The MAP1B-LC1/UBE2L3 complex catalyzes degradation of cell surface CaV2.2 channels

We reported recently a new mechanism by which the neuronal N-type Ca²⁺ (Ca_V2.2) channel expression may be regulated by ubiquitination. This mechanism involves the interaction between the channel and the light chain (LC1) of the microtubule associated protein B (MAP1B). We also showed that MAP1B-LC1 could interact with the ubiquitin-conjugating E2 enzyme UBE2L3 and that the ubiquitination/degradation mechanism triggered by MAP1B-LC1 could be prevented by inhibiting the ubiquitin-proteasome proteolytic pathway. We now report that MAP1B-LC1 can interact with the 2 main variants of the Ca_V2.2 channels (Ca_V2.2e37a and Ca_V2.2e37b) and that the MAP1B-LC1-mediated regulation most likely involves an internalization of the channels via a dynamin and clathrin-dependent pathway. In addition, here we propose that this novel mechanism of Ca_V channel regulation might be conserved among N-type and P/Q-type channels.     

The MAP1B-LC1/UBE2L3 complex catalyzes degradation of cell surface CaV2.2 channels

We reported recently a new mechanism by which the neuronal N-type Ca²⁺ (Ca_V2.2) channel expression may be regulated by ubiquitination. This mechanism involves the interaction between the channel and the light chain (LC1) of the microtubule associated protein B (MAP1B). We also showed that MAP1B-LC1 could interact with the ubiquitin-conjugating E2 enzyme UBE2L3 and that the ubiquitination/degradation mechanism triggered by MAP1B-LC1 could be prevented by inhibiting the ubiquitin-proteasome proteolytic pathway. We now report that MAP1B-LC1 can interact with the 2 main variants of the Ca_V2.2 channels (Ca_V2.2e37a and Ca_V2.2e37b) and that the MAP1B-LC1-mediated regulation most likely involves an internalization of the channels via a dynamin and clathrin-dependent pathway. In addition, here we propose that this novel mechanism of Ca_V channel regulation might be conserved among N-type and P/Q-type channels.     

Apoptosis induced by doxorubicin in neurotumor cells is divorced from drug effects on ceramide accumulation and may involve cell cycle-dependent caspase activation

Doxorubicin (0.5 microgram/ml) induced caspase-dependent apoptosis in SH-SY5Y neuroblastoma and CHP-100 neuroepithelioma cells. The apoptotic response started to be evident approximately 15 h after drug administration and, as monitored over a 48-h period, was more pronounced in CHP-100 than in SH-SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B(1) (25 microM); this compound, however, did not prevent drug-induced apoptosis. Untreated cells from both lines expressed negligible p53 levels; on the other hand, whereas p53 and p21(Cip1/Waf1) were rapidly up-regulated in doxorubicin-treated SH-SY5Y cells, such a response was not observed in CHP-100 cells. Doxorubicin induced a G(2)/M phase block in both cell lines, but whereas the G(1) phase was markedly depleted in CHP-100 cells, it was substantially retained in SH-SY5Y cells. In the latter system, double G(1) and G(2)/M block largely preceded cell death; however, as apoptosis underwent completion, it selectively targeted late S and G(2)/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G(1) cell population. These results support the notion that doxorubicin-induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G(1) cells appear to be refractory to doxorubicin-induced apoptosis, p53 up-regulation and p21(Cip1/Waf1) expression may provide an unfavorable setting for the apoptotic action of the drug.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

Microtubule-associated protein 1B: a neuronal binding partner for gigaxonin

Giant axonal neuropathy (GAN), an autosomal recessive disorder caused by mutations in GAN, is characterized cytopathologically by cytoskeletal abnormality. Based on its sequence, gigaxonin contains an NH2-terminal BTB domain followed by six kelch repeats, which are believed to be important for protein-protein interactions (Adams, J., R. Kelso, and L. Cooley. 2000. Trends Cell Biol. 10:17-24.). Here, we report the identification of a neuronal binding partner of gigaxonin. Results obtained from yeast two-hybrid screening, cotransfections, and coimmunoprecipitations demonstrate that gigaxonin binds directly to microtubule-associated protein (MAP)1B light chain (LC; MAP1B-LC), a protein involved in maintaining the integrity of cytoskeletal structures and promoting neuronal stability. Studies using double immunofluorescent microscopy and ultrastructural analysis revealed physiological colocalization of gigaxonin with MAP1B in neurons. Furthermore, in transfected cells the specific interaction of gigaxonin with MAP1B is shown to enhance the microtubule stability required for axonal transport over long distance. At least two different mutations identified in GAN patients (Bomont, P., L. Cavalier, F. Blondeau, C. Ben Hamida, S. Belal, M. Tazir, E. Demir, H. Topaloglu, R. Korinthenberg, B. Tuysuz, et al. 2000. Nat. Genet. 26:370-374.) lead to loss of gigaxonin-MAP1B-LC interaction. The devastating axonal degeneration and neuronal death found in GAN patients point to the importance of gigaxonin for neuronal survival. Our findings may provide important insights into the pathogenesis of neurodegenerative disorders related to cytoskeletal abnormalities.     

TIMP-1基因缄默削弱SH-SY5Y细胞的增殖潜能并诱导其凋亡增强

前期研究发现,人基质金属蛋白酶组织抑制剂-1(tissue inhibitors of metalloproteinases-1,TIMP-1)在唐氏综合征（Down’s syndrome ,DS)胎儿脑组织内表达下调．为了探讨TIMP-1表达下调参与DS脑病变发生的可能机制,本研究以人神经母细胞瘤细胞（SH-SY5Y）为模型,观察TIMP-1基因沉默后对其增殖和凋亡的影响．应用LipofectaminTM2000将TIMP-1特异性短发卡 RNA( short hairpin RNA,shRNA)导入SH-SY5Y细胞,经嘌呤霉素筛选获得稳定表达TIMP-1-shRNA细胞株;应用RT-PCR、real-time PCR和Western 印迹对干扰效率进行鉴定：与SH-SY5Y细胞相比,无论在mRNA水平还是蛋白水平,SH-SY5Y-TIMP-1-shRNA细胞中TIMP-1的表达显著下调（下调率接近100%）．结果显示,已成功构建了TIMP-1基因沉默的SH-SY5Y细胞模型．在此基础上,通过MTT检测发现,TIMP-1基因沉默后SH-SY5Y细胞增殖减慢;流式细胞仪和荧光显微镜凋亡检测显示,TIMP-1基因沉默后SH-SY5Y细胞凋亡明显增加．这些研究结果表明,TIMP-1基因沉默能削弱SH-SY5Y细胞的增殖能力并增强SH-SY5Y的凋亡效应,提示TIMP-1可能是通过影响神经细胞的增殖和凋亡参与DS智力低下的发病过程．     

Cu,Zn-superoxide dismutase-dependent apoptosis induced by nitric oxide in neuronal cells

Nitric oxide (NO) challenge to human neuroblastoma cells (SH-SY5Y) ultimately results in apoptosis. Tumor suppressor protein p53 and cell cycle inhibitor p21 accumulate as an early sign of S-nitrosoglutathione-mediated toxicity. Cytochrome c release from mitochondria and caspase 3 activation also occurred. Cells transfected with either wild type (WT) or mutant (G93A) Cu, Zn-superoxide dismutase (Cu,Zn-SOD) produced comparable amounts of nitrite/nitrate but showed different degree of apoptosis. G93A cells were the most affected and WT cells the most protected; however, Cu, Zn-SOD content of these two cell lines was 2-fold the SH-SY5Y cells under both resting and treated conditions. We linked decreased susceptibility of the WT cells to higher and more stable Bcl-2 and decreased reactive oxygen species. Conversely, we linked G93A susceptibility to increased reactive oxygen species production since simultaneous administration of S-nitrosoglutathione and copper chelators protects from apoptosis. Furthermore, G93A cells showed a significant decrease of Bcl-2 expression and, as target of NO-derived radicals, showed lower cytochrome c oxidase activity. These results demonstrate that resistance to NO-mediated apoptosis is strictly related to the level and integrity of Cu,Zn-SOD and that the balance between reactive nitrogen and reactive oxygen species regulates neuroblastoma apoptosis.     

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

Aims

This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.

Main methods

CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit.

Key findings

Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC₅₀ of 45 μM. Ft1 not only arrested the cell cycle at S, G₂/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1.

Significance

Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.     

LncRNA MALAT1 targeting miR-124-3p regulates DAPK1 expression contributes to cell apoptosis in Parkinson's Disease

Death associated protein kinase 1 (DAPK1) was initially discovered in the progress of gamma-interferon induced programmed cell death, it is a key factor in the central nervous system, including Parkinson's disease (PD). However, the underlying mechanisms of DAPK1 in PD remain unclear and this research work aims to explore the potential mechanisms of DAPK1 in PD. In the study, we exposed SH-SY5Y cells to MPP⁺ and treated mice with MPTP to investigate the roles of DAPK1 in PD and the underlying mechanisms. The results indicated that the expression of DAPK1 is significantly upregulated and negatively correlated with miR-124-3p levels in SH-SY5Y cells treated by MPP⁺, and miR-124-3p mimics could effectively inhibit DAPK1 expressions and alleviate MPP⁺-induced cell apoptosis. In addition, knockdown MALAT1 reduces the levels of DAPK1 and the ratio of SH-SY5Y cell apoptosis, which is reversed via miR-124-3p inhibitor in vitro. Similarly, knockdown MALAT1 could improve behavioral changes and reduce apoptosis by miR-124-3p upregulation and DAPK1 downregulation in MPTP induced PD mice. Taken together, our data showed that lncRNA MALAT1 positively regulates DAPK1 expression by targeting miR-124-3p, and mediates cell apoptosis and motor disorders in PD. In summary, these results suggest that MALAT1/miR-124-3p /DAPK1 signaling cascade mediates cell apoptosis in vitro and in vivo, which may provide experimental evidence of developing potential therapeutic strategies for PD.     

(R)-roscovitine (CYC202, Seliciclib) sensitizes SH-SY5Y neuroblastoma cells to nutlin-3-induced apoptosis

In this study, we have analyzed the consequences, on several neuroblastoma cell lines, of combined treatments with (R)-roscovitine (CYC202, Seliciclib), a CDK inhibitory drug, and nutlin-3, a p53 activating drug. Both compounds were found to synergize, causing significant levels of apoptosis in cultured cells when combined at sublethal concentrations. In SH-SY5Y cells, Bcl-XL protein overexpression protected from apoptosis induced by either nutlin-3 alone or the (R)-roscovitine plus nutlin-3 association but failed to prevent apoptosis triggered by (R)-roscovitine alone. Moreover, Western blot studies showed that (R)-roscovitine increased nutlin-3-mediated p53 stabilization. Therefore, we conclude the contribution of (R)-roscovitine to the synergism is basically the sensitization of SH-SY5Y cells to the action of nutlin-3 on p53. The relevance of this pharmacological synergism with respect to the treatment of neuroblastoma is discussed.     

CHD5 is a tumor suppressor at human 1p36

Cancer gene discovery has relied extensively on analyzing tumors for gains and losses to reveal the location of oncogenes and tumor suppressor genes, respectively. Deletions of 1p36 are extremely common genetic lesions in human cancer, occurring in malignancies of epithelial, neural, and hematopoietic origin. Although this suggests that 1p36 harbors a gene that drives tumorigenesis when inactivated, the identity of this tumor suppressor has remained elusive. Here we use chromosome engineering to generate mouse models with gain and loss of a region corresponding to human 1p36. This approach functionally identifies chromodomain helicase DNA binding domain 5 (Chd5) as a tumor suppressor that controls proliferation, apoptosis, and senescence via the p19(Arf)/p53 pathway. We demonstrate that Chd5 functions as a tumor suppressor in vivo and implicate deletion of CHD5 in human cancer. Identification of this tumor suppressor provides new avenues for exploring innovative clinical interventions for cancer.     

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.     

Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.     

An endogenous electrophile that modulates the regulatory mechanism of protein turnover: inhibitory effects of 15-deoxy-Delta 12,14-prostaglandin J2 on proteasome

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield electrophilic PGs, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We have previously shown that 15d-PGJ(2) potently induces apoptosis of SH-SY5Y human neuroblastoma cells via accumulation of the tumor suppressor gene product p53. In the study presented here, we investigated the molecular mechanisms involved in the 15d-PGJ(2)-induced accumulation of p53. It was observed that 15d-PGJ(2) potently induced p53 protein expression but scarcely induced p53 gene expression. In addition, exposure of the cells to 15d-PGJ(2) resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that 15d-PGJ(2) acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. The effects of 15d-PGJ(2) on the protein turnover were attributed to its electrophilic feature, based on the observations that (i) the reduction of the double bond in the cyclopentenone ring of 15d-PGJ(2) virtually abolished the effects on protein turnover, (ii) overexpression of an endogenous redox regulator, thioredoxin 1, significantly retarded the inhibition of proteasome activities and accumulations of p53 and ubiquitinated proteins induced by 15d-PGJ(2), and (iii) treatment of SH-SY5Y cells with biotinylated 15d-PGJ(2) indeed resulted in the formation of a 15d-PGJ(2)-proteasome conjugate. These data suggest that the modulation of proteasome activity may be involved in the mechanism responsible for the accumulation of p53 and subsequent induction of apoptotic cell death induced by 15d-PGJ(2).