not receive GDNF always served as control. Ototoxicity was induced systemically either by intraperitoneal cisplatin injections (1 mg/kg/day for 15 days or two injections of 7.5 mg/kg at a 5-day interval or by a combination of kanamycin (200-300 mg/kg, administered subcutaneously) and ethacrinic acid (40 mg/kg, intravenous). It was found that the number of surviving hair cells in GDNF-treated ears was about twice that of control ears in animals exposed to the ototoxins. The transducing GDNF receptor (ret) is expressed in the inner ear.

CNTF promotes the survival of neonatal rat corticospinal neurons in vitro.

Corticospinal neurons were identified in cell cultures of neonatal rat cortex by immunostaining of cholera toxin B subunit (CTB), retrogradely transported from the cervical part of the spinal cord. The CTB-immunoreactive neurons were larger than the neurons in the overall (unstained) neuronal population and represented a small fraction of it (average of 0.3%) after 6 hours in vitro. The number of both total and CTB-labeled neurons declined progressively with time in culture. The neuronal death was, however, markedly faster in the CTB-labeled neuronal population than in the overall neuronal population. Ciliary neurotrophic factor (CNTF) promoted the survival of CTB-positive corticospinal neurons in a dose-dependent manner; with CNTF, the death rate of the CTB-labeled neurons became identical to that of the overall population.

GDNF protects the cochlea against noise damage.

Glial-derived neurotrophic factor (GDNF) was tested for its ability to prevent hearing and sensory cell loss in guinea pigs exposed to acoustic trauma. Hearing was measured prior to any treatment. Animals were exposed to damaging levels of noise either before or after local application of GDNF to one ear. Four weeks later, hearing and sensory cell loss was greater in the control ear than in the ear receiving GDNF before acoustic trauma or 2 h after trauma, but not 4 or 6 h after trauma. The results indicate that GDNF treatment in vivo can prevent cochlear sensory cell damage and hearing loss if present during or shortly after acoustic trauma.

Reduction of protein kinase C activity in the adult rat brain following transient forebrain ischemia.

Acute forebrain ischemia reduced protein kinase C (PKC) activity in the adult rat cortex, striatum and hippocampus by 60-70% after 20 min ischemia episodes, followed by 48 h of recirculation. Ischemia of 1 min, followed by recirculation, produced a less pronounced but significant decrease in PKC activity. The ischemia-induced decrease of PKC affected both the soluble and the membrane-bound kinase. Alterations of PKC predate neuronal death following ischemia.

Long-term culture of neurones from human cerebral cortex in serum-free medium.

A method for cultivating neurones from the fetal human central nervous system in the absence of glial cells is described. Brain cells from 15-18-week-old human fetuses are plated on polylysine-coated surfaces and grown in a serum-free hormonally-defined medium. About 98% of the cells were identified as neurones using tetanus toxin as a marker. The cultures survive for up to 7 weeks and develop an extremely complex network of neurites.

Neuronal and glial localization of guanylate cyclase. Immunohistochemical evidence in cultured cells.

A study has been carried out on the localization of guanylate cyclase employing cultured brain cells. Guanylate cyclase has been found to be located in neurons as well as in glial cells. This has been supported by the immunohistochemical as well as biochemical data.

Influence of adrenocorticotropic hormone on the growth of isolated neurons in culture.

The present study reports the influence of adrenocorticotropic hormone (ACTH) on the development of cultured neurons from chick embryo cerebral hemispheres. Cultures were initiated in serum-supplemented medium and then transferred to serum-free hormonally-defined medium containing various concentrations of ACTH1--24. The effects of ACTH on the light microscopic features, metabolic activity and permeation properties were examined. The results demonstrate that ACTH exerts a trophic action on the neurons.

Efflux of choline from neurons and glia in culture.

The efflux of radioactive choline from exclusively neuronal or glial cell cultures was dependent upon the concentrations of choline present in the cells and in the incubation medium, suggesting the possible presence of a homoexchange phenomenon between influx and efflux. The ionic dependence of the outward movement of choline from these cells showed that is could be stimulated by high K+ concentrations and by the absence of Ca2+. In glial cells, however, the efflux of choline was increased with a much lower concentration of K+ compared to neurons. The result may suggest that during nerve stimulation the release of K+ from neurons could stimulate, from glia, the efflux of choline which would then be taken up in neurons.