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Nature Reviews. Molecular Cell Biology Nov 2021Autophagy is a versatile degradation system for maintaining cellular homeostasis whereby cytosolic materials are sequestered in a double-membrane autophagosome and... (Review)
Review
Autophagy is a versatile degradation system for maintaining cellular homeostasis whereby cytosolic materials are sequestered in a double-membrane autophagosome and subsequently delivered to lysosomes, where they are broken down. In multicellular organisms, newly formed autophagosomes undergo a process called 'maturation', in which they fuse with vesicles originating from endolysosomal compartments, including early/late endosomes and lysosomes, to form amphisomes, which eventually become degradative autolysosomes. This fusion process requires the concerted actions of multiple regulators of membrane dynamics, including SNAREs, tethering proteins and RAB GTPases, and also transport of autophagosomes and late endosomes/lysosomes towards each other. Multiple mechanisms modulate autophagosome maturation, including post-translational modification of key components, spatial distribution of phosphoinositide lipid species on membranes, RAB protein dynamics, and biogenesis and function of lysosomes. Nutrient status and various stresses integrate into the autophagosome maturation machinery to coordinate the progression of autophagic flux. Impaired autophagosome maturation is linked to the pathogenesis of various human diseases, including neurodegenerative disorders, cancer and myopathies. Furthermore, invading pathogens exploit various strategies to block autophagosome maturation, thus evading destruction and even subverting autophagic vacuoles (autophagosomes, amphisomes and autolysosomes) for survival, growth and/or release. Here, we discuss the recent progress in our understanding of the machinery and regulation of autophagosome maturation, the relevance of these mechanisms to human pathophysiology and how they are harnessed by pathogens for their benefit. We also provide perspectives on targeting autophagosome maturation therapeutically.
Topics: Autophagosomes; Autophagy; Endosomes; Humans; Lysosomes; Neurodegenerative Diseases; Phagosomes; Protein Processing, Post-Translational; SNARE Proteins; Transport Vesicles; rab GTP-Binding Proteins
PubMed: 34302147
DOI: 10.1038/s41580-021-00392-4 -
Cellular and Molecular Life Sciences :... Feb 2021Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide... (Review)
Review
Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and sequentially triggers two signaling cascades: the first one involving TIRAP and MyD88 adaptor proteins is induced in the plasma membrane, whereas the second engaging adaptor proteins TRAM and TRIF begins in early endosomes after endocytosis of the receptor. The LPS-induced internalization of TLR4 and hence also the activation of the TRIF-dependent pathway is governed by a GPI-anchored protein, CD14. The endocytosis of TLR4 terminates the MyD88-dependent signaling, while the following endosome maturation and lysosomal degradation of TLR4 determine the duration and magnitude of the TRIF-dependent one. Alternatively, TLR4 may return to the plasma membrane, which process is still poorly understood. Therefore, the course of the LPS-induced pro-inflammatory responses depends strictly on the rates of TLR4 endocytosis and trafficking through the endo-lysosomal compartment. Notably, prolonged activation of TLR4 is linked with several hereditary human diseases, neurodegeneration and also with autoimmune diseases and cancer. Recent studies have provided ample data on the role of diverse proteins regulating the functions of early, late, and recycling endosomes in the TLR4-induced inflammation caused by LPS or phagocytosis of E. coli. In this review, we focus on the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and also of LPS, in immune cells and discuss how dysregulation of the endo-lysosomal compartment contributes to the development of diverse human diseases.
Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Humans; Inflammation; Lipopolysaccharide Receptors; Lipopolysaccharides; Membrane Glycoproteins; Myeloid Differentiation Factor 88; Phagocytosis; Receptors, Interleukin-1; Toll-Like Receptor 4
PubMed: 33057840
DOI: 10.1007/s00018-020-03656-y -
Science (New York, N.Y.) Aug 2020Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest...
Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest that Huntington's disease might affect neurodevelopment. To determine whether this is actually the case, we examined tissue from human fetuses (13 weeks gestation) that carried the Huntington's disease mutation. These tissues showed clear abnormalities in the developing cortex, including mislocalization of mutant huntingtin and junctional complex proteins, defects in neuroprogenitor cell polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. We observed the same phenomena in Huntington's disease mouse embryos, where we linked these abnormalities to defects in interkinetic nuclear migration of progenitor cells. Huntington's disease thus has a neurodevelopmental component and is not solely a degenerative disease.
Topics: Animals; Cell Cycle; Endosomes; Fetus; Humans; Huntingtin Protein; Huntington Disease; Mice; Mice, Mutant Strains; Mitosis; Mutation; Nervous System; Neuroepithelial Cells; Tight Junctions; Zonula Occludens-1 Protein
PubMed: 32675289
DOI: 10.1126/science.aax3338 -
Nature Communications Jul 2021Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully...
Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.
Topics: Cell Communication; Cell Membrane; Endosomes; Exosomes; Extracellular Vesicles; Fusion Regulatory Protein 1, Heavy Chain; Gene Knockout Techniques; HeLa Cells; Humans; Membrane Proteins; Protein Transport; Proteomics; Tetraspanin 29; Tetraspanin 30
PubMed: 34282141
DOI: 10.1038/s41467-021-24384-2 -
The Biochemical Journal May 2021Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during... (Review)
Review
Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during a sequential maturation process of autophagosomes before their ultimate fusion with lysosomes for cargo degradation. This process is highly regulated with multiple protein machineries, such as SNAREs, Rab GTPases, tethering complexes, and ESCRTs, are involved to facilitate autophagic flux to proceed. In neurons, autophagosomes are robustly generated in axonal terminals and then rapidly fuse with late endosomes to form amphisomes. This fusion event allows newly generated autophagosomes to gain retrograde transport motility and move toward the soma, where proteolytically active lysosomes are predominantly located. Amphisomes are not only the products of autophagosome maturation but also the intersection of the autophagy and endo-lysosomal pathways. Importantly, amphisomes can also participate in non-canonical functions, such as retrograde neurotrophic signaling or autophagy-based unconventional secretion by fusion with the plasma membrane. In this review, we provide an updated overview of the recent discoveries and advancements on the molecular and cellular mechanisms underlying amphisome biogenesis and the emerging roles of amphisomes. We discuss recent developments towards the understanding of amphisome regulation as well as the implications in the context of major neurodegenerative diseases, with a comparative focus on Alzheimer's disease and Parkinson's disease.
Topics: Animals; Autophagosomes; Autophagy; Endosomes; Humans; Neurodegenerative Diseases; Neurons
PubMed: 34047789
DOI: 10.1042/BCJ20200917 -
Metabolic Brain Disease Dec 2021Niemann-Pick type C (NPC) disease is a genetically determined neurodegenerative metabolic disease. It belongs to the lysosomal storage diseases and its main cause is... (Review)
Review
Niemann-Pick type C (NPC) disease is a genetically determined neurodegenerative metabolic disease. It belongs to the lysosomal storage diseases and its main cause is impaired cholesterol transport in late endosomes or lysosomes. It is an autosomal recessive inherited disease that results from mutations in the NPC1 or NPC2 genes. The treatment efforts are focused on the slowing its progression. The only registered drug, devoted for NPC patients is Miglustat. Effective treatment is still under development. NPC disease mainly affects the nervous system, and the crossing of the blood-brain barrier by medicines is still a challenge, therefore the combination therapies of several compounds are increasingly being worked on. The aim of this paper is to present the possibilities in treatment of Niemann-Pick type C disease. The discussed research results relate to animal studies.
Topics: Animals; Cholesterol; Endosomes; Humans; Lysosomes; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C
PubMed: 34596813
DOI: 10.1007/s11011-021-00842-0 -
Nature Sep 2022Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly. The...
Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly. The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments. How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism that is likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries.
Topics: Animals; Axon Initial Segment; Caenorhabditis elegans; Cell Membrane; Cell Polarity; Dendrites; Diffusion; Endocytosis; Endosomes; Humans; Mice; Protein Transport; Proteolysis; Rats; Receptors, Cell Surface
PubMed: 35978188
DOI: 10.1038/s41586-022-05074-5 -
Journal of Controlled Release :... Jul 2022Delivery of nucleic acids, such as mRNA, to immune cells has become a major focus in the past decade with ionizable lipid nanoparticles (LNPs) emerging as a...
Delivery of nucleic acids, such as mRNA, to immune cells has become a major focus in the past decade with ionizable lipid nanoparticles (LNPs) emerging as a clinically-validated delivery platform. LNPs-typically composed of ionizable lipids, cholesterol, phospholipids, and polyethylene glycol lipids -have been designed and optimized for a variety of applications including cancer therapies, vaccines, and gene editing. However, LNPs have only recently been investigated for delivery to T cells, which has various therapeutic applications including the engineering of T cell immunotherapies. While several LNP formulations have been evaluated for mRNA delivery, recent work has demonstrated that the utilization of cholesterol analogs may enhance mRNA delivery. Other studies have shown that cholesterols modified with hydroxyl groups can alter endocytic recycling mechanisms. Here, we engineered a library of LNPs incorporating hydroxycholesterols to evaluate their impact on mRNA delivery to T cells by leveraging endosomal trafficking mechanisms. Substitution of 25% and 50% 7α-hydroxycholesterol for cholesterol in LNPs enhanced mRNA delivery to primary human T cells ex vivo by 1.8-fold and 2.0-fold, respectively. Investigation of endosomal trafficking revealed that these modifications also increase late endosome production and reduce the presence of recycling endosomes. These results suggest that hydroxyl modification of cholesterol molecules incorporated into LNP formulations provides a mechanism for improving delivery of nucleic acid cargo to T cells for a range of immunotherapy applications.
Topics: Cholesterol; Humans; Hydroxycholesterols; Lipids; Liposomes; Nanoparticles; RNA, Messenger; T-Lymphocytes
PubMed: 35569584
DOI: 10.1016/j.jconrel.2022.05.020 -
Developmental Cell Oct 2022Gasdermin D (GSDMD)-mediated pyroptosis induces immunogenic cell death and promotes inflammation. However, the functions of GSDMD in tissue homeostasis remain unclear....
Gasdermin D (GSDMD)-mediated pyroptosis induces immunogenic cell death and promotes inflammation. However, the functions of GSDMD in tissue homeostasis remain unclear. Here, we identify a physiological function of GSDMD in osteoclasts via a non-lytic p20-generated protein, which prevents bone loss to maintain bone homeostasis. In the late stage of RANKL-induced osteoclastogenesis, GSDMD underwent cleavage, which is dependent on RIPK1 and caspase-8/-3, to yield this p20 product. Gsdmd-deficient osteoclasts showed normal differentiation but enhanced bone resorption with excessive lysosomal activity. Mice with complete or myeloid-specific Gsdmd deletion exhibited increased trabecular bone loss and more severe aging/ovariectomy-induced osteoporosis. GSDMD p20 was preferentially localized to early endosomes and limited endo-lysosomal trafficking and maturation, relying on its oligomerization and control of phosphoinositide conversion by binding to phosphatidylinositol 3-phosphate (PI(3)P). We have thus identified an anti-osteoclastic function of GSDMD as a checkpoint for lysosomal maturation and secretion and linked this to bone homeostasis and endosome-lysosome biology.
Topics: Animals; Female; Mice; Bone Resorption; Caspase 8; Intracellular Signaling Peptides and Proteins; Lysosomes; Mice, Inbred C57BL; Phosphate-Binding Proteins; Phosphatidylinositol Phosphates
PubMed: 36243012
DOI: 10.1016/j.devcel.2022.09.013 -
Developmental Cell Jul 2021Mitochondria are critical metabolic and signaling hubs, and dysregulated mitochondrial homeostasis is implicated in many diseases. Degradation of damaged mitochondria by...
Mitochondria are critical metabolic and signaling hubs, and dysregulated mitochondrial homeostasis is implicated in many diseases. Degradation of damaged mitochondria by selective GABARAP/LC3-dependent macro-autophagy (mitophagy) is critical for maintaining mitochondrial homeostasis. To identify alternate forms of mitochondrial quality control that functionally compensate if mitophagy is inactive, we selected for autophagy-dependent cancer cells that survived loss of LC3-dependent autophagosome formation caused by inactivation of ATG7 or RB1CC1/FIP200. We discovered rare surviving autophagy-deficient clones that adapted to maintain mitochondrial homeostasis after gene inactivation and identified two enhanced mechanisms affecting mitochondria including mitochondrial dynamics and mitochondrial-derived vesicles (MDVs). To further understand these mechanisms, we quantified MDVs via flow cytometry and confirmed an SNX9-mediated mechanism necessary for flux of MDVs to lysosomes. We show that the autophagy-dependent cells acquire unique dependencies on these processes, indicating that these alternate forms of mitochondrial homeostasis compensate for loss of autophagy to maintain mitochondrial health.
Topics: Autophagy; Autophagy-Related Protein 7; Autophagy-Related Proteins; Endosomes; Humans; Lysosomes; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Dynamics; Mitophagy; Sorting Nexins; Transport Vesicles
PubMed: 34171288
DOI: 10.1016/j.devcel.2021.06.003