Thirteen AD patients who fulfilled inclusion criteria for a clinical diagnosis of AD were randomly selected within a large database of patients consecutively admitted between January 2017 and September 2019 by the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico in Milano, Italy. All patients were enrolled in a cognitive rehabilitation experimental program and underwent complete medical and neurological evaluation, laboratory analysis, CT scan or MRI, and other investigations (e.g., EEG, SPET scan, CSF examination, etc.) to exclude reversible causes of dementia. The clinical diagnosis of AD was performed according to the NINCDS-ADRDA work group criteria  and the DMS IV–R . Neuropsychological evaluation and psychometric assessment were performed with a Neuropsychological Battery that included MiniMental State Examination (MMSE) . Digit Span Forward and Backward, Logical Memory and Paired Associated Words Tests, Token Test, supra Span Corsi Block Tapping Test, Verbal Fluency Tasks, Raven Colored Matrices, the Rey Complex Figure, Clinical Dementia Rating Scale (CDR) ; the study conformed to the ethical principles of the Helsinki Declaration.
All patients underwent lumbar puncture (LP) for quantification of Aβ, total tau (Τ-tau), and tau phosphorylated at position 181 (P-tau) in the CSF. Normality cut-off thresholds were: Aβ ≥600 pg/ml; tau ≤500 pg/ml for individuals older than 70 and ≤450 pg/ml for individuals aged between 50 and 70 years; P-tau ≤61 pg/ml. The Institutional Review Board of the Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico (Milan, Italy) approved this study. All patients (or their legal guardians) gave their written informed consent for this research before entering the study.
APO ε4 genotyping
Apoε genotype was determined by allelic discrimination as previously described .
CSF collection and Aβ and tau determination
CSF was collected between 8 and 10 a.m. after one-night fasting by LP in the L3/L4 or L4/L5 interspace according to standardized local procedures. CSF samples were centrifuged in 1500 rpm for 10 minutes at 4C°. The supernatants were aliquoted in polypropylene tubes and stored at -80 °C. CSF cells, glucose, and proteins were determined. CSF Aβ, tau and P tau were measured using commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits (Fujirebio, Ghent, Belgium).
Blood sample collection and processing
Whole blood was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson & Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMC) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA) and washed twice in PBS at 1500 RPM for 10 min; viable leukocytes were determined using a TC20 Automated Cell Counter (Biorad Hercules,California, USA).
d4T Cellular toxicity
Viability of PBMC incubated with d4T was determined using the MTT 3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) assay, as previously described .
PBMC were resuspended in RPMI 1640 (PAN-Biotech GmbH, Am Gewerbepark, DE) supplemented with 10% human serum, L-glutamine (2 mM), and 1% penicillin (Invitrogen, Ltd, Paisley, UK) in 6-wells plate and incubated at 37 °C in a humidified 5% CO2 atmosphere. Cells (7x106/well) were either cultured in medium alone (Medium) or were primed with Lypopolisaccaride (LPS) (1µg/ml)(Sigma-Aldrich, St. Louis, MO) for 2 hours and stimulated with Aβ42 (10µg/ml Sigma-Aldrich, St. Louis, MO) for 22 hours in the absence/presence of d4T (50µM)(Sigma-Aldrich) .
Enzyme-Linked Immunosorbent Assay (ELISA)
IL-1β, IL-18, and activated Caspase-1 production was analyzed in supernatants of PBMC that were resuspendend in medium alone or were stimulated (see above) by sandwich immunoassays according to the manufacturer’s instructions (Quantikine Immunoassay; R&D Systems, Minneapolis, MN, USA).
Image Stream Analysis by FlowSight AMNIS
PBMCs (2x106), stimulated as described above, were fixed with 100 μl of PFA (1%) (BDH, UK), permeabilized with 100 μl of Saponine (0.1%) (Life Science VWR, Lutterworth, Leicestershire, LE), and stained with FITC-anti human NLRP3 (Clone #768319, isotype Rat IgG2a, R&D Systems, Minneapolis, MN, USA) and PE-anti human ASC (clone HASC-71, isotype mouse IgG1, Biolegend, San Diego, CA, USA) for 1 h at room temperature; cells were then washed with PBS, centrifuged at 1,500 rpm for 10 min, resuspended in 50 μl of PBS, and examined using the AMNIS FlowSight. Results were analyzed by IDEAS analysis software (Amnis Corporation, Seattle, WA, USA). The analysis of NLRP3 expression was performed by internalization feature utilizing a mask representing the whole cell, defined by the brightfield (BF) image, and an internal mask defined by eroding the whole cell mask. Apoptosis-associated speck-like protein containing CARD speck formation was analyzed using the same mask of internalization feature, differentiating diffuse or spot (speck) fluorescenceinside of cells. Threshold mask was used to separate all ASC positive cells population in ASC-Speck spot cells or ASC-diffuse cells by the different diameter of the spot area: in ASC-speck, the spot shows a small area and high max pixel vice versa in cell with ASC-diffuse.
Cytosol protein-extraction was performed in cultured PBMC by Cell Extraction Buffer (BioSource) containing 1mM PMSF, protease and phosphatase inhibitor cocktail (Sigma-Aldrich) (1:200 and 1:100). After incubation for 30min on ice, lysates were centrifuged at 12,000g for 10 minutes at 4°C. Protein concentration was determined by Bradford assay at 595nm.
Proteins (15µg) were separated by electrophoresis into 4-12% NuPAGE® Bis-Tris gels (Invitrogen) and blotted on nitrocellulose filter (GE Healthcare). Blots were blocked 1 hour at room temperature on a shaker in 5% fat-free dried milk in TBS-T buffer (50mM Tris-HCl pH 7.6, 200mM NaCl, 0.1% Tween 20). Blots were incubated over night on a shaker at 4°C with the following rabbit Antibody (Ab): anti-beclin1 (1:300, Cell Signaling), anti-LAMP2A (1:400, Abcam) anti-p-p38 (T180/Y182 1:350, Cell Signaling) anti-pERK1/2 (T202/Y204 1:300, Cell-Signaling), anti-pAKT (S473 1:500, Cell Signaling), anti-p-p70S6K (1:500 Cell Signaling), anti-pCREB (1:800, Biosource) and mouse anti-Bax (1:300, Chemicon). Abs were diluted in 5% fat-free dried milk in TBS-T buffer. A mouse anti-actin Ab (1:20000, Sigma-Aldrich) was used as internal standard. A peroxidase-linked anti-rabbit/mouse (1:5,000; Sigma-Aldrich) IgG secondary Ab was incubated for 1 hour, at room temperature on an orbital shaker in TBS-T buffer containing 3% fat-free dried milk. Signals were detected by chemiluminescent reagents (ECL Plus Kit; Amersham), visualized on X-ray film, quantified using ImageJ software and expressed as the ratio between the target and the actin signals.
Quantitative data were not normally distributed (Shapiro–Wilk test) and are thus summarized as median and interquartile range. Comparisons between the different cultural conditions were made using: 1) a 2-tailed Mann–Whitney U test performed for independent samples for NLRP3 complex, cytokines and caspases production quantified by Elisa; 2) One-way repeated ANOVA test, followed by Tukey’s multiple comparison test for protein extracts evaluated by WB analysis. Data analysis was performed using the MedCalc statistical package (MedCalc Software bvba, Mariakerke Belgium). p-Values of less than 0.05 were considered statistically significant.