Research Article: New Research Neuronal Excitability Proteasomal-Mediated Degradation of AKAP150 Accompanies AMPAR Endocytosis during cLTD Wenwen Cheng,1,2 Dolores Siedlecki-Wullich,1,2 Judit Català-Solsona,1,2 Cristina Fábregas,1,2 Rut Fadó,3 Núria Casals,3,4 Montse Solé,1,5 Mercedes Unzeta,1 Carlos A. Saura,1,2 José Rodríguez-Alvarez,1,2,6 and Alfredo J. Miñano-Molina1,2 https://doi.org/10.1523/ENEURO.0218-19.2020 1 Institut de Neurociències and Dpt. Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Cerdanyola del Vallès 08193, Spain, 2Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid 28031, Spain, 3Basic Sciences Department, Facultat de Medicina i Ciències de la Salut, Universitat Internacional de Catalunya (UIC), Sant Cugat del Vallès 08195, Spain, 4Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid E-28029, Spain, 5Neurovascular Research Laboratory, Vall d’Hebron Institute of Research, Universitat Autònoma de Barcelona, Barcelona 08035, Spain, and 6Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461 Abstract The number and function of synaptic AMPA receptors (AMPARs) tightly regulates excitatory synaptic transmis- sion. Current evidence suggests that AMPARs are inserted into the postsynaptic membrane during long-term potentiation (LTP) and are removed from the membrane during long-term depression (LTD). Dephosphorylation of GluA1 at Ser-845 and enhanced endocytosis are critical events in the modulation of LTD. Moreover, changes in scaffold proteins from the postsynaptic density (PSD) could be also related to AMPAR regulation in LTD. In the present study we analyzed the effect of chemical LTD (cLTD) on A-kinase anchoring protein (AKAP)150 and AMPARs levels in mouse-cultured neurons. We show that cLTD induces AKAP150 protein degradation via proteasome, coinciding with GluA1 dephosphorylation at Ser-845 and endocytosis of GluA1- containing AMPARs. Pharmacological inhibition of proteasome activity, but not phosphatase calcineurin (CaN), reverted cLTD-induced AKAP150 protein degradation. Importantly, AKAP150 silencing induced dephosphoryl- ation of GluA1 Ser-845 and GluA1-AMPARs endocytosis while AKAP150 overexpression blocked cLTD-medi- ated GluA1-AMPARs endocytosis. Our results provide direct evidence that cLTD-induced AKAP150 degradation by the proteasome contributes to synaptic AMPARs endocytosis. Key words: AKAP150; AMPAR; LTD; LTP; plasticity; trafficking Significance Statement Multiple evidences have shown that changes in glutamatergic synaptic AMPA receptors (AMPARs) modu- late excitatory synaptic plasticity, playing a fundamental role in learning and memory-related processes such as long-term potentiation (LTP) or long-term depression (LTD). AMPARs are inserted in the synapses during LTP and removed during LTD although the mechanisms involved in synaptic AMPARs dynamics are not fully understood. Here, we show that chemical LTD (cLTD)-dependent proteasome degradation of A-ki- nase anchoring protein (AKAP)150, a scaffold protein from the postsynaptic density (PSD), is related to syn- aptic AMPARs removal during cLTD whereas restored AKAP150 levels blocks cLTD-mediated endocytosis of AMPARs. Thus, our findings expand our understanding of the regulatory role of AKAP150 on AMPARs dynamics at glutamatergic synapses. March/April 2020, 7(2) ENEURO.0218-19.2020 1–19 Research Article: New Research 2 of 19 Introduction dependent AMPAR exocytosis at extrasynaptic sites dur- NMDA receptor-dependent long-term potentiation ing LTP also depends on the binding of complexin to a (LTP) and long-term depression (LTD) in excitatory hip- postsynaptic unique SNARE complex (Ahmad et al., pocampal synapses are believed to play a fundamental 2012; Jurado et al., 2013). However, extrasynaptic role in learning and memory. Several reports have shown AMPARs delivery and lateral diffusion to the synapse are that changes in synaptic AMPA receptor (AMPAR) num- necessary but not sufficient for LTP because AMPARs ber, properties or subunits composition are involved in reaching the synapse are not necessarily retained (Ehlers LTP and LTD (Huganir and Nicoll, 2013). It is now well es- et al., 2007; Petrini et al., 2009). Synaptic retention of tablished that LTP involves the modulation of AMPARs AMPARs occurs mainly through binding with transmem- that are already present at the synapse and/or the rapid brane AMPAR regulatory proteins (TARPs) and PDZ-con- recruitment of new AMPARs from extrasynaptic sites to taining scaffolding proteins present in the postsynaptic the synapse (Benke et al., 1998; Park et al., 2004; Makino density (PSD) such as Stargazin or PSD95 (Ehrlich and and Malinow, 2009; Petrini et al., 2009). On the other hand, Malinow, 2004; Kim and Sheng, 2004; Elias et al., 2006; removal of synaptic AMPARs from synapses and even Opazo et al., 2010). their degradation, seem to be fundamental issues for LTD In contrast to LTP, removal of synaptic AMPARs during (Beattie et al., 2000; Ehlers 2000; Lee et al., 2000). LTD seems to be mediated by clathrin-dependent endo- It has been described that membrane insertion of cytosis (Beattie et al., 2000; Man et al., 2000) followed by GluA1-containing AMPARs is regulated by cAMP-de- activation of degradation or recycling pathways (Ehlers pendent protein kinase (PKA), Ca21/calmodulin-depend- 2000). LTD triggered by NMDA receptor activation is Ca21- ent protein kinase II (CaMKII), and protein kinase C (PKC) dependent and requires the activation of phosphatases phosphorylation of Ser-845, Ser-831, and Ser-818, re- that dephosphorylate AMPARs and their associated pro- spectively (Mammen et al., 1997; Song and Huganir, teins to promote the destabilization of synaptic AMPARs, 2002; Esteban et al., 2003; Boehm et al., 2006). Among lateral diffusion to extrasynaptic sites and endocytosis them, PKA-mediated phosphorylation of Ser-845 seems (Mulkey et al., 1994; Lee et al., 1998, 2000; Tomita et al., to be essential for new AMPARs recruitment to extrasy- 2004). For instance, dephosphorylation of GluA1 Ser-845 naptic sites and was proposed to represent a necessary by PP2B (calcineurin; CaN) is necessary for LTD-mediated event for the establishment of LTP (Esteban et al., 2003; synaptic AMPARs removal (Oh et al., 2006; Lee et al., Makino and Malinow, 2009; Lee et al., 2010). Activity- 2010). Since PKA-mediated phosphorylation of Ser-845 is needed for LTP priming, fine-tuning of PKA/CaN activity is determinant for the insertion or removal of synaptic Received June 6, 2019; accepted March 2, 2020; First published March 16, AMPARs during LTP/LTD. On the other hand, CaMKII is 2020. The authors declare no competing financial interests. also emerging as an additional requirement for LTD, reduc- Author contributions: J.R.-A. and A.J.M.-M. designed research; W.C., D.S.- ing synaptic location of GluA1-containing AMPAR and W., J.C.-S., C.F., and A.J.M.-M. performed research; R.F., N.C., M.S., and M. thus, producing synaptic depression (Lu et al., 2010; U. contributed unpublished reagents/analytic tools; W.C., D.S.-W., C.A.S., J. Coultrap et al., 2014). R.-A., and A.J.M.-M. analyzed data; J.R.-A. and A.J.M.-M. wrote the paper. This work was partially supported by grants from Ministerio de Economia y A-kinase anchoring protein 79 (AKAP79; in human) or Competitividad (SAF2014-59697-R, SAF2017-89271-R, and SAF2017-83813- AKAP150 (the rodent homolog) is a postsynaptic scaffold C3-3-R), the Centro de Investigación Biomédica en Red sobre Enfermedades protein that anchors PKA, PKC, and CaN to the PSD, reg- Neurodegenerativas Grant CB06/05/0042, the Centro de Investigación ulating AMPAR phosphorylation, trafficking and activity Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición Grant CB06/ 03/0001, Fundació La Marató de TV3 Grants 2014-3610 and 201627.30.31, associated with LTP and LTD (Tavalin et al., 2002; Smith and Generalitat de Catalunya Grants SGR2009-1231 and SGR2014-0984 (to et al., 2006; Lu et al., 2007, 2008; Tavalin, 2008; Jurado et J.R.-A. and N.C.). W.C. was a recipient of a PhD fellowship from the China al., 2010; Sanderson et al., 2012). AKAP79/150 is linked Scholarship Council. J.C.-S. is supported by a FPU (Formación del to both AMPAR and NMDAR by additional binding to the Profesorado Universitario) fellowship from Ministerio de Economía y membrane-associated guanylate kinase (MAGUK) scaf- Competitividad. C.F. is supported by a PIF (Personal Investigador en Formación) fellowship from Unitat de Bioquímica, Departament de Bioquímica fold proteins SAP97 and PSD95 (Colledge et al., 2000; I Biologia Molecular, in UAB (Universitat Autònoma de Barcelona). UC Davis/ Bhattacharyya et al., 2009; Robertson et al., 2009). All of National Institutes of Health (NIH) NeuroMabs Facility provided the SAP97 them are S-palmitoylated in an activity-dependent man- monoclonal antibody supported by NIH Grant U24NS050606 and maintained ner controlling their targeting to dendritic spines and regu- by the Department of Neurobiology, Physiology, and Behavior, College of Biological Sciences, University of California, Davis, CA 95616. lating trafficking, spine enlargement and synaptic function Acknowledgements: We thank Dr. John D. Scott (Howard Hughes Medical (Fukata and Fukata, 2010; Keith et al., 2012; Purkey et al., Institute investigator, University of Washington, Seattle) for kindly providing 2018). Recently, it has been reported that LTD induces re- pcDNA3-AKAP150 construct. We also thank Helena Montón and Núria Barba moval of synaptic AKAP79/150 via CaMKII (Woolfrey et for their unvaluable technical assistance at Servei de Microscopia, Universitat Autònoma de Barcelona. al., 2018) contributing, together with the regulation of Correspondence should be addressed to Alfredo J. Miñano-Molina at alfredo. PKA/CaN, to synaptic GluA1-AMPARs removal. Thus, an- minano@uab.cat or José Rodriguez-Alvarez at jose.rodriguez@uab.cat tagonistic regulation of PKA, CaN and CaMKII activities https://doi.org/10.1523/ENEURO.0218-19.2020 together with the cycling of AKAP79/150 in and out the Copyright © 2020 Cheng et al. synapses are major molecular mechanisms controlling This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, synaptic AMPARs. However, it is still unknown whether distribution and reproduction in any medium provided that the original work is degradation of AKAP79/150 is triggered by LTD and properly attributed. whether it could be associated to AMPARs endocytosis. March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 3 of 19 In the present study, we show that AKAP150 is targeted RNAi interference experiments, a shAKAP150 construct to the ubiquitin-proteasome system (UPS) and degraded was generated using the Thermo Fisher Scientific RNAi under chemical LTD (cLTD) conditions. Interestingly, designer webtool (https://rnaidesigner.thermofisher.com/ pharmacological inhibition of proteasome restores basal rnaiexpress/) using specific oligonucleotides against mouse AKAP150 levels and blocks AKAP150 removal from syn- AKAP150 (NM_001101471.1) indicated by capital letter as apses. This degradation of AKAP150 goes in parallel follows: RNAi, forward, 5’-agatctcccGGAAAGTGCTTTCA with LTD-mediated GluA1 dephosphorylation at Ser-845 TTCAAAGttcaagagaCTTTGAATGAAAGCACTTTCC ttttta-39 and GluA1-AMPARs endocytosis. Indeed, maintaining and reverse, 5’-aagcttaaaaaGGAAAGTGCTTTCATTCAAAG AKAP150 levels blocks LTD-mediated GluA1-AMPARs tctcttgaaTTTGAATGAAAGCACTTTCCgggg-39. Oligonul- endocytosis. ceotides were purchased at Thermo Fisher Scientific and cloned between BglII and HindIII sites of the pSUPER. Materials and Methods retro.puro vector (OligoEngine), under the control of the DNA Pol III promoter of H1 (Brummelkamp et al., 2002). Cell culture, transfection, and transduction Lentiviral constructs were achieved by digestion at the Dissociated hippocampal cultures were prepared from EcoRI-ClaI sites to replace H1 promoter in the lentiviral newborn C57BL/6J wild-type mice as previously de- vector pLVTHM with H1-short hairpin RNA cassette from scribed (Ahmad et al., 2012). Briefly, hippocampi were pSUPER. pLVTHM-H1-RNAi was used for lentiviral knock- isolated and incubated with a digestion solution contain- down experiments. pLVTHM vector was a gift from Didier ing papain (10 U/ml; Worthington) and DNase I (300–450 Trono (Addgene plasmid #12247). Lentiviruses were pro- Kunitz/ml; Sigma) for 30 min at 37°C. A papain inactiva- duced in HEK293T cells using calcium phosphate transfec- tion solution containing fetal bovine serum and bovine tion of a 20 mg of pLVTHM-shAKAP150 or 20 mg of pWPI- serum albumin was then applied. Tissue was triturated AKAP150 (for AKAP down-regulation or over-expression, and cells were plated on poly-D-lysine (0.01 mg/ml in 0.1 respectively), together with 15 mg of psPax2 and 6 mg of M borate buffer, pH 8.4; both from Sigma)-coated 24- pMD2.G (gifts from Didier Trono, Addgene, #12260 and well plates with 12-mm coverslip (Paul Marienfeld) at a #12259, respectively); 24, 36, and 48 h after transfection, density of 75,000 cells per cover slip (for imaging stud- lentivirus-containing media were harvested, filtered with ies) or from 300,000 to 900,000 cells per 35- to 60-mm 0.45 mm filter units and centrifuged at 25,000 rpm for 2 h at dishes, respectively (for Western blotting). Wells con- 4°C. Pelleted viruses were resuspended in ice-cold PBS tained Neurobasal-A media (Thermo Fisher Scientific) buffer by shaking at 175 rpm overnight at 4°C. Lentiviruses supplemented with B-27 (Thermo Fisher Scientific) and were aliquoted and stored at 80°C until use. Biological GlutaMAX (Thermo Fisher Scientific). Glial growth was titers of the viral preparations expressed as a number of inhibited by 5-fluorodeoxyuridine (FDUR) at 3 days in TUs per milliliter were determined by infecting HEK293T vitro (DIV). Neurons were maintained in a humidified in- cells with serial dilutions. After 48 h, the percentage of cubator at 37°C with 5% CO2, and half medium was re- GFP-positive cells was detected by flow cytometry placed with fresh medium every week until use at 17–21 (Cytomics FC 500, Beckman Coulter) and analyzed with DIV. All animal procedures were performed in accord- RXT software. ance with Universitat Autònoma de Barcelona animal care committee’s regulations. When necessary, neurons were Cell stimulation and lysate preparation transduced with lentivirus [2–3 transducing units (TU)] on cLTD was induced as previously described (Oh et al., day 7 in culture and assayed as indicated in results. Direct 2006). Cultures were first incubated in ACSF for 30 min at counting of GFP-positive cells was used to monitor the effi- room temperature: 125 mM NaCl, 2.5 mM KCl, 1 mM ciency of infection in each experiment. The percentage of MgCl2, 2 mM CaCl2, 33 mM D-glucose, and 25 mM infected cells reached at least 75%. Overexpression or HEPES (pH 7.3), followed by stimulation with 50 mM NMDA down-regulation of AKAP was assessed by immunoblot- in ACSF (no MgCl2) for 5 min. After NMDA treatment, neu- ting and immunocytochemistry. rons were replaced in regular ACSF and then subjected to HEK293T cells were maintained in DMEM supplemented the corresponding procedure at indicated time points. with 10% fetal bovine serum and 1 pen/strep (Thermo Cultures were then washed with ice-cold PBS once and Fisher Scientific). Cells at 90% confluence were passaged lysed in cold 1% NP-40 homogenization buffer (20 mM every 6 d using 1 Trypsin-EDTA solution (T3924-Sigma) Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, replacing the media every 3 d. HEK293T cells were trans- 1 mM Na2VO4, and 1 Sigma protease inhibitor and phos- fected using the calcium phosphate method with 20 mg phatase inhibitor cocktails). When indicated, cultures plasmid DNA. were pre-treated or post-treated with BAPTA-AM (20 mM), FK506 (10 mM), MG132 (10 mM), or MK801 (10 mM; all from Plasmids and lentivirus production Tocris) and were present until cells were lysed. cLTP was AKAP150 was cloned by PCR from pcDNA3-AKAP150 induced as previously described (Otmakhov et al., 2004) vector (kindly provided by J. D. Scott; Howard Hughes by application of forskolin (50 mM) and rolipram (0.1 mM; Medical Institute, University of Washington, Seattle) and in- both from Tocris), 60 min before or after cLTD. Cells were serted into the lentiviral vector pWPI-IRES-GFP at the PmeI lysed 60 min after cLTP or cLTD treatment. Lysates were site for lentiviral overexpression experiments. pWPI vector centrifuged at 12,000  g for 10 min at 4°C and protein in was a gift from Didier Trono (Addgene plasmid #12 254). For the supernatant was quantified by Bradford method assay March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 4 of 19 kit (Bio-Rad Laboratories). The intensity of the cLTD proto- membranes (GE Healthcare Life Sciences). Blots were col may vary depending on the cultures. blocked at room temperature for 1 h with 10% dry milk, 0.1% BSA (fraction V), pH 7.4 in PBS and incubated at 4° Surface biotinylation C overnight with primary antibody in PBS 0.1% BSA, pH Treated cells were transferred to ice-cold PBS-Ca21- 7.4. Primary antibodies were: anti-phospho-Ser-845- Mg21 buffer (pH 7.4; 1 mM CaCl2; 0.1 mM MgCl2), followed GluA1 (1:1000; clone EPR2148; 04–1073; RRID: AB_ by biotinylation in 1 mg/ml of biotin (EZ-Link Sulfo-NHS- 1977219; Merck-Millipore), anti-GluA1 (1:1000; AB1504; SS-Biotin; Thermo Fisher Scientific) for 30 min with slow RRID: AB_2113602; Merck-Millipore), anti-AKAP150 agitation. Free biotin was quenched by wash (3) in cold (1:1000; 07–210; RRID: AB_310430; Merck-Millipore), PBS-Ca21-Mg21 1 glycine 0.1 M. Cell cultures were im- anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_ mediately lysed in cold 1% Triton X-100 homogenization 303248; Abcam), anti-SAP97 (1:1000; clone K64/15; 75– 030; RRID: AB_2091920; UC Davis/NIH NeuroMab), anti- buffer (150 ml/35 mm well; two well per condition; 50 mM Ubiquitin (1:5000; U5379; RRID: AB_477667), anti- b -tu- NaCl, 10 mM EDTA, 10 mM EGTA, 1 mM Na3VO4, 50 mM bulin (1:20,000; clone 5H1; 556321; RRID: AB_396360; NaF, 25 mM NaPPi, 1 mM b -glycerphosphate, 1 mM BD Biosciences), anti- b -actin (1:20,000; clone AC-74; phenylmethylsulfonyl fluoride, 1 protease inhibitor cock- A2228; RRID: AB_476697; Sigma), and anti-GAPDH tail, 1 phosphatase inhibitor cocktail, and 50 mM HEPES; (1:40 000; clone 6C5; AM4300; RRID: AB_437392; pH 7.5). Cell lysates were centrifuged at 10,000  g for Thermo Fisher Scientific). After washing, blots were incu- 20 min to pellet insoluble fraction. A total of 75 ml of the su- bated with horseradish peroxidase-conjugated secondary pernatant were mixed and heated with 25 ml of 4 SDS antibodies, goat anti-mouse or goat anti-rabbit (554002 sample buffer to determine total fraction of GluA1 (surface and 554021; RRID: AB_395198 and RRID: 395213, re- plus internal). Biotinylated surface proteins in the remaining spectively; BD Biosciences), diluted in blocking buffer, supernatant (225 ml) were pulled-down with 40 ml of 50% and developed using the ECL Western Blotting Detection avidin-agarose beads (ImmunoPure Immobilized Avidin; Reagents (GE Healthcare Life Sciences). Semi-quantita- Thermo Fisher Scientific) overnight at 4°C. The beads were tive analysis of immunoblots was performed by densitom- pelleted and 75 ml of the supernatant (internal fraction) were etry using ImageJ 2.0v (https://imageJ.nih.gov/ij/) and mixed and heated with 25 ml of 4 SDS sample buffer. The protein levels were corrected for corresponding loading beads were then rinsed three times with 1% Triton X-100 control. homogenization buffer and heated in 100 ml of 2 SDS sample buffer (surface fraction). Equal volumes of total and biotinylated fractions were subjected to 10% SDS-PAGE, Immunocytochemistry probed by immunoblot for total GluA1 levels and normal- Neuronal cultures on coverslips were washed with ice- ized to GAPDH. cold 1 PBS, fixed with ice-cold 4% paraformaldehyde with 4% sucrose in 1 PBS for 15 min at 4°C and then permeabilized with 0.1% Triton X-100 for 20 min at room Immunoprecipitation temperature. For surface GluA1-containing AMPARs de- Neurons were washed in ice-cold PBS 1 and immedi- tection, no permeabilization was done. Afterwards, plates ately lysed in cold immunoprecipitation buffer (200 ml  60 were washed twice with PBS, blocked with 2% normal mm plate; four plates per condition; 0.1% SDS, 1% Triton Goat serum (Sigma) in 1 PBS (blocking buffer) for 1 h at X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM NaPPi, 37°C and then incubated for 1 h at 37°C with specific anti- 10 mM N-ethylmaleimide, 1  protease inhibitor cocktail, and 1  phosphatase inhibitor cocktail). Homogenates bodies detecting AKAP150 (1:200; clone N-19; sc-6446; from cultures were centrifuged at 16,000  g for 10 min at RRID: AB_2225903 and 1:200; clone R-300; sc-10 765; 4°C and the protein in the supernatant was quantified by AB_2289482, Santa Cruz Biotechnology), PSD95 (1:1000; BCA protein assay kit (Bio-Rad Laboratories); 50 mg of clone 6G6-1C9; ab2723; RRID: AB_303248; Abcam), GluA1 protein from each supernatant were mixed and heated (1:1000; AB1504; RRID: AB_2113602; Merck-Millipore), with 4 SDS sample buffer to keep total homogenate GFP (1:1000; ab13970; RRID: 300798; Abcam), and fraction (1 mg/ml). The remaining supernatants were immu- VGLUT1 (1:500; AB5905; RRID: 2301751; Merck-Millipore). noprecipitated with 5 mg of either rabbit anti-AKAP150 Surface GluA1-containing AMPARs were measured using a (clone R-300; sc-10 765; RRID: AB_2289482, Santa Cruz rabbit polyclonal antibody directed against the N terminus Biotechnology) or control rabbit IgG (011-000-003; RRID: of the GluA1 subunit (1:30; PC246; RRID: 564636; Merck- AB_2337118; Jackson ImmunoResearch) antibodies Millipore). Neurons were washed with warmed PBS-MC-su- overnight at 4°C, with gentle rocking. Immune complexes crose (PBS 1, 0.5 mM CaCl2, 1 mM MgCl2, 4% sucrose) were precipitated for 1 h at 4°C using 40 ml protein G and incubated with N-term-GluA1 antibody in PBS-MC-su- Sepharose beads (GE Healthcare Life Sciences). The crose at 37°C for 5 min. Cells were then washed with ice- beads were pelleted, washed and denatured in 100 ml of cold PBS-MC and fixed with ice-cold 4% paraformaldehyde 2 SDS sample buffer, and 20 ml were loaded on an SDS- with 4% sucrose in 1 PBS for 15 min at 4°C. Next, plates PAGE gel for immunoblotting. were washed twice with ice-cold PBS-MC, blocked for 1 h, and incubated with Alexa Flour-conjugated secondary anti- Immunoblotting body (1:300) in blocking buffer for 1 h, both at room temper- Samples were separated on 7.5% or 10% SDS-PAGE ature. Cells were washed, postfixed with methanol at 20°C and transferred onto Hybond-C Extra, Nitrocellulose for 1 min, blocked, and incubated with specific antibodies March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 5 of 19 A H mature cLTD cultured neurons 30 min 5 min 55 min + ACSF Basal cLTD BAPTA BAPTA ACSF ACSF(Ca2+) (Ca2+,Mg2+) Exp procedures 2+ 2+ (Ca ,Mg ) +NMDA 50PM FK506 or + BAPTA 150kDa AKAP150 FK506 FK506 or + BAPTA or + BAPTA 50kDa E-tubulin cLTD 60min (or+Inh) B C I ACSF 0 mM Mg2+ 100kDa pS845-GluA1 1.5 1.5 pS845-GluA1 Fold change (vs Basal) 100kDa GluA1 AKAP150/GAPDH Protein levels normalized GluA1 # Fold change (vs Basal) 37kDa 1.0 GAPDH 1.0 * * 15' 30' 60' 120' * 0.5 cLTD 0.5 * * * * 100kDa pS845-GluA1 * 0.0 Basal cLTD cLTD BAPTA 100kDa GluA1 0.0 + 15' 30' 60' 120' BAPTA 37kDa GAPDH 15' 30' 60' 120' D E J cLTD 1.5 D TD cLT st-cL Fold change (vs Basal) Basal 15' 30' 60' 120' p re- p o 06 06 sGluA1/ tGluA1 K5 K506 1.0 l sa D K5 100kDa sGluA1 * Ba cLT +F +F F * 150kDa AKAP150 100kDa tGluA1 0.5 37kDa * 42kDa GAPDH GAPDH 0.0 Basal 15' 30' 60' 120' F G K 2+ 1.5 Fold change (vs Basal) ACSF 0 mM Mg 1.5 AKAP150/GAPDH Fold change (vs Basal) 150kDa AKAP150 AKAP150/GAPDH 1.0 1.0 37kDa GAPDH * * * * cLTD 0.5 0.5 * 150kDa AKAP150 * 0.0 l D D D 06 37kDa sa GAPDH 0.0 Ba cLT e-cLT t-cLT FK5 15' 30' 60' 120' p r o s 06 p 15' 30' 60' 120' K5 06 +F K5 +F Figure 1. cLTD reduces AKAP150. A, Cultured neurons were incubated in ACSF buffer for 30 min to equilibrate cells before 50 mM NMDA treatment for 5 min (cLTD). Cells were lysed after cLTD induction at indicated times (or 60 min if no time is indicated) in the presence or absence of inhibitors, antagonists, or cell-permeable Ca21 chelators as indicated in the experimental design scheme. B, Representative blot images showing reduced levels of Ser-845 GluA1 and GluA1 after treatment (cLTD) compared with cells in ACSF buffer. Ser-845 GluA1 levels (pS845-GluA1; ;110-kDa band, top panel) were related to total GluA1 (GluA1; ;110-kDa band, middle panel). GAPDH was used as a loading control (;36-kDa band, bottom panel). C, Quantification of pS845-GluA1 and GluA1 changes in response to NMDA stimulation compared with basal condition (n = 5–17, pp , 0.0001 vs basal). D, Representative blot images showing reduced levels of surface GluA1 after treatment (sGluA1; ;110-kDa band, top panel) related to total GluA1 (tGluA1; ;110-kDa band, middle panel). GAPDH was used as a loading control (;36-kDa band, bottom panel). E, Quantification of surface GluA1 changes in response to cLTD compared with basal condition (n = 6, pp = 0.0310 for 30 min and pp , 0.0001 for 60 min and 120 min vs basal). F, Representative blot images showing reduced levels of AKAP150 after cLTD (;150-kDa band, top panel) re- lated to GAPDH (bottom panel). G, Quantification of AKAP150 changes in response to cLTD compared with baseline (n = 5–20, March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 6 of 19 continued pp = 0.0021 for 30 min and pp , 0.0001 for 60 min and 120 min vs basal). H, J, Representative blot images showing the effect of the presence of Ca21 chelator BAPTA (H; 20 mM) or Ca21-dependent phosphatase CaN inhibitor FK506 (J; 10 mM) on AKAP150 protein levels after cLTD (top panel) related to b -tubulin (;51-kDa band, bottom panel) or GAPDH (bottom panel) used as a loading control. I, K, Quantification of AKAP150 changes in response to cLTD in presence of BAPTA (I) or FK506 (K) compared with cLTD treatment (n = 3 in BAPTA experiments, pp = 0.0187 vs basal and #p = 0.0377 vs cLTD, n = 3–10 in FK506 pre-cLTD, pp , 0.0001 vs basal and FK506 post-cLTD experiments, pp = 0.0073 vs basal). Bar represent mean 6 SEM. against desired protein. Overall, primary antibodies were stimulation, to observe the characteristics features of Ser- detected after washing with 1 PBS by incubation (1 h at 845-GluA1 dephosphorylation and decrease of GluA1- 37°C) with Alexa Fluor-conjugated secondary antibodies AMPARs levels (Kamal et al., 1999; Oh et al., 2006; (1:500 or 1:1000; A405, A488, A568 and/or A633; Thermo Bhattacharyya et al., 2009; Li et al., 2010; Keith et al., Fisher Scientific) in blocking buffer. Cells were washed in 1 2012; Shehata et al., 2012; Bacaj et al., 2015; Yap et al., PBS and when necessary, treated with HOECHST 33258 2017). Since consistent results (data not shown) were ob- for nuclei staining (1:10,000 in PBS; H3569; Thermo Fisher served when hippocampal cultures were treated with Scientific). Following, coverslips were washed with 1 PBS, 50 mM NMDA for 5 min in 0 mM Mg2, we decided to use and mounted on glass slides on Fluoromount-G as an anti- these conditions in our experiments. Western blot analysis quenching reagent (Southern Biotech). revealed a time-dependent decrease of Ser-845 GluA1 phosphorylation in hippocampal cultured neurons after Confocal imaging and analysis cLTD induction [50 mM for 5 min; cLTD (Fig. 1A–C); cLTD: Confocal z-stacks were acquired on a Zeiss LSM700 0.449 6 0.020 for 15 min, 0.283 6 0.014 for 30 min, confocal microscope using the 63/1.40 NA oil objective 0.365 6 0.047 for 60 min, and 0.335 6 0.125 for 120 min; or Olympus FV3000 confocal microscope using the 60/ pp , 0.0001, respectively, vs basal) in accordance to previ- 1.5 NA oil High Resolution objective. Sequential frame ac- ously reported data (Beattie et al., 2000; Ehlers 2000; He et quisition was set to acquire an average of 10 planes per al., 2011). Moreover, significant time-dependent changes stack at 16 bits and a minimum of 1024  1024 resolution. were also found in GluA1 total levels after NMDA stimulus Channel gain settings were optically adjusted to minimize (cLTD: 0.791 6 0.034 for 30 min; pp = 0.0189, 0.594 6 saturation of punctae and were maintained across experi- 0.045 for 60 min and 0.415 6 0.077 for 120 min; pp , mental groups. Unmodified images were used for all anal- 0.0001, respectively, vs basal) as previously reported yses that were done blind and linear scaling was applied (Fernández-Monreal et al., 2012). A reduction in surface on images only for presentation purposes using ImageJ software. Fluorescent signal on the single planes from a GluA1-containing AMPARs after NMDA application was stack, quantification of punctae number and punctae inte- also observed by biotinylation and Western blot analyses. grated fluorescence intensity over area measurements The surface reduction in GluA1-containing AMPARs was were performed with ImageJ software. Untreated and also time-dependent starting with 30-min NMDA applica- treated cells from the same culture preparation were al- tion (Fig. 1D,E; 0.726 6 0.095 for 30 min; pp = 0.0310, ways compared. For each experimental group an average 0.478 6 0.077 for 60 min and 0.161 6 0.005 for 120 of . 200 mm dendrite was quantified for 8–12 images per min; pp , 0.0001, respectively, vs basal). Previous reports experiment in duplicate and repeated for a total of three showed that cLTD causes a redistribution of the scaffold- or four independent experiments. ing protein AKAP150 away from synapses (Gomez et al., 2002). However, it has not been reported whether this re- Statistical analysis distribution is also associated to changes in total AKAP150 Statistical analysis was performed with Prism 6.0 levels. Our results indicate that cLTD significantly reduces (GraphPad Software). One-way ANOVA with Bonferroni’s AKAP150 levels in a time-dependent manner, as shown in multiple comparison post hoc test or unpaired Student’s t the immunoblot analysis (Fig. 1F,G; cLTD: 0.799 6 0.024 test was used for mean comparisons between experimen- for 30 min; pp = 0.0021, 0.410 6 0.041 for 60 min, 0.232 6 tal conditions as required. Differences were considered 0.034 for 120 min; pp , 0.0001 vs basal). As expected, ap- significant when p , 0.05. Depending on the type of ex- plication of Ca21 chelator BAPTA-AM blocked cLTD-medi- periment the number of samples used for statistical analy- ated AKAP150 levels decrease (Fig. 1H,I; cLTD: 0.525 6 ses, n refers to the accumulated number of dendrites, 0.111; cLTD 1 BAPTA: 0.935 6 0.066; pp = 0.0187 vs punctae, or independent cultures assayed per group (as indicated in figure legend) being always three or more in- basal, #p = 0.0377 vs cLTD). dependent biological replicates. Data in bar graphs are re- Moreover, the phosphatase CaN has been described to ported as mean 6 SEM. form a complex with AKAP150 that is required for cLTD (Jurado et al., 2010) and to play a major role in AMPAR Results endocytosis by dephosphorylation of GluA1 Ser-845 (Oh et al., 2006; Lee et al., 2010). Therefore, we examined cLTD triggers a decrease in Ser-845 GluA1 whether CaN activity is necessary for cLTD NMDA-de- phosphorylation, GluA1 protein levels, GluA1-AMPAR pendent AKAP150 degradation. Pharmacological inhibi- surface expression, and AKAP150 degradation tion of CaN with FK506 after or during cLTD induction did First, we analyzed different cLTD paradigms used in the not prevent the degradation of AKAP150 (Fig. 1J,K; cLTD: literature, changing NMDA dose and time of NMDAR 0.454 6 0.034; cLTD 1 FK506 post-cLTD: 0.652 6 0.043; March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 7 of 19 A B 1.5 Fold change (vs Basal) AKAP150/GAPDH cLTD + # 1.0 Basal cLTD MK801 MK801 150kDa * AKAP150 0.5 37kDa GAPDH 0.0 Basal cLTD cLTD MK801 + MK801 C Basal MK801 AKAP150 PSD95 AKAP150/PSD95/Hoechst Merge cLTD cLTD + MK801 D E AKAP150 intensity at dendrite # AKAP150 intesity at PSD95 1.5 2.0 # (Relative to Basal) (Relative to Basal) 1.5 1.0 * 1.0 * 0.5 0.5 0.0 0.0 l l sa TD 01 01 sa TD 01 01 Ba cL MK8 MK8 Ba cL MK8 MK8 + + TD TD cL cL Figure 2. cLTD-mediated reduction of AKAP150 requires calcium influx through NMDA receptors. A, Representative blot images showing the effect of NMDAR antagonist MK801 (10 mM) on AKAP150 protein levels after cLTD (top panel) related to GAPDH (bot- tom panel) used as a loading control. B, Quantification of AKAP150 changes in response to NMDA stimulation in presence of MK801 compared with cLTD (n = 4, pp = 0.0036 vs basal and #p = 0.0395 vs cLTD). C, Representative confocal images of primary neurons showing AKAP150 (green), postsynaptic marker PSD95 (magenta) and nuclei (blue) staining. Separated color panels for in- dividual marker from the boxed regions have been magnified for dendrite clarity (right panels). Lower right panels show the merge March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 8 of 19 continued for AKAP150 (green)/PSD95 (magenta), where cLTD reduced dendritic and synaptic intensity in AKAP150 staining. The presence of MK801 (10 mM) blocks this reduction. D, Quantification of AKAP150 intensity at dendrites. cLTD reduced AKAP150 dendritic inten- sity and the presence of MK801 avoids this reduction (n . 15 dendrites of different neurons from four independent cultures, pp , 0.0001 vs basal; #p , 0.0001 vs cLTD). E, Quantification of AKAP150 intensity at the PSD95. cLTD reduction of synaptic AKAP150 intensity was blocked by MK801 (n . 50 puncta from .15 dendrites from four independent cultures, pp = 0.0150 vs basal; #p , 0.0001 vs cLTD). Scale bars = 20 mm (C, left panels) and 5 mm (C, right panels). Bars represent mean 6 SEM. pp= 0.0073 vs basal; cLTD 1 FK506 pre-cLTD: 0.499 6 (Fig. 3E,H; cLTD 1 MG132: 1.088 6 0.013; #p = 0.0002 vs 0.066; pp , 0.0001 vs basal). These results confirm that cLTD). However, we did not observe changes in PSD95 although CaN mediates cLTD-dependent AKAP150 redis- punctae number at dendrites after cLTD treatment (Fig. tribution away from the synapse (Smith et al., 2006), it is 3I; basal: 17.164 6 2.002 punctae vs cLTD: 14.929 6 not involved in cLTD-dependent AKAP150 degradation. 2.428 punctae per 10 mm). Our data in cultured neurons suggest that not only redistribution of AKAP150 but also Ubiquitin-proteasome system mediates cLTD- its degradation may play a role in the early stages of LTD. dependent AKAP150 degradation To determine whether cLTD-mediated AKAP150 de- crease required AKAP150 ubiquitination, we check ubiq- After our finding that cLTD produced a decrease in uitination levels after cLTD induction in the presence of AKAP150 levels we wondered whether this effect was MG132 in cultured neurons. Surprisingly, we observed mediated by proteasome activation. The reduction in less ubiquitin-conjugated pattern after cLTD induction in AKAP150 levels was blocked by pretreatment with the whole lysate compared with basal levels (Fig. 4A,B; cLTD: NMDA antagonist MK801 (Fig. 2A,B; cLTD 1 MK801: 0.666 6 0.066; pp = 0.0355), despite we expected an in- 0.847 6 0.133; #p = 0.0395 vs cLTD) and also by the spe- crease in ubiquitination levels since inhibition of ubiquiti- cific inhibition of proteasome activity with MG132 (Fig. nation attenuates NMDA-dependent cLTD (Citri et al., 3A,B; cLTD 1 MG132: 1.228 6 0.184; #p = 0.0014 vs 2009). Proteasome inhibition with MG132 restored ubiqui- cLTD). Since cLTD-induced NMDAR activation causes a tination levels (Fig. 4A,B; cLTD1MG132: 1.039 6 0.079; persistent loss of synaptic AKAP150 in cultured neurons #p = 0.0183 vs cLTD), suggesting that cLTD could be in- (Smith et al., 2006), we analyzed whether changes in ducing rapid protein degradation via ubiquitin-protea- AKAP150 levels go beyond synapse relocation and some system. Previous studies surveying substrates of whether its degradation is participating on AKAP150 re- ubiquitin ligation in the PSD identified AKAP150 as a moval from synapses. We assessed AKAP150 levels by highly ubiquitinated protein under basal conditions which confocal microscopy pretreating neurons with both is highly regulated by synaptic activity (Ehlers 2003; Jia et MK801 and MG132. We observed a significant reduction al., 2008). Accordingly, while cLTD reduced AKAP150 lev- in AKAP150 immunoreactivity at dendrites after cLTD that els (Fig. 4C; cLTD: 0.614 6 0.059; pp = 0.0121 vs basal), was blocked by MK801 and MG132 application (Figs. pull-down results showed a significant increase in total 2C,D, 3E,F; cLTD: 0.504 6 0.042; pp , 0.0001; cLTD 1 ubiquitin conjugated-AKAP150 observed in AKAP150 MK801: 1.31 6 0.061; #p , 0.0001 vs cLTD; cLTD 1 shift when overexposed (Fig. 4C,D; cLTD: 1.813 6 0.125; MG132: 1.140 6 0.0471; #p , 0.0001 vs cLTD). Moreover, pp = 0.0026 vs basal). These results suggest that synaptic we analyzed AKAP150 levels at PSD95 clusters and we AKAP150 degradation by ubiquitin-proteasome system observed that both MK801 and MG132 were able to could be an important factor in cLTD-induced synapse maintain AKAP150 at synapses after cLTD (Figs. 2C,E, regulation. It is known that AKAP150 participates in 3E,G; cLTD: 0.666 6 0.031; pp = 0.0150; cLTD 1 MK801: the regulation of LTP processes and even it expression 1.435 6 0.073; #p , 0.0001 vs cLTD; cLTD 1 MG132: is upregulated in this conditions (Génin et al., 2003; 1.239 6 0.086; #p , 0.0001 vs cLTD). Previous work by Sanderson and Dell’Acqua, 2011). Since our results show Colledge et al., (2003) indicated that cLTD treatment of that cLTD induce AKAP150 degradation, we studied whether LTP could restore AKAP150 levels in our neuro- cultured neurons induces proteasomal degradation of nal cultures. For this purpose we used a chemical stimula- PSD95, leading its decreased synaptic clustering and tion protocol of forskolin and rolipram (Fig. 5A; cLTP; 50 contributing to the removal of synaptic AKAP150 and mM /0.1 mM) that results in LTP (Grey and Burrell, 2008) AMPARs (Colledge et al., 2003; Smith et al., 2006). and produces and increase in both GluA1 phosphoryla- Therefore, we analyzed PSD95 levels after cLTD by tion at Ser-845 and cell surface GluA1 levels (Fig. 5B,C; Western blotting and we observed that protein levels cLTD: 0.418 6 0.085; pp , 0.0001; cLTP 1.325 6 0.041; were reduced and treatment with MG132 was able to re- pp = 0.0009 vs basal; Oh et al., 2006; Miñano-Molina et store them (Fig. 3C,D; cLTD 1 MG132: 1.001 6 0.078; al., 2011). When cLTP was induced in cultured neurons #p = 0.0091 vs cLTD). We also quantified both PSD95 after cLTD no reversion of cLTD-mediated effects on cluster intensity and punctae number after cLTD treat- AKAP150 levels was observed by immunoblotting (Fig. ment of cultured hippocampal neurons. Consistent with 5D,E; cLTD: 0.545 6 0.049; cLTD 1 cLTP: 0.400 6 0.052; previously reported data (Colledge et al., 2003; Smith et pp , 0.0001 vs basal) or by immunocytochemistry (Fig. 5F,G; al., 2006; Bhattacharyya et al., 2009), we observed a sig- cLTD: 0.444 6 0.046; pp = 0.0004 vs basal; cLTD 1 cLTP: nificant decrease in PSD95 cluster intensity after 1 h of 0.467 6 0.065; #p = 0.0017 vs basal). Similarly, AKAP150 treatment (Fig. 3E,H; cLTD: 0.659 6 0.011; pp = 0.0005 vs was not observed even when we tried to favor AKAP150 basal) that was not observed in the presence of MG132 retention at the synapses by inducing cLTP before cLTD March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 9 of 19 A cLTD C cLTD + + Basal cLTD MG132 MG132 Basal cLTD MG132 MG132 100kDa 150kDa AKAP150 PSD95 37kDa 37kDa GAPDH GAPDH B 2.0 D 2.0 Fold change (vs Basal) Fold change (vs Basal) AKAP150/GAPDH # PSD95/GAPDH 1.5 1.5 # 1.0 1.0 * * 0.5 0.5 0.0 0.0 Basal cLTD cLTD MG132 Basal cLTD cLTD MG132 + + MG132 MG132 E Basal MG132 AKAP150 PSD95 AKAP150/PSD95/Hoechst Merge cLTD cLTD + MG132 F G H I AKAP150 intensity at dendrite 1.5 2.0 1.5 25 AKAP150 intesity at PSD95 # (Relative to Basal) PSD95 puncta /10Pm (Relative to Basal) (Relative to Basal) # 20 PSD95 intensity 1.5 # 1.0 1.0 15 * 1.0 * * 10 0.5 0.5 0.5 5 0.0 0.0 0.0 0 l l l l sa TD 32 32 sa TD 32 32 sa TD 32 32 sa TD Ba cL MG1 MG1 Ba cL MG1 MG1 Ba cL MG1 MG1 Ba cL + + + TD TD TD cL cL cL Figure 3. cLTD-mediated reduction of AKAP150 requires proteasome activity. A, Representative blot images showing the effect of proteasome inhibitor MG132 (10 mM) on AKAP150 protein levels after cLTD (top panel) related to GAPDH (bottom panel) used as loading control. B, Quantification of AKAP150 changes in response to NMDA stimulation in presence of MG132 compared with cLTD (for n = 4, pp = 0.0125 vs basal and #p = 0.0014 vs cLTD). C, Representative blot images showing the effect of proteasome in- hibitor MG132 (10 mM) on PSD95 protein levels after cLTD (top panel) related to GAPDH (bottom panel) used as loading control. D, Quantification of PSD95 changes in response to NMDA stimulation in presence of MG132 compared with cLTD (for n = 3, March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 10 of 19 continued pp = 0.0092 vs basal and #p = 0.0091 vs cLTD). E, Representative confocal images of primary neurons showing AKAP150 (green), postsynaptic marker PSD95 (magenta) and nuclei (blue) staining. Separated color panels for individual marker from the boxed re- gions have been magnified for dendrite clarity (right panels). Lower right panels show the merge for AKAP150 (green)/PSD95 (ma- genta), where cLTD reduced dendritic and synaptic intensity in AKAP150 staining. The presence of MG132 (10 mM) blocks this reduction. F, Quantification of AKAP150 intensity at dendrites. cLTD reduced AKAP150 dendritic intensity and the presence of MG132 avoids this reduction (n . 15 dendrites of different neurons from four independent cultures, pp , 0.0001 vs basal; #p , 0.0001 vs cLTD). G, Quantification of AKAP150 intensity at the PSD95. cLTD reduction of synaptic AKAP150 intensity was blocked by MG132 (n . 50 puncta from .15 dendrites from four independent cultures, pp = 0.0150 vs basal; #p , 0.0001 vs cLTD). H, Quantification of PSD95 intensity at dendrites. cLTD reduction of PSD95 intensity was blocked by MG132 (n . 15 dendrites of different neurons from four independent cultures, pp = 0.0005 vs basal; #p = 0.0002 vs cLTD). I, Quantification of PSD95 puncta at dendrites normalized to basal. There is no difference in puncta number between basal and cLTD conditions (n . 50 puncta from .15 dendrites from four independent cultures). Scale bars = 20 mm (A, left panels) and 5 mm (A, right panels). Bars represent mean 6 SEM. (Fig. 5D,E; cLTP: 0.869 6 0.051; cLTP 1 cLTD: 0.245 6 internalization (Fig. 6J,L; shAKAP150: 0.279 6 0.018; pp = 0.044; pp , 0.0001 vs basal). 0.0003, cLTD: 0.517 6 0.070; pp = 0.0080 and shAKAP150 1 cLTD: 0.131 6 0.027; pp , 0.0001 vs basal). Endogenous AKAP150 knock-down triggers dephosphorylation and endocytosis of AMPARs Overexpression of AKAP150 blocks cLTD-mediated Synaptic distribution of AKAP150 is a key factor for syn- AMPARs endocytosis aptic expression of AMPARs (Tavalin et al., 2002). Previous Above results show that a decrease in AKAP150 endog- reports have shown that removal of AKAP150 from synap- enous levels either by cLTD induction, shRNA-mediated ses, toward the cytosol in dendritic shafts or soma, elicits knock-down or both, triggers GluA1-containing AMPARs synaptic AMPARs endocytosis (Bhattacharyya et al., 2009; endocytosis. Therefore, we studied whether overexpres- Keith et al., 2012). In this context, we explored AKAP150 si- sion of AKAP150 was able to rescue AMPARs endocyto- lencing effect on AMPAR. Using shRNA-mediated acute sis produced after cLTD. According to previous studies knock-down of endogenous AKAP150 (shAKAP150), we showing that AKAP150 overexpression somehow in- were able to effectively reduce AKAP150 expression in cul- creases basal AMPAR surface expression (Bhattacharyya tured neurons (Fig. 6A). Interestingly, whereas cLTD pro- et al., 2009), neurons overexpressing AKAP150 showed a duces the degradation of AKAP150 (Fig. 1F,G), PSD95 slight no significant increase in Ser-845 phosphorylation (Fig. 3C,D), and SAP97 (data not shown), knock-down of without affecting GluA1 total levels (Fig. 6F,G; ovAKAP150: endogenous AKAP150 did not induce significant changes 1.180 6 0.047 and 0.963 6 0.125, respectively). However, in PSD95 or SAP97 levels (Fig. 6B,C; shAKAP150: 1.070 6 AKAP150 overexpression did elevate surface GluA1 label- 0.112 and 0.873 6 0.145, respectively). However, AKAP150 ing (;24%) compared with basal (Fig. 7A,B; ovAKAP150: silencing decreased S845-GluA1 phosphorylation and 1.274 6 0.098; #p = 0.0419). When cLTD was induced in GluA1 total levels (Fig. 6D,E; shAKAP150: 0.327 6 0.101; AKAP150 overexpressing neurons, surface GluA1 levels pp = 0.0057 and 0.516 6 0.073; #p = 0.0070, respectively, vs remained unchanged (Fig. 7C,D and E,F; ovAKAP1501 Ø). Consistent with the results observed by immunoblotting, cLTD: 0.996 6 0.112; #p = 0.0185 vs cLTD and ovAKAP1501 AKAP150 knock-down reduced both synaptic and dendritic cLTD: 0.893 6 0.125; #p = 0.0016 vs cLTD). GluA1 levels (Fig. 6H,I; shAKAP150: 0.432 6 0.088; pp , 0.0001 and 0.445 6 0.023; #p , 0.0001, respectively), Discussion followed by a decrease in surface GluA1 expression (Fig. It is well known that modulation of AMPAR function and 7A,B; shAKAP150: 0.401 6 0.109; pp , 0.0001 vs basal). membrane trafficking, in and out the synapses, is a major Next, we wanted to test whether this reduction in surface contributor to synaptic plasticity processes, such as LTP GluA1 by AKAP150 silencing could occlude further or LTD, that are believed to underlie learning and memory AMPAR removal after cLTD induction. We induced cLTD in (Huganir and Nicoll, 2013). Whereas LTP has been associ- AKAP150-silenced neurons and surface GluA1 levels were ated to the insertion of AMPARs in the postsynaptic mem- analyzed. We observed that after cLTD in shAKAP150 con- brane, the synaptic AMPARs endocytosis drives NMDA- ditions, further reduction of GluA1 surface levels was ob- dependent LTD (Lledo et al., 1998; Carroll et al., 1999; served (Fig. 6J,K; shAKAP150: 0.472 6 0.115; pp = 0.0141, Takumi et al., 1999; Shi et al., 2001). It has been previ- cLTD: 0.287 6 0.081; pp = 0.0030 and shAKAP150 1 cLTD: ously described that the phosphorylated state of 0.152 6 0.053; pp = 0.0012 vs basal). These results clearly AMPARs modulates their insertion or removal from syn- show that silencing endogenous AKAP150 decreases Ser- aptic membranes (Lee et al., 1998, 2000, 2003, 2010; 845 phosphorylation, GluA1 levels and consequently re- Chung et al., 2000; Esteban et al., 2003; Boehm et al., duces GluA1-containing AMPARs recruitment to synapses 2006; Oh et al., 2006; Delgado et al., 2007; Lu et al., 2010; and surface expression. Remaining levels of AKAP150, Coultrap et al., 2014). Among AMPARs subunits, it is be- under shAKAP150 conditions, seem to be enough to par- lieved that GluA1 trafficking in and out the synapse de- tially keep anchored AMPAR to the membrane and, after pends on neuronal activity, while GluA2 subunit seems to cLTD, its degradation still contribute to increase GuA1 be constitutively regulated in a neuronal activity- March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 11 of 19 independent manner (Passafaro et al., 2001; Shi et al., A cLTD 2001). Phosphorylation status of GluA1 on Ser-845 seems + to determine synaptic localization of GluA1 AMPARs dur- Basal cLTD MG132 MG132 ing LTP or LTD processes. In this way, PKA-mediated Ser- 250 kDa 845 phosphorylation facilitates AMPARs trafficking to the 150 kDa synapses favoring LTP (Oh et al., 2006; Makino et al., Ub 2011) whereas Ser-845 dephosphorylation by CaN has 100 kDa been shown to promote AMPARs endocytosis associated 75 kDa to hippocampal LTD (Mulkey et al., 1994; Beattie et al., 2000; Lee et al., 2000). 37 kDa GAPDH Several studies have reported that the scaffolding pro- tein AKAP79/150 has a prominent role in the reciprocal modulation of PKA and CaN activities at the synapse and B therefore in the regulation of AMPARs cycling in and out Ub conjugation/GAPDH Fold change (vs Basal) 2.0 the synapses during LTP and LTD (Tavalin et al., 2002; Lu 1.5 # et al., 2008; Jurado et al., 2010). AKAP79/150 forms a 1.0 complex with PKA and CaN (Bauman et al., 2004; Jurado * et al., 2010) that binds the MAGUK scaffold proteins 0.5 PSD95 and SAP97, linking the AKAP79/150 complex to 0.0 AMPARs (Colledge et al., 2000; Bhattacharyya et al., Basal cLTD cLTD MG132 + 2009; Robertson et al., 2009). Thus, a disruption in the in- MG132 teraction between these proteins in the PSD could lead to an alteration in plasticity of glutamatergic synapses. For C cLTD instance, PSD95 knock-down by shRNA or inhibition of + Control PSD95 binding to AKAP150, blocks NMDA-dependent Basal cLTD MG132 MG132 IgG Input LTD endocytosis of synaptic AMPARs in cultured neurons 150 kDa AKAP150 (Bhattacharyya et al., 2009). In the present work, we stud- ied the eventual changes in AKAP150 levels caused by 150 kDa Ub cLTD and whether these changes are involved in synaptic 37 kDa AMPARs endocytosis triggered by cLTD in neuronal GAPDH cultures. Previous results suggested that during LTD, AKAP79/ IP: AKAP150 150, together with PKA, translocate away from PSD95 150 kDa IB: AKAP150 and synaptic spines (Gomez et al., 2002; Smith et al., 2006). This translocation is believed to favor CaN activity promoting the dephosphorylation of postsynaptic sub- IB: AKAP150 shifted strates, such as GluA1 and leading to synaptic AMPARs 150 kDa (overexposed) endocytosis (Smith et al., 2006). Our results show that be- sides AKAP79/150 redistribution away from the synapses, IB: Ub cLTD produces an additional and significant proteasome- 150 kDa dependent decrease in total AKAP150 levels that it is ac- companied by a reduction in surface and total GluA1 lev- D els. Furthermore, our data also show that the addition of shAKAP150 does not blocks NMDA-dependent LTD en- Ub conjugation/AKAP150 Fold change (vs Basal) 2.5 * docytosis of synaptic AMPAR, indicating a differential role 2.0 of AKAP150, PSD95, and its interaction on AMPAR 1.5 1.0 continued 0.5 ubiquitination after cLTD. Lysates from neurons after cLTD 0.0 were immunoprecipitated with anti-AKAP150 or control rabbit Basal cLTD cLTD MG132 IgG (control IgG) and blotted with anti-AKAP150 and anti-ubiq- + MG132 uitin (Ub). The three top panels show representative blot images (input) for AKAP150 and 150-kDa ubiquitinated proteins (Ub) re- Figure 4. cLTD-mediated AKAP150 degradation requires lated to GAPDH (bottom panel of three) used as a loading con- AKAP150 ubiquitination. A, Representative blot images showing trol. The three bottom panels show representative blot images the ubiquitination (Ub) of high molecular weight protein after of immunoprecipitated Ub-AKAP150 protein (IP:AKAP150; in- cLTD (top panel) related to GAPDH (bottom panel) used as a cluding overexposed panel to observe AKAP150-shifted) and loading control. B, Quantification of total ubiquitination signal in total ubiquitination levels (Ub). D, Quantification of ubiquiti- response to cLTD in presence of MG132 (10 mM) compared nated-AKAP150 levels related to total AKAP150 signal in re- with cLTD (n = 6, pp = 0.0355 vs basal and #p = 0.0183 vs cLTD). sponse to cLTD compared with basal (n = 3 for each group, C, Representative blot images showing an increase in AKAP150 pp = 0.0026 vs basal). Bars represent mean 6 SEM. March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 12 of 19 A mature cultured neurons 30 min 5 or 10min 55 or 50 min 5 or 10min 55 or 50 min ACSF ACSF(Ca2+) ACSF ACSF(Ca2+) ACSF Exp procedures (Ca2+,Mg2+) (Ca2+,Mg2+) (Ca2+,Mg2+) +NMDA 50PM +Forskolin 50PM or +Rolipram 0,1PM (cLTP) ACSF(Ca2+) or +Forskolin 50PM ACSF(Ca2+) +Rolipram 0,1PM (cLTP) +NMDA 50PM cLTD or cLTP 60min cLTP or cLTD 120min B D Basal cLTD cLTP TP D in in + cL + cLT 120m 120m l sa LTP TD LTD LTP TP TD 100kDa sGluA1 Ba c cL c c cL cL 100kDa tGluA1 150kDa AKAP150 37kDa GAPDH 37kDa GAPDH C E 1.5 Fold change (vs Basal) 1.5 * Fold change (vs Basal) AKAP150/GAPDH sGluA1/tGluA1 1.0 1.0 * * 0.5 * * 0.5 * 0.0 0.0 l n n sa TP LTD LTP LTD mi mi Basal cLTD cLTP Ba cL c c c 20 20 TD + TP + TP 1 TD 1 cL cL cL cL F G 1.5 Basal cLTD cLTP cLTD + cLTP AKAP150 intensity (Relative to Basal) 1.0 * # 0.5 0.0 l sa LTD LTP LTP Ba c c +c TD cL Figure 5. cLTP neither rescue nor prevents cLTD-mediated reduction of AKAP150 levels. A, Hippocampal neurons were cultured during 17–21 DIV. cLTP was induced with forskolin/rolipram (50 mM/0.1 mM) for 60 min (10-min stimulation plus 50-min incubation in ACSF buffer) before or after cLTD as indicated in experimental design scheme. B, Representative blot images showing increased levels of surface GluA1 after cLTP treatment (sGluA1; ;110-kDa band, top panel) related to total GluA1 (tGluA1; ;110-kDa band, middle panel). GAPDH was used as a loading control (;36-kDa band, bottom panel). C, Quantification of surface GluA1 changes in response to cLTD and cLTP compared with basal condition (n = 4–9, pp , 0.0001 for cLTD and pp = 0.0009 for cLTP vs basal). D, Representative blot images showing levels of AKAP150 in response to cLTP before and after cLTD treatment (top panel) and GAPDH (bottom panel) as a loading control. E, Quantification of AKAP150 changes in response to cLTP before and after cLTD treat- ment compared with basal (n = 3–9, pp , 0.0001 vs basal). F, Dendritic staining for AKAP150 after cLTD with or without cLTP. Representative confocal images showing AKAP150 (gray). Separated panels from the boxed regions have been magnified for den- drite clarity (bottom panels). cLTD reduced AKAP150 staining. The presence of cLTP (50 mM; 0.1 mM) does not rescue AKAP150 lev- els. G, Quantification of AKAP150 intensity at dendrites normalized to basal condition. cLTD reduced AKAP150 dendritic intensity and cLTP induction does not prevent this reduction (n . 25 dendrites of different neurons from three independent cultures, pp = 0.0004 vs basal; #p = 0.0017 vs basal). Scale bars = 20 mm (F, top panels) and 5 mm (F, bottom panels). Bars represent mean 6 SEM. March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 13 of 19 A B C 1.5 SAP97 PSD95 Protein levels normalized 0 0 Fold change (vs Basal) 15 15 a l K AP KAP s pLVTHM pWPI Ba shA A 1.0 ov Basal ø sh ø ov 150kDa AKAP150 150kDa AKAP150 150kDa 0.5 SAP97 42kDa E-actin 100kDa PSD95 0.0 37kDa GAPDH Basal sh ov AKAP150 D E F G 5 0 5 0 P1 1.5 P1 GluA1 1.5 GluA1 KA Protein levels normalized KA Protein levels normalized A pS845-GluA1 A pS845-GluA1 ø sh ø ov Fold change (vs ø) Fold change (vs ø) 1.0 1.0 150kDa AKAP150 150kDa AKAP150 # 100kDa pS845-GluA1 100kDa pS845-GluA1 0.5 * 0.5 100kDa GluA1 100kDa GluA1 37kDa GAPDH 0.0 42kDa E-actin 0.0 ø shAKAP150 ø ovAKAP150 H I Basal ø shAKAP150 1.5 Dendritic GluA1 Synaptic GluA1 GFP (Relative to Basal) GluA1 intensity 1.0 GluA1 * # 0.5 PSD95 VGlut1 0.0 Basal ø shAKAP150 Merge J K L D 1.5 1.5 cLT 0+ Fold Change (vs Basal) Fold Change (vs Basal) 15 50 P1 AKAP150/GAPDH P D l A cLT A sGluA1/tGluA1 sa LTD hAK AK Ba c s ø+ ø sh 1.0 1.0 100kDa sGluA1 * * * 100kDa tGluA1 0.5 0.5 * * * 150kDa AKAP150 * * 37kDa 0.0 0.0 GAPDH AP l + D + D TD AP l + D + D TD ø sa sa ø ø LT ø LT AP cLT AP cLT AK Ba Ba AK cL cL c c sh sh AK AK sh sh Figure 6. Silencing endogenous AKAP150 reduces AMPARs-GluA1 subunit levels. A, AKAP150 was silenced (sh or shAKAP150) or overexpressed (ov or ovAKAP150) in neuronal cultures after transduction with lentiviral vectors. Representative blot images showing the efficiency of shAKAP150 or ovAKAP150 transduction in primary neurons. Empty lentiviral vector (Ø; pLVTHM or pWPI) was used March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 14 of 19 continued as a control. B, Silencing AKAP150 does not affect related scaffold proteins like PSD95 (;95-kDa band) or SAP97 (;140-kDa band). Representative blot images showing levels of AKAP150 (top panel; as a control of silencing or overexpressing), PSD95 and SAP97 when endogenous AKAP150 was knocked down (shAKAP150) or overexpressed (ovAKAP150). GAPDH (bottom panel) was used as a loading control. C, Quantification of PSD95 or SAP97 levels when AKAP150 was silenced (shAKAP150) or overexpressed (overAKAP150) normalized to basal (n = 4). D, Endogenous AKAP150 silencing reduces S845-GluA1 phosphorylation and total GluA1 protein. Representative blot images showing AKAP150 levels (top panel; as a control of silencing), phosphorylated GluA1 (pS845-GluA1), total GluA1 and GAPDH (bottom panel; loading control). E, Quantification in pS845-GluA1 and GluA1 levels in re- sponse to AKAP150 knock-down compared with basal (n = 3, pp = 0.0057 vs basal; #p = 0.0070 vs basal). F, AKAP150 overexpres- sion does not modify S845-GluA1 phosphorylation and total GluA1 protein levels. Representative blot images showing AKAP150 (top panel; as a control of overexpression), phosphorylated GluA1 (pS845-GluA1), total GluA1 and GAPDH (bottom panel; loading control) levels. G, Quantification of pS845-GluA1 and GluA1 in response to AKAP150 overexpression compared with basal (n . 3). H, Representative confocal images of dendrites show GluA1 (magenta), presynaptic VGlut1 (gray), and postsynaptic PSD95 (blue) levels after knocking down AKAP150 compared with basal condition (Ø; empty vector). GFP expression (green) is used as a control of transduction. Down-regulation of endogenous AKAP150 reduced dendritic and synaptic GluA1 intensity. Bottom panels in H show the merge for GFP/GluA1/PSD95/VGlut1. I, Quantification of GluA1 (GluA1) intensity normalized to basal. AKAP150 knock- down expression reduced dendritic (o) and synaptic () GluA1 (n . 30 dendrites of different neurons from five independent cultures, pp , 0.0001 vs basal; #p , 0.0001 vs basal). J, Surface GluA1 levels on cLTD in presence of shAKAP150. Representative blot im- ages showing decreased levels of surface GluA1 after cLTD in presence of shAKAP150 (sGluA1; ;110-kDa band, top panel) related to total GluA1 (tGluA1; ;110-kDa band, middle panel). GAPDH was used as a loading control (;36-kDa band, bottom panel). K, Quantification of surface GluA1 changes in response to cLTD in presence of shAKAP150 compared with basal condition (n = 3, pp = 0.0030 for cLTD, pp = 0.0012 for shAKAP1501cLTD, pp = 0.0018 for empty1cLTD and pp = 0.0141 for shAKAP150 vs basal). L, Quantification of AKAP150 in response to cLTD in presence of shAKAP150 compared with basal condition (n = 3, pp = 0.0080 for cLTD, pp , 0.0001 for shAKAP1501cLTD, pp = 0.0080 for empty1cLTD and pp = 0.0003 for shAKAP150 vs basal). Scale bars = 5 mm. Bars represent mean 6 SEM. endocytosis in LTD (Bhattacharyya et al., 2009). Moreover, possibility at different time points. Based on these data, we the maintenance of AKAP150 levels by overexpression is propose that cLTD has a double effect on AKAP79/150 sufficient to prevent GluA1-AMPARs endocytosis associ- that is responsible for AMPARs dephosphorylation and ated to cLTD in cultured neurons. A causative relationship endocytosis: a translocation of AKAP79/150 out of the syn- between AKAP150 levels decrease and AMPARs endocy- apses that precedes its synaptic or extra-synaptic degra- tosis is also supported by our data using shAKAP150 to- dation by proteasome (Fig. 8). Since, cLTD would prevent gether with AKAP150 overexpression; whereas AKAP150 cLTP induction depending on AKAP150 levels, it is feasible silencing produced the expected dephosphorylation of to hypothesize that the intensity of cLTD stimulus could de- GluA1 Ser-845 and the endocytosis of GluA1-AMPARs, termine the extent of AKAP150 degradation and the recov- overexpression of AKAP150 increased GluA1 Ser-845 ery time necessary for cLTP induction, although additional phosphorylation and surface GluA1 levels. Precisely, over- experiments are needed. expression of AKAP150 not only drives more AMPAR to Our results indicating that AKAP150 levels influence the membrane but can also block cLTD-mediated GluA1- surface expression and phosphorylation of AMPARs, sup- AMPARs endocytosis, indicating that the presence of port previous reports showing that the expression levels AKAP150 is increasing retention/stabilization at synaptic of other components of the PSD complex regulate synap- locations. Further work remains to be done to address tic distribution and function of AMPARs. For instance, it whether changes in AKAP150 could, and how, also affect has been described that overexpression of PSD95 in cul- other scaffolding proteins. Moreover, since LTD triggers tured neurons increases synaptic AMPARs delivery (El- AKAP150 degradation, AKAP150 plays an important role Husseini et al., 2000). By contrast, shRNA-mediated regulating LTP processes (Sanderson and Dell’Acqua, knock-down of PSD95 decreases synaptic AMPARs and 2011) and LTP is associated with up-regulation of enhances LTD (Elias et al., 2006; Schlüter et al., 2006; AKAP150 gene expression (Génin et al., 2003), we postu- Ehrlich et al., 2007; Bhattacharyya et al., 2009). Similar re- late that AKAP150 degradation would impair subsequent sults have been observed with SAP97 (Rumbaugh et al., LTP. This is supported by our data showing that cLTD pre- 2003; Schlüter et al., 2006; Howard et al., 2010). vent cLTP induction due to a failure in normal AKAP150 Interestingly, we have also observed that PSD95 and levels recovery. Surprisingly, prior cLTP induction as a SAP97 (unpublished data) levels decrease after cLTD in- strategy to retain AKAP150 levels and block cLTD effect on duction. However, shRNA-mediated silencing of endoge- GluA1-AMPAR was unproductive according to our data. It nous AKAP150 did not change PSD95 and SAP97 levels is possible that cLTP induction 1 h before cLTD is not in cultured neurons, suggesting that although cLTD trig- enough to protect AKAP150 from cLTD. However, from our gers a coordinated down-regulation of these proteins, an images it seems that there may be more AKAP150 concen- indirect cross-regulation mechanism may occur between trated at the plasma membrane after cLTP induction, with them. bigger and more intense clusters than in basal conditions It has been reported a key role of CaN in the modulation in agreement with the notion that LTP recruits more of AMPARs trafficking in and out the synapses mediated AKAP150 to the plasma membrane (Purkey et al., 2018). by its capacity to dephosphorylate AMPARs and other Additional experiments are needed to explore this proteins in the PSD (Smith et al., 2006; Jurado et al., March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 15 of 19 A Basal pLVTHM-ø shAKAP150 pWPI-ø ovAKAP150 AKAP150 sGluA1 Merge B C D 1.5 # Fold change (vs Basal) 1.5 LT D # +c (Relative to Basal) sGluA1/tGluA1 sGluA1 intensity 1 50 150 1.0 l AP AP -ø 1.0 sa LTD vAK vAK WPI * Ba c o o p * 100kDa sGluA1 0.5 0.5 100kDa tGluA1 0.0 0.0 37kDa tGAPDH l l sa -ø 50 I-ø 50 sa LTD LTD 150 PI-ø Ba THM AP1 pWP AP1 Ba c +c KAP pW V K K 50 pL shA ov A A P1 ovA AK ov E F Basal cLTD cLTD + ovAKAP150 ovAKAP150 1.5 & GFP # (Relative to Basal) sGluA1 intensity 1.0 * 0.5 Surface GluA1 0.0 l sa LTD 150 150 Ba c AP AP K K o vA ovA T D+ cL Figure 7. Overexpression of AKAP150 blocked cLTD-mediated GluA1-AMPARs endocytosis. A, Representative confocal images of dendrites showing AKAP150 silencing (shAKAP150) and overexpressing (ovAKAP150) effect on surface GluA1 expression. AKAP150 (green) and surface GluA1 (magenta). Bottom panels, Merge for AKAP150/sGluA1. Knocking down endogenous AKAP150 reduced and AKAP150 overexpression increased surface GluA1 intensity. Empty lentiviral vector (Ø; pLVTHM or pWPI) was used as a control. Scale bar = 5 mm. B, Quantification of surface GluA1 intensity (sGluA1) after silencing or overexpressing AKAP150 (n . 12 dendrites of different neurons from four independent cultures, pp , 0.0001, #p = 0.0419 vs basal). C, cLTD was induced in primary neurons overexpressing AKAP150 and surface proteins were biotinylated and pulled-down. Representative blot images show sur- face GluA1 (top panel; sGluA1), total GluA1 (middle panel; tGluA1) and GAPDH levels (bottom panel; loading control). D, Quantification of surface GluA1 intensity (sGluA1) in response to cLTD without or with overexpression of AKAP150 compared with basal (n = 6, pp = 0.0173 vs basal; #p = 0.0185 vs cLTD). E, Representative confocal images show surface GluA1 and GFP staining on primary neurons overexpressing AKAP150 after cLTD induction. Separated panels from the boxed regions have been magnified for dendrite clarity (bottom panels). AKAP150 overexpression prevents cLTD-dependent surface GluA1 reduction compared with control cells expressing GFP. F, Quantification of surface GluA1 intensity (sGluA1) in response to cLTD without or with overexpres- sion of AKAP150 (n = 10 dendrites from 10 independent cultures, pp , 0.0001 vs basal; #p = 0.0016 vs cLTD; &p = 0.0285 vs basal). Scale bars = 20 mm (top panels) and 5 mm (bottom panels). Bars represent mean 6 SEM. March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 16 of 19 Figure 8. Model for LTD-mediated AKAP150 degradation through the proteasome system. Under basal conditions, a pool of palmitoylated AKAP150 is anchoring and regulating PKA and CaN function in dendritic spines. After LTD induction, a weak and extended Ca 21 influx depalmitoylate and remove AKAP150 from spines. This removal promotes CaN-mediated dephos- phorylation and consequent endocytosis of GluA1-AMPARs. During this process AKAP150 is ubiquitinated and degraded via proteasome. The presence of 20S proteasome inhibitor MG132 blocks not only LTD-mediated AKAP150 degradation but also its removal from spines. Maintenance of AKAP150 levels is sufficient to prevent LTD-induced GluA1-AMPARs endocyto- sis suggesting that AKAP150 degradation by the proteasome could be an important factor in the regulation of surface GluA1-AMPARs at spines. 2010). Here, we have reported that CaN activation is not of cLTD on ubiquitination-proteasome system and synap- necessary for cLTD-mediated AKAP150 degradation by tic function. Previous data from Citri and colleagues proteasome. Therefore, CaN would be able to favor cLTD showed that cLTD-mediated AMPAR endocytosis re- by enhancing synaptic AMPARs endocytosis but it would quires ubiquitination but not proteasomal degradation not participate in the induction of AKAP79/150 degrada- (Citri et al., 2009), while our results suggest that both tion. In this context, It has been described that activity-de- ubiquitin and proteasome processes are involved in pendent palmitoylation of AKAP79/150 is necessary for cLTD-mediated AKAP150 degradation. AKAP150 seems synaptic targeting of AKAP79/150 and cLTP induction to be part of a complex of synaptic proteins regulated by (Keith et al., 2012). In addition, cLTD activates CaMKII proteasome system in the molecular mechanisms of LTD. regulating depalmitoylation and synaptic removal of Here, we also find involved PSD95 and GluA1 (data not AKAP79/150 (Woolfrey et al., 2018) being feasibly a pre- shown) in proteasomal-mediated degradation indicating requisite for posterior degradation in the proteasome. that it could be a more general effect on synaptic proteins. At present, the mechanism through which cLTD indu- Furthermore, restoration of AKAP150 levels by overex- ces proteasome-dependent AKAP150 degradation is un- pression was able to rescue cLTD-dependent GluA1- known. However, It is well known that AKAP150 is highly AMPAR endocytosis. This finding highlights the impor- ubiquitinated and can be regulated by ubiquitin-protea- tance of AKAP150 levels in excitatory synapses along some system depending on synaptic activity (Ehlers with their interaction to other scaffolding proteins through 2003; Jia et al., 2008). Moreover, it has recently been de- the MAGUK binding domain (Robertson et al., 2009). scribed that Nedd4 ubiquitinates (Schwarz et al., 2010; Nevertheless, further work should be addressed interfer- Lin et al., 2011) and USP46 deubiquitinates (Huo et al., ing selectively with AKAP150 cLTD-induced ubiquitina- 2015) AMPARs. Some controversy exists about the role tion and to prevent aspects of LTD expression or whether March/April 2020, 7(2) ENEURO.0218-19.2020 eNeuro.org Research Article: New Research 17 of 19 Nedd4 and/or USP46 proteins also regulate AKAP79/150 Ehlers MD (2000) Reinsertion or degradation of AMPA receptors de- ubiquitination and their activities under cLTD condition to termined by activity-dependent endocytic sorting. Neuron 28:511– further support an AKAP150-degradation centric model in 525. Ehlers MD (2003) Activity level controls postsynaptic composition control of AMPAR endocytosis in LTD. and signaling via the ubiquitin-proteasome system. 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