肢体缺血预处理通过激活有丝分裂原激活蛋白激酶p38减轻脑缺血导致的大鼠海马CA1区神经元凋亡和脑水肿(英文)

目的旨在探讨肢体缺血预处理(LIP)能否减轻脑缺血过程中海马CA1区神经元凋亡和脑水肿。方法72只永久凝闭椎动脉的Wistar大鼠随机分为6组:假手术,LIP(双侧股动脉夹闭10min,间歇10min,3次循环),脑缺血,LIP+脑缺血,DMSO和SB 203580+LIP+脑缺血组。各组大鼠中6只在脑缺血后3d处死,TUNEL染色计数凋亡细胞;6只在脑缺血后24h处死,测定脑组织含水量。结果TUNEL染色显示,假手术和LIP组海马CA1区偶有TUNEL阳性细胞;脑缺血组海马CA1区可见大量棕黄色着色的TUNEL阳性细胞,与假手术组及LIP组相比,细胞数量明显增加;LIP+脑缺血组,TUNEL阳性神经元数与脑缺血组相比明显减少,提示LIP明显抑制缺血引起的海马CA1区锥体细胞凋亡;有丝分裂原激活蛋白激酶p38拮抗剂SB 203580+LIP+脑缺血组,海马CA1区阳性染色锥体细胞明显增加,与DMSO+LIP+脑缺血组相比有显著性差别,表明SB 203580可拮抗LIP抑制凋亡的作用。与假手术和LIP组比较,脑缺血组脑组织含水量明显增加,表明LIP降低了脑缺血引起的脑组织含水量增加;LIP前应用SB 203580可抑制LIP的脑保护作用,使脑组织含水量较LIP+脑缺血组显著增加。结论LIP能够减轻脑缺血过程中海马CA1区神经元凋亡和脑水肿,可能与活化有丝分裂原激活蛋白激酶p38有关。     

Functional proteins involved in regulation of intracellular Ca(2+) for drug development: role of calcium/calmodulin-dependent protein kinases in ischemic neuronal death

Excessive elevation of intracellular calcium level seems to be a trigger of ischemic neuronal injury. Calcium/calmodulin (CaM)-dependent protein kinase kinase (CaM-KK) is an upstream kinase for CaM kinase IV (CaM-KIV) that was reported to prevent apoptosis through phosphorylation of CREB (cyclic AMP responsive element-binding protein). We here observed that CaM-KK could directly activate Akt, thereby preventing apoptosis in cultured cells. Then we examined changes in Akt and CaM-KIV activities in gerbil forebrain ischemia. In 5-min-ischemia-caused delayed neuronal death in hippocampal CA1 neurons, Akt and CaM-KIV activities were decreased after reperfusion. On the other hand, during induction of ischemic tolerance, Akt activity gradually and persistently increased in the CA1 neurons with transient increase in CREB phosphorylation. Inhibition of Akt activity with wortmannin or CREB-DNA binding with CRE-decoy injection resulted in failure of generation of ischemic tolerance. These results indicated activation of Akt and CaM-KIV play important roles in induction of the ischemic tolerance. Activation of CaM-KK may provide a new strategy for overcoming the ischemic stress.     

Berberry extract reduces neuronal damage and N-Methyl-D-aspartate receptor 1 immunoreactivity in the gerbil hippocampus after transient forebrain ischemia

In the present study, we studied the neuroprotective effects of berberry extract (BE) against ischemic damage and the temporal and spatial alterations of N-methyl-D-aspartate receptor type 1 (NR1) and NR2A/2B immunoreactivities in the gerbil hippocampal CA1 region after transient ischemia to examine anti-ischemic effects and its role in transient forebrain ischemia. In the vehicle-treated group, the percentage of cresyl violet positive pyramidal cells in the CA1 region was about 11.4% compared to the sham-operated group 4 d after ischemic insult. BE showed neuroprotective effects against ischemic damage after ischemia-reperfusion. In the BE-treated groups, about 60-75% of CA1 pyramidal cells were stained with cresyl violet 4 d after ischemic insult. We observed the percentage of berberine (7.45+0.85 mg/g in BE) by HPLC, which is active ingredient of BE. NR1 immunoreactivity in the stratum pyramidale of the CA1 region in the vehicle-treated group was significantly increased at 30 min after transient forebrain ischemia, while at this time the NR1 immunoreactivity in the BE-treated groups was significantly low compared to the vehicle-treated group. The pattern of NR2A/B immunoreactivity in the stratum pyramidale of the BE-treated group and its protein levels were similar to that in the vehicle-treated group after ischemic insult. These results suggest that BE has potent neuroprotective effects against ischemic damage via the reduction of NR1 activity.     

Insulin-like growth factor inhibits vascular contraction to 5-hydroxytryptamine: involvement of tyrosine phosphatase

This study tests the hypothesis that insulin-like growth factor 1 (IGF-1)-induced vasodilation is due to the stimulation of tyrosine phosphatase. Rat aortic segments (endothelium intact) were placed in muscle baths for force measurement. Segments were contracted to serotonin [5-hydroxytyptamine (5-HT), 10(-7)-10(-5) M] before and after incubation with IGF-1 (10-100 nM; 90 min). IGF-1 caused a 20% inhibition of 5-HT-induced contractions. This inhibition was reversed by the tyrosine phosphatase inhibitors sodium orthovanadate and molybdate. Orthovanadate did not alter inhibitory properties of the calcium channel antagonist verapamil, suggesting that the phosphatase inhibitors were relatively specific. IGF-1-induced inhibition was not altered by blockade of nitric oxide synthase. Western blot analysis confirmed that the 5-HT-induced stimulation of tyrosine phosphorylation of the 42-kDa extracellular signal-regulated mitogen-activated protein kinase protein was reduced by IGF-1 (52% inhibition), an inhibition that was attenuated by orthovanadate. These data are consistent with the hypothesis that the vasodilator activity of IGF-1 is mediated by the activation of a tyrosine phosphatase.     

Evidence for neuroprotective effect of sulbutiamine against oxygen-glucose deprivation in rat hippocampal CA1 pyramidal neurons

Hippocampus is one of the earliest brain regions that gets affected by ischemia, however, no pharmacological therapy exists yet that can fully counteract the ischemic damage. Here we study the effect of sulbutiamine, a synthetic thiamine analogue that can cross the blood-brain barrier easily, on hippocampal neurons under an in vitro model of ischemia, oxygen-glucose deprivation (OGD). We find that exposure to OGD in the presence of sulbutiamine significantly increases neuronal viability and enhances electrophysiological properties such as excitatory synaptic transmissions and intrinsic neuronal membrane input resistance in a concentration-dependent manner. Overall, here we report, for the first time, the neuroprotective evidence of sulbutiamine on hippocampal CA1 pyramidal neurons under OGD, which may have beneficial implications as a possible therapeutic agent/substance against ischemic insult.     

Effects of hepatocyte growth factor on phosphorylation of extracellular signal-regulated kinase and hippocampal cell death in rats with transient forebrain ischemia

Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischemia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IkappaB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischemia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischemia could be related to an ERK-dependent pathway.     

A protein tyrosine phosphatase inhibitor accelerates angiogenesis in a rat model of hindlimb ischemia

Vascular endothelial growth factor (VEGF) receptor-2 (KDR/flk-1) has a tyrosine kinase domain and, once activated, induces the autophosphorylation of the tyrosine residues. The phosphorylated KDR/flk-1 can be a substrate for intracellular protein tyrosine phosphatases (PTPs). In the present study, we have examined whether the PTP inhibitor sodium orthovanadate (SOV) activates KDR/flk-1 and accelerates angiogenesis in a rat model of hindlimb ischemia. The left femoral artery was exposed and excised to induce limb ischemia. The PTP activity in ischemic adductors increased, whereas SOV significantly suppressed the increase in the activity. Tyrosine phosphorylation of KDR/flk-1 and Akt phosphorylation significantly increased in the muscles injected with SOV compared with those injected with saline. The amount of VEGF increased in both the muscles injected with SOV and those injected with the saline but did not differ significantly. At 21 days after the induction of ischemia, immunohistochemical studies demonstrated that muscles injected with SOV showed significantly increased capillary density compared with those injected with saline. In a rat model of hindlimb ischemia, not only VEGF but also PTP, which might impair angiogenesis, increased. SOV activated KDR/flk-1 and accelerated angiogenesis. Thus, a PTP inhibitor can be a new drug for therapeutic angiogenesis in peripheral ischemic diseases.     

Neuronal death mode switch and neurogenesis as in vivo neuroprotection

The brain has various in vivo neuroprotective mechanisms that allow it to survive for an entire lifetime. As well as neurotrophic factor-mediated inhibition of in vivo apoptotic mechanisms through various protein kinases including Akt and MAP kinase, we propose adding the neuronal death mode switch mechanism observed under the brain ischemic stress to the list of neuroprotective mechanisms. Necrosis occurs when energy or ATP levels are markedly reduced. Lowered ATP levels cause a Na(+)-K(+)-ATPase failure, leading to an osmolysis. On the other hand, sufficient ATP is required for the apoptosome activation. Under the serum-free condition, cortical neurons rapidly die in necrosis. High-glucose treatment converted the cell death mode to apoptosis through an elevation of cellular ATP levels. This treatment also rescued the cell from death due to retinal ischemic injury. These findings suggest the possibility that ischemia-induced neuronal death could be inhibited by some drugs to elevate cellular ATP levels. Neurogenesis in the adult brain is now an important topic in neuroscience. As brain injury is reported to enhance the neurogenesis, this might be also included in the ways of in vivo neuroprotection. As lysophosphatidic acid has various activities to drive neurogenesis, the neurogenesis could also be managed by other drugs to compensate for functions lost by neuronal death.     

The post-ischemic administration of 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel calmodulin antagonist, prevents delayed neuronal death in gerbil hippocampus

The novel calmodulin (CaM) antagonist DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) with an apparent neuroprotective effect in vivo preferentially inhibits neuronal nitric oxide synthase (nNOS), Ca2+/CaM-dependent protein kinase IIalpha (CaMKIIalpha), and calcineurin in vitro. In the present study, we investigated the molecular mechanism underlying its neuroprotective effect with the gerbil transient forebrain ischemia model, by focusing on its inhibition of these Ca2+/CaM-dependent enzymes. Post-ischemic DY-9760e treatment (5 mg/kg, i.p.) immediately after 5-min ischemia significantly reduced the delayed neuronal death in the hippocampal CA1 region. CaMKIIalpha was transiently autophosphorylated immediately after reperfusion with concomitant sustained decrease in its total amounts in the Triton X-100-soluble fractions. Calcineurin activity, accessed by the phosphorylation state of dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) at Thr34, was elevated at 6 h after reperfusion. Post-treatment of DY-9760e had no effects on both CaMKIIalpha and DARPP-32 phosphorylation at 6 h after reperfusion. However, DY-9760e significantly inhibited nitrotyrosine formation, as a biomarker of NO, and in turn, peroxynitrite (ONOO-) production. These results suggest that DY-9760e primarily inhibits Ca2+/CaM-dependent neuronal NOS, without any effects on CaMKII and calcineurin, and the inhibition of NO production possibly accounts for its neuroprotective action in brain ischemic injury.     

Tyrosine dephosphorylation underlies DHPG-induced LTD

A form of long-term depression (LTD) of synaptic transmission can be induced by bath application of the group I metabotropic glutamate (mGlu) receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). The mechanisms responsible for the induction and expression of DHPG-induced LTD in the CA1 region of the hippocampus are currently the subject of intense investigation. Here we show that two protein tyrosine kinase (PTK) inhibitors (10 microM lavendustin A or 30 microM genistein) have little effect on DHPG-induced LTD. In contrast two protein tyrosine phosphatase (PTP) inhibitors (1 mM orthovanadate or 15 microM phenyl-arsine oxide) significantly inhibited DHPG-induced LTD. These data suggest that DHPG-induced LTD involves activation of a protein tyrosine phosphatase.     

Mechanisms of control of neuron survival by the endocannabinoid system

Endocannabinoids act as retrograde messengers that, by inhibiting neurotransmitter release via presynaptic CB(1) cannabinoid receptors, regulate the functionality of many synapses. In addition, the endocannabinoid system participates in the control of neuron survival. Thus, CB(1) receptor activation has been shown to protect neurons from acute brain injury as well as in neuroinflammatory conditions and neurodegenerative diseases. Nonetheless, some studies have reported that cannabinoids can also exert neurotoxic actions. Cannabinoid neuroprotective activity relies on the inhibition of glutamatergic neurotransmission and on other various mechanisms, and is supported by the observation that the brain overproduces endocannabinoids upon damage. Coupling of neuronal CB(1) receptors to cell survival routes such as the phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase pathways may contribute to cannabinoid neuroprotective action. These pro-survival signals occur, at least in part, by the cross-talk between CB(1) receptors and growth factor tyrosine kinase receptors. Besides promoting neuroprotection, a role for the endocannabinoid system in the control of neurogenesis from neural progenitors has been put forward. In addition, activation of CB(2) cannabinoid receptors on glial cells may also participate in neuroprotection by limiting the extent of neuroinflammation. Altogether, these findings support that endocannabinoids constitute a new family of lipid mediators that act as instructive signals in the control of neuron survival.     

Therapeutic window of opportunity for the neuroprotective effect of valproate versus the competitive AMPA receptor antagonist NS1209 following status epilepticus in rats

Epileptogenesis, i.e., the process leading to epilepsy, is a presumed consequence of brain insults including head trauma, stroke, infections, tumors, status epilepticus (SE), and complex febrile seizures. Typically, brain insults produce morphological and functional alterations in the hippocampal formation, including neurodegeneration in CA1, CA3, and, most consistently, the dentate hilus. Most of these alterations develop gradually, over several days, after the insult, providing a therapeutic window of opportunity for neuroprotective agents in the immediate post-injury period. We have previously reported that prolonged (four weeks) treatment with the antiepileptic drug valproate (VPA) after SE prevents hippocampal damage and most of the behavioral alterations that occur after brain insult, but not the development of spontaneously occurring seizures. These data indicated that VPA, although not preventing epilepsy, might be an effective disease-modifying treatment following brain insult. The present study was designed to (1) determine the therapeutic window for the neuroprotective effect of VPA after SE; (2) compare the efficacy of different intermittent i.p. versus continuous i.v. VPA treatment protocols; and (3) compare VPA with the glutamate (AMPA) receptor antagonist NS1209. As in our previous study with VPA, SE was induced by sustained electrical stimulation of the basolateral amygdala in rats and terminated after 4 h by diazepam. In vehicle controls, >90% of the animals developed significant neurodegeneration in the dentate hilus, whereas damage in CA1 and CA3 was more variable. Hilar parvalbumin-expressing interneurons were more sensitive to the effects of seizures than somatostatin-stained hilar interneurons or hilar mossy cells. Among the various VPA treatment protocols, continuous infusion of VPA for 24 immediately following the SE was the most effective neuroprotective treatment, preventing most of the neuronal damage. Infusion with NS1209 for 24 h exhibited similar neuroprotective efficacy. These data demonstrate that short treatment after SE with either VPA or NS1209 is powerfully neuroprotective, and may be disease-modifying treatments following brain insult.     

bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways

Extensive research has focused on finding effective strategies to prevent or improve recovery from brain ischemia and reperfusion (I/R) injury. The basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some central nervous system (CNS) disorders, including ischemic injury. In this study, we demonstrate that bFGF administration can improve locomotor activity and inhibit the ER stress induced in the CA1 region of the hippocampus in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response proteins CHOP, XBP-1, ATF-6 and caspase-12 that are induced by H(2)O(2) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signaling pathways, PI3K/Akt and ERK1/2. Inhibition of the PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and U0126, respectively, partially reduce the protective effect of bFGF. Taken together, our results indicate that the neuroprotective role of bFGF involves the suppression of ER stress in the ischemic oxidative damage models and oxidative stress-induced PC12 cell injury, and these effects is underlying the activation of the PI3K/Akt and ERK1/2 signal pathway.     

Delta opioid peptide [D-Ala2, D-Leu5] enkephalin (DADLE) triggers postconditioning against transient forebrain ischemia

Preconditioning with selective delta opioid peptide [d-Ala2, d-Leu5] enkephalin (DADLE) provides ischemic tolerance following transient forebrain ischemia in rats. However, whether DADLE postconditioning retains its neuroprotective efficacy and the underlying molecular mechanism in ischemic brain is largely unknown. We investigated DADLE postconditioning protection of hippocampal CA1 neurons against transient forebrain ischemia. 6 days after being implanted with cannula at the right lateral ventricle, rats underwent 10 min of forebrain ischemia by four vessel occlusion. Hippocampal CA1 neuronal survival and degeneration were measured in the hippocampi of rats at 3 days after ischemia. The behavioral and cognitive improvements of DADLE treatment in rats were also evaluated on days 5-9 using open-field and Morris water maze tests. The results showed that DADLE at doses of 0.25 and 2.5 nmol, but not 25 nmol, could significantly protect CA1 neurons against ischemia/reperfusion injury. Co-administration with the delta-opioid receptor antagonist naltrindole or pretreatment with the Akt antagonist LY294002 completely abolished the DADLE postconditioning effect. Furthermore, DADLE postconditioning exhibited cognitive benefits in rats with transient forebrain ischemia. The study thus suggested a therapeutic opportunity of postconditioning neuroprotection by DADLE and also provided important information in understanding the mechanism of DADLE action in the ischemic brain.     

Nitric oxide donors induce neurotrophin-like survival signaling and protect neurons against apoptosis

Our previous results showed that inhibition of protein tyrosine phosphatases (PTP) by orthovanadate is an appropriate strategy to mimic nerve growth factor (NGF) effects in neurons, including enhanced phosphorylation of TrkA, stimulation of downstream survival signaling pathways, and protection against apoptotic stress. In this study, we wanted to trigger such NGF-like survival signaling in primary hippocampal neurons with the more specific PTP inhibitors ethyl-3,4-dephostatin (DPN), 4-O-methyl-ethyl-3,4-dephostatin (Me-DPN), and methoxime-3,4-dephostatin. It was striking that only the nitric oxide (NO)-releasing dephostatin analogs DPN and Me-DPN, but not the nitrosamine-free methoxime derivative (which did not release NO), enhanced TrkA phosphorylation and protected the neurons against staurosporine (STS)-induced apoptosis. The established NO donor S-nitroso-N-acetylpenicillamine (SNAP) also enhanced TrkA phosphorylation and prevented apoptosis similarly to DPN and Me-DPN. Analysis of the major signaling pathways downstream of TrkA revealed that both SNAP and DPN enhanced phosphorylation of Akt and the mitogen-activated kinases (MAPK) Erk1/2. Blocking of these signaling pathways by the PI3-K inhibitor wortmannin or the MAPK kinase inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] equally abolished the neuroprotective effect of the NO donors. It was striking that inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or protein kinase G (PKG) inhibition by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823) also blocked the neuroprotective effect of the NO donors, and ODQ clearly attenuated SNAP-induced phosphorylation of TrkA, Akt, and MAPK. In conclusion, NO release by the dephostatin derivatives and subsequent stimulation of sGC and PKG is essential for their neuroprotective effects. In primary neurons, such NO-activated survival signaling involves NGF-like effects, including enhanced phosphorylation of TrkA and activation of PI3-K/Akt and MAPK pathways.     

JNK通路在缺血预处理诱导海马神经元保护中的作用

目的探讨JNK通路在缺血预处理诱导海马神经元保护中的作用。方法♂蒙古沙土鼠,随机分为假手术组(SH)、缺血/再灌注组(I/R)、缺血预处理组(IP)、Anisomy-cin组(AN)、Curcumin组(CU)、Anisomycin复合IP组(AP)、Curcumin复合IP组(CP)及溶剂对照组(VE),每组据再灌注15min、2、4、6h、1、3、5及7d又分8个亚组。预定时间点行TUNEL海马CA1区凋亡细胞检测、免疫组化SP法检测p-JNK及Jun蛋白在海马CA1区的表达变化。结果IP、CU及CP可减少海马CA1区凋亡锥体细胞数(vsI/R,P<0.01),减弱CA1区再灌注各点p-JNK及Jun蛋白的表达水平(vsIR,P<0.01),该效应CP组>IP组>CU组。AN增加CA1区凋亡锥体细胞数(vsIR,P<0.01),增强CA1区再灌后1d内各点p-JNK及再灌后1～7d各点Jun蛋白表达水平(vsIR,P<0.01)。AP部分抵消IP保护效应。结论JNK通路激活参与沙土鼠海马CA1区缺血性神经元凋亡,缺血预处理可通过抑制CA1区JNK磷酸化、减少Jun蛋白表达而保护海马细胞和功能。抑制JNK通路激活可发挥缺血预处理相似的保护作用。     

热休克蛋白与神经保护

对脑中风患者,治疗时给予神经保护剂能挽救半暗带(penumbra),改善患者预后。缺血区ATP匮乏是脑损伤的始动因素,而神经元内蛋白质翻译停滞是脑缺血引起迟发性神经元死亡的标志。研究认为Hsp70高表达处于神经保护通路的上游关口,可以对抗缺血区的两大变化:蛋白质翻译停滞和ATP供给匮乏,抑制细胞凋亡和坏死,其机制与Hsp70参与蛋白折叠,抑制异常蛋白聚集,减少细胞毒性相关。因此,研究诱导Hsp70蛋白表达的药物对脑缺血的保护作用、阐明Hsp70诱导剂保护神经元的机制是很有意义的。     

Insulin-like growth factor type-I receptor-dependent phosphorylation of extracellular signal-regulated kinase 1/2 but not Akt (protein kinase B) can be induced by picropodophyllin

The initial event upon binding of insulin-like growth factor 1 to the insulin-like growth factor type-I receptor (IGF-1R) is auto-phosphorylation of tyrosine residues within the activation loop of the kinase domain followed by phosphorylation of other receptor tyrosine residues and the subsequent activation of the intracellular signaling cascades. We found recently that the cyclolignan picropodophyllin (PPP) inhibits phosphorylation of IGF-1R and phosphatidyl-3 kinase/Akt (protein kinase B) signaling molecules without interfering with the highly homologous insulin receptor. Furthermore, PPP causes regression of tumor grafts and substantially prolongs the survival of animals with systemic tumor disease. It is of interest that we show here that short treatments with PPP activate the intracellular extracellular signal-regulated kinase (ERK) signaling. Our data suggest that PPP induces IGF-1R ubiquitination and in turn activates ERK1/2. The PPP-induced ERK activation requires IGF-1R because PPP is not able to induce ERK phosphorylation in IGF-1R-negative cells or in cells in which the receptor is knocked down by small interfering RNA. Moreover, in the absence of Mdm2, an E3 ligase that has been shown previously to be involved in IGF-1R ubiquitination, the phosphorylation of ERK did not occur. Thus, apart from inhibiting the receptor activity, PPP can induce IGF-1R ubiquitination and stimulate ERK in an Mdm2-dependent manner. This response could contribute to the apoptotic effect of PPP.     

Transient oxygen-glucose deprivation induces rapid morphological changes in rat hippocampal dendrites.

Previous studies have revealed that pyramidal neurons in the CA1 region of the hippocampus are extremely susceptible to ischemia-induced cell damage and undergo selective degeneration 2-4 days after the insult. Little is known about early morphological changes in neurons occurring immediately after ischemic insult. Using two-photon laser scanning microscopy we monitored dendritic morphology of cells expressing enhanced green fluorescent protein in response to a transient hypoxic-ischemic episode in organotypic hippocampal slice preparations. This type of vital imaging provides direct evidence of dendritic rearrangements in rat CA1 pyramidal neurons occurring as soon as 20 min after oxygen-glucose deprivation. We propose that dendritic reorganization, resembling that occurring after tetanic stimulation, may be an early stage response to compensate the loss of synapses caused by ischemia-induced neuronal injury.