Randolph Mellus Johnson

Regulation of glial cell line-derived neurotrophic factor release from rat C6 glioblastoma cells.

Abstract: We have monitored glial cell line‐derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24‐h treatment with fibroblast growth factor‐1, −2, or −9 elevated secreted GDNF protein levels five‐ to 10‐fold. The proinflammatory cytokines interleukin‐1β, interleukin‐6, tumor necrosis factor‐α, and lipopolysaccharide elevated GDNF release 1.5‐ to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13‐didecanoate (10 nM) activation of protein kinase C, the Ca2+ ionophore A23187 (1 µM), okadaic acid (10 nM) inhibition of type‐2A protein phosphatases, nitric oxide donors (1 mM), and H2O2 (1 mM)‐induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 µM) or dibutyryl cyclic AMP (1 mM) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 µM). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.

Differential regulation of glial cell line–derived neurotrophic factor (GDNF) expression in human neuroblastoma and glioblastoma cell lines

Human SK‐N‐AS neuroblastoma and U‐87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line–derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24‐hr exposure to tumor necrosis factor‐α (TNFα; 10 ng/ml) or interleukin‐1β (IL‐1β; 10 ng/ml) induced GDNF release in U‐87MG cells, but repressed GDNF release from SK‐N‐AS cells. Fibroblast growth factors (FGF)‐1, ‐2, and ‐9 (50 ng/ml), the prostaglandins PGA2, PGE2, and PGI2 (10 μM), phorbol 12,13‐didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 μM), and vitamin D3 (1 μm) also differentially effected GDNF release from U‐87MG and SK‐N‐AS cells. A result shared by both cell lines, was a two‐ to threefold increase in GDNF release by db‐cAMP (1 mM), or forskolin (10 μM). In general, analysis of steady‐state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U‐87MG cells but remained static in SK‐N‐AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous facto, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U‐87MG) and neuronal (SK‐N‐AS) origin. J. Neurosci. Res. 55:187–197, 1999. 

Hyperplastic Changes within the Leptomeninges of the Rat and Monkey in Response to Chronic Intracerebroventricular Infusion of Nerve Growth Factor

Recombinant human nerve growth factor (rhNGF) was delivered for up to 6 months by continuous intracerebroventricular (i.c.v.) infusion to CD (Sprague-Dawley derived) rats and cynomolgus monkeys. Rats (n = 15/sex/group) received doses of 0 (vehicle), 6, 60, or 300 ng/day; monkeys (n = 5/sex/group) received 0, 0.6, 6, or 60 microg/day of rhNGF. Animals tolerated i.c.v. infusion with no behavioral signs attributable to rhNGF. Body weight was transiently decreased in female rats at the highest dose. At the completion of dosing, histological examination in both species revealed an increase in the thickness of the leptomeninges along the ventral and lateral surfaces of the hindbrain and extending over the dorsal aspect of the spinal cord. The change was present to varying degrees at all doses of rhNGF and tended to be more severe at higher doses. At the light microscopic level, the leptomeninges contained layers of well-differentiated, spindle-shaped cells and a plexus of axonal fibers. Cells were immunoreactive for S-100 protein and were associated with an accumulation of Type IV collagen, suggesting Schwann cell origin. Electron microscopy revealed numerous fine caliber axons ensheathed by the presumptive Schwann cells, with myelination of individual axonal segments. These findings suggest that chronic i.c.v. delivery of rhNGF has stimulated axonal sprouting and secondary hyperplasia of Schwann or Schwann-like support cells within the pia-arachnoid.

Linopirdine (DuP 996) improves performance in several tests of learning and memory by modulation of cholinergic neurotransmission

The actions of linopirdine (DuP 996; 3,3-bis[4-pyrindinylmethyl]-1-phenylindolin-2-one) were evaluated in rats and mice in several cognitive behavioral tests, and for its effects on hippocampal acetylcholine (ACh) overflow in rats. Using mice treated with the muscarinic receptor antagonist, scopolamine, we studied the effects of linopirdine on retention of a passive avoidance task. Linopirdine (0.1 and 1 mg/kg) ameliorated the scopolamine-induced deficit, but at doses ranging from 0.01-1 mg/kg, it did not affect passive avoidance retention in normal (untreated) mice. In a scopolamine-induced hyperactivity test, linopirdine (1 mg/kg) decreased the motoric stimulation associated with the cholinergic hypofunction, without affecting locomotor activity on its own. Using rats, we studied the effects of linopirdine on performance in the Morris water maze spatial memory task. Young rats treated with atropine (30 mg/kg), a muscarinic receptor antagonist, took significantly longer to locate the submerged platform across 12 trials. Linopirdine (0.01 and 0.1, but not 1 mg/kg) ameliorated the atropine deficit. In addition, linopirdine (0.1 mg/kg) ameliorated the deficit in cognition-impaired aged rats (23-24 mo), but did not affect unimpaired aged rats. In terms of neurochemical action, linopirdine (1, 10, and 100 microM) produced a concentration-dependent increase in K(+)-evoked ACh overflow from superfused rat hippocampal slices. Also, linopirdine (10 microM) similarly increased ACh release in young control rats and cognition-impaired and nonimpaired aged rats. Our results confirm and extend findings from other studies that demonstrate the cognition-enhancing action of linopirdine in rodent models.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of Radioiodinated Recombinant Human Nerve Growth Factor in Primate Brain Following Intracerebroventricular Infusion

The distribution of radioiodinated recombinant human nerve growth factor ([125I]rhNGF) was evaluated in adult cynomolgus monkeys following unilateral intracerebroventricular (icv) administration. Animals were cannulated into the right ventricle and recovered for 7 days. Monkeys were infused with 1.2 micrograms of [125I]rhNGF or [125I]rhNGF with a 140-fold excess of rhNGF. Twenty-four hours after infusion, animals were anesthetized and transcardially perfused with an aldehyde fixative. Coronal brain sections were processed for quantitative film autoradiography or for choline-acetyltransferase immunohistochemistry and then emulsion dipped. Specific radiolabel was distributed bilaterally and, with equal density, throughout the basal forebrain and was colocalized with choline acetyltransferase-positive neurons. Specific labeling was also present in the superficial ventral cortex. Nonspecific binding was observed surrounding the ventricles and lining blood vessels. These results demonstrate that unilateral icv infusion is an effective approach for delivering NGF to basal forebrain cholinergic neurons in primates and represents a viable drug delivery strategy for the therapeutic use of NGF in Alzheimers Disease.

5-HT3 receptor ligands lack modulatory influence on acetycholine release in rat entorhinal cortex

SummaryThe objective of this study was to explore the role of 5-HT3 receptors in modulating potassium (K+)-evoked release of [3H]-acetylcholine ([3H]-ACh) from superfused slices of rat entorhinal cortex previously loaded with [3H]-choline. Rat entorhinal cortices were cross-chopped into 300 μm slices, superfused with oxygenated Krebs buffer containing 2.5 mmol/1 Ca2+ and stimulated with two consecutive exposures of 20 mmol/l K+ for 4 min (S1 and S2, respectively). Compounds were added 20 min before S2 stimulation and remained in the superfusion buffer for the duration of the experiment. The S2/S1 ratio was then calculated.Stimulated release of [3H]-ACh was dependent on extracellular Ca2+ and K+ concentration. In Sprague Dawley rats, 2-methyl-5-HT (10-9−10-6 mol/l), in the presence of 1 μmol/l ritanserin or 1 gmmol/l ondansetron, had no influence on K+-evoked release of [3H]-ACh. In slices prepared from Hooded Lister rats, 2 μmol/l 5-HT but not 2-Me-5-HT significantly (P<0.05) inhibited K+-evoked [3H]-ACh release only 17% in the presence of 1 μmol/l ritanserin. However, 2 μmol/l 2-Me-5-HT plus 1 nmol/l ondansetron had no effect. High performance liquid chromatography coupled to electrochemical detection (HPLC-ECD) was used to monitor endogenous release of ACh in the above conditions to confirm data from the radiolabelled experiments. No significant inhibition or increase in K+-evoked ACh release was observed with either 5-HT3 receptor agonists or antagonists. 2-Me-5-HT (10−9 – 10−5 mol/l) or 1-(m-chlorophenyl)-biguanide (10−9 – 10−5 mol/l), when added simultaneously at the S2 stimulation, in the presence of 1 όl/l methysergide, also showed no effect on [3H]ACh release.In entorhinal cortex slices from aged Wistar rats, neither 1-(m-chlorophenyl)-biguanide (2 or 10 μol/l) nor 2-Me-5-HT (2 μmol/l) in combination with ritanserin (1 μmol/l) or ondansetron (1 nmol/l) elicited any effect on K+-evoked [3H]-ACh release. However, release of [3H]-ACh was inhibited by carbachol (10 μmol/l) and adenosine (10 μmol/l). DuP 996 (3,3-bis(4- pyridinyl-methyl)-1-phenylindolin-2-one) (10−7 – 10−5 mol/l), a known releaser of ACh, markedly augmented K+-evoked [3H]-ACh release.These studies have failed to confirm the postulated role of 5-HT3 receptors in modulating cortical ACh release in rat entorhinal cortex slices and suggest that a critical reexamination of the interaction of 5-HT3 receptor and cortical cholinergic function needs to be addressed.

Dose-response comparison of recombinant human nerve growth factor and recombinant human basic fibroblast growth factor in the fimbria fornix model of acute cholinergic degeneration

Both nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) have been proposed for the treatment of Alzheimers disease. This study describes a comparative, dose-response analysis of recombinant human (rh)NGF and rhbFGF in a rat unilateral fimbria-fornix model of acute cholinergic neuronal degeneration. Doses for rhNGF were 0.6, 6, 60, 600 and 1,800 ng/rat/day and for rhbFGF were 600, 1,800, 3,000 and 6,000 ng/rat/day, delivered for 4 weeks. The number of surviving septal cholinergic neurons was evaluated using ChAT immunohistochemistry. In control animals, the number of ChAT-positive neurons remaining on the lesioned side was between 22 and 18% compared to the non-lesioned side. Infusion with either neurotrophic factor increased the number of ChAT-positive neurons on the lesioned side in a dose-dependent manner. The maximal response to rhbFGF peaked at 3,000 ng/rat/day with a cell savings of 47%. However, there was evidence of neuropathological changes associated with rhbFGF. In contrast, rhNGF produced a maximal response with an infusion of 600 ng rhNGF/rat/day and a cell savings of 70% and no evidence of neuropathology, indicating that rhNGF was better tolerated and more efficacious than rhbFGF.

EVALUATION OF HUMAN ASTROCYTOMA AND GLIOBLASTOMA CELL LINES FOR NERVE GROWTH FACTOR RELEASE

Nerve growth factor (NGF) prevents degeneration of cholinergic neurons in the central nervous system (CNS), and has potential as a therapeutic treatment for Alzheimers disease. The inability of NGF to cross the blood-brain barrier has prompted pharmacological approaches investigating peripherally administered compounds that stimulate release of endogenous NGF. This study describes the NGF-releasing properties of six human astrocytoma and glioblastoma cell lines (SW 1088, SW 1783 and CRL 1718 astrocytomas, and U-138, U-373, and T98G glioblastomas). Using a highly specific two-site ELISA for human NGF, basal NGF release could be detected in all cell lines, with the lowest level in the T98G line (approximately 80 pg NGF/ml). Cell lines tested with a variety of compounds for 24 h in serum-free media demonstrated stimulation of NGF release by distinct mechanisms. NGF levels were markedly elevated (up to 8-fold above vehicle-treated cells) when stimulated with the cytokine interleukin-1 beta (IL-1 beta). Phorbol ester stimulated NGF release 4-fold. Clenbuterol, 4-methyl catechol, and propentofylline had little activity, while 6-(4-hydroxybutyl)-2,3,5,-trimethyl-1,4,benzoquinone (TMQ), dexamethasone and 1,25-dihydroxyvitamin D3 elevated NGF levels 3-fold. These data indicate differences in the ability of human astrocytoma and glioblastoma cells to release NGF when stimulated with mechanistically distinct compounds.

Recombinant human ciliary neurotrophic factor stimulates the metabolic activity of SH-SY5Y cells as measured by a cytosensor microphysiometer

Information on the transmembrane signaling events and subsequent biochemical processes initiated by ciliary neurotrophic factor (CNTF) receptor activation in neurons is lacking. SH-SY5Y cells, a human neuroblastoma cell line expressing CNTF receptors, were used to study metabolic changes associated with functional ligand-receptor interactions. Real-time measurements quantifying the rate of extracellular acidification by SH-SY5Y cells (a measure of metabolic activity) were made using a silicon-based cytosensor. Application of recombinant human CNTF (rhCNTF) to resting SH-SY5Y cells increased their acidification rate in a concentration and time-dependent manner with an apparent EC50 of 60 ng/ml. Pretreatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) prevented the CNTF, but not an NGF-stimulated increase in acidification rate. Collectively, these results demonstrate that: (1) SH-SY5Y cells express functional CNTF receptors; and (2) the initial signal transduction mechanism activated by the CNTF receptor in SH-SY5Y cells is distinct from that activated by the NGF receptor; however, both may ultimately stimulate the same downstream biochemical messengers to increase cellular metabolism.

PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE INHIBITOR D609 DIFFERENTIALLY AFFECTS MAP KINASES AND IMMEDIATE-EARLY GENES IN PC12 CELLS

The effects of tricyclodecan-9-yl xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipases, on PC12 cells were investigated. D609 repressed nerve growth factor (NGF)-mediated induction of c-fos mRNA with an IC50 approximately 50 microg/ml. Interestingly, maximal c-fos-suppressing doses of D609 did not affect activity of extracellular signal-regulated kinases. Surprisingly, D609 enhanced the extracellular acidification rate of PC12 cells, even in the absence of NGF. D609 alone induced c-jun mRNA with the same potency as it repressed the NGF-induced expression of c-fos. Like NGF, D609 alone induced c-jun even in the presence of dominant-negative Ras. Immediate-early induction of c-jun mRNA by NGF and D609 was abrogated by pretreatment with the kinase inhibitor olomoucine. Jun kinase, which is inhibited by olomoucine, was found to be activated by D609. Thus, D609 might induce c-jun in PC12 cells as a consequence of Jun kinase activation through a Ras-independent pathway. Under the same conditions, D609 repressed NGF-mediated induction of c-fos mRNA.