Protein phosphatase inhibitor-1

Human brain tissues from four sporadic AD patients, three Down syndrome patients with abundant tau pathology qualified for AD (referred to as AD/DS), and two normal controls were used in this study (Table S1). All cases used were histologically confirmed. Two of the AD/DS cases were provided by the University of Washington brain bank. The use of postmortem brain tissues for research was approved by the University of Pennsylvania’s Institutional Review Board with informed consent from patients or their families. For each purification, 6–14 g of frontal cortical gray matter was homogenized using a Dounce homogenizer in nine volumes (v/w) of high-salt buffer (10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 1 mM EDTA, and 2 mM dithiothreitol [DTT], with protease inhibitor cocktail, phosphatase inhibitor, and PMSF) with 0.1% sarkosyl and 10% sucrose added and centrifuged at 10,000 g for 10 min at 4°C. Pellets were reextracted once or twice using the same buffer conditions as the starting materials, and the supernatants from all two to three initial extractions were filtered and pooled. Additional sarkosyl was added to the pooled low-speed supernatant to reach 1%. After 1-h nutation at room temperature, samples were centrifuged again at 300,000 g for 60 min at 4°C. The resulted 1% sarkosyl-insoluble pellets, which contain pathological tau, were washed once in PBS and then resuspended in PBS (∼100 µl/g gray matter) by passing through 27-G 0.5-in. needles. The resuspended sarkosyl-insoluble pellets were further purified by a brief sonication (20 pulses at ∼0.5 s/pulse) using a hand-held probe (QSonica) followed by centrifugation at 100,000 g for 30 min at 4°C, whereby the majority of protein contaminants were partitioned into the supernatant, with 60–70% of tau remaining in the pellet fraction. The pellets were resuspended in PBS at one fifth to one half of the precentrifugation volume, sonicated with 20–60 short pulses (∼0.5 s/pulse), and spun at 10,000 g for 30 min at 4°C to remove large debris. The final supernatants, which contained enriched AD PHFs, were used in the study and referred to as AD-tau. In a subset of the experiments, the samples were boiled for 10 min right before the final 10,000-g spin to get rid of contaminating protease activity. The same purification protocol was used to prepare brain extracts from the two normal controls. The different fractions from PHF purification were characterized by Ponceau S staining, Western blotting (refer to Table S3 for antibodies), and sandwich ELISA for tau. The final supernatant fraction was further analyzed by transmission EM, BCA assay (Thermo Fisher Scientific), silver staining (SilverQuest Silver Staining kit; Thermo Fisher Scientific), and sandwich ELISA for Aβ 1–40, Aβ 1–42, and α-syn. The frontal cortex from one AD/DS case was purified using the traditional procedure with sucrose gradient fractionation as previously reported (Boluda et al., 2015 (link)). Enriched AD PHFs prepared using both methods showed similar seeding activity in primary hippocampal neurons from CD1 (non-Tg) mice.