Rat Primary Neuronal Cultures Research Articles

Peroxiredoxin 2 overexpression protects cortical neuronal cultures from ischemic and oxidative injury but not glutamate excitotoxicity, whereas Cu/Zn superoxide dismutase 1 overexpression protects only against oxidative injury

We previously reported that peroxiredoxin 2 (PRDX2) and Cu/Zn superoxide dismutase 1 (SOD1) proteins are up-regulated in rat primary neuronal cultures following erythropoietin (EPO) preconditioning. In the present study, we have demonstrated that adenovirally mediated overexpression of PRDX2 in cortical neuronal cultures can protect neurons from in vitro ischemia (oxygen-glucose deprivation) and an oxidative insult (cumene hydroperoxide) but not glutamate excitotoxicity. We have also demonstrated that adenovirally mediated overexpression of SOD1 in cortical neuronal cultures protected neurons only against the oxidative insult. Interestingly, we did not detect up-regulation of PRDX2 or SOD1 protein in the rat hippocampus following exposure to either 3 min or 8 min of global cerebral ischemia. Further characterization of PRDX2's neuroprotective mechanisms may aid in the development of a neuroprotective therapy.

Neurotoxicity of HIV-1 Tat protein: Involvement of D1 dopamine receptor

Neurotoxic viral proteins released from HIV-infected cells are believed to play a major role in the pathogenesis of the dementia displayed in a significant number of AIDS patients. HIV-1 associated neuropathology severely affects dopaminergic regions of the brain. Growing evidence indicates that HIV-1 neurotoxic proteins, such as Tat may affect the function of the dopamine transmission system. In turn, molecular components of dopamine neurotransmission may participate in a complex network of Tat-induced cell responses which result in neurodegeneration. In this study we investigated whether D1 dopamine receptors are involved in the mechanism of Tat neurotoxicity in primary rat neuronal cell cultures. We found that in rat midbrain cell cultures, which express significant levels of D1 dopamine receptors, the specific D1 antagonist SCH 23390 attenuates the cell death caused by HIV-1 Tat. In rat hippocampal cell cultures, where the expression of D1 receptors is low, SCH 23390 did not change the toxicity of Tat. Thus, the protective effect of SCH 23390 in rat primary neuronal cell cultures is a function of the level of D1 receptor protein expression. Our results provide further evidence for the involvement of the dopaminergic transmission system in the mechanism of HIV-1 Tat neurotoxicity.

Neuroprotective Effects of Leptin Against Ischemic Injury Induced by Oxygen-Glucose Deprivation and Transient Cerebral Ischemia

Leptin is the major adipose hormone that regulates body weight and energy expenditure by activating leptin receptors in the hypothalamus. Leptin receptors are also present in other cell types, and a potent antiapoptotic effect for leptin has recently been reported. We investigated whether leptin was neuroprotective against ischemic brain injury. In vitro ischemic injury was induced in rat primary neuronal culture by oxygen-glucose deprivation for 90 minutes. In vivo ischemic brain injury was induced by middle cerebral artery occlusion in mice for 60 minutes. Leptin receptors were detected in cultured rat cortical neurons, as well as in the mouse cortex, striatum, and hippocampus. In vitro results showed that leptin, 50 to 100 mug/mL, protected primary cortical neurons against death induced by oxygen-glucose deprivation in a concentration-dependent manner. In vivo studies in the mouse brain demonstrated that the intraperitoneal administration of leptin, 2 to 8 mg/kg, dose-dependently reduced infarct volume induced by middle cerebral artery occlusion. Leptin was effective when injected 5 minutes before or 30 to 90 minutes after reperfusion, but not 2 hours after reperfusion. Leptin improved animal body weight recovery and behavioral parameters after cerebral ischemia. Leptin enhanced the phosphorylation of extracellular signal-related kinase 1/2. Both extracellular signal-related kinase 1/2 activation and neuroprotection were abolished by the administration of PD98059 in vitro and in vivo. Leptin is neuroprotective against ischemic neuronal injury. Our findings suggest that leptin is a legitimate candidate for the treatment of ischemic stroke.

Effects of estrogen against LPS-induced inflammation and toxicity in primary rat glial and neuronal cultures

Several lines of evidence link inflammation with neurodegenerative diseases, which are aggravated by the age-related decline in estrogen levels in postmenopausal women. Lipopolysaccharide (LPS) is used widely to stimulate glial cells to produce pro-inflammatory mediators such as NO, PGE(2), and TNF-alpha, and was found to be toxic in high doses. We examined the effects of a physiological dose of 17beta-estradiol (E2) against LPS-induced inflammation and toxicity (cell death) in rat primary glial and neuronal cultures. Cultures were treated with 0.1 nM E2 for 24 h and then exposed to LPS 0.5-200 microg/ml for another 24 h. Levels of NO, PGE(2), and TNF-alpha in the culture medium were determined by the Griess reaction assay, radio-immunoassay, and enzyme-linked immunoassay, respectively. Cell death was quantified by measuring the leakage of lactate dehydrogenase (LDH) into the medium from dead or dying cells using the non-radioactive cytotoxicity assay. E2 significantly reduced the LPS-induced increase in NO and TNF-alpha (but not PGE(2)) production in glial cells. PGE(2) and TNF-alpha were undetectable in neuronal cultures, while only basal levels of NO were detected, even after stimulation with LPS. Moreover, pretreatment with E2 significantly reduced LPS-induced cell death, as measured by the release of LDH, in both glial and neuronal cultures. These results suggest that the neuroprotective effects attributed to E2 are derived, at least in part, from its anti-inflammatory and cytoprotective effects in both glial and neuronal cells.

IL-8 Induces Expression of Matrix Metalloproteinases, Cell Cycle and Pro-Apoptotic Proteins, and Cell Death in Cultured Neurons

Neuronal cell loss is a critical feature of age-related neurodegenerative diseases such as Alzheimer's disease (AD). In the AD brain, a marked increase in pro-inflammatory cytokines and chemokines, including IL-8, has been documented. The objective of this study was to determine the effect of IL-8 on cell viability and expression of neurotoxic, apoptotic, and cell cycle proteins in cultured neurons. Incubation of cultured neurons with IL-8 for 24 h resulted in neuronal cell death. RT-PCR analysis of primary rat neuronal cultures treated with IL-8 for 24 h showed induction of genes for matrix metalloproteinases (MMP-2 and MMP-9), proinflammatory proteases with neurotoxic properties. Gelatin zymography demonstrated IL-8 induced MMP-2 and MMP-9 activity. Western blot analysis showed that IL-8 also increased levels of the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death). In addition, message levels of the cell cycle protein cyclin D1, an early marker for G1/S transition and a protein implicated as a regulator of neuronal apoptosis, were elevated after IL-8 exposure. These results suggest that IL-8 could be an important mediator of neuronal death in AD both via its effects on release of neurotoxins such as MMPs as well as by induction of cell cycle and pro-apoptotic proteins.

Effects of PACAP and VIP on cyclic AMP formation in rat neuronal and astrocyte cultures under normoxic and hypoxic condition

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) concentration (0.001–1000 nM)-dependently stimulated cyclic AMP production in rat primary neuronal and glial cell (astrocyte) cultures. The actions of both peptides were much more pronounced in astrocytes than in neuronal cultures. Stimulatory effects of PACAP and VIP on cyclic AMP formation were significantly smaller in cell cultures subjected to 24 h lasting hypoxic conditions, induced either chemically (100 μM cobalt chloride) or by low 3% oxygen hypoxia, compared to the normoxic condition (95% air and 5% CO 2). This picture contrasted with the effects of forskolin that were similar under normoxic and hypoxic conditions. It is suggested that hypoxia leads to changes in PACAP- and VIP-driven cyclic AMP-dependent signaling in the rat brain by influencing molecular processes likely occurring at the level of receptor protein or receptor-Gs protein coupling.

Chronic ethanol exposure causes mitochondrial dysfunction and oxidative stress in immature central nervous system neurons

Cerebellar hypoplasia in experimental fetal alcohol syndrome (FAS) is associated with impaired insulin-stimulated survival signaling. In vitro studies demonstrated that ethanol inhibition of neuronal survival is mediated by apoptosis and mitochondrial dysfunction. Since insulin and insulin-like growth factors (IGFs) regulate energy metabolism, and ethanol can exert its toxic effects by causing oxidative damage to DNA and proteins, we further characterized the effects of chronic gestational exposure to ethanol on mitochondrial gene expression, and the degree to which ethanol inhibition of mitochondrial function is mediated by impaired insulin/IGF responsiveness. Pregnant Long-Evans rats were fed isocaloric liquid diets containing 0, 2, 4.5, 6.5, or 9.25% v/v ethanol from gestation day 6 through delivery. Cerebella harvested on postnatal day 1 were examined for indices of oxidative stress, and mRNA levels of mitochondrial, pro-oxidant, and pro-apoptosis gene expression. Rat primary cerebellar neuron cultures were used to characterize the effects of ethanol (50 mM for 96 h) on insulin and IGF stimulated mitochondrial function and ATP production. Ethanol-exposed cerebella had significantly reduced mRNA levels of mitochondrial genes encoding Complexes II-A, IV, and V, increased expression of p53 and NADPH oxidase (NOX) 1 and 3, and increased immunoreactivity for 4-hydroxy-2,3-nonenal (HNE) and 8-OHdG in cerebellar granule cells. The activations of p53 and NOX genes were highest in cerebella from pups exposed to the 6.5 or 9.25% ethanol containing diet, whereas the impairments in mitochondrial Complex IV and V expression were similar at low and high levels of ethanol exposure. In vitro experiments confirmed that ethanol treatment reduces neuronal expression of mitochondrial genes encoding Complexes IV and V, impairs mitochondrial function and ATP production, and increases HNE and 8-OHdG immunoreactivity, but they also showed that these effects were not insulin- or IGF-dependent. Together, the results suggest that mitochondrial dysfunction, oxidative stress, and DNA damage in FAS may be largely due to the toxic effects of ethanol rather than specific impairments in insulin or IGF signaling.

S18986: A positive modulator of AMPA-receptors enhances ( S)-AMPA-mediated BDNF mRNA and protein expression in rat primary cortical neuronal cultures

The present study describes the effect of ( S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986), a positive allosteric modulator of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, on ( S)-AMPA-mediated increases in brain-derived neurotrophic factor (BDNF) mRNA and protein expression in rat primary cortical neuronal cultures. ( S)-AMPA (0.01–300 μM) induced a concentration-dependent increase in BDNF mRNA and protein expression (EC 50 = 7 μM) with maximal increases (50-fold) compared to untreated cultures observed between 5 and 12 h, whereas for cellular protein levels, maximal expression was detected at 24 h. S18986 alone (≤ 300 μM) failed to increase basal BDNF expression. However, S18986 (300 μM) in the presence of increasing concentrations of ( S)-AMPA maximally enhanced AMPA-induced expression of BDNF mRNA and protein levels (3–5-fold). S18986 (100–300 μM) potentiated BDNF mRNA induced by 3 μM ( S)-AMPA (2–3-fold). Under similar conditions, the AMPA allosteric modulator cyclothiazide induced a potent stimulation of ( S)-AMPA-mediated BDNF expression (40-fold; EC 50 = 18 μM), whereas IDRA-21 was inactive. Kinetic studies indicated that S18986 (300 μM) in the presence of 3 μM ( S)-AMPA was capable of enhancing BDNF mRNA levels for up to 25 h, compared to 3 μM ( S)-AMPA alone. On the other hand, S18986 only partially enhanced kainate-mediated expression of BDNF mRNA, but failed to significantly enhance N-methyl- d-aspartate-stimulated BDNF expression levels. In support of these observations, the competitive AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) but not the selective NMDA-receptor antagonist, (+)-MK-801 [(5R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cyclohepten-5,10-imine], abrogated S18986-induced effects on BDNF expression. S18986-mediated enhancement of ( S)-AMPA-evoked BDNF protein expression was markedly attenuated in Ca 2+-free culture conditions. Furthermore, from a series of kinase inhibitors only the Calmodulin–Kinase II/IV inhibitor (KN-62, 25 μM) significantly inhibited (− 85%, P < 0.001) AMPA + S18986 stimulated expression of BDNF mRNA. The present study supports the observations that AMPA receptor allosteric modulators can enhance the expression of BDNF mRNA and protein expression via the AMPA receptor in cultured primary neurones. Consequently, the long-term elevation of endogenous BDNF expression by pharmacological intervention with this class of compounds represents a potentially promising therapeutic approach for behavioural disorders implicating cognitive deficits.

Genistein and daidzein induce neurotoxicity at high concentrations in primary rat neuronal cultures

It is known that estrogen can protect neurons from excitotoxicity. Since isoflavones possess estrogen-like activity, it is of interest to determine whether isoflavones can also protect neurons from glutamate-induced neuronal injury. Morphological observation and lactate dehydrogenase (LDH) release assay were used to estimate the cellular damage. It is surprising that, contrary to estrogen, isoflavones, specifically genistein and daidzein, are toxic to primary neuronal culture at high concentration. Treatment of neurons with 50 microM genistein and daidzein for 24 h increased LDH release by 90% and 67%, respectively, indicating a significant cellular damage. Under the same conditions, estrogen such as 17beta-estradiol did not show any effect on primary culture of brain cells. At 100 microM, both genistein and daidzein increased LDH release by 2.6- and 3-fold, respectively with a 30-min incubation. Furthermore, both genistein and daidzein at 50 microM increased the intracellular calcium level, [Ca(2+)](i), significantly. To determine their mode of action, genistein and daidzein were tested on glutamate and GABA(A)receptor binding. Both genistein and daidzein were found to have little effect on glutamate receptor binding, while the binding of [(3)H]muscimol to GABA(A) receptors was markedly inhibited. However, 17beta-estradiol did not affect GABA(A) receptor binding suggesting that the toxic effect of genistein and daidzein could be due to their inhibition of the GABA(A) receptor resulting in further enhancement of excitation by glutamate and leading to cellular damage.

Glutamate stimulates glutamate receptor interacting protein 1 degradation by ubiquitin-proteasome system to regulate surface expression of GluR2

The glutamate receptor interacting protein 1 (GRIP1) is a scaffolding protein in postsynaptic density (PSD), tethering AMPA receptors to other signaling proteins. Here we report that glutamate stimulation caused a rapid reduction in protein levels of GRIP1, but not that of glutamate receptor (GluR) 1, GluR2 and protein interacting with C kinase 1 (PICK1) in rat primary cortical neuron cultures. Down-regulation of GRIP1 by glutamate was blocked by carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), a proteasome inhibitor and by expression of K48R-ubiquitin, a dominant negative form of ubiquitin. The GRIP1 reduction was inhibited by MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist, but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra acetic acid tetrakis (BAPTA), two Ca2+ chelators, but not nifedipine, an L-type Ca2+ channel blocker, prevented GRIP1 degradation. Furthermore, MG132 prevented glutamate-stimulated reduction in surface amount of GluR2, and knockdown of GRIP1 by RNAi against GRIP1 reduced surface GluR2 in neurons. Our results suggest that glutamate induces GRIP1 degradation by proteasome through an NMDA receptor–Ca2+ pathway and that GRIP1 degradation may play an important role in regulating GluR2 surface expression.

Neuroprotective actions of a histidine analogue in models of ischemic stroke

Histidine is a naturally occurring amino acid with antioxidant properties, which is present in low amounts in tissues throughout the body. We recently synthesized and characterized histidine analogues related to the natural dipeptide carnosine, which selectively scavenge the toxic lipid peroxidation product 4-hydroxynonenal (HNE). We now report that the histidine analogue histidyl hydrazide is effective in reducing brain damage and improving functional outcome in a mouse model of focal ischemic stroke when administered intravenously at a dose of 20 mg/kg, either 30 min before or 60 min and 3 h after the onset of middle cerebral artery occlusion. The histidine analogue also protected cultured rat primary neurons against death induced by HNE, chemical hypoxia, glucose deprivation, and combined oxygen and glucose deprivation. The histidine analogue prevented neuronal apoptosis as indicated by decreased production of cleaved caspase-3 protein. These findings suggest a therapeutic potential for HNE-scavenging histidine analogues in the treatment of stroke and related neurodegenerative conditions.

Serotonin modulation of cell excitability and of [ 3H]GABA and [ 3H] d-aspartate efflux in primary cultures of rat cortical neurons

The effects of 5-hydroxytryptamine (5-HT) on neuronal excitability, evaluated as depolarization-induced firing rate, and on amino acid release, measured as electrically-evoked [ 3H]GABA and [ 3H] d-aspartate efflux, were investigated in rat primary cortical neuronal cultures. 5-HT displayed a concentration-dependent, bimodal effect on neuronal excitability: at 3–10 μM it increased excitability through 5-HT 2A receptors, and was blocked by the selective 5-HT 2A antagonist MDL 100907, whereas at 30–100 μM it reduced excitability through 5-HT 1A receptors, and was, in turn, blocked by the selective 5-HT 1A antagonist WAY 100135. The electrically-evoked [ 3H]GABA efflux was concentration-dependently inhibited by 5-HT (pEC 50 = 4.74) and such inhibition was prevented by WAY 100135, but not by GR 55562, a selective 5-HT 1D/B receptor antagonist. Conversely, 5-HT concentration-dependently increased stimulus-evoked [ 3H] d-aspartate efflux (pEC 50 = 4.71). The increase was facilitated by methiothepin and was reversed into inhibition by ICS 205930, a selective 5-HT 3 receptor antagonist. In the presence of ICS 205930, the inhibition induced by 5-HT was prevented by the selective 5-HT 1D/B receptor antagonist GR 55562, but not by WAY 100135. These findings suggest that 5-HT inhibits GABA release through 5-HT 1A receptors and exerts a dual modulation on glutamate release, mostly facilitatory (through 5-HT 3 receptors) but also inhibitory (through 5-HT 1D/B receptors), leading to a prevalently positive modulation of the excitatory signal by amino acid neurotransmitter containing neurons.

Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin

Nerve cell injury by unconjugated bilirubin (UCB) has been implicated in brain damage during neonatal hyperbilirubinemia, particularly in the preterm newborn. Recently, it was shown that UCB is a substrate for the multidrug resistance-associated protein 1 (Mrp1), an ATP-dependent efflux pump, which may decrease UCB intracellular levels. To obtain a further insight into the role of Mrp1 in the increased vulnerability of immature cells to UCB, we evaluated the mRNA and the protein levels of Mrp1 throughout differentiation in primary cultures of rat neurons and astrocytes. Furthermore, in order to provide supportive evidence for the role of Mrp1 in the protection of nerve cells from UCB-induced effects, we evaluated cell susceptibility to UCB when Mrp1 was inhibited with MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl) ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid). The results are the first to demonstrate that Mrp1 is expressed in neurons and that both mRNA and protein levels of Mrp1 increase with cell differentiation. Additionally, inhibition of Mrp1 was associated with an increase in UCB toxic effects, namely cell death, cell dysfunction, and secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, as well as of glutamate. These results point to a novel role of Mrp1 in the susceptibility of premature babies to UCB encephalopathy, and provide a startup point for the development of a new therapeutic strategy.

No apoptosis is induced in rat cortical neurons exposed to GSM phone fields

The aim of this study was to investigate the radiofrequency (RF) electromagnetic fields (EMF) effects on neuronal apoptosis in vitro. Primary cultured neurons from cortices of embryonic Wistar rats were exposed to a 900-MHz global system for mobile communication (GSM) RF field for 24 h in a wire-patch cell. The average-specific absorption rate (SAR) used was 0.25 W/kg. Apoptosis rate was assessed immediately or 24 h after exposure using three methods: (i) DAPI staining; (ii) flow cytometry using double staining with TdT-mediated dUTP nick-end labeling (TUNEL) and propidium iodide (PI); and (iii) measurement of caspase-3 activity by fluorimetry. No statistically significant difference in the apoptosis rate was observed between controls and 24 h GSM-exposed neurons, either 0 h or 24 h post-exposure. All three methods used to assess apoptosis were concordant. These results showed that, under the conditions of experiment used, GSM-exposure does not significantly increase the apoptosis rate in rat primary neuronal cultures. This work is in accordance with other studies performed on cell lines and, to our knowledge, is the first one performed on cultured cortical neurons.

Expression of the mRNA and protein of serine racemase in primary cultures of rat neurons

Real-time quantitative PCR, Western blot and in situ hybridization techniques were employed to clarify the presence of serine racemase in the primary cultures of rat neurons. We have detected both serine racemase mRNA and protein in the cultured neurons. Both the mRNA and the protein levels in the neurons are higher than those in the astrocytes. Sequential detection of serine racemase mRNA and MAP2 immunoreactivity also revealed that serine racemase and MAP2 are co-localized in the cultured neurons. These data are the first to demonstrate that a substantial amount of serine racemase exists in the cultured neurons.

Calpain- and caspase-mediated αII-spectrin and tau proteolysis in rat cerebrocortical neuronal cultures after ecstasy or methamphetamine exposure

Abuse of 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) and methamphetamine (Meth or Speed) is a growing international problem with an estimated 250 million users of psychoactive drugs worldwide. It is important to demonstrate and understand the mechanism of neurotoxicity so potential prevention and treatment therapies can be designed. In this study rat primary cerebrocortical neuron cultures were challenged with MDMA and Meth (1 or 2 mM) for 24 and 48 h and compared to the excitotoxin N-methyl-D-aspartate (NMDA). The neurotoxicity of these drugs, as assessed by microscopy, lactate dehydrogenase release and immunoblot, was shown to be both dose- and time-dependent. Immunoblot analysis using biomarkers of cell death showed significant proteolysis of both alphaII-spectrin and tau proteins. Breakdown products of alphaII-spectrin (SBDPs) of 150, 145, and 120 kDa and tau breakdown products (TBDPs) of 45, 32, 26, and 14 kDa were observed. The use of the protease inhibitors calpain inhibitor SJA6017 and caspase inhibitors z-VAD-fmk and Z-D-DCB, attenuated drug-induced alphaII-spectrin and tau proteolysis. The calpain inhibitor reduced the calpain-induced breakdown products SBDP145 and TBDP14, but there was an offset increase in the caspase-mediated breakdown products SBDP120 and TBDP45. The caspase inhibitors, on the other hand, decreased SBDP120 and TBDP45. These data suggest that both MDMA and Meth trigger concerted proteolytic attacks of the structural proteins by both calpain and caspase family of proteases. The ability of the protease inhibitors to reduce the damage caused by these drugs suggests that the treatment arsenal could include similar drugs as possible tools to combat the drug-induced neurotoxicity in vivo.

Inhibition of nuclear factor κB is associated with neuroprotective effects of glycyrrhizic acid on glutamate-induced excitotoxicity in primary neurons

Glycyrrhizic acid is an herbal drug with a broad spectrum of antiviral activities and pharmacological effects and multiple sites of action. We investigated whether glycyrrhizic acid protects against glutamate-induced excitotoxicity and the underlying mechanisms. We found that glycyrrhizic acid protected against neurotoxicity in rat primary neuronal cultures and hippocampal slices by suppression of the glutamate-induced apoptosis. Glycyrrhizic acid conferred neuroprotective properties in a concentration-dependent manner, as determined by cell survival, apoptosis, and Ca 2+ influx. Glycyrrhizic acid selectively inhibited the Ca 2+ influx activated through N-methyl- d-aspartate (NMDA) receptor by glutamate, but not through membrane depolarization elicited by high K + induction. Glycyrrhizic acid treatment also diminished glutamate-induced DNA fragmentation and cleavage of poly (ADP-ribose) polymerase (PARP). Electrophoretic mobility shift assay (EMSA) indicated that glycyrrhizic acid inhibited the binding activity of nuclear factor κB (NF- κB) to its target elements. Western blot analysis of NF- κB inhibitor (I κBα) protein revealed that the inhibitory effect of glycyrrhizic acid on glutamate-induced activation of NF- κB activity was attributable to the inhibition of I κB kinase activity. Thus, the site of action of glycyrrhizic acid could be a downstream consequence of Ca 2+entry through NMDA receptors and that NF- κB may be one downstream target in this process. These observations suggest that glycyrrhizic acid may be of therapeutic value for the prevention of cerebral damage elicited by the glutamate.

P3-326: Expression of human truncated tau protein in long term culture of rat hippocampal neurons derived from Axon transgenic AD rat model

Truncated and hyperphosphorylated tau protein is one of the most important molecular and histopathological hallmarks found in Alzheimer‘s disease. Mutual occurrence of both events – truncation and hyperphosphorylation – is essential for disease progression and therefore transgenic models expressing non–mutated truncated tau could be successfully employed for detailed analysis of molecular pathways leading to neurodegeneration. To investigate the role of human truncated tau in neurofibrillary degeneration in long term rat primary hippocampal neuronal cultures. All experiments were performed using long–term hippocampal neuronal cultures prepared from transgenic rats expressing truncated human AlzTau. Neurons were cultured in vitro for 6–84 days (DIV). Distribution of AlzTau within the cell body and neurites was analyzed by staining with anti–human tau mAb – HT7. Tau protein phosphorylation at epitope 199/202,205 was detected with AT8 mAb. Pictures of neurons were taken at constant exposure times using confocal microscope (Olympus–Fluoview). Expression of transgenic human AlzTau in hippocampal neurons was observed from 6DIV. Intraneuronal AlzTau was not evenly distributed within the cell body and neurites. In the time period of 6th – 37th DIV AlzTau was present exclusively in bigger (wider) processes. However, on 52nd DIV it emerged also in peripheral neurites of neurons. Alzheimer‘s tau expression (gene dose dependent) pattern was enhanced in homozygous animals. Neurons derived from these animals displayed AlzTau in all neurites already on 25th DIV. Embryonic tau protein phosphorylation was detected up to 6th DIV in both, wild type and transgenic cultures. Strikingly AT–8 phosphorylation in transgenic neurons persisted up to 84 DIV. Detailed analysis of aged AT8 positive neurons (84DIV) showed the presence of granular and filamentous tau protein structures, localized within the cell bodies, suggesting early stage of NFT maturation. We have shown the expression pattern and intraneuronal distribution of human truncated tau protein in transgenic hippocampal neurons. Transgenic neuronal primary cultures (6 – 84 DIV) strongly suggest that truncated tau induced phosphorylation and formation of altered tau that leads to assembly into filamentous and granular structures.

Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures

In order to investigate protein function in rat primary cortical neuronal cultures, we modified an adenoviral vector expression system and assessed the strength and specificity of the cytomegalovirus (CMV), rous sarcoma virus (RSV), and rat and human synapsin 1 (SYN1) promoters to drive DsRed-X expression. We also incorporated the woodchuck post-transcriptional regulatory element (WPRE) and a CMV promoter-enhanced green fluorescent protein (EGFP) reporter cassette. We observed that the RSV promoter activity was strong in neurons and moderate in astrocytes, while the CMV promoter activity was weak-to-moderate in neurons and very strong in astrocytes. The rat and human SYN1 promoters exhibited similar but weak activity in neurons, despite inclusion of the WPRE. We confirmed that the WPRE enhanced RSV promoter-mediated DsRed-X expression in a time-dependent fashion. Interestingly, we observed very weak SYN1-mediated DsRed-X expression in astrocytes and HEK293 cells suggesting incomplete neuronal-restrictive behavior for this promoter. Finally, using our adenoviral expression system, we demonstrated that RSV promoter-mediated Bcl-X L overexpression attenuated neuronal death caused by in vitro ischemia and oxidative stress.

PTEN, Akt, and GSK3β signalling in rat primary cortical neuronal cultures following tumor necrosis factor‐α and trans‐4‐hydroxy‐2‐nonenal treatments

PTEN is a dual phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway important for cell survival. We determined effects of the inflammation and oxidative stresses of tumor necrosis factor-alpha (TNFalpha) and trans-4-hydroxy-2-nonenal (HNE), respectively, on PTEN, Akt, and GSK3beta signalling in rat primary cortical neurons. The inhibitors bisperoxovanadium [bpV(Pic)] and LY294002 were also used to determine PTEN and PI3K involvement in TNFalpha and HNE modulation of neuronal cell death. PTEN inhibition with bpV(Pic) alone did not affect Ser(473)Akt or Ser(9)GSK3beta phosphorylation. Instead, effects of this inhibitor were manifest when it was used together with TNFalpha and to a lesser extent with HNE. TNFalpha together with PTEN inhibition increased phosphorylation of Ser(473)Akt and Ser(9)GSK3beta. TNFalpha and HNE both gave decreased numbers of viable and increased numbers of early apoptotic neurons. PTEN inhibition partially reversed the toxic effect of TNFalpha as shown by an increased number of viable and a decreased number of early apoptotic neurons. All effects were reversed by PI3K inhibition. HNE together with inhibition of PTEN gave increased Ser(473)Akt but not Ser(9)GSK3beta phosphorylation and no effects on the number of viable or early apoptotic cells. In conclusion, PTEN inhibition gives a mild reversal of TNFalpha- but not HNE-induced cell death via the PI3K pathway.