Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263

Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP₃ receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP₃Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2–IP₃R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2.Lung cancer accounts for 12% of all new cancers worldwide and is a leading cause of cancer-related mortality in the United States.^{1, 2, 3} Although small cell lung cancer (SCLC) comprises only 15% of lung cancer cases,^{2, 3} it has an annual mortality rate approaching that of breast and prostate cancer.⁴ Compared with the more common non-small cell lung cancer (NSCLC), SCLC is more aggressive and associated with rapid development of metastasis.² Moreover, although SCLC is more responsive to chemotherapy and radiation therapy initially, it typically relapses quickly with treatment-resistant disease.² In contrast to dramatic advances in chemotherapy and personalized medicine in other malignancies, the life expectancy of SCLC patients has remained <2 years for decades and is <1 year for patients with extensive disease.^{5, 6} The lethality of SCLC is attributed in part to the development of resistance to standard combination chemotherapies, underscoring the need to develop novel therapeutic approaches based on understanding the molecular and cellular biology of SCLC.^{5, 6}Evasion from apoptosis is a major hallmark of cancer and a prominent factor underlying drug resistance in SCLC.³ Multiple mechanisms contribute to apoptosis resistance in SCLC, including elevated expression of the antiapoptotic Bcl-2 protein³ (Supplementary Figure S1). Tsujimoto and colleagues discovered elevated levels of Bcl-2 mRNA and protein in SCLC cells not long after their identification of Bcl-2 as the protein product of the bcl-2 gene in follicular lymphoma.^{7, 8} Subsequently, immunohistochemistry of 164 primary SCLC samples revealed 76% were positive for Bcl-2, a finding substantiated by microarray detection of increased BCL-2 mRNA levels in 84% of SCLC samples^{9, 10} and by genomic sequencing of circulating SCLC tumor cells.¹¹ Moreover, proteomic profiling documented that Bcl-2 is more highly expressed in SCLC than in NSCLC, reflecting the vastly different biology of these lung cancer subtypes.¹²The major known function of Bcl-2 is to bind and sequester BH3-only proteins such as Bim, preventing these proteins from inducing apoptosis.^{13, 14, 15} Therefore, a major investment has been made in targeting this interaction for cancer treatment. The interaction takes place in a hydrophobic groove on Bcl-2 and the therapeutic strategy for targeting this interaction has been to develop small molecules, BH3-mimetic agents, which bind in the hydrophobic groove and induce apoptosis by displacing the BH3-only proteins. This approach has been reviewed in detail.^{14, 15, 16}Among BH3-mimetic agents advancing through clinical trials for both hematological malignancies^{15, 17} and solid tumors¹⁸ are ABT-737 and its orally bioavailable derivative ABT-263 (Navitoclax). Reported studies of ABT-199, a selective inhibitor of Bcl-2, are at present limited to hematological malignancies.¹⁸ In screening a large number of cancer cell lines, the pioneering work of Oltersdorf et al.¹⁹ demonstrated potent single-agent activity of ABT-737 against cell lines representative of lymphoid malignancies and SCLC. Clinical trials of ABT-263, an orally bioavailable version of ABT-737, achieved overall response rates ranging from as high as 35% in relapsed/refractory chronic lymphocytic leukemia (CLL) and 22% in follicular lymphoma.¹⁷ Reported responses are generally less in solid tumors with the notable exception of SCLC.¹⁸ But even in SCLC, activity of ABT-263 is limited in comparison to hematological malignancies, with 1 of the 39 (3%) of patients achieving a partial response to ABT-263 and 9 of the 37 (23%) achieving stable disease in a phase I clinical trial.²⁰ This experience suggests a need to develop additional ways of targeting Bcl-2 for cancer treatment.A potential alternative therapeutic target for Bcl-2-positive malignancies involves interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP₃R), an IP₃-gated Ca²⁺ channel located on the endoplasmic reticulum (ER). Bcl-2 is located not only on the outer mitochondrial membrane but also on the ER, and at both of these locations, it functions as a potent inhibitor of apoptosis.^{21, 22, 23} ER-localized Bcl-2 interacts with IP₃Rs and inhibits apoptosis by preventing excessive IP₃R-mediated Ca²⁺ transfer from the ER lumen into the cytoplasm and nearby mitochondria.^{24, 25, 26} Notably, regions of Bcl-2 involved in binding BH3-only proteins and IP₃Rs are entirely different. Whereas BH3-only proteins and their BH3-mimetic counterparts bind in a hydrophobic groove composed of BH3 domains 1–3 of Bcl-2,^{13, 14} the BH4 domain of Bcl-2 is necessary for interaction with IP₃Rs.²⁷ To develop a peptide inhibitor of Bcl-2–IP₃R interaction, we identified the Bcl-2-binding region on the IP₃R and developed a small synthetic 20 amino-acid peptide corresponding to this region.²⁸ This peptide, when fused to the cell-penetrating peptide of HIV TAT, binds to the BH4 domain of Bcl-2 and functions as a decoy peptide, inhibiting Bcl-2–IP₃R interaction.^{29, 30} We currently refer to this peptide as BIRD-2 (Bcl-2-IP₃ Receptor Disruptor-2), having formerly named it TAT-IDP_DD/AA.³¹ By disrupting the Bcl-2–IP₃R interaction, BIRD-2 abrogates Bcl-2 control over IP₃R-mediated Ca²⁺ elevation and induces Ca²⁺-mediated apoptosis in primary human CLL cells²⁹ and diffuse large B-cell lymphoma cells.³² Notably, BIRD-2 does not kill normal cells, including human lymphocytes isolated from peripheral blood²⁹ and normal murine embryonic fibroblasts (F Zhong and C Distelhorst, unpublished data).The present investigation was undertaken to determine whether Bcl-2–IP₃R interaction is a potentially useful therapeutic target in SCLC. In support of this concept, we find the majority of SCLC lines tested are sensitive to BIRD-2-induced apoptosis and that BIRD-2 induces apoptosis in several ABT-263-resistant SCLC lines. BIRD-2, we find, lacks generalized cytotoxicity as it does not induce cell death in NSCLC lines or a normal lung epithelial line. On the other hand, we find that BIRD-2 and ABT-263 synergize in killing SCLC cells. These findings for the first time provide preclinical evidence of the potential value of targeting both antiapoptotic mechanisms of Bcl-2 for the treatment of SCLC.     

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer

Lung cancer is the most common cause of cancer-related mortality worldwide, urging the discovery of novel molecular targets and therapeutic strategies. Stem cells have been recently isolated from non-small cell lung cancer (NSCLC), thus allowing the investigation of molecular pathways specifically active in the tumorigenic population. We have found that Bcl-X_L is constantly expressed by lung cancer stem cells (LCSCs) and has a prominent role in regulating LCSC survival. Whereas chemotherapeutic agents were scarcely effective against LCSC, the small molecule Bcl-2/Bcl-X_L inhibitor ABT-737, but not the selective Bcl-2 inhibitor ABT-199, induced LCSC death at nanomolar concentrations. Differently from gemcitabine, which preferentially eliminated proliferating LCSC, ABT-737 had an increased cytotoxic activity in vitro towards quiescent/slow-proliferating LCSC, which expressed high levels of Bcl-X_L. In vivo, ABT-737 as a single agent was able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors. Altogether, these results indicate that quiescent/slow-proliferating LCSC strongly depend on Bcl-X_L for their survival and indicate Bcl-X_L inhibition as a potential therapeutic avenue in NSCLC.Lung cancer is the leading cause of cancer-related death in men and is expected to become the main cause of cancer death for women in the near future.^{1, 2} There is increasing evidence that cancer stem cells (CSCs) have a key role in drug resistance, tumor progression and metastasis in multiple tumor types, including lung cancer.³ Lung cancer stem cells (LCSCs) have been previously identified through different criteria including surface expression of CD133, c-kit or through functional properties such as selective drug survival, elevated aldehyde dehydrogenase (ALDH) activity, increased glycolysis and glycine/serine metabolism or low concentrations of reactive oxygen species and ATP.^{4, 5, 6, 7, 8, 9} Importantly, when inoculated into immunocompromised mice, LCSCs give rise to xenografts that histologically reproduce the tumor of origin, thus representing an improved model for in vivo testing of new targeted therapies.¹⁰ Several tumors express elevated levels of anti-apoptotic Bcl-2 family proteins such as Bcl-2, Bcl-X_L and Mcl-1, which affect the apoptotic threshold of neoplastic cells contributing to chemotherapy resistance.¹¹ Inhibition of anti-apoptotic Bcl-2 family members has been for long time regarded as a promising strategy to induce cancer cell death through approaches of increasing specificity. BH3 mimetics such as ABT-737, the related orally available ABT-263 (navitoclax) and the recently developed Bcl-2-selective inhibitor ABT-199 have been shown to exert an antitumor effect in preclinical and clinical settings either as single agents or in combination with conventional or targeted drugs.¹² Recently, a new role for Bcl-2 has emerged in acute myeloid leukemia (AML), where quiescent stem cells characterized by low levels of reactive oxygen species were found to overexpress Bcl-2 and rely on this factor for survival.¹³ Similarly, in chronic myeloid leukemia (CML), quiescent therapy-resistant stem cells were sensitized to tyrosine kinase inhibitors by treatment with a pan-Bcl-2 inhibitor.¹⁴ In solid tumors, the role of Bcl-2 family members in regulating the stem cell compartment is less clear. By analyzing the expression and relative function of Bcl-2 and Bcl-X_L in LCSC, we identified a prevalent role of Bcl-X_L in LCSC survival. Differently from chemotherapy, ABT-737 showed a preferential cytotoxic activity towards quiescent/slowly proliferating LCSC in vitro indicating a potential use of this inhibitor to eradicate chemotherapy-resistant LCSC. In vivo, ABT-737 blocked the progression of LCSC-derived xenografts and reduced CSC content, substantiating its specific effect on the CSC compartment. Altogether, these results indicate for the first time a key role of Bcl-X_L in LCSC, opening new perspectives for the elimination of therapy-resistant cells.     

The ratio of Mcl-1 and Noxa determines ABT737 resistance in squamous cell carcinoma of the skin

Tumour progression and therapy resistance in squamous cell carcinoma of the skin (SCC) is strongly associated with resistance to intrinsic mitochondrial apoptosis. We thus investigated the role of various anti-apoptotic Bcl-2 proteins for apoptosis protection in SCC using the BH3 agonist ABT737 that can overcome multidomain Bcl-2 protein protection. Sensitive SCC cells underwent rapid loss of mitochondrial membrane potential (MMP), subsequent apoptosis concomitant with caspase-3 activation and an early release of mitochondria-derived cytochrome c and smac/DIABLO. In contrast, ABT737 resistance in subsets of SCC cells was not explained by XIAP, important for protection from DR-induced apoptosis in SCC. Of note, ABT737 did not prime SCC cells to DR-induced apoptosis. Interestingly, the ratio of Mcl-1 and Noxa determined sensitivity to ABT737: loss of Mcl-1 rendered resistant cells sensitive to ABT737, whereas loss of Noxa promoted resistance in sensitive cells. In line, suppression of Mcl-1 by the pan-Bcl-2 inhibitor Obatoclax or overexpression of Noxa rendered resistant SCC cells sensitive to BH3 mimetics. Our data indicate that targeting of the Mcl-1/Noxa axis is important to overcome resistance to mitochondrial apoptosis in SCC. Therefore, combination treatment of ABT737 or derivatives with Mcl-1 inhibitors, or inducers of Noxa, may represent a novel option of targeted therapy in metastatic SCC of the skin.Apoptosis is an indispensible process to maintain cellular homeostasis, in particular in highly dynamic tissues. Apoptosis can be induced by activation of death receptors (DRs; such as TRAIL-R1/R2 or cluster of differentiation 95 (CD95)) or by intrinsic disturbance of mitochondria.¹ Death ligands (DLs; TNF-related apoptosis-inducing ligand (TRAIL) or CD95L), when bound to their respective DRs, induce apoptosis by activation of procaspase-8 within the death-inducing signalling complex (DISC).² Caspase-8 activation is followed by proteolytic cleavage of caspase-3.³ Extrinsic and intrinsic cell death is negatively controlled by caspase inhibitors such as X-linked inhibitor of apoptosis protein (XIAP)⁴ or by B-cell lymphoma 2 (Bcl-2) proteins that suppress the mitochondria outer membrane permeability (MOMP) by limiting Bax (Bcl-2-associated X protein)/Bak (Bcl-2 homologous antagonist/killer) translocation into the mitochondrial outer membrane.⁵ The extrinsic signalling cascade communicates with the intrinsic death pathway by cleavage of Bid (BH3 interacting-domain death agonist), a pro-apoptotic member of the BH3 (Bcl-2 homology domain 3)-only subfamily of Bcl-2 proteins.¹ Other stimuli such as genotoxic stress allow for translocation and pore formation of pro-apoptotic multidomain Bcl-2 proteins Bax and Bak in the outer mitochondrial membrane.^{6, 7, 8} This process promotes release of mitochondria-derived apoptogenic proteins, in particular cytochrome c,⁹ or smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI).¹⁰ Within the apoptosome,¹¹ active caspase-9 finally leads to activation of caspase-3,¹² and subsequent cell death.Anti-apoptotic multidomain Bcl-2 proteins (Bcl-2, Bcl-2-like protein 2 (Bcl-w), B-cell lymphoma-extra large (Bcl-X_L), induced myeloid leukaemia cell differentiation protein (Mcl-1) and Bcl-2-related protein A1 (A1)) with four Bcl-2 homology domains (BH1, BH2, BH3 and BH4) suppress the pro-apoptotic function of Bax-like proteins such as Bax, Bak and Bok (that contain BH1–BH3 domains) or the BH3-only proteins Bad (Bcl-2-associated death promoter), Bim (Bcl-2-like protein 11), Bid, Noxa (phorbol-12-myristate-13-acetate-induced protein 1) and Puma (p53 upregulated modulator of apoptosis).¹³ Regulation of mitochondria-mediated apoptosis is determined by the balance between pro- and anti-apoptotic Bcl-2 proteins.¹⁴In a variety of cancer types, a decrease of BH3-only protein or upregulation of pro-survival Bcl-2 proteins is associated with poor prognosis.¹⁵ In metastatic squamous cell carcinoma (SCC) of the skin or the so-called ‘head and neck SCC'' (HNSCC), high expression of pro-survival Bcl-2 proteins conferred radio- and chemotherapy resistance.^{16, 17} These findings mark Bcl-2 proteins as regulators of SCC apoptosis and indicate that BH3 mimetics may hold therapeutic potential for metastatic SCC. The BH3 mimetics navitoclax (ABT263) and ABT199 are currently under investigation in clinical studies.^{18, 19, 20} Mechanistically, their lead compound ABT737 suppresses Bcl-2 activity by binding to the hydrophobic groove of Bcl-2, Bcl-w and Bcl-X_L.¹⁸ As ABT263 upregulates Mcl-1, resistance to a number of Bcl-2 inhibitors (ABT737 and ABT263) has been described.²¹ Another compound, Obatoclax, was developed to block all anti-apoptotic Bcl-2 proteins including Mcl-1.²² Obatoclax blocks the interaction of Bim or Bax with Mcl-1.²³ In this report, we have studied the effect of ABT737 for cell death in SCC of the skin and investigated the molecular mechanisms of resistance to different BH3 mimetics.     

Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

Mitochondrial inhibitor sensitizes non-small-cell lung carcinoma cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-XL/p53-mediated amplification mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-X_L overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-X_L downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-X_L and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-X_L and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising cancer therapeutic because it can selectively induce apoptosis in tumor cells in vitro, and most importantly, in vivo with little adverse effect on normal cells.¹ However, a number of cancer cells are resistant to TRAIL, especially highly malignant tumors such as lung cancer.^{2, 3} Lung cancer, especially the non-small-cell lung carcinoma (NSCLC) constitutes a heavy threat to human life. Presently, the morbidity and mortality of NSCLC has markedly increased in the past decade,⁴ which highlights the need for more effective treatment strategies.TRAIL has been shown to interact with five receptors, including the death receptors 4 and 5 (DR4 and DR5), the decoy receptors DcR1 and DcR2, and osteoprotegerin.⁵ Ligation of TRAIL to DR4 or DR5 allows for the recruitment of Fas-associated protein with death domain (FADD), which leads to the formation of death-inducing signaling complex (DISC) and the subsequent activation of caspase-8/10.⁶ The effector caspase-3 is activated by caspase-8, which cleaves numerous regulatory and structural proteins resulting in cell apoptosis. Caspase-8 can also cleave the Bcl-2 inhibitory BH3-domain protein (Bid), which engages the intrinsic apoptotic pathway by binding to Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (BAK). The oligomerization between Bcl-2 and Bax promotes the release of cytochrome c from mitochondria to cytosol, and facilitates the formation of apoptosome and caspase-9 activation.⁷ Like caspase-8, caspase-9 can also activate caspase-3 and initiate cell apoptosis. Besides apoptosis-inducing molecules, several apoptosis-inhibitory proteins also exist and have function even when apoptosis program is initiated. For example, cellular FLICE-like inhibitory protein (c-FLIP) is able to suppress DISC formation and apoptosis induction by sequestering FADD.^{8, 9, 10, 11}Until now, the recognized causes of TRAIL resistance include differential expression of death receptors, constitutively active AKT and NF-κB,^{12, 13} overexpression of c-FLIP and IAPs, mutations in Bax and BAK gene.² Hence, resistance can be overcome by the use of sensitizing agents that modify the deregulated death receptor expression and/or apoptosis signaling pathways in cancer cells.⁵ Many sensitizing agents have been developed in a variety of tumor cell models.² Although the clinical effectiveness of these agents needs further investigation, treatment of TRAIL-resistant tumor cells with sensitizing agents, especially the compounds with low molecular weight, as well as prolonged plasma half-life represents a promising trend for cancer therapy.Mitochondria emerge as intriguing targets for cancer therapy. Metabolic changes affecting mitochondria function inside cancer cells endow these cells with distinctive properties and survival advantage worthy of drug targeting, mitochondria-targeting drugs offer substantial promise as clinical treatment with minimal side effects.^{14, 15, 16} Rotenone is a potent inhibitor of NADH oxidoreductase in complex I, which demonstrates anti-neoplastic activity on a variety of cancer cells.^{17, 18, 19, 20, 21} However, the neurotoxicity of rotenone limits its potential application in cancer therapy. To avoid it, rotenone was effectively used in combination with other chemotherapeutic drugs to kill cancerous cells.²²In our previous investigation, we found that rotenone was able to suppress membrane Na⁺,K⁺-ATPase activity and enhance ouabain-induced cancer cell death.²³ Given these facts, we wonder whether rotenone may also be used as a sensitizing agent that can restore the susceptibility of NSCLC cells toward TRAIL-induced apoptosis, and increase the antitumor efficacy of TRAIL on NSCLC. To test this hypothesis, we initiated this study.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim

Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.Variola virus (VAR), the causative agent of smallpox, is a member of the poxvirus family and belongs to the orthopoxviridae. Despite its successful eradication nearly 30 years ago, VAR remains an ongoing concern because of its potential use as a bioterrorism agent.¹ The threat of intentional use of VAR coupled with the absence of an FDA-approved drug for the prevention or treatment of smallpox infection is cause for considerable interest in the development of small-molecule therapeutics against VAR. Current strategies for dealing with smallpox are based on vaccination using live vaccinia virus (VV),^{2, 3} a closely related member of the orthopoxvirus genus, which shares >90% sequence identity with VAR. Vaccination using live VV, however, can cause serious complications,⁴ underscoring the need for effective anti-viral treatments, particularly since anti-viral treatment may be a more efficacious strategy compared with vaccination.⁵ Recent strategies to target VAR for small-molecule therapeutics included the use of polymerase inhibitors,⁶ notably Cidofovir, inhibitors of extracellular virus formation⁷ and tyrosine kinase inhibitors including Gleevec.^{8, 9} Cidofovir is currently the only approved antiviral drug for the treatment of orthopoxviruses, although it is not approved for smallpox treatment. Other host–virus interactions have been identified that may be suitable drug targets^{10, 11} but currently require further investigation.Several poxvirus members other than VAR have been shown to rely on virulence factors that prevent premature host cell demise via programmed cell death or apoptosis,^{12, 13, 14, 15, 16} thus ensuring survival and proliferation. The B-cell lymphoma 2 (Bcl-2) protein family is a key mediator for maintaining cell survival or to drive apoptosis, thereby removing infected, damaged or unwanted cells,¹⁷ and sequence, structural and functional orthologs of Bcl-2 have been found in a number of poxviruses.¹⁸ Certain viral Bcl-2-like proteins were only identified as family members after their 3D structures were determined, owing to their complete lack of sequence identity to mammalian Bcl-2 proteins. This group of proteins include the myxoma virus M11L¹² and VV F1L¹⁵ and N1L.¹⁹ Myxoma virus M11L was shown to adopt the classical Bcl-2 fold^{20, 21} that utilizes the canonical Bcl-2 homology 3 (BH3)-binding groove to engage BH3 ligands to exert its pro-survival effect. VV F1L also adopts a Bcl-2 fold, but unlike M11L it exists as a domain-swapped dimer,^{22, 23} whereas N1L also adopted a dimeric Bcl-2 fold but with a different dimeric arrangement.^{24, 25}Although F1L from VAR has not previously been investigated, the VV homolog is well characterized. VV F1L has been shown to inhibit the mitochondrial pathway of apoptosis by replacing Mcl-1²⁶ and interacts with the isolated BH3 domains of Bim, Bax and Bak,²³ which are bound in the canonical Bcl-2-binding groove.²² Furthermore, an F1L-deficient VV potently causes Bak/Bax-mediated apoptosis.^{15, 27} Functionally, VV F1L appears to rely primarily on neutralization of Bim in the context of a viral infection.²² Given the close similarity between VAR and VV, VAR may also rely on inhibition of host cell apoptosis for successful infection and proliferation. Disruption of VAR ability to inhibit apoptosis thus may constitute an attractive strategy for small-molecule-based intervention. To investigate this possibility, we performed a biochemical, structural and functional characterization of VAR F1L. Here we report that despite possessing a nearly identical 3D structure and sequence, VAR F1L inhibits apoptosis via a different mechanism compared with its homolog in VV.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.