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1.  Hypothermia Blocks β-catenin Degradation after Focal Ischemia in Rats 
Brain research  2008;1198:182-187.
Dephosphorylated and activated glycogen synthase kinase (GSK) 3β hyperphophorylates β-catenin, leading to its ubiquitin-proteosome-mediated degradation. β-catenin-knockdown increases while β-catenin overexpression prevents neuronal death in vitro; in addition, protein levels of β-catenin are reduced in the brain of Alzheimer’s patients. However, whether β-catenin degradation is involved in stroke-induced brain injury is unknown. Here we studied activities of GSK3 β and β-catenin, and the protective effect of moderate hypothermia (30 °C) on these activities after focal ischemia in rats. The results of Western blot showed that GSK3 β was dephosphorylated at 5 and 24 hours after stroke in the normothermic (37 °C) brain; hypothermia augmented GSK3β dephosphorylation. Because hypothermia reduces infarction, these results contradict with previous studies showing that GSK3β dephosphorylation worsens neuronal death. Nevertheless, hypothermia blocked degradation of total GSK3β protein. Corresponding to GSK3β activity in normothermic rats, β-catenin phosphorylation transiently increased at 5 hours in both the ischemic penumbra and core, and the total protein level of β-catenin degraded after normothermic stroke. Hypothermia did not inhibit β-catenin phosphorylation, but it blocked β-catenin degradation in the ischemic penumbra. In conclusion, moderate hypothermia can stabilize β-catenin, which may contribute to the protective effect of moderate hypothermia.
doi:10.1016/j.brainres.2008.01.007
PMCID: PMC2350209  PMID: 18241848
Focal ischemia; hypothermia; GSK-3β; β-catenin
2.  Lipoprotein Receptor–Related Protein-6 Protects the Brain From Ischemic Injury 
Background and Purpose
Loss-of-function mutations of the lipoprotein receptor–related protein-6 (LRP6), a coreceptor in the Wingless-related integration site-β–catenin prosurvival pathway, have been implicated in myocardial ischemia and neurodegeneration. However, it remains to be established whether LRP6 is also involved in ischemic brain injury. We used LRP6+/− mice to examine the role of this receptor in the mechanisms of focal cerebral ischemia.
Methods
Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery. Motor deficits and infarct volume were assessed 3 days later. Glycogen-synthase-kinase-3β (GSK-3β) phosphorylation was examined by Western blotting with phosphospecific antibodies, and the mitochondrial membrane potential changes induced by Ca2+ were also assessed.
Results
LRP6+/− mice have larger stroke and more severe motor deficits, effects that were independent of intraischemic cerebral blood flow, vascular factors, or cytosolic β-catenin levels. Rather, LRP6 haploinsufficiency increased the activating phosphorylation and decreased the inhibitory phosphorylation of GSK-3β, a kinase involved in proinflammatory signaling and mitochondrial dysfunction. Accordingly, postischemic inflammatory gene expression was enhanced in LRP6+/− mice. Furthermore, the association of mitochondria with activated GSK-3β was increased in LRP6+/− mice, resulting in a reduction in the Ca2+ handling ability of mitochondria. The mitochondrial dysfunction was reversed by pharmacological inhibition of GSK-3β.
Conclusions
LRP6 activates an endogenous neuroprotective pathway that acts independently of β-catenin by controlling GSK-3β activity and preventing its deleterious mitochondrial and proinflammatory effects. The findings raise the possibility that emerging treatment strategies for diseases attributable to LRP6 loss-of-function mutations could also lead to new therapeutic avenues for ischemic stroke.
doi:10.1161/STROKEAHA.113.001320
PMCID: PMC3933931  PMID: 23743975
cerebral ischemia; glycogen-synthase-kinase-3; mitochondria; stroke; Wnt signaling pathway
3.  Targeting the oncogenic protein beta-catenin to enhance chemotherapy outcome against solid human cancers 
Molecular Cancer  2010;9:310.
Background
Beta-catenin is a multifunctional oncogenic protein that contributes fundamentally to cell development and biology. Elevation in expression and activity of β-catenin has been implicated in many cancers and associated with poor prognosis. Beta-catenin is degraded in the cytoplasm by glycogen synthase kinase 3 beta (GSK-3β) through phosphorylation. Cell growth and proliferation is associated with β-catenin translocation from the cytoplasm into the nucleus.
This laboratory was the first to demonstrate that selenium-containing compounds can enhance the efficacy and cytotoxicity of anticancer drugs in several preclinical xenograft models. These data provided the basis to identify mechanism of selenium action focusing on β-catenin as a target. This study was designed to: (1) determine whether pharmacological doses of methylseleninic acid (MSeA) have inhibitory effects on the level and the oncogenic activity of β-catenin, (2) investigate the kinetics and the mechanism of β-catenin inhibition, and (3) confirm that inhibition of β-catenin would lead to enhanced cytotoxicity of standard chemotherapeutic drugs.
Results
In six human cancer cell lines, the inhibition of total and nuclear expression of β-catenin by MSeA was dose and time dependent. The involvement of GSK-3β in the degradation of β-catenin was cell type dependent (GSK-3β-dependent in HT-29, whereas GSK-3β-independent in HCT-8). However, the pronounced inhibition of β-catenin by MSeA was independent of various drug treatments and was not reversed after combination therapy.
Knockout of β-catenin by ShRNA and its inhibition by MSeA yielded similar enhancement of cytotoxicity of anticancer drugs.
Collectively, the generated data demonstrate that β-catenin is a target of MSeA and its inhibition resulted in enhanced cytotoxicity of chemotherapeutic drugs.
Conclusions
This study demonstrates that β-catenin, a molecule associated with drug resistance, is a target of selenium and its inhibition is associated with increased multiple drugs cytotoxicity in various human cancers. Further, degradation of β-catenin by GSK-3β is not a general mechanism but is cell type dependent.
doi:10.1186/1476-4598-9-310
PMCID: PMC3014904  PMID: 21126356
4.  Phosphorylation and ubiquitination of oncogenic mutants of β-catenin containing substitutions at Asp32 
Oncogene  2004;23(28):4839-4846.
β-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3β (GSK-3β)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3β region of β-catenin stabilize the protein and activate β-catenin/TCF/LEF target genes. This study examined three oncogenic β-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3β. Compared with wild-type β-catenin (WT), the β-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of β-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the β-catenin mutants. Specifically, antiphospho-β-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated β-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant β-catenins, as well as on WT β-catenin. The relative order of ubiquitination was WT > D32G > D32N > D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of β-catenin.
doi:10.1038/sj.onc.1207634
PMCID: PMC2267883  PMID: 15064718
β-catenin; TCF/LEF; APC; Wnt signaling; colorectal cancer; CTNNB1; human β-catenin gene; Ctnnb1; rat β-catenin gene
5.  δ-Catenin/NPRAP: A New Member of the Glycogen Synthase Kinase-3β Signaling Complex That Promotes β-catenin Turnover in Neurons 
Journal of neuroscience research  2010;88(11):10.1002/jnr.22414.
Through a multiprotein complex, glycogen synthase kinase-3β (GSK-3β) phosphorylates and destabilizes β-catenin, an important signaling event for neuronal growth and proper synaptic function. δ-Catenin, or NPRAP (CTNND2), is a neural specific member of the β-catenin superfamily, and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ-catenin expression is also affected by GSK-3β signaling, and it participates in the molecular complex regulating β-catenin turnover in neurons. Immunofluorescent light microscopy revealed co-localization of δ-catenin with members of the molecular destruction complex: GSK-3β, β-catenin, and APC in rat primary neurons. GSK-3β formed a complex with δ-catenin, and its inhibition resulted in increased δ-catenin and β-catenin expression levels. LY294002 and amyloid peptide, known activators of GSK-3β signaling, reduced δ-catenin expression levels. Furthermore, δ-catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ-catenin, like that of β-catenin, is regulated by proteasome-mediated degradation. Co-immunoprecipitation experiments showed that δ-catenin overexpression promoted GSK-3β and β-catenin interactions. Primary cortical neurons and PC12 cells expressing δ-catenin treated with proteasome inhibitors showed increased ubiquitinated β-catenin forms. Consistent with the hypothesis that δ-catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ-catenin showed enhanced β-catenin turnover. These studies identify δ-catenin as a new member of the GSK-3β signaling pathway and further suggest that δ-catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β-catenin in neuronal cells.
doi:10.1002/jnr.22414
PMCID: PMC3813950  PMID: 20623542
Glycogen synthase kinase-3β; δ-catenin/NPRAP; β-catenin; proteasome; ubiquitination
6.  Reperfusion Differentially Induces Caspase-3 Activation in Ischemic Core and Penumbra After Stroke in Immature Brain 
Background and Purpose
Different strategies for neuroprotection of neonatal stroke may be required because the developing brain responds differently to hypoxia-ischemia than the mature brain. This study was designed to determine the role of caspase-dependent injury in the pathophysiology of pure focal cerebral ischemia in the immature brain.
Methods
Postnatal day 7 rats were subjected to permanent or transient middle cerebral artery (MCA) occlusion. Diffusion-weighted MRI was used during occlusion to noninvasively map the evolving ischemic core. The time course of caspase-3 activation in ischemic brain tissue was determined with the use of an Asp-Glu-Val-Asp-aminomethylcoumarin cleavage assay. The anatomy of caspase-3 activation in the ischemic core and penumbra was mapped immunohistochemically with an anti–activated caspase-3 antibody in coronal sections that matched the imaging planes on diffusion-weighted MRI.
Results
A marked increase in caspase-3 activity occurred within 24 hours of reperfusion after transient MCA occlusion. In contrast, caspase-3 activity remained significantly lower within 24 hours of permanent MCA occlusion. Cells with activated caspase-3 were prominent in the penumbra beginning at 3 hours after reperfusion, while a more delayed but marked caspase-3 activation was observed in the ischemic core by 24 hours after reperfusion.
Conclusions
In the neonate, caspase-3 activation is likely to contribute substantially to cell death not only in the penumbra but also in the core after ischemia with reperfusion. Furthermore, persistent perfusion deficits result in less caspase-3 activation and appear to favor caspase-independent injury.
doi:10.1161/01.STR.0000047101.87575.3C
PMCID: PMC2262098  PMID: 12511776
apoptosis; caspases; cerebral ischemia; magnetic resonance imaging, diffusion-weighted; neonate
7.  A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling 
PLoS ONE  2009;4(9):e6892.
The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.
doi:10.1371/journal.pone.0006892
PMCID: PMC2731218  PMID: 19727391
8.  The EDD E3 ubiquitin ligase ubiquitinates and up-regulates β-catenin 
Molecular Biology of the Cell  2011;22(3):399-411.
The effector protein of the canonical Wnt pathway is β-catenin, which is regulated by the ubiquitin system. This study shows that the E3 ubiquitin ligase EDD ubiquitinates β-catenin, leading to up-regulation of β-catenin's expression levels and activity. Thus the results demonstrate a role for the ubiquitin system in up-regulation of the Wnt pathway.
Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. β-Catenin activity is tightly regulated via a multiprotein complex that includes the kinase glycogen synthase kinase-3β (GSK-3β). GSK-3β phosphorylates β-catenin, marking it for ubiquitination and degradation via the proteasome. Thus in regulation of the Wnt pathway, the ubiquitin system is known to be involved mostly in mediating the turnover of β-catenin, resulting in reduced Wnt signaling levels. Here we report that an arm of the ubiquitin system increases β-catenin protein levels. We show that GSK-3β directly interacts with the E3 ubiquitin ligase identified by differential display (EDD) that also binds β-catenin. Expression of EDD leads to enhanced nuclear accumulation of both GSK-3β and β-catenin and results in up-regulation of β-catenin expression levels and activity. Importantly, EDD ubiquitinates β-catenin through Lys29- or Lys11-linked ubiquitin chains, leading to enhanced stability of β-catenin. Our results demonstrate a role for the ubiquitin system in up-regulation of the Wnt signaling pathway, suggesting that EDD could function as a colorectal oncogene.
doi:10.1091/mbc.E10-05-0440
PMCID: PMC3031469  PMID: 21118991
9.  Caspase-3 Contributes to ZO-1 and Cl-5 Tight-Junction Disruption in Rapid Anoxic Neurovascular Unit Damage 
PLoS ONE  2011;6(2):e16760.
Background
Tight-junction (TJ) protein degradation is a decisive step in hypoxic blood-brain barrier (BBB) breakdown in stroke. In this study we elucidated the impact of acute cerebral ischemia on TJ protein arrangement and the role of the apoptotic effector protease caspase-3 in this context.
Methodology/Principal Findings
We used an in vitro model of the neurovascular unit and the guinea pig whole brain preparation to analyze with immunohistochemical methods the BBB properties and neurovascular integrity. In both methodological approaches we observed rapid TJ protein disruptions after 30 min of oxygen and glucose deprivation or middle cerebral artery occlusion, which were accompanied by strong caspase-3 activation in brain endothelial cells (BEC). Surprisingly only few DNA-fragmentations were detected with TUNEL stainings in BEC. Z-DEVD-fmk, an irreversible caspase-3 inhibitor, partly blocked TJ disruptions and was protective on trans-endothelial electrical resistance.
Conclusions/Significance
Our data provide evidence that caspase-3 is rapidly activated during acute cerebral ischemia predominantly without triggering DNA-fragmentation in BEC. Further we detected fast TJ protein disruptions which could be partly blocked by caspase-3 inhibition with Z-DEVD-fmk. We suggest that the basis for clinically relevant BBB breakdown in form of TJ disruptions is initiated within minutes during ischemia and that caspase-3 contributes to this process.
doi:10.1371/journal.pone.0016760
PMCID: PMC3043060  PMID: 21364989
10.  A divergent canonical WNT-signaling pathway regulates microtubule dynamics 
The Journal of Cell Biology  2004;164(2):243-253.
Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability.
doi:10.1083/jcb.200309096
PMCID: PMC2172322  PMID: 14734535
β-catenin; GSK-3β; neurons; cytoskeleton; axin
11.  Physiological regulation of β-catenin stability by Tcf3 and CK1ε 
The Journal of Cell Biology  2001;154(5):983-994.
The wnt pathway regulates the steady state level of β-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit β-catenin turnover via its competition with axin and adenomatous polyposis for β-catenin binding. A mutant of β-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of β-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between β-catenin and Tcf3 stimulate β-catenin degradation, indicating this interaction normally plays an important role in regulating β-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1ε, and phosphorylation of Tcf3 by CKIε stimulates its binding to β-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1ε to inhibit β-catenin degradation, whereas CKI-7, an inhibitor of CK1ε, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1ε stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1ε can modulate wnt signaling in vivo by regulating both the β-catenin-Tcf3 and the GBP-dsh interfaces.
doi:10.1083/jcb.200102074
PMCID: PMC2196183  PMID: 11524435
β-catenin; Tcf; wnt; casein kinase; Xenopus
12.  Sex Differences in Caspase Activation after Experimental Stroke 
Background and purpose
Over the past five years, experimental data has emerged that ischemia-induced cell death pathways may differ in males and females. Cell death in males is triggered by Poly(ADP-ribose) polymerase (PARP) activation and nuclear translocation of apoptosis-inducing factor (AIF). We have previously shown that interference with this pathway benefits males but not females after an experimental stroke. In contrast caspase activation may be the major pathway activated after ischemic injury in females. The aim of this study is to examine whether sex differences exist in caspase activation in adult mice after stroke and to determine if interference with stroke-induced caspase activation preferentially protects females.
Methods
Focal stroke was induced by reversible middle cerebral artery occlusion (MCAO; 90 minutes) in young and aging C57BL6 mice of both sexes. The pan-caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh) was administered at reperfusion. Histological outcomes was assessed 48 hours after MCAO. Separate cohorts were utilized for protein analysis of key cell death proteins including caspase-3, caspase-8, cytochrome C and Apoptosis Inducing Factor (AIF).
Results
Drug-treated female mice had significantly decreased infarct volumes and improved neurological deficits after stroke compared to vehicle-treated mice. Q-VD-OPh administration had no effect in male mice. The expression of cytochrome C and nuclear caspase-8 levels were increased in females after stroke.
Conclusions
Female mice had an early release of cytochrome C and enhanced caspase activation after MCAO. Caspase inhibition benefited females but not males. Sex differences exist in both the response to ischemic injury and the efficacy of neuroprotective agents.
doi:10.1161/STROKEAHA.108.538686
PMCID: PMC2674515  PMID: 19265047
MCAO; cytochrome C; caspase; sex differences; stroke
13.  The Akt pathway is involved in rapid ischemic tolerance in focal ischemia in Rats 
Translational stroke research  2010;1(3):202-209.
Although the protective mechanisms of delayed ischemic preconditioning have received extensive studies, few have addressed the mechanisms associated with rapid ischemic postconditioning. We investigated whether ischemic tolerance induced by rapid preconditioning is regulated by the Akt survival signaling pathway. Stroke was generated by permanent occlusion of the left distal middle cerebral artery (MCA) plus 30 min or 1 h occlusion of the bilateral common carotid artery (CCA) in male rats. Rapid preconditioning performed 1h before stroke onset reduced infarct size by 69% in rats with 30 min CCA occlusion, but by only 19% with 1 h occlusion. After control ischemia with 30 min CCA occlusion, Western Blot showed that P-Akt was transiently increased while Akt kinase assay showed that Akt activity was decreased. Although preconditioning did not change P-Akt levels at 1h and 5h compared with control ischemia, it attenuated reduction in Akt activity at 5h in the penumbra. However, preconditioning did not change the levels of P-PDK1, P-PTEN, and P-GSK3β in the Akt pathway, all of which were decreased after stroke. At last, the PI3K kinase inhibitor, LY294002, completely reversed the protection from ischemic preconditioning. In conclusion, Akt contributes to the protection of rapid preconditionin against stroke.
doi:10.1007/s12975-010-0017-5
PMCID: PMC3144475  PMID: 21804899
rapid preconditioning; ischemic tolerance; cerebral ischemia; focal ischemia; neuroprotection; Akt
14.  Dual Role of Caspase-11 in Mediating Activation of Caspase-1 and Caspase-3 under Pathological Conditions 
The Journal of Cell Biology  2000;149(3):613-622.
Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.
PMCID: PMC2174843  PMID: 10791975
caspase-11; initiator caspase; stroke; middle cerebral artery occlusion; apoptosis
15.  Caspase-3-dependent and -independent apoptosis in focal brain ischemia. 
Molecular Medicine  2002;8(7):347-352.
BACKGROUND: Although extensive caspase-3 activation has been demonstrated in experimental brain ischemia produced in neonatal rat, the role this caspase plays in the focal ischemia of adult brain is not clear, as the levels of caspase-3 in adult rat brain are extremely low. This raises the question whether caspase-3 synthesis and activation are essential for execution of the apoptotic program and DNA fragmentation in permanent brain ischemia, a condition that impairs cellular protein synthesis. MATERIALS AND METHODS: Rat middle cerebral artery was permanently occluded and histochemical detection of procaspase-3, active caspase-3 and DFF 40/CAD and apoptotic morphology analysis were performed at 6, 24, 48, and 72 hours after occlusion. RESULTS: Necrosis and two types of programmed cell death (PCD) are identified in this study of permanent focal brain ischemia. The first type of PCD is represented by active caspase-3 and DFF 40/CAD-positive cells. The second type of PCD is represented by caspase-3 and DFF40/CAD negative cells, which display morphological signs of apoptosis-like PCD: namely, nuclear chromatin condensation in lump masses and apoptotic body formation. The cells of the first type have a maximum number noted after 24 hours of ischemia. The cells of the second type are primarily seen after 48 and 72 hours of ischemia. Necrotic cells, which are also detected in the stroke, are caspase-3 negative, and have swollen nuclei, without chromatin condensation and apoptotic body formation. CONCLUSIONS: Our results indicate that in permanent brain ischemia in adult rats, PCD processes occur differently in various parts of ischemic zone. In conditions of severe energy depletion, the reactions of cellular disassembly and packaging into apoptotic bodies are accomplished without either caspase-3 expression or the activation of caspase-3-dependent deoxyribonuclease.
PMCID: PMC2040004  PMID: 12393932
16.  Limb remote ischemic postconditioning protects against focal ischemia in rats 
Brain research  2009;1288:88-94.
Remote ischemic postconditioning (RIP) refers to an ischemia conducted in a distant organ that protects against a prior ischemia in another organ. We tested whether RIP protects against focal ischemia in the rat brain. Stroke was generated by a permanent occlusion of the left distal middle cerebral artery combined with a 30 min occlusion of the bilateral common carotid arteries (CCA) in male rats. After CCA release, RIP was generated by 3 cycles of 15 min occlusion/15 min release of the left hind femoral artery. The results showed that rapid RIP performed immediately after CCA release reduced infarction by 67% measured at 2 d after stroke. In addition, delayed RIP initiated as late as 3 h, but not 6 h, still robustly reduced infarction by 43% 2 d after stroke. RIP's protective effect was abolished by injecting the protein synthesis inhibitor, cycloheximide, and the afferent nerve blocker, capsaicin, suggesting that RIP blocks ischemic injury by modulating protein synthesis and nerve activity. Nevertheless, rapid RIP did not reduce infarction size 2 months after stroke while it ameliorated the outcome of the behavioral test. In conclusion, RIP attenuates brain injury after focal ischemia.
doi:10.1016/j.brainres.2009.07.029
PMCID: PMC2744502  PMID: 19631625
stroke; cerebral ischemia; preconditioning; remote postconditioning
17.  Sex differences in the response to PARP-1 deletion and caspase inhibition after stroke 
Background and purpose
Emerging data suggests that the molecular cell death pathways triggered by ischemic insults differ in male and female brain. Cell death in males is initiated by poly (ADP-ribose) polymerase-1 (PARP-1) activation; however manipulation of this pathway paradoxically increases ischemic damage in females. In contrast, females are exquisitely sensitive to caspase mediated cell death. The effect of caspase inhibition in PARP-1 knockout mice was evaluated to determine if the detrimental effects of PARP deletion in females was secondary to increased caspase activation.
Methods
Focal stroke was induced by transient or permanent middle cerebral artery occlusion (MCAO) in wild type (WT) and PARP-1−/− mice of both sexes. The pan-caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh) was administered 90 minutes after MCAO. Infarct size and neurological sores were assessed. Separate cohorts were utilized for protein analysis for PAR, AIF, caspase-9 and -3.
Results
WT mice of both sexes had increased nuclear AIF after stroke compared to PARP-1−/− mice. PARP-1−/− females had higher mitochondrial cytochrome C, activated caspase-9 and -3 levels than WT female mice. PARP-1−/− females also had an increase in stroke-induced cytosolic C release compared to WT females which was not seen in males. Q-VD-OPh decreased caspase-9 in both males and females but only led to reduction of infarct in females. PARP-1−/− males had smaller infarcts whereas PARP-1−/− females had larger strokes compared to WT. Q-VD-OPh significantly decreased infarct in both WT and PARP-1−/− females in both transient and permanent MCAO models, but had no effect in males.
Conclusions
Deletion of PARP-1 reduces infarct in males, but exacerbates injury in females. PARP−/− females have enhanced caspase activation. The detrimental effects of PARP loss in females can be reversed with caspase inhibition.
doi:10.1161/STROKEAHA.110.594861
PMCID: PMC3066270  PMID: 21311064
AIF; caspase; MCAO; PARP; sex differences
18.  Interaction among Gsk-3, Gbp, Axin, and APC in Xenopus Axis Specification 
The Journal of Cell Biology  2000;148(4):691-702.
Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is β-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal–ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and β-catenin form a complex that promotes the GSK-3–mediated phosphorylation and subsequent degradation of β-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates β-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3–mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic β-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in β-catenin levels and establishment of the dorsal axis.
PMCID: PMC2169372  PMID: 10684251
Wnt pathway; dorsal/ventral; β-catenin
19.  Phosphorylation of β-Catenin by Cyclic AMP-Dependent Protein Kinase Stabilizes β-Catenin through Inhibition of Its Ubiquitination 
Molecular and Cellular Biology  2005;25(20):9063-9072.
The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E1 (PGE1), isoproterenol, and dibutyryl cAMP (Bt2cAMP), all of which activate PKA, increased the cytoplasmic and nuclear β-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE1 and Bt2cAMP also increased T-cell factor (Tcf)-dependent transcription through β-catenin. Bt2cAMP suppressed degradation of β-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3β (GSK-3β), β-catenin, and Axin, phosphorylation of β-catenin by PKA inhibited ubiquitination of β-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in β-catenin attenuated inhibition of the ubiquitination of β-catenin by PKA, PKA-induced stabilization of β-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of β-catenin by phosphorylating β-catenin, thereby causing β-catenin to accumulate and the Wnt signaling pathway to be activated.
doi:10.1128/MCB.25.20.9063-9072.2005
PMCID: PMC1265785  PMID: 16199882
20.  Progesterone is neuroprotective against ischemic brain injury through its effects on the PI3K/Akt signaling pathway 
Neuroscience  2012;210:442-450.
We tested the hypothesis that the phosphatidylinositol-3 kinase (PI3K/Akt) pathway mediates some of the neuroprotective effects of progesterone (PROG) after ischemic stroke. We examined whether PROG acting through the PI3K/Akt pathway could affect the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Rats underwent permanent focal cerebral ischemia (pMCAO) by electro-coagulation and received intraperitoneal injections of PROG (8mg/kg) or vehicle at 1h post-occlusion and subcutaneous injections at 6, 24, and 48h. PAkt/Akt levels, apoptosis and apoptosis-related proteins (pBAD, BAD, caspase-3, and cleaved caspase-3) were analyzed by TUNEL assays, Western blotting and immunohistochemistry at 24h post-pMCAO. VEGF and BDNF were analyzed at 24, 72h and 14 days post-pMCAO with Western blots. Following pMCAO, PROG treatment significantly (p<0.05) reduced ischemic lesion size and edema. Treatment with PROG significantly (p<0.05) decreased VEGF at 24 and 72h but increased VEGF expression 14d after injury. The treatment also increased BDNF, and attenuated apoptosis by increasing Akt phosphorylation compared to vehicle-alone. The selective PI3K inhibitor Wortmannin compromised PROG-induced neuroprotective effects and reduced the elevation of pAkt levels in the ischemic penumbra. Our findings lead us to suggest that the PI3K/Akt pathway can play a role in mediating the neuroprotective effects of PROG after stroke by altering the expression of trophic factors in the brain.
doi:10.1016/j.neuroscience.2012.03.008
PMCID: PMC3358507  PMID: 22450229
apoptosis; growth factors; ischemic stroke; neuroprotection; phosphoinositide-3-kinase/Akt pathway; progesterone
21.  Delayed Postconditioning Protects against Focal Ischemic Brain Injury in Rats 
PLoS ONE  2008;3(12):e3851.
Background
We and others have reported that rapid ischemic postconditioning, interrupting early reperfusion after stroke, reduces infarction in rats. However, its extremely short therapeutic time windows, from a few seconds to minutes after reperfusion, may hinder its clinical translation. Thus, in this study we explored if delayed postconditioning, which is conducted a few hours after reperfusion, offers protection against stroke.
Methods and Results
Focal ischemia was generated by 30 min occlusion of bilateral common carotid artery (CCA) combined with permanent occlusion of middle cerebral artery (MCA); delayed postconditioning was performed by repetitive, brief occlusion and release of the bilateral CCAs, or of the ipsilateral CCA alone. As a result, delayed postconditioning performed at 3h and 6h after stroke robustly reduced infarct size, with the strongest protection achieved by delayed postconditioning with 6 cycles of 15 min occlusion/15 min release of the ipsilateral CCA executed from 6h. We found that this delayed postconditioning provided long-term protection for up to two months by reducing infarction and improving outcomes of the behavioral tests; it also attenuated reduction in 2-[18F]-fluoro-2-deoxy-D-glucose (FDG)-uptake therefore improving metabolism, and reduced edema and blood brain barrier leakage. Reperfusion in ischemic stroke patients is usually achieved by tissue plasminogen activator (tPA) application, however, t-PA's side effect may worsen ischemic injury. Thus, we tested whether delayed postconditioning counteracts the exacerbating effect of t-PA. The results showed that delayed postconditioning mitigated the worsening effect of t-PA on infarction.
Conclusion
Delayed postconditioning reduced ischemic injury after focal ischemia, which opens a new research avenue for stroke therapy and its underlying protective mechanisms.
doi:10.1371/journal.pone.0003851
PMCID: PMC2588536  PMID: 19066627
22.  dl-3-n-Butylphthalide Prevents Neuronal Cell Death after Focal Cerebral Ischemia in Mice via the JNK Pathway 
Brain research  2010;1359:216-226.
dl-3-n-Butylphthalide (NBP) has shown cytoprotective effects in animal models of stroke and has passed clinical trails as a therapeutic drug for stroke in China. Hence, as a potential clinical treatment for stroke, understanding the mechanism(s) of action of NBP is essential. This investigation aimed to delineate the cellular and molecular mechanism of NBP protection in neuronal cultures and in the ischemic brain. NBP (10 M) attenuated serum deprivation-induced neuronal apoptosis and the production of reactive oxygen species (ROS) in cortical neuronal cultures. Adult male 129 S2/sv mice were subjected to permanent occlusion of the middle cerebral artery (MCA). NBP (100 mg/kg, i.p.) administrated 2 hrs before or 1 hr after ischemia reduced ischemia-induced infarct formation, attenuated caspase-3 and caspase-9 activation in the ischemic brain. TUNEL-positive cells and mitochondrial release of cytochrome c and apoptosis-inducing-factor (AIF) in the penumbra region were reduced by NBP. The pro-apoptotic signaling mediated by phospho-JNK and p38 expression was down-regulated by NBP treatment in vitro and in vivo. It is suggested that NBP protects against ischemic damage via multiple mechanisms including mitochondria associated caspase-dependent and -independent apoptotic pathways. Previous and current studies and recent clinical trials encourage exploration of NBP as a neuroprotective drug for the treatment of ischemic stroke.
doi:10.1016/j.brainres.2010.08.061
PMCID: PMC3099257  PMID: 20800583
dl-3-n-butylphthalide; Ischemic stroke; Apoptosis; Caspase; AIF; Cytochrome C; Mitochondria; MAP kinase
23.  Changes in Wnt/β-Catenin Pathway During Regulated Growth in Rat Liver Regeneration 
Hepatology (Baltimore, Md.)  2001;33(5):1098-1109.
The wnt/β-catenin pathway is important during embryogenesis and carcinogenesis. β-Catenin interaction with E-cadherin has been shown to be crucial in cell-cell adhesion. We report novel findings in the wnt pathway during rat liver regeneration after 70% partial hepatectomy using Western blot analyses, immunoprecipitation studies, and immunofluorescence. We found wnt-1 and β-catenin proteins to be predominantly localized in hepatocytes. Immediately following partial hepatectomy, we observed an initial increase in β-catenin protein during the first 5 minutes with its translocation to the nucleus. We show this increase to be the result of decreased degradation of β-catenin (decrease in serine phosphorylated β-catenin) as seen by immunoprecipitation studies. We observed activation of β-catenin degradation complex comprising of adenomatous polyposis coli gene product (APC) and serine-phosphorylated axin protein, beginning at 5 minutes after hepatectomy, leading to its decreased levels after this time. Quantitative changes observed in E-cadherin protein during liver regeneration are, in general, reverse to those seen in β-catenin. In addition, using immunoprecipitation, we observe elevated levels of tyrosine-phosphorylated β-catenin at 6 hours onward. Thus, changes in the wnt pathway during regulated growth seem to tightly regulate cytosolic β-catenin levels and may be contributing to induce cell proliferation and target gene expression. Furthermore, these changes might also be intended to negatively regulate cell-cell adhesion for structural reorganization during the process of liver regeneration.
doi:10.1053/jhep.2001.23786
PMCID: PMC1821078  PMID: 11343237
24.  Exosome release of β-catenin: a novel mechanism that antagonizes Wnt signaling 
The Journal of Cell Biology  2010;190(6):1079-1091.
The tetraspanins CD9 and CD82 suppress Wnt signaling by promoting the discharge of β-catenin from cells.
CD82 and CD9 are tetraspanin membrane proteins that can function as suppressors of tumor metastasis. Expression of CD9 and CD82 in transfected cells strongly suppresses β-catenin–mediated Wnt signaling activity and induces a significant decrease in β-catenin protein levels. Inhibition of Wnt/β-catenin signaling is independent of glycogen synthase kinase-3β and of the proteasome- and lysosome-mediated protein degradation pathways. CD82 and CD9 expression induces β-catenin export via exosomes, which is blocked by a sphingomyelinase inhibitor, GW4869. CD82 fails to induce exosome release of β-catenin in cells that express low levels of E-cadherin. Exosome release from dendritic cells generated from CD9 knockout mice is reduced compared with that from wild-type dendritic cells. These results suggest that CD82 and CD9 down-regulate the Wnt signaling pathway through the exosomal discharge of β-catenin. Thus, exosomal packaging and release of cytosolic proteins can modulate the activity of cellular signaling pathways.
doi:10.1083/jcb.201002049
PMCID: PMC3101591  PMID: 20837771
25.  Inhibition of GSK3 Phosphorylation of β-Catenin via Phosphorylated PPPSPXS Motifs of Wnt Coreceptor LRP6 
PLoS ONE  2009;4(3):e4926.
The Wnt/β-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated β-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, β-catenin phosphorylation by GSK3 is inhibited and β-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of β-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent β-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit β-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of β-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of β-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/β-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of β-catenin. This model provides a possible mechanism to account, in part, for inhibition of β-catenin phosphorylation by Wnt-activated LRP6.
doi:10.1371/journal.pone.0004926
PMCID: PMC2654145  PMID: 19293931

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