Carvedilol Attenuates 6-Hydroxydopamine-Induced Cell Death in PC12 Cells: Involvement of Akt and Nrf2/ARE Pathways

Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinson’s disease. Carvedilol, a nonselective β-adrenergic receptor blocker with pleiotropic activity has been shown to exert neuroprotective effect due to its antioxidant property. However, the neuroprotective mechanism of carvedilol is still not fully uncovered. The phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway plays key role in cell survival and the nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is the major cellular defense mechanism against oxidative stress. Here we investigated the effects of carvedilol on 6-hydroxydopamine (6-OHDA)-induced cell death as well as the Akt and Nrf2/ARE pathways in PC12 cells. We found that carvedilol significantly increased cell viability and decreased reactive oxygen species in PC12 cells exposed to 6-OHDA. Furthermore, carvedilol activated the Akt and Nrf2/ARE pathways in a concentration-dependent manner, and increased the protein levels of heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase-1(NQO-1), two downstream factors of the Nrf2/ARE pathway. In summary, our results indicate that carvedilol protects PC12 cells against 6-OHDA-induced neurotoxicity possibly through activating the Akt and Nrf2/ARE signaling pathways.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Localization of Murine X and Autosomal Sequences Homologous to the Human Y Located Testis-Determining Region

Recently a candidate gene for the primary testis-determining factor (TDF) encoding a zinc finger protein (ZFY) has been cloned from the human Y chromosome. A highly homologous X-linked copy has also been identified. Using this human sequence it is possible to identify two Y loci, an X and an autosomal locus in the mouse (Zfy-1, Zfy-2, Zfx and Zfa, respectively). Suprisingly ZFY is more homologous to the mouse X and autosomal sequences than it is to either of the Y-linked loci. Both Zfy-1 and Zfy-2 are present in the Sxr region of the Y but Zfy-2 is absent in the Sxr deletion variant Sxrb (or Sxr") suggesting it is not necessary for male determination. Extensive backcross analyses map Zfa to mouse chromosome 10 and Zfx to a 5-cM interval between anonymous X probe MDXS120 and the tabby locus (Ta). We also show that the mouse androgen receptor locus (m-AR) believed to underlie the testicular feminization mutation (Tfm) shows complete linkage to Zfx. Comparative mapping indicates that in man these genes lie in separate conserved DNA segments.     

Increased LRRK2 kinase activity alters neuronal autophagy by disrupting the axonal transport of autophagosomes

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Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO^LRRK2 self-interaction. Interestingly, ROCO^LRRK2 fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO^LRRK2 reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO^LRRK2 fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.     

Exon-Skipping Strategy by Ratio Modulation between Cytoprotective versus Pro-Apoptotic Clusterin Forms Increased Sensitivity of LNCaP to Cell Death

Background

In prostate cancer the secreted form of clusterin (sCLU) has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties.

Methodology

In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress.

Results and Conclusions

We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line “LNCaP” after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an “on demand alternative splicing” strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.     

Defects in DNA Lesion Bypass Lead to Spontaneous Chromosomal Rearrangements and Increased Cell Death

Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1Δ is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3Δ mutant and are essential for survival of rev3Δ cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1Δ or rrm3Δ helicase mutations, whereas mph1Δ is hypostatic to sgs1Δ. Previously reported slow growth of mph1Δ srs2Δ cells is accompanied by G₂/M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.Nonreplicative DNA helicases play an important role in the maintenance of genome stability from bacteria to humans, most likely by affecting the formation and/or resolution of recombination intermediates and by facilitating replication fork progression through chromosomal regions with a propensity to adopt unusual DNA structures or those bound by proteins. In Saccharomyces cerevisiae, this group of DNA helicases includes the 3′-to-5′ helicases Sgs1 and Srs2 and the 5′-to-3′ DNA helicase Rrm3. In the absence of any two of these three helicases, unresolved recombination intermediates accumulate and lead to extremely slow growth that is fully suppressed by deletion of genes encoding early homologous recombination (HR) factors (4, 6, 17, 20, 37, 46). In the absence of Sgs1, cells exhibit increased rates of mitotic recombination, frequent chromosome missegregation, accumulation of extrachromosomal ribosomal DNA (rDNA) circles, and increased rates of gross chromosomal rearrangements (GCRs) involving nonhomologous chromosomes (5, 24, 25, 38, 40, 43, 49, 50). Based on the increased crossover frequency during HO endonuclease-induced double-strand breaks (DSBs) in cells lacking Sgs1, it has also been proposed that Sgs1 may function in decatenation of Holliday junctions (HJs) to yield noncrossovers (12, 22). Like Sgs1, Srs2 acts to favor noncrossover outcomes during DSB repair but appears to act earlier than Sgs1 in regulating recombination outcomes through its ability to dislodge Rad51 from recombinogenic 3′ overhangs, thereby promoting a noncrossover synthesis-dependent single-strand annealing (SDSA) pathway (12, 33, 35). In contrast, Rrm3 has not been implicated in DNA repair but is thought to be important for avoidance of recombination substrate formation by removal of DNA protein complexes in certain chromosomal locations, such as chromosome ends and replication fork barriers at the rDNA locus, thus facilitating replication fork progression (13, 14).In addition to Sgs1, Rrm3, and Srs2, the yeast genome encodes two other nonreplicative DNA helicases with proposed functions in DNA repair, Mph1 and Chl1. Mph1 possesses 3′-to-5′ helicase activity, and its ATPase activity requires a relatively long fragment of single-stranded DNA (ssDNA) (≥40 nucleotides [nt]) for full activity in vitro (32). Mph1 is also necessary for resistance to the DNA damaging agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) and suppresses spontaneous mutations toward canavanine resistance (3, 41). The modest mutator phenotype of the mph1Δ mutant is enhanced by additional mutations in base excision repair (apn1Δ and apn2Δ) and is suppressed by mutations in translesion DNA synthesis (TLS) (rev3Δ) (36, 41). These findings, in combination with the observation of an epistatic relationship between mph1Δ and homologous recombination mutations, have led to the proposal that Mph1 may act in Rad52-dependent, error-free bypassing of DNA lesions (41). Like the 3′-to-5′ DNA helicases Sgs1 and Srs2, Mph1 was recently shown to affect crossover frequency during repair of an HO endonuclease-induced DNA DSB, favoring noncrossovers as the outcome (33). The authors showed that Mph1 can unwind intermediates of homologous recombination in vitro, specifically D loops that are thought to form early during homologous recombination when a homoduplex is invaded by a Rad51 filament. While Srs2 has been shown to be able to disassemble Rad51 filaments in vitro, it does not appear to possess Mph1''s ability to dissociate D loops once they have formed (19, 47).Although Chl1 has been shown to be required for the establishment of sister chromatid cohesion, a possible role in DNA repair by homologous recombination has also been proposed (11, 28, 30, 42). While Chl1 possesses a conserved helicase domain, helicase activity has so far been shown only for its putative human homolog, hCHLR1 (10).To further elucidate the functional interaction between nonreplicative DNA helicases and DNA repair pathways, we generated a series of mutants with combinations of mph1Δ, chl1Δ, rrm3Δ, srs2Δ, and sgs1Δ mutations and mutations in translesion DNA synthesis (TLS), base excision repair (BER), homologous recombination (HR), and DNA damage checkpoints. In addition to synthetic fitness defects due to aberrant HR and checkpoint activation, we identified epistatic and synergistic relationships with regard to fitness, the accumulation of gross chromosomal rearrangements (GCRs), and sensitivity to DNA damage. We propose that Mph1 functions in a Rad51-dependent, Rad59-independent pathway of HR for DNA lesion bypass and interacts genetically with REV3 in the suppression of gross chromosomal rearrangements.     

Peroxiredoxins and the Regulation of Cell Death

Cell death pathways such as apoptosis can be activated in response to oxidative stress, enabling the disposal of damaged cells. In contrast, controlled intracellular redox events are proposed to be a significant event during apoptosis signaling, regardless of the initiating stimulus. In this scenario oxidants act as second messengers, mediating the post-translational modification of specific regulatory proteins. The exact mechanism of this signaling is unclear, but increased understanding offers the potential to promote or inhibit apoptosis through modulating the redox environment of cells. Peroxiredoxins are thiol peroxidases that remove hydroperoxides, and are also emerging as important players in cellular redox signaling. This review discusses the potential role of peroxiredoxins in the regulation of apoptosis, and also their ability to act as biomarkers of redox changes during the initiation and progression of cell death.     

Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 μM NMDA. Within 2 h after switching to 5 mM K⁺, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax −/− neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K⁺ deprivation. Wild-type granule cells in 5 mM K⁺ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.     

Caveolin-1 Deficiency Leads to Increased Susceptibility to Cell Death and Fibrosis in White Adipose Tissue: Characterization of a Lipodystrophic Model

Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1^−/− mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1^−/− adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1^+/+ mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1^−/− mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.     

The Root Hair Assay Facilitates the Use of Genetic and Pharmacological Tools in Order to Dissect Multiple Signalling Pathways That Lead to Programmed Cell Death

The activation of programmed cell death (PCD) is often a result of complex signalling pathways whose relationship and intersection are not well understood. We recently described a PCD root hair assay and proposed that it could be used to rapidly screen genetic or pharmacological modulators of PCD. To further assess the applicability of the root hair assay for studying multiple signalling pathways leading to PCD activation we have investigated the crosstalk between salicylic acid, autophagy and apoptosis-like PCD (AL-PCD) in Arabidopsis thaliana. The root hair assay was used to determine rates of AL-PCD induced by a panel of cell death inducing treatments in wild type plants treated with chemical modulators of salicylic acid synthesis or autophagy, and in genetic lines defective in autophagy or salicylic acid signalling. The assay demonstrated that PCD induced by exogenous salicylic acid or fumonisin B1 displayed a requirement for salicylic acid signalling and was partially dependent on the salicylic acid signal transducer NPR1. Autophagy deficiency resulted in an increase in the rates of AL-PCD induced by salicylic acid and fumonisin B1, but not by gibberellic acid or abiotic stress. The phenylalanine ammonia lyase-dependent salicylic acid synthesis pathway contributed only to death induced by salicylic acid and fumonisin B1. 3-Methyladenine, which is commonly used as an inhibitor of autophagy, appeared to influence PCD induction in all treatments suggesting a possible secondary, non-autophagic, effect on a core component of the plant PCD pathway. The results suggest that salicylic acid signalling is negatively regulated by autophagy during salicylic acid and mycotoxin-induced AL-PCD. However, this crosstalk does not appear to be directly involved in PCD induced by gibberellic acid or abiotic stress. This study demonstrates that the root hair assay is an effective tool for relatively rapid investigation of complex signalling pathways leading to the activation of PCD.     

在血清饥饿条件下CHP2调节NHE活性减少细胞死亡

钠氢离子交换蛋白（NHE）是维持细胞内pH值等内环境稳定的重要蛋白;钙调磷酸酶B同源蛋白（CHP）是NHE的一个活性调节亚单位。研究CHP2对NHE1的调节作用时发现,在血清饥饿的条件下,PS120细胞依赖于CHP2的表达来调节外源性NHE1的活性,使细胞维持必要的钠氢交换生理活性和较高水平的细胞内pH值（pHi 7．4）,明显减少细胞因自身的胞浆酸性化而死亡,延长细胞存活时间（70％以上的细胞存活时间超过7天）。实验结果提示,通过研究减少CHP2表达或抑制其活性,可望找到加速细胞死亡的新方法。     

Sequences within the VP6 molecule of bluetongue virus that determine cytoplasmic and nuclear targeting of the protein.

Genome segment 9 of bluetongue virus serotype 10 encodes the minor protein VP6. The protein is abundant with basic residues particularly in two regions of the carboxy half of the molecule. A series of amino- and carboxy-terminal deletion mutants was expressed in mammalian cells by using a vaccinia virus T7 polymerase-driven transient expression system, and the intracellular fate of the products was monitored by both immunofluorescence staining and cell fractionation techniques. Data obtained indicated clearly that VP6 has nuclear transportation signals which may be correlated with positively charged domains of the molecule. In the intact molecule, though, these signals are masked and the protein is retained in the cytoplasm. The biochemical and immunofluorescence data obtained indicate that sequences in the region of residues 33 to 80 of the 328-amino acid protein are required for the retention of VP6 within the cell cytoplasm while amino acids 303 to 308 in the carboxy-terminal half of the molecule appear to possess nuclear localization capabilities.     

LRRK2 phosphorylates tubulin-associated tau but not the free molecule: LRRK2-mediated regulation of the tau-tubulin association and neurite outgrowth

Leucine-rich repeat kinase 2 (LRRK2), a large protein kinase containing multi-functional domains, has been identified as the causal molecule for autosomal-dominant Parkinson's disease (PD). In the present study, we demonstrated for the first time that (i) LRRK2 interacts with tau in a tubulin-dependent manner; (ii) LRRK2 directly phosphorylates tubulin-associated tau, but not free tau; (iii) LRRK2 phosphorylates tau at Thr181 as one of the target sites; and (iv) The PD-associated LRRK2 mutations, G2019S and I2020T, elevated the degree of tau-phosphorylation. These results provide direct proof that tau is a physiological substrate for LRRK2. Furthermore, we revealed that LRRK2-mediated phosphorylation of tau reduces its tubulin-binding ability. Our results suggest that LRRK2 plays an important role as a physiological regulator for phosphorylation-mediated dissociation of tau from microtubules, which is an integral aspect of microtubule dynamics essential for neurite outgrowth and axonal transport.