A guideline on the molecular ecosystem regulating ferroptosis.

Ferroptosis, an intricately regulated form of cell death characterized by uncontrolled lipid peroxidation, has garnered substantial interest since this term was first coined in 2012. Recent years have witnessed remarkable progress in elucidating the detailed molecular mechanisms that govern ferroptosis induction and defence, with particular emphasis on the roles of heterogeneity and plasticity. In this Review, we discuss the molecular ecosystem of ferroptosis, with implications that may inform and enable safe and effective therapeutic strategies across a broad spectrum of diseases.

Regulation and Functions of Autophagy During Animal Development.

Autophagy is used to degrade cytoplasmic materials, and is critical to maintain cell and organismal health in diverse animals. Here we discuss the regulation, utilization and impact of autophagy on development, including roles in oogenesis, spermatogenesis and embryogenesis in animals. We also describe how autophagy influences postembryonic development in the context of neuronal and cardiac development, wound healing, and tissue regeneration. We describe recent studies of selective autophagy during development, including mitochondria-selective autophagy and endoplasmic reticulum (ER)-selective autophagy. Studies of developing model systems have also been used to discover novel regulators of autophagy, and we explain how studies of autophagy in these physiologically relevant systems are advancing our understanding of this important catabolic process.

Mass isolation of staged Drosophila pupal intestines for analysis of protein ubiquitylation.

Large quantities of developmentally synchronized pupal intestines are required for biochemistry experiments. Here, we present a protocol for the mass isolation of staged pupal intestines during Drosophila melanogaster development based on buoyancy in sucrose for biochemical evaluation of protein ubiquitylation. We describe steps for crossing flies, preparation of samples, immunoprecipitation of proteins from staged isolated tissues, and analysis of samples by western blot. This protocol can be extended to investigate biochemical changes in other tissues. For complete details on the use and execution of this protocol, please refer to Wang et al. (2023).1.

PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development.

Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. Here, we describe a developmentally programmed selective ER clearance by autophagy. We show that Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy.

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.

Atg6 promotes organismal health by suppression of cell stress and inflammation.

Autophagy targets cytoplasmic materials for degradation, and influences cell health. Alterations in Atg6/Beclin-1, a key regulator of autophagy, are associated with multiple diseases. While the role of Atg6 in autophagy regulation is heavily studied, the role of Atg6 in organism health and disease progression remains poorly understood. Here, we discover that loss of Atg6 in Drosophila results in various alterations to stress, metabolic and immune signaling pathways. We find that the increased levels of circulating blood cells and tumor-like masses in atg6 mutants vary depending on tissue-specific function of Atg6, with contributions from intestine and hematopoietic cells. These phenotypes are suppressed by decreased function of macrophage and inflammatory response receptors crq and drpr. Thus, these findings provide a basis for understanding how Atg6 systemically regulates cell health within multiple organs, and highlight the importance of Atg6 in inflammation to organismal health.

ESCRT dysfunction compromises endoplasmic reticulum maturation and autophagosome biogenesis in Drosophila.

Autophagy targets cytoplasmic materials for degradation and influences cell health. Organelle contact and trafficking systems provide membranes for autophagosome formation, but how different membrane systems are selected for use during autophagy remains unclear. Here, we report a novel function of the endosomal sorting complex required for transport (ESCRT) in the regulation of endoplasmic reticulum (ER) coat protein complex II (COPII) vesicle formation that influences autophagy. The ESCRT functions in a pathway upstream of Vps13D to influence COPII vesicle transport, ER-Golgi intermediate compartment (ERGIC) assembly, and autophagosome formation. Atg9 functions downstream of the ESCRT to facilitate ERGIC and autophagosome formation. Interestingly, cells lacking either ESCRT or Vps13D functions exhibit dilated ER structures that are similar to cranio-lenticulo-sutural dysplasia patient cells with SEC23A mutations, which encodes a component of COPII vesicles. Our data reveal a novel ESCRT-dependent pathway that influences the ERGIC and autophagosome formation.

Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis.

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 â€‹hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. Here we show that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of larval salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. We also show that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Vps13D functions in a Pink1-dependent and Parkin-independent mitophagy pathway.

Defects in autophagy cause problems in metabolism, development, and disease. The autophagic clearance of mitochondria, mitophagy, is impaired by the loss of Vps13D. Here, we discover that Vps13D regulates mitophagy in a pathway that depends on the core autophagy machinery by regulating Atg8a and ubiquitin localization. This process is Pink1 dependent, with loss of pink1 having similar autophagy and mitochondrial defects as loss of vps13d. The role of Pink1 has largely been studied in tandem with Park/Parkin, an E3 ubiquitin ligase that is widely considered to be crucial in Pink1-dependent mitophagy. Surprisingly, we find that loss of park does not exhibit the same autophagy and mitochondrial deficiencies as vps13d and pink1 mutant cells and contributes to mitochondrial clearance through a pathway that is parallel to vps13d. These findings provide a Park-independent pathway for Pink1-regulated mitophagy and help to explain how Vps13D regulates autophagy and mitochondrial morphology and contributes to neurodegenerative diseases.

Vmp1, Vps13D, and Marf/Mfn2 function in a conserved pathway to regulate mitochondria and ER contact in development and disease.

Mutations in Vps13D cause defects in autophagy, clearance of mitochondria, and human movement disorders. Here, we discover that Vps13D functions in a pathway downstream of Vmp1 and upstream of Marf/Mfn2. Like vps13d, vmp1 mutant cells exhibit defects in autophagy, mitochondrial size, and clearance. Through the relationship between vmp1 and vps13d, we reveal a novel role for Vps13D in the regulation of mitochondria and endoplasmic reticulum (ER) contact. Significantly, the function of Vps13D in mitochondria and ER contact is conserved between fly and human cells, including fibroblasts derived from patients suffering from VPS13D mutation-associated neurological symptoms. vps13d mutants have increased levels of Marf/MFN2, a regulator of mitochondrial fusion. Importantly, loss of marf/MFN2 suppresses vps13d mutant phenotypes, including mitochondria and ER contact. These findings indicate that Vps13d functions at a regulatory point between mitochondria and ER contact, mitochondrial fusion and autophagy, and help to explain how Vps13D contributes to disease.

Histological assessment of developmental cell death in Drosophila pupae.

This protocol describes the embedding and processing of Drosophila pupae in paraffin to monitor tissue changes during development. Although multiple methods are available to evaluate developmental changes in Drosophila embryos, imaging detailed changes during metamorphosis is challenging as the animal is enclosed in the cuticle, rendering it inaccessible to whole mount imaging. Here, we present a protocol that focuses on developmental clearance of the larval salivary glands in Drosophila pupae that can be extended to examine other tissues/stages for similar purposes. For complete details on the use and execution of this protocol, please refer to Velentzas et al. (2018).

VPS13D promotes peroxisome biogenesis.

The VPS13 gene family consists of VPS13A-D in mammals. Although all four genes have been linked to human diseases, their cellular functions are poorly understood, particularly those of VPS13D. We generated and characterized knockouts of each VPS13 gene in HeLa cells. Among the individual knockouts, only VPS13D-KO cells exhibit abnormal mitochondrial morphology. Additionally, VPS13D loss leads to either partial or complete peroxisome loss in several transformed cell lines and in fibroblasts derived from a VPS13D mutation-carrying patient with recessive spinocerebellar ataxia. Our data show that VPS13D regulates peroxisome biogenesis.

A conserved myotubularin-related phosphatase regulates autophagy by maintaining autophagic flux.

Macroautophagy (autophagy) targets cytoplasmic cargoes to the lysosome for degradation. Like all vesicle trafficking, autophagy relies on phosphoinositide identity, concentration, and localization to execute multiple steps in this catabolic process. Here, we screen for phosphoinositide phosphatases that influence autophagy in Drosophila and identify CG3530. CG3530 is homologous to the human MTMR6 subfamily of myotubularin-related 3-phosphatases, and therefore, we named it dMtmr6. dMtmr6, which is required for development and viability in Drosophila, functions as a regulator of autophagic flux in multiple Drosophila cell types. The MTMR6 family member MTMR8 has a similar function in autophagy of higher animal cells. Decreased dMtmr6 and MTMR8 function results in autophagic vesicle accumulation and influences endolysosomal homeostasis.

Autophagy in animal development.

Macroautophagy (autophagy) delivers intracellular constituents to the lysosome to promote catabolism. During development in multiple organisms, autophagy mediates various cellular processes, including survival during starvation, programmed cell death, phagocytosis, organelle elimination, and miRNA regulation. Our current understanding of autophagy has been enhanced by developmental biology research during the last quarter of a century. Through experiments that focus on animal development, fundamental mechanisms that control autophagy and that contribute to disease were elucidated. Studies in embryos revealed specific autophagy molecules that mediate the removal of paternally derived mitochondria, and identified autophagy components that clear protein aggregates during development. Importantly, defects in mtDNA inheritance, or removal of paternal mtDNA via mitochondrial autophagy, can contribute to mitochondrial-associated disease. In addition, impairment of the clearance of protein aggregates by autophagy underlies neurodegenerative diseases. Experiments in multiple organisms also reveal conserved mechanisms of tissue remodeling that rely on the cooperation between autophagy and apoptosis to clear cell corpses, and defects in autophagy and apoptotic cell clearance can contribute to inflammation and autoimmunity. Here we provide an overview of key developmental processes that are mediated by autophagy in multiple animals.

Autophagy, Inflammation, and Metabolism (AIM) Center in its second year.

The NIH-funded center for autophagy research named Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center is now completing its second year as a working center with a mission to promote autophagy research locally, nationally, and internationally. The center has thus far supported a cadre of 6 junior faculty (mentored PIs; mPIs) at a near-R01 level of funding. Two mPIs have graduated by obtaining their independent R01 funding and 3 of the remaining 4 have won significant funding from NIH in the form of R21 and R56 awards. The first year and a half of setting up the center has been punctuated by completion of renovations and acquisition and upgrades for equipment supporting autophagy, inflammation and metabolism studies. The scientific cores usage, and the growth of new studies is promoted through pilot grants and several types of enablement initiatives. The intent to cultivate AIM as a scholarly hub for autophagy and related studies is manifested in its Vibrant Campus Initiative, and the Tuesday AIM Seminar series, as well as by hosting a major scientific event, the 2019 AIM symposium, with nearly one third of the faculty from the International Council of Affiliate Members being present and leading sessions, giving talks, and conducting workshop activities. These and other events are often videostreamed for a worldwide scientific audience, and information about events at AIM and elsewhere are disseminated on Twitter and can be followed on the AIM web site. AIM intends to invigorate research on overlapping areas between autophagy, inflammation and metabolism with a number of new initiatives to promote metabolomic research. With the turnover of mPIs as they obtain their independent funding, new junior faculty are recruited and appointed as mPIs. All these activities are in keeping with AIM's intention to enable the next generation of autophagy researchers and help anchor, disseminate, and convey the depth and excitement of the autophagy field.

The NF-κB Factor Relish Regulates Atg1 Expression and Controls Autophagy.

Macroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. Here, we show that the nuclear factor κB (NF-κB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation. Surprisingly, of the classic Imd pathway components, only Relish and the PGRP receptors were involved in salivary gland degradation. Significantly, Relish controls salivary gland degradation by regulating autophagy but not caspases. In addition, expression of either Relish or PGRP-LC causes premature autophagy induction and subsequent gland degradation. Relish controls autophagy by regulating the expression of Atg1, a core component and activator of the autophagy pathway. Together these findings demonstrate that a NF-κB pathway regulates autophagy during developmentally programmed cell death.

Autophagy Promotes Tumor-like Stem Cell Niche Occupancy.

Adult stem cells usually reside in specialized niche microenvironments. Accumulating evidence indicates that competitive niche occupancy favors stem cells with oncogenic mutations, also known as tumor-like stem cells. However, the mechanisms that regulate tumor-like stem cell niche occupancy are largely unknown. Here, we use Drosophila ovarian germline stem cells as a model and use bam mutant cells as tumor-like stem cells. Interestingly, we find that autophagy is low in wild-type stem cells but elevated in bam mutant stem cells. Significantly, autophagy is required for niche occupancy by bam mutant stem cells. Although loss of either atg6 or Fip200 alone in stem cells does not impact their competitiveness, loss of these conserved regulators of autophagy decreases bam mutant stem cell niche occupancy. In addition, starvation enhances the competition of bam mutant stem cells for niche occupancy in an autophagy-dependent manner. Of note, loss of autophagy slows the cell cycle of bam mutant stem cells and does not influence stem cell death. In contrast to canonical epithelial cell competition, loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influences the competition of bam mutant stem cells for niche occupancy. Additionally, autophagy promotes the tumor-like growth of bam mutant ovaries. Autophagy is known to be induced in a wide variety of tumors. Therefore, these results suggest that specifically targeting autophagy in tumor-like stem cells has potential as a therapeutic strategy.

The Proton-Coupled Monocarboxylate Transporter Hermes Is Necessary for Autophagy during Cell Death.

Nutrient availability influences the production and degradation of materials that are required for cell growth and survival. Autophagy is a nutrient-regulated process that is used to degrade cytoplasmic materials and has been associated with human diseases. Solute transporters influence nutrient availability and sensing, yet we know little about how transporters influence autophagy. Here, we screen for solute transporters that are required for autophagy-dependent cell death and identify CG11665/hermes. We show that hermes is required for both autophagy during steroid-triggered salivary gland cell death and TNF-induced non-apoptotic eye cell death. hermes encodes a proton-coupled monocarboxylate transporter that preferentially transports pyruvate over lactate. mTOR signaling is elevated in hermes mutant cells, and decreased mTOR function suppresses the hermes salivary gland cell death phenotype. Hermes is most similar to human SLC16A11, a protein that was recently implicated in type 2 diabetes, thus providing a link between pyruvate, mTOR, autophagy, and possibly metabolic disorders.

Life, death and autophagy.

Autophagy influences cell survival through maintenance of cell bioenergetics and clearance of protein aggregates and damaged organelles. Several lines of evidence indicate that autophagy is a multifaceted regulator of cell death, but controversy exists over whether autophagy alone can drive cell death under physiologically relevant circumstances. Here, we review the role of autophagy in cell death and examine how autophagy interfaces with other forms of cell death including apoptosis and necrosis.