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1.
Front Mol Neurosci ; 15: 870085, 2022.
Article in English | MEDLINE | ID: mdl-35592112

ABSTRACT

Astrocytes respond to injury, infection, and inflammation in the central nervous system by acquiring reactive states in which they may become dysfunctional and contribute to disease pathology. A sub-state of reactive astrocytes induced by proinflammatory factors TNF, IL-1α, and C1q ("TIC") has been implicated in many neurodegenerative diseases as a source of neurotoxicity. Here, we used an established human induced pluripotent stem cell (hiPSC) model to investigate the surface marker profile and proteome of TIC-induced reactive astrocytes. We propose VCAM1, BST2, ICOSL, HLA-E, PD-L1, and PDPN as putative, novel markers of this reactive sub-state. We found that several of these markers colocalize with GFAP+ cells in post-mortem samples from people with Alzheimer's disease. Moreover, our whole-cells proteomic analysis of TIC-induced reactive astrocytes identified proteins and related pathways primarily linked to potential engagement with peripheral immune cells. Taken together, our findings will serve as new tools to purify reactive astrocyte subtypes and to further explore their involvement in immune responses associated with injury and disease.

2.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Article in English | MEDLINE | ID: mdl-35058357

ABSTRACT

After injury, severed dendrites and axons expose the "eat-me" signal phosphatidylserine (PS) on their surface while they break down. The degeneration of injured axons is controlled by a conserved Wallerian degeneration (WD) pathway, which is thought to activate neurite self-destruction through Sarm-mediated nicotinamide adenine dinucleotide (NAD+) depletion. While neurite PS exposure is known to be affected by genetic manipulations of NAD+, how the WD pathway coordinates both neurite PS exposure and self-destruction and whether PS-induced phagocytosis contributes to neurite breakdown in vivo remain unknown. Here, we show that in Drosophila sensory dendrites, PS exposure and self-destruction are two sequential steps of WD resulting from Sarm activation. Surprisingly, phagocytosis is the main driver of dendrite degeneration induced by both genetic NAD+ disruptions and injury. However, unlike neuronal Nmnat loss, which triggers PS exposure only and results in phagocytosis-dependent dendrite degeneration, injury activates both PS exposure and self-destruction as two redundant means of dendrite degeneration. Furthermore, the axon-death factor Axed is only partially required for self-destruction of injured dendrites, acting in parallel with PS-induced phagocytosis. Lastly, injured dendrites exhibit a unique rhythmic calcium-flashing that correlates with WD. Therefore, both NAD+-related general mechanisms and dendrite-specific programs govern PS exposure and self-destruction in injury-induced dendrite degeneration in vivo.


Subject(s)
Dendrites/metabolism , Phagocytosis , Sensory Receptor Cells/metabolism , Wallerian Degeneration/etiology , Wallerian Degeneration/metabolism , Animals , Drosophila , Drosophila Proteins/deficiency , Fluorescent Antibody Technique , Gene Knockdown Techniques , Nerve Degeneration , Nicotinamide-Nucleotide Adenylyltransferase/deficiency , Phosphatidylserines/metabolism , Wallerian Degeneration/pathology
3.
J Genet Genomics ; 46(4): 187-199, 2019 04 20.
Article in English | MEDLINE | ID: mdl-31080046

ABSTRACT

Dendrites and axons are delicate neuronal membrane extensions that undergo degeneration after physical injuries. In neurodegenerative diseases, they often degenerate prior to neuronal death. Understanding the mechanisms of neurite degeneration has been an intense focus of neurobiology research in the last two decades. As a result, many discoveries have been made in the molecular pathways that lead to neurite degeneration and the cell-cell interactions responsible for the subsequent clearance of neuronal debris. Drosophila melanogaster has served as a prime in vivo model system for identifying and characterizing the key molecular players in neurite degeneration, thanks to its genetic tractability and easy access to its nervous system. The knowledge learned in the fly provided targets and fuel for studies in other model systems that have further enhanced our understanding of neurodegeneration. In this review, we will introduce the experimental systems developed in Drosophila to investigate injury-induced neurite degeneration, and then discuss the biological pathways that drive degeneration. We will also cover what is known about the mechanisms of how phagocytes recognize and clear degenerating neurites, and how recent findings in this area enhance our understanding of neurodegenerative disease pathology.


Subject(s)
Drosophila melanogaster , Neurites/pathology , Animals , Humans , Neurodegenerative Diseases/pathology
4.
Genetics ; 211(2): 459-472, 2019 02.
Article in English | MEDLINE | ID: mdl-30504366

ABSTRACT

Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. Here, we present a simple, yet highly efficient, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. We describe convenient toolkits for making enhancer-driven Cas9 lines and multi-gRNAs that are optimized for mutagenizing somatic cells. We demonstrate that insertions or deletions in coding sequences more reliably cause somatic mutations than DNA excisions induced by two gRNAs. We further show that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasks redundant soluble N-ethylmaleimide-sensitive factor attachment protein receptor gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage reveal the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISPR tools can be applied to analyze tissue-specific gene function in many biological processes.


Subject(s)
CRISPR-Cas Systems , Gene Dosage , Genetic Techniques , Mutagenesis , Animals , Drosophila/genetics , Epidermis/metabolism , Loss of Function Mutation , Neurons/metabolism , Organ Specificity , RNA, Guide, Kinetoplastida/genetics , Transgenes
5.
Cell Rep ; 24(9): 2273-2286, 2018 08 28.
Article in English | MEDLINE | ID: mdl-30157423

ABSTRACT

Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an "eat-me" signal allowing recognition of degenerating neurites by phagocytes. Here we show that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. Our findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo.


Subject(s)
Drosophila Proteins/metabolism , Drosophila/pathogenicity , Nerve Degeneration/pathology , Neurites/metabolism , Phosphatidylserines/metabolism , Animals
6.
Proc Natl Acad Sci U S A ; 114(38): E8062-E8071, 2017 09 19.
Article in English | MEDLINE | ID: mdl-28874572

ABSTRACT

Neurons sometimes completely fill available space in their receptive fields with evenly spaced dendrites to uniformly sample sensory or synaptic information. The mechanisms that enable neurons to sense and innervate all space in their target tissues are poorly understood. Using Drosophila somatosensory neurons as a model, we show that heparan sulfate proteoglycans (HSPGs) Dally and Syndecan on the surface of epidermal cells act as local permissive signals for the dendritic growth and maintenance of space-filling nociceptive C4da neurons, allowing them to innervate the entire skin. Using long-term time-lapse imaging with intact Drosophila larvae, we found that dendrites grow into HSPG-deficient areas but fail to stay there. HSPGs are necessary to stabilize microtubules in newly formed high-order dendrites. In contrast to C4da neurons, non-space-filling sensory neurons that develop in the same microenvironment do not rely on HSPGs for their dendritic growth. Furthermore, HSPGs do not act by transporting extracellular diffusible ligands or require leukocyte antigen-related (Lar), a receptor protein tyrosine phosphatase (RPTP) and the only known Drosophila HSPG receptor, for promoting dendritic growth of space-filling neurons. Interestingly, another RPTP, Ptp69D, promotes dendritic growth of C4da neurons in parallel to HSPGs. Together, our data reveal an HSPG-dependent pathway that specifically allows dendrites of space-filling neurons to innervate all target tissues in Drosophila.


Subject(s)
Dendrites/metabolism , Drosophila Proteins/metabolism , Heparin/analogs & derivatives , Nociceptors/metabolism , Proteoglycans/metabolism , Receptor-Like Protein Tyrosine Phosphatases/metabolism , Signal Transduction , Animals , Drosophila Proteins/genetics , Drosophila melanogaster , Heparin/genetics , Heparin/metabolism , Nociceptors/cytology , Proteoglycans/genetics , Receptor-Like Protein Tyrosine Phosphatases/genetics
7.
Microb Cell ; 3(2): 79-88, 2016 Jan 18.
Article in English | MEDLINE | ID: mdl-28357337

ABSTRACT

Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae. Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2∆ yeast, and extends the replicative lifespan and cellular health span of sir2∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast.

8.
Stem Cell Reports ; 1(4): 350-9, 2013.
Article in English | MEDLINE | ID: mdl-24319669

ABSTRACT

Chromosomal integrity has been known for many years to affect the ability of mouse embryonic stem cells (mESCs) to contribute to the germline of chimeric mice. Abnormal chromosomes are generally detected by standard cytogenetic karyotyping. However, this method is expensive, time consuming, and often omitted prior to blastocyst injection, consequently reducing the frequency of mESC-derived offspring. Here, we show a fast, accurate, and inexpensive screen for identifying the two most common aneuploidies (Trisomy 8 and loss of chromosome Y) in genetically manipulated mESCs using quantitative real-time PCR (qPCR). Screening against these two aneuploidies significantly increases the fraction of normal mESC clones. Our method is extremely sensitive and can detect as low as 10% aneuploidy among a large population of mESCs. It greatly expedites the generation of mutant mice and provides a quick tool for assessing the aneuploidy percentages of any mESC line.


Subject(s)
Aneuploidy , Embryonic Stem Cells , Karyotyping/methods , Real-Time Polymerase Chain Reaction/methods , Animals , Chromosome Deletion , Chromosomes, Human, Pair 8 , Computer Simulation , Mice , Trisomy , Y Chromosome
9.
PLoS One ; 8(9): e74941, 2013.
Article in English | MEDLINE | ID: mdl-24086401

ABSTRACT

Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the ß2 adrenergic receptor (ß2AR). We have characterized these ß2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound ß2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All ß2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality.


Subject(s)
Cell Membrane/metabolism , Green Fluorescent Proteins/metabolism , Receptors, Adrenergic, beta-2/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Arrestins/metabolism , Cell Line , Cell Membrane/drug effects , Endocytosis/drug effects , Isoproterenol/pharmacology , Ligands , Mice , Protein Transport/drug effects , Pseudopodia/drug effects , Pseudopodia/metabolism , Time Factors , Transferrin/metabolism , beta-Arrestins
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