Animal care, diets and termination
C57BL/6N (Charles River, Lyon, France), BALB/c (Harlan Laboratories, Horst, the Netherlands) and C57BL/6N × BALB/c F2 hybrid mice were housed in a temperature controlled room (21 °C) with a 12:12 h light–dark cycle (dawn: 5.30–6.00 am, dusk: 5.30–6 pm) and controlled humidity (45–55%). They were checked daily had free access to water and regular chow diet (R3; Lactamin, Kimstad Sweden) containing 12% fat, 62% carbohydrates, and 26% protein (energy percentage), with a total energy content of 3 kcal/g. At termination, mice were euthanized under 5% isoflurane anesthesia and decapitated. Blood samples for hematology were collected by intra-cardiac puncture, tissues were isolated, directly transferred to dry ice, and kept at − 80 °C until biochemical analyses.
Generation of PA28αOE mice
A knock-in strategy was used to target the murine Rosa26 locus in order to generate mice carrying a murine PA28α overexpression cassette at this site. The targeting vector was built using homologous recombination in bacteria [51] and a C57 mouse BAC served as template for the extraction of Rosa26 homology arms. The targeting vector contained the CAG promoter [52] driving the expression of the coding region of murine PA28α and a rabbit β-globin poly (A) signal (CAG-PA28α-pA) and a neomycin phosphotransferase (Neo) selectable marker cassette. The PGK-gb2-neo cassette with CAG-PA28α-pA was inserted into a Rosa26 targeting vector comprised of a 1.5 kb 5′ and 5 kb 3′ homology arms of Rosa26, and a PGK-diphtheria toxin A (DTA) gene for negative selection (Fig. 1a). The Neo selectable marker cassette, which was flanked by loxp sites, was deleted in the germline of the chimeric mice generating the KI allele using a self-excising Neo strategy. After linearization, the targeting construct was electroporated into C57BL/6N mouse embryonic stem (ES) cells which were then grown in media containing G418 (200 μg/ml). Thus, the PA28αOE mouse line was established on a pure C57BL/6 genetic background. PCR screens and Targeted Locus Amplification (Cergentis, Utrecht, the Netherlands) analyses revealed clones that had undergone the desired homologous recombination event. Several of these clones were expanded and injected into Balb/cOlaHsd blastocysts to generate chimeric males which were then bred to C57BL/6JOlaHsd females and black-coated offspring were genotyped on both sides of the homology arms for correct integration into the Rosa26 locus.
SDS–PAGE and Western blot analysis
MEFs, brain sample containing frontal cortex and striatum, left ventricle of the heart, and hippocampi were lysed with a modified RIPA buffer (50 mM Na2HPO4 pH 7.8; 150 mM NaCl; 1% Nonidet P-40; 0.5% deoxycholate; 0.1% SDS; 1 mM DTPA; 1 mM pefablock). Cell debris was removed by centrifugation at 5000 g for 10 min and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Eye lenses were lysed by sonication (Branson Ultrasonic Corp., Danbury, CT, USA) in PBS [53]. Samples were prepared for SDS–PAGE as described [54], separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred onto a nitrocellulose membrane (Invitrogen, Bleiswjik, the Netherlands) and probed with rabbit mAb PA28α (#9643; Cell Signaling Technology, Inc., Leiden, the Netherlands), rabbit pAb PA28β (#2409), rabbit mAb GluA2 (#13607), rabbit pAb N-Cadherin (#4061), rabbit pAb GluA1 (#13185), rabbit pAb β5/PSMB5 (#11903), rabbit mAb Phospho-CaMKII Thr286 (#12716), rabbit mAb Phospho-CREB Ser133 (#9198), rabbit pAb polyubiquitin K48-linkage specific (ab190061; Abcam, Cambridge UK), goat pAb PSD-95 (ab12093), rabbit pAb GluA1 (ab31232), rabbit mAb Synaptophysin (ab32127), rabbit pAb Spinophilin (ab203275), rabbit mAb NeuN (ab177487), rabbit pAb Estrogen Receptor beta phospho S105 (ab62257), rabbit pAb Rpt2/S4 (ab3317), or rabbit pAb β5i/LMP7 (ab3329). IRDye 800CW-labelled goat anti-rabbit, 680CW-labelled goat anti-mouse, 800CW-labelled donkey anti-goat IgG antibodies (LI-COR Biosciences, Cambridge, UK) were used for detection and blots were analyzed with the Odyssey infrared imaging system and software (LI-COR Biosciences), except for lens samples for which HRP-conjugated secondary Ab was used and luminescence after ECL reaction was imaged using ImageQuant LAS 500 (GE Healthcare, Piscataway, NJ, USA). Blots were quantified using the ImageJ software. Equal total protein present of each sample on the membrane was confirmed using the Novex reversible membrane protein stain (IB7710, Invitrogen) according to manufacturer’s instructions.
RNA extraction and quantitative (qPCR) analysis
Total RNA was extracted using Stat60 (CS-502, Tel-Test Inc) as per manufacturer’s recommendations. cDNA was synthesized on 1 µg total RNA using the High-Capacity cDNA Reverse Transcription Kit (#4368814 Applied Biosystems, Thermo Fisher Scientific) according to manufacturer’s instructions. Synthesized cDNA was analyzed in triplicates by qPCR using iQTM SYBRH Green Supermix and the QuantStudio 7 Flex system (Applied Biosystems, Thermo Fisher Scientific). For primer sequences, see Additional file 14.
Study design of physiological and behavioral phenotypic profiling
10 Wildtype (WT) and 6 PA28αOE heterozygous female F2 hybrid littermates at the age of 6 months were subjected to a 2-month protocol of physiological and behavioral phenotypic profiling as outlined in Fig. 2a. The mice were housed 4 in each cage with 2 WT-cages, 1 PA28αOE-cage, and 1 mixed cage. The genotype was not indicated on the cage, and the animal number to genotype was not decoded until after data analysis. The animals were analyzed cage by cage in the following order: WT-cage 1, PA28αOE-cage, WT-cage 2, mixed cage. Shuttle box and zeromaze were performed in 3 rounds with 6 mice each round as follows: (1) 4 WT from WT-cage 1 and 2 PA28αOE from PA28αOE-cage, (2) 2 PA28αOE from PA28αOE-cage and 2 WT from WT-cage 2, (3) 2 WT from WT-cage 2 and 2 PA28αOE and 2 WT from mixed cage. Activity box was performed in 2 rounds with 8 mice each round as follows: (1) 4 WT from WT-cage 1 and 4 PA28αOE from PA28αOE-cage, (2) 4 WT from WT-cage 2 and 2 WT from WT-cage 2 and 2 PA28αOE and 2 WT from mixed cage. All animal experiments were carried out at 10–11 am, except activity box that was carried out at 10–12 am.
Activity box
Activity box is an open field activity-like test to study general activity, exploratory behavior, signs of anxiety, stress and depression [55]. The mice are three dimensionally recorded by infrared sensors built into the walls (8Lx8Bx8H) of a sound-proof opaque box (50 × 50 × 50 cm) with a low intensity lamp into the lid of the box (Kungsbacka mät och regler, Fjärrås, Sweden). The mice were placed in the middle of the box and recorded for 1 h in this novel environment. On the following day, they were recorded again in the—now considered—acquainted environment. The parameters recorded as events/5 min were horizontal activity, peripheral activity, rearing activity, peripheral rearing, rearing time, locomotion, and corner time.
Forced swim test
This test is performed to analyze mice for signs of depression [55, 56]. The assembly consists of a transparent plexiglas cylinder with 25 cm inner diameter and 60 cm in length with a grey, circular plastic platform hanging from wires on the outside of the cylinder, approximately 20 cm from the top (bespoke construction, AstraZeneca Gothenburg), and filled with room tempered (22 °C) water in level with the platform. A single mouse is placed on the water surface inside the cylinder and its behavior is monitored by a video camera placed directly above the cylinder for 6 min and 20 s, of which the last 4 min are used in calculation (MouseTracker analysis software).
Oral glucose tolerance test (OGTT)
Oral glucose tolerance test (OGTT) baseline measurements were obtained after 5 h of fasting, followed by oral glucose dosing (6.7 ml/kg). Insulin levels was measured with Ultra-sensitive mouse insulin ELISA kit (Crystal Chem, Zaandam, Netherlands) according to manufacturer’s instructions and glucose levels by AccuChek mobile blood glucose meter (Roche Diagnostics Scandinavia, Solna, Sweden) at baseline and after 15, 30, 60, 120 min from dosing.
Shuttle box passive avoidance test
Passive avoidance testing was performed using the shuttle box system (Accuscan Instruments Inc., Columbus, OH, USA). This test is used to study memory performance in mice and is carried out over 2 days [55, 57, 58]. The system consists of a cage centrally divided by a wall into two compartments, one of which has transparent walls (the bright compartment) while the other is covered from all sides with opaque walls (the dark compartment). Both chambers are equipped with sensors that determine the location of the mouse and the central wall has a mechanical sliding door that can be programmed to open or close. The cage floor is made of stainless steel grid, which can deliver a mild electric shock to the mouse upon certain stimuli. On the first day, a mouse is released into the well-lit compartment and tends to migrate to the dark compartment when the central door opens (30 s after mouse entry). Upon entry to the dark compartment, the central door closes and the mouse is exposed to a mild electric shock (0.3 mA). Intensity of pain response was monitored. All mice responded to the electric shock by a vocal response (“beep”) and a jump, indicating similar strength of discomfort. On the second day, the mouse is released as before into the well-lit compartment and when the central door opens, may or may not enter the dark compartment. The time taken to enter the dark compartment is recorded on both days and a longer interval or no entry on the second day indicates memory response. Maximum assay time is 300 s each day.
Elevated zeromaze monitoring system
The elevated zeromaze system (Accuscan Instruments Inc.) was used to study anxiety-related behavior [55, 59, 60]. The maze is made up of a circular Perspex platform, elevated 75 cm above the floor, 5 cm wide and 40 cm inner diameter, equally divided into four quadrants, of which two quadrants on opposite sides of the platform are closed by 30 cm high Perspex transparent walls with photocell transceivers, while the other two quadrants are open and bordered by a Perspex lip (0.5 cm high), a security and tactile guide on the open quadrants. During testing, a mouse is placed at the entrance of a closed quadrant and monitored for 5 min. Activity in closed arm, latency to enter open arm, and time spent in open and closed arm are the parameters analyzed.
Body composition and core temperature
Core body temperature of the mice was obtained with a rectal probe thermometer (ELFA AB, Sweden). Under 2% isoflurane sedation the mice were analyzed by dual energy X-ray absorptiometry (DEXA) using Lunar PIXImus Densitometer (GE Medical Systems, Madison, WI, USA) to determine body fat (g), body fat (%), lean body mass (g), and total BMD (g/cm2) [55].
Immunoprofiling of peripheral blood
Blood samples for hematology (in EDTA tubes, Microvette CB300, Sarstedt, Nürnbrecht, Germany) were collected from the left atrium of the heart under isoflurane anesthesia, prior to necropsy. Leucocytes and erythrocytes were isolated by centrifugation and stained with 1:50 dilutions of MS CD45 HRZN V500 mAb (#561487; BD Diagnostics, Stockholm, Sweden), MS F4/80 PE T45-2342 (#565410), MS CD4 PERCP mAb (#561090), MS CD19 APC mAb (#561738), CD8 APC-Cy7 mAb (#561967), NK1.1 FITC mAb (#553164), and CD3e conjugated to BD Horizon V450 (#560804). Erythrocytes were lysed with BD FACS lysis buffer and analyzed using flow cytometry (FACS Fortessa, BD Bioscience, Stockholm, Sweden) with appropriate filter settings, gating on live cells.
Blood serum preparation and β-estradiol detection
Blood samples for blood serum preparation were collected from the left atrium of the heart under isoflurane anesthesia, prior to necropsy, incubated at room temperature for 30–45 min, and coagulates were removed by centrifugation. Relative β-estradiol serum levels were detected by the Mouse/Rat Estradiol ELISA-Kit (SKU: ES180S-100, Calbiotech, Spring Valley, CA USA) according to manufacturer’s instructions.
Cell culture and IFN-γ treatment of embryonic fibroblasts
MEFs from C57BL/6N females that had been mated with C57BL/6N PA28αOE heterozygote males, were isolated at E13.5 as described [61], with the following exceptions: embryos were isolated individually, heads were used for genotyping and the trypsin treatment was for 45 min in 0.05% trypsin–EDTA solution with 1% chicken serum (Gibco, Thermo Fisher Scientific, Gothenburg, Sweden) under gentle agitation. Cells were cultivated in DMEM (Dulbecco’s modified Eagle’s medium, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/streptomycin and 1% non-essential amino acids at 37 °C under 5% CO2 and ambient oxygen. For positive control in the analysis of PA28αβ-dependent proteasome capacity, 150 U/mL recombinant mouse IFN-γ (Thermo Fisher Scientific) was added to the culture media 24 h prior harvest.
Proteasome capacity assays
PA28–20S or 20S proteasome capacity was analyzed as previously described [32] with some modifications. Cells were lysed in 25 mM Tris/HCl (pH 8.3) by 4 cycles of high-speed centrifugation (20,000 g) and resuspension at 4 °C, cell debris was removed by centrifugation at 5000 g for 10 min and protein concentration was determined using the BCA Protein Assay kit (Pierce, Thermo Fisher Scientific). The chemotryptic activity was assayed by hydrolysis of the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (suc-LLVY-AMC; Calbiochem Merck-Millipore, Darmstadt, Germany). 10 µg total protein was incubated with 200 µM suc-LLVY-AMC in 50 mM Tris/HCl (pH 8.3) and 0.5 mM DTT for PA28–20S activity or 50 mM Tris/HCl (pH 8.3), 0.5 mM DTT and 0.02% SDS for 20S activity in a total volume of 100 µL; fluorescence was monitored using 390 nm excitation and 460 nm emission filters with free AMC as standard (Molekula Ltd., Gillingham, UK) and activity was determined as the slope of fluorescence over time divided by total protein. Protein levels in the assay were determined by SDS–PAGE, InstantBlueTM (Expedeon Ltd., Cambridge UK) staining, and analysis using the Odyssey infrared imaging system and software (LI-COR Biosciences). Activity upon proteasome inhibition with 5 µM epoxomicin (Sigma-Aldrich, Stockholm, Sweden) is considered non-specific/background activity. Epoxomicin inhibited the PA28–20S proteasome capacity to 70 ± 9% (mean ± SD) of WT, 77 ± 10% of PA28αOE, 98.3% of untreated MEFs and 99.8% of interferon-γ treated MEFs; and epoxomicin inhibited the 20S proteasome capacity to 94 ± 3% of WT, 95 ± 3% of PA28αOE, 85% of untreated MEFs and 81% of interferon-γ treated MEFs.
Luciferase aggregation prevention
Luciferase aggregation prevention capacity was analyzed as previously described [62] with some modifications. To increase the number of n in the analysis, hippocampi from females of similar age (5–6 months) of the C57BL/6N background, 6 WT and 6 PA28αOE, were included to the 4 WT and 3 PA28αOE hippocampi from the C57BL/6N × BALB/c F2 hybrids. Right hippocampi were lysed in extraction buffer (25 mM Tris/HCl, 100 mM NaCl, 5 mM MgCl2, 1 mM ATP, and 5% glycerol, pH 7.4) by 4 cycles of high-speed centrifugation (20,000 g) and resuspension at 4 °C. Cell debris was removed by centrifugation at 5000 g for 10 min and 1 mM DTT was added after an aliquot was set aside for protein concentration determination with the BCA Protein Assay kit (Pierce, Thermo Fisher). Heat-sensitive luciferase (200 nM; L9506; Sigma-Aldrich) was heat-denatured at 42 °C in 50 mM Tris pH 7.6, 2 mM EDTA, in the presence of 4.5 µg protein extracts or corresponding volume of extraction buffer. Aggregation of luciferase was determined as light scattering at 340 nm at 42 °C. At around 80% of maximum, the increase in turbidity of the positive control (without protein extract) started to plateau, and the closest time point was chosen for analysis (40 min in the experiments on hybrid hippocampal extracts and 20 min at 42 °C in the experiments with C57). The turbidity of the positive control was considered maximum aggregation (100%). Turbidity of the negative control with no addition of heat-sensitive luciferase did not change over time and was considered background. Luciferase aggregation prevention capacity was calculated as percentage of non-aggregated luciferase. Extracts that had been incubated at 99 °C for 45 min served as negative control to the cell extract and did not prevent aggregation.
Statistical analysis
Comparisons between two groups were performed with unpaired t test assuming two-tailed distribution and equal variances and differences were considered significant at P < 0.05. Statistical analysis of the activity box corner time day 2 (Fig. 3a) by two-way ANOVA repeated measurements followed by Sidak multiple comparisons test and of the shuttle box PAT (Fig. 3b) was done by Mantel–Cox survival test; both in GraphPad Prism and the null hypothesis was rejected at the 0.05 level.