Abundant Aβ fibrils in ultracentrifugal supernatants of aqueous extracts from Alzheimer’s disease brains.

Resource Availability

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dennis Selkoe ( dselkoe@bwh.harvard.edu ).

Materials availability

Lecanemab and bapineuzumab were produced recombinantly from publicly available patent sequences. Human 1C22 (h1C22) was produced recombinantly after sequencing 1C22 and cloned with a human IgG1 backbone. 21F12, 2G3, and 3D6 were gifts from Elan Pharmaceuticals. 266 was produced from hybridoma cells purchased from ATCC. 71A1 was supplied by Abyssinia Biologics. No new unique reagents were generated in this study, but all antibodies are available for sharing upon request of the lead contact.

Data and code availability

All biochemical data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-15770, EMD-15771 and EMD-15772. Corresponding refined atomic models have been deposited in the Protein Data Bank (PDB) under accession numbers 8AZS, 8AZT and 8AZU.

Experimental Model and Study Participant Details

Brain donor demographic and pathologic information is described in Table S1 . Data on gender, race, ancestry, ethnicity, and socioeconomic status were not available for the deidentified brain tissue samples. Low sample size precluded subgroup analysis by sex. Both subjects undergoing lumbar puncture were consented according to protocols approved by the Mass General Brigham Institutional Review Board. All animal procedures were performed in accordance with the National Institutes of Health Policy on the Use of Animals in Research and were approved by the Harvard Medical School Standing Committee on Animals. Studies of mouse hippocampal slice recording used wild-type C57bl/6 mice aged 1–3 months purchased from Jackson Labs. The mice were group-housed 4 mice in each cage in a controlled room with 22–25 °C, 50% humidity and a 12 h light/dark circadian cycle with ad libitum access to food and water. Male and female mice were randomly assigned to treatment groups. Method Details Aqueous soaking extraction of fibrils The soaking method for aqueous extraction of diffusible Aβ aggregates from AD brain has been described previously 20 , 21 and is depicted in Table 1 . Brain donor demographic and pathologic information is described in Table S1 . Grey matter was dissected from fresh or frozen cortex (pooled tissue from frontal, parietal, and temporal lobes) of individuals with AD and minced using a McIlwain tissue chopper (razor blade) set at 0.05 mm. The resultant brain bits were soaked for 30 min in a 1:5 ratio (w:v) of TBS extraction buffer (25 mM Tris supplemented with 150 mM NaCl, as well as protease inhibitors 5 μg/ml leupeptin, 5 μg/ml aprotinin, 2 μg/ml pepstatin, 120 μg/ml 4-benzenesulfonyl fluoride hydrochloride, and phosphatase inhibitor 5 mM NaF, pH 7.2) in 50-ml Eppendorf Protein LoBind tubes, and then spun at 2,000 g in a Fiberlite F14–14 × 50cy rotor in a Sorvall Lynx 6000 centrifuge for 10 min at 4°C (pelleting distance ~4 cm depending on volume). TBS was chosen because its physiologic pH and salt concentration. We chose this over artificial CSF because the latter is not as well buffered. The top ~90% of the supernatants were transferred to thin-wall polypropylene tubes (Beckman catalog #331372) and spun using an SW41Ti rotor at 40,000 rpm for 110 minutes in an Optima L90K ultracentrifuge at 4°C (pelleting distance ~9 cm). The top ~90% supernatants were retained and frozen at −80°C in 1 ml aliquots. For re-centrifugation (see Results ), the frozen aliquots were thawed and spun in an Eppendorf 5417R centrifuge equipped with an F-45–30-11 rotor or else a TOMY MX-307 centrifuge equipped with an AR015–24 rotor at 20,000 g for 2 h at 4°C (pelleting distance ~1 cm). Prior to this re-centrifugation, a 50 μl portion of each aliquot was retained for Aβ ELISA. The final supernatants, as well as the associated pellets, were collected and retained for ELISAs and electron microscopy (EM). For Aβ42 monomer ELISAs, pellets were resuspended and denatured in 50 μl TBS containing 5 M GuHCl and 5 mM EDTA. Similarly, for Aβ42 monomer ELISAs on the original soaking extracts (inputs) and their recentrifuged supernatants, each was denatured by mixing them at a 2:5 ratio (v:v) with TBS containing 7 M GuHCl and 5 mM EDTA, to reach a final GuHCl concentration of 5 M. For analysis by negative-staining EM, final pellets were resuspended in 15 μl cold water.

CSF Analysis

In order to obtain sufficient CSF for differential centrifugation experiments, we consented two subjects undergoing lumbar puncture for evaluation of normal pressure hydrocephalus (NPH) during which >30 cc of CSF is withdrawn. The CSF was frozen on dry ice within 15 minutes of lumbar puncture. It was later thawed and spun in 1-ml duplicates in a TLA120.2 rotor in thick-wall polycarbonate tubes (Beckman) for one hour at 4°C. The supernatants were then analyzed by Aβ aggregate-preferring ELISA. ELISAs Aβ monomer-preferring ELISAs were home-brew immunoassays run on the Meso Scale Discovery (MSD) platform, as described. 22 Samples containing GuHCl were diluted before ELISA, such that the GuHCl concentration was 30 cc of CSF is withdrawn. The CSF was frozen on dry ice within 15 minutes of lumbar puncture. It was later thawed and spun in 1-ml duplicates in a TLA120.2 rotor in thick-wall polycarbonate tubes (Beckman) for one hour at 4°C. The supernatants were then analyzed by Aβ aggregate-preferring ELISA. ELISAs Aβ monomer-preferring ELISAs were home-brew immunoassays run on the Meso Scale Discovery (MSD) platform, as described. 22 Samples containing GuHCl were diluted before ELISA, such that the GuHCl concentration was