Mera: A scalable high throughput automated micro-physiological system.

There is an urgent need for scalable Microphysiological Systems (MPS's)1 that can better predict drug efficacy and toxicity at the preclinical screening stage. Here we present Mera, an automated, modular and scalable system for culturing and assaying microtissues with interconnected fluidics, inbuilt environmental control and automated image capture. The system presented has multiple possible fluidics modes. Of these the primary mode is designed so that cells may be matured into a desired microtissue type and in the secondary mode the fluid flow can be re-orientated to create a recirculating circuit composed of inter-connected channels to allow drugging or staining. We present data demonstrating the prototype system Mera using an Acetaminophen/HepG2 liver microtissue toxicity assay with Calcein AM and Ethidium Homodimer (EtHD1) viability assays. We demonstrate the functionality of the automated image capture system. The prototype microtissue culture plate wells are laid out in a 3 × 3 or 4 × 10 grid format with viability and toxicity assays demonstrated in both formats. In this paper we set the groundwork for the Mera system as a viable option for scalable microtissue culture and assay development.

Non-Canonical Roles of Apoptotic Caspases in the Nervous System.

Caspases are a family of cysteine proteases that predominantly cleave their substrates after aspartic acid residues. Much of what we know of caspases emerged from investigation a highly conserved form of programmed cell death called apoptosis. This form of cell death is regulated by several caspases, including caspase-2, caspase-3, caspase-7, caspase-8 and caspase-9. However, these "killer" apoptotic caspases have emerged as versatile enzymes that play key roles in a wide range of non-apoptotic processes. Much of what we understand about these non-apoptotic roles is built on work investigating how "killer" caspases control a range of neuronal cell behaviors. This review will attempt to provide an up to date synopsis of these roles.

Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form.

The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of the inactive "closed" form of Apaf-1. Here, a split luciferase complementation assay was used to monitor the effect of disrupting this ionic bond on apoptosome activation and caspase-3 activity in cells. The K192E mutation, predicted to disrupt the ionic interaction with D616, increased apoptosome formation and caspase activity, suggesting that this mutation favors the "open"/active form of Apaf-1. However, mutation of D616 to alanine or lysine had different effects. While both mutants favored apoptosome formation such as K192E, D616K cannot activate caspases and D616A activates caspases poorly, and not as well as wild-type Apaf-1. Thus, our data show that the ionic bond between K192 and D616 is critical for maintaining the closed form of Apaf-1 and that disrupting the interaction enhances apoptosome formation. However, our data also reveal that after apoptosome formation, D616 and K192 play a previously unsuspected role in caspase activation. The molecular explanation for this observation is yet to be elucidated.

Non-lethal message from the Holy Land: The first international conference on nonapoptotic roles of apoptotic proteins.

Apoptosis is a major form of programmed cell death (PCD) that eliminates unnecessary and potentially dangerous cells in all metazoan organisms, thus ensuring tissue homeostasis and many developmental processes. Accordingly, defects in the activation of the apoptotic pathway often pave the way to disease. After several decades of intensive research, the molecular details controlling the apoptosis program have largely been unraveled, as well as the regulatory mechanisms of caspase activation during apoptosis. Nevertheless, an ever-growing list of studies is suggesting the essential role of caspases and other apoptotic proteins in ensuring nonlethal cellular functions during normal development, tissue repair, and regeneration. Moreover, if deregulated, these novel nonapoptotic functions can also instigate diseases. The difficulty of identifying and manipulating the caspase-dependent nonlethal cellular processes (CDPs), as well as the nonlethal functions of other cell death proteins (NLF-CDPs), meant that CDPs and NLF-CDPs have been only curiosities within the apoptotic field; however, the recent technical advancements and the latest biological findings are assigning an unanticipated biological significance to these nonapoptotic functions. Here, we summarize the various talks presented in the first international conference fully dedicated to discuss CDPs and NFL-CDPs and named 'The Batsheva de Rothschild Seminar on Non-Apoptotic Roles of Apoptotic Proteins'. The conference was organized between September 22, 2019, and 25, 2019, by Eli Arama (Weizmann Institute of Science), Luis Alberto Baena-Lopez (University of Oxford), and Howard O. Fearnhead (NUI Galway) at the Weizmann Institute of Science in Israel, and hosted a large international group of researchers.

Loss of WD2 subdomain of Apaf-1 forms an apoptosome structure which blocks activation of caspase-3 and caspase-9.

Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1-921) on Apaf-1 interactions and caspase activation. Apaf-1 1-921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway.

Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

Apoptosome-dependent myotube formation involves activation of caspase-3 in differentiating myoblasts.

Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic myoblasts inducing differentiation in neighboring cells. Here we show for the first time that caspase-3 is activated in the myoblasts undergoing differentiation. Using RNAi, we also demonstrate that differentiation requires both cytochrome c and Apaf-1, and by using a new pharmacological approach, we show that apoptosome formation is required. We also show that Bid, whose cleavage links caspase-2 to the mitochondrial death pathway, was required for differentiation, and that the caspase cleavage product, tBid, was generated during differentiation. Taken together, these data suggest that myoblast differentiation requires caspase-2 activation of the mitochondrial death pathway, and that this occurs in the cells that differentiate. Our data also reveal a hierarchy of caspases in differentiation with caspase-2 upstream of apoptosome activation, and exerting a more profound control of differentiation, while caspases downstream of the apoptosome primarily control cell fusion.

Droplet Combinations: A Scalable Microfluidic Platform for Biochemical Assays.

Droplet-based microfluidics holds enormous potential for transforming high-throughput drug screening. Miniaturization through droplets in combination with automation contributes to reduce reagent use and analysis time as well as minimizing or eliminating labor-intensive steps leading to associated reductions in cost. In this paper, we demonstrate the potential of automated and cost-effective microfluidic droplet-generating technology in the context of an enzymatic activity assay for screening collagenase inhibitors. Experimental results show reproducible and accurate creation and mixing of droplet combinations resulting in biochemical data comparable to data produced by an industry standard instrument. This microfluidic platform that can generate and combine multiple droplets represents a promising tool for high-throughput drug screening.

A new split-luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation.

The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase-based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease-Activating Factor-1 (Apaf-1) molecules and induces cell death by activating caspase-9. Apaf-1-dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf-1 fused to an N-terminal fragment of luciferase binds to Apaf-1 fused to a C-terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill-defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.

How do we fit ferroptosis in the family of regulated cell death?

In the last few years many new cell death modalities have been described. To classify different types of cell death, the term 'regulated cell death' was introduced to discriminate it from 'accidental cell death'. Regulated cell death involves the activation of genetically encoded molecular machinery that couples the presence of some signal to cell death. These forms of cell death, like apoptosis, necroptosis and pyroptosis have important physiological roles in development, tissue repair, and immunity. Accidental cell death occurs in response to physical or chemical insults and occurs independently of molecular signalling pathways. Ferroptosis, an emerging and recently (re)discovered type of regulated cell death occurs through Fe(II)-dependent lipid peroxidation when the reduction capacity of a cell is insufficient. Ferroptosis is coined after the requirement for free ferrous iron. Here, we will consider the extent to which ferroptosis is similar to other regulated cell deaths and explore emerging ideas about the physiological role of ferroptosis.

Viral hijacking of host caspases: an emerging category of pathogen-host interactions.

Viruses co-evolve with their hosts, and many viruses have developed mechanisms to suppress or modify the host cell apoptotic response for their own benefit. Recently, evidence has emerged for the opposite strategy. Some viruses have developed the ability to co-opt apoptotic caspase activity to facilitate their own proliferation. In these strategies, viral proteins are cleaved by host caspases to create cleavage products with novel activities which facilitate viral replication. This represents a novel and interesting class of viral-host interactions, and also represents a new group of non-apoptotic roles for caspases. Here we review the evidence for such strategies, and discuss their origins and their implications for our understanding of the relationship between viral pathogenesis and programmed cell death.

DNA-PK activity is associated with caspase-dependent myogenic differentiation.

Differentiation of myoblasts into myotubes is essential for skeletal muscle development and regeneration. Caspase-3 and caspase-9 are required for efficient myoblast differentiation. The caspase-activated endonuclease activity, CAD, and the DNA-damage repair protein XRCC1 have also been shown to be required to complete differentiation. DNA-damage associated with differentiation is accompanied by phosphorylation of Histone 2AX, an event normally catalysed by kinases ATR, ATM or DNA-PK. However, the kinase responsible for phosphorylation during differentiation is not known. Here we show that inhibition of DNA-PK, but not of ATR or ATM, blocked histone phosphorylation during differentiation. We also show that DNA-PK inhibition and siRNA-mediated DNA-PK knockdown blocked cell fusion. These data implicate a new role for DNA-PK in myogenic differentiation.

Mesenchymal stem cells and a vitamin D receptor agonist additively suppress T helper 17 cells and the related inflammatory response in the kidney.

Mesenchymal stem cells (MSCs) suppress T helper (Th)17 cell differentiation and are being clinically pursued for conditions associated with aberrant Th17 responses. Whether such immunomodulatory effects are enhanced by coadministration of MSCs with other agents is not well known. In the present study, individual and combined effects of MSCs and the vitamin D receptor (VDR) agonist paricalcitol on Th17 induction were investigated in vitro and in a mouse model of sterile kidney inflammation (unilateral ureteral obstruction). In vitro, MSCs and paricalcitol additively suppressed Th17 differentiation, although only MSCs suppressed expression of Th17-associated transcriptions factors. Combined administration of MSCs and paricalcitol resulted in an early (day 3) reduction of intrarenal CD4(+) and CD8(+) T cells, CD11b(+)/lymphocyte antigen 6G(+) neutrophils, and inflammatory (lymphocyte antigen 6C(hi)) monocytes as well as reduced transcript for IL-17 compared with untreated animals. Later (day 8), obstructed kidneys of MSC/paricalcitol double-treated mice, but not mice treated with either intervention alone, had reduced tubular injury and interstitial fibrosis as well as lower numbers of neutrophils and inflammatory monocytes and an increase in the ratio between M2 (CD206(+)) and M1 (CD206(-)) macrophages compared with control mice. Adjunctive therapy with VDR agonists may enhance the immunosuppressive properties of MSCs in the setting of pathogenic Th17-type immune responses and related inflammatory responses.

New roles for old enzymes: killer caspases as the engine of cell behavior changes.

It has become increasingly clear that caspases, far from being merely cell death effectors, have a much wider range of functions within the cell. These functions are as diverse as signal transduction and cytoskeletal remodeling, and caspases are now known to have an essential role in cell proliferation, migration, and differentiation. There is also evidence that apoptotic cells themselves can direct the behavior of nearby cells through the caspase-dependent secretion of paracrine signaling factors. In some processes, including the differentiation of skeletal muscle myoblasts, both caspase activation in differentiating cells as well as signaling from apoptotic cells has been reported. Here, we review the non-apoptotic outcomes of caspase activity in a range of different model systems and attempt to integrate this knowledge.

Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues.

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.

"Dead Cells Talking": The Silent Form of Cell Death Is Not so Quiet.

After more than twenty years of research, the molecular events of apoptotic cell death can be succinctly stated; different pathways, activated by diverse signals, increase the activity of proteases called caspases that rapidly and irreversibly dismantle condemned cell by cleaving specific substrates. In this time the ideas that apoptosis protects us from tumourigenesis and that cancer chemotherapy works by inducing apoptosis also emerged. Currently, apoptosis research is shifting away from the intracellular events within the dying cell to focus on the effect of apoptotic cells on surrounding tissues. This is producing counterintuitive data showing that our understanding of the role of apoptosis in tumourigenesis and cancer therapy is too simple, with some interesting and provocative implications. Here, we will consider evidence supporting the idea that dying cells signal their presence to the surrounding tissue and, in doing so, elicit repair and regeneration that compensates for any loss of function caused by cell death. We will discuss evidence suggesting that cancer cell proliferation may be driven by inappropriate or corrupted tissue-repair programmes that are initiated by signals from apoptotic cells and show how this may dramatically modify how we view the role of apoptosis in both tumourigenesis and cancer therapy.

p53-mediated induction of Noxa and p53AIP1 requires NFkappaB.

The intricate regulation of cell survival and cell death is critical for the existence of both normal and transformed cells. Two factors central to these processes are p53 and NFkappaB, with both factors having ascribed roles in both promoting and repressing cell death. Not surprisingly, a number of studies have previously reported interplay between p53 and NFkappaB. The mechanistic basis behind these observations, however, is currently incomplete. We report here further insights into this interplay using a system where blockade of NFkappaB inhibits cell death from p53, but at the same time sensitizes cells to death by TNFalpha. We found in agreement with a recent report showing that NFkappaB is required for the efficient activation of the BH3-only protein Noxa by the p53 family member p73, that p53's ability to induce Noxa is also impeded by inhibition of NFkappaB. In contrast to the regulation by p73, however, blockade of NFkappaB downstream of p53 decreases Noxa protein levels without effects on Noxa mRNA. Our further analysis of the effects of NFkappaB inhibition on p53 target gene expression revealed that while most target genes analysed where unaffected by blockade of NFkappaB, the p53-mediated induction of the pro-apoptotic gene p53AIP1 was significantly dependent on NFkappaB. These studies therefore add further insight into the complex relationship of p53 and NFkappaB. In addition, since both Noxa and p53AIP1 have been shown to be important components of p53-mediated cell death responses, these findings may also indicate critical points where NFkappaB plays a pro-apoptotic role downstream of p53.

The Apaf-1*procaspase-9 apoptosome complex functions as a proteolytic-based molecular timer.

During stress-induced apoptosis, the initiator caspase-9 is activated by the Apaf-1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase-9 is paradoxically observed outside the complex. We show herein that apoptosome-mediated cleavage of procaspase-9 occurs exclusively through a CARD-displacement mechanism, so that unlike the effector procaspase-3, procaspase-9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase-9 possessed higher affinity for the apoptosome and could displace the processed caspase-9 from the complex, thereby facilitating a continuous cycle of procaspase-9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase-9 per se contributed little to the activation of procaspase-3. Thus, the Apaf-1 apoptosome functions as a proteolytic-based 'molecular timer', wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which the processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer 'ticks' over.

Activation of p73 and induction of Noxa by DNA damage requires NF-kappa B.

Although the transcription factor NF-kappaB is most clearly linked to the inhibition of extrinsic apoptotic signals such as TNFalpha by upregulating known anti-apoptotic genes, NF-kappaB has also been proposed to be required for p53-induced apoptosis in transformed cells. However, the involvement of NF-kappaB in this process is poorly understood. Here we investigate this mechanism and show that in transformed MEFs lacking NF-kappaB (p65-null cells) genotoxin-induced cytochrome c release is compromised. To further address how NF-kappaB contributes to apoptosis, gene profiling by microarray analysis of MEFs was performed, revealing that NF-kappaB is required for expression of Noxa, a pro-apoptotic BH3-only protein that is induced by genotoxins and that triggers cytochrome c release. Moreover, we find that in the absence of NF-kappaB, genotoxin treatment cannot induce Noxa mRNA expression. Noxa expression had been shown to be regulated directly by genes of the p53 family, like p73 and p63, following genotoxin treatment. Here we show that p73 is activated after genotoxin treatment only in the presence of NF-kappaB and that p73 induces Noxa gene expression through the p53 element in the promoter. Together our data provides an explanation for how loss of NF-kappaB abrogates genotoxin-induced apoptosis.