Primary Neuronal Cell Cultures Research Articles

Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro

The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7±9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15–17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5–80 μM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life–death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose- and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77–1.86 μM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13 h after ethylcholine aziridinium application. The present data indicate that in a model of delayed apoptotic neurodegeneration as induced by ethylcholine aziridinium neuronal cell death in vitro and in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of nitric oxide production and absence of inducible nitric oxide synthase expression. In contrast, evidence for a neuroprotective role of nitric oxide was obtained in vivo as indicated by the exaggeration of the cholinergic lesion after unspecific nitric oxide synthase inhibition by N-nitro- l-arginine methylester.

Establishment of an in vitro screening model for neurodegeneration induced by antimalarial drugs of the artemisinin-type..

The establishment of an in vitro screening model for neurodegeneration inducing antimalarial drugs was conducted in stepwise fashion. Firstly, the in vivo selective neurotoxic potency of artemisinin was tested in neuronal cells in vitro in relation to the cytotoxic potency in other organ cell cultures such as liver and kidney or versus glial cells. Secondly, a comparison between different parts of the brain (cortex vs. brain stem) was performed and in the last step, a fast and sensitive screening endpoint was identified. In summary, non-neuronal cell lines such as hepatocytes (HEP-G2), liver epithelial cells (IAR), proximal tubular cells (LLC-PK(1)) and glial cells from the rat (C6) and human (GO-G-IJKT) displayed only moderate sensitivity to artemisinin and its derivatives. The same was found in undifferentiated neuronal cell lines from the mouse (N-18) and from human (Kelly), whereas during differentiation, these cells became much more sensitive. Primary astrocytes from the rat also were not specifically involved. In the comparison of primary neuronal cell cultures from the cortex and brain stem of the rat, the brain stem was found to be more sensitive than the cortex. The neurotoxic potential was determined by cytoskeleton elements (neurofilaments), which were degradated in vitro by diverse neurodegenerative compounds. In comparison of dog and rat primary brain stem cultures, the dog cells were found to be more sensitive to artemisinin than the rat cells. In addition to the primary brain stem cell cultures it was shown that the sprouting assay, which determines persistent delayed neurotoxic effects, is also useful for screening antimalarial drugs. To other compounds, artemether and artesunate, showed that use of the sprouting assay followed by primary brain stem cultures of the rat will be a good strategy to select candidate compounds.

Apoptosis Induced by Manganese in Basal Ganglia Primary Neuronal Cell Culture: Morphological Findings

Apoptosis Induced by Manganese in Basal Ganglia Primary Neuronal Cell Culture: Morphological Findings

A brain derived peptide preparation reduces the translation dependent loss of a cytoskeletal protein in primary cultured chicken neurons.

Neuronal cytoskeletal proteins like the microtubule associated protein 2 (MAP2) are objected to pathological proteolysis in case of Alzheimer's disease and brain ischemia. The neurotrophic peptidergic drug Cerebrolysin (EBEWE Arzneimittel, Austria, Europe) is produced by a standardized enzymatic break-down of lipid free porcine brain proteins. Cerebolysin protected MAP2 in primary neuronal cultures after a brief histotoxic hypoxia and in a rat model of acute brain ischemia. Furthermore the drug was shown to inhibit the proteases mu- and m-calpain dose dependently in several cell free protease activity assays. The question if the higher MAP2 levels are due to an alleviation of proteolysis, to a higher synthesis rate or both is addressed in the current investigation: Monitoring the MAP2 content of primary neuronal cell cultures over a period of eight days revealed MAP2 to reach a peak level on day six in vitro followed by a degradation phase. In other experiments the protein synthesis of Cerebrolysin treated and untreated cells was blocked with cycloheximide at that moment when all cells exhibited the same MAP2 content. After the following MAP2 degradation phase--i.e. after eight days in vitro--the MAP2 contents were determined by western blotting. Cerebrolysin treated cells contained more MAP2 than untreated controls proving that the drug protects MAP2 independently from de novo synthesis, although further work is in progress to investigate if the drug supplementary boosts this effect by increasing MAP2 synthesis.

Tau Pathology Generated by Overexpression of Tau

Neurofibrillary changes of abnormally hyperphosphorylated tau are the key lesion in Alzheimer’s disease (AD) and a number of other tauopathies. Recent developments in the field of autosomal dominantly inherited dementias, in particular the frontotemporal dementias and Parkinsonism linked to chromosome 17 (FTDP-17) group, have shown that abnormalities in the tau gene result in neurofibrillary degeneration and cell death. Clinically this disorder presents with behavioral abnormalities, which are followed by dementia and, depending on the affected areas, by motor dysfunction. The location of the lesions does not seem to depend so much on the type of mutation as on the individual’s genetic background and may vary even in the same family with the identical mutation. For instance, in one family with a P 301 S mutation in exon 10 of tau, the father presented with frontotemporal dementia, whereas the son had corticobasal degeneration. 1 FTDP-17 is associated with both exonic and intronic mutations of the tau gene. The microtubule-associated protein (MAP) tau is a family of six proteins derived by alternative mRNA splicing 2,3 from a single gene located on chromosome 17. These molecular isoforms of tau differ in whether they contain three or four tubulin binding domains/repeats of 31 or 32 amino acids each near the C-terminal end and no, one, or two inserts of 29 amino acids each at the N-terminal end of the molecule. There are nine missense mutations on tau exons 9 to 13; all but three are on exon 10. 4-7 Exon 10 codes for the additional insert of the three 4-repeat tau isoforms. The resulting mutated taus possess an altered conformation 8 and a somewhat reduced ability to bind to and assemble microtubules. 9,10 In addition to the exonic mutations, mutations at several sites have been found in the predicted stem loop structure in the 5′ splice site to exon 10. These intronic mutations and certain mutations in exon 10 that are close to the stem loop, and thus able to disrupt it, lead to two- to sixfold higher proportion of tau mRNA containing exon 10 than in control brains. 5 The tau protein resulting from intronic mutations is normal, but the ratio of 4-repeat to 3-repeat isoforms is increased. Presently it is believed that due to the increased proportion of 4-repeat tau in the case of intronic mutations and the compromised biological activity of the tau with missense mutations, excess tau is not bound to the microtubules, which can then be hyperphosphorylated and would lead to neurofibrillary degeneration. In contrast to the FTDP-17 group of diseases, no mutations in the tau gene have been reported in AD at the time of writing this Commentary. In more than 90% of the AD patients the disease occurs sporadically above 60 years of age. In less than 5% of the cases the disease segregates with mutations in the amyloid precursor protein (APP), presenilin-1 (PS-1), or presenilin-2 (PS-2) genes. 11 Frameshift mutations of APP and ubiquitin at the level of transcription have been reported to be associated with sporadic and familial AD and Down’s syndrome. 12 The occurrence of the apolipoprotein E4 allele 13 and, most recently, mutations in the β2 macroglobulin gene 14 have been reported to be risk factors for the development of the late onset, sporadic AD. AD has two prominent neuropathological lesions, the extracellular deposits of the amyloid β peptide (Aβ) as plaques and the intraneuronal paired helical filaments (PHF) of abnormally hyperphosphorylated tau, which accumulate in the neuronal cell body as neurofibrillary tangles, in the neuropil (the so-called neuropil threads), 15 and in the dystrophic neurites surrounding the neuritic plaques. The direct relationship, if any, between the tangles and β-amyloid is not yet understood. At the one extreme, in the normal aged brain there is the significant β-amyloid accumulation and minimal neurofibrillary degeneration, whereas in the early onset familial AD with mutations in the β-APP or presenilin gene, massive β-amyloid deposits, tau hyperphosphorylation, and tangle formation are always seen. The other extreme situation is represented by extensive neurofibrillary degeneration and minimal β-amyloidosis. These include tauopathies like the tangle-predominant form of senile dementia (tangle-only dementia), Guam Parkinsonism dementia complex, dementia with argyrophilic grains, Nieman Pick’s disease type C, subacute sclerosing panencephalitis, Pick’s disease, and the dementia group with mutations in the tau gene. 16 Although at present the exact role of PHF and β-amyloid in the pathogenesis of AD is not established, there is growing evidence from a number of laboratories that the intellectual deterioration in AD patients is associated with neurofibrillary degeneration. 17-20 A third and well characterized phenomenon is synaptic loss and cell death exceeding 50% in certain areas of the brain. 21,22

Effect of 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8) on neuronal calcium homeostasis, protein synthesis, and energy metabolism.

It has been suggested recently that disturbances of endoplasmic reticulum calcium homeostasis plays a major role in ischaemic cell injury of the brain. Depletion of endoplasmic reticulum calcium stores induces suppression of the initiation process of protein synthesis, a prominent feature of ischaemic cell damage. The benzoic acid derivative 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8), an established inhibitor of calcium release from endoplasmic reticulum, would be an ideal tool for elucidating the role of endoplasmic reticulum dysfunction in this pathological process. The present investigation was performed to study the effects of TMB-8 on neuronal metabolism (cytoplasmic calcium activity, ATP levels and protein synthesis) using hippocampal slices and primary neuronal cell cultures. In addition, we investigated whether the rise in cytoplasmic calcium activity and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion, is reversed by this agent. Exposure of neurones to TMB-8 (100 microM) induced a small transient increase in cytoplasmic calcium activity ([Ca2+]i), whereas a second dose of TMB-8 (200 microM) produced a marked and sustained rise in [Ca2+]i. The increase in [Ca2+]i evoked by blocking endoplasmic reticulum Ca(2+)-ATPase was only transiently suppressed and then aggravated by TMB-8. The dose-dependent suppression of protein synthesis by TMB-8, observed both in neuronal cultures and hippocampal slices, indicates that TMB-8 has a pathological effect on neuronal metabolism. This inhibition was not reversed after washing-off of the drug. TMB-8 did not reverse the inhibition of protein synthesis evoked by caffeine, which depletes endoplasmic reticulum calcium stores by activating the ryanodine receptor. The results indicate that TMB-8 is not a suitable investigative tool for blocking in neuronal cell cultures the depletion of endoplasmic reticulum calcium stores and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion.

Primary cultures of neuronal and non-neuronal rat brain cells secrete similar proportions of amyloid beta peptides ending at A beta40 and A beta42.

To determine the types of brain cells responsible for the production of amyloid beta peptides (A beta), as well as their carboxyl-terminal properties, we studied the secretion of A beta in rat neuronal, astrocytic, microglial and meningeal primary cell cultures. All four types of cells produced A beta, among which neurons secreted approximately 4 times more A beta than other cell types. The percentage of A beta42 ending at position 42 as a fraction of total A beta was similar between different cell types, ranging from 10 to 15%. These results suggest that neurons might be the most potent source for A beta production in the brain, although other non-neuronal type cells could also contribute to this process.

Catechol‐O‐methyltransferase activity in rat brain primary neuronal and glial cell cultures and its inhibitation by novel drugs

Catechol‐O‐methyltransferase activity in rat brain primary neuronal and glial cell cultures and its inhibitation by novel drugs

Catechol-O-methyltransferase activity in rat brain primary neuronal and glial cell cultures and its inhibitation by novel drugs

To analyze the intracellular catechol-O-methyltransferase (COMT) activity in cultured cells, a substrate (dihydroxybenzoic acid) was added to the tissue culture plates and the product (vanillic acid) was anlayzed with reversed-phase HPLC with coulometric detection. The cultures, prepared from various regions of the brain, were immunohistochemically characterized. Basal COMT activites were similar in all glial cultures while in some neuron-enriched cultures the enzyme activity was lower. Preincubation with nitrocatechol COMR inhibitors, entacapone or tolcapone (30-300 nM), decreased COMT activity in all culture types with slightly different potencies. An atypical inhibitor of O-methylation, CGP 28104, had no oeffect on COMT activity in any of these cultures. In conclusion: 1) this method enables straight-forward analysis of a large number of COMT activity samples in cultured cells, 2) COMT activity is almost equally distributed throughout the brain, and 3) tolcapone was a more potent COMT inhibitor than entacapone in cultures containing glial cells.

Silencer-Mediated Repression and Non-Mediated Activation of BDNF and c-fos Gene Promoters in Primary Glial or Neuronal Cells

Although the neuron-restrictive silencer element (NRSE/Regard) has been shown to function as a negative-acting DNA regulatory element to prevent the expression of neuron-specific genes in non-neuronal cells, little is known about its silencing effect on transcription in primary glial cells nor its effect on transcriptional activation in primary neurons. By DNA transfection in primary cultures of rat cortical neuronal or glial cells, we investigated the effect of NRSE on transcription mediated by the BDNF promoter I or c-fos promoter to which NRSE sequences derived from the SCG10 gene were linked. Transfection of plasmid DNAs to NIH3T3 fibroblasts resulted in a marked repressive effect of NRSE on BDNF promoter I- or c-fos promoter-mediated transcription. In primary neuronal cells, however, NRSE did not repress the basal promoter activities of BDNF and c-fos genes and allowed the transcriptional activation of these genes induced by membrane depolarization although NRSE slightly reduced the magnitude of BDNF promoter I activation. In contrast to neuronal cells, a marked repression of basal promoter activities of both genes was detected in primary glial culture and a two base pair-mutation of NRSE partially recovered the repression. These results indicate that NRSE negatively acts on its linked promoters in primary glial cells and does not interfere an activation of linked promoters in neuronal cells.

Astroglia contain a specific transport mechanism for N-acetyl-L-aspartate.

N-Acetylaspartate (NAA) is the second most abundant amino acid in the adult brain. It is located and synthesized in neurons and probably degraded in the glia compartment, but the transport mechanisms are unknown. Rat primary neuron and astrocyte cell cultures were exposed to the L isomer of [3H]NAA and demonstrated concentration-dependent uptake of [3H]NAA with a Km approximately 80 microM. However, Vmax was 23+/-6.4 pmol/mg of protein/min in astrocytes but only 1.13+/-0.4 pmol/mg of protein/min in neurons. The fact that neuron cultures contain 3-5% astrocytes suggests that the uptake mechanism is expressed only in glial cells. The astrocyte uptake was temperature and sodium chloride dependent and specific for L-NAA. The affinity for structural analogues was (IC50 in mM) as follows: L-NAA (0.12) > N-acetylaspartylglutamate (0.4) > N-acetylglutamate (0.42) > L-aspartate (>1) > L-glutamate (>1) > or = DL-threo-beta-hydroxyaspartate > N-acetyl-L-histidine. The naturally occurring amino acids showed no inhibitory effect at 1 mM. The glutamate transport blocker trans-pyrrolidine-2,4-dicarboxylate exhibited an IC50 of 0.57 mM, whereas another specific glutamate transport inhibitor, DL-threo-beta-hydroxyaspartate, had an IC50 of >1 mM. The experiments suggest that NAA transport in brain parenchyma occurs by a novel type of sodium-dependent carrier that is present only in glial cells.

Heme oxygenase immunoreactive neurons in the rat intestine and their relationship to nitrergic neurons

Background: Carbon monoxide (CO), like nitric oxide (NO), is a putative gaseous neurotransmitter. CO is produced by the enzyme heme oxygenase (HO) acting on a family of heme-containing compounds. Two isomers of HO have been characterized (HO-1, HO-2). In the CNS and in peripheral ganglia HO-2 occurs in a majority of neurons. NO and CO function as transmitters of enteric neurons but the relative distribution of enteric neurons utilizing these gaseous transmitters is unknown in rodent. We have studied the distribution of HO-2 immunoreactivity and NO synthase (NOS) activity within the rat ileum. Methods: Tissue sections and primary neuronal cell cultures were incubated with a HO-2 specific antibody, and then assessed or reprocessed for NOS activity using NADPH-dependent diaphorase staining. Results: HO-2 immunoreactivity was expressed in subpopulations of myenteric and submucosal neurons. Approximately 45% of the ganglion cells in tissue section were HO-2 positive. This was similar in proportion to those found to stain for NOS activity, and 10% of HO-2 positive neurons also contained NOS. HO-2 immunoreactivity was also found in epithelial cells within the villi, and in interstitial cells around the myenteric plexus and within the smooth muscle. In culture, the distribution and colocalisation of HO-2 and NOS positive neurons was similar to that in tissue sections. We identified labelled neurons as either Dogiel Type I or II; only Type II cells colocalized NOS and HO-2. Conclusion: Neurons, endocrine-like cells and interstitial cells with the capacity for CO production are distributed throughout the ileum and some neurons have the capacity to synthesize both NO and CO as gaseous messengers.

Interaction of Neuronal Nitric-oxide Synthase with α1-Syntrophin in Rat Brain

Neuronal nitric-oxide synthase (nNOS) has a PSD-95/Dlg/ZO-1 (PDZ) domain that can interact with multiple proteins. nNOS has been known to interact with PSD-95 and a related protein, PSD-93, in brain and with alpha1-syntrophin in skeletal muscle in mammals. In this study, we have purified an nNOS-interacting protein from bovine brain using an affinity column made of Sepharose conjugated with glutathione S-transferase-rat nNOS fusion protein and identified it as alpha1-syntrophin by microsequencing. Immunostaining of primary cultures of rat embryonic brain neuronal cells with antibodies against these proteins showed that nNOS and alpha1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicated that the nNOS- and alpha1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and alpha1-syntrophin-like immunoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts with alpha1-syntrophin in specific neurons of the SCN and paraventricular nucleus and that this interaction might play a physiological role in functions of these neurons.

Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the V max (chelerythrine: −61%, P<0.06; H89: −59%, P<0.05) without affecting the K m of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

Ischemia-induced changes in 2′-5′-oligoadenylate synthethase mRNA levels in rat brain: comparison with changes produced by perturbations of endoplasmic reticulum calcium homeostasis in neuronal cell cultures

2′-5′ Oligoadenylate synthetase (OAS) expression is induced by interferon or viral infection of cells. To better understand ischemia-induced changes in gene expression and to elucidate the possible underlying mechanisms, changes in OAS mRNA levels were evaluated after 30 min four-vessel occlusion and 2, 4, 8 or 24 h recovery and compared to the temporal profile of changes in mRNA levels induced by a transient depletion of endoplasmic reticulum (ER) calcium stores in primary neuronal cell cultures. OAS mRNA levels dropped during early recovery both in vivo and in vitro. After 6 h recovery from ER calcium pool depletion, OAS mRNA levels increased to about 350% of controls and returned to control levels after 24 h of recovery. After 24 h recovery from ischemia, OAS mRNA levels rose to about 390% of controls in the hippocampus and striatum and to 210% of the control value in the cortex. It is concluded that transient ischemia place cells into an antiviral state, most pronounced in the hippocampus and striatum, and that disturbances of ER calcium homeostasis may contribute to this process.

Nicotinic receptor binding sites in rat primary neuronal cells in culture: characterization and their regulation by chronic nicotine

We have characterized high affinity neuronal nicotinic acetylcholine receptors labeled by [ 3 H ]cytisine in primary neuronal cell cultures from fetal rat brains. After 15 days in culture, the highest density of [ 3 H ]cytisine binding sites ( B max≈57 fmol/mg protein) was found in cells from the brainstem, which includes the following subcortical brain areas: the septum, thalamus, hypothalamus, midbrain, pons and medulla. A lower density of sites was found in cells from the cerebral cortex, hippocampus, and caudate nucleus. [ 3 H ]Cytisine binds to receptors in primary cells from the brainstem and cerebral cortex with a K d of ≈0.5 nM, and the binding is inhibited by the agonists nicotine, acetylcholine, and epibatidine with IC 50 values of 1 to 20 nM, and by carbachol and the antagonist dihydro-β-erythroidine with IC 50 values of 0.5 to 1.5 μM. Chronic treatment of neuronal cultures with nicotine for 7 days differentially affected the number of nicotinic receptors in cells from different brain areas; it significantly increased the number of nicotinic binding sites in cells from the cerebral cortex, hippocampus, and caudate, but not in cells from the brainstem. The nicotine-induced increase of receptors in cerebral cortical cultures was not blocked by either mecamylamine or dihydro-β-erythroidine. These results indicate that primary cultures of rat neuronal cells provide a good model system in which to study and compare the properties and regulation of native neuronal nicotinic acetylcholine receptors.

Effect of highly halogenated ?-carbolines on dopaminergic cells in culture and on mitochondrial respiration

Pyridoindoles (carbolines) are relatively common indole alkaloids in most diets and in our ecosystem. Besides 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite, MPP + , several psychotropic β-carbolines have been described to exhibit neurotoxic effects on the dopaminergic system. In this work, we have investigated a new class of neurotoxic β-carbolines, the highly halogenated tetrahydro-β-carbolines. The present compounds are derived from the condensation of endogenous tryptamine with the hypnotic drug chloral hydrate or by exposure to the industrial solvent trichloroethylene (which can be metabolized to chloral). These tetrahydro-β-carbolines inhibit the mitochondrial respiratory chain, acting as strong inhibitors of Complex I and partial inhibitors of Complex II. They are also neurotoxic to dopaminergic neurons in primary cell culture.

Effect of highly halogenated β‐carbolines on dopaminergic cells in culture and on mitochondrial respiration

Pyridoindoles (carbolines) are relatively common indole alkaloids in most diets and in our ecosystem. Besides 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite, MPP+, several psychotropic β-carbolines have been described to exhibit neurotoxic effects on the dopaminergic system. In this work, we have investigated a new class of neurotoxic β-carbolines, the highly halogenated tetrahydro-β-carbolines. The present compounds are derived from the condensation of endogenous tryptamine with the hypnotic drug chloral hydrate or by exposure to the industrial solvent trichloroethylene (which can be metabolized to chloral). These tetrahydro-β-carbolines inhibit the mitochondrial respiratory chain, acting as strong inhibitors of Complex I and partial inhibitors of Complex II. They are also neurotoxic to dopaminergic neurons in primary cell culture. Drug Dev. Res. 46:51–56, 1999. © 1999 Wiley-Liss, Inc.

Activation of MYD116 (gadd34) expression following transient forebrain ischemia of rat: implications for a role of disturbances of endoplasmic reticulum calcium homeostasis

MyD116 is the murine homologue of growth arrest- and DNA damage-inducible genes (gadd34), a gene family implicated in growth arrest and apoptosis induced by endoplasmic reticulum dysfunction. The present study investigated changes in MyD116 mRNA levels induced by transient forebrain ischemia. MyD116 mRNA levels were measured by quantitative PCR. After 2 h of recovery following 30 min forebrain ischemia, MyD116 mRNA levels rose to about 550% of control both in the cortex and hippocampus. In the cortex, MyD116 mRNA levels gradually declined to 290% of control 24 h after ischemia, whereas in the hippocampus they remained high (538% of control after 24 h of recovery). To elucidate the possible mechanism underlying this activation process, MyD116 mRNA levels were also quantified in primary neuronal cell cultures under two different experimental conditions, both leading to a depletion of endoplasmic reticulum (ER) calcium pools. Changes in cytoplasmic calcium activity were assessed by fluorescence microscopy of fura-2-loaded cells, and protein synthesis (PS) was evaluated by measuring the incorporation of l-[4,5- 3 H ]leucine into proteins. The first procedure, exposure to thapsigargin (Tg), an irreversible inhibitor of ER Ca 2+-ATPase, produced a parallel increase in cytoplasmic calcium activity and a long-lasting suppression of PS, while the second, immersion in a calcium-free medium supplemented with the calcium chelator EGTA, caused a parallel decrease in cytoplasmic calcium levels and a short-lasting suppression of PS. Exposure of neurons to Tg induced a permanent increase in MyD116 mRNA levels. Exposure of cells to calcium-free medium supplemented with EGTA produced only a transient rise in MyD116 mRNA levels peaking after 6 h of recovery. The results demonstrate that depletion of ER calcium stores without any increase in cytoplasmic calcium activity is sufficient to activate MyD116 expression. A similar mechanism may be responsible for the increase in MyD116 mRNA levels observed after transient forebrain ischemia. It is concluded that those pathological disturbances triggering the activation of MyD116 expression after transient forebrain ischemia are only transient in the cerebral cortex but permanent in the hippocampus.

Inducibility of BDNF Gene Promoter I Detected by Calcium-Phosphate-Mediated DNA Transfection Is Confined to Neuronal but Not to Glial Cells

Although DNA transfection with calcium-phosphate/DNA precipitates for promoter analysis of genes has been previously applied to primary cultures of neuronal cells, it remains uncertain whether the expression of the introduced genes in glial cells, which are also in primary culture, affects the transcriptional signals obtained. Using plasmid DNA containing the brain-derived neurotrophic factor (BDNF) gene promoter I or c-fospromoter, we investigated the optimum conditions for calcium-phosphate-mediated DNA transfection in primary culture of rat cortical neuronal cells. Normalizing the firefly luciferase activity of the experimental reporter gene to theRenillaluciferase activity of the internal control increased experimental reliability. Maximum expression of firefly luciferase activity in the cells stimulated by membrane depolarization required 6–12 hrs of incubation after stimulation. Differences in the ratio of the experimental reporter gene to the internal control did not affect experimental gene expression. Under our optimal conditions, the activation of the BDNF gene promoter I was detected in neuronal but not in glial cells. Calcium-phosphate-mediated DNA transfection should be widely applicable for promoter analysis of inducible genes in neurons.