Antibodies and reagents
For immunocytochemical staining (ICC) and Western blotting (WB) the following primary antibodies were used: mouse antibodies against flag (ICC and WB 1:1000, Sigma-Aldrich, F1804), SNAP25 (WB 1:1000, Synaptic Systems, 111 011), β-actin (WB 1:1000, Sigma), PSMA7 (ICC and WB 1:1000, MCP34; PW8120), PSMA5 (WB 1:1000, MCP196), PSMA6 (WB 1:1000, MCP20; PW8100), PSMA3 (WB 1:1000, MCP72; PW8110), PSMB7 (WB 1:1000, MCP168; PW8145), PSMB3 (WB 1:1000, MCP102), PSMB4 (WB 1:1000, MCP205; PW8135),
PSMC5 (WB 1:1000, p45-110) and FK2 (ICC and WB 1:1000; PW8810). All antibodies against proteasome subunits were purchased from Enzo Life Sciences. Rabbit antibodies against RIM 1,2 (ICC 1:500, Synaptic System, # 140203), Munc13-1(ICC 1:1000, Synaptic System, # 126103), β- tubulin III (WB 1:1000, Sigma-Aldrich), PSMG1/PAC1 (WB 1:1000, Cell Signaling, #13378), PSMC4 (WB 1:1000, Bethyl Labolatories Inc), GFP (WB 1:1000, Abcam, ab 6556), RFP (ICC and WB 1:1000, Rockland Immunochemicals Inc.). Secondary antibodies from goat or donkey coupled with Cy3 (ICC 1:1000) or peroxidase- (1:10000) were obtained from Jackson ImmunoResearch Laboratories. MG132 and epoxomicin were purchased from Enzo Life Sciences.
Cells and tissues used in the study were obtained from Wistar rats and bassoon gene trap (BsnGT)  mouse strains backcrossed to C57BL/6 N. BsnGT mice were obtained from Omnibank ES cell line OST486029 by Lexicon Pharmaceuticals, Inc. (The Woodlands, TX). All experiments were performed in accordance with the European Committees Council Directive (86/609/EEC) and approved by the local animal care committee (Landesverwaltungsamt Sachsen-Anhalt, AZ: 42502-2-1303 LIN).
N-terminally truncated PSMB4 covering nucleotides (nt) 118-820 and amino acids (aa) 32- 264, 26 kDa, ΔNPSMB4: nt 161–820, aa 45–264, 24 kDa as well as ΔNΔC-PSMB4: nt 160–764, aa 45–246, 22 kDa of rat Psmb4 (NM_031629.2, NP_113817.2) were produced by PCR using the pACT2 rat brain Matchmaker cDNA library (Clontech Laboratories, Inc.) as a template with extended primers, adding EcoRI and XhoI restriction sites at the 5′ and 3′ ends of the fragments, respectively. The introduced restriction sites were used for in-frame cloning of the fragments into the pCMV-Tag2B and pCMV-Tag3B vectors (Agilent Technologies). mRFP-PSMB4 was created by insertion of N-terminally truncated PSMB4 (aa 32–264) into mRFP-C2 vector that was generated as previously described  RFP-CtermPSMB4 (nt 767–820, aa 247–264, 2 kDa) construct was created by insertion of annealed synthetic oligonucleotides into pmRFP-C2 vector. Bsn fragments Bsn4 (aa 2715–3013, 33 kDa) and Bsn1 (aa 1692–3263, 169 kDa)  as well as Bsn2 (aa 1653–2082, 47 kDa)  were described previously. pEGFP-Bsn2 and pEGFP-Bsn4 were subcloned into RFP-C2 vector as well as FUGW lentiviral transfer vector . Bsn5 (aa 2715–2820 of NP_062019.2, 11 kDa) was created from pEGFP-Bsn4 as a template using PCR with extended primers. EcoRI and BamHI sites were introduced at 5′ and 3′ ends, respectively, and used for in-frame cloning of the fragment into the pBS-SK (+) and pEGFP-C2 vector. Bsn3 and Bsn6 were generated using PCR on rat cDNA of Bsn as a template. EcoRI and XhoI restriction sites were added at the 5′ and 3′ ends of the fragments, respectively. Bsn constructs Bsn7 (aa 1653–1878, 24 kDa), Bsn8 (aa 1964–2087, 14 kDa), Bsn9 (aa 1653–1963, 33 kDa) and Bsn10 (aa 2013–2087, 8 kDa) were generated using PCR on Bsn2 as a template, with extended primers to add EcoRI and XhoI (Bsn9, Bsn7, and Bsn8) or EcoRV and XhoI (Bsn10) restriction sites at the 5′ and 3′ ends of the fragments, which were used for in-frame cloning of fragments into pGADT7, pCMV-3B or pBS-SK(+) vectors. All the fragments were also inserted into pEGFP-C2 vector. All constructs were verified by sequencing. HA-Ubiquitin (#18712) , Ub-G76V-YFP (#11949), Ub-R-YFP (#11948),), CD3delta YFP (#11951)  and CMV-d2EGFP-empty (#26164) , were purchased from Addgene.
Primary neuronal cultures
Primary cultures of hippocampal and cortical neurons from P0 to P1 BsnGT mice and their wild-type siblings were prepared as described previously . Briefly, after trypsin treatment of the hippocampus and mechanical trituration, cells were plated in densities of 3.5*104 cells per coverslip (18 mm diameter). 1 h after plating, coverslips were transferred into dishes containing 60–70% confluent monolayer of astrocytes and Neurobasal A medium supplemented with B27, 1 mM sodium pyruvate, 4 mM Glutamax and antibiotics (100U/ml penicillin, 100ug/ml streptomycin). At 1 and 3 days in vitro (DIV) AraC (Sigma Aldrich) was added to the cells (0,6 µM each time) to reach a final concentration of 1.2 µM. For cortical culture preparation, the brains were dissected, and meninges removed. Tissue was treated with 0.25% trypsin for 20 min and triturated in the presence of 0.1% DNAse. 2*106 cells were plated in DMEM with 10% FCS (fetal calf serum), 1 mM glutamine and antibiotics (100U/ml penicillin, 100 µg/ml streptomycin, Life Technologies) into poly-d-lysine coated 75 cm2 flasks. After 7–8 h the medium was changed to Neurobasal A supplemented with B27, 1 mM sodium pyruvate, 4 mM Glutamax and antibiotics (100 U/ml penicillin, 10 μg/ml streptomycin). At 4 DIV 0.6 μM AraC was added.
Lentiviral particles production and infection of neuronal cells
Lentiviral particles were generated in HEK293T cells (ATTC, Manassas, VA, USA) using FUGW-based transfer, psPAX2 packaging and pVSVG pseudotyping vectors . HEK293T cells were grown in media containing 10% FCS to 60% confluence in the 75 cm2 flasks. Cells were transfected with 20 µg of total DNA per flask using calcium phosphate method . Molar ratio of FUGW: psPAX2: pVSVG was 2:1:1. 6–8 h after transfection, medium was changed to 10 ml production medium containing Neurobasal A supplemented with antibiotics, 1 mM sodium pyruvate (Life Technologies), B27 and 1 mM Glutamax (Life Technologies). 48 h after transfection, virus-containing media was collected and cleared from large cellular debris by centrifugation for 20 min at 2000g. Virus-containing supernatant was aliquoted and stored at − 80 °C. For infection of hippocampal and cortical cultures, viral particles were applied overnight at 4 DIV. Neurons were stained or collected for proteasome activity assays at 14–18 DIV.
Immunoprecipitation and Western blotting
HEK293T cells were transfected using the standard calcium phosphate method . One day after transfection, cells were lysed in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, complemented with complete protease inhibitor (Roche) for 10 min on ice and cleared by centrifugation for 10 min at 15,000 g. Co-immunoprecipitation was performed using MicroMACS anti-GFP Microbeads and Micro Columns (Miltenyi Biotec) according to the manufacturer’s instructions, except for the washing steps where lysis buffer was used. Bound proteins were eluted in the SDS-loading buffer, heated for 5 min at 95 °C and analyzed by immunoblotting. Briefly, the samples were separated on 5%-20% Tris- glycine gradient polyacrylamide gels and blotted onto PVDF membrane (Millipore) by wet electroblotting system (Hoefer). The membranes were incubated with indicated antibodies diluted in PBS containing either 5% BSA or 5% non-fat dry milk and supplemented with 0.1% Tween-20. Immunodetection was performed using Pierce ECL WB Substrate (Thermo Scientific) and ChemoCam Imager (Intas). Size markers in Western blot images are indicated in kDa.
Co-recruitment assay in COS7 cells
COS-7 cells grown on the glass coverslips were transfected using Polyfect reagent (QIAGEN) according to the manufacturer’s protocol. After 24 h, cells were fixed, blocked and stained as described elsewhere . Images were acquired with Zeiss Axio Imager A2 microscope with Cool Snap EZ camera and VisiView Software (Visitron Systems).
Mouse brain fractionation
For the preparation of subcellular fractions, 6–7 weeks mice were sacrificed by cervical fracture. Cortex and hippocampi were dissected and homogenized in the buffer containing 0.32 M sucrose, 5 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 1 mM DTT and 2 mM ATP, with a Potter glass-Teflon homogenizer using 12 strokes at 900 rpm. This and all the following procedures were carried out at 4 °C. The homogenate (H) was centrifuged at 1000g for 10 min to sediment nuclear fraction and cell debris (P1). The supernatant (S1) was collected and centrifuged at 10,000g for 15 min, yielding pellet (P2, membrane-enriched fraction) and supernatant (S2, cytosol). For isolation of synaptosomes (Syn), the P2 fraction was resuspended in a buffer containing 0.32 M sucrose, 5 mM Tris–HCl pH 8.0 and 1 mM ATP, laid atop a discontinuous sucrose gradient (0.8/1.0/1.2 M; 3 ml per step) and centrifuged for 2 h at 85,000g. The Syn fraction was collected from the 1.0/1.2 M sucrose interface.
Chymotrypsin, Caspase and Trypsin-like peptidase activity assay on lysates from HEK293T cells, cortical neurons, or brain samples
One day after transfection or 14–16 days after infection, HEK293T cells or mice cortical neurons, respectively, were lysed in 50 mM Tris–HCl, pH 7.5, 250 mM sucrose, 5 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.5 mM EDTA, 0.025% digitonin for 10 min at 4 °C. The lysate was cleared by centrifugation for 15 min at 20,000 g. Protein concentration was measured by Coomassie Plus Bradford Assay according to manufacturer instructions (Thermo Scientific). Protein concentration after subcellular brain fractionation was determined using the BCA kit (Pierce BSA). For the assessment of the proteasome activity from HEK239T cells or cortical mice cultures, equal amounts of total protein (5 μg/100 µl well) were incubated with the assay buffer (50 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 40 mM KCl, 1 mM DTT, 2 mM ATP) containing 100 μM N- Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) for chymotrypsin-like activity, 100 μM Z-LLE-7-Amino-4-methylcoumarin (Z-Leu-Leu-Glu-AMC) for Caspase-like activity and 100 μM Boc-LRR-7-Amino-4-methylcoumarin (Boc-Leu-Arg–Arg-AMC) for Trypsin-like activity (all fluorogenic peptides were purchased from Enzo Life Science). For determination of the chymotrypsin-like proteasome activity in different brain fractions, equal amounts of protein extracts (5 μg/100 µl well) were incubated with the assay buffer (0.5 mM EDTA, 50 mM Tris–HCl, pH 8) containing 40 μM Suc-LLVY-AMC . 30 min at 37 °C and the proteasome activity was recorded as a fluorescent signal of AMC release. Fluorescence (380 nm/440 nm excitation/emission) was detected using the Fluostar Omega microplate reader with appropriate fluorescence filters (BMG Labtech). The assays were performed in quintupled for HEK cells and in quadruplicate for cortical neurons. After background subtraction from each data point, the values were normalized to the proteasome activity of control-GFP transfected HEK cells or WT FUGW-GFP infected neurons and expressed in %. Treatment with 20 μM Epoxomicin for 30 min at 37 °C was used to verify the validity of the assay.
Native gel electrophoresis and in-gel activity assay (zymography)
To resolve proteasome complexes either HEK293T cell lysate (25 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.025% digitonin) or brain fraction lysate were subjected to the native gel electrophoresis. 50 μg of the brain fractions lysate or 20 µg of HEK293T cell lysate was separated on 3%-8% NuPAGE Tris–Acetate Mini Gels (Life Technologies). The gels were run at 4 °C, first at 150 V for 1.30 h, thereafter, the voltage was increased to 200 V for the next 2.30 h. The in-gel activity of the 26S proteasome was revealed by the incubation of the gel in the buffer (20 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP) containing 100 μM Suc-LLVY-AMC for 20 min at 37 °C. Proteasome activity was detected upon illumination with UV light (excitation 366 nm, emission 440/40 nm) using ChemoCam Imager (Intas). Fluorescence intensity was quantitated using ImageJ software.
Measurement of the UPS activity in HEK293T cells using fluorescent proteasome substrates
HEK293T cells were co-transfected at 70% confluency with an equimolar amount of each fluorescent UPS substrates together with RFP or RFP-tagged Bsn constructs using the jetPEI (Polyplus) transfection reagent according to the manufacturer´s instructions.48 h post-transfection, cells were resuspended, counted and plated at the density of 70,000 cells/well in 96-well poly-l-lysine-coated clear-bottom black well plates (Costar). YFP or GFP fluorescence was measured in a Clariostar microplate reader (BMG Labtech) (YFP: excitation 497-15; emission 540-20; GFP: excitation 470-15; emission 515-20) one day later. The background fluorescence (i.e. mean fluorescence of non-transfected cells) was subtracted from each raw data. Eight wells were quantified for each condition and all data were normalized to the control (RFP) and expressed in percentage.
Quantitative immunostaining, image acquisition and analysis
Cultured hippocampal neurons from newborn WT and BsnGT mice were fixed in 4% paraformaldehyde (PFA), 4% sucrose in PBS for 5 min and washed twice with PBS. Then, cells were permeabilized for 30 min with blocking solution (10% FCS, 0.1% glycine and 0.3% Triton X-100 in PBS). Primary antibodies were applied overnight at 4 °C. Afterwards, cells were washed four times with PBS (10 min each) and incubated with secondary antibodies for 1 h at room temperature. Both primary and secondary antibodies were diluted in PBS containing 3% FCS. Finally, coverslips were dipped in water and mounted on glass slices with FluorSave™ (Calbiochem). Images of stainings were acquired on a Zeiss Axio Imager A2 microscope with Cool Snap EZ camera (Visitron Systems) controlled by VisiView (Visitron Systems GmbH) software. For quantifications, settings of camera were applied identically to all coverslips quantified in one experiment. For each IF quantification in each experiment, images from at least two different coverslips (5–7 cells each) were acquired and quantified to avoid effects given by experimental variance. Unspecific background was removed using threshold subtraction in ImageJ software (NIH, http://rsb.info.nih.gov/ij/). In all experiments, synaptic puncta were defined semiautomatically by setting rectangular regions of interest (ROI) with dimensions of about 0.8 X 0.8 um around local intensity maxima in the channel with staining for RIMs, and Munc13-1 using OpenView software (written and kindly provided by N.E. Ziv . Mean IF intensities were measured in synaptic ROIs in all corresponding channels using the same software and normalized to the mean IF intensities of the control group for each of the experiments.
All the results of quantitative analyses are expressed as means ± standard errors of the mean (s.e.m) and in graphs, represented as boxes indicating the interquartile distance with median, whiskers minimum and maximum values with mean showed as +. Statistical analysis was done with Prism 8 software (GraphPad Software, Inc.), Test were used as indicated specifically for each experiment. The normal distribution of the data was verified before choosing the appropriate test. In all graphs, numbers within bars depict the number of independent values used for statistics. Statistical significance is marked as non-significant (ns) or with stars as follows: *p < 0.0332, ** or ##p < 0.0021, *** or ###p < 0.0002 ****p < 0.0001, in the graphs.