As the number of patients with cardioembolic ischemic stroke is predicted to be double by 2030, increased burden of warfarin-associated hemorrhagic transformation (HT) after cerebral ischemia is an expected consequence. However, thus far, no effective treatment strategy is available for HT prevention in routine clinical practice. While the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) is known to protect against oxidative stress and neuronal cell death caused by ischemic brain damage, its effect on preventing warfarin-associated HT after cerebral ischemia is yet unknown. Therefore, we hypothesized that Ex-4 would stabilize the blood-brain barrier (BBB) and suppress neuroinflammation through PI3K-Akt-induced inhibition of glycogen synthase kinase-3β (GSK-3β) after warfarin-associated HT post-cerebral ischemia.
We used male C57BL/6 mice for all experiments. A 5-mg warfarin sodium tablet was dissolved in animals’ drinking water (effective warfarin uptake 0.04 mg (2 mg/kg) per mouse). The mice were fed for 0, 6, 12, and 24 h with ad libitum access to the treated water. To study the effects of Ex-4, temporary middle cerebral artery occlusion (MCAO) was performed. Then, either Ex-4 (10 mg/kg) or saline was injected through the tail vein, and in the Ex-4 + wortmannin group, PI3K inhibitor wortmannin was intravenously injected, after reperfusion. The infarct volume, neurological deficits, and integrity of the BBB were assessed 72 h post MCAO. One- or two-way ANOVA was used to test the difference between means followed by Newman–Keuls post hoc testing for pair-wise comparison.
We observed that Ex-4 ameliorated warfarin-associated HT and preserved the integrity of the BBB after cerebral ischemia through the PI3K/Akt/GSK-3β pathway. Furthermore, Ex-4 suppressed oxidative DNA damage and lipid peroxidation, attenuated pro-inflammatory cytokine expression levels, and suppressed microglial activation and neutrophil infiltration in warfarin-associated HT post-cerebral ischemia. However, these effects were totally abolished in the mice treated with Ex-4 + the PI3K inhibitor—wortmannin. The PI3K/Akt-GSK-3β signaling pathway appeared to contribute to the protection afforded by Ex-4 in the warfarin-associated HT model.
GLP-1 administration could reduce warfarin-associated HT in mice. This beneficial effect of GLP-1 is associated with attenuating neuroinflammation and BBB disruption by inactivating GSK-3β through the PI3K/Akt pathway.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-016-0661-0) contains supplementary material, which is available to authorized users.
Cerebral ischemia; Exendin-4; Hemorrhagic transformation; Blood-brain barrier; Neuroinflammation; PI3K/Akt-GSK-3β signaling pathway; Warfarin
Glioblastoma stem cells (GSCs) are a subpopulation of highly tumorigenic and stem-like cells that are responsible for resistance to conventional therapy. Bcl-2-intreacting cell death suppressor (BIS; also known as BAG3) is an anti-apoptotic protein that is highly expressed in human cancers with various origins, including glioblastoma. In the present study, to investigate the role of BIS in GSC subpopulation, we examined the expression profile of BIS in A172 and U87-MG glioblastoma cell lines under specific in vitro culture conditions that enrich GSC-like cells in spheres. Both BIS mRNA and protein levels significantly increased under the sphere-forming condition as compared with standard culture conditions. BIS depletion resulted in notable decreases in sphere-forming activity and was accompanied with decreases in SOX-2 expression. The expression of STAT3, a master regulator of stemness, also decreased following BIS depletion concomitant with decreases in the nuclear levels of active phosphorylated STAT3, while ectopic STAT3 overexpression resulted in recovery of sphere-forming activity in BIS-knockdown glioblastoma cells. Additionally, immunoprecipitation and confocal microscopy revealed that BIS physically interacts with STAT3. Furthermore, BIS depletion increased STAT3 ubiquitination, suggesting that BIS is necessary for STAT3 stabilization in GSC-like cells. BIS depletion also affected epithelial-to-mesenchymal transition-related genes as evidenced by decrease in SNAIL and MMP-2 expression and increase in E-cadherin expression in GSC-like cells. Our findings suggest that high levels of BIS expression might confer stem-cell-like properties on cancer cells through STAT3 stabilization, indicating that BIS is a potential target in cancer therapy.
BIS; glioblastoma; CSCs; STAT3; ubiquitination
MicroRNAs (miRNAs) have been identified as key players in cardiomyocyte hypertrophy, which is associated with significant risks of heart failure. However, many microRNAs are still not recognized for their functions in pathophysiological processes. In this study, we evaluated effects of miR-218 in cardiomyocyte hypertrophy using both in vitro and in vivo models. We found that miR-218 was evidently downregulated in a transverse aortic constriction (TAC) mouse model. Overexpression of miR-218 is sufficient to reduce hypertrophy, whereas the suppression of miR-218 aggravates hypertrophy in primary cardiomyocytes induced by isoprenaline (ISO). In addition, we identified RE1-silencing transcription factor (REST) as a novel target of miR-218; it negatively regulated the expression of REST in hypertrophic cardiomyocytes and the TAC model. These results showed that miR-218 plays a crucial role in cardiomyocyte hypertrophy, likely via targeting REST, suggesting a potential candidate target for interfering hypertrophy.
miR-218; cardiomyocyte; hypertrophy; REST
As the main bioactive metabolites of Ganoderma lucidum, triterpenoids have various pharmacological effects. In this paper, the nutritional requirements and culture conditions of a submerged culture of G. lucidum were optimized using the response surface methodology; maximum mycelia biomass and intracellular triterpenoid production reached 1.87 g/100 ml and 93.21 mg/100 ml, respectively, for a culture consisting of wort 4.10% (0.041 g/ml) and yeast extract 1.89% (0.0189 g/ml), pH 5.40. For the first time, we established that wort, which is cheap and abundant, can replace the more commonly used glucose as the sole source of carbohydrate. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), 10 major ganoderic acids were tentatively identified based on the predominant fragmentation pathways with the elimination of H2O and CO2, as well as cleavage of the D-ring.
Ganoderma lucidum; Wort; Response surface methodology; Ganoderic acids; HPLC-ESI-MS
γ -secretase-mediated processing of the amyloid-β protein precursor (AβPP) is a crucial step in the formation of the amyloid-β peptide (Aβ), but little is known about how the substrate AβPP interacts with the γ-secretase complex. To understand the molecular events involved in γ-secretase-mediated AβPP processing and Aβ formation, in the present study we determined the role of a well conserved GxxxG motif in the transmembrane domain of AβPP. Our data clearly demonstrate that substitution of aspartic acid for the key glycine residues in the GxxxG motif almost completely abolished the formation of Aβ. Furthermore, our data revealed that substitution of aspartic acid for the glycine in this GxxxG motif disrupts the interaction of AβPP with the γ-secretase complex. Thus, the present study revealed an essential role for the GxxxG motif in the interaction of AβPP with the γ-secretase complex and the formation of Aβ.
Alzheimer’s disease; amyloid-β peptide; amyloid-β precursor protein; GxxxG motif; γ-secretase
Overwhelming evidence supports the amyloid hypothesis of Alzheimer’s disease that stipulates that the relative level of the 42 amino acid β-amyloid peptide (Aβ42) in relationship to Aβ40 is critical to the pathogenesis of the disease. While it is clear that the multi-subunit gamma secretase is responsible for cleavage of the amyloid precursor protein (APP) into Aβ42 and Aβ40, the exact molecular mechanisms regulating the production of the various Aβ species remain elusive. To elucidate the underlying mechanisms, we replaced individual amino acid residues from positions 43 to 52 of Aβ with phenylalanine to examine the effects on the production of Aβ40 and Aβ42. All mutants, except for V50F, resulted in a decrease in total Aβ with a more prominent reduction in Aβ for residues 45, 48, and 51, following an every three residue repetition pattern. In addition, the mutations with the strongest reductions in total Aβ had the largest increases in the ratio of Aβ42/Aβ40. Curiously, the T43F, V44F, and T48F mutations caused a striking decrease in the accumulation of membrane bound Aβ46, albeit by a different mechanism. Our data suggest that initial cleavage of APP at the ε site is crucial in the generation of Aβ. The implicated sequential cleavage and an α-helical model may lead to a better understanding of the γ-secretase-mediated APP processing and may also provide useful information for therapy and drug design aimed at altering Aβ production.
Alzheimer’s disease; amyloid; amyloid precursor protein processing
Presenilin-associated protein (PSAP) has been identified as a mitochondrial proapoptotic protein. However, the mechanism by which PSAP induces apoptosis remains unknown. To this end, we have established an inducible expression system. Using this system, we have examined the roles of B-cell lymphoma 2 (Bcl-2) family proteins, cytochrome c, Smac (Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein with low PI), and Apaf-1 (apoptotic protease-activating factor) in PSAP-induced apoptosis. Our results demonstrate that knockdown of Apaf-1 abolished PSAP-induced caspase activation and poly(ADP ribose) polymerase (PARP) cleavage, indicating that the apoptosome formation triggered by cytochrome c is crucial for PSAP-induced apoptosis. Our data also demonstrate that knockdown of Smac abolished PSAP-induced caspase activation and PARP cleavage, indicating that, in addition to Apaf-1 or apoptosome formation, Smac is also essential for PSAP-induced apoptosis. However, interestingly, our data demonstrate that overexpression of Bcl-2 and Bcl-xL did not protect cells from PSAP-induced apoptosis, and that knockdown of Bid, Bax, and Bak had no effect on PSAP-induced cytochrome c and Smac release, indicating that PSAP-induced apoptosis is not regulated by Bcl-2 family proteins. These results strongly suggest that PSAP evokes mitochondrial apoptotic cascades via a novel mechanism that is not regulated by Bcl-2 family proteins, but that both the formation of cytochrome c-Apaf-1 apoptosome and the presence of Smac are absolutely required for PSAP-induced apoptosis.
Alzheimer’s disease; Apoptosis; Mitochondria; Bcl-2; Oxidative stress
Mitochondrial dysfunction has been reported in both familial and sporadic Parkinson’s disease (PD). However, effective therapy targeting this pathway is currently inadequate. Recent studies suggest that manipulating the processes of mitochondrial fission and fusion has considerable potential for treating human diseases. To determine the therapeutic impact of targeting these pathways on PD, we used two complementary mouse models of mitochondrial impairments as seen in PD. We show here that blocking mitochondrial fission is neuroprotective in the PTEN-induced putative kinase-1 deletion (PINK1−/−) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models. Specifically, we show that inhibition of the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) using gene-based and small molecule approaches attenuates neurotoxicity and restores pre-existing striatal dopamine release deficits in these animal models. These results suggest Drp1 inhibition as a potential treatment for PD.
Congenital heart disease (CHD), the most common type of birth defect, is still the leading non-infectious cause of infant morbidity and mortality in humans. Aggregating evidence demonstrates that genetic defects are involved in the pathogenesis of CHD. However, CHD is genetically heterogeneous and the genetic components underpinning CHD in an overwhelming majority of patients remain unclear. In the present study, the coding exons and flanking introns of the PITX2 gene, which encodes a paired-like homeodomain transcription factor 2essential for cardiovascular morphogenesis as well as maxillary facial development, was sequenced in 196 unrelated patients with CHD and subsequently in the mutation carrier’s family members available. As a result, a novel heterozygous PITX2 mutation, p.Q102X for PITX2a, or p.Q148X for PITX2b, or p.Q155X for PITX2c, was identified in a family with endocardial cushion defect (ECD) and Axenfeld-Rieger syndrome (ARS). Genetic analysis of the pedigree showed that the nonsense mutation co-segregated with ECD and ARS transmitted in an autosomal dominant pattern with complete penetrance. The mutation was absent in 800 control chromosomes from an ethnically matched population. Functional analysis by using a dual-luciferase reporter assay system revealed that the mutant PITX2 had no transcriptional activity and that the mutation eliminated synergistic transcriptional activation between PITX2 and NKX2.5, another transcription factor pivotal for cardiogenesis. To our knowledge, this is the first report on the association of PITX2 loss-of-function mutation with increased susceptibility to ECD and ARS. The findings provide novel insight into the molecular mechanisms underpinning ECD and ARS, suggesting the potential implications for the antenatal prophylaxis and personalized treatment of CHD and ARS.
To investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of Streptococcus agalactiae (S. agalactiae) in Beijing to provide references for the prevention and treatment of S. agalactiae infections.
All isolates were identified using the CAMP test and the latex-agglutination assay and serotyped using a Strep-B-Latex kit, after which they were assessed for antibiotic susceptibility, macrolide-resistance genes, and MLST profiles.
In total, 56 S. agalactiae isolates were identified in 863 pregnant women (6.5%). Serotypes Ia, Ib, II, III, and V were identified, among which types III (32.1%), Ia (17.9%), Ib (16.1%), and V (14.3%) were the predominant serotypes. All isolates were susceptible to penicillin and ceftriaxone. The nonsusceptiblity rates measured for erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, tetracycline, and levofloxacin were 85.7%, 92.9%, 98.2%, 30.4%, 73.2%, 91%, and 39.3%, respectively. We identified 14 sequence types (STs) for the 56 isolates, among which ST19 (30.4%) was predominant. The rate of fluoroquinolone resistance was higher in serotype III than in the other serotypes. Among the 44 erythromycin-resistant isolates, 32 (72.7%) carried ermB.
S. agalactiae isolates of the serotypes Ia, Ib, III, and V are common in Beijing. Among the S. agalactiae isolates, the macrolide and clindamycin resistance rates are extremely high. Most of the erythromycin-resistant isolates carry ermB.
Background: Diabetes is proved to be one of the independent risk factors for cognitive dysfunction. The pathophysiologic changes caused by diabetes including hyperglycemia and tissue hypoxia may contribute greatly to cognitive decline. In the present study, we demonstrate E3 Ubiquitin Ligase Siah-1 downregulates the key synaptic protein Synaptophysin expression under high glucose and hypoxia condition which may be the underlying factor leading to cognitive dysfunction in diabetic patients. Methods: In this study, hypoxia (2% oxygen) and high glucose (50 mM) were used to treat primary neuronal culture. By using quantitative PCR and western blotting we determined the influence of hypoxia and high glucose on the expression of synaptophysin and Siah-1 and the phosphorylated forms of extracellular signal-regulated kinase (ERK). Knockdown of Siah-1, inhibitors for proteasome, lysosome and ERK kinase was employed to evaluate the role of Siah-1 and ERK activity on the expression of synaptophysin. By immunoprecipitation we also examined the role of Siah-1 in the ubiquitination of synaptophysin under hypoxic and hyperglycemic condition. Results: We demonstrated that hypoxia and high glucose together but not hypoxia or high glucose along mediated posttranscriptional reduction of synaptophysin with increased ERK phosphorylation and Siah-1 expression. The downregulation of synaptophysin was reversed by inhibition of ERK and Siah-1 knockdown. Overexpression of Siah-1 accelerated the degradation of synaptophysin under hypoxia and high glucose conditions and promoted the ubiquitination of synaptophysin. Conclusions: The present results demonstrate that Siah-1 is the key factor that contributes to hypoxia and high glucose mediated synaptophysin degradation.
Siah-1; diabetes; synaptophysin; ERK; cognitive dysfunction
Mitochondrial dysfunction has been reported in both familial and sporadic Parkinson’s disease (PD). However, effective therapy targeting this pathway is currently inadequate. Recent studies suggest that manipulating the processes of mitochondrial fission and fusion has considerable potential for treating human diseases. To determine the therapeutic impact of targeting these pathways on PD, we used two complementary mouse models of mitochondrial impairments as seen in PD. We show here that blocking mitochondrial fission is neuroprotective in the PTEN-induced putative kinase-1 deletion (PINK1−/−) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse models. Specifically, we show that inhibition of the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) using gene-based and small-molecule approaches attenuates neurotoxicity and restores pre-existing striatal dopamine release deficits in these animal models. These results suggest Drp1 inhibition as a potential treatment for PD.
Mitochondrial dysfunction has been associated with Parkinson’s disease but effective therapies targeting this pathway are yet to be developed. Here the authors show that inhibition of the mitochondrial fission protein Drp-1 using genetic or small-molecule approaches in mouse models of the disease, leads to improvements in the pathology.
Cyclic AMP inhibits vascular smooth muscle cell (VSMC) proliferation which is important in the aetiology of numerous vascular diseases. The anti-mitogenic properties of cAMP in VSMC are dependent on activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), but the mechanisms are unclear.
Methods and results
Selective agonists of PKA and EPAC synergistically inhibited Egr1 expression, which was essential for VSMC proliferation. Forskolin, adenosine, A2B receptor agonist BAY60-6583 and Cicaprost also inhibited Egr1 expression in VSMC but not in endothelial cells. Inhibition of Egr1 by cAMP was independent of cAMP response element binding protein (CREB) activity but dependent on inhibition of serum response element (SRE) activity. SRF binding to the Egr1 promoter was not modulated by cAMP stimulation. However, Egr1 expression was dependent on the SRF co-factors Elk1 and 4 but independent of MAL. Inhibition of SRE-dependent Egr1 expression was due to synergistic inhibition of Rac1 activity by PKA and EPAC, resulting in rapid cytoskeleton remodelling and nuclear export of ERK1/2. This was associated with de-phosphorylation of the SRF co-factor Elk1.
cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. This occurs, at least in part, via inhibition of Rac1 activity leading to rapid actin-cytoskeleton remodelling, nuclear export of ERK1/2, impaired Elk1-phosphorylation and inhibition of SRE activity. This identifies one of the earliest mechanisms underlying the anti-mitogenic effects of cAMP in VSMC but not in endothelial cells, making it an attractive target for selective inhibition of VSMC proliferation.
•cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression.•PKA and Epac synergise to inhibit Egr1 expression.•cAMP-mediated inhibition of Egr1 is cell-type specific.•cAMP inhibits SRE-dependent Egr1 transcription.•cAMP inhibits Egr1 via nuclear export of ERK1/2 and de-phosphorylation of Elk1.
Zif268; Early growth response gene 1; Exchange protein activated by cAMP; 3'-5'-Cyclic adenosine monophosphate; Serum response factor
Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future.
Unclassified renal cell carcinoma; comparative genomic hybridization; exon GeneChip; chromosome imbalance; gene mutation
Bcl-2 interacting cell death suppressor (Bis) has been shown to have anti-apoptotic and anti-stress functions. Recently, increased Bis expression was reported to correlate with glioma aggressiveness. Here, we investigated the effect of Bis knockdown on the acquisition of the invasive phenotype of A172 glioma cells, induced by 12-O-Tetradecanoylphorbol- 3-acetate (TPA), using a Transwell assay. Bis knockdown resulted in a significant decrease in the migration and invasion of A172 cells. Furthermore, Bis knockdown notably decreased TPAinduced matrix metalloproteinase-9 (MMP-9) activity and mRNA expression, as measured by zymography and quantitative real time PCR, respectively. A luciferase reporter assay indicated that Bis suppression significantly down-regulated NF-κB-driven transcription. Finally, we demonstrated that the rapid phosphorylation and subsequent degradation of IκB-α induced by TPA was remarkably delayed by Bis knockdown. These results suggest that Bis regulates the invasive ability of glioma cells elicited by TPA, by modulating NF-κB activation, and subsequent induction of MMP-9 mRNA. [BMB Reports 2014; 47(5): 262-267]
Bis; Glioma; Invasion; MMP-9; NF-κB
The abnormal production and accumulation of β-amyloid peptide (Aβ), which is produced from amyloid precursor protein (APP) by the sequential actions of β-secretase and γ-secretase, are thought to be the initial causative events in the development of Alzheimer’s disease (AD). Accumulating evidence suggests that vascular factors play an important role in the pathogenesis of AD. Specifically, studies have suggested that one vascular factor in particular, oxidized low density lipoprotein (oxLDL), may play an important role in regulating Aβ formation in AD. However, the mechanism by which oxLDL modulates Aβ formation remains elusive. In this study, we report several new findings that provide biochemical evidence suggesting that the cardiovascular risk factor oxLDL may contribute to Alzheimer’s disease by increasing Aβ production. First, we found that lysophosphatidic acid (LPA), the most bioactive component of oxLDL induces increased production of Aβ. Second, our data strongly indicate that LPA induces increased Aβ production via upregulating β-secretase expression. Third, our data strongly support the notion that different isoforms of protein kinase C (PKC) may play different roles in regulating APP processing. Specifically, most PKC members, such as PKCα, PKCβ, and PKCε, are implicated in regulating α-secretase-mediated APP processing; however, PKCδ, a member of the novel PKC subfamily, is involved in LPA-induced upregulation of β-secretase expression and Aβ production. These findings may contribute to a better understanding of the mechanisms by which the cardiovascular risk factor oxLDL is involved in Alzheimer’s disease.
Alzheimer’s disease; Lysophosphatidic acid; Oxidized LDL; β-secretase; Beta-amyloid peptide; Amyloid precursor protein
γ-secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C-terminal fragment (PS1C) caused by knockdown of pen-2 or knockout of NCT or Aph-1 was recovered by the addition of proteasome inhibitors, indicating that Pen-2, as well as NCT and Aph-1α , is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin-Aph-1 subcomplex plays not only an important role in γ-secretase complex assembly but also in recruiting substrate C-terminal fragment of amyloid precursor protein generated by β-cleavage (CTFβ). Ablating any one component resulted in the instability of other components of the γ-secretase complex, and the presence of all three of the other components is required for full maturation of NCT.
Alzheimer’s disease; gamma-secretase; presenilin; Pen-2; APP
Malpighian tubules (MTs) are highly specific organs of arthropods (Insecta, Myriapoda and Arachnida) for excretion and osmoregulation. In order to highlight the important genes and pathways involved in multi-functions of MTs, we performed a systematic proteomic analysis of silkworm MTs in the present work. Totally, 1,367 proteins were identified by one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry, and as well as by Trans Proteomic Pipeline (TPP) and Absolute protein expression (APEX) analyses. Forty-one proteins were further identified by two-dimensional gel electrophoresis. Some proteins were revealed to be significantly associated with various metabolic processes, organic solute transport, detoxification and innate immunity. Our results might lay a good foundation for future functional studies of MTs in silkworm and other lepidoptera.
Huntington disease (HD) is an inherited neurodegenerative disease resulting from an abnormal expansion of polyglutamine in huntingtin (Htt). Compromised oxidative stress defense systems have emerged as a contributing factor to the pathogenesis of HD. Indeed activation of the Nrf2 pathway, which plays a prominent role in mediating antioxidant responses, has been considered as a therapeutic strategy for the treatment of HD. Given the fact that there is an interrelationship between impairments in mitochondrial dynamics and increased oxidative stress, in this present study we examined the effect of mutant Htt (mHtt) on these two parameters. STHdhQ111/Q111 cells, striatal cells expressing mHtt, display more fragmented mitochondria compared to STHdhQ7/Q7 cells, striatal cells expressing wild type Htt, concurrent with alterations in the expression levels of Drp1 and Opa1, key regulators of mitochondrial fission and fusion, respectively. Studies of mitochondrial dynamics using cell fusion and mitochondrial targeted photo-switchable Dendra revealed that mitochondrial fusion is significantly decreased in STHdhQ111/Q111 cells. Oxidative stress leads to dramatic increases in the number of STHdhQ111/Q111 cells containing swollen mitochondria, while STHdhQ7/Q7 cells just show increases in the number of fragmented mitochondria. mHtt expression results in reduced activity of Nrf2, and activation of the Nrf2 pathway by the oxidant tBHQ is significantly impaired in STHdhQ111/Q111 cells. Nrf2 expression does not differ between the two cell types, but STHdhQ111/Q111 cells show reduced expression of Keap1 and p62, key modulators of Nrf2 signaling. In addition, STHdhQ111/Q111 cells exhibit increases in autophagy, whereas the basal level of autophagy activation is low in STHdhQ7/Q7 cells. These results suggest that mHtt disrupts Nrf2 signaling which contributes to impaired mitochondrial dynamics and may enhance susceptibility to oxidative stress in STHdhQ111/Q111 cells.
Achieving a prolonged neuroprotective state following transient ischemic attacks (TIAs) is likely to effectively reduce the brain damage and neurological dysfunction associated with recurrent stroke. HPC is a phenomenon in which advanced exposure to mild hypoxia reduces the stroke volume produced by a subsequent TIA. However, this neuroprotection is not long-lasting, with the effects reaching a peak after 3 days. Therefore, in this study, we investigated the use of multiple episodes of hypoxic exposure at different time intervals to induce longer-term protection in a mouse stroke model. C57BL/6 mice were subjected to different hypoxic preconditioning protocols: a single episode of HPC or five identical episodes at intervals of 3 days (E3d HPC) or 6 days (E6d HPC). Three days after the last hypoxic exposure, temporary middle cerebral artery occlusion (MCAO) was induced. The effects of these HPC protocols on hypoxia-inducible factor (HIF) regulated gene mRNA expression were measured by quantitative PCR. Changes in extracellular adenosine concentrations, known to exert neuroprotective effects, were also measured using in vivo microdialysis and high pressure liquid chromatography (HPLC). Neuroprotection was provided by E6d HPC but not E3d HPC. HIF-regulated target gene expression increased significantly following all HPC protocols. However, E3d HPC significantly decreased extracellular adenosine and reduced cerebral blood flow in the ischemic region with upregulated expression of the adenosine transporter, equilibrative nucleoside transporter 1 (ENT1). An ENT1 inhibitor, propentofylline increased the cerebral blood flow and re-established neuroprotection in E3d HPC. Adenosine receptor specific antagonists showed that adenosine mainly through A1 receptor mediates HPC induced neuroprotection. Our data indicate that cooperation of HIF-regulated genes and extracellular adenosine is necessary for HPC-induced neuroprotection.
Most of the Alzheimer’s disease (AD)-linked mutations in amyloid precursor protein (APP), which cause abnormal production of β-amyloid (Aβ), are localized at the major β-secretase- and γ-secretase cleavage sites. In this study, using an APP-knockout mouse neuronal cell line, our data demonstrated that at the P2-P1 positions of the ε-cleavage site at Aβ49 and the ζ-cleavage site at Aβ46, aromatic amino acids caused a strong reduction in total Aβ. On the other hand, residues with a long side chain caused a decrease in Aβ40 and a concomitant increase in Aβ42 and Aβ38. These findings indicate that the structures of the substituting residues at these key positions strongly determine the efficiency and preference of γ-secretase-mediated APP processing, which determines the ratio of different secreted Aβ species, a crucial factor in the disease development. Our findings provide new insight into the mechanisms of γ-secretase-mediated APP processing and, specifically, into why most AD-linked APP mutations are localized at major γ-secretase cleavage sites. This information may contribute to the development of methods of prevention and treatment of Alzheimer’s disease aimed at modulating γ-secretase activity.
Alzheimer’s disease; β-amyloid; γ-secretase; APP; Intramembrane processing
Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start condon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.
Prostate cancer cell migration is an essential event both in the progression of prostate cancer and in the steps leading to metastasis. We report here that lysophosphatidic acid (LPA), a potent bioactive phospholipid, induces prostate cancer PC3 cell migration via the activation of the LPA1 receptor, which is linked to a PTX-sensitive activation mechanism of the mitogen-activated protein kinases (MAPK). Our results demonstrate that parallel activation of ERK1/2 and p38, but not JNK, is responsible for LPA-stimulated PC3 cell migration. Furthermore, using small interfering RNA (siRNA) technology, and overexpressing dominant-negative mutants of p38 MAPK isotypes of α, β, γ and δ, we have identified that the activation of ERK2 (p42) and p38α, but not of ERK1 and the other isoforms of p38 MAPK, is required for LPA-induced migration. Our study provides the first evidence for a functional role of p42 and p38α in LPA-induced mammalian cell migration, and also demonstrates, for the first time, that the receptor LPA1 mediates prostate cancer cell migration. The results of the present study suggest that LPA, the receptor LPA1, ERK2 and p38α are important regulators for prostate cancer cell invasion and thus could play a significant role in the development of metastasis.
Lysophosphatidic acid; Receptors; Cell migration; Protein kinases and prostate cancer cells
Lysophosphatidic acid (LPA) has been found to accumulate in high concentrations in atherosclerotic lesions. LPA is a bioactive phospholipid produced by activated platelets and formed during the oxidation of LDL. Accumulating evidence suggests that this lipid mediator may serve as an important risk factor for development of atherosclerosis and thrombosis. The role of LPA in atherogenesis is supported by the evidence that LPA: stimulates endothelial cells to produce adhesion molecules and chemoattractants; induces smooth muscle cells to produce inflammatory cytokines; stimulates smooth muscle cell dedifferentiation, proliferation, and migration; increases monocyte migration and decreases monocyte-derived cell emigration from the vessel wall; induces hypertension and vascular neointimal formation in vivo; and promotes plaque progression in a mouse atherosclerosis model. The role of LPA in thrombogenesis is supported by the evidence that LPA markedly induces the aggregation of platelets and the expression of tissue factor, which is the principal initiator of blood coagulation. Recent experimental data indicate that LPA is produced by specific enzymes and that LPA binds to and activates multiple G-protein-coupled receptors, leading to intracellular signaling. Therapeutics targeting LPA biosynthesis, metabolism and signaling pathways could be viable for prevention and treatment of atherosclerosis and thrombosis.
atherosclerosis; lysophosphatidic acid; lysophosphatidic acid receptor; lysophospholipase D; thrombosis; vascular disease and therapeutics; vascular lesion
Although gene therapy was regarded as a promising approach for glioma treatment, its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems. Mesenchymal stem cells (MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy. Therefore, in this study, we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus. We firstly compared the infectivity of type 3, type 5, and type 35 fiber-modified adenoviruses in MSCs. We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo. Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus. MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups. In conclusion, MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma, making it a potential therapeutic strategy for treating malignant glioma.
Mesenchymal stem cell; replication-competent adenovirus; gene therapy; glioma