p53-dependent programmed necrosis controls germ cell homeostasis during spermatogenesis.

The importance of regulated necrosis in pathologies such as cerebral stroke and myocardial infarction is now fully recognized. However, the physiological relevance of regulated necrosis remains unclear. Here, we report a conserved role for p53 in regulating necrosis in Drosophila and mammalian spermatogenesis. We found that Drosophila p53 is required for the programmed necrosis that occurs spontaneously in mitotic germ cells during spermatogenesis. This form of necrosis involved an atypical function of the initiator caspase Dronc/Caspase 9, independent of its catalytic activity. Prevention of p53-dependent necrosis resulted in testicular hyperplasia, which was reversed by restoring necrosis in spermatogonia. In mouse testes, p53 was required for heat-induced germ cell necrosis, indicating that regulation of necrosis is a primordial function of p53 conserved from invertebrates to vertebrates. Drosophila and mouse spermatogenesis will thus be useful models to identify inducers of necrosis to treat cancers that are refractory to apoptosis.

A new MEIOB mutation is a recurrent cause for azoospermia and testicular meiotic arrest.

STUDY QUESTION: Are there genetic variants that can be used for the clinical evaluation of azoospermic men? SUMMARY ANSWER: A novel homozygous frame-shift mutation in the MEIOB gene was identified in three azoospermic patients from two different families. WHAT IS KNOWN ALREADY: Up to 1% of all men have complete absence of sperm in the semen, a condition known as azoospermia. There are very few tools for determining the etiology of azoospermia and the likelihood of sperm cells in the testis. The MEIOB gene codes for a single-strand DNA binding protein required for DNA double-strand breaks repair during meiosis. MEIOB appears to be exclusively expressed in human and mouse testis, and MeioB knockout mice are azoospermic due to meiotic arrest. STUDY DESIGN, SIZE, DURATION: Two brothers with non-obstructive azoospermia (NOA) underwent whole-exome sequencing followed by comprehensive bioinformatics analyses. Candidate variations were further screened in infertile and fertile men, as well as in public and local reference databases. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 159 infertile and 77 fertile men. The exomes of two Arab men were completely sequenced. In addition, 213 other men of the same Arab ethnicity (136 infertile and 77 fertile men) underwent restriction fragment length polymorphism (RFLP) screening, as did 21 NOA men, of other ethnicities, with testicular impairment of spermatocyte arrest. All of the infertile men underwent Y-chromosome microdeletion and CFTR gene mutation assessments. Comprehensive bioinformatics analyses were designed to uncover candidate mutations associated with azoospermia. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous frame-shift mutation in the MEIOB gene was identified in two brothers of Arab ethnicity. This frame-shift is predicted to result in a truncated MEIOB protein, which lacks the conserved C-terminal DNA binding domain. RFLP screening of the mutation in 157 infertile men, including 112 NOA patients of Arab ethnicity, identified an additional unrelated NOA patient with the same homozygous mutation and a similar testicular impairment. This mutation was not found in available public databases (n > 160 000), nor in the 77 proven fertile men, nor in our database of local Israeli population variations derived from exome and genome sequencing data (n = 500). LIMITATIONS, REASONS FOR CAUTION: We have thus far screened for only two specific MEIOB probable pathogenic mutations in a relatively small local cohort. Therefore, the relative incidence of MEIOB mutations in azoospermia should be further assessed in larger and diverse cohorts in order to determine the efficiency of MEIOB sequence screening for clinical evaluations. WIDER IMPLICATIONS OF THE FINDINGS: The relatively high incidence of likely NOA-causing mutations in MEIOB that was found in our cohort supports the idea that a complete screening of this gene might be beneficial for clinical evaluation of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This research was supported in part by a grant to EA from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC grant agreement (616088). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

International conference on Cell Death in Cancer and Toxicology 2018 (CDCT-2018).

The International Conference on Cell Death in Cancer and Toxicology 2018 (February 20-22, 2018) provided an international forum for scientific collaborations across multiple disciplines in cancer, cell death, and toxicology. During the three-day symposium, researchers and clinicians shared recent advances in basic, clinical, and translational research in cancer. Several student poster abstracts were selected for platform talks and many young investigators participated in the meeting. Together, this highly interactive meeting showcased the rapid expansion in biomedical research in India and paved the way for future meetings on cell death and cancer throughout India.

Egg multivesicular bodies elicit an LC3-associated phagocytosis-like pathway to degrade paternal mitochondria after fertilization.

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.

A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila.

Terminal differentiation of male germ cells in Drosophila and mammals requires extensive cytoarchitectural remodeling, the elimination of many organelles, and a large reduction in cell volume. The associated process, termed spermatid individualization, is facilitated by the apoptotic machinery, including caspases, but does not result in cell death. From a screen for genes defective in caspase activation in this system, we isolated a novel F-box protein, which we termed Nutcracker, that is strictly required for caspase activation and sperm differentiation. Nutcracker interacts through its F-box domain with members of a Cullin-1-based ubiquitin ligase complex (SCF): Cullin-1 and SkpA. This ubiquitin ligase does not regulate the stability of the caspase inhibitors DIAP1 and DIAP2, but physically binds Bruce, a BIR-containing giant protein involved in apoptosis regulation. Furthermore, nutcracker mutants disrupt proteasome activity without affecting their distribution. These findings define a new SCF complex required for caspase activation during sperm differentiation and highlight the role of regulated proteolysis during this process.

A pathogenic variant in the uncharacterized RNF212B gene results in severe aneuploidy male infertility and repeated IVF failure.

Quantitative and qualitative spermatogenic impairments are major causes of men's infertility. Although in vitro fertilization (IVF) is effective, some couples persistently fail to conceive. To identify causal variants in patients with severe male infertility factor and repeated IVF failures, we sequenced the exome of two consanguineous family members who underwent several failed IVF cycles and were diagnosed with low sperm count and motility. We identified a rare homozygous nonsense mutation in a previously uncharacterized gene, RNF212B, as the causative variant. Recurrence was identified in another unrelated, infertile patient who also faced repeated failed IVF treatments. scRNA-seq demonstrated meiosis-specific expression of RNF212B. Sequence analysis located a protein domain known to be associated with aneuploidy, which can explain multiple IVF failures. Accordingly, FISH analysis revealed a high aneuploidy rate in the patients' sperm cells and their IVF embryos. Finally, inactivation of the Drosophila orthologs significantly reduced male fertility. Given that members of the evolutionary conserved RNF212 gene family are involved in meiotic recombination and crossover maturation, our findings indicate a critical role of RNF212B in meiosis, genome stability, and in human fertility. Since recombination is completely absent in Drosophila males, our findings may indicate an additional unrelated role for the RNF212-like paralogs in spermatogenesis.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

The NAB-Brk signal bifurcates at JNK to independently induce apoptosis and compensatory proliferation.

Apoptosis operates to eliminate damaged or potentially dangerous cells. This loss is often compensated by extra proliferation of neighboring cells. Studies in Drosophila imaginal discs suggest that the signal for the additional growth emanates from the dying cells. In particular, it was suggested that the initiator caspase Dronc mediates compensatory proliferation (CP) through Dp53 in wing discs. However, the exact mechanism that governs this CP remained poorly understood. We have previously shown that elimination of misspecified cells due to reduced Dpp signaling is achieved by the interaction of the co-repressor NAB with the transcriptional repressor Brk, which in turn induces Jun N-terminal kinase-dependent apoptosis. Here, we performed a systematic in vivo loss- and gain-of-function analysis to study NAB-induced death and CP. Our findings indicate that the NAB primary signal activates JNK, which in turn transmits two independent signals. One triggers apoptosis through the pro-apoptotic proteins Reaper and Hid, which in turn promote activation of caspases by the apoptosome components Ark and Dronc. The other signal induces CP in a manner that is independent of the death signal, Dronc, or Dp53. Once induced, the apoptotic pathway further activates a CP response. Our data suggest that JNK is the candidate factor that differentiates between apoptosis that involves CP and apoptosis that does not.

Axonal degeneration is regulated by the apoptotic machinery or a NAD+-sensitive pathway in insects and mammals.

Selective degeneration of neuronal projections and neurite pruning are critical for establishment and maintenance of functional neural circuits in both insects and mammals. However, the molecular mechanisms that govern developmental neurite pruning versus injury-induced neurite degeneration are still mostly unclear. Here, we show that the effector caspases 6 and 3 are both expressed within axons and that, on trophic deprivation, they exhibit distinct modes of activation. Surprisingly, inhibition of caspases is not sufficient for axonal protection and a parallel modulation of a NAD(+)-sensitive pathway is required. The proapoptotic protein BAX is a key element in both pathways as its genetic ablation protected sensory axons against developmental degeneration both in vitro and in vivo. Last, we demonstrate that both pathways are also involved in developmental dendritic pruning in Drosophila. More specifically, the mouse Wld(S) (Wallerian degeneration slow) protein, which is mainly composed of the full-length sequence of the NAD(+) biosynthetic Nmnat1 enzyme, can suppress dendritic pruning in C4da (class IV dendritic arborization) sensory neurons in parallel to the fly effector caspases. These findings indicate that two distinct autodestruction pathways act separately or in concert to regulate developmental neurite pruning.

Editor Profile: Eli Arama.

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus, perspectives on the journal and future directions in their field. Eli Arama is an Associate Professor at the Weizmann Institute of Science in Rehovot, Israel. He has served as an editorial board member of The FEBS Journal since 2018.

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.

DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells.

During Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. Here, we demonstrate that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila.

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3-dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3(Testis)), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3(Testis) that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis-like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation.

Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila.

The final stage of spermatid terminal differentiation involves the removal of their bulk cytoplasm in a process known as spermatid individualization. Here we show that apoptotic proteins play an essential role during spermatid individualization in Drosophila melanogaster. Several aspects of sperm terminal differentiation, including the activation of caspases, are reminiscent of apoptosis. Notably, caspase inhibitors prevent the removal of bulk cytoplasm in spermatids and block sperm maturation in vivo, causing male sterility. We further identified loss-of-function mutations in one of the two Drosophila cyt-c genes, cyt-c-d, which block caspase activation and subsequent spermatid terminal differentiation. Finally, a giant ubiquitin-conjugating enzyme, dBruce, is required to protect the sperm nucleus against hypercondensation and degeneration. These observations suggest that an apoptosis-like mechanism is required for spermatid differentiation in Drosophila.

Can't live without them, can live with them: roles of caspases during vital cellular processes.

Since the pioneering discovery that the genetic cell death program in C. elegans is executed by the cysteine-aspartate protease (caspase) CED3, caspase activation has become nearly synonymous with apoptosis. A critical mass of data accumulated in the past few years, have clearly established that apoptotic caspases can also participate in a variety of non-apoptotic processes. The roles of caspases during these processes and the regulatory mechanisms that prevent unrestrained caspase activity remain to be fully investigated, and may vary in different cellular contexts. Significantly, some of these processes, such as terminal differentiation of vertebrate lens fiber cells and red blood cells, as well as spermatid terminal differentiation and dendritic pruning of sensory neurons in Drosophila, all involve proteolytic degradation of major cellular compartments, and are conceptually, molecularly, biochemically, and morphologically reminiscent of apoptosis. Moreover, some of these model systems bear added values for the study of caspase activation/apoptosis. For example, the Drosophila sperm differentiation is the only system known in invertebrate which absolutely requires the mitochondrial pathway (i.e. Cyt c). The existence of testis-specific genes for many of the components in the electron transport chain, including Cyt c, facilitates the use of the Drosophila sperm system to investigate possible roles of these otherwise essential proteins in caspase activation. Caspases are also involved in a wide range of other vital processes of non-degenerative nature, indicating that these proteases play much more diverse roles than previously assumed. In this essay, we review genetic, cytological, and molecular studies conducted in Drosophila, vertebrate, and cultured cells, which underlie the foundations of this newly emerging field.

Caspases maintain tissue integrity by an apoptosis-independent inhibition of cell migration and invasion.

Maintenance of tissue integrity during development and homeostasis requires the precise coordination of several cell-based processes, including cell death. In animals, the majority of such cell death occurs by apoptosis, a process mediated by caspase proteases. To elucidate the role of caspases in tissue integrity, we investigated the behavior of Drosophila epithelial cells that are severely compromised for caspase activity. We show that these cells acquire migratory and invasive capacities, either within 1-2 days following irradiation or spontaneously during development. Importantly, low levels of effector caspase activity, which are far below the threshold required to induce apoptosis, can potently inhibit this process, as well as a distinct, developmental paradigm of primordial germ cell migration. These findings may have implications for radiation therapy in cancer treatment. Furthermore, given the presence of caspases throughout metazoa, our results could imply that preventing unwanted cell migration constitutes an ancient non-apoptotic function of these proteases.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

A Krebs Cycle Component Limits Caspase Activation Rate through Mitochondrial Surface Restriction of CRL Activation.

How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. We investigate caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. We show that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase ß subunit (A-Sß), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sß and the GTP-specific SCSß (G-Sß), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sß in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation.

The de-ubiquitylating enzyme DUBA is essential for spermatogenesis in Drosophila.

De-ubiquitylating enzymes (DUBs) reverse protein ubiquitylation and thereby control essential cellular functions. Screening for a DUB that counteracts caspase ubiquitylation to regulate cell survival, we identified the Drosophila ovarian tumour-type DUB DUBA (CG6091). DUBA physically interacts with the initiator caspase death regulator Nedd2-like caspase (Dronc) and de-ubiquitylates it, thereby contributing to efficient inhibitor of apoptosis-antagonist-induced apoptosis in the fly eye. Searching also for non-apoptotic functions of DUBA, we found that Duba-null mutants are male sterile and display defects in spermatid individualisation, a process that depends on non-apoptotic caspase activity. Spermatids of DUBA-deficient flies showed reduced caspase activity and lack critical structures of the individualisation process. Biochemical characterisation revealed an obligate activation step of DUBA by phosphorylation. With genetic rescue experiments we demonstrate that DUBA phosphorylation and catalytic activity are crucial in vivo for DUBA function in spermatogenesis. Our results demonstrate for the first time the importance of de-ubiquitylation for fly spermatogenesis.