Cultured Brain Neurons Research Articles

Surface Phosphorylation by Ecto‐Protein Kinase C in Brain Neurons: A Target for Alzheimer's β‐Amyloid Peptides

The powerful regulatory machinery of protein phosphorylation operates in the extracellular environment of the brain. Enzymatic activity with the catalytic specificity of protein kinase C (PKC) was detected on the surface of brain neurons, where it can serve as a direct target for neurotrophic and neurotoxic substances that control neuronal development and cause neurodegeneration. This activity fulfilled all the criteria required of an ecto-protein kinase (ecto-PK). Detailed analysis of surface protein phosphorylation in cultured brain neurons using specific exogenous substrates (casein, histones, and myelin basic protein), inhibitors (PKC-pseudosubstrate 19-36; K252b) and antibodies (anti-PKC catalytic region M.Ab.1.9, antibodies to the carboxy-terminus of eight PKC isozymes) revealed several types of ecto-PK activity, among them ecto-PKs with catalytic specificity of the PKC isozymes zeta and delta. The activity of the neuronal ecto-PKC is constitutive and not stimulated by phorbol esters. the phosphorylation of a 12K/13K surface protein duplex by ecto-PKC-delta was found to be developmentally regulated, with peak activity occurring during the onset of neuritogenesis. Alzheimer's amyloid peptides beta 1-40 and beta 25-35 applied at neurotrophic concentrations stimulated the phosphorylation of endogenous substrates of ecto-PKC activity in brain neurons but inhibited specifically this surface phosphorylation activity with the same dose-response relationships that cause neurodegeneration. As may be expected from a relevant pathophysiological activity, beta-amyloid peptide 1-28 did not inhibit this surface phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human fetal central neurons in culture: voltage- and ligand-gated currents.

1. The functional properties of sodium, potassium, calcium, N-methyl-D-aspartate (NMDA), kainate, and gamma-aminobutyric acid (GABA) currents were studied in dissociated monolayer cultures of fetal human brain neurons, using the whole cell patch-clamp technique. 2. Sodium currents were characterized with respect to the following properties: current density, voltage dependence of activation, voltage dependence of inactivation, and sensitivity to tetrodotoxin (TTX). All sodium currents exhibited voltage dependencies of activation and inactivation, and sensitivities to TTX that are characteristic of the neuronal form of the sodium current. 3. At least two types of potassium current were present, resembling the delayed rectifier and fast-inactivating potassium current. These two types of potassium current were distinguishable by their different kinetics, voltage dependencies of activation and inactivation, and sensitivities to 4-aminopyridine and tetraethylammonium. 4. High-voltage-activated calcium channel currents were present and were characterized with respect to current density, voltage dependencies of activation and inactivation, and sensitivity to cadmium. Low-voltage-activated calcium channel currents were also present. 5. NMDA- and kainate-gated currents were studied with respect to current density, time course, and current-voltage relationship. Kainate currents were also characterized with respect to inhibition by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In addition, NMDA and kainate responses were compared for cortical versus cerebellar neurons. NMDA responses, which are only found in neurons, were present, confirming the neuronal phenotype suggested by the presence of the neuronal form of the sodium current. Nondesensitizing kainate currents were also present, with a half-maximally effective concentration (EC50) of approximately 200 microM for kainate; CNQX inhibited the kainate current with a half-inactivating concentration of 0.55 microM. 6. GABA-gated currents were characterized with respect to current density, time course, receptor subtype, desensitization, dose response, current-voltage relationship, ionic selectivity, pharmacology, and potentiation by the neurosteroid 5 alpha-pregnan-3 alpha-ol-11,20-dione (alfaxalone). Desensitizing GABAA currents were selective for chloride, inhibited by bicuculline and tert-butyl-bicyclophosphorothionate, and potentiated by diazepam, pentobarbital sodium, and alfaxalone. The EC50 for GABA was 15 microM.

Nuclear localization of initiation factor 2 in neuron primary cultures

The dominant mechanism that controls protein synthesis is the phosphorylation/dephosphorylation of initiation and elongation factors, with a translational control function. Each phase of protein synthesis is promoted by some of these factors that transiently interact with ribosomes, mRNAs and aminoacyltRNAs. Eukaryotic initiation factor-2 (eIF2, 130 kD) is one of these proteins and it is composed of three subunits: alpha, beta and gamma. eIF2 forms a ternary complex (GTP-eIF2-Met tRNAi) that can then interact with the 40S ribosomal subunit which in turn binds mRNA and the 60S ribosomal subunit to form the 80S initiation complex. The relation between eIF2 and the ribosomes is then a well established aspect of protein synthesis, but there are no previous studies about the distribution of eIF2 within the cell.Using immunocytochemical techniques, we show the distribution of eIF2 within the cell found in primary cultures of rat embryo brain neurons, in which eIF2 and eIF2-kinases have been identified. Primary culture neuron cells were grown in D15 and N2 mediums for 8 days.

The Effects of Thiamine and Oxythiamine on the Survival of Cultured Brain Neurons

The effects of treatment with thiamine (Vitamin B1) alone or together with its antagonist oxythiamine on the survival of brain neurons in primary culture were investigated. Treatment with thiamine significantly promoted the survival of hippocampal neurons in high cell density culture, but had no effects on the neuronal survival in low cell density culture. In addition, the survival-promoting activity exerted by thiamine was remarkably decreased by the co-application of oxythiamine, although oxythiamine used alone revealed neither a trophic nor toxic effect on the neurons of examined brain regions. The neurotrophic function of thiamine may be due to its coenzymatic role in a biochemical reaction and/or its specific function on neurotransmission and nerve conduction.

Antagonistic Effect of N-(3-Aminopropyl)cyclohexylamine on Neurotrophic Action of Spermine in Primary Cultured Rat Hippocampal and Cerebellar Neurons

We previously found that spermine potently promotes the neuronal survival and regeneration of primary cultured brain neurons. N-(3-Aminopropyl)cyclohexylamine (APCHA) was originally developed as a spermine synthase inhibitor. To test if endogenous spermine biosynthesis contributes to neuronal survival and morphogenesis, we examined the effects of APCHA in primary cultured rat hippocampal and cerebellar neurons. APCHA at concentrations up to 10-6 M did not affect the neuronal survival, but significantly blocked the survival-promoting effect of spermine (10-8 M). APCHA also blocked the spermine-induced promotion of neurite regeneration following axotomy. Unlike APCHA, another cyclohexylamine derivative trans-4-methylcyclohexylamine did not affect the neurotrophic effect of spermine. These results suggest that in primary cultured brain neurons, APCHA works as a spermine antagonist rather than as a spermine synthesis inhibitor.

Novel expression of the tyrosine hydroxylase gene requires both acidic fibroblast growth factor and an activator

Substances found in the soluble extract of muscle can alter the differentiative fate of certain brain neurons in culture by triggering novel expression of the gene for the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) (Iacovitti et al., 1989; Iacovitt, 1991). In this study, we demonstrate that TH induction in cultured noncatecholamine neurons from the mouse striatum requires the cooperative interaction of at least two substances found in muscle. Purification studies, combined with biological assay, revealed that one necessary component is acidic fibroblast growth factor (aFGF), and the other, an unidentified molecule(s) of < 10 kDa molecular weight that activated aFGF. Thus, muscle-derived aFGF, if incubated in the presence but not the absence of the < 10 kDa fraction of muscle, induced a dose-dependent increase in the number of striatal neurons that novelly express TH. This expression was blocked by prior incubation and protein A precipitation of the factor with polyclonal antibodies to aFGF (1:200-1:1000). Similar to muscle-purified aFGF, commercial preparations of native bovine and human recombinant aFGF (0.1-100 ng/ml) were potent inducers of TH when coincubated with the < 10 kDa activator. In contrast, basic FGF produced little and FGF-7 no induction of TH. Unlike the unidentified activating agent in muscle, heparin (20-500 mU), a known activator of aFGF, did not potentiate the factor's TH-inducing activity. Nonetheless, heparatinase (100 mU) prevented TH induction by aFGF and its activator, indicating that binding of heparan sulfated proteoglycans is necessary for the effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation and inactivation of endogenous cannabinoid anandamide in central neurons.

Anandamide (N-arachidonoyl-ethanolamine) was recently identified as a brain arachidonate derivative that binds to and activates cannabinoid receptors, yet the mechanisms underlying formation, release and inactivation of this putative messenger molecule are still unclear. Here we report that anandamide is produced in and released from cultured brain neurons in a calcium ion-dependent manner when the neurons are stimulated with membrane-depolarizing agents. Anandamide formation occurs through phosphodiesterase-mediated cleavage of a novel phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine. A similar mechanism also governs the formation of a family of anandamide congeners, whose possible roles in neuronal signalling remain unknown. Our results and those of others indicate therefore that multiple biochemical pathways may participate in anandamide formation in brain tissue. The life span of extracellular anandamide is limited by a rapid and selective process of cellular uptake, which is accompanied by hydrolytic degradation to ethanolamine and arachidonate. Our results thus strongly support the proposed role of anandamide as an endogenous neuronal messenger.

Ethanol attenuation of ganglioside sialylation and neuritogenesis

Neurite-producing cultured embryonic chick brain neurons in 96-h culture were exposed chronically to 25 mM ethanol for a 72-h period. Neurital plasma membrane extension was markedly attenuated in the ethanol-exposed neurons as compared with ethanol-free control cultures. The rate of biosynthetic sialylation of gangliosides, which are major structural sialoglycosphingolipid components in the exofacial lipid bilayer of the neurital plasma membrane, decreased to about half that in the alcohol-free neurons. The findings show that ganglioside sialylation in the morphodifferentiating central nervous system neuron is significantly attenuated by chronic exposure to a moderate dose of ethanol. Depression of ganglioside-dependent neuritogenesis may represent a mechanism for development of the central nervous system component of the fetal alcohol syndrome.

2-mercaptoethanol-independent survival of fetal mouse brain neurons cultured in a medium of human serum

In primary cultures of fetal mouse brain neurons, medium supplemented with fetal calf serum required 2-mercaptoethanol to support the survival and maturation of neurons, while medium containing human serum did not require the drug. These findings suggest that human serum is more active than fetal calf serum in reducing oxidative stress of neurons.

Effect of α1-antichymotrypsin on the toxicity of β-amyloid fragment 25–40 in rat primary cultured neurons

α 1-Antichymotrypsin (ACT) and β-amyloid have been reported to be present in senile plaques. We investigated the combination effects of ACT and a novel synthetic β-amyloid peptide, on rat primary cultured brain neurons. ACT had no effect in high density culture and decreased the number of surviving neurons in low density cell culture. Combination of ACT with β 25–40 did not affect the toxicity of β 25–40 in high and low density culture.

Isolation of a neuronal cell surface receptor of heparin binding growth-associated molecule (HB-GAM). Identification as N-syndecan (syndecan-3).

HB-GAM (heparin binding growth-associated molecule; pleiotrophin) is a secretory, extracellular matrix-associated protein that is strongly expressed in developing nervous tissues and belongs to a novel family of differentiation/growth factors. It promotes axonal growth from perinatal rat brain neurons and is suggested to be mitogenic for some cell types and to display cell-transforming activity. Since the receptors of HB-GAM in cells are unknown, we have started isolation of putative cell surface receptors from brain neurons and from perinatal rat brain. For this purpose, recombinant HB-GAM was produced with the aid of a baculovirus vector and used as an affinity matrix in receptor isolation. A detergent-solubilized component from cultured brain neurons and from brain was identified that binds specifically to HB-GAM and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad smear with an apparent molecular mass of about 200 kDa. This cell surface component was found to contain heparan sulfate chains, which are bound to a core protein with an apparent molecular mass of 120 kDa. Gel electrophoretic characteristics, immunochemical analysis, and partial peptide sequencing revealed that the cell surface component isolated as an HB-GAM receptor is N-syndecan (syndecan-3). In a solid phase binding assay, N-syndecan was found to bind to HB-GAM in a similar manner as to basic fibroblast growth factor (KD = 0.6 nM). Immunofluorescence microscopy indicated that in brain neurons, N-syndecan occurs at the surface of the cell soma and of the neurites that grow along HB-GAM-coated substrates. Anti-N-syndecan antibodies added to culture media had an inhibitory effect on HB-GAM-induced neurite outgrowth. We suggest that N-syndecan mediates the neurite outgrowth-promoting signal from HB-GAM to the cytoskeleton of growing neurites.

Effects of retinoic acid on NB 69 human neuroblastoma cells and fetal rat mid brain neurons.

Retinoids are chemical compounds which play important roles in ontogenetic development and cranio-caudal differentiation in animals, but their effect on phenotypic expression of neurotransmitters are unknown. We studied the pharmacological and morphological effects of retinoic acid (RA) on two types of immature vertebrate neurons, the human derived neuroblastoma cells, NB69, and fetal rat mid brain neurons in culture. The pharmacological effects of RA on the cultures and their relation to catecholamine and acetylcholine neurotransmission were evaluated according the levels of catecholamines, tyrosine hydroxylase (TH) activity, TH immunostaining, and choline acetyltransferase (CAT) activity, respectively. RA reduces catecholamine levels and TH activity in NB69 cells and the number of dopamine neurons in cultures derived from rat fetal mid brain. The detrimental effect of RA on mid brain neurons is dose- dependent; limited to TH+ cells at low concentrations (100 to 500 nM) and toxic for all types of cells at high concentrations (1 to 2 microM). RA increases CAT activity in NB 69 cells and produces phenotypic differentiation of these to a more mature neuronal phenotype with more prolonged neurite extensions. Therefore, RA may play a trophic positive role in the differentiation of immature cells to cholinergic neurons; this contrasts with the detrimental effects of RA on catecholamine neurons.

Chronic L-alpha-glyceryl-phosphoryl-choline increases inositol phosphate formation in brain slices and neuronal cultures.

Repeated, but not single injections of L-alpha-glyceryl-phosphorylcholine (alpha GPC) significantly increased basal [3H]inositol monophosphate (InsP) formation in hippocampal, cortical, and striatal slices of male rats. The effect was dose-dependent and was accompanied by an increased incorporation of [3H]inositol into the phospholipid fraction. Incubation of brain slices with different neurotransmitter antagonists, such as atropine, prazosin, or L-2-amino-4-phosphonobutanoate (L-AP4) did not modify the increase in [3H]InsP formation produced by alpha GPC, suggesting that the effect is not mediated by an increased availability of a specific neurotransmitter. Similar results were obtained in cerebellar and cortico-striatal neurones in primary culture exposed to daily addition of alpha GPC since the second day of maturation in vitro. We suggest that alpha GPC treatment may result in an increased rate of phospholipid synthesis, including the phosphoinositides available for signal transduction at central nervous system level.

Piperine, a pungent alkaloid, is cytotoxic to cultured neurons from the embryonic rat brain.

The present study reveals that piperine, a pungent alkaloid present in various Piper species, is cytotoxic to cultured brain neurons. Exposure to piperine (12.5-100 microM) for 72 h caused a concentration-dependent reduction in the survival of primary cultured neurons from various regions of the embryonic rat brain under high density cell culture conditions. There were relative regional differences in the susceptibility to cytotoxic effects of piperine in which septum and hippocampus showed higher vulnerability among the eight regions. The primary cultures of septal and hippocampal neurons under low density cell culture condition were performed to evaluate the contribution of non-neuronal cells. The concentration-response profiles in both high and low density cell culture conditions were comparable (septum: EC50 = 43 and 27 microM, hippocampus: EC50 = 50 and 44 microM, under high and low density cell culture conditions, respectively) suggesting a minor role of non-neuronal cells on cytotoxicity of piperine to developing neurons.

Effects of 2-mercaptoethanol on survival and differentiation of fetal mouse brain neurons cultured in vitro

Effects of 2-mercaptoethanol on primary cultures of fetal mouse brain neurons have been investigated. The addition of 2-mercaptoethanol to the culture medium increased 6- or 200-fold the survival rates of embryonic day-16 murine striatum neurons and day-18 cerebral cortical neurons cultured in serum-free medium, respectively, and also induced neurite outgrowth, particularly being prominent in cortical neurons. Moreover, this drug enhanced trophic activities of the conditioned medium of VR-2g or BIM cells. These findings indicate that 2-mercaptoethanol can support the viability and differentiation of fetal mouse brain neurons.

Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain

We investigated the effects of interleukin-2 (IL-2) on the survival and morphology of primary cultured neurons from fetal rat brain. Addition of recombinant human IL-2 significantly supported the survival of brain neurons in high cell density culture, but did not show any effect on the neuronal survival in low cell density culture. In addition, IL-2 significantly promoted the neurite elongation and branching of hippocampal neurons in low cell density culture. These results suggest that IL-2 supports neuronal survival indirectly and promotes neuritogenesis by directly acting on brain neurons.

Modulation of neuropeptide Y expression in rat brain neuronal cultures

Neuropeptide Y (NPY) and its encoding mRNA were measured in neurons co-cultured from rat basal forebrain and cerebral cortex. NPY was synthesized and released in a manner consistent with secretion-synthesis coupling; depolarization increased each in a calcium-dependent manner. The accumulation of NPY encoding mRNA was elevated by a muscarinic receptor blocker, without changes in transmitter release or peptide synthesis, thereby consistent with a membrane potential-independent mechanism. Changes in intrinsic muscarinic transmission could nonetheless be expressed rapidly as an elevation in NPY levels by depolarizing the neurons subsequent to muscarinic receptor blockade. This depolarization-induced elevation of NPY subsequent to muscarinic receptor blockade was dependent on the presence of extracellular calcium ions. Forskolin and pertussis toxin also increased NPY encoding mRNA levels in a manner that was not additive with muscarinic receptor blockade. These results suggest that one or more muscarinic receptors may tonically modulate NPY synthesis via changes in adenylate cyclase activity, providing a model for the non-homeostatic modulation of neuropeptide turnover.

Down-regulation of phosphatidylinositol response to BDNF and NT-3 in cultures of cortical neurons

The hydrolysis of phosphatidyl 4,5-biphosphate (PI), which is involved in the transduction mechanism of neurotransmitters and growth factors, is stimulated by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in primary cultures of fetal brain neurons. In the present study we sought to examine the effect of pretreatment with these factors on their acute stimulation capabilities and, furthermore, to substantiate that the effects of BDNF and NT-3 reflect actions on neurons rather than glial cells. Pretreatment with BNDF and NT-3 for 4 days followed by 1 day without growth factor abolished the effect of an acute stimulation with these factors. The growth factors were mutually effective so that BDNF pretreatment abolished the acute response to NT-3 and vice versa. In contrast, the effects of bFGF (basic fibroblast growth factor, a non-neurotrophin growth factor) also stimulating PI hydrolysis in these culture systems, were not reduced by neurotrophin pretreatment. Pretreatment with K-252b, at concentrations known to inhibit trk receptors, did not alter the acute stimulation of PI hydrolysis induced by the neurotrophins. PI hydrolysis stimulated by BDNF and NT-3 in cultures grown in presence of cytosine arabinoside C, containing > 95% neurons, was higher than in cultures containing non-neuronal cells, indicating that the neurotrophin stimulation occurs in neuronal cells. No stimulatory effect was detected in bFGF treated pure neuronal cultures. The findings suggest that prolonged exposure of responsive neurons to BDNF and NT-3 down-regulates their stimulatory effects on PI hydrolysis.

Towards an improved survival of rat brain neurons in culture by cerebrospinal fluid of patients with senile dementia of Alzheimer's type

Neuronal cell survival was investigated in rat brain cortical cultures in the presence of increaseing concentrations of human brain extracts or cerebrospinal fluid (CSF) from control and Senile Dementia of Alzheimer's type (SDAT) patients. Using hippocampal brain extracts, converted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was compared to the content of the neuronal marker MAP2 in foeral rat brain neuronal cultures in order to test converted MTT as a quantitative parameter for neuronal cell survival. A significant correlatio was found between both parameters. SDAT frontal cortex brain extracts induced a two to four-fold increase in neuronal cell survival at 25 to 125 μg protein extract, whereas control brain extracts induced at similar protein concentrations a decline in neuronal cell survival. The enhanced survival yielded by SDAT brain extracts was fully abolished in the presence of control brain extract. Control CSF concentration-dependently increased neuronal cell survival in postnatal rat brain neuronal cultures independent of the difference in the protein content of CSF samples and age of the patients. SDAT CSF also concentration-dependently enhanced neuronal cell survival, however, the effect was more pronounced compared to control CSF. These observations are in favour of the hypothesis that there might a higher neurotrophic activity in SDAT brain tissue.

Contribution of organic and inorganic osmolytes to volume regulation in rat brain cells in culture

In this work we examined the time course and the amount released, by hyposmolarity, for the most abundant free amino acids (FAA) in rat brain cortex astrocytes and neurons in culture. The aim was to evaluate their contribution to the process of cell volume regulation. Taurine, glutamate, and D-aspartate in the two types of cells, beta-alanine in astrocytes and GABA in neurons were promptly released by hyposmolarity, reaching a maximum within 1-2 min. after an osmolarity change. A substantial amount of the intracellular pool of these amino acids was mobilized in response to hyposmolarity. The amount released in media with osmolarity reduced from 300 mOsm to 150 mOsm or 210 mOsm, represented 50%-65% and 13%-31%, respectively, of the total amino acid content in cells. In both astrocytes and neurons, the efflux of glutamine and alanine was higher under isosmotic conditions and increased only marginally during hyposmotic conditions. 86Rb+, used as tracer for K+, was released from astrocytes, 30% and 11%, respectively, in hyposmotic media of 150 mOsm or 210 mOsm but was not transported in neurons. From these results it was calculated that FAA contribute 54% and inorganic ions 46% to the process of volume regulation in astrocytes exposed to a 150 mOsm hyposmotic medium. This contribution was 55% for FAA and 45% for K+ and Cl- in cells exposed to 210 mOsm hyposmotic solutions. These results indicate that the contribution of FAA to the process of cell volume regulation is higher in astrocytes than in other cell types including renal and blood cells.