title: Shank3 is localized in axons and presynaptic specializations of developing hippocampal neurons and involved in the modulation of NMDA receptor levels at axon terminals.
abstract: Autism-related Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses. A few studies, however, have already indicated that within a neuron, the presence of Shank family members is not limited to the postsynaptic density. By separating axons from dendrites of developing hippocampal neurons in microfluidic chambers, we show that RNA of all three Shank family members is present within axons. Immunostaining confirms these findings as all three Shanks are indeed found within separated axons and further co-localize with well-known proteins of the presynaptic specialization in axon terminals. Therefore, Shank proteins might not only serve as postsynaptic scaffold proteins, but also play a crucial role during axonal outgrowth and presynaptic development and function. This is supported by our findings that shRNA-mediated knockdown of Shank3 results in up-regulation of the NMDA receptor subunit GluN1 in axon terminals. Taken together, our findings will have major implications for the future analysis of neuronal Shank biology in both health and disease. Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses strongly related to several neuropsychiatric disorders. However, a few studies have already implicated a functional role of the Shanks beyond the postsynaptic density (PSD). We here show that all three Shanks are localized in both axons and pre-synaptic specializiations of developing hippocampal neurons in culture. We further provide evidence that Shank3 is involved in the modulation of NMDA receptor levels at axon terminals. Taken together, our study will open up novel avenues for the future analysis of neuronal Shank biology in both health and disease.
authors.affiliation: Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany.|International Graduate School in Molecular Medicine Ulm, IGradU, Ulm University, Ulm, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Halbedl
authors.affiliation: Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schoen
authors.affiliation: International Graduate School in Molecular Medicine Ulm, IGradU, Ulm University, Ulm, Germany.|Department of Neurology, Ulm University, Ulm, Germany.
authors.forename: Marisa S
authors.initials: MS
authors.lastname: Feiler
authors.affiliation: Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany.
authors.forename: Tobias M
authors.initials: TM
authors.lastname: Boeckers
authors.affiliation: Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany.|Department of Neurology, Ulm University, Ulm, Germany.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Schmeisser
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism*; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental; D020439:Growth Cones / Q000737:chemistry; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D046210:Microfluidic Analytical Techniques; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D016501:Neurites / Q000737:chemistry; D055495:Neurogenesis; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000235:genetics; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016194:Receptors, N-Methyl-D-Aspartate; C120531:Shank1 protein, rat; C485686:Shank2 protein, rat; C120532:Shank3 protein, rat
keywords: NMDA receptor; Shank1; Shank2; Shank3; axon; synapse