Ülkan Kilic

Delayed post-ischaemic neuroprotection following systemic neural stem cell transplantation involves multiple mechanisms

Recent evidence suggests that neural stem/precursor cells (NPCs) promote recovery in animal models with delayed neuronal death via a number of indirect bystander effects. A comprehensive knowledge of how transplanted NPCs exert their therapeutic effects is still lacking. Here, we investigated the effects of a delayed transplantation of adult syngenic NPCs--injected intravenously 72 h after transient middle cerebral artery occlusion--on neurological recovery, histopathology and gene expression. NPC-transplanted mice showed a significantly improved recovery from 18 days post-transplantation (dpt) onwards, which persisted throughout the study. A small percentage of injected NPCs accumulated in the brain, integrating mainly in the infarct boundary zone, where most of the NPCs remained undifferentiated up to 30 dpt. Histopathological analysis revealed a hitherto unreported very delayed neuroprotective effect of NPCs, becoming evident at 10 and 30 dpt. Tissue survival was associated with downregulation of markers of inflammation, glial scar formation and neuronal apoptotic death at both mRNA and protein levels. Our data highlight the relevance of very delayed degenerative processes in the stroke brain that are intimately associated with inflammatory and glial responses. These processes may efficaciously be antagonized by (stem) cell-based strategies at time-points far beyond established therapeutic windows for pharmacological neuroprotection.

Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective functions in brain hypoxia and ischemia. To examine the mechanisms mediating Epos neuroprotective activity in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo in brain without inducing excessive erythrocytosis. We show that human Epo is expressed in tg21 brains and that cortical and striatal neurons carry the Epo receptor. After middle cerebral artery occlusion, human Epo potently protected brains of tg21 mice against ischemic injury, both when severe (90 min) and mild (30 min) ischemia was imposed. Histochemical studies revealed that Epo induced an activation of JAK‐2, ERK‐1/‐2, and Akt pathways in the ischemic brain. This activation was associated with elevated Bcl‐XL and decreased NO synthase‐1 and ‐2 levels in neurons. Intracerebroventricular injections of selective inhibitors of ERK‐1/‐2 (PD98059) or Akt (wortmannin) pathways revealed that both ERK‐1/‐2 and Akt were required for Epos neuroprotective function, antagonization of either pathway completely abolishing tissue protection. On the other hand, ERK‐1/‐2 and Akt blockade did not reverse the neuronal NO synthase‐1/‐2 inhibition, indicating that Epo down‐regulates these NO synthases in an ERK‐1/‐2 and Akt independent manner. On the basis of our data, the dual activation of ERK‐1/‐2 and Akt is crucial for Epos neuroprotective activity.

The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF’s neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia

Based on its trophic influence on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for stroke treatment. VEGFs survival‐promoting effects are purchased at the expense of an increased blood brain barrier permeability, which potentially compromises tissue survival. The mechanisms via which VEGF protects the brain against ischemia remained unknown. We examined signaling pathways underlying VEGFs neuroprotective activity in our transgenic mouse line, which expresses human VEGF165 under a neuron‐specific enolase (NSE) promoter. We show that VEGF receptor‐2 (Flk‐1) is expressed on ischemic neurons and astrocytes and is activated by VEGF. Following 90‐min episodes of middle cerebral artery occlusion, VEGF increased phosphorylated (but not total) Akt and ERK‐1/‐2 and reduced phosphorylated mitogen activated protein kinase/p38 and c‐Jun NH2‐terminal kinase (JNK)‐1/‐2 levels, at the same time decreasing inducible NO synthase expression in ischemic neurons. Inhibition of Akt with Wortmannin reversed VEGFs neuroprotective properties, diminished brain swelling, and restored the vascular permeability induced by VEGF to below levels in WT animals. The aggravation of brain injury by Wortmannin was associated with the restitution of p38, but not of JNK‐1/‐2, ERK‐1/‐2, or inducible NOS (iNOS). Our data demonstrate that VEGF mediates both neuroprotection and blood brain barrier permeability via the phosphatidylinositol‐3 kinase (PI3K)/Akt pathway. Based on our observation that VEGF neuroprotection and vascular leakage depend on PI3K/Akt, which is putatively regulated by VEGF receptor‐2, we predict that it may not easily be possible to make use of VEGFs neuroprotective function without accepting its unfavorable consequence, the increased vascular permeability.—Kilic, E., Kilic, Ü., Wang, Y., Bassetti, C. L., Marti, H. H., Hermann, D. M. The phosphatidylinositol‐3 kinase/Akt pathway mediates VEGFs neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia. FASEB J. 20, E307–E314 (2006)

Intravenous TAT-GDNF Is Protective After Focal Cerebral Ischemia in Mice

Background and Purpose— Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by their size and biochemical properties. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to cross cell membranes and the blood-brain barrier, even when coupled with larger peptides. The present studies were done to evaluate whether TAT–glial line-derived neurotrophic factor (GDNF) fusion protein is protective in focal cerebral ischemia. Methods— Anesthetized male C57BL/6j mice were submitted to intraluminal thread occlusion of the middle cerebral artery. Reperfusion was initiated 30 minutes later by thread retraction. Laser Doppler flow was monitored during the experiments. TAT-GDNF, TAT-GFP (0.6 nmol each), or vehicle was intravenously applied over 10 minutes immediately after reperfusion. After 3 days (30 minutes of ischemia), animals were reanesthetized and decapitated. Brain injury was evaluated by histochemical stainings. Results— Immunocytochemical experiments confirmed the presence of TAT-GDNF protein in the brains of fusion protein–treated nonischemic control animals 3 to 4 hours after TAT fusion protein delivery. TAT-GDNF significantly reduced the number of caspase-3–immunoreactive and DNA-fragmented cells and increased the number of viable neurons in the striatum, where disseminated tissue injury was observed, compared with TAT-GFP– or vehicle-treated animals. Conclusions— Our results demonstrate that TAT fusion proteins are powerful tools for the treatment of focal ischemia when delivered both before and after an ischemic insult. This approach may be of clinical interest because such fusion proteins can be intravenously applied and reach the ischemic brain regions. This approach may therefore offer new perspectives for future strategies in stroke therapy.

Human Vascular Endothelial Growth Factor Protects Axotomized Retinal Ganglion Cells In Vivo by Activating ERK-1/2 and Akt Pathways

Based on its trophic effects on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for the treatment of neurodegenerative diseases. To evaluate the therapeutic potential of VEGF, we here examined effects of this growth factor on the degeneration of axotomized retinal ganglion cells (RGCs), which, as CNS-derived neurons, offer themselves in an excellent way to study neuroprotection in vivo. Making use of a transgenic mouse line that constitutively expresses human VEGF under a neuron-specific enolase promoter, we show that (1) the VEGF-transgenic retina overexpresses human VEGF, (2) RGCs carry the VEGF receptor-2, and (3) vascular networks in normal and axotomized VEGF-transgenic (tg) retinas do not differ from control animals. After axotomy, RGCs of VEGF-tg mice were protected against delayed degeneration, as compared with wild-type littermates. Western blots revealed increased phosphorylated ERK-1/2 and Akt and reduced phosphorylated p38 and activated caspase-3 levels in axotomized VEGF-transgenic retinas. Intravitreous injections of pharmacological ERK-1/2 (PD98059) or Akt (LY294002) inhibitors showed that VEGF exerts neuroprotection by dual activation of ERK-1/2 and Akt pathways. In view that axotomy-induced RGC death occurs slowly and considering that RGCs are CNS-derived neurons, we predict the clinical implementation of VEGF in neurodegenerative diseases of both brain and retina.

When Melatonin Gets on Your Nerves: Its Beneficial Actions in Experimental Models of Stroke:

This article summarizes the evidence that endogenously produced and exogenously administered melatonin reduces the degree of tissue damage and limits the biobehavioral deficits associated with experimental models of ischemia/reperfusion injury in the brain (i.e., stroke). Melatonins efficacy in curtailing neural damage under conditions of transitory interruption of the blood supply to the brain has been documented in models of both focal and global ischemia. In these studies many indices have been shown to be improved as a consequence of melatonin treatment. For example, when given at the time of ischemia or reperfusion onset, melatonin reduces neurophysio-logical deficits, infarct volume, the degree of neural edema, lipid peroxidation, protein carbonyls, DNA damage, neuron and glial loss, and death of the animals. Melatonins protective actions against these adverse changes are believed to stem from its direct free radical scavenging and indirect antioxidant activities, possibly from its ability to limit free radical generation at the mitochondrial level and because of yet-undefined functions. Considering its high efficacy in overcoming much of the damage associated with ischemia/reperfusion injury, not only in the brain but in other organs as well, its use in clinical trials for the purpose of improving stroke outcome should be seriously considered.

TLR-4 deficiency protects against focal cerebral ischemia and axotomy-induced neurodegeneration

The pattern recognition receptor toll-like receptor (TLR)-4 mediates innate danger signaling in the brain, being activated in response to lipopolysaccharide. Until now, its role in the degenerating brain remained unknown. We here examined effects of a loss-of-function mutation of TLR-4 in mice submitted to transient focal cerebral ischemia and retinal ganglion cell (RGC) axotomy, which are highly reproducible and clinically relevant in vivo models of acute and subacute neuronal degeneration. We show that TLR-4 deficiency protects mice against ischemia and axotomy-induced RGC degeneration. Decreased phosphorylation levels of the mitogen-activated kinases ERK-1/-2, JNK-1/-2 and p38 together with reduced inducible NO synthase levels in injured neurons of TLR-4 mutant mice suggests that TLR-4 deficiency downscales parenchymal stress responses, thereby enhancing neuronal survival. At the same time, densities of MPO+ neutrophils and Iba1+ microglial cells were increased in the brains of TLR-4 mutant animals, pointing towards a futile inflammatory response aiming to compensate lost functions. Our data indicate that innate immunity may represent an attractive target for neuroprotective treatments in stroke and neurodegeneration.

Aggravation of ischemic brain injury by prion protein deficiency : Role of ERK-1/-2 and STAT-1

The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp(0/0)), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury and cell signaling. In immunohistochemical experiments and Western blots, we show that PrPc expression is absent in the brains of Prnp(0/0) mice, detectable in wild-type controls and approximately 4.0-fold elevated in tga20 mice. We provide evidence that PrPc deficiency increases infarct size by approximately 200%, while transgenic PrPc restores tissue viability, albeit not above levels in wild-type animals. To elucidate the mechanisms underlying Prnp(0/0)-induced injury, we performed Western blots, which revealed increased activities of ERK-1/-2, STAT-1 and caspase-3 in ischemic brains of Prnp(0/0)mice. Our data suggest a role of cytosolic signaling pathways in Prnp(0/0)-induced cell death.

Erythropoietin protects from axotomy-induced degeneration of retinal ganglion cells by activating ERK-1/-2

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective activity upon reduced oxygenation or ischemia of brain, retina, and spinal cord. To examine whether Epo has an impact on the retrograde degeneration of retinal ganglion cells (RGCs) following optic nerve transection in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo preferentially in neuronal cells without inducing polycythemia. We show that the tg21 retina expresses human Epo and that RGCs in this mouse line carry the Epo receptor. Upon axotomy, the RGCs of Epo transgenic tg21 mice were protected against degeneration, as compared with wild‐type control animals. Western blot analysis revealed decreased phosphorylation levels of STAT‐5 and reduced expression of Bcl‐XL in RGCs of axotomized tg21 animals, suggesting that the corresponding pathways are not crucial for Epos neuroprotective activity. Increased phosphorylation levels of ERK‐1/‐2 and Akt, as well as decreased caspase‐3 activity, however, were observed in injured tg21 retinae. Injection of selective inhibitors of ERK‐1/‐2 (PD98059) or Akt (Wortmannin) pathways into the vitreous space revealed that transgenic Epo protected the RGCs by a pathway involving ERK‐1/‐2 but not Akt. In view that axotomy‐induced degeneration of RGC occurs slowly, and considering the earlier data on the safety and efficacy of Epo in human stroke patients, we predict the clinical implementation of recombinant human Epo not only in patients with acute ischemic stroke, but also with more delayed degenerative neurological diseases.

Signal transduction pathways involved in melatonin-induced neuroprotection after focal cerebral ischemia in mice

Abstract:  Because of its favorable action profile in humans, melatonin is a particularly interesting candidate as a neuroprotectant in acute ischemic stroke. Until now, the signaling mechanisms mediating melatonins neuroprotective actions remained essentially uninvestigated. Herein, we examined the effects of melatonin, administered either orally for 9 wk as a stroke prophylactic (4 mg/kg/day) or intraperitoneally immediately after reperfusion onset (4 mg/kg), on the activation of signal transduction pathways in mice submitted to 90 min of intraluminal middle cerebral artery occlusion, followed by 24 hr of reperfusion. In these studies, melatonin significantly reduced ischemic infarct size by ∼30–35%, as compared with animals receiving diluent (sham) treatment, independent of whether the indole was administered prior to or after ischemia. Under both conditions, animals receiving melatonin exhibited elevated phosphorylated Akt levels in their brains, as determined by Western blots. Additionally, phosphorylation levels of mitogen‐activated protein kinase/extracellular‐regulated kinase (ERK)‐1/‐2 and Jun kinase (JNK)‐1/‐2 were increased following prophylactic, but not acute, melatonin treatment. Our data suggest a role of phosphatidyl inositol‐3 kinase/Akt signaling in acute melatonin‐induced neuroprotection, while ERK‐1/‐2 and/or JNK‐1/‐2 rather appear to be involved in melatonins long‐term effects.