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Compromised Fidelity of Endocytic Synaptic Vesicle Protein Sorting In Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2 Natalia L. Kononenkoa,b, M. Kasim Dirilc, Dmytro Puchkova, Michael Kintscherb, Seong Joo Kooa, Gerit Pfuhld, York Winterd, Martin Wienische, Jürgen Klingaufe, Jörg Breustedtb, Dietmar Schmitzb, Tanja Maritzena,1,2, and Volker Hauckea,b,c,1,2 aDepartment of Molecular Pharmacology and Cell Biology, Leibniz Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; bNeuroCure Cluster of Excellence, Charité Berlin, 10117 Berlin, Germany; cFreie Universität Berlin, Institut für Chemie und Biochemie, 14195 Berlin, Germany; dDepartment of Cognitive Neurobiology, Humboldt Universität zu Berlin, 10117 Berlin, Germany; and eInstitute for Medical Physics and Biophysics, University of Münster, 48149 Münster, Germany Edited by Pietro De Camilli, Yale University and Howard Hughes Medical Institute, New Haven, CT, and approved December 28, 2012 (received for review October 23, 2012) Neurotransmission depends on the exocytic fusion of synaptic Several components of the endocytic machinery may function as vesicles (SVs) and their subsequent reformation either by clathrin- adaptors for SV protein sorting. These components include the mediated endocytosis or budding from bulk endosomes. How syn- heterotetrameric adaptor complex AP-2, a key component of apses are able to rapidly recycle SVs to maintain SV pool size, yet the clathrin machinery involved in sorting of constitutive cargo in preserve their compositional identity, is poorly understood. We nonneuronal cells (17), AP180, an abundant factor of synaptic demonstrate that deletion of the endocytic adaptor stonin 2 (Stn2) clathrin-coated vesicles (18–20), the BAR-SH3 domain pro- in mice compromises the fidelity of SV protein sorting, whereas tein endophilin (8, 21), and the AP-2μ–related protein stonin 2 the apparent speed of SV retrieval is increased. Loss of Stn2 leads (Stn2), a binding partner of the SV calcium sensor synaptotagmin to selective missorting of synaptotagmin 1 to the neuronal surface, (22–24). AP180 together with its ubiquitously expressed paralog an elevated SV pool size, and accelerated SV protein endocytosis. Clathrin Assembly Lymphoid Myeloid Leukemia Protein (CALM) The latter phenotype is mimicked by overexpression of endocytosis- has been proposed to facilitate sorting of the SV SNARE defective variants of synaptotagmin 1. Increased speed of SV synaptobrevin 2 (19), whereas endophilin may regulate the protein retrieval in the absence of Stn2 correlates with an up- speed of vesicular glutamate transporter 1 (VGLUT1) retrieval regulation of SV reformation from bulk endosomes. Our results (8). Genetic studies have been inconclusive with respect to the are consistent with a model whereby Stn2 is required to preserve mechanisms of SV protein sorting because all adaptor mutants SV protein composition but is dispensable for maintaining the studied thus far exhibit generalized defects in SV endocytosis (4, 5) speed of SV recycling. leading to gross alterations in synapse structure and functionality. For example, loss of AP-2 is associated with early embryonic le- pHluorin | hippocampus | mossy fibers thality in mice (25), whereas mouse mutants lacking endophilin A are perinatally lethal due to strongly impaired synaptic trans- eurotransmission involves the calcium-regulated fusion of mission (21). Nsynaptic vesicles (SVs), a process that requires the SV calcium Conflicting data exist regarding the physiological function of sensor synaptotagmin (Syt) (1) and components of the active stonins in vivo: knockdown studies in transfected neurons (26) fi zone (AZ) that de ne sites of neurotransmitter release (2). Post- and data from invertebrates suggest a general essential role of exocytic fusion SV membranes are retrieved by endocytosis from the plasma membrane (2–5) to regenerate SVs of the correct size Significance and composition (6). Alternatively, SVs can also be reformed from large plasma membrane infoldings and from endosomes (7) via a brefeldin A–sensitive pathway (8) that may become par- Brain function depends on neurotransmission, and alterations ticularly important under conditions of sustained high-level in this process are linked to neuropsychiatric disorders. Neu- activity and involves endosomal adaptor complexes such as rotransmitter release requires the rapid recycling of synaptic adaptor protein complex 1 (AP-1) (9, 10). Maintenance of the vesicles (SVs) by endocytosis. How synapses can rapidly re- SV pool requires that the number of recycled SVs closely mat- generate SVs, yet preserve their molecular composition, is ches those having undergone exocytosis. As SVs are character- poorly understood. We demonstrate that mice lacking the endo- ized by a precise protein composition (11) that at least for some cytic protein stonin 2 (Stn2) show changes in exploratory behavior SV proteins including Syt1, VGLUT1, and SV2A displays little and defects in SV composition, whereas the speed at which SVs intervesicle variation (12), molecular mechanisms must exist to are regenerated is increased. As Stn2 is implicated in schizo- control the fidelity of SV protein sorting while maintaining the phrenia and autism in humans, our findings bear implications speed of exo-endocytosis. for neuropsychiatric disorders. The mechanisms by which exo-endocytic balance and the fidelity of SV protein sorting are maintained are unknown. One Author contributions: N.L.K., M.K.D., D.P., M.K., G.P., Y.W., J.K., J.B., D.S., T.M., and V.H. designed research; N.L.K., D.P., M.K., S.J.K., G.P., M.W., J.B., and T.M. performed research; possibility is that retrieval of SV proteins involves clustering M.K.D., S.J.K., and J.K. contributed new reagents/analytic tools; N.L.K., M.K.D., D.P., M.K., (13, 14), which would alleviate a need for specific sorting, even S.J.K., G.P., Y.W., M.W., J.K., J.B., D.S., and T.M. analyzed data; and N.L.K., D.S., and V.H. if multiple pathways of SV reformation are used (9, 10). How- wrote the paper. ever, data based on imaging of SV proteins tagged with the GFP- The authors declare no conflict of interest. derived pH sensor pHluorin indicate that exocytosed and newly This article is a PNAS Direct Submission. endocytosed SV proteins are not identical, suggesting that Freely available online through the PNAS open access option. intermixing between exocytosed and preexisting surface pools 1T.M. and V.H. contributed equally to this work. of vesicle proteins occurs (15, 16). If SVs lose their identity over 2To whom correspondence may be addressed. E-mail: [email protected] or multiple rounds of exo-endocytosis, specific mechanisms should [email protected]. fi exist for the cargo-speci c recognition and sorting of SV proteins, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. e.g., by adaptors (4, 15). 1073/pnas.1218432110/-/DCSupplemental. E526–E535 | PNAS | Published online January 23, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1218432110 Downloaded by guest on September 27, 2021 stonins (termed Stn2 in mammals, stoned B in Drosophila mel- a lower, albeit greater, than baseline steady-state level in slices PNAS PLUS anogaster, and unc-41 in Caenorhabditis elegans) in SV endocy- from both WT and Stn2 KO mice (Fig. 1I). Significantly higher tosis, which is evidenced by a reduced SV pool size and impaired responses were elicited in slices from Stn2 KO mice during the neurotransmission in loss-of-function alleles (27–31). On the steady-state phase (Fig. 1J) in striking contrast to the depression other hand, the evolutionary conserved association of stonins seen in other endocytic mutants (4, 21, 32). Thus, loss of Stn2 with the endocytic machinery and with Syt (22–24) has been leads to increased short-term facilitation, likely reflecting a re- taken as evidence in favor of a more specific role of Stn2 in Syt duced release probability of MF synapses and a partial resistance sorting, although this postulate has never been tested at the or- of KO synapses to high frequency–induced depression. ganismic level in mammals in vivo. Synaptic plasticity and hippocampal function have been Here we demonstrate that deletion of Stn2 in mice com- linked to exploratory behavior (33), with a particularly im- promises the fidelity of SV protein sorting, whereas the apparent portant role for dentate granule cells and the MF pathway in speed of SV retrieval is increased. Loss of Stn2 leads to selective exploratory learning (34). In agreement with the observed missorting of synaptotagmin 1 (Syt1) to the neuronal surface, an changes in MF short-term plasticity, Stn2 KO mice displayed elevated SV pool size, and accelerated SV endocytosis. The increased explorative activity in the open arena (Fig. S3A), as latter phenotype is mimicked by overexpression of endocytosis- well as increased vertical locomotion (Fig. S3B), whereas defective variants of Syt1. Increased speed of SV protein re- spontaneous alternation performance in the Y-maze, considered trieval in the absence of Stn2 correlates with an up-regulation as a test for working memory, did not differ significantly (Fig. of SV reformation from bulk endosomes. Our results are con- S3D). Moreover, Stn2 KO mice had a shorter latency to ap- sistent with a model according to which Stn2 is required to proach a new object than WT mice (Fig. S3C). The increased preserve SV protein composition but is dispensable for main- explorative drive in Stn2 KO mice was not due to reduced taining the speed of SV recycling. anxiety or fear (Fig. S3 F and G), consistent with their un- altered behavior in the dark-light box (Fig. S3E). Stn2 KO mice Results also did not display memory deficits in object habituation Increased SV Pool Size and Altered Short-Term Plasticity in the experiments (Fig. S3H). Thus, loss of Stn2 causes increased lo- Absence of Stn2. To address the question of whether and how comotion- and exploration-related behaviors in mice. This phe- the fidelity of SV protein sorting may be linked to the speed of notype resembles arousal seeking and impulsivity observed in retrieval of SV membranes, we focused on Stn2, the mammalian patients suffering from Tourette syndrome (35) or schizophre- homolog of Drosophila stoned B(27, 30) and of C.
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