title: Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition.
abstract: Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.
authors.affiliation: 1 Hans-Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany 2 Department of Neurology, University of Würzburg, Josef-Schneider Str. 11, 97080 Würzburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Werner
authors.affiliation: 3 Department of Neurophysiology, Institute of Physiology, University of Würzburg, Roentgenring 9, 97070 Würzburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Pauli
authors.affiliation: 4 Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Sören
authors.initials: S
authors.lastname: Doose
authors.affiliation: 2 Department of Neurology, University of Würzburg, Josef-Schneider Str. 11, 97080 Würzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Weishaupt
authors.affiliation: 1 Hans-Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany 2 Department of Neurology, University of Würzburg, Josef-Schneider Str. 11, 97080 Würzburg, Germany 5 Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Haselmann
authors.affiliation: 1 Hans-Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany 2 Department of Neurology, University of Würzburg, Josef-Schneider Str. 11, 97080 Würzburg, Germany 5 Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Grünewald
authors.affiliation: 4 Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
authors.affiliation: 3 Department of Neurophysiology, Institute of Physiology, University of Würzburg, Roentgenring 9, 97070 Würzburg, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Heckmann
authors.affiliation: 2 Department of Neurology, University of Würzburg, Josef-Schneider Str. 11, 97080 Würzburg, Germany.
authors.forename: Klaus V
authors.initials: KV
authors.lastname: Toyka
authors.affiliation: 6 Institute for Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany.
authors.forename: Esther
authors.initials: E
authors.lastname: Asan
authors.affiliation: 2 Department of Neurology, University of Würzburg, Josef-Schneider Str. 11, 97080 Würzburg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Sommer
authors.affiliation: 1 Hans-Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany 2 Department of Neurology, University of Würzburg, Josef-Schneider Str. 11, 97080 Würzburg, Germany 5 Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany christian.geis@med.uni-jena.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Geis
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000008:administration & dosage* / Q000097:blood; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007278:Injections, Spinal; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000008:administration & dosage* / Q000097:blood; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011247:Pregnancy; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D051381:Rats; D011917:Rats, Inbred Lew; D016750:Stiff-Person Syndrome / Q000097:blood / Q000175:diagnosis; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D001323:Autoantibodies; D009419:Nerve Tissue Proteins; C075637:amphiphysin
keywords: amphiphysin; autoantibody; clathrin-mediated endocytosis; dSTORM super-resolution microscopy; stiff-person syndrome