Gro H. Mathisen

Mono-2-ethylhexylphthalate (MEHP) induces TNF-α release and macrophage differentiation through different signalling pathways in RAW264.7 cells.

Epidemiological studies have associated indoor phthalate exposure with increased incidences and severity of asthma in children and adults, and inflammatory effects have been suggested as a possible mechanism. Recent studies report that phthalates may activate mitogen-activated protein (MAP) kinase p38 and various peroxisome proliferator-activated receptor (PPAR) isoforms. Here we confirm and extend these findings by investigating possible signalling pathways activated in the murine monocyte-macrophage cell line RAW264.7, using mono-2-ethylhexylphthalate (MEHP) as a model compound. MEHP exposure (0.3-1.0 mM) for 3h increased tumour necrosis factor (TNF)-α release and changed the cellular morphology into elongated spindle-like appearance, resembling more differentiated anti-inflammatory macrophages (M2). This was accompanied by increased expression of the macrophage differentiation marker CD163. Western analysis showed phosphorylation of p38 and Akt after 30 min exposure. Experiments using specific inhibitors suggested that MEHP-induced activation of both p38 and the phosphoinositide-3 (PI3) kinase/Akt pathway were involved in the release of TNF-α; whereas only PI3kinase seemed to be involved in differentiation. In contrast, inhibitors of PPARα and γ reduced differentiation, but did not affect TNF-α release. In conclusion, MEHP induced cytokine release and triggered differentiation of RAW264.7 cells, possibly into M2-like macrophages, but different signalling pathways appear to be involved in these responses.

Chicken cerebellar granule neurons rapidly develop excitotoxicity in culture

Rat cerebellar granule cell culture is widely used as a model to study factors that control neuronal differentiation and death (e.g. excitotoxicity). However, a main drawback of this model is its dependence on depolarizing culture condition (25 mM potassium). In addition, it is quite expensive to maintain and requires animal facilities. Here we report that cerebellar granule neuron cultures from chicken may be used as an alternative model to study excitotoxicity. Surprisingly, fetal chicken cells may be grown in a physiological potassium concentration (5 mM potassium). They develop excitotoxicity rapidly in culture (fully developed at 3 days in vitro), and respond to glutamate excitotoxicity similar to rat cultures (ROS production and activation of caspase-3).

Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken.

In mice, prenatal exposure to low doses of bisphenol A has been shown to affect neurogenesis and neuronal migration in cortex, resulting in disturbance of both neuronal positioning and the network formation between thalamus and cortex in the offspring brain. In the present study we investigated whether prenatal exposure to bisphenol A disturbs the neurodevelopment of the cerebellum. Two different model systems were used; offspring from two strains of mice from mothers receiving bisphenol A in the drinking water before mating, during gestation and lactation, and chicken embryos exposed to bisphenol A (in the egg) on embryonic day 16 for 24 h before preparation of cerebellar granule cell cultures. In the cerebellum, tight regulation of the level of transcription factor Pax6 is critical for correct development of granule neurons. During the development, the Pax6 level in granule neurons is high when these cells are located in the external granule layer and during their migration to the internal granule layer, and it is then reduced. We report that bisphenol A induced an increase in the thickness of the external granule layer and also an increase in the total cerebellar Pax6 level in 11 days old mice offspring. In cultured chicken cerebellar granule neurons from bisphenol A injected eggs the Pax6 level was increased day 6 in vitro. Together, these findings indicate that bisphenol A may affect the granule neurons in the developing cerebellum and thereby may disturb the correct development of the cerebellum.

Differences in NGFI-B, Nurr1, and NOR-1 expression and nucleocytoplasmic translocation in glutamate-treated neurons

NGFI-B (NR4A1, Nur77 or TR3) together with Nurr1 (NR4A2) and NOR-1 (NR4A3) constitute the NR4A subgroup of orphan nuclear receptors. They play critical roles in proliferation, differentiation, survival and apoptosis in different cell types, including neurons, immature T-cells, and different cancer cells. As ligand-independent and constitutively active receptors, the diverse biological activities of NGFI-B, Nurr1 and NOR-1 depend on their levels of expression, post-translational modifications and subcellular localization. Nuclear localization of the NR4A proteins leads to transcriptional activity, whereas NGFI-B and recently also NOR-1 have been shown to induce apoptosis by a more direct mechanism when localized at mitochondria. In the present study we investigated mRNA expression and subcellular translocation of the NR4A proteins during glutamate excitotoxicity in rat cerebellar granule neurons. NGFI-B and Nurr1 mRNA, but not NOR-1 mRNA, were induced by treatments associated with calcium influx, although their regulation seemed to be different. NR4A(gfp) fusion proteins showed a predominant nuclear localization in untreated cells. After glutamate treatment NGFI-B(gfp) translocated to cytosol and mitochondria within a few hours, whereas Nurr1(gfp) translocation was delayed, and NOR-1(gfp) mainly stayed in the nucleus. Subcellular targeting of NGFI-B seems to be tightly regulated, as a single mutation of threonine 142 altered NGFI-B(gfp) localization. Differences in expression and subcellular translocation of NGFI-B, Nurr1, and NOR-1 may reflect different functions in neurons in glutamate excitotoxicity.

Delayed translocation of NGFI-B/RXR in glutamate stimulated neurons allows late protection by 9-cis retinoic acid

Nuclear receptor and apoptosis inducer NGFI-B translocates out of the nucleus as a heterodimer with RXR in response to different apoptosis stimuli, and therefore represents a potential pharmacological target. We found that the cytosolic levels of NGFI-B and RXRα were increased in cultures of cerebellar granule neurons 2h after treatment with glutamate (excitatory neurotransmitter in the brain, involved in stroke). To find a time-window for potential intervention the neurons were transfected with gfp-tagged expressor plasmids for NGFI-B and RXR. The default localization of NGFI-Bgfp and RXRgfp was nuclear, however, translocation out of the nucleus was observed 2-3h after glutamate treatment. We therefore hypothesized that the time-window between treatment and translocation would allow late protection against neuronal death. The RXR ligand 9-cis retinoic acid was used to arrest NGFI-B and RXR in the nucleus. Addition of 9-cis retinoic acid 1h after treatment with glutamate reduced the cytosolic translocation of NGFI-B and RXRα, the cytosolic translocation of NGFI-Bgfp observed in live neurons, as well as the neuronal death. However, the reduced translocation and the reduced cell death were not observed when 9-cis retinoic acid was added after 3h. Thus, late protection from glutamate induced death by addition of 9-cis retinoic acid is possible in a time-window after apoptosis induction.

Effects of 4-methylimidazole on cerebral glutamate decarboxylase activity and specific GABA receptor binding in mice

4-Methylimidazole (4MeI) is a tremorogenic and convulsive agent of concern both in human and veterinary toxicology. The in vitro effects of 4MeI (5 μM–20 mM) on cerebral glutamate decarboxylase (GAD) activity and (in concentrations up to 50 mM) on binding of [3H]GABA to cerebral GABA receptors were tested in brain tissue from B6D2 mice. The effects of 1-methylimidazole (1MeI), 2-methylimidazole (2MeI), 4-methylhydroxy-imidazole (4MeOHI), imidazole-4-acetic acid (4AcI) (all in concentrations of 5–20 mM) and imidazole (20 mM) on GAD activity were also tested. In addition, the effect of a lethal dose of 4MeI (250 mg/kg ip) to B6D2 mice in vivo on the postmortem concentrations of γ-aminobutyric acid (GABA) and glutamate in their brains were measured. In all experiments, student’s t-test was used for statistical comparison. 4MeI in concentrations of 2 mM and above did inhibit GAD activity significantly in vitro, but glutamate and GABA concentrations in mouse brains after lethal 4MeI poisoning were not significantly different from control values. The effect of 2MeI on GAD activity was stronger than the effect of 4MeI. Binding of [3H]GABA to cerebral GABA receptors in vitro was significantly inhibited only at 4MeI concentrations of 5 mM and above. The results indicate that neither inhibition of GABA synthesis nor competitive inhibition of the binding of GABA to its receptors are likely mechanisms for the excitation and convulsions seen in 4MeI poisoning in animals.

Combretastatin A-4 and structurally related triazole analogues induce caspase-3 and reactive oxygen species-dependent cell death in PC12 cells.

Cancer cells are more sensitive to oxidative stress due to higher levels of reactive oxygen species. Therefore, the ability of anti-cancer agent combretastatin A-4 (CA-4) and triazole analogues to induce reactive oxygen species may be important for selectivity against cancer cells. The purpose of the present study was to investigate the structural requirements for reactive oxygen species production by CA-4 and the triazole analogues Ana-2, Ana-3 and Ana-4. Ana-2 and Ana-3 mimic the cis configuration in CA-4; Ana-3 lacks the phenolic hydroxyl group, while Ana-4 mimics a trans configuration. The rat pheochromocytoma cancer cell line PC12 was used as model system. CA-4 and Ana-2 were highly toxic; Ana-3 was less toxic, whereas Ana-4 was non-toxic. The probe dihydroethidium detected reactive oxygen species production from CA-4, Ana-2, and Ana-3. CA-4 and Ana-2 also induced oxidation of the reactive oxygen species probe dihydrorhodamine and activation of caspase-3. Thus, the phenolic hydroxyl group in CA-4 and Ana-2 was necessary for dihydrorhodamine oxidation, caspase-3 activation, and increased cytotoxicity.

17α-Estradiol down-regulates glutathione synthesis in serum deprived PC-12 cells

Abstract During the last decades it has been shown that estrogen may have neuroprotective functions in the CNS. However, we have previously reported that pretreatment with estradiol abolishes its protection of cultured cerebellar granule neurons from glutamate-induced cell death due to down-regulation of endogenous glutathione. 17α-Estradiol is considered a hormonally inactive isomer of 17β-estradiol still containing its antioxidant potential. Here, we demonstrate that 17α-estradiol enhanced serum deprivation-induced cell death in the rat pheochromocytoma cell line PC-12, while antioxidants vitamins C and E in combination (vitamins C/E) tended to protect. We further examined mechanisms behind the glutathione lowering effect of 17α-estradiol in serum deprived PC-12 cells. Endogenous glutathione levels were reduced in the serum deprived cells. Serum deprivation-induced cell death seemed to depend partly on this reduction as supplemented N-acetylcysteine, a cysteine precursor with potential to restore glutathione levels, reduced cell death. 17α-Estradiol down-regulated glutathione, promoter activity of the rate-limiting enzyme in glutathione production, glutamate cysteine ligase (GCL), as well as c-Fos protein levels in serum deprived cells. The c-Fos transcription factor normally binds to the AP-1 response element in the GCL promoter resulting in increased production of glutathione as a stress response. Over-expression of AP-1 proteins partly restored the GCL promoter activity in serum deprived cells treated with 17α-estradiol. Nrf2, a transcription factor binding another response element in the GCL promoter was unaffected by 17α-estradiol. Conclusively, 17α-estradiol may have a long-term negative effect on the endogenous glutathione level through its ability to down-regulate the glutathione synthesis during serum deprivation.

Transfection of chicken cerebellar granule neurons used to study glucocorticoid receptor regulation by nuclear receptor 4A (NR4A).

Transfection is a useful tool for studying molecular signalling pathways. However, neurons have proven hard to transfect. In the present paper we have optimized a new electroporation procedure using the Cellaxess(®) system for transient transfection of adherent primary neurons from chicken (Gallus gallus) and compared it to a liposome based procedure using Metafectene(®) Pro. In order to evaluate the two methods, glucocorticoid receptor (GR) function was chosen as a test. GRs are expressed in high amounts in the cerebellum. GR is regulated by another nuclear receptor (NGFI-B, the first member found in the NR4A family). We first showed that forskolin and phorbol ester activated an NR4A-dependent reporter gene indicating that members of the NR4A nuclear receptor family are present endogenously and upregulated by external stimuli. Then, transfected NGFI-B was shown to antagonize the dexamethasone-activated transcriptional activation by endogenous GR, leading to the conclusion that NR4A-family members are important modulators of GR mediated regulatory processes in the cerebellum, as in other cell types. Both transfection methods proved useful. While the electroporation technique yielded small rings with many transfected cells optimal for microscopy studies, the liposome based method resulted in transfected cells evenly distributed in the dish rendering this method well suited for biochemical studies.

Calcium-induced apoptosis of developing cerebellar granule neurons depends causally on NGFI-B

Immediate early gene nerve growth factor‐induced clone B (NGFI‐B), a nuclear receptor important for differentiation and apoptosis, is expressed in mice and rat cerebellum from an early stage of postnatal development. Following apoptotic stimuli NGFI‐B translocates to mitochondria to initiate cell death processes. Controlled cell death is critical for correct cerebellar development. Immunohistochemical analysis of NGFI‐B in sections of mice cerebella showed NGFI‐B to be expressed in granule neurons in vivo at a time (P8‐11) when apoptosis is known to occur. The importance of NGFI‐B for apoptosis of cultured rat cerebellar granule neurons was investigated by inducing apoptosis with calcium ionophore A23187 (CaI, 0.1 μM). Imaging studies of gfp‐tagged NGFI‐B confirmed that mitochondrial translocation of NGFI‐B occurred following treatment with CaI and was reduced by addition of 9‐cis‐retinoic acid (1 μM), a retinoid X receptor (RXR) agonist that prevents dimerization of RXR and NGFI‐B that is known to occur before translocation. Consequently, 9‐cis‐retinoic acid partly reduced cell death. To address the causality of NGFI‐B in apoptosis further, knock‐down by siRNA was performed and it removed 85% of the NGFI‐B protein. This resulted in a complete inhibition of apoptosis after CaI exposure. Together these findings suggest that NGFI‐B plays a role in controlling correct cerebellar development.