S1PR1 inhibition induces proapoptotic signaling in T cells and limits humoral responses within lymph nodes.

Effective immunity requires a large, diverse naive T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this proapoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naive T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.

Sphingosine 1-phosphate receptor 1 inhibition induces a pro-apoptotic signaling cascade in T cells.

Effective immunity requires a large, diverse naïve T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we address how S1P enables T cell survival, and the implications for patients treated with S1PR1 antagonists. Contrary to expectations, we found that S1PR1 limits apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization are required to prevent this pro-apoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naïve T cells limited B cell responses. Our findings highlight an unexpected effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggest both limitations and novel uses of this important class of drugs.

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.

A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma.

Tumors exhibit enhancer reprogramming compared to normal tissue. The etiology is largely attributed to cell-intrinsic genomic alterations. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon, we find divergent epigenetic landscapes between CRC tumors and cell lines. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over normal. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating cell-extrinsic etiology. We demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway. We show this activation enables efficient growth under oxidative conditions, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the significance of epigenomic profiling on primary specimens.

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

Mitochondrial localization and moderated activity are key to murine erythroid enucleation.

Mammalian red blood cells (RBCs), which primarily contain hemoglobin, exemplify an elaborate maturation process, with the terminal steps of RBC generation involving extensive cellular remodeling. This encompasses alterations of cellular content through distinct stages of erythroblast maturation that result in the expulsion of the nucleus (enucleation) followed by the loss of mitochondria and all other organelles and a transition to anaerobic glycolysis. Whether there is any link between erythroid removal of the nucleus and the function of any other organelle, including mitochondria, remains unknown. Here we demonstrate that mitochondria are key to nuclear clearance. Using live and confocal microscopy and high-throughput single-cell imaging, we show that before nuclear polarization, mitochondria progressively move toward one side of maturing erythroblasts and aggregate near the nucleus as it extrudes from the cell, a prerequisite for enucleation to proceed. Although we found active mitochondrial respiration is required for nuclear expulsion, levels of mitochondrial activity identify distinct functional subpopulations, because terminally maturing erythroblasts with low relative to high mitochondrial membrane potential are at a later stage of maturation, contain greatly condensed nuclei with reduced open chromatin-associated acetylation histone marks, and exhibit higher enucleation rates. Lastly, to our surprise, we found that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation, primarily through pyruvate but independent of in situ glycolysis. These findings demonstrate the critical but unanticipated functions of mitochondria during the erythroblast enucleation process. They are also relevant to the in vitro production of RBCs as well as to disorders of the erythroid lineage.

Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness.

Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes.

High-Throughput Cell Death Assays with Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling (SPARKL).

High-throughput cytostatic and cell death assays are a critical component of pharmacological screens and mechanism-based interrogations into cellular biology. We developed a method for single-cell and population-level analyses using real-time kinetic labeling (abbreviated "SPARKL") with non-toxic fluorescent probes and high-content live-cell imagers. The protocols herein detail the steps, specifics, and suggested utilization of the SPARKL method within several "label-and-go" zero-handling workflows. For complete details on the use and execution of this protocol, please refer to Gelles et al. (2019).

Cell death through the ages: The ICDS 25th Anniversary Meeting.

In June of 2019, the International Cell Death Society (ICDS) held its 25th anniversary meeting in New York City at the Icahn School of Medicine at Mount Sinai organized by Drs. Richard A. Lockshin (St. John's University, USA), Zahra Zakeri (Queens College, USA), and Jerry Edward Chipuk (Icahn School of Medicine at Mount Sinai, USA). The three-day event, entitled 'Cell death through the ages: The ICDS 25th anniversary meeting', hosted ninety-one delegates including thirty-four speakers and twenty-two poster presentations. Additionally, the organizers gave special recognition to the twenty-one previous ICDS Lifetime Achievement awardees-those who have significantly contributed to the field of cell death and the growth of the organization. Here, we provide a summary of the meeting and highlight trending research in the fields of cell death, autophagy, immunology, and their impact on health and disease.

Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling Couples Proliferation and Cell Death Mechanisms.

Quantifying cytostatic and cytotoxic outcomes are integral components of characterizing perturbagens used as research tools and in drug discovery pipelines. Furthermore, data-rich acquisition, coupled with robust methods for analysis, is required to properly assess the function and impact of these perturbagens. Here, we present a detailed and versatile method for single-cell and population-level analyses using real-time kinetic labeling (SPARKL). SPARKL integrates high-content live-cell imaging with automated detection and analysis of fluorescent reporters of cell death. We outline several examples of zero-handling, non-disruptive protocols for detailing cell death mechanisms and proliferation profiles. Additionally, we suggest several methods for mathematically analyzing these data to best utilize the collected kinetic data. Compared to traditional methods of detection and analysis, SPARKL is more sensitive, accurate, and high throughput while substantially eliminating sample processing and providing richer data.

Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool.

Caloric restriction is known to improve inflammatory and autoimmune diseases. However, the mechanisms by which reduced caloric intake modulates inflammation are poorly understood. Here we show that short-term fasting reduced monocyte metabolic and inflammatory activity and drastically reduced the number of circulating monocytes. Regulation of peripheral monocyte numbers was dependent on dietary glucose and protein levels. Specifically, we found that activation of the low-energy sensor 5'-AMP-activated protein kinase (AMPK) in hepatocytes and suppression of systemic CCL2 production by peroxisome proliferator-activator receptor alpha (PPARα) reduced monocyte mobilization from the bone marrow. Importantly, we show that fasting improves chronic inflammatory diseases without compromising monocyte emergency mobilization during acute infectious inflammation and tissue repair. These results reveal that caloric intake and liver energy sensors dictate the blood and tissue immune tone and link dietary habits to inflammatory disease outcome.

Mitochondrial origins of fractional control in regulated cell death.

Individual cells in clonal populations often respond differently to environmental changes; for binary phenotypes, such as cell death, this can be measured as a fractional response. These types of responses have been attributed to cell-intrinsic stochastic processes and variable abundances of biochemical constituents, such as proteins, but the influence of organelles is still under investigation. We use the response to TNF-related apoptosis inducing ligand (TRAIL) and a new statistical framework for determining parameter influence on cell-to-cell variability through the inference of variance explained, DEPICTIVE, to demonstrate that variable mitochondria abundance correlates with cell survival and determines the fractional cell death response. By quantitative data analysis and modeling we attribute this effect to variable effective concentrations at the mitochondria surface of the pro-apoptotic proteins Bax/Bak. Further, our study suggests that inhibitors of anti-apoptotic Bcl-2 family proteins, used in cancer treatment, may increase the diversity of cellular responses, enhancing resistance to treatment.

Late-onset megaconial myopathy in mice lacking group I Paks.

BACKGROUND: Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase ß (Chk ß) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKß are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. METHODS: We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase ß (Chk ß) were analyzed for Pak1/2 phosphorylation. RESULTS: dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkß and are a model for human diseases associated with CHKß deficiency. Pak1/2 and Chkß activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. CONCLUSIONS: Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKß deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

Activation of Nrf2 Is Required for Normal and ChREBPα-Augmented Glucose-Stimulated ß-Cell Proliferation.

Patients with both major forms of diabetes would benefit from therapies that increase ß-cell mass. Glucose, a natural mitogen, drives adaptive expansion of ß-cell mass by promoting ß-cell proliferation. We previously demonstrated that a carbohydrate response element-binding protein (ChREBPα) is required for glucose-stimulated ß-cell proliferation and that overexpression of ChREBPα amplifies the proliferative effect of glucose. Here we found that ChREBPα reprogrammed anabolic metabolism to promote proliferation. ChREBPα increased mitochondrial biogenesis, oxygen consumption rates, and ATP production. Proliferation augmentation by ChREBPα required the presence of ChREBPß. ChREBPα increased the expression and activity of Nrf2, initiating antioxidant and mitochondrial biogenic programs. The induction of Nrf2 was required for ChREBPα-mediated mitochondrial biogenesis and for glucose-stimulated and ChREBPα-augmented ß-cell proliferation. Overexpression of Nrf2 was sufficient to drive human ß-cell proliferation in vitro; this confirms the importance of this pathway. Our results reveal a novel pathway necessary for ß-cell proliferation that may be exploited for therapeutic ß-cell regeneration.

FBXW7 regulates a mitochondrial transcription program by modulating MITF.

FBXW7 is well characterized as a tumor suppressor in many human cancers including melanoma; however, the mechanisms of tumor-suppressive function have not been fully elucidated. We leveraged two distinct RNA sequencing datasets: human melanoma cell lines (n = 10) with control versus silenced FBXW7 and a cohort of human melanoma tumor samples (n = 51) to define the transcriptomic fingerprint regulated by FBXW7. Here, we report that loss of FBXW7 enhances a mitochondrial gene transcriptional program that is dependent on MITF in human melanoma and confers poor patient outcomes. MITF is a lineage-specific master regulator of melanocytes and together with PGC-1alpha is a marker for melanoma subtypes with dependence for mitochondrial oxidative metabolism. We found that inactivation of FBXW7 elevates MITF protein levels in melanoma cells. In vitro studies examining loss of FBXW7 and MITF alone or in combination showed that FBXW7 is an upstream regulator for the MITF/PGC-1 signaling.

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.

Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells.

Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P1 receptor (S1P1R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P1R on T cells, and that the requirement for S1P1R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAFV600E), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAFV600E is pharmacologically inhibited by targeted therapies (e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAFV600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

Mitochondrial Fission in Human Diseases.

Mitochondria are an essential component of multicellular life - from primitive organisms, to highly complex entities like mammals. The importance of mitochondria is underlined by their plethora of well-characterized essential functions such as energy production through oxidative phosphorylation (OX-PHOS), calcium and reactive oxygen species (ROS) signaling, and regulation of apoptosis. In addition, novel roles and attributes of mitochondria are coming into focus through the recent years of mitochondrial research. In particular, over the past decade the study of mitochondrial shape and dynamics has achieved special significance, as they are found to impact mitochondrial function. Recent advances indicate that mitochondrial function and dynamics are inter-connected, and maintain the balance between health and disease at a cellular and an organismal level. For example, excessive mitochondrial division (fission) is associated with functional defects, and is implicated in multiple human diseases from neurodegenerative diseases to cancer. In this chapter we examine the recent literature on the mitochondrial dynamics-function relationship, and explore how it impacts on the development and progression of human diseases. We will also highlight the implications of therapeutic manipulation of mitochondrial dynamics in treating various human pathologies.