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1.
Nat Commun ; 14(1): 3077, 2023 05 29.
Article in English | MEDLINE | ID: mdl-37248218

ABSTRACT

Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. Here we show that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. Our results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system.


Subject(s)
Drosophila , Microtubule-Associated Proteins , Animals , Drosophila/metabolism , Microtubule-Associated Proteins/metabolism , Phagocytosis/genetics , Autophagy/genetics , Axons/metabolism , Neuroglia/metabolism
2.
Front Neurosci ; 16: 954949, 2022.
Article in English | MEDLINE | ID: mdl-36278016

ABSTRACT

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.

3.
PLoS Genet ; 18(6): e1010257, 2022 06.
Article in English | MEDLINE | ID: mdl-35737721

ABSTRACT

Elucidating signal transduction mechanisms of innate immune pathways is essential to defining how they elicit distinct cellular responses. Toll-like receptors (TLR) signal through their cytoplasmic TIR domains which bind other TIR domain-containing adaptors. dSARM/SARM1 is one such TIR domain adaptor best known for its role as the central axon degeneration trigger after injury. In degeneration, SARM1's domains have been assigned unique functions: the ARM domain is auto-inhibitory, SAM-SAM domain interactions mediate multimerization, and the TIR domain has intrinsic NAD+ hydrolase activity that precipitates axonal demise. Whether and how these distinct functions contribute to TLR signaling is unknown. Here we show divergent signaling requirements for dSARM in injury-induced axon degeneration and TLR-mediated developmental glial phagocytosis through analysis of new knock-in domain and point mutations. We demonstrate intragenic complementation between reciprocal pairs of domain mutants during development, providing evidence for separability of dSARM functional domains in TLR signaling. Surprisingly, dSARM's NAD+ hydrolase activity is strictly required for both degenerative and developmental signaling, demonstrating that TLR signal transduction requires dSARM's enzymatic activity. In contrast, while SAM domain-mediated dSARM multimerization is important for axon degeneration, it is dispensable for TLR signaling. Finally, dSARM functions in a linear genetic pathway with the MAP3K Ask1 during development but not in degenerating axons. Thus, we propose that dSARM exists in distinct signaling states in developmental and pathological contexts.


Subject(s)
Armadillo Domain Proteins , NAD , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolism , Cytoskeletal Proteins/genetics , Hydrolases/metabolism , Phagocytosis/genetics , Signal Transduction/genetics
4.
J Vis Exp ; (157)2020 03 16.
Article in English | MEDLINE | ID: mdl-32225164

ABSTRACT

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.


Subject(s)
Axons/physiology , Drosophila melanogaster/physiology , Synapses/physiology , Animals , Mutation , Neurodegenerative Diseases/metabolism , Neurons/physiology , Optogenetics , Signal Transduction , Wings, Animal/anatomy & histology , Wings, Animal/physiology
5.
Open Biol ; 9(8): 190118, 2019 08 30.
Article in English | MEDLINE | ID: mdl-31455157

ABSTRACT

Axon loss is a shared feature of nervous systems being challenged in neurological disease, by chemotherapy or mechanical force. Axons take up the vast majority of the neuronal volume, thus numerous axonal intrinsic and glial extrinsic support mechanisms have evolved to promote lifelong axonal survival. Impaired support leads to axon degeneration, yet underlying intrinsic signalling cascades actively promoting the disassembly of axons remain poorly understood in any context, making the development to attenuate axon degeneration challenging. Wallerian degeneration serves as a simple model to study how axons undergo injury-induced axon degeneration (axon death). Severed axons actively execute their own destruction through an evolutionarily conserved axon death signalling cascade. This pathway is also activated in the absence of injury in diseased and challenged nervous systems. Gaining insights into mechanisms underlying axon death signalling could therefore help to define targets to block axon loss. Herein, we summarize features of axon death at the molecular and subcellular level. Recently identified and characterized mediators of axon death signalling are comprehensively discussed in detail, and commonalities and differences across species highlighted. We conclude with a summary of engaged axon death signalling in humans and animal models of neurological conditions. Thus, gaining mechanistic insights into axon death signalling broadens our understanding beyond a simple injury model. It harbours the potential to define targets for therapeutic intervention in a broad range of human axonopathies.


Subject(s)
Axons/metabolism , Neurons/metabolism , Signal Transduction , Wallerian Degeneration/metabolism , Animals , Biomarkers , Cell Death , Disease Progression , Disease Susceptibility , Humans , Species Specificity , Wallerian Degeneration/diagnosis , Wallerian Degeneration/etiology
6.
Neuron ; 103(1): 52-65.e6, 2019 07 03.
Article in English | MEDLINE | ID: mdl-31101394

ABSTRACT

Mitochondria are essential in long axons to provide metabolic support and sustain neuron integrity. A healthy mitochondrial pool is maintained by biogenesis, transport, mitophagy, fission, and fusion, but how these events are regulated in axons is not well defined. Here, we show that the Drosophila glutathione S-transferase (GST) Gfzf prevents mitochondrial hyperfusion in axons. Gfzf loss altered redox balance between glutathione (GSH) and oxidized glutathione (GSSG) and initiated mitochondrial fusion through the coordinated action of Mfn and Opa1. Gfzf functioned epistatically with the thioredoxin peroxidase Jafrac1 and the thioredoxin reductase 1 TrxR-1 to regulate mitochondrial dynamics. Altering GSH:GSSG ratios in mouse primary neurons in vitro also induced hyperfusion. Mitochondrial changes caused deficits in trafficking, the metabolome, and neuronal physiology. Changes in GSH and oxidative state are associated with neurodegenerative diseases like Alzheimer's. Our demonstration that GSTs are key in vivo regulators of axonal mitochondrial length and number provides a potential mechanistic link.


Subject(s)
Axons/physiology , Carrier Proteins/physiology , Glutathione/metabolism , Mitochondria/physiology , Animals , Axons/ultrastructure , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Female , Membrane Proteins/genetics , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Neurons/metabolism , Oxidation-Reduction , Peroxidases/genetics , Peroxidases/physiology , Pregnancy , Primary Cell Culture , Thioredoxin Reductase 1/genetics , Thioredoxin Reductase 1/physiology
7.
Curr Opin Neurobiol ; 53: 162-173, 2018 12.
Article in English | MEDLINE | ID: mdl-30241058

ABSTRACT

Adult, circuit-integrated neurons must be maintained and supported for the life span of their host. The attenuation of either maintenance or plasticity leads to impaired circuit function and ultimately to neurodegenerative disorders. Over the last few years, significant discoveries of molecular mechanisms were made that mediate the formation and maintenance of axons. Here, we highlight intrinsic and extrinsic mechanisms that ensure the health and survival of axons. We also briefly discuss examples of mutations associated with impaired axonal maintenance identified in specific neurological conditions. A better understanding of these mechanisms will therefore help to define targets for therapeutic interventions.


Subject(s)
Axons/metabolism , Biological Transport/physiology , Mitochondria/metabolism , Neurodegenerative Diseases/metabolism , Neurodevelopmental Disorders/metabolism , Neuroglia/metabolism , Animals , Humans
8.
Proc Natl Acad Sci U S A ; 115(6): 1358-1363, 2018 02 06.
Article in English | MEDLINE | ID: mdl-29295933

ABSTRACT

Genetic studies of Wallerian degeneration have led to the identification of signaling molecules (e.g., dSarm/Sarm1, Axundead, and Highwire) that function locally in axons to drive degeneration. Here we identify a role for the Drosophila C2H2 zinc finger transcription factor Pebbled [Peb, Ras-responsive element binding protein 1 (RREB1) in mammals] in axon death. Loss of Peb in Drosophila glutamatergic sensory neurons results in either complete preservation of severed axons, or an axon death phenotype where axons fragment into large, continuous segments, rather than completely disintegrate. Peb is expressed in developing and mature sensory neurons, suggesting it is required to establish or maintain their competence to undergo axon death. peb mutant phenotypes can be rescued by human RREB1, and they exhibit dominant genetic interactions with dsarm mutants, linking peb/RREB1 to the axon death signaling cascade. Surprisingly, Peb is only able to fully block axon death signaling in glutamatergic, but not cholinergic sensory neurons, arguing for genetic diversity in axon death signaling programs in different neuronal subtypes. Our findings identify a transcription factor that regulates axon death signaling, and peb mutant phenotypes of partial fragmentation reveal a genetically accessible step in axon death signaling.


Subject(s)
Axons/pathology , Drosophila Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Wallerian Degeneration/pathology , Animals , Animals, Genetically Modified , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolism , Axons/metabolism , Cholinergic Neurons/pathology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Wallerian Degeneration/genetics , Wallerian Degeneration/metabolism , Wings, Animal/innervation , Wings, Animal/metabolism , Zinc Fingers/genetics
9.
Neuron ; 95(1): 78-91.e5, 2017 Jul 05.
Article in English | MEDLINE | ID: mdl-28683272

ABSTRACT

Axon degeneration is a hallmark of neurodegenerative disease and neural injury. Axotomy activates an intrinsic pro-degenerative axon death signaling cascade involving loss of the NAD+ biosynthetic enzyme Nmnat/Nmnat2 in axons, activation of dSarm/Sarm1, and subsequent Sarm-dependent depletion of NAD+. Here we identify Axundead (Axed) as a mediator of axon death. axed mutants suppress axon death in several types of axons for the lifespan of the fly and block the pro-degenerative effects of activated dSarm in vivo. Neurodegeneration induced by loss of the sole fly Nmnat ortholog is also fully blocked by axed, but not dsarm, mutants. Thus, pro-degenerative pathways activated by dSarm signaling or Nmnat elimination ultimately converge on Axed. Remarkably, severed axons morphologically preserved by axon death pathway mutations remain integrated in circuits and able to elicit complex behaviors after stimulation, indicating that blockade of axon death signaling results in long-term functional preservation of axons.


Subject(s)
Armadillo Domain Proteins/genetics , Axons/metabolism , Cytoskeletal Proteins/genetics , Drosophila Proteins/genetics , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Wallerian Degeneration/genetics , Animals , Animals, Genetically Modified , Armadillo Domain Proteins/metabolism , Arthropod Antennae/injuries , Arthropod Antennae/innervation , Axotomy , Behavior, Animal , Blotting, Western , Cell Line , Cytoskeletal Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster , Grooming , Immunity, Active , NAD/metabolism , Neurons/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Optogenetics , Wallerian Degeneration/metabolism , Wings, Animal/injuries , Wings, Animal/innervation
10.
Nat Commun ; 8: 14355, 2017 02 06.
Article in English | MEDLINE | ID: mdl-28165006

ABSTRACT

Draper/Ced-1/MEGF-10 is an engulfment receptor that promotes clearance of cellular debris in C. elegans, Drosophila and mammals. Draper signals through an evolutionarily conserved Src family kinase cascade to drive cytoskeletal rearrangements and target engulfment through Rac1. Glia also alter gene expression patterns in response to axonal injury but pathways mediating these responses are poorly defined. We show Draper is cell autonomously required for glial activation of transcriptional reporters after axonal injury. We identify TNF receptor associated factor 4 (TRAF4) as a novel Draper binding partner that is required for reporter activation and phagocytosis of axonal debris. TRAF4 and misshapen (MSN) act downstream of Draper to activate c-Jun N-terminal kinase (JNK) signalling in glia, resulting in changes in transcriptional reporters that are dependent on Drosophila AP-1 (dAP-1) and STAT92E. Our data argue injury signals received by Draper at the membrane are important regulators of downstream transcriptional responses in reactive glia.


Subject(s)
Axons/pathology , Drosophila Proteins/metabolism , Membrane Proteins/metabolism , Nerve Degeneration/metabolism , Neuroglia/pathology , Signal Transduction/physiology , Animals , Animals, Genetically Modified , Axons/metabolism , Cell Membrane/metabolism , Cell Membrane/pathology , Drosophila melanogaster/metabolism , Female , JNK Mitogen-Activated Protein Kinases/metabolism , Male , Nerve Degeneration/pathology , Neuroglia/cytology , Neuroglia/metabolism , Phagocytosis , STAT Transcription Factors/metabolism , TNF Receptor-Associated Factor 4/metabolism , Transcription Factor AP-1/metabolism
11.
Curr Biol ; 25(16): 2130-6, 2015 Aug 17.
Article in English | MEDLINE | ID: mdl-26234214

ABSTRACT

The RNA-processing protein TDP-43 is central to the pathogenesis of amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron (MN) disease. TDP-43 is conserved in Drosophila, where it has been the topic of considerable study, but how TDP-43 mutations lead to age-dependent neurodegeneration is unclear and most approaches have not directly examined changes in MN morphology with age. We used a mosaic approach to study age-dependent MN loss in the adult fly leg where it is possible to resolve single motor axons, NMJs and active zones, and perform rapid forward genetic screens. We show that expression of TDP-43(Q331K) caused dying-back of NMJs and axons, which could not be suppressed by mutations that block Wallerian degeneration. We report the identification of three genes that suppress TDP-43 toxicity, including shaggy/GSK3, a known modifier of neurodegeneration. The two additional novel suppressors, hat-trick and xmas-2, function in chromatin modeling and RNA export, two processes recently implicated in human ALS. Loss of shaggy/GSK3, hat-trick, or xmas-2 does not suppress Wallerian degeneration, arguing TDP-43(Q331K)-induced and Wallerian degeneration are genetically distinct processes. In addition to delineating genetic factors that modify TDP-43 toxicity, these results establish the Drosophila adult leg as a valuable new tool for the in vivo study of adult MN phenotypes.


Subject(s)
Aging , Amyotrophic Lateral Sclerosis/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/physiology , Motor Neurons/physiology , Nerve Degeneration/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , DNA-Binding Proteins/metabolism , Disease Models, Animal , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Humans , Larva/genetics , Larva/physiology , Nerve Degeneration/physiopathology
12.
Proc Natl Acad Sci U S A ; 111(27): 9965-70, 2014 Jul 08.
Article in English | MEDLINE | ID: mdl-24958874

ABSTRACT

Axons damaged by acute injury, toxic insults, or neurodegenerative diseases execute a poorly defined autodestruction signaling pathway leading to widespread fragmentation and functional loss. Here, we describe an approach to study Wallerian degeneration in the Drosophila L1 wing vein that allows for analysis of axon degenerative phenotypes with single-axon resolution in vivo. This method allows for the axotomy of specific subsets of axons followed by examination of progressive axonal degeneration and debris clearance alongside uninjured control axons. We developed new Flippase (FLP) reagents using proneural gene promoters to drive FLP expression very early in neural lineages. These tools allow for the production of mosaic clone populations with high efficiency in sensory neurons in the wing. We describe a collection of lines optimized for forward genetic mosaic screens using MARCM (mosaic analysis with a repressible cell marker; i.e., GFP-labeled, homozygous mutant) on all major autosomal arms (∼95% of the fly genome). Finally, as a proof of principle we screened the X chromosome and identified a collection eight recessive and two dominant alleles of highwire, a ubiquitin E3 ligase required for axon degeneration. Similar unbiased forward genetic screens should help rapidly delineate axon death genes, thereby providing novel potential drug targets for therapeutic intervention to prevent axonal and synaptic loss.


Subject(s)
Axons , Drosophila/genetics , Alleles , Animals , Genes, Dominant , Genes, Insect , Genes, Recessive , Mosaicism , Wings, Animal/blood supply
13.
Trends Cell Biol ; 24(9): 515-23, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24780172

ABSTRACT

The elimination of large portions of axons is a widespread event in the developing and diseased nervous system. Subsets of axons are selectively destroyed to help fine-tune neural circuit connectivity during development. Axonal degeneration is also an early feature of nearly all neurodegenerative diseases, occurs after most neural injuries, and is a primary driver of functional impairment in patients. In this review we discuss the diversity of cellular mechanisms by which axons degenerate. Initial molecular characterization highlights some similarities in their execution but also argues that unique genetic programs modulate each mode of degeneration. Defining these pathways rigorously will provide new targets for therapeutic intervention after neural injury or in neurodegenerative disease.


Subject(s)
Axons/metabolism , Neurodegenerative Diseases/metabolism , Neuroglia/metabolism , Axons/pathology , Humans , Nerve Degeneration/pathology , Nervous System/metabolism , Nervous System/pathology , Neurodegenerative Diseases/pathology , Neuroglia/pathology
14.
Genome Res ; 22(7): 1360-71, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22454234

ABSTRACT

MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression. As miRNAs are involved in a wide range of biological processes and diseases, much effort has been invested in identifying their mRNA targets. Here, we present a novel combinatorial approach, RIP-chip-SRM (RNA-binding protein immunopurification + microarray + targeted protein quantification via selected reaction monitoring), to identify de novo high-confidence miRNA targets in the nematode Caenorhabditis elegans. We used differential RIP-chip analysis of miRNA-induced silencing complexes from wild-type and miRNA mutant animals, followed by quantitative targeted proteomics via selected reaction monitoring to identify and validate mRNA targets of the C. elegans bantam homolog miR-58. Comparison of total mRNA and protein abundance changes in mir-58 mutant and wild-type animals indicated that the direct bantam/miR-58 targets identified here are mainly regulated at the level of protein abundance, not mRNA stability.


Subject(s)
Caenorhabditis elegans/genetics , MicroRNAs/metabolism , Proteomics/methods , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/metabolism , Caenorhabditis elegans/metabolism , Crosses, Genetic , Immunologic Techniques/methods , MicroRNAs/genetics , Oligonucleotide Array Sequence Analysis/methods , Plasmids/genetics , Plasmids/metabolism , Protein Biosynthesis , RNA Interference , RNA Stability , RNA, Helminth/genetics , RNA, Helminth/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transgenes
15.
PLoS Biol ; 10(1): e1001245, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22272187

ABSTRACT

Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3)P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K) Vps34 is regarded as the only kinase that produces PtdIns(3)P in phagosomal membranes. We report here that, in the nematode C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3)P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3)P in a manner dependent on the large GTPase dynamin (DYN-1). PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3)P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3)P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3)P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3)P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3)P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events.


Subject(s)
Apoptosis , Caenorhabditis elegans/cytology , Caenorhabditis elegans/enzymology , Phagosomes/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphoprotein Phosphatases/metabolism , Animals , Caenorhabditis elegans/ultrastructure , Caenorhabditis elegans Proteins/metabolism , Down-Regulation , Enzyme Activation , Phagosomes/ultrastructure , Protein Binding , rab GTP-Binding Proteins/metabolism
16.
Proc Natl Acad Sci U S A ; 108(11): 4441-6, 2011 Mar 15.
Article in English | MEDLINE | ID: mdl-21368173

ABSTRACT

Frontotemporal lobar degeneration is a progressive neurodegenerative syndrome that is the second most common cause of early-onset dementia. Mutations in the progranulin gene are a major cause of familial frontotemporal lobar degeneration [Baker M, et al. (2006) Nature 442:916-919 and Cruts M, et al. (2006) Nature 442:920-924]. Although progranulin is involved in wound healing, inflammation, and tumor growth, its role in the nervous system and the mechanism by which insufficient levels result in neurodegeneration are poorly understood [Eriksen and Mackenzie (2008) J Neurochem 104:287-297]. We have characterized the normal function of progranulin in the nematode Caenorhabditis elegans. We found that mutants lacking pgrn-1 appear grossly normal, but exhibit fewer apoptotic cell corpses during development. This reduction in corpse number is not caused by reduced apoptosis, but instead by more rapid clearance of dying cells. Likewise, we found that macrophages cultured from progranulin KO mice displayed enhanced rates of apoptotic-cell phagocytosis. Although most neurodegenerative diseases are thought to be caused by the toxic effects of aggregated proteins, our findings suggest that susceptibility to neurodegeneration may be increased by a change in the kinetics of programmed cell death. We propose that cells that might otherwise recover from damage or injury are destroyed in progranulin mutants, which in turn facilitates disease progression.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/genetics , Caenorhabditis elegans Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , Mutation/genetics , Neurodegenerative Diseases/genetics , Animals , Apoptosis Regulatory Proteins/metabolism , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Granulins , Intercellular Signaling Peptides and Proteins/metabolism , Intestinal Mucosa/metabolism , Intestines/cytology , Kinetics , Longevity , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, Knockout , Models, Biological , Neurons/cytology , Neurons/metabolism , Phagocytosis , Progranulins
17.
Nat Cell Biol ; 13(1): 79-86, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21170032

ABSTRACT

Multicellular animals rapidly clear dying cells from their bodies. Many of the pathways that mediate this cell removal are conserved through evolution. Here, we identify srgp-1 as a negative regulator of cell clearance in both Caenorhabditis elegans and mammalian cells. Loss of srgp-1 function results in improved engulfment of apoptotic cells, whereas srgp-1 overexpression inhibits apoptotic cell corpse removal. We show that SRGP-1 functions in engulfing cells and functions as a GTPase activating protein (GAP) for CED-10 (Rac1). Interestingly, loss of srgp-1 function promotes not only the clearance of already dead cells, but also the removal of cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults. In contrast, impaired engulfment allows damaged cells to escape clearance, which results in increased long-term survival. We propose that C. elegans uses the engulfment machinery as part of a primitive, but evolutionarily conserved, survey mechanism that identifies and removes unfit cells within a tissue.


Subject(s)
Apoptosis , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , GTPase-Activating Proteins/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Cell Line , GTPase-Activating Proteins/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Phagocytosis , Protein Binding , RNA Interference , Sequence Homology, Amino Acid , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolism
18.
PLoS Biol ; 8(2): e1000297, 2010 Feb 02.
Article in English | MEDLINE | ID: mdl-20126385

ABSTRACT

Wnt signalling pathways have extremely diverse functions in animals, including induction of cell fates or tumours, guidance of cell movements during gastrulation, and the induction of cell polarity. Wnt can induce polar changes in cellular morphology by a remodelling of the cytoskeleton. However, how activation of the Frizzled receptor induces cytoskeleton rearrangement is not well understood. We show, by an in depth 4-D microscopy analysis, that the Caenorhabditis elegans Wnt pathway signals to CED-10/Rac via two separate branches to regulate modulation of the cytoskeleton in different cellular situations. Apoptotic cell clearance and migration of the distal tip cell require the MOM-5/Fz receptor, GSK-3 kinase, and APC/APR-1, which activate the CED-2/5/12 branch of the engulfment machinery. MOM-5 (Frizzled) thus can function as an engulfment receptor in C. elegans. Our epistatic analyses also suggest that the two partially redundant signalling pathways defined earlier for engulfment may act in a single pathway in early embryos. By contrast, rearrangement of mitotic spindles requires the MOM-5/Fz receptor, GSK-3 kinase, and beta-catenins, but not the downstream factors LIT-1/NLK or POP-1/Tcf. Taken together, our results indicate that in multiple developmental processes, CED-10/Rac can link polar signals mediated by the Wnt pathway to rearrangements of the cytoskeleton.


Subject(s)
Apoptosis/physiology , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans , Signal Transduction/physiology , Spindle Apparatus/metabolism , Wnt Proteins/metabolism , rac GTP-Binding Proteins/metabolism , Animals , Apoptosis/genetics , Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Signal Transduction/genetics , Two-Hybrid System Techniques , Wnt Proteins/genetics , beta Catenin/genetics , beta Catenin/metabolism , rac GTP-Binding Proteins/genetics
19.
Curr Biol ; 17(11): 994-9, 2007 Jun 05.
Article in English | MEDLINE | ID: mdl-17540571

ABSTRACT

Phospholipids are distributed asymmetrically across the plasma-membrane bilayer of eukaryotic cells: Phosphatidylserine (PS), phosphatidylethanolamine, and phosphoinositides are predominantly restricted to the inner leaflet, whereas phophatidylcholine and sphingolipids are enriched on the outer leaflet [1, 2]. Exposure of PS on the cell surface is a conserved feature of apoptosis and plays an important role in promoting the clearance of apoptotic cells by phagocytosis [3]. However, the molecular mechanism that drives PS exposure remains mysterious. To address this issue, we studied cell-surface changes during apoptosis in the nematode C. elegans. Here, we show that PS exposure can readily be detected on apoptotic C. elegans cells. We generated a transgenic strain expressing a GFP::Annexin V reporter to screen for genes required for this process. Although none of the known engulfment genes was required, RNAi knockdown of the putative aminophospholipid transporter gene tat-1 abrogated PS exposure on apoptotic cells. tat-1(RNAi) also reduced the efficiency of cell-corpse clearance, suggesting that PS exposure acts as an "eat-me" signal in worms. We propose that tat-1 homologs might also play an important role in PS exposure in mammals.


Subject(s)
Apoptosis/physiology , Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/cytology , Cell Membrane/metabolism , Phosphatidylserines/metabolism , Phospholipid Transfer Proteins/physiology , Animals , Biomarkers , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/metabolism , Cells, Cultured , Embryonic Development/genetics , Germ Cells/metabolism , Green Fluorescent Proteins/analysis , Organisms, Genetically Modified/metabolism , Phospholipid Transfer Proteins/antagonists & inhibitors , Phospholipid Transfer Proteins/metabolism , RNA Interference
20.
Mol Cell Biochem ; 256-257(1-2): 407-24, 2004.
Article in English | MEDLINE | ID: mdl-14977199

ABSTRACT

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na(+)/Cl(-)-dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain. In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (alpha-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the alpha-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our alpha-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of approximately 60 kDa was enriched. This latter is most likely representing the genuine plasma membrane CrT. Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the approximately 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions. In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with 14C-Cr-isotope tracer studies and a number of [31P]-NMR magnetization transfer studies, as well as with recent [1H]-NMR spectroscopy data.


Subject(s)
Membrane Transport Proteins/metabolism , Amino Acid Metabolism, Inborn Errors/genetics , Amino Acid Metabolism, Inborn Errors/metabolism , Amino Acid Sequence , Animals , Creatine/metabolism , Humans , Male , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Mitochondria/metabolism , Molecular Sequence Data , Rats , Rats, Wistar , Sequence Homology, Amino Acid
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