Barbara Ahlemeyer

In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.

A comparative in vitro cytotoxicity study with different water-soluble, cationic macromolecules which have been described as gene delivery systems was performed. Cytotoxicity in L929 mouse fibroblasts was monitored using the MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observations were carried out as indicators for cell viability. Furthermore, hemolysis of erythrocytes was quantified spectrophotometrically. To determine the nature of cell death induced by the polycations, the nuclear morphology after DAPI staining and the inhibition of the toxic effects by the caspase inhibitor zVAD.fmk were investigated. All assays yielded comparable results and allowed the following ranking of the polymers with regard to cytotoxicity: Poly(ethylenimine)=poly(L-lysine)>poly(diallyl-dimethyl-ammonium chloride)>diethylaminoethyl-dextran>poly(vinyl pyridinium bromide)>Starburst dendrimer>cationized albumin>native albumin. The magnitude of the cytotoxic effects of all polymers were found to be time- and concentration dependent. The molecular weight as well as the cationic charge density of the polycations were confirmed as key parameters for the interaction with the cell membranes and consequently, the cell damage. Evaluating the nature of cell death induced by poly(ethylenimine), we did not detect any indication for apoptosis suggesting that the polymer induced a necrotic cell reaction. Cell nuclei retained their size, chromatin was homogenously distributed and cell membranes lost their integrity very rapidly at an early stage. Furthermore, the broad spectrum caspase inhibitor zVAD.fmk did not inhibit poly(ethylenimine)-induced cell damage. Insights into the structure-toxicity relationship are necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems.

Transforming Growth Factor-β1 Increases Bad Phosphorylation and Protects Neurons Against Damage

Despite the characterization of neuroprotection by transforming growth factor-β1 (TGF-β1), the signaling pathway mediating its protective effect is unclear. Bad is a proapoptotic member of the Bcl-2 family and is inactivated on phosphorylation via mitogen-activated protein kinase (MAPK). This study attempted to address whether MAPK signaling and Bad phosphorylation were influenced by TGF-β1 and, furthermore, whether these two events were involved in the antiapoptotic effect of TGF-β1. We found a gradual activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and MAPK-activated protein kinase-1 (also called Rsk1) and a concomitant increase in Bad phosphorylation at Ser112 in mouse brains after adenovirus-mediated TGF-β1 transduction under nonischemic and ischemic conditions induced by transient middle cerebral artery occlusion. Consistent with these effects, the ischemia-induced increase in Bad protein level and caspase-3 activation were suppressed in TGF-β1-transduced brain. Consequently, DNA fragmentation, ischemic lesions, and neurological deficiency were significantly reduced. In cultured rat hippocampal cells, TGF-β1 inhibited the increase in Bad expression caused by staurosporine. TGF-β1 concentration- and time-dependently activated Erk1/2 and Rsk1 accompanied by an increase in Bad phosphorylation. These effects were blocked by U0126, a mitogen-activated protein kinase/Erk kinase 1/2 inhibitor, suggesting an association between Bad phosphorylation and MAPK activation. Notably, U0126 and a Rsk1 inhibitor (Ro318220) abolished the neuroprotective activity of TGF-β1 in staurosporine-induced apoptosis, indicating that activation of MAPK is necessary for the antiapoptotic effect of TGF-β1 in cultured hippocampal cells. Together, we demonstrate that TGF-β1 suppresses Bad expression under lesion conditions, increases Bad phosphorylation, and activates the MAPK/Erk pathway, which may contribute to its neuroprotective activity.

Neuroprotection by Estrogens in a Mouse Model of Focal Cerebral Ischemia and in Cultured Neurons: Evidence for a Receptor-Independent Antioxidative Mechanism

Estrogens have been suggested for the treatment of neurodegenerative disorders, including stroke, because of their neuroprotective activities against various neurotoxic stimuli such as glutamate, glucose deprivation, iron, or β-amyloid. Here, the authors report that 17β-estradiol (0.3 to 30 mg/kg) and 2-OH-estradiol (0.003 to 30 mg/kg) reduced brain tissue damage after permanent occlusion of the middle cerebral artery in male NMRI mice. In vitro, 17β-estradiol (1 to 10 μmol/L) and 2-OH-estradiol (0.01 to 1 μmol/L) reduced the percentage of damaged chick embryonic neurons treated with FeSO4. In these primary neurons exposed to FeSO4, the authors also found reactive oxygen species to be diminished after treatment with 17β-estradiol (1 to 10 μmol/L) or 2-OH-estradiol (0.01 to 10 μmol/L), suggesting a strong antioxidant activity of the estrogens that were used. Neither the neuroprotective effect nor the free radical scavenging properties of the estrogens were influenced by the estrogen receptor antagonist tamoxifen. The authors conclude that estrogens protect neurons against damage by radical scavenging rather than through estrogen receptor activation.

S-100β protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT1A-receptor agonist, Bay x 3702

The serotonin (5-HT)(1A) receptor agonists have already been shown to protect cultured neurons from excitotoxic as well as from apoptotic damage [B. Ahlemeyer, J. Krieglstein, Stimulation of 5-HT(1A) receptors inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo, Brain Res. 777 (1997) 179-186. ; B. Ahlemeyer, A. Glaser, C. Schaper, I. Semkova, J. Krieglstein, The 5-HT(1A) receptor agonist, Bay x 3702, inhibited apoptosis induced by serum deprivation in cultured neurons, Eur. J. Pharmacol. 370 (1999) 211-216.; J.H.M. Prehn, M. Welsch, C. Backhauss, J. Nuglisch, F. Ausmeier, C. Karkoutly, J. Krieglstein, Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia, Brain Res. 630 (1993) 110-120.; I. Semkova, P. Wolz, J. Krieglstein, Neuroprotective effect of 5-HT(1A) receptor agonist, Bay x 3702, demonstrated in vitro and in vivo, Eur. J. Pharmacol. 359 (1998) 251-260.; B. Suchanek, H. Struppeck, T. Fahrig, The 5-HT(1A) receptor agonist, Bay x 3702, prevents staurosporine-induced apoptosis, Eur. J. Pharmacol. 355 (1998) 95-101.] and to increase the release of the neurotrophic protein, S-100beta [P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, Stimulation of astroglial 5-HT(1A) receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology, Brain Res. 497 (1989) 80-86. ; P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, S-100 protein is released from astroglial cells by stimulation of 5-HT(1A) receptors, Brain Res. 528 (1990) 155-158.]. In this study, we tried to find out whether S-100beta can protect cultured neurons from glutamate- and staurosporine-induced damage and whether the neuroprotective activity of the highly selective 5-HT(1A) receptor agonist, Bay x 3702, is mediated by an induction of S-100beta. Extracellularly added S-100beta (1-10 ng/ml) reduced staurosporine-induced damage in pure neuronal cultures from chick embryo telencephalon as well as in mixed neuronal/glial cultures from neonatal rat hippocampus. In addition, S-100beta (1 ng/ml) reduced neuronal death induced by exposure to glutamate (0.25 mM, 30 min) in mixed neuronal/glial cultures from neonatal rat hippocampus. In cultured rat cortical astrocytes, a 24 h-treatment with Bay x 3702 (1 nM) increased the S-100beta content in the culture medium from 2.2+/-0.3 (controls) to 6.2+/-0.7 ng/ml. In the adult rat, a 4 h-infusion of 4 microg/kg Bay x 3702 (i.v.) was found to increase the S-100beta content in the striatum 6 h after the beginning of the infusion to 153+/-37 microg/g compared with 60+/-20 microg/g in vehicle-treated rats. Bay x 3702 had no effect on the S-100beta content in the rat hippocampus. Finally, we tried to block the protective effect of Bay x 3702 against staurosporine-induced damage in mixed neuronal/glial cultures from rat neonatal hippocampus by anti-S-100beta antibodies. We found only a partial blockade, although the antibodies fully blocked the antiapoptotic effect of S-100beta itself demonstrating that the antibody was effective in blocking neuroprotection by S-100beta. Thus, we conclude that S-100beta was able to protect cultured neurons against glutamate- and staurosporine-induced damage. Furthermore, S-100beta mediated partially the protective effect of the 5-HT(1A) receptor agonist, Bay x 3702, against staurosporine-induced apoptosis in mixed neuronal/glial cultures from neonatal rat hippocampus.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-κB

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor‐κB (NF‐κB) and how this activation contributes to preconditioning‐induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine‐induced (200 nm, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF‐κB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IκBα as well as by electrophoretic mobility shift assay. Preconditioning‐mediated neuroprotection was abolished by antioxidants, inhibitors of NF‐κB activation and cycloheximide suggesting the involvement of ROS, an activation of NF‐κB and de novo protein synthesis in preconditioning‐mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn‐superoxide dismutase which could be blocked by antioxidants, cycloheximide and κB decoy DNA. Our data suggest that inhibition of staurosporine‐induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF‐κB and an increase in the protein level of Mn‐superoxide dismutase.

Retinoic acid reduces apoptosis and oxidative stress by preservation of SOD protein level.

Retinoic acid (RA) has already been shown to exert antiapoptotic and antioxidative activity in various cells. In this study, we determined the effect of RA on the mRNA and protein levels of the Cu-,Zn-superoxide dismutase (SOD-1) and Mn-superoxide dismutase (SOD-2) during staurosporine-induced apoptosis in primary cultures from neonatal rat hippocampus. Exposure to staurosporine (300 nM, 24 h) increased the percentage of apoptotic neurons to 62% compared with 18% in controls. We determined an increase in the reactive oxygen species (ROS) content from 4 up to 48 h after the induction of the injury. Treatment with staurosporine did not significantly change the mRNA levels of SOD-1 and SOD-2. However, the SOD-1 and SOD-2 protein levels markedly decreased 24 and 48 h after the addition of staurosporine. Compared with staurosporine-exposed controls, RA (10 nM)-treated cultures showed a significant increase in neuronal survival, a reduced neuronal ROS content, and enhanced protein levels of SOD-1 and SOD-2 24 and 48 h after the start of the exposure to staurosporine. The results suggest that RA reduced staurosporine-induced oxidative stress and apoptosis by preventing the decrease in the protein levels of SOD-1 and SOD-2, and thus supported the antioxidant defense system.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species

The current study was performed to determine the role of reactive oxygen species (ROS) in preconditioning against different forms of neuronal damage. Primary cultures of chick embryonic neurons were treated with either FeSO(4) (100 microM; 15 min) to generate hydroxyl radicals or xanthine/xanthinoxidase (10 microM/0.5 mU ml(-1); 15 min; =X/XO (pre)) to produce superoxide radicals. Both stimuli moderately enhanced ROS formation as measured by fluorescence microscopy. This preconditioning significantly protected the neurons against subsequent glutamate (1 mM)-induced excitotoxic damage, staurosporine (200 nM)-induced neuronal apoptosis and oxidative damage caused by exposure to xanthine/xanthinoxidase (500 microM/5 mU ml(-1); 1 h; =X/XO (dam)). The antioxidants vitamin E (10 microM) and 2-OH-estradiol (1 microM), present during the 15-min preconditioning period, completely abolished the protective effect of X/XO (pre). Furthermore, glutamate, staurosporine or X/XO (dam) markedly enhanced oxygen radical formation. Preceding preconditioning by mild ROS stimulation with X/XO (pre) or Fe(2+) reduced this oxygen radical burst. Again, the effect of X/XO (pre) could be blocked by coadministration of vitamin E or 2-OH-estradiol. However, the FeSO(4)-mediated preconditioning was not abolished by the radical scavengers. To address this phenomenon, the effect of vitamin E and 2-OH-estradiol on Fe(2+)- and X/XO (pre)-induced ROS formation kinetics within the 15 min of preconditioning was monitored. The moderate rise of intracellular ROS content during preconditioning was only reduced permanently by the antioxidants, when the neurons were treated with X/XO (pre), but not when Fe(2+) was used. Thus, an immediate and constant radical scavenging seems to be indispensable to abolish the ROS-induced neuronal preconditioning. The current results indicate that preconditioning by moderate ROS-stimulation protects cultured neurons against different damaging agents and prevents against the subsequent massive oxygen radical formation.

Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents.

Previous studies have already demonstrated that some constituents of an extract of Ginkgo biloba (EGb), such as ginkgolide B and bilobalide, protect cultured neurons from hypoxia- and glutamate-induced damage. This prompted us to investigate whether they were also able to inhibit neuronal apoptosis. We induced apoptosis in cultured chick embryonic neurons as well as in mixed cultures of neurons and astrocytes from neonatal rat hippocampus by serum deprivation and staurosporine. The increase in the percentage of apoptotic chick neurons from 12% in controls to 30% after 24 h of serum deprivation was reduced to control level by EGb (10 mg/l), ginkgolide B (10 microM), ginkgolide J (100 microM) and bilobalide (1 microM). After treatment with staurosporine (200 nM) for 24 h we observed 74% apoptotic chick neurons. This percentage of apoptotic neurons was reduced to 24%, 62% and 31% in the presence of EGb (100 mg/l), ginkgolide J (100 microM) and ginkgolide B (10 microM), respectively. Bilobalide (10 microM) decreased apoptotic damage induced by staurosporine treatment for 12 h nearly to the control level. In mixed neuronal/glial cultures, the extract of EGb (100 mg/l) and bilobalide (100 microM) rescued rat neurons from apoptosis caused by serum deprivation, whereas, bilobalide (100 microM) and ginkgolide B (100 microM) reduced staurosporine-induced apoptotic damage. Ginkgolide A revealed no anti-apoptotic effect in either serum-deprived or staurosporine-treated neurons. Our results suggest that EGb and some of its constituents possess anti-apoptotic capacity and that bilobalide is the most potent constituent.

NMDA receptor activation and respiratory chain complex V inhibition contribute to neurodegeneration in d‐2‐hydroxyglutaric aciduria

The inherited neurometabolic disease d‐2‐hydroxyglutaric aciduria is complicated by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, frequently presenting with hypotonia, epilepsy and psychomotor retardation. Here, we report that the pathogenetic role of the endogenously accumulating metabolite d‐2‐hydroxyglutarate (D‐2), which is structurally similar to the excitatory amino acid glutamate, is mediated by at least three mechanisms. (i) D‐2‐induced excitotoxic cell damage in primary neuronal cultures from chick and rat involved N‐methyl‐d‐aspartate (NMDA) receptor activation. Indeed, D‐2 activated recombinant NMDA receptors (NR1/NR2A, NR1/NR2B) but not recombinant alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole (AMPA) receptors in HEK293 cells. (ii) Fluorescence microscopy using fura‐2 as a calcium indicator and the oxidant‐sensitive dye dihydrorhodamine‐123 revealed that D‐2 disturbed intracellular calcium homeostasis and elicited the generation of reactive oxygen species. (iii) D‐2 reduced complex V (ATP synthase) activity of the mitochondrial respiratory chain, reflecting an impaired energy metabolism due to inhibition of ATP synthesis but without affecting the electron‐transferring complexes I–IV. Thus, D‐2 stimulates neurodegeneration by mechanisms well‐known for glutamate, NMDA or mitochondrial toxins. In conclusion, excitotoxicity contributes to the neuropathology of d‐2‐hydroxyglutaric aciduria, highlighting new neuroprotective strategies.

Contribution of Reactive Oxygen Species to 3-Hydroxyglutarate Neurotoxicity in Primary Neuronal Cultures from Chick Embryo Telencephalons

Glutaryl-CoA dehydrogenase deficiency is an autosomal recessively inherited neurometabolic disorder with a distinct neuropathology characterized by acute encephalopathic crises during a vulnerable period of brain development. 3-Hydroxyglutarate (3-OH-GA), which accumulates in affected patients, has been identified as an endogenous neurotoxin mediating excitotoxicity via N-methyl-d-aspartate receptors. As increased generation of reactive oxygen species (ROS) and nitric oxide (NO) plays an important role in excitotoxic neuronal damage, we investigated whether ROS and NO contribute to 3-OH-GA neurotoxicity. 3-OH-GA increased mitochondrial ROS generation in primary neuronal cultures from chick embryo telencephalons, which could be prevented by MK-801, confirming the central role of N-methyl-d-aspartate receptor stimulation in 3-OH-GA toxicity. ROS increase was reduced by α-tocopherol and—less effectively—by melatonin. α-Tocopherol revealed a wider time frame for neuroprotection than melatonin. Creatine also reduced neuronal damage and ROS formation but only if it was administered ≥6 h before 3-OH-GA. NO production revealed only a slight increase after 3-OH-GA incubation. NO synthase inhibitor Nω-nitro-l-arginine prevented NO increase but did not protect neurons against 3-OH-GA. The NO donor S-nitroso-N-acetylpenicillamine revealed no effect on 3-OH-GA toxicity at low concentrations (0.5–5 μM), whereas it potentiated neuronal damage at high concentrations (50–500 μM), suggesting that weak endogenous NO production elicited by 3-OH-GA did not affect neuronal viability. We conclude from our results that ROS generation contributes to 3-OH-GA neurotoxicity in vitro and that radical scavenging and stabilization of brain energy metabolism by creatine are hopeful new strategies in glutaryl-CoA dehydrogenase deficiency.