Drugs: SIRT2 Inhibitors

The compound 33i (2-benzamide) was gently provided by Dr. Suzuki and prepared in saline with 5% dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint Louis, MO, USA) and 18% Tween 80 (Sigma-Aldrich) for in vivo experiments. For in vitro studies, 33i was dissolved in DMSO. The SIRT2-peripheral inhibitor AGK-2 (2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide) was purchased from Selleck Chemicals (Houston, TX, USA) and prepared in saline with 5% DMSO (Sigma-Aldrich) and 30% PEG-300 (Merck KGaA, Darmstadt, Germany).

Ames Test

33i compound was assessed for mutagenicity by Ames test using TA98 Salmonella typhimurium strain. Four different concentrations of 33i dissolved in DMSO were tested in triplicates with and without S9 fraction. Briefly, 500 µL of PBS or 10% S9 fraction were mixed with 100 µL of bacteria (2 × 109 bacteria/mL) and 50 µL of the corresponding 33i solution to obtained the concentrations to be tested (i.e., 0.5, 5, 50 and 500 µg/plate), and incubated for 1 h at 37 °C. Afterwards, 2 mL of agar containing biotin and traces of histidine was added to each mix and verted into a plaque containing solidified minimal agar medium. Positive controls were also included: 20 µg/plaque 4-Nitro-o-phenylenediamine (NPD) when no-metabolic activation was used and (con PBS) y 10 µg/plaque 2-aminofluorene (2-AF) when metabolic activation was used. Once plated, bacteria were incubated for 48 h at 37 °C and afterwards, the number of colonies observed, called revertant colonies, was counted with a laser bacterial colony counter (500A, Interscience, Saint Nom la Brétèche, France). Two independent experiments were performed.

Cell Culture

SH-SY5Y human neuroblastoma cells were obtained from American Type Culture Collection (CRL-2266™, ATCC, VA, USA) and cultured according to standards procedures. SH-SY5Y cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with high glucose and supplemented with GlutaMAX™, 10% fetal bovine serum (FBS) and Penicillin–Streptomycin (10,000 U/mL; Gibco). Cells in a passage number lower than 20, were maintained in culture for no longer than 2 months since thawed. Cells were grown at 37 °C in a humidified atmosphere of 95% air / 5% CO2.

Survival and Proliferation Assays

SH-SY5Y cells were seeded in 6-well plates and after 24 h, they were treated with 33i compound at 0.1–20 µM concentration range for 3 h. A negative control (i.e., cells treated with the 33i solvent -saline with 5% DMSO-) was also included. Two identical 6-well plates were seeded per independent experiment. After the treatment, cells were washed with PBS. Cells from on one of the plates, were trypsinized and counted (and used for the comet assay; see next section) using a Neubauer chamber. Fresh cell medium were added to the cells in the second plate and after 48 h were trypsinized and counted. Total suspension growth (TSG) was calculated for each condition dividing the number of cells after 48 h by the number of cells treated (before the treatment; cells plates in an extra well were trypsinized and counted just after the treatment to obtain this data). The Relative suspension growth (RSG) was calculated by dividing the TSG from each concentration tested by the TSG of the negative control. Three independent experiments were carried out.

MTT

SH-SY5Y cells were seeded in 48-well plates and treated with 33i compound for 24 h at 37 °C. A negative control (i.e., cells treated with the 33i solvent -saline with 5% DMSO-) was also included. Afterwards, the medium was replaced by MTT reagent (0.5 mg/mL; Sigma-Aldrich) and cultures were incubated for 2 h at 37 °C. MTT was then removed, 200 µL of DMSO was added to each well and absorbance was read at 595 nm with Multiskan FC microplate reader (Thermo Fisher Sicentific).

The absorbance in the control group was assumed to represent the 100% of cell survival. The % of survival corresponding to each of the concentrations tested was calculated in relation to the negative control. Three independent experiments we performed.

Comet Assay

The genotoxicity of the compound 33i was assessed using the standard alkaline comet assay (single-cell gel electrophoresis) and in combination with the formamidopyrimidine DNA-glycosylase (Fpg). SH-SY5Y cells were treated with different concentrations of 33i (0.1, 1, 5, 10 and 20 µM) for 3 h. A negative control (i.e., cells treated with the 33i solvent -saline with 5% DMSO-) and a positive control (i.e., cells treated with 20 µM methyl methanesulfonate (MMS) of). After the treatment cells were washed, trypsinized and counted (see previous section). Thirty 30 µL of each cellular suspension (1 × 106 cells/mL) were mixed with 140 µL of 1% low melting point agarose in PBS at 37 °C. Two drops of 70 µL of the mix were placed on 1% standard agarose pre-coated slides and covered with 20 × 20 mm coverslips. Three sets of identical slides were prepared per culture called ‘Lysis’, ‘Buffer F’ and ‘Fpg’ slides. They were all immersed for 1 h in lysis solution (2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Trizma® base, 1% Triton X-100, pH 10.0) at 4 °C. Afterwards, ‘Buffer F’ and ‘Fpg’ slides were washed with Buffer F (40 mM HEPES, 0.1 M KCl, 0.5 mM Na2EDTA, 0.2 mg/mL BSA, pH 8.0) three times (5 min each) at 4 °C. Then, 45 µL of Buffer F or Fpg enzyme was added to each gel of their corresponding set of slides. Gels were then covered with 22 × 22 mm coverslips and incubated in a humidified chamber at 37 °C for 1 h. During this time, “Lysis” slides were kept immersed in the lysis solution at 4 °C. After that, all the slides were immersed for 40 min in alkaline solution (0.3 M NaOH, 1 mM Na2EDTA, pH > 13.0) at 4 °C, and then, electrophoresis was performed at 1.1 V/cm and 4 °C for 20 min. Slides were firstly neutralized with PBS and secondly with distilled water (10 min, 4 °C each wash). Finally, they were air-dried at RT.

The next day, each gel was stained with 50 µL of 1 µg/mL of DAPI solution (Sigma-Aldrich) and comets were visualized under a fluorescence microscope (Nikon Eclipse 50 i, Tokyo, Japan). DNA damage was analyzed in 100 randomly selected cells per slide (50 in each gel) by measuring tail DNA intensity (% DNA in tail) using the image analysis software Comet Assay IV (Perceptive Instruments, Cambridge, UK). The median value of the % DNA in tail was calculated for each slide. DNA strand breaks (SBs) and alkali labile sites (ALS) were measured in the “Lysis” slides, whereas Fpg-sensitive sites were calculated by subtracting the median value of % DNA in tail of the buffer F-treated slides from the Fpg-treated ones. Two independent experiments we performed.

Animals

Experiments were carried out in male and female WT and APP/PS1 transgenic mice (5 months of age) on a C57BL/6;C3H genetic background. For microglial phagocytosis of Aβ plaques analysis, 8 months-old male and female APP/PS1 mice were used. Animals were housed in groups in standard breeding cages and had access to food and water ad libitum. Temperature and humidity were constant (23 ± 1 °C and 55 ± 10%, respectively), and lights were maintained on a 12 h light/dark cycle (light–dark: 8:00AM–8:00PM). All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments. All the procedures followed in this study and animal husbandry were conducted according to the principles of laboratory animal care as detailed in the European Communities Council Directive (2013/53/EC), are reported in compliance with the ARRIVE guidelines and were approved by the ethical committee of the University of Navarra (#051–18).

33i and AGK-2 Administration

WT and APP/PS1 mice were treated intraperitoneally once a day with 33i (5mg/kg) or vehicle (5% DMSO, 18% Tween 80) for 12 consecutive weeks (n = 14–16 animals per group). In a second set of experiments, the rodents were treated intraperitoneally once a day with AGK-2 (5mg/kg) or vehicle (5% DMSO, 30% PEG-300) for 12 consecutive weeks (n = 5–8 animals per group). Behavioral tests were carried out at 8th week of treatment. Sample size for the studies were chosen following previous studies in our laboratory (Diaz-Perdigon et al. 2020) and using one of the available interactive web sites (http://www.biomath.info/power/index.html).

Elevated Plus Maze Test

In order to take into account any possible effect of the 33i treatment on the anxious state of the animals, elevated plus maze (EPM) was performed. It is an elevated platform with two close and two open arms, crossed in the center oppositely one to another with a middle region. Mice were placed inside one of the arms and were permitted to move freely between them for 5 min. The frequency to enter in the open arms and the total time spent in them were used as a measure of an anxiety-like behavior.

All trials were monitored by a video camera set above the mazes and connected to a video tracking system (Ethovision XT 5.0, Noldus, Wageningen, The Netherlands).

Morris Water Maze

The Morris Water Maze (MWM), a hippocampus-dependent learning task used to analyze the spatial memory and to assess the working and reference memory, was performed as previously described (Sola-Sevilla et al. 2021) with minor modifications. The water maze consisted of a circular pool (diameter of 145 cm) filled with water (21–22 °C) and virtually divided into four equal quadrants (northeast, northwest, southeast, and southwest). To guide the mice, visual cues were placed in the room.

Firstly, mice underwent visible-platform training for 2 days (6 trials per day), in which a platform was located in the southwest quadrant raised above the water with an object placed on top to facilitate its location (Habituation phase). In this phase, it was confirmed that all mice exhibited a normal swimming pattern and were able to reach the platform.

For assessing learning capacity (Acquisition phase), a hidden platform (1 cm below the water surface) was placed in the northeast quadrant of the pool. The trial was finished when the animal reached the platform (escape latency) or after 60 s in the pool. After each trial, mice remained on the platform for 15 s. The test was conducted over 7 consecutive days (4 trials per day). In both Habituation and Acquisition phases, mice were placed pseudo-randomly from different locations in each trial, facing towards the wall of the pool to eliminate the potentially confounding contribution of extra maze spatial cues. Moreover, the order of the entry positions varied each day.

To test memory retention, the platform was removed, and animals were allowed to swim for 60 s (Retention phase). This trial was performed on days 5th and 8th (last day) of the test and the percentage of time spent in the northeast quadrant was recorded.

All trials were monitored by a video camera set above the center of the pool and connected to a video tracking system (Ethovision XT 5.0, Noldus).

Electrophysiology

Synaptic transmission in hippocampal slices of vehicle and 33i treated WT and APP/PS1 mice was analysed as previously described (Zamora-Moratalla and Martín 2021). Briefly, transverse brain slices of 400 μm thick were cut with a vibratome and incubated for at least 1 h at RT in artificial cerebrospinal fluid (aCSF) gassed with a 95% O2/5% CO2 mixture at pH 7.3–7.4. Individual slices were then transferred to an immersion recording chamber and perfused with oxygenated warmed aCSF (32 ± 2 °C). Field postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 pyramidal layer by a carbon fiber microelectrode (Carbostar-1, Kation Scientific, Minneapolis, MN, USA). Evoked fEPSPs were elicited by stimulation of the Schaffer collateral fibers with an extracellular bipolar tungsten electrode placed in the stratum radiatum. At the beginning of each experiment, basal synaptic transmission was analyzed by applying isolating stimuli of increasing intensity to reach a maximal fEPSP response. For Long-term potentiation (LTP) experiments, the stimulus intensity was adjusted to elicit 50% of the maximum response signal and kept constant throughout the experiment. After recording stable baseline responses for 30 min, LTP was induced by a single train of theta burst stimulation (TBS; 5 bursts of 5 pulses at 100 Hz, with an interval of 200 ms between bursts). Potentiation was measured for 1 h after LTP induction at 0.033 Hz.

Glucose- and Insulin-Tolerance Tests

In Glucose-Tolerance Test (GTT), mice were fasted for 6 h with free access to water. Animals were intraperitoneally administered with 20% of D-glucose (Merck KGaA). Blood glucose concentrations were measured before (baseline) and after 15, 30, 60 and 120 min of glucose administration by venous tail puncture using glucometer and Accu-Check Aviva glucose strips (Hoffmann-La Roche, Basel, Switzerland).

For Insulin-Tolerance Test (ITT), animals were fasted for 1 h with free access to water. After basal glucose in blood was determined (baseline), insulin was intraperitoneally administered (0.75 UI per kg of body weight; Actrapid, Novo Nordisk, Bagsværd, Denmark) and glycaemia was measured after 15, 30 and 60 min using a glucometer and Accu-Check Aviva glucose strips (Hoffmann-La Roche).

Analysis of Microglial Phagocytosis of Aβ Plaques

In vivo Aβ phagocytosis was determined following a flow cytometry-based protocol described by Lau et al. (2021) with some modifications. 8 month-old APP/PS1 mice were treated intraperitoneally once a day with 33i (5mg/kg) or vehicle (5% DMSO, 18% Tween 80) for 1 week (n = 6 animals per group). The 8th day, methoxy-X04 (MeX04) was injected intraperitoneally (10mg/kg; Tocris Bioscience, Bristol, UK). After 3 h, mice were perfused transcardially with ice-cold PBS under xylazine/ketamine anesthesia. Hippocampus and cortex were collected, chopped into pieces and digested together with papain (0.4 mg/mL; Worthington Biochemical Corporation, Lakewood, NJ, USA) in a 37 °C water bath with shaking for 30 min. Then, the samples were mechanically dissociated, and the cellular suspension was filtered through a 70 μm cup Filcon cell suspension filter (BD, Franklin Lakes, NJ, USA) into a solution of 20% FBS/80% HBSS (Gibco). After a centrifugation of 200 g for 5 min, the pellets were resuspended in 5 mL of 80% HBSS/20% Percoll solution. After creating an interphase with HBSS, samples were centrifuged at 200 g for 20 min. This interphase was removed, and cells were centrifuged at 4500 rpm for 5min. Cells were washed and incubated with anti-mouse CD45 (1:100; eBioscience, Thermo Fisher Scientific) and anti-mouse CD11b (1:200; BioLegend, PerkinElmer, Waltham, MA, US) monoclonal antibodies for 30 min on ice. After washing the cells, they were resuspended in 350 µL of sorting buffer (0.5% bovine serum albumin or BSA, 2.5mM EDTA in PBS). Samples were analyzed in a FACS Canto II flow cytometer (BD) and using FlowJo software (BD). For analysis, the CD11b+CD45low population was gated. WT mice injected with methoxy-X04 were used to determine the methoxy-X04-threshold for non-phagocytosing cells, and unstained wild-type cells were used to determine background fluorescence.

RNA Extraction and Quantitative PCR

Total RNA was isolated from hippocampus and epididymal white adipose (Epi-WAT) samples using TRI Reagent® (Sigma-Aldrich). One microgram of RNA was retrotranscribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA), and quantitative real-time PCR was carried out on CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using iQTM SYBR® Green Supermix reagent (Bio-Rad). Primers used are detailed in the following table (Table 1). For hippocampus samples, Gapdh was used as an internal control, whereas 36b4 gene expression was used for Epi-WAT samples.

Table 1 Primers sequences used for SYBR Green qPCR analysis

For Abca1 determination, quantitative real-time PCR was carried out using Taqman® Universal PCR Master Mix (Applied Biosystems) and ViiA™ 7 Real-Time PCR System (Applied Biosystems). Both Abca1 and Gapdh (internal control) primers were from Applied Biosystems (cat# Mm00442646_m1 and Mm99999915_g1, respectively).

For gene expression quantification, the double delta CT (ΔΔCT) method was used where delta CT (ΔCT) values represent normalized target genes levels with respect the internal control (Gapdh or 36b4). The relative quantification of all targets was carried out using the comparative cycle threshold method, 2−ΔΔCt, where ΔΔCt = (Ct target gene − Ct endogenous control) treated/(Ct target gene − Ct endogenous control) untreated.

Western Blot

For Western blot analysis, hippocampal and Epi-WAT tissues were sonicated in cold lysis buffer with protease inhibitors (0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 200 mM NaF, 2 mM Na4P2O7, 5 mM EDTA, 1 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 1 mM Na3VO4, 1 mM benzamidine, 10 mg/mL leupeptin, 400 U/mL aprotinin) and incubated on ice for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatant was collected. In the case of Epi-WAT samples, the upper fat layer was also removed.

In order to measure total protein concentration, Bio-Rad protein assay was performed, following the manufacturer’s protocol (Bio-Rad). Equal amounts of protein (30 μg for hippocampal tissues, and 20 μg for Epi-WAT) were separated by electrophoresis on a sodium dodecyl sulphate–polyacrylamide gel (7.5%) under reducing conditions and transferred onto a nitrocellulose membrane (Hybond-ECl; Amersham Bioscience, Amersham, UK) for 16 h at 4 °C. The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-GluA1 (1:1000; cat# AB1504, Sigma-Aldrich). As internal control, mouse monoclonal anti-β-Actin was used (1:1000; cat# A1978, Sigma-Aldrich).

The next day, membranes were incubated with goat polyclonal anti-rabbit (cat# 926–68021) and anti-mouse (cat# 926–32210) secondary antibodies (1:5000; Odyssey, LI-COR Biosciences, Lincoln, NE, USA) for 2 h at RT. Bands were visualized using Odyssey Infrared Imaging System (LICOR Biosciences). Results were calculated as the optical density values of WT-Vehicle mice.

Immunofluorescence

Brains of six mice per experimental group were histologically processed for Aβ plaques and Iba1 determination. After dissection, one brain hemisphere was postfixed for 24 h with 4% paraformaldehyde and conserved in 30% sucrose for 1 week. Serial coronal brain slices (thickness: 40 μm) were cut with a freezing microtome and stored in cryoprotectant solution.

Free-floating slices were washed 3 times in PBS and incubated in 70% formic acid for 10 min to expose the Aβ epitope. Then, brain sections were incubated in blocking solution (PBS containing 0.5% Triton X-100, 0.1% BSA, and 2% normal donkey serum) for 2 h at RT. Afterwards, slices were incubated with mouse monoclonal anti-β-amyloid (1:200; cat# 803001, BioLegend) and rabbit polyclonal anti-Iba1 (1:1000; cat# 019–19741, Fujifilm Wako, Osaka, Japan) primary antibodies overnight at 4 °C. Sections were washed with PBS and incubated with the secondary antibody Alexa Fluor donkey anti-mouse 488 (1:200) and Alexa Fluor goat anti-rabbit 568 (1:250) for 2 h at RT, protected from light (cat# A-21202 and A-11011, respectively; Thermo Fisher Sicentific). Finally, sections were washed with PBS and mounted with DAPI Fluoromount-G® Mounting Medium (Southern Biotech, Birmingham, AL, USA).

In order to ensure comparable immunostaining, sections were processed together under same conditions. Images were acquired with the Vectra Polaris scanner (Perkin Elmer). Quantification of fluorescent signal in brain sample images was carried out using ImageJ program (NIH, Bethesda, MD, USA).

Quantification of Aβ-42 Levels in Brain Cortex

For Aβ-42 levels quantification, 20 mg of brain cortex were homogenized in 8 volumes of cold 5 M guanidine-HCl in 50 mM Tris buffer. The homogenate was incubated for 3 h at RT on an orbital shaker and then, it was diluted ten-fold with cold PBS supplemented with 1X protease inhibitor cocktail (Calbiochem, Merck KGaA). Samples were centrifuged 20 min at 16,000 g at 4 °C and the supernatant was diluted 1:2000 with Standard Diluent Buffer provided with the ELISA kit. Fifty microliters of the resultant solution were assayed using the Ultrasensitive Amyloid-β 42 Human ELISA Kit (cat# KHB3544; Invitrogen) following the manufacturer’s instructions. Each sample was analyzed in duplicate.

Quantification of IL-1β In Epididymal White Adipose Tissues

25 mg of Epi-WAT from each animal was sonicated in Cell Lysis Buffer 2 (cat# 895347; R&D systems, Minneapolis, MN, USA) at 1:5 dilution and incubated on ice for 30 min. Samples were then centrifuged 12 min at 13,000 rpm at 4 °C and fifty microliters of the resultant supernatant were assayed in the Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (cat# MLB00C; R&D systems) following the manufacturer’s protocol.

Quantification of Cytokines in Serum

For the quantitative measurement of cytokines in serum samples, BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (cat# 552364; BD) was used. Interleukin-6 (IL-6), Interleukin-10 (IL-10), Monocyte Chemoattractant Protein-1 (MCP-1), Interferon-γ (IFN-γ), Tumor Necrosis Factor (TNF), and Interleukin-12p70 (IL-12p70) protein levels were assayed following the manufacturer’s instructions. Briefly, fifty microliters of serum were incubated with Capture Beads and Mouse Inflammation PE Detection Reagent for 2 h at RT, protected from light. After washing the samples, data acquisition was performed with a FACS Canto II flow cytometer (BD) and analyzed using FlowJo software (BD).

SIRT2 Expression in Postmortem Brain Tissue Samples from AD Patients

Brain tissues were obtained from the Oxford Project to Investigate Memory and Ageing (OPTIMA, see www.medsci.ox.ac.uk/optima). Subjects for this study constituted a randomly selected subset of the participants, now part of the Thomas Willis Oxford Brain Collection within the Brains for Dementia Research Initiative (BDR). At death, informed consent had been obtained from the patients’ next-of-kin before collection of brains and the study was approved by the UK National Research Ethics Service. All cases were selected based on clinic-pathological consensus diagnoses. Participants classified as normal controls (n = 10), did not have dementia or other neurological diseases, did not meet CERAD criteria for AD diagnosis, and were staged at Braak 0-II. AD cases (n = 10) were clinically diagnosed based on meeting the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) criteria for a diagnosis of probable or definite AD. Frontal (Brodmann Area, BA10) cortex were dissected free of meninges. In the control group, the average age was 69.8 years (SD 3.06) and the sex distribution was 6/4 men/women. In the AD group, the average age was 80.4 years (SD 2.12) and sex distribution was 3/7 men/women. All tissue used had a brain pH > 6.1, condition used as an indication of tissue quality in post-mortem research.

mRNA extraction from 20 mg of human brain tissue was performed using the Nucleospin RNA kit (Macherey–Nagel, Düren, Germany) according to the manufacturer's instructions. Afterwards, 500 ng of RNA was retrotranscribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and quantitative real-time PCR was carried out using Taqman® Universal PCR Master Mix (Applied Biosystems) and ViiA™ 7 Real-Time PCR System (Applied Biosystems). Both SIRT2 and β-ACTIN (internal control) primers were from Applied Biosystems (cat# Hs01560289_m1 and Hs01060665_g1, respectively).

For Western blot analysis, 10 mg of human brain tissue were sonicated in cold lysis buffer with protease inhibitors (0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 200 mM NaF, 2 mM Na4P2O7, 5 mM EDTA, 1 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 1 mM Na3VO4, 1 mM benzamidine, 10 mg/mL leupeptin, 400 U/mL aprotinin) and incubated on ice for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatant was collected. Equal amounts of protein (50 μg per sample) were separated by electrophoresis on a sodium dodecyl sulphate–polyacrylamide gel (7.5%) under reducing conditions and transferred onto a nitrocellulose membrane (Hybond-ECl; Amersham Bioscience, Amersham, UK) for 16 h at 4 °C. The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich). The next day, membranes were incubated with goat polyclonal anti-rabbit (cat# 926–68021) secondary antibody (1:5000; Odyssey, LI-COR Biosciences, Lincoln, NE, USA) for 2 h at RT. Bands were visualized using Odyssey Infrared Imaging System (LICOR Biosciences). As internal control, mouse monoclonal anti-β-Actin was used (1:1000; cat# A1978, Sigma-Aldrich).

Quantification Of SIRT2 in Human Serum Samples

Human serum samples were obtained from the Karolinska University Hospital Memory Clinic in Huddinge (Sweden) including control (n = 26) and AD (n = 28) patients, for a total of 54 samples. The average age was 68 years (SD 9.42), and the sex distribution was 23/77% men/women (n = 6/20) in the control group, and 36/64% men/women (n = 10/18) in the AD group. The clinical and diagnostic data of the GEDOC cohort are described in detail in Goikolea et al. (2022) and Rosenberg et al. (2019).

For the quantitative measurement of SIRT2 protein in human serum samples from control and AD patients, Human SIRT2 SimpleStep ELISA Kit (cat# ab227895; Abcam, Cambridge, UK) was used. Serum samples were 2X-diluted in the assay diluent buffer. Briefly, fifty microliters of diluted serum samples were incubated with the Antibody Cocktail for 1 h at RT on a plate shaker. Next, samples were incubated with TMB Development Solution, the reaction was stopped after 10 min and the OD was recorded at 450 nm. The concentration of SIRT2 protein was determined by interpolating the sample absorbance values (blank subtracted) against the standard curve and multiplying by the dilution factor.

Statistical Analysis

In vitro experiments were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. In the habituation and acquisition phase of the MWM, body weight, GTT and ITT, strain and treatment effects were analysed by repeated-measures two-way ANOVA followed by multiple comparisons with Tukey’s test. The rest of the behavioral tests and biochemical results were analyzed using two-way ANOVA (strain*treatment) followed by multiple comparisons with Tukey’s test was used. Post hoc test was applied only if F on interaction was significant. In figure legends, the F values represent the F of interaction followed by the p-value of the corresponding post hoc test. In those cases where the F of interaction was not statistically significant, the F value shown represents the main effect observed strain or treatment. Microglial Phagocytosis of Aβ plaques, Aβ-42 levels and Aβ plaques quantification as well as SIRT2 analysis in human samples were analyzed by unpaired parametric Student’s t test.

Results were expressed as mean ± standard error of the mean (SEM), and differences among groups were considered statistically significant at p < 0.05. All the statistics were performed by GraphPad Prism software (San Diego, CA, USA).