Ulrike Harms

Neuroprotective effects of atorvastatin against glutamate-induced excitotoxicity in primary cortical neurones

Statins [3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase inhibitors] exert cholesterol‐independent pleiotropic effects that include anti‐thrombotic, anti‐inflammatory, and anti‐oxidative properties. Here, we examined direct protective effects of atorvastatin on neurones in different cell damage models in vitro. Primary cortical neurones were pre‐treated with atorvastatin and then exposed to (i) glutamate, (ii) oxygen–glucose deprivation or (iii) several apoptosis‐inducing compounds. Atorvastatin significantly protected from glutamate‐induced excitotoxicity as evidenced by propidium iodide staining, nuclear morphology, release of lactate dehydrogenase, and mitochondrial tetrazolium metabolism, but not from oxygen–glucose deprivation or apoptotic cell death. This anti‐excitototoxic effect was evident with 2–4 days pre‐treatment but not with daily administration or shorter‐term pre‐treatment. The protective properties occurred independently of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibition because co‐treatment with mevalonate or other isoprenoids did not reverse or attenuate neuroprotection. Atorvastatin attenuated the glutamate‐induced increase of intracellular calcium, which was associated with a modulation of NMDA receptor function. Taken together, atorvastatin exerts specific anti‐excitotoxic effects independent of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibition, which has potential therapeutic implications.

Neuronal gelsolin prevents apoptosis by enhancing actin depolymerization

Gelsolin (gsn), an actin-severing protein, protects neurons from excitotoxic cell death via inactivation of membranous Ca(2+) channels. Its role during apoptotic cell death, however, has remained unclear. Using several models of neuronal cell death, we demonstrate that endogenous gelsolin has anti-apoptotic properties that correlate to its dynamic actions on the cytoskeleton. We show that neurons lacking gelsolin (gsn(-/-)) have enhanced apoptosis following exposure to staurosporine, thapsigargin, or the cholinergic toxin ethylcholine aziridinium (AF64A). AF64A-induced loss of mitochondrial membrane potential and activation of caspase-3 was specifically enhanced in gsn(-/-) neurons and could be reversed by pharmacological inhibition of mitochondrial permeability transition. Moreover, increased caspase-3 activation and cell death in AF64A-treated gsn(-/-) neurons were completely reversed by pharmacological depolymerization of actin filaments and further enhanced by their stabilization. In conclusion, actin remodeling by endogenous gelsolin or analogues protects neurons from apoptosis mediated by mitochondria and caspase-3.

Phosphatidylinositol 3-Akt-kinase-dependent phosphorylation of p21(Waf1/Cip1) as a novel mechanism of neuroprotection by glucocorticoids.

The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21Waf1/Cip1. In primary cortical neurons, corticosterone leads to a dose- and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21Waf1/Cip1 at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21Waf1/Cip1 preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21Waf1/Cip1 and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3- and Akt-kinase. Both germline and somatically induced p21Waf1/Cip1 deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21Waf1/Cip1 suffices to protect neurons from apoptosis. We identify p21Waf1/Cip1 as a novel antiapoptotic factor for postmitotic neurons and implicate p21Waf1/Cip1 as the molecular target of neuroprotection by high-dose glucocorticoids.

Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation.

Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre‐treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin‐binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up‐regulated gelsolin messenger RNA and protein expression in a dose‐ and time‐dependent manner. Double‐label confocal immunocytochemistry visualized the up‐regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up‐regulation, TSA pre‐treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.

Role of NAD(P)H:quinone oxidoreductase in the progression of neuronal cell death in vitro and following cerebral ischaemia in vivo.

A direct involvement of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1) in neuroprotection has not yet been shown. The aim of this study was to examine changes, localization and role of NQO1 after different neuronal injury paradigms. In primary cultures of rat cortex the activity of NQO1 was measured after treatment with ethylcholine aziridinium (AF64A; 40 µm), inducing mainly apoptotic cell death, or oxygen‐glucose deprivation (OGD; 120 min), which combines features of apoptotic and necrotic cell death. After treatment with AF64A a significant NQO1 activation started after 24 h. Sixty minutes after OGD a significant early induction of the enzyme was observed, followed by a second increase 24 h later. Enzyme activity was preferentially localized in glial cells in control and injured cultures, however, expression also occurred in injured neuronal cells. Inhibition of the NQO1 activity by dicoumarol, cibacron blue or chrysin (1–100 nm) protected the cells both after exposure to AF64A or OGD as assessed by the decreased release of lactate dehydrogenase. Comparable results were obtained in vivo using a mouse model of focal cerebral ischaemia. Dicoumarol treatment (30 nmol intracerebroventricular) reduced the infarct volume by 29% (p = 0.005) 48 h after the insult. After chemical induction of NQO1 activity by t‐butylhydroquinone in vitro neuronal damage was exaggerated. Our data suggest that the activity of NQO1 is a deteriorating rather than a protective factor in neuronal cell death.

Vascular Signal Transducer and Activator of Transcription-3 Promotes Angiogenesis and Neuroplasticity Long-Term After Stroke

Center for Stroke Research Berlin, Charite-Universitatsmedizin Berlin, Germany; Dept of Neurology, Charite-Universitatsmedizin Berlin, Germany; Max-Delbruck Center for Molecular Medicine, Berlin, Germany; Institute of Clinical Neurobiology, University Hospital, University of Wurzburg, Germany; Cluster of Excellence NeuroCure, Charite-Universitatsmedizin Berlin, Germany; German Center for Neurodegenerative Diseases (DZNE), Partner Site, Berlin, Germany; German Center for Cardiovascular Diseases (DZHK), Partner Site, Berlin, GermanyBackground— Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. Methods and Results— We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3floxed/KO and Tie2-CreERT2 mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. Conclusions— Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.

Vascular Stat3 Promotes Angiogenesis and Neuroplasticity Long-Term After Stroke

Center for Stroke Research Berlin, Charite-Universitatsmedizin Berlin, Germany; Dept of Neurology, Charite-Universitatsmedizin Berlin, Germany; Max-Delbruck Center for Molecular Medicine, Berlin, Germany; Institute of Clinical Neurobiology, University Hospital, University of Wurzburg, Germany; Cluster of Excellence NeuroCure, Charite-Universitatsmedizin Berlin, Germany; German Center for Neurodegenerative Diseases (DZNE), Partner Site, Berlin, Germany; German Center for Cardiovascular Diseases (DZHK), Partner Site, Berlin, GermanyBackground— Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. Methods and Results— We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3floxed/KO and Tie2-CreERT2 mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. Conclusions— Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.

Certain types of iron oxide nanoparticles are not suited to passively target inflammatory cells that infiltrate the brain in response to stroke

Intravenous administration of iron oxide nanoparticles during the acute stage of experimental stroke can produce signal intensity changes in the ischemic region. This has been attributed, albeit controversially, to the infiltration of iron-laden blood-borne macrophages. The properties of nanoparticles that render them most suitable for phagocytosis is a matter of debate, as is the most relevant timepoint for administration. Both of these questions are examined in the present study. Imaging experiments were performed in mice with 30 minutes of middle cerebral artery occlusion (MCAO). Iron oxide nanoparticles with different charges and sizes were used, and mice received 300 μmol Fe/kg intravenously: either superparamagnetic iron oxide nanoparticles (SPIOs), ultrasmall SPIOs, or very small SPIOs. The particles were administered 7 days before MCAO, at the time of reperfusion, or 72 hours after MCAO. Interestingly, there was no observable signal change in the ischemic brains that could be attributed to iron. Furthermore, no Prussian blue-positive cells were found in the brains or blood leukocytes, despite intense staining in the livers and spleens. This implies that the nanoparticles selected for this study are not phagocytosed by blood-borne leukocytes and do not enter the ischemic mouse brain.

Combined temozolomide and sunitinib treatment leads to better tumour control but increased vascular resistance in O6-methylguanine methyltransferase-methylated gliomas

INTRODUCTION Combined antiangiogenic and cytotoxic treatment represents an appealing treatment approach for malignant glioma. In this study we characterised the antitumoural and microvascular consequences of sunitinib (Su) and temozolomide (TMZ) therapy and verified the ideal treatment protocol, with special focus on a potential therapeutic window for combined scheduling. MATERIALS AND METHODS O(6)-Methylguanine methyltransferase (MGMT) status was analysed by pyrosequencing. Tumour growth of subcutaneous xenografts was assessed under different treatment protocols (TMZ, SU, SU followed by TMZ, TMZ followed by SU, combined TMZ/SU). Intravital microscopy (dorsal skinfold chamber model) assessed microvascular consequences. Immunohistochemistry included tumour and endothelial cell proliferation, apoptosis and vascular pericyte coverage. Real-time polymerase chain reaction (RT-PCR) analysed the expression of angiogenesis-related pathways in response to therapy. RESULTS Combined TMZ/SU resulted in significantly reduced tumour growth compared to either monotreatment (TMZ: 106 ± 13 mm(3); SU: 114 ± 53 mm(3); TMZ/SU: 34 ± 7 mm(3)) by additional antiangiogenic effects and synergistic induction of apoptosis versus TMZ monotreatment. Sequential treatment protocols did not show additive antitumour responses. TMZ/SU aggravated vascular resistance mechanisms characterised by significantly higher blood flow rate (TMZ: 74 ± 34 μl/s; SU: 164 ± 36 μl/s; TMZ/SU: 254 ± 95 μl/s), reduced permeability (TMZ: 1.05 ± 0.02; SU: 0.99 ± 0.07; TMZ/SU: 0.89 ± 0.05) and recovery of pericyte-endothelial interactions (TMZ: 89 ± 7%; SU: 67 ± 9%, TMZ/SU:80 ± 10%) versus either monotreatment. Vascular resistance was paralleled by an increase in Ang-1 and Tie-2 and by the downregulation of Dll4. CONCLUSION Sequential application of TMZ and SU in the angiogenic window does not add antitumour efficacy to monotherapy. Simultaneous application yields beneficial tumour control due to additive antiangiogenic and proapoptotic effects. Combined treatment may aggravate pericyte-mediated vascular resistance mechanisms by altering Ang-1-Tie-2 and Dll4/Notch pathways.

Interaction of ARC and Daxx: A Novel Endogenous Target to Preserve Motor Function and Cell Loss after Focal Brain Ischemia in Mice

The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 μg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX–ASK1–JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1–JNK activation. Our work identifies for the first time ARC–DAXX binding to block ASK1–JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. SIGNIFICANCE STATEMENT Up to now, the only successful pharmacological target of human ischemic stroke is thrombolysis. Neuroprotective pharmacological strategies are needed to accompany therapies aiming to achieve reperfusion. We describe that apoptosis repressor with CARD (ARC) interacts and inhibits DAXX and proximal signals of cell death. In a murine stroke model mimicking human malignant infarction in the territory of the middle cerebral artery, TAT.ARC salvages brain tissue when given during occlusion or 3 h delayed with sustained functional benefits (28 d). This is a promising novel therapeutic approach because it appears to be effective in a model producing severe injury by interfering with an array of proximal signals and effectors of the ischemic cascade, upstream of JNK, caspases, and BIM and BAX activation.