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      Defining the binding interface of Amyloid Precursor Protein (APP) and Contactin3 (CNTN3) by site-directed mutagenesis

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          Abstract

          The Amyloid Precursor Protein (APP) and Contactin (CNTN) families of cell-surface proteins have been intensively studied in the context of neural development and neuropsychiatric diseases. Earlier studies demonstrated both genetic and biochemical interactions between the extracellular domains of APP and CNTN3, but their precise binding interfaces were not defined. In the present study, we have used binding assays between APP-alkaline phosphatase (AP) fusion proteins and CNTN-Fc fusion proteins, together with alanine substitution mutagenesis, to show that: (i) the second Fibronectin domain (Fn(2)) in CNTN3 mediates APP binding; (ii) the copper binding domain (CuBD) in APP mediates CNTN3 binding; and (iii) the most important amino acids for APP-CNTN3 binding reside on one face of CNTN3-Fn(2) and on one face of APP-CuBD. These experiments define the regions of direct contact that mediate the binding interaction between APP and CNTN3.

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          Most cited references28

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          Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone.

          The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.
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            Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2.

            Biochemical and genetic studies place the amyloid precursor protein (APP) at the center stage of Alzheimer's disease (AD) pathogenesis. Although mutations in the APP gene lead to dominant inheritance of familial AD, the normal function of APP remains elusive. Here, we report that the APP family of proteins plays an essential role in the development of neuromuscular synapses. Mice deficient in APP and its homolog APP-like protein 2 (APLP2) exhibit aberrant apposition of presynaptic marker proteins with postsynaptic acetylcholine receptors and excessive nerve terminal sprouting. The number of synaptic vesicles at presynaptic terminals is dramatically reduced. These structural abnormalities are accompanied by defective neurotransmitter release and a high incidence of synaptic failure. Our results identify APP/APLP2 as key regulators of structure and function of developing neuromuscular synapses.
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              APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP.

              Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock-in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα-DM) by crossing APPsα-KI mice onto an APLP2-deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα-DM muscle showed fragmented post-synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABA(A) receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: Writing – original draftRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                18 July 2019
                2019
                : 14
                : 7
                : e0219384
                Affiliations
                [1 ] Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
                [2 ] Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
                [3 ] Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
                [4 ] Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
                Torrey Pines Institute for Molecular Studies, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-2189-1415
                http://orcid.org/0000-0001-8106-5460
                Article
                PONE-D-19-11402
                10.1371/journal.pone.0219384
                6638891
                31318883
                c93cf1aa-c24e-43e3-8dfb-e3277b05728e
                © 2019 Peng et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 April 2019
                : 22 June 2019
                Page count
                Figures: 3, Tables: 0, Pages: 12
                Funding
                This work was supported by Howard Hughes Medical Institute.
                Categories
                Research Article
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Research and Analysis Methods
                Chemical Characterization
                Binding Analysis
                Biology and Life Sciences
                Genetics
                Mutation
                Substitution Mutation
                Biology and Life Sciences
                Biochemistry
                Proteins
                Protein Domains
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Amino Acids
                Amino Acid Substitution
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Amino Acid Substitution
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Amino Acid Substitution
                Biology and Life Sciences
                Genetics
                Mutagenesis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblotting
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblotting
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Protein Sequencing
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Protein Sequencing
                Custom metadata
                Data are presented in the manuscript.

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