Decoding Cell Death: From a Veritable Library of Babel to Vade Mecum?

Programmed cell death (PCD) is a requisite feature of development and homeostasis but can also be indicative of infections, injuries, and pathologies. In concordance with these heterogeneous contexts, an array of disparate effector responses occur downstream of cell death and its clearance-spanning tissue morphogenesis, homeostatic turnover, host defense, active dampening of inflammation, and tissue repair. This raises a fundamental question of how a single contextually appropriate response ensues after an event of PCD. To explore how complex inputs may together tailor the specificity of the resulting effector response, here we consider (a) the varying contexts during which different cell death modalities are observed, (b) the nature of the information that can be passed on by cell corpses, and (c) the ways by which efferocyte populations synthesize signals from dying cells with those from the surrounding microenvironment.

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes.

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.

Neutralizing antibodies evolve to exploit vulnerable sites in the HCV envelope glycoprotein E2 and mediate spontaneous clearance of infection.

Individuals who clear primary hepatitis C virus (HCV) infections clear subsequent reinfections more than 80% of the time, but the mechanisms are poorly defined. Here, we used HCV variants and plasma from individuals with repeated clearance to characterize longitudinal changes in envelope glycoprotein E2 sequences, function, and neutralizing antibody (NAb) resistance. Clearance of infection was associated with early selection of viruses with NAb resistance substitutions that also reduced E2 binding to CD81, the primary HCV receptor. Later, peri-clearance plasma samples regained neutralizing capacity against these variants. We identified a subset of broadly NAbs (bNAbs) for which these loss-of-fitness substitutions conferred resistance to unmutated bNAb ancestors but increased sensitivity to mature bNAbs. These data demonstrate a mechanism by which neutralizing antibodies contribute to repeated immune-mediated HCV clearance, identifying specific bNAbs that exploit fundamental vulnerabilities in E2. The induction of bNAbs with these specificities should be a goal of HCV vaccine development.

Mechanisms and Functions of Spatial Protein Quality Control.

A healthy proteome is essential for cell survival. Protein misfolding is linked to a rapidly expanding list of human diseases, ranging from neurodegenerative diseases to aging and cancer. Many of these diseases are characterized by the accumulation of misfolded proteins in intra- and extracellular inclusions, such as amyloid plaques. The clear link between protein misfolding and disease highlights the need to better understand the elaborate machinery that manages proteome homeostasis, or proteostasis, in the cell. Proteostasis depends on a network of molecular chaperones and clearance pathways involved in the recognition, refolding, and/or clearance of aberrant proteins. Recent studies reveal that an integral part of the cellular management of misfolded proteins is their spatial sequestration into several defined compartments. Here, we review the properties, function, and formation of these compartments. Spatial sequestration plays a central role in protein quality control and cellular fitness and represents a critical link to the pathogenesis of protein aggregation-linked diseases.

ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei.

The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.

Interleukin-33 Promotes Serotonin Release from Enterochromaffin Cells for Intestinal Homeostasis.

The gastrointestinal tract is known as the largest endocrine organ that encounters and integrates various immune stimulations and neuronal responses due to constant environmental challenges. Enterochromaffin (EC) cells, which function as chemosensors on the gut epithelium, are known to translate environmental cues into serotonin (5-HT) production, contributing to intestinal physiology. However, how immune signals participate in gut sensation and neuroendocrine response remains unclear. Interleukin-33 (IL-33) acts as an alarmin cytokine by alerting the system of potential environmental stresses. We here demonstrate that IL-33 induced instantaneous peristaltic movement and facilitated Trichuris muris expulsion. We found that IL-33 could be sensed by EC cells, inducing release of 5-HT. IL-33-mediated 5-HT release activated enteric neurons, subsequently promoting gut motility. Mechanistically, IL-33 triggered calcium influx via a non-canonical signaling pathway specifically in EC cells to induce 5-HT secretion. Our data establish an immune-neuroendocrine axis in calibrating rapid 5-HT release for intestinal homeostasis.

Fluid transport in the brain.

The brain harbors a unique ability to, figuratively speaking, shift its gears. During wakefulness, the brain is geared fully toward processing information and behaving, while homeostatic functions predominate during sleep. The blood-brain barrier establishes a stable environment that is optimal for neuronal function, yet the barrier imposes a physiological problem; transcapillary filtration that forms extracellular fluid in other organs is reduced to a minimum in brain. Consequently, the brain depends on a special fluid [the cerebrospinal fluid (CSF)] that is flushed into brain along the unique perivascular spaces created by astrocytic vascular endfeet. We describe this pathway, coined the term glymphatic system, based on its dependency on astrocytic vascular endfeet and their adluminal expression of aquaporin-4 water channels facing toward CSF-filled perivascular spaces. Glymphatic clearance of potentially harmful metabolic or protein waste products, such as amyloid-ß, is primarily active during sleep, when its physiological drivers, the cardiac cycle, respiration, and slow vasomotion, together efficiently propel CSF inflow along periarterial spaces. The brain's extracellular space contains an abundance of proteoglycans and hyaluronan, which provide a low-resistance hydraulic conduit that rapidly can expand and shrink during the sleep-wake cycle. We describe this unique fluid system of the brain, which meets the brain's requisites to maintain homeostasis similar to peripheral organs, considering the blood-brain-barrier and the paths for formation and egress of the CSF.

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Zygotic genome activation during the maternal-to-zygotic transition.

Embryogenesis depends on a highly coordinated cascade of genetically encoded events. In animals, maternal factors contributed by the egg cytoplasm initially control development, whereas the zygotic nuclear genome is quiescent. Subsequently, the genome is activated, embryonic gene products are mobilized, and maternal factors are cleared. This transfer of developmental control is called the maternal-to-zygotic transition (MZT). In this review, we discuss recent advances toward understanding the scope, timing, and mechanisms that underlie zygotic genome activation at the MZT in animals. We describe high-throughput techniques to measure the embryonic transcriptome and explore how regulation of the cell cycle, chromatin, and transcription factors together elicits specific patterns of embryonic gene expression. Finally, we illustrate the interplay between zygotic transcription and maternal clearance and show how these two activities combine to reprogram two terminally differentiated gametes into a totipotent embryo.

Targeting Efferocytosis in Inflammaging.

Rapid removal of apoptotic cells by phagocytes, a process known as efferocytosis, is key for the maintenance of tissue homeostasis, the resolution of inflammation, and tissue repair. However, impaired efferocytosis can result in the accumulation of apoptotic cells, subsequently triggering sterile inflammation through the release of endogenous factors such as DNA and nuclear proteins from membrane permeabilized dying cells. Here, we review the molecular basis of the three key phases of efferocytosis, that is, the detection, uptake, and degradation of apoptotic materials by phagocytes. We also discuss how defects in efferocytosis due to the alteration of phagocytes and dying cells can contribute to the low-grade chronic inflammation that occurs during aging, described as inflammaging. Lastly, we explore opportunities in targeting and harnessing the efferocytic machinery to limit aging-associated inflammatory diseases.

Mitochondrial defects triggered by amg-1 mutation elicit UPRmt and phagocytic clearance during spermatogenesis in C. elegans.

Mitochondria are the powerhouses of many biological processes. During spermatogenesis, post-transcriptional regulation of mitochondrial gene expression is mediated by nuclear-encoded mitochondrial RNA-binding proteins (mtRBPs). We identified AMG-1 as an mtRBP required for reproductive success in Caenorhabditis elegans. amg-1 mutation led to defects in mitochondrial structure and sperm budding, resulting in mitochondria being discarded into residual bodies, which ultimately delayed spermatogenesis in the proximal gonad. In addition, mitochondrial defects triggered the gonadal mitochondrial unfolded protein response and phagocytic clearance to ensure spermatogenesis but ultimately failed to rescue hermaphroditic fertility. These findings reveal a previously undiscovered role for AMG-1 in regulating C. elegans spermatogenesis, in which mitochondrial-damaged sperm prevented the transmission of defective mitochondria to mature sperm by budding and phagocytic clearance, a process which may also exist in the reproductive systems of higher organisms.

Transfer of Cell-Surface Antigens by Scavenger Receptor CD36 Promotes Thymic Regulatory T Cell Receptor Repertoire Development and Allo-tolerance.

The development of T cell tolerance in the thymus requires the presentation of host proteins by multiple antigen-presenting cell (APC) types. However, the importance of transferring host antigens from transcription factor AIRE-dependent medullary thymic epithelial cells (mTECs) to bone marrow (BM) APCs is unknown. We report that antigen was primarily transferred from mTECs to CD8α+ dendritic cells (DCs) and showed that CD36, a scavenger receptor selectively expressed on CD8α+ DCs, mediated the transfer of cell-surface, but not cytoplasmic, antigens. The absence of CD8α+ DCs or CD36 altered thymic T cell selection, as evidenced by TCR repertoire analysis and the loss of allo-tolerance in murine allogeneic BM transplantation (allo-BMT) studies. Decreases in these DCs and CD36 expression in peripheral blood of human allo-BMT patients correlated with graft-versus-host disease. Our findings suggest that CD36 facilitates transfer of mTEC-derived cell-surface antigen on CD8α+ DCs to promote tolerance to host antigens during homeostasis and allo-BMT.

Foraging mechanisms in excavate flagellates shed light on the functional ecology of early eukaryotes.

The phagotrophic flagellates described as "typical excavates" have been hypothesized to be morphologically similar to the Last Eukaryotic Common Ancestor and understanding the functional ecology of excavates may therefore help shed light on the ecology of these early eukaryotes. Typical excavates are characterized by a posterior flagellum equipped with a vane that beats in a ventral groove. Here, we combined flow visualization and observations of prey capture in representatives of the three clades of excavates with computational fluid dynamic modeling, to understand the functional significance of this cell architecture. We record substantial differences amongst species in the orientation of the vane and the beat plane of the posterior flagellum. Clearance rate magnitudes estimated from flow visualization and modeling are both like that of other similarly sized flagellates. The interaction between a vaned flagellum beating in a confinement is modeled to produce a very efficient feeding current at low energy costs, irrespective of the beat plane and vane orientation and of all other morphological variations. Given this predicted uniformity of function, we suggest that the foraging systems of typical excavates studied here may be good proxies to understand those potentially used by our distant ancestors more than 1 billion years ago.

Roles of Individual Human Cytochrome P450 Enzymes in Drug Metabolism.

Our knowledge of the roles of individual cytochrome P450 (P450, CYP) enzymes in drug metabolism has developed considerably in the past 30 years, and this base has been of considerable use in avoiding serious issues with drug interactions and issues due to variations. Some newer approaches are being considered for "phenotyping" of metabolism reactions with new drug candidates. Endogenous biomarkers are being used for non-invasive estimation of levels of individual P450 enzymes. There is also the matter of some remaining "orphan" P450s, which have yet to be assigned reactions. Practical problems that continue in drug development include predicting drug-drug interactions, predicting the effects of polymorphic and other P450 variations, and evaluating inter-species differences in drug metabolism, particularly in the context of "metabolism in safety testing" (MIST) regulatory issues ("disproportionate (human) metabolites"). Significance Statement Cytochrome P450 enzymes are the major catalysts involved in drug metabolism. The characterization of their individual roles has major implications in drug development and clinical practice.

Apoptotic cell clearance components in inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints that affects ~1% of the human population. Joint swelling and bone erosion, hallmarks of RA, contribute to disability and, sometimes, loss of life. Mechanistically, disease is driven by immune dysregulation characterized by circulating autoantibodies, inflammatory mediators, tissue degradative enzymes, and metabolic dysfunction of resident stromal and recruited immune cells. Cell death by apoptosis has been therapeutically explored in animal models of RA due to the comparisons drawn between synovial hyperplasia and paucity of apoptosis in RA with the malignant transformation of cancer cells. Several efforts to induce cell death have shown benefits in reducing the development and/or severity of the disease. Apoptotic cells are cleared by phagocytes in a process known as efferocytosis, which differs from microbial phagocytosis in its "immuno-silent," or anti-inflammatory, nature. Failures in efferocytosis have been linked to autoimmune disease, whereas administration of apoptotic cells in RA models effectively inhibits inflammatory indices, likely though efferocytosis-mediated resolution-promoting mechanisms. However, the nature of signaling pathways elicited and the molecular identity of clearance mediators in RA are understudied. Furthermore, canonical efferocytosis machinery elements also play important non-canonical functions in homeostasis and pathology. Here, we discuss the roles of efferocytosis machinery components in models of RA and discuss their potential involvement in disease pathophysiology.

Erythropoietin signaling in peripheral macrophages is required for systemic ß-amyloid clearance.

Impaired clearance of beta-amyloid (Aß) is a primary cause of sporadic Alzheimer's disease (AD). Aß clearance in the periphery contributes to reducing brain Aß levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aß-degrading enzymes, and Aß clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aß-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aß levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aß clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Tissue-Resident Macrophages Are Locally Programmed for Silent Clearance of Apoptotic Cells.

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.

Dual-color live imaging unveils stepwise organization of multiple basal body arrays by cytoskeletons.

For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.

c-JUN-mediated transcriptional responses in lymphatic endothelial cells are required for lung fluid clearance at birth.

Fluid clearance mediated by lymphatic vessels is known to be essential for lung inflation and gas-exchange function during the transition from prenatal to postnatal life, yet the molecular mechanisms that regulate lymphatic function remain unclear. Here, we profiled the molecular features of lymphatic endothelial cells (LECs) in embryonic and postnatal day (P) 0 lungs by single-cell RNA-sequencing analysis. We identified that the expression of c-JUN is transiently upregulated in P0 LECs. Conditional knockout of Jun in LECs impairs the opening of lung lymphatic vessels at birth, leading to fluid retention in the lungs and neonatal death. We further demonstrated that increased mechanical pressure induces the expression of c-JUN in LECs. c-JUN regulates the opening of lymphatic vessels by modulating the remodeling of the actin cytoskeleton in LECs. Our study established the essential regulatory function of c-JUN-mediated transcriptional responses in facilitating lung lymphatic fluid clearance at birth.