Cell Death Discovery

Fish erythrocytes remain nucleated for their life-span, unlike mammalian erythrocytes which undergo enucleation. Asides transportation of oxygen, fish erythrocytes are capable of several immune defence processes. Nucleated fish erythrocytes represent prime candidates for carrying out ETotic responses. ETosis is an evolutionary conserved innate immune defence process found in both vertebrates and invertebrates, which involves the extrusion of DNA studded with antimicrobial proteins into the extracellular space serving to trap and kill microorganisms. In this report, we demonstrate that fish erythrocytes isolated from Danio rerio (zebrafish) produce ETotic-like responses when exposed to chemical and physiological stimuli. Furthermore, we found Salmo salar (Atlantic salmon) erythrocytes produce similar ETotic responses. We have termed these ET-like formations Fish Erythrocyte Extracellular Traps (FEETs). Interestingly, we discovered that mammalian inducers of NETosis, such as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate and the calcium ionophore ionomycin, induced FEETs. Moreover, we found that FEETs are dependent upon activation of PKC and generation of mitochondrial reactive oxygen species. Thus, this brief report represents the first demonstration that fish erythrocytes can exhibit ETotic-like responses, unveiling a previously unknown function of nucleated erythrocytes, which sheds new light on the innate immune arsenal of erythrocytes.

Ferroptosis, an intracellular iron-catalyzed form of programmed cell death (PCD) driven by lipid reactive oxygen species induced membrane damage, is mechanistically uncharacterized in its execution process. Here, we investigated ferroptosis execution in mesenchymal-like ovarian cancer cells treated with ferroptosis inducers ML162 and erastin. We showed that YVAD (a pyroptosis-associated inflammatory caspase inhibitor) and disulfiram (preventing gasdermin pore formation on plasma membrane) deterred ferroptotic cell death. Moreover, we also observed LDH release and IL-1{beta} secretion from ferroptotic cells, suggesting that ferroptosis involves a pore-forming process. Intriguingly, ferroptosis is independent of the canonical inflammasome pathway because caspase-1 is dispensable and not activated upon ferroptosis induction. In contrast, we found that caspase-5 was activated while caspase-4 was not during ferroptosis. In addition, depletion of caspase-5 rendered cells not responding to ferroptosis inducers. Also intriguingly, GSDMD, the well-established caspase-5 substrate, was not involved in ferroptosis. We instead detected GSDME cleavage upon ferroptosis induction and knockdown of GSDME reduced cell death induced by ferroptosis inducers. As caspase-5 activity was necessary for ferroptosis and caspase-5 directly cleaved GSDME, we conclude that the axis of caspase-5/GSDME executes ferroptosis in ovarian cancer cells.

Dependence receptors (DRs) induce cell death by apoptosis when unbound by their cognate ligands. Among them, Kremen1 was first described to induce cancer cell death in the absence of its ligand, DKK1. However, the precise mechanism of Kremen1-induced cell death remains unclear. In this study, we demonstrate that Kremen1 induces cell death with autophagic features, contrasting with the apoptotic process typically associated with dependence receptors. Specifically, the pharmacological inhibition of autophagy, or genetic silencing of key autophagy effectors, efficiently suppresses this cell death process. A biotin proximity labeling for protein-protein interactions identified SEC24C, a component of the COP-II complex, as a critical effector in Kremen1-induced autophagy and cell death. Our findings further reveal that Kremen1 is in proximity with SEC24C and ATG9A after vesicular trafficking and fosters the interaction of SEC24C with ATG8, ERGIC and ATG9A. This potentially underlies the increased number of autophagosomes leading to cell death. The induction of aberrant autophagy by Kremen1 deserves particular attention, especially as the Kremen1/DKK1 pair is frequently altered in cancers. Thus, targeting this pathway may offer a potential strategy for treating cancers resistant to current therapies.

Errors in mitosis can contribute to aneuploidy and CIN and play a pivotal role in cancer. So the identification of altered mitotic regulators can contribute to the understanding of the development and progression of breast cancer. In the present study we used an in vitro model of disease progression (the MCF10A series of BC continuum) and analyzed the errors of chromosome segregation that occur during the progression of the disease. Our findings indicated that the MCF10A series exhibited several abnormalities in chromosome segregation and its frequency increased with the disease progression. These errors included anaphase lagging chromosomes, micronuclei, nuclear buds, nucleoplasmic bridges, errors of chromosome alignment, and centrosome loss/amplification. Moreover, the presence of centrosome amplification disrupted the proper orientation of the mitotic spindle, resulting in the generation asymmetrical cell lines and aneuploidy in the MCF10A series. Hyper stable kinetochore-microtubule (kt-MT) attachment was also found in premalignant, preinvasive, and invasive cell lines, which can also explain the presence of errors of chromosome alignment. The human transcriptome array also determined possible negative regulators of ciliogenesis that can explain the mechanism of chromosome missegregation that lead to CIN found in the MCF10A series. Collectively, these findings highlight the importance of mitotic defects in the progression of breast cancer.

We have previously identified sphingolipid metabolism as a key intracellular process associated with immunogenic cell death (ICD) induced by mitoxantrone in colon cancer cells. Specifically, we have demonstrated that inhibition of the sphingosine kinases (SphKs) synergistically enhanced production of hallmarks of ICD including ectoCRT production. To better understand the mechanism associated with ICD enhanced by SphK1-inhibition, we focused on the ER stress-associated intracellular signaling pathways leading to ectoCRT production. It is known that ABT-263 and AZD-5991 (ABT/AZD) are inhibitors of Bcl-2/Bcl-XL and MCL-1, respectively, leading to activation of Bak/Bax. Herein, we now provide evidence that treatment of DLD-1 colon cancer cells with ABT/AZD results in the production of ectoCRT indicative of ICD. Additionally, our data show that ABT/AZD-induced ectoCRT production is significantly enhanced by combination treatment with the SphK1 inhibitor, PF-543. Mechanistically, we demonstrate that combined treatment of ABT/AZD+PF-543 induces ectoCRT exposure in a caspase 8-dependent manner. Accordingly, we have identified a Bak/Bax activation-dependent pathway that leads to activation of a pro-survival SphK1/sphingosine-1-phosphate (S1P) signaling that attenuates ectoCRT production. Additionally, we have identified a regulatory role of ceramide synthase 6 (CerS6)- C16:0 Cer in transporting of dimeric CRT to the cell surface (ectoCRT). Together, these results indicate that sphingolipid metabolites, such as S1P and C16:0 Cer, have a key regulatory role for survival/death decisions of cancer cells in response to ICD-inducing chemotherapeutic agents such as mitoxantrone and ABT/AZD. Hence, targeting SphKs may be an innovative means to enhance the efficacy of ICD-inducing chemotherapeutic agents promoting anti-tumor innate/adaptive immune response since SphK inhibition blocks the anti-ICD effects of S1P while simultaneously accumulating sphingosine (Sph) leading to pro-ICD C16:0 Cer synthesis.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB-mediated acetylation serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to extrinsic pro-apoptotic stimuli, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway impacts apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

Autophagic elimination of depolarized mitochondria (mitophagy) depends on Ubiquitin proteasome complex to expose the inner mitochondrial membrane-resident protein-Prohibitin 2 (PHB2). This uncovering facilitates its interaction with autophagosomal membrane-associated protein LC3. It remains unclear whether PHB2 is uncovered randomly through mitochondrial rupture sites. Prior knowledge and initial screening indicated that Voltage-dependent anion-selective channel protein 1 (VDAC1) might play a role in this process. Through in vitro biochemical assays and imaging, we have found that VDAC1-PHB2 interaction increases during mitochondrial depolarization. Subsequently, this interaction enhances the efficiency of PHB2 exposure and mitophagy. To investigate the relevance in vivo, we utilized a Porin (equivalent to VDAC1) knockout Drosophila line. Our findings demonstrate that during rotenone-induced mitochondrial stress, Porin is essential for PHB2 exposure, PHB2-LC3 interaction, and mitophagy. This study highlights that VDAC1 predominantly synchronizes efficient PHB2 exposure through mitochondrial rupture sites during mitophagy. These findings may provide insights to understand progressive neurodegeneration.

In the animal kingdom, proteins of the Bcl-2 family are widely recognized as regulators of mitochondrial outer membrane permeabilization (MOMP), leading to apoptotic cell death. These proteins were recently also shown to control IP3-dependent calcium fluxes at the level of the endoplasmic reticulum (ER). However, the origin and evolution of these pleiotropic functions remain elusive. Here, we molecularly characterized the four members of the Bcl-2 family (trBcl-2L1 to -2L4) in the most primitive metazoan, namely Trichoplax adhaerens. Primary structure and phylogenetic analyses demonstrated that all four trBcl-2 homologs belong to the multidomain Bcl-2 group and presented a conserved C-terminus transmembrane (TM) domain. TrBcl-2L1 and trBcl-2L2 are highly divergent proteins clustering with the anti-apoptotic Bcl-2 members, whereas trBcl-2L3 and trBcl-2L4 were homologous to the pro-apoptotic Bax (trBax) and Bak (trBak). Interestingly, at the functional level, trBak operates as a BH3 only sensitizer repressing the anti-apoptotic activities of trBcl-2L1 and trBcl-2L2, whereas trBax leads to MOMP, similarly to the well-known indirect model of Bax activation. Finally, we found that trBcl-2L1 had a dual ER and mitochondrial subcellular localization and was able to bind to IP3R. By generating two TM domain mutants we demonstrated that trBcl-2L1 targeted to the ER was able to control IP3-dependent calcium fluxes, whereas Mito-trBcl-2L1 represses trBax-dependent MOMP, suggesting that Bcl-2 pleiotropy appeared early and was conserved throughout metazoan evolution.

Atypically expressed transglutaminase 2 (TG2) has been identified as a poor prognostic factor in a variety of cancers. In this study, we evaluated the contribution of TG2 to the prolonged cell survival of differentiated acute promyelocytic leukaemia (APL) cells in response to the standard treatment with combined retinoic acid (ATRA) and arsenic trioxide (ATO). We report that one advantage of ATRA + ATO treatment compared to ATRA alone diminishes the amount of activated and non-activated CD11b/CD18 and CD11c/CD18 cell surface integrin receptors. These changes suppress ATRA-induced TG2 docking on the cytosolic part of CD18 {beta}2-integrin subunits and reduce cell survival. In addition, TG2 overexpresses and hyperactivates the phosphatidylinositol-3-kinase (PI3K), phospho-AKT S473, and phospho-mTOR S2481 signalling axis. mTORC2 acts as a functional switch between cell survival and death by promoting the full activation of AKT. We show that TG2 presumably triggers the formation of a signalosome platform, hyperactivates downstream mTORC2-AKT signalling, which in turn phosphorylates and inhibits the activity of FOXO3, a key pro-apoptotic transcription factor. In contrast, the absence of TG2 restores basic phospho-mTOR S2481, phospho-AKT S473, PI3K, and PTEN expression and activity, thereby sensitising APL cells to ATO-induced cell death. We conclude, that atypically expressed TG2 may serve as a hub, facilitating signal transduction via signalosome formation by the CD18 subunit with both PI3K hyperactivation and PTEN inactivation through the PI3K PTEN cycle in ATRA-treated APL cells.

The fundamental transcription factor p53 regulates cellular processes and integrates signals of cellular stress, triggering a coordinated response to ensure survival of cells restored to healthy function and programmed death of those that couldnt be repaired. Unsurprisingly, this is one of the most mutated genes in human cancers, with most changes occurring in the DNA-binding domain of the protein. In this work, we take a genome-wide approach and use available resources to identify high confidence p53-target genes, that we examine in three breast cancer cell lines with different p53 status, wild type (MCF-7) and different mutations in the DNA-binding domain (MDA-MB231, T47D). Comparison of p53-targets expression in response to DNA damage by RNAseq and cellular assays reveals that MDA-MB231 have a severely impaired p53-dependent pathway functionality while T47D are much less affected. MDA-MB231 are more resistant to DNA damage yet unable to repair and able to override cell cycle arrest leading to survival while T47D are sensitive only to high dose and exposure to genotoxic agents. This data shows the variability of effects of different p53 mutations and highlight the importance of understanding the mechanisms of p53 in the context of genotoxicity-based treatment.

BackgroundLipocalin 2 (Lcn2), a small-secreted protein, is an established sensitive biomarker of kidney injury. In Cisplatin (CDDP) induced acute kidney injury (AKI), Lcn2 expression is swiftly and strongly increased in suffering renal medullary tubules. While recent reports correlates Lcn2 expression in cancer cells with CDDP resistance, the role of Lcn2 in kidney tubule damaged by CDDP remains unknown. MethodsTo better understand the role of Lcn2 in CDDP-induced AKI, experiments on Lcn2+/+ or Lcn2-/- mice as well as immortalized kidney cells knock-down (KD) for Lcn2 were conducted. Kidney function and injury were assessed using standard techniques. Cellular and molecular mechanisms were studied in WT and Lcn2 KD cells in combination with pathways inhibitors in order to gain insight Lcn2 driven mechanisms. ResultsIn animal injected with CDDP, Lcn2 was upregulated mostly in kidney inner medulla collecting duct while it was reabsorbed in the proximal tubules. Lcn2-/- in mice significantly decreased kidney function compare to WT mice, while it increased parenchymal damage due to increased cell death and cast formation. Interestingly, while little to no damage were present in the medulla of CDDP injected WT animal, a clear increase of medulla tubular lesions was observed in Lcn2-/- mice. Using Inner Medullary Collecting Duct cells (mIMDC-3), we showed that Lcn2 KD induces a p53-dependent apoptosis upon Cisplatin exposure while no effect on necrosis was observed. Finally, we demonstrated that Lcn2 drives CDDP resistance to apoptosis through the activation of the pro-survival EGFR pathway. ConclusionsWe established that Lcn2 is a renoprotective protein in CDDP-induced AKI via the activation of EGFR pathway. This new mechanism might represent a new approach for the treatment of AKI.

Senescence is a non-proliferative cellular state derived from aging or in response to exogenous insults, such as those that cause DNA damage. As a result of cancer treatments like cisplatin, certain tumor cells may undergo senescence. However, rather than being beneficial for patients, this is detrimental because these cells might proliferate again under specific conditions and, more importantly, because they synthesize and secrete molecules that promote the proliferation of nearby cells. Therefore, to achieve complete tumor remission, it is necessary to develop senolytic compounds to eliminate senescent cells. Here, we studied the role of {beta}TrCP1 in cell proliferation and senescence and found that lentiviral overexpression of {beta}TrCP1 induces the death of senescent cells obtained after cisplatin treatment in both two-dimensional cell cultures and tumorspheres. Mechanistically, we demonstrated that overexpression of {beta}TrCP1 triggers proteasome- dependent degradation of p21 CIP1, allowing damaged cells to progress through the cell cycle and consequently die. Furthermore, we identified nucleophosmin 1 (NPM1) as the intermediary molecule involved in the effect of {beta}TrCP1 on p21 CIP1. We determined that increased amounts of {beta}TrCP1 partially retains NPM1 in the nucleoli, preventing it from associating with p21 CIP1, thus leaving it unprotected from degradation by the proteasome. These results have allowed us to discover a potential new target for senolytic drugs, as retaining NPM1 in the nucleoli under senescent conditions induces cell death.

The cadherin family of cell surface glycoproteins plays a fundamental role in cell-cell recognition, thereby participating in diverse biological process such as embryonic morphogenesis and oncogenic transformation. The subset of clustered protocadherin (PCDH) genes generated from the , {beta}, and {gamma} loci, have been widely studied for their potential role in neuronal cell-cell recognition and neurogenesis, however their broader role in normal embryonic development and cancer has not been examined in detail. We utilized human embryonic stem (hES) cells to model early human development in vitro, comparing PCDH isoform transcription in diverse types of embryonic progenitors with normal adult-derived and cancer counterparts. Embryonic progenitors express genes from the and {beta} cluster at levels comparable to that seen in the CNS, while fetal and adult-derived cells express primarily from the {gamma} cluster. Replicative senescence left fibroblasts with markedly lower expression of all isoforms. We observe that an embryonic pattern of clustered protocadherin gene expression and associated CpG island methylation is commonly associated with cancer cell lines from diverse tissue types. The differential regulation of the , {beta}, and {gamma} loci coincide with alternate regions of DNA accessibility at CTCF binding sites and lamina-associated domains and CPL expression correlated with the expression of LMNA and LMNB1. These observations support a potential role for the differential regulation of genes within the clustered protocadherin locus in selective cell-cell adhesion during embryogenesis, regeneration, cancer and aging.

Targeted protein degradation (TPD) destroys proteins of interest (POIs) by hijacking the cellular proteolytic machinery. Most proteins in cells exist and function as part of multi-protein or macromolecular complexes, thereby allowing a single protein to control multiple biological processes. Therefore, when a small molecule degrader induces proximity between an E3 ligase and the POI, the macromolecular context of the POI potentially influences the degradation outcomes of the POI and of the complex components. Here, we explore degradation of the eight CK1-SACK1(A-H) (formerly known as FAM83A-H) complexes initiated by molecular glue degraders primarily designed to target Ser/Thr kinase CK1. We demonstrate that lenalidomide-derived degraders DEG-77 and SJ3149, which selectively target the CK1 isoform, co-degrade multiple SACK1(A-H) proteins. We show that the degradation of SACK1(A-H) proteins by DEG-77 and SJ3149 requires CK1, the CUL4ACRBN E3 ligase complex and the proteasome. In cells derived from palmoplantar keratoderma patients harbouring the CK1-binding deficient SACK1GR265P mutation, DEG-77 targets CK1 and mitotic SACK1D but not SACK1GR265P, highlighting the requirement for CK1-SACK1(A-H) interaction to achieve co-degradation. Our study underscores the importance of POI context in TPD and reinforces the potential for selectively targeting specific protein complexes for degradation.

Cellular material in apoptotic cells must be efficiently cleared by phagocytes to maintain tissue homeostasis. Defects in this process can lead to the onset of secondary necrosis and the release of intracellular contents such as damage associated molecular patterns (DAMPs) and autoantigens that are often derived from the nucleus. Therefore, appropriate handling and clearance of apoptotic material is vital to prevent unwanted inflammatory response and the onset of autoimmune disorders. However, how nuclear material is packaged by apoptotic cells for effective clearance by phagocytes is not well understood. By utilising murine models of apoptosis, we observed that a distinct subset of large extracellular vesicles generated from apoptotic thymocytes, known as apoptotic bodies (ApoBDs), can harbour the majority of nuclear contents. Mechanistically, we discovered that apoptotic cells can asymmetrically partition the nucleus into a single large membrane bleb located at one side of the cell, with other cellular contents such as mitochondria and acid organelles distributed to the opposite side. Whilst this newly observed apoptotic morphology, coined as asymmetric cell death morphology (AsyCDM), is morphologically similar to the process of erythroblast enucleation, pharmacological compounds that could interfere with erythroblast enucleation did not block the establishment of AsyCDM during apoptosis. Notably, AsyCDM was reliant on the contractile forces generated by ROCK1-dependent plasma membrane blebbing. Taken together, this study suggests that intracellular contents are partitioned into different ApoBD subsets during apoptosis through a regulated process driven by ROCK1-dependent actomyosin contraction.

Fas-associated death domain protein (FADD) plays a vital role in the extrinsic apoptotic pathway, where it forms an essential component of the death-inducing signaling complex (DISC). However, the precise early molecular events that facilitate recruitment of FADD to the DISC remain poorly defined. Using affinity purification and mass spectrometry we investigated the FADD interactome in untreated cells and following death receptor stimulation to identify novel FADD-interacting proteins. As expected, in death receptor-stimulated samples our analysis identified key components of the DISC such as Caspase-8. In addition, we identified novel binding partners including Transferrin Receptor 1 (TfR1) and Myosin Light Chain Kinase 2 (MYLK2) that are able to modulate FADD recruitment to the DISC and consequently downstream apoptotic signaling. TfR1 is pre-associated with FADD and recruited into the DISC; moreover, our data reveal that TfR1 is also pre-associated with the death receptors, TRAIL-R1 and TRAIL-R2, thereby functioning as a key regulator of DISC formation. In the case of MYLK2, specific binding of FADD to MYLK2 in non-apoptotic cells sequesters FADD from other DISC components ensuring aberrant apoptosis is not initiated. Furthermore, MYLK2 enzymatic activity is required to for it to translocate, in complex with FADD, to sites of DISC-mediated death receptor oligimerization. Taken together, our study highlights the important role that additional novel FADD binding partners play in the regulation of death receptor-mediated apoptotic cell death, in part by modulating FADD recruitment to the DISC.

High-risk neuroblastoma is one of the most lethal childhood cancers. Half of these tumors are driven by MYCN gene amplification (MNA). Despite intensive chemo- and radiotherapy, only 40% of patients survive, and they often suffer from severe long-term side effects of these genotoxic treatments. Thus, new therapies are needed that are less toxic and more efficacious. Here, we identified diphenyleneiodonium (DPI) as a tool compound that preferentially targets MNA neuroblastoma. Using proteomic assays we investigated the DPI mode of action, finding that DPI induces the proteasomal degradation of MYCN and could reverse some alterations induced by high levels of MYCN. These include profound changes in the expression of proteins participating in the mitochondrial electron transport chain. Metabolic and biological assays suggested that alterations in mitochondrial function and the associated production of reactive oxygen species (ROS) are critical DPI targets in the context of MNA. DPI reduced the survival, and malignant transformation of neuroblastoma across a panel of cell lines at clinically achievable concentrations. DPI also shrank tumors and prevented metastatic spread in zebrafish models of MYCN-driven neuroblastoma. These findings suggest that processes impacted by complex I inhibitors could be valuable new targets for the development of non-genotoxic drugs against high-risk MNA neuroblastoma. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/619268v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@167e953org.highwire.dtl.DTLVardef@10749e4org.highwire.dtl.DTLVardef@1842598org.highwire.dtl.DTLVardef@c05d82_HPS_FORMAT_FIGEXP M_FIG C_FIG

It has been reported that caspase activation by the proapoptotic ligand Apo2L/TRAIL disrupts the clathrin-mediated endocytosis machinery (CMEM). To confirm whether TRAIL induces caspase-mediated cleavage of specific CMEM components, we examined the temporal and functional relationship between TRAIL-induced processing of the apoptosis-initiating protease, caspase-8, and the apoptosis-executing protease, caspase-3, versus cleavage of specific adaptin (AP) and clathrin heavy chain (CHC) proteins. TRAIL induced time-dependent proteolytic processing of caspase-8 and caspase-3, which coincided with cleavage of AP2 and CHC. The pan caspase inhibitor zVAD-FMK, which blocked caspase processing in response to TRAIL, also prevented the cleavage AP2. Whereas FADD-deficient or caspase-8-deficient cells showed little or no TRAIL-driven cleavage of either AP2 or CHC, Bax-deficient or caspase-3-deficient cell lines retained AP2 cleavage but failed to cleave CHC in response to TRAIL. The DNA-damaging agent doxorubicin also led to processing of caspase-8 and caspase-3 in conjunction ith cleavage of AP2 and CHC. Together, these results confirm that TRAIL induces caspase-dependent cleavage of AP2 and CHC. Whereas TRAIL-driven cleavage of both AP2 and CHC requires caspase-8, cleavage of CHC, but not of AP2, requires caspase-3. MATERIALS AND METHODS Cell lines and cell cultureAll cell lines were obtained from ATCC or as kind gifts from Dr. Bert Vogelstein (HCT116 Bax-/-) and Dr. John Blenis (Jurkat I9.2 and E1). Cell lines were cultured with standard media as previously described (1). Immunoblot analysisCells were lysed in RIPA lysis buffer (20-188, Millipore) supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific) and kept on ice for 30 min. Lysates were cleared by centrifugation at 13,600 x g for 15 min at 4 {degrees}C, and protein amount was determined by BCA protein assay (ThermoFisher Scientific). Protein was denatured by adding NuPAGE LDS buffer and DTT reducing buffer (Invitrogen) and incubating the samples at 95 {degrees}C for 5 min. Equal amounts of denatured protein were loaded into each well of NuPAGE pre-cast gels (Invitrogen), resolved by SDS-PAGE, and electro-transferred to nitrocellulose membranes using the iBLOT2 system (Invitrogen). Membranes were blocked in a 5% nonfat milk solution for 1 hr at room temperature and probed with the corresponding primary antibody at 1:1,000 dilution overnight at 4 {degrees}C. This was followed by incubation with the corresponding horseradish peroxidase (HRP)- conjugated secondary antibody at 1:10,000 dilution during 1 hr at room temperature. All secondary antibodies were from Jackson Laboratories. The primary antibodies and secondary HRP-conjugated antibodies are listed respectively in Table 1 and Table 2. {beta}-actin IB was used to verify uniformity of protein loading and electro-transfer. O_TBL View this table: org.highwire.dtl.DTLVardef@ed5a65org.highwire.dtl.DTLVardef@c9aecdorg.highwire.dtl.DTLVardef@f31b4aorg.highwire.dtl.DTLVardef@849deaorg.highwire.dtl.DTLVardef@9b847b_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONPrimary antibodies used for IB analysis C_TABLECAPTION C_TBL O_TBL View this table: org.highwire.dtl.DTLVardef@16c33forg.highwire.dtl.DTLVardef@6c6b81org.highwire.dtl.DTLVardef@5efb4org.highwire.dtl.DTLVardef@1bc354forg.highwire.dtl.DTLVardef@15007aa_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONHorseradish peroxidase conjugated secondary antibodies used for IB analysis C_TABLECAPTION C_TBL Additional reagentsRecombinant soluble non-tagged human TRAIL (Apo2L.0) and Flag-tagged TRAIL were prepared at Genentech. Flag-tagged TRAIL was crosslinked with anti-Flag M2 antibody at a ligand-to-antibody molar ratio of 1:2. zVAD-FMK was purchased from R & D Systems, FMK001. Doxorubicin was purchased from Sigma Aldrich, D1515.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts (MPSF) ionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+-dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted wound closure in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down NAD+, it is also allosterically inhibited by NAD+. However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a "biological trigger" of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP1 inhibitor, PJ34 restored mitochondrial homeostasis and prevented subsequent Sarm1 activation in rotenone treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone mediated loss of locomotor abilities and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncovers a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders mediated by Sarm1.