20 results on '"Josh Dubnau"'
Search Results
2. TDP-43 pathology in Drosophila induces glial-cell type specific toxicity that can be ameliorated by knock-down of SF2/SRSF1.
- Author
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Sarah Krupp, Isabel Hubbard, Oliver Tam, Gale M Hammell, and Josh Dubnau
- Subjects
Genetics ,QH426-470 - Abstract
Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.
- Published
- 2023
- Full Text
- View/download PDF
3. A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia.
- Author
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Jorge Azpurua, Enas Gad El-Karim, Marvel Tranquille, and Josh Dubnau
- Subjects
Genetics ,QH426-470 - Abstract
Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer's disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear/import export- pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator.
- Published
- 2021
- Full Text
- View/download PDF
4. Intercellular viral spread and intracellular transposition of Drosophila gypsy.
- Author
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Richard M Keegan, Lillian R Talbot, Yung-Heng Chang, Michael J Metzger, and Josh Dubnau
- Subjects
Genetics ,QH426-470 - Abstract
It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome.
- Published
- 2021
- Full Text
- View/download PDF
5. Nuclear Transcriptomes of the Seven Neuronal Cell Types That Constitute the Drosophila Mushroom Bodies
- Author
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Meng-Fu Maxwell Shih, Fred Pejman Davis, Gilbert Lee Henry, and Josh Dubnau
- Subjects
Drosophila ,mushroom body ,transcriptome ,RNA-seq ,Genetics ,QH426-470 - Abstract
The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The Drosophila melanogaster MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes (γ, α′/β′ and α/β) and 7 cell subtypes (γd, γm, α′/β′ap, α′/β′m, α/βp, α/βs and α/βc) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. Here, we used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. We identified 350 MB class- or subtype-specific genes, including the widely used α/β class marker Fas2 and the α′/β′ class marker trio. Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, our data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the Drosophila MB provides a valuable resource for the fly neuroscience community.
- Published
- 2019
- Full Text
- View/download PDF
6. Postmortem Cortex Samples Identify Distinct Molecular Subtypes of ALS: Retrotransposon Activation, Oxidative Stress, and Activated Glia
- Author
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Oliver H. Tam, Nikolay V. Rozhkov, Regina Shaw, Duyang Kim, Isabel Hubbard, Samantha Fennessey, Nadia Propp, Delphine Fagegaltier, Brent T. Harris, Lyle W. Ostrow, Hemali Phatnani, John Ravits, Josh Dubnau, Molly Gale Hammell, Justin Kwan, Dhruv Sareen, James R. Broach, Zachary Simmons, Ximena Arcila-Londono, Edward B. Lee, Vivianna M. Van Deerlin, Neil A. Shneider, Ernest Fraenkel, Frank Baas, Noah Zaitlen, James D. Berry, Andrea Malaspina, Pietro Fratta, Gregory A. Cox, Leslie M. Thompson, Steve Finkbeiner, Efthimios Dardiotis, Timothy M. Miller, Siddharthan Chandran, Suvankar Pal, Eran Hornstein, Daniel J. MacGowan, Terry Heiman-Patterson, Molly G. Hammell, Nikolaos.A. Patsopoulos, Oleg Butovsky, Joshua Dubnau, Avindra Nath, Robert Bowser, Matt Harms, Eleonora Aronica, Mary Poss, Jennifer Phillips-Cremins, John Crary, Nazem Atassi, Dale J. Lange, Darius J. Adams, Leonidas Stefanis, Marc Gotkine, Robert Baloh, Suma Babu, Towfique Raj, Sabrina Paganoni, Ophir Shalem, Colin Smith, and Bin Zhang
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Summary: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several pathogenic mutations have been identified, the vast majority of ALS cases have no family history of disease. Thus, for most ALS cases, the disease may be a product of multiple pathways contributing to varying degrees in each patient. Using machine learning algorithms, we stratify the transcriptomes of 148 ALS postmortem cortex samples into three distinct molecular subtypes. The largest cluster, identified in 61% of patient samples, displays hallmarks of oxidative and proteotoxic stress. Another 19% of the samples shows predominant signatures of glial activation. Finally, a third group (20%) exhibits high levels of retrotransposon expression and signatures of TARDBP/TDP-43 dysfunction. We further demonstrate that TDP-43 (1) directly binds a subset of retrotransposon transcripts and contributes to their silencing in vitro, and (2) pathological TDP-43 aggregation correlates with retrotransposon de-silencing in vivo. : Tam et al. present transcriptome profiling results from a large set of amyotrophic lateral sclerosis (ALS) patient cortex samples, finding 3 distinct groups. Two ALS subtypes are marked by gene pathways previously associated with ALS disease, while a third group shows elevated retrotransposon expression linked to TDP-43 pathology. Keywords: amyotrophic lateral sclerosis, retrotransposons, transposable elements, TDP-43, neurodegenerative disease, neurodegeneration, genetics and genomics of ALS
- Published
- 2019
- Full Text
- View/download PDF
7. Cellular labeling of endogenous retrovirus replication (CLEVR) reveals de novo insertions of the gypsy retrotransposable element in cell culture and in both neurons and glial cells of aging fruit flies.
- Author
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Yung-Heng Chang, Richard M Keegan, Lisa Prazak, and Josh Dubnau
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging, such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. We describe a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses.
- Published
- 2019
- Full Text
- View/download PDF
8. Retrotransposon activation contributes to neurodegeneration in a Drosophila TDP-43 model of ALS.
- Author
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Lisa Krug, Nabanita Chatterjee, Rebeca Borges-Monroy, Stephen Hearn, Wen-Wei Liao, Kathleen Morrill, Lisa Prazak, Nikolay Rozhkov, Delphine Theodorou, Molly Hammell, and Josh Dubnau
- Subjects
Genetics ,QH426-470 - Abstract
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.
- Published
- 2017
- Full Text
- View/download PDF
9. Endogenous Retroviruses and TDP-43 Proteinopathy Form a Sustaining Feedback to Drive the Intercellular Spread of Neurodegeneration
- Author
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Yung-Heng Chang and Josh Dubnau
- Abstract
Neurodegenerative disorders are thought to initiate focally and then spread over time through neural circuits. A mechanism that has been proposed to explain this is the inter-cellular movement of misfolded “prion-like” proteins that then recruit normally folded proteins in recipient cells to also adopt pathological conformations. Such a mechanism has been proposed, for example, to explain the propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) during the progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases such as mad cow disease, which can be caused by ingestion or by injection into animal brains of misfolded PrP, ALS and FTD are not infectious and the injection of phosphorylated and aggregated TDP-43 protein into rodent brains is not sufficient to cause disease phenotypes unless those animals also express high levels of transgenic TDP-43 protein. This suggests a missing component of a positive feedback that is necessary to sustain disease progression. We provide evidence that endogenous retroviruses (ERVs) are that missing component. We demonstrate that ERV expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either the Drosophila ERV, gypsy (mdg4) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Importantly. We also demonstrate that viral ERV transmission causes propagation of such TDP-43 pathology to cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism underlies the toxicity of glial cells to neurons and other glia in a Drosophila in vivo model.
- Published
- 2022
- Full Text
- View/download PDF
10. Josh Dubnau
- Author
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Josh Dubnau
- Subjects
General Agricultural and Biological Sciences ,General Biochemistry, Genetics and Molecular Biology - Published
- 2021
- Full Text
- View/download PDF
11. A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia
- Author
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Josh Dubnau, Jorge Azpurua, Enas Gad El-Karim, and Marvel Tranquille
- Subjects
Cancer Research ,Aging ,Genetic Screens ,Physiology ,Gene Identification and Analysis ,Gene Expression ,RNA-binding protein ,QH426-470 ,Toxicology ,Pathology and Laboratory Medicine ,Biochemistry ,Suppressor Genes ,Small hairpin RNA ,Animals, Genetically Modified ,Guide RNA ,C9orf72 ,Animal Cells ,Medicine and Health Sciences ,Genetics (clinical) ,Motor Neurons ,Neurons ,Gene knockdown ,Serine-Arginine Splicing Factors ,Chromosome Biology ,Drosophila Melanogaster ,Neurodegeneration ,Eukaryota ,Animal Models ,Chromatin ,Cell biology ,DNA-Binding Proteins ,Nucleic acids ,Insects ,Experimental Organism Systems ,Epigenetics ,Drosophila ,Cellular Types ,Neuroglia ,Research Article ,Arthropoda ,Biology ,Research and Analysis Methods ,Model Organisms ,Gene Types ,mental disorders ,medicine ,Genetics ,Animals ,Humans ,Nuclear export signal ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,Toxicity ,Biological Locomotion ,Organisms ,Biology and Life Sciences ,Cell Biology ,medicine.disease ,Invertebrates ,Cellular Neuroscience ,Animal Studies ,RNA ,CRISPR-Cas Systems ,Trinucleotide repeat expansion ,Zoology ,Entomology ,Genetic screen ,Neuroscience - Abstract
Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer’s disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear/import export- pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator., Author summary Loss of nuclear function of TDP-43 and its mislocalization into cytoplasmic inclusions are central features to a suite of neurodegenerative disorders. We screened 2700 Drosophila genes to identify downstream mediators that suppress an age-dependent motor dysfunction phenotype when they are knocked down by RNA interference. We identified both previously implicated pathways and several novel genes whose knock down is sufficient to dramatically and robustly rescue TDP-43 toxicity both in neuronal and glial contexts. Notably, we demonstrate that SF2/SRSF1, which was previously reported as a suppressor of C9orf72 hexanucleotide expansion repeat toxicity, also is a potent suppressor of TDP-43 mediated neurodegeneration.
- Published
- 2021
12. Intercellular viral spread and intracellular transposition of Drosophila gypsy
- Author
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Michael J. Metzger, Richard M. Keegan, Yung-Heng Chang, Josh Dubnau, and Lillian R Talbot
- Subjects
RNA viruses ,Cancer Research ,European People ,Somatic cell ,Endogenous retrovirus ,Retrotransposon ,Artificial Gene Amplification and Extension ,QH426-470 ,Pathology and Laboratory Medicine ,Polymerase Chain Reaction ,Mice ,0302 clinical medicine ,Neoplasms ,Invertebrate Genomics ,Medicine and Health Sciences ,Ethnicities ,Genetics (clinical) ,Neurons ,0303 health sciences ,Schneider 2 cells ,Drosophila Melanogaster ,Eukaryota ,Animal Models ,Genomics ,Long terminal repeat ,Cell biology ,Insects ,Experimental Organism Systems ,Medical Microbiology ,Viral Pathogens ,Viruses ,Romani People ,Drosophila ,Drosophila melanogaster ,Pathogens ,Research Article ,Arthropoda ,Retroelements ,Imaging Techniques ,Biology ,Transfection ,Research and Analysis Methods ,Microbiology ,Evolution, Molecular ,03 medical and health sciences ,Model Organisms ,Viral envelope ,Virology ,Retroviruses ,Fluorescence Imaging ,Genetics ,Animals ,Humans ,Molecular Biology Techniques ,Molecular Biology ,Microbial Pathogens ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Endogenous Retroviruses ,Organisms ,Terminal Repeat Sequences ,Biology and Life Sciences ,biology.organism_classification ,Invertebrates ,Viral Replication ,Viral replication ,Animal Genomics ,People and Places ,Nerve Degeneration ,Animal Studies ,Population Groupings ,Zoology ,Entomology ,030217 neurology & neurosurgery - Abstract
It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome., Author summary The genomes of animals and plants contain a vast quantity of so called “junk DNA” that does not provide obvious function to the organism. But it is increasingly clear that “junk DNA” has more important contributions to both normal function and to dysfunction that can cause disease. A great deal of this “junk” is made up by so called “retrotransposons”, which have many similarities to viruses. Many of them are actually evolutionary relatives of retroviruses, and are able to replicate themselves and insert new copies into the host genome. But unlike retroviruses, which are infectious and replicate by moving from one cell to another, retrotransposons replicate within one cell and re-insert their new copies back into the chromosomes of the cell in which they originated. In this publication, we studied replication of gypsy, which is a well known retrotransposon in fruit flies. We found that gypsy has the ability to replicate within a cell, like a retrotransposon and also has the ability to replicate by moving to a new cell like a retrovirus.
- Published
- 2021
13. Intercellular viral spread and intracellular transposition ofDrosophilagypsy
- Author
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Yung-Heng Chang, Michael J. Metzger, Josh Dubnau, and Richard M. Keegan
- Subjects
Retrovirus ,Viral envelope ,Somatic cell ,Schneider 2 cells ,Endogenous retrovirus ,Retrotransposon ,Biology ,biology.organism_classification ,Long terminal repeat ,Germline ,Cell biology - Abstract
It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-retrotransposons are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) that derive from an exogenous retrovirus that have integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. The fact that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration raises the question of whether their replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome.
- Published
- 2020
- Full Text
- View/download PDF
14. The gypsy endogenous retrovirus drives non-cell autonomous propagation in a Drosophila TDP-43 model of neurodegeneration
- Author
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Yung-Heng Chang and Josh Dubnau
- Subjects
0301 basic medicine ,DNA damage ,Endogenous retrovirus ,Degeneration (medical) ,Protein aggregation ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,mental disorders ,medicine ,Animals ,Drosophila Proteins ,Amyotrophic lateral sclerosis ,Mechanism (biology) ,Neurodegeneration ,Endogenous Retroviruses ,nutritional and metabolic diseases ,Neurodegenerative Diseases ,medicine.disease ,nervous system diseases ,DNA-Binding Proteins ,Disease Models, Animal ,030104 developmental biology ,Drosophila melanogaster ,General Agricultural and Biological Sciences ,Neuroscience ,Neuroglia ,030217 neurology & neurosurgery ,Frontotemporal dementia - Abstract
A hallmark of neurodegenerative disease is focal onset of pathological protein aggregation, followed by progressive spread of pathology to connected brain regions. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), pathology is often associated with aggregation of TAR DNA-binding protein 43 (TDP-43). Although aggregated TDP-43 protein moves between cells, it is not clear whether and how this movement propagates the degeneration. Here, we have established a Drosophila model of human TDP-43 in which we initiated toxic expression of human TDP-43 focally within small groups of glial cells. We found that this focal onset kills adjacent neurons. Surprisingly, we show that this spreading death is caused by an endogenous retrovirus within the glia, which leads to DNA damage and death in adjacent neurons. These findings suggest a possible mechanism by which human retroviruses such as HERV-K might contribute to TDP-43-mediated propagation of neurodegeneration.
- Published
- 2019
15. Cellular labeling of endogenous retrovirus replication (CLEVR) reveals de novo insertions of the gypsy retrotransposable element in cell culture and in both neurons and glial cells of aging fruit flies
- Author
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Lisa Prazak, Yung-Heng Chang, Josh Dubnau, and Richard M. Keegan
- Subjects
0301 basic medicine ,Aging ,Somatic cell ,Cell Culture Techniques ,Endogenous retrovirus ,Retrotransposon ,Artificial Gene Amplification and Extension ,Virus Replication ,Polymerase Chain Reaction ,Biochemistry ,0302 clinical medicine ,Animal Cells ,Genes, Reporter ,Mobile Genetic Elements ,Ethnicities ,Biology (General) ,Cellular Senescence ,Neurons ,General Neuroscience ,Drosophila Melanogaster ,Methods and Resources ,Eukaryota ,Animal Models ,Genomics ,Long terminal repeat ,3. Good health ,Cell biology ,Insects ,Retrotransposons ,Experimental Organism Systems ,Romani People ,Drosophila ,Biological Cultures ,Drosophila melanogaster ,Cellular Types ,Cellular Structures and Organelles ,General Agricultural and Biological Sciences ,Genetic Engineering ,Neuroglia ,Arthropoda ,Retroelements ,QH301-705.5 ,Biology ,Research and Analysis Methods ,Green Fluorescent Protein ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Model Organisms ,Genetic Elements ,Nuclear Membrane ,Genetics ,Animals ,Molecular Biology Techniques ,Gene ,Molecular Biology ,Cell Nucleus ,General Immunology and Microbiology ,Staining and Labeling ,Endogenous Retroviruses ,Organisms ,Transposable Elements ,Biology and Life Sciences ,Proteins ,Cell Biology ,Cell Cultures ,biology.organism_classification ,Invertebrates ,Long interspersed nuclear element ,Luminescent Proteins ,030104 developmental biology ,Cell culture ,Cellular Neuroscience ,People and Places ,Mutation ,Animal Studies ,Population Groupings ,030217 neurology & neurosurgery ,Neuroscience - Abstract
Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging, such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. We describe a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses., Using gypsy elements in aging fruit flies, this paper describes cellular labeling of endogenous retrovirus replication (CLEVR), an in vivo reporter system that allows the tracing of virus or LTR retrotransposon replication in tissues.
- Published
- 2019
16. Postmortem Cortex Samples Identify Distinct Molecular Subtypes of ALS: Retrotransposon Activation, Oxidative Stress, and Activated Glia
- Author
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Isabel Hubbard, Nadia Propp, Josh Dubnau, Lyle W. Ostrow, Duyang Kim, Regina Shaw, Oliver H. Tam, John Ravits, Nikolay V. Rozhkov, Hemali Phatnani, Samantha Fennessey, Delphine Fagegaltier, and Molly Hammell
- Subjects
Genetics ,0303 health sciences ,Retrotransposon ,Disease ,Biology ,medicine.disease ,medicine.disease_cause ,3. Good health ,Cortex (botany) ,Pathogenesis ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Amyotrophic lateral sclerosis ,Family history ,030217 neurology & neurosurgery ,Oxidative stress ,030304 developmental biology - Abstract
SummaryAmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several inherited pathogenic mutations have been identified as causative, the vast majority of cases are sporadic with no family history of disease. Thus, for the majority of ALS cases, a specific causal abnormality is not known and the disease may be a product of multiple inter-related pathways contributing to varying degrees in different ALS patients. Using unsupervised machine learning algorithms, we stratified the transcriptomes of 148 ALS decedent cortex tissue samples into three distinct and robust molecular subtypes. The largest cluster, identified in 61% of patient samples, displayed hallmarks of oxidative and proteotoxic stress. Another 20% of the ALS patient samples exhibited high levels of retrotransposon expression and other signatures of TDP-43 dysfunction. Finally, a third group showed predominant signatures of glial activation (19%). Together these results demonstrate that at least three distinct molecular signatures contribute to ALS disease. While multiple dysregulated components and pathways comprising these clusters have previously been implicated in ALS pathogenesis, unbiased analysis of this large survey demonstrated that sporadic ALS patient tissues can be segregated into distinct molecular subsets.
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- 2019
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17. Nuclear Transcriptomes of the Seven Neuronal Cell Types That Constitute the
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Meng-Fu Maxwell, Shih, Fred Pejman, Davis, Gilbert Lee, Henry, and Josh, Dubnau
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Neurons ,Behavior, Animal ,Sequence Analysis, RNA ,Cell Adhesion Molecules, Neuronal ,Neuropeptides ,Brain ,Gene Expression Regulation, Developmental ,Investigations ,mushroom body ,Drosophila melanogaster ,Animals ,Cell Lineage ,Drosophila ,RNA-seq ,Transcriptome ,Mushroom Bodies - Abstract
The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The Drosophila melanogaster MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes (γ, α′/β′ and α/β) and 7 cell subtypes (γd, γm, α′/β′ap, α′/β′m, α/βp, α/βs and α/βc) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. Here, we used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. We identified 350 MB class- or subtype-specific genes, including the widely used α/β class marker Fas2 and the α′/β′ class marker trio. Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, our data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the Drosophila MB provides a valuable resource for the fly neuroscience community.
- Published
- 2018
18. Cellular labeling of endogenous virus replication (CLEVR) reveals de novo insertions of the gypsy endogenous retrovirus in cell culture and in both neurons and glial cells of aging fruit flies
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Josh Dubnau, Yung-Heng Chang, Richard M. Keegan, and Lisa Prazak
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Transposable element ,0303 health sciences ,Small interfering RNA ,Somatic cell ,Neurodegeneration ,Endogenous retrovirus ,Retrotransposon ,Biology ,medicine.disease ,Long terminal repeat ,3. Good health ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Gene silencing ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Evidence is rapidly mounting that transposable element expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, successful reporters of LINE element mobilization have been in use for some time, but similar strategies have not been developed to report Long Terminal Repeat (LTR) retrotransposons and endogenous retroviruses. We describe Cellular Labeling of Endogenous Virus Replication (CLEVR), which reports replication of the gypsy element in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR-retrotransposons, endogenous retroviruses and exogenous retroviruses.
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- 2018
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19. The Retrotransposon Storm and the dangers of a Collyer’s genome
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Josh Dubnau
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0301 basic medicine ,Aging ,Retroelements ,Heterochromatin ,Genome, Human ,Brain ,Retrotransposon ,Neurodegenerative Diseases ,Normal aging ,Biology ,Environmental stress ,Genome ,Noncoding DNA ,Human genetics ,Article ,03 medical and health sciences ,030104 developmental biology ,Gene Expression Regulation ,Evolutionary biology ,Genetics ,Gene silencing ,Animals ,Humans ,Developmental Biology - Abstract
Like the famous Collyer’s mansion in NY, our genomes have accumulated vast quantities of sequences that have been referred to as ‘junk DNA’, much of which consists of retrotransposons. A recent literature establishes the phenomenology that many retrotransposons become expressed at progressively higher levels during the course of normal aging. This seems to reflect gradual loss of heterochromatin in old age. In addition, retrotransposons appear to be precociously expressed in brains of younger animals that are experiencing neurodegenerative decline. Although it is difficult to distinguish cause from consequence, several recent studies support the contention that retrotransposon expression, and even perhaps transposition, causally contribute to both the normal deterioration seen with age and to the precipitous decline in some neurodegenerative disorders. This may reflect a two hit model in which normal age-dependent loss of heterochromatin synergizes with a disruption to post transcriptional silencing of RTEs caused by genetic and environmental stress.
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- 2018
20. Retrotransposon Activation Contributes to Neurodegeneration in aDrosophilaTDP-43 Model of ALS
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Stephen Hearn, Nabanita Chatterjee, Lisa Krug, Wen-Wei Liao, Lisa Prazak, Nikolay V. Rozhkov, Delphine Theodorou, Rebeca Borges-Monroy, Molly Hammell, Kathleen Morrill, and Josh Dubnau
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0301 basic medicine ,Male ,Cancer Research ,Small interfering RNA ,Endogenous retrovirus ,Apoptosis ,Toxicology ,Pathology and Laboratory Medicine ,Biochemistry ,Animals, Genetically Modified ,0302 clinical medicine ,RNA interference ,Animal Cells ,Invertebrate Genomics ,Medicine and Health Sciences ,Small interfering RNAs ,Amyotrophic lateral sclerosis ,Genetics (clinical) ,Neurons ,Genetics ,0303 health sciences ,Microscopy, Confocal ,Cell Death ,Reverse Transcriptase Polymerase Chain Reaction ,Drosophila Melanogaster ,Neurodegeneration ,Neurodegenerative Diseases ,Animal Models ,Genomics ,Frontotemporal lobar degeneration ,Immunohistochemistry ,3. Good health ,Cell biology ,Nucleic acids ,Insects ,DNA-Binding Proteins ,Experimental Organism Systems ,Cell Processes ,Drosophila ,RNA Interference ,Cellular Types ,Neuroglia ,Research Article ,Programmed cell death ,Cell type ,lcsh:QH426-470 ,Arthropoda ,Retroelements ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Model Organisms ,Microscopy, Electron, Transmission ,medicine ,Animals ,Humans ,Gene silencing ,Non-coding RNA ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Biology and life sciences ,Toxicity ,Gene Expression Profiling ,Amyotrophic Lateral Sclerosis ,Organisms ,DNA ,Cell Biology ,medicine.disease ,Invertebrates ,Gene regulation ,lcsh:Genetics ,Disease Models, Animal ,030104 developmental biology ,Animal Genomics ,Cellular Neuroscience ,RNA ,DNA damage ,Gene expression ,Frontotemporal Lobar Degeneration ,030217 neurology & neurosurgery ,Neuroscience - Abstract
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD., Author summary Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, and mutations in TDP-43 are causal in a subset of familial ALS cases. Although cytoplasmic inclusions of this mostly nuclear protein are a hallmark of the disease, the cascade of events leading to cell death are not understood. We demonstrate that expression of human TDP-43 (hTDP-43) in Drosophila neurons or glial cells, which results in toxic cytoplasmic accumulation of TDP-43, causes broad expression of retrotransposons. In the case of glial hTDP-43 expression, the endogenous retrovirus (ERV) gypsy causally contributes to degeneration because inhibiting gypsy genetically or pharmacologically is sufficient to rescue the phenotypic effects. Moreover, we demonstrate that activation of DNA damage-mediated programmed cell death underlies hTDP-43 and gypsy mediated toxicity. Finally, we find that hTDP-43 pathology impairs small interfering RNA silencing, which is an essential system that normally protects the genome from RTEs. These findings suggest a novel mechanism in which a storm of retrotransposon activation drives neurodegeneration in TDP-43 mediated diseases such as ALS and FTLD.
- Published
- 2016
- Full Text
- View/download PDF
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