Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Stephen Strittmatter ( stephen.strittmatter@yale.edu ).

Mice were cared for by the Yale Animal Resource Center and all experiments were approved by Yale’s Institutional Animal Care and Use Committee and performed in accordance with the American Association for Accreditation of Laboratory Animal Care (AAALAC). Wild-type and APPswe/PS1ΔE9 mice (APP/PS1) () were purchased from Jackson Laboratory and maintained on a C57/Bl6J background as described previously (). All experiments were conducted in a blinded fashion with respect to genotype and treatment, and groups were matched for age and sex, and groups contained 45%–55% of each sex.

Method Details

Aßo preparation Um et al., 2012 Um J.W.

Nygaard H.B.

Heiss J.K.

Kostylev M.A.

Stagi M.

Vortmeyer A.

Wisniewski T.

Gunther E.C.

Strittmatter S.M. Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons. Laurén et al., 2009 Laurén J.

Gimbel D.A.

Nygaard H.B.

Gilbert J.W.

Strittmatter S.M. Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Biotinylated and unlabeled synthetic Aβ1-42 peptide are obtained as lyophilized powder from The ERI Amyloid Laboratory, LLC (Oxford, CT). Preparation and characterization of Aβ1-42 oligomers (Aβo) have been described previously (). Aβ monomer is dissolved at 10 mg/ml in HFIP and boiled in water bath 1 h at 70C, then cooled on ice, transferred to 2 mL microfuge and tubes spun 7 min at 12,000 x g. Avoiding pellet, 50 μl (0.5 mg) is aliquoted in a 1.6 mL microfuge tube and allowed to evaporate completely in chemical hood (24 h), followed by a minimum of 1 h in a speed vac. An observable clear film surrounds the inside tip of the tube. Closed tubes are stored at RT for later oligomer preparation. To prepare oligomers, add 40 μl DMSO to tube, allow to stand 20 min with occasional flicking/trituration to suspend peptide, wiping down tube sides to insure no un-dissolved peptide remains. Aliquot 20 μl/1.6 mL microfuge tube. Add 1 mL phenol red-free F12 (Atlanta Biologicals cat # M15350) to tubes for 0.25 mg/ml final conc. Let stand O/N at RT. Spin 15 min at 14000 rpm - there is usually no pellet; a large pellet indicates an unsuccessful prep. Aliquots can be frozen for later use. Binding assays show consistent results over at least 72 h post F12 addition. Concentrations of Aβo are expressed in monomer equivalents, with 1 μM total Aβ1-42 peptide corresponding to approximately 10 nM oligomeric species ().

Cell-based Screen for small molecule inhibitors of Aßo/PrPC interaction CV1 cells stably transfected with rat PrP were plated in 96 well tissue culture plates (Corning, 354461) 24 h prior to application of small molecule library components dissolved at 10 mM in DMSO (10 μM final concentration) for 1 h prior to addition of biotinylated Aßo (1 μM final concentration). Wells were fixed 25 min in 4% paraformaldehyde, washed twice with PBS, blocked 1 h in PBS containing 5% goat serum (GIBCO, 16210-064). 50 μl 1:1000 Eu-labeled streptavidin (PerkinElmer, 1244-360) in DELFIA assay buffer (PerkinElmer Life Sciences) was added per well for 30 min. After washing five times in PBST, 50 μL of DELFIA Enhancement Solution (PerkinElmer Life Sciences) was applied to each well, and time-resolved europium fluorescence was measured using a Victor 3 plate reader (PerkinElmer Life Sciences). Compounds were assayed in singlet, with hits defined as inhibition exceeding 50% of Aßo-dependent signal, with assay plates exhibiting a Z’ over 0.5 relative to anti-PrP positive control antibody 6D11 (Covance). Hits were validated at 10 μM in triplicate in a similar assay, with visual inspection of each treatment for cell toxicity and detachment. Compounds inducing obvious cellular toxicity were eliminated from further study. Non-toxic active compounds were subjected to dose-response evaluation in triplicate in the same cell-based format. Libraries screened were Enzo FDA Approved Drugs Library (Enzo), Microsource Pharm 1600 (Microsource) and Yale Small Molecule Discovery Center compound collection.

Immunocytochemistry COS-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. COS-7 cells transfected 2 days earlier with PrPC expression vector were pre-incubated with inhibitor in F12 media at 22°C for 30 min. Biotin–Aβ42 oligomers were added to a concentration of 500 nM monomer equivalent for 2 h. Cells were washed twice with PBS, fixed 25 min with 4% paraformaldehyde in PBS, washed twice with PBS, blocked 1 h with 5% goat serum in PBS, incubated in 0.1% 488-streptavidin (Life technologies, S11223) in PBS 1 h, washed twice and visualized on an ImageExpress Micro (Molecular Devices). Images were analyzed using ImageJ. Alternatively, higher resolution images were capture from cells cultured on poly-lysine coated glass with a Zeiss confocal microscope.

Size-exclusion Chomatography (SEC) Aged ceftazidime (as 14-day reconstituted Fortaz) was separated on a Superdex 75 10/300 GL gel filtration column (GE Healthcare Bio-Sciences) using AKTA purifier FPLC system (GE Healthcare). 200 μl of sample was injected at a flow rate of 0.75 ml/min. PBS, pH 7.4, was used as a mobile phase. Fractions of 0.5 mL were continuously collected thoughout the run and analyzed for PLISA activity or selected for Z quantitation.

Z concentration measurement The SEC fraction of aged Fortaz corresponding to 15 kDa was collected, exchanged into water though extensive washes with a 3 kDa filter (Amicon, UFC500396), and desiccated. Weighed material (a light brown powder) was resolubilized in PBS and absorbance determined at 280 nm to determine concentration.

Anion-exchange Chomatography Aged ceftazidime (as 14-day 440 mM reconstituted Fortaz in sodium carbonate) was separated on an XK 50/20 column (GE Healthcare Life Sciences) packed with Q Sepharose Fast Flow (GE Healthcare, 17-0510-01) with a 0.5-2.0 M NaCl gradient. Fractions eluting at 100-120 millisiemens were used as Z.

Biolayer Interferometry C biotinylated by incubation in 2-fold molar excess biotin-NHS (Thermo Scientific, 21329) 2 h in PBS at RT were coated onto streptavidin Biosensors (ForteBio, 18-5019) and exposed to dilution series of solutes as described in Biotinylated Aßo or full-length human PrPbiotinylated by incubation in 2-fold molar excess biotin-NHS (Thermo Scientific, 21329) 2 h in PBS at RT were coated onto streptavidin Biosensors (ForteBio, 18-5019) and exposed to dilution series of solutes as described in Figures 2 A, 5 F, and 5G in PBS with 0.05% Tween 20 (Sigma, P7949) and 0.1% BSA. Association curves were detected and analyzed using an Octet biolayer interferometer (ForteBio).

Blue Native PAGE shift assay Recombinant human PrPC (1 μM) was incubated in the presence or absence of 2 μM Z for 10 min in 0.25X PBS at room temperature. Following the incubation, the samples were loaded on 4%–16% NOVEX Bis-Tris gel (ThermoFisher) and separated according to manufacturer’s recommendations. To demonstrate the requirement of native PrPC structure for complexation with compound Z, some samples were heated in 65C heatblock for 10 minutes prior to BN-PAGE separation to denature the protein. Following the BN-PAGE, the gels were transferred onto PVDF membranes using iBlot semi-dry transfer (ThermoFisher), the membranes were dried and the excess of Coomassie dye was removed by washing the membrane in methanol thee times for 5 min to avoid interference with subsequent immunoblotting. The membranes were then washed 3X with water, blocked using fluorescent western blot blocking buffer (Rockland) and immunoblotted with 1:500 SAF-32 mouse anti-PrPC antibody (Cayman Chemical) in TBS-T followed by 800CW-conjugated donkey anti-mouse secondary antibody (Li-Cor). Immunoblots were imaged using Li-Cor Odyssey near-infrared scanner.

Assay for Aßo binding to immobilized PrPC (PLISA) MaxiSorp 384 well white microplates (ThermoFisher Scientific, 460372) were coated overnight with 20 μl/well of 250 nM human full length PrPC in 30 mM Na 2 CO 3 , 80 mM NaHCO 3 , pH 9.6, at 4°C. After washing two times with PBST (PBS, 0.05% Tween 20), the plates were blocked with 100 μl/well protein-free T20 PBS blocking buffer (Pierce, 37573) for 1 h at 23C. After thee PBST washes, 20 μl samples diluted in PBST (PBS, 0.05% Tween 20) were applied to microplates in triplicate and incubated 1 h at RT. 20 μl of synthetic biotinylated Aβo in PBSTB (5 nM monomer equivalent) was added for 2 h. Plates were then washed four times with PBST and incubated 1 h with 20 μl 1:1000 Eu-labeled streptavidin (PerkinElmer, 1244-360) in DELFIA assay buffer (PerkinElmer Life Sciences). Finally, after washing five times in PBST, 20 μL of DELFIA Enhancement Solution (PerkinElmer Life Sciences) was applied, and time-resolved europium fluorescence measured using a Victor 3 plate reader (PerkinElmer Life Sciences).

Biochemical small molecule screen PLISA was used to screen 56,610 unique compounds not included in the initial cell-based screen. Libraries screened were MicroSource GenPlus (Microsource), Yale compound collection, and ChemDiv Diversity Library (ChemDiv). Small molecule library components are stored dissolved at 10 mM in DMSO (10 μM final concentration) and added in singlet to PrP-coated wells of a 384-well plate containing PBST for a final concentration of 10 μM. After 30 min, PBST containing biotinylated Aβo was added for a final Aβo concentration of 5 nM, incubated @ RT 2 h and developed per PLISA protocol. Hits exceeding 50% signal inhibition were evaluated further.

Z-binding PrP-epitope mapping A MaxiSorp 384 well white microplate (ThermoFisher Scientific, 460372) was coated overnight with 20 μl/well of 250 nM human full length PrPC in 30 mM Na 2 CO 3 , 80 mM NaHCO 3 , pH 9.6, at 4°C. After two PBST washes, the plates were blocked with 100 μl/well protein-free T20 PBS blocking buffer (Pierce, 37573) for 1 h at 23°C. After 3 PBST washes, 20 μL of anti-PrP antibodies (8B4, Santa Cruz Biotech, sc-47729; 5058, Millipore Sigma, AB5058; 8G8, Cayman chemical,189760; 6D11, Covance, SIG-399810; Pri 308, Cayman Chemical, 189750; 8H4, abcam, ab61409; SAF70, Cayman Chemical, 189770) diluted 1:50 in PBST were applied to wells in triplicate and incubated 1 h at RT. Twenty μl of 50 nM Z biotinylated by incubation in 10-fold molar excess biotin-NHS (Thermo Scientific, 21329) 2 h in PBS at RT was added per well for 2 h. Plates were washed four times with PBST and incubated 1 h with 20 μl 1:1000 Eu-labeled streptavidin (PerkinElmer, 1244-360) in DELFIA assay buffer (PerkinElmer Life Sciences). Finally, after 3 PBST washes, 20 μL of DELFIA Enhancement Solution (PerkinElmer Life Sciences) was applied, and time-resolved europium fluorescence \ measured (Victor 3V PerkinElmer Life Sciences).

Melanin Synthesis Norepinephine bitartrate salt (Sigma Aldrich A0937) was dissolved at 50 mg/ml in H 2 O, brought to pH 8.5 with ammonium hydroxide and allowed to stand at RT at least 72 h, forming a black solution.

PrPSc Propagation Assay Chonically PrPSc-infected scN2a cells, RML strain, were cultured in Delbucco’s Modified Eagle Medium (DMEM) with L-glutamine and 4.5 g/L glucose plus 10% fetal bovine serum (FBS) and 50 U/ml penicillin, 50 μg/ml streptomycin. 10 mM and 5 mM stock solutions of compounds Z and PSCMA, respectively, were prepared in PBS and stored at 4°C for no longer than 1 week prior to use. Compound stock solutions or PBS alone (vehicle) were added to culture medium and working concentrations obtained via serial dilution. Trypsinized ScN2a cells were split 1:10 and allowed to adhere to plates in compound-free media for 12 h, at which point medium containing the treatment compound was added. Cells were grown for 3 days to confluence with a media exchange at 36 h. Cells were trypsinized, split 1:10 and again allowed to adhere in compound-free media for 12 h, and then returned to compound-containing medium for an additional 3 days prior to processing. Cells were lysed in ice-cold lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5% w/v Na deoxycholate, 0.5% v/v NP-40). Lysate was centrifuged at 2,100 x g for 30 s to pellet DNA. 10% of the resulting supernatant was added to an equal volume of 2x SDS-PAGE loading buffer, boiled at 95°C for 10 min and saved as the minus proteinase K (–PK) sample. To the remaining supernatant, PK was added to a final concentration of 20 μg/ml and samples were digested shaking at 37°C for 30 min prior to quenching with the addition of phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 5 mM. PK-digested samples were centrifuged at 100,000 x g for 1 hour at 4°C and the pellet was resuspended in equal volumes of lysis buffer and 2x SDS-PAGE loading buffer prior to boiling and analysis via SDS-PAGE and western blot. Western blotting was carried out with GE8 primary antibody at 1:2000 and HP-conjugated sheep anti-mouse secondary antibody at 1:5000.

Immunoblots Proteins were electrophoresed though precast 4%–20% tris-glycine gels (Bio-Rad) and transferred with an iBlotTM Gel Transfer Device (Novex-Life Technologies) onto nitrocellulose membranes (Invitrogen). Membranes were blocked in blocking buffer for fluorescent western blotting (Rockland MB-070-010) for 1 hour at room temperature and incubated overnight in primary antibodies at 4°C. The following primary antibodies were used: anti-Fyn (Cell Signaling Technology 4023; 1:1,000), anti-phospho-Src (Cell Signaling Technology 2101; 1:1,000). appropriate secondary antibodies were applied for 1 h at room temperature (Odyssey donkey anti-mouse or donkey anti-rabbit conjugated to IRDye 680 or IRDye 800, LI-COR Biosciences) and proteins were visualized with a LI-COR Odyssey infrared imaging system. Quantification of band intensities was performed within a linear range of exposure.

Neuronal culture and Aßo binding Brain cortices and hippocampi were dissected from embryonic day 13 pups removed from CO2-euthanized pregnant C57/Bl6 mice, dissociated by incubating in 0.25% trypsin 10 min at 37C, followed by gentle trituration in Neurobasal A medium supplemented with 2% B27, 1% Glutamax, 1% sodium pyruvate, 1% pen/strep and 0.2% FBS, filtration though a 40 um filter and plated at 30,000 cells/well in a polylysine-coated 96 well plate. After DIV 14, wells were treated with Z or PSCMA at the specified concentrations 30 min by adding to the conditioned media, followed by addition of biotinylated Aßo for 2 h, after which cells were washed once with PBS, fixed 25 min in 4% formaldehyde in PBS, washed 3 times with PBS, blocked 1 h in PBST with 5% goat serum, incubated overnight at 4°C with designated antibody in PBST (SV2a, Abcam 32942, 1:250; NeuN, Millipore, mab377, 1:500 or actin, Cell Signaling Technology, 4967S, 1:500), followed by washing twice with PBS and incubating 2 h in cognate secondary antibodies, DAPI and 0.1% 555-streptavidin (Invitrogen, S32355) in PBST, followed by washing twice in PBS and imaging with an ImagExpress (Molecular Devices). Signal was quantitated with ImageJ. Alternatively, stained neurons cultured on poly-D-lysine-coated glass were imaged with a Nikon Eclipse Ti Spinning Disk Confocal Microscope.

Cell Transfection and Aßo Staining Human embryonic kidney cells (HEK293) were plated in 24-well plates and transfected with full length human PrPC or human SCARF1 (Transomic, clone ID pCS6(BC039735)) using lipofectamine 3000 transfection reagent. Thee days post transfection, cells in conditioned media were treated with 1 μM PSCMA or PBS vehicle for 30 min prior to addition of biotinylated Aßo at final 1 μM monomer equivalent concentration for an additional 2 h. Cells were washed 1 x with PBS, fixed 25 min in 4% formaldehyde, washed 3x in PBS, blocked 1 h in 5% normal goat serum, incubated 1 h in 488-streptavidin (Molecular Probes), washed 3x in PBS and imaged with an ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices).

Imaging of Dendritic Spine Stability (Z) Um et al., 2012 Um J.W.

Nygaard H.B.

Heiss J.K.

Kostylev M.A.

Stagi M.

Vortmeyer A.

Wisniewski T.

Gunther E.C.

Strittmatter S.M. Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons. 2 for 30 min), the neurons were transfected with myristoyl-GFP expression vector by Amaxa Nucleofector. Cells were plated at 100,000 cells per well on poly-D-lysine-coated glass 8 well plates (Lab-Tek Chambered Coverslip 155411). The culture medium was Neurobasal A supplemented with 1X penicillin/streptomycin, 1 mM Na-pyruvate, 2 mM GlutaMax, and B27 supplement with weekly replenishment. After 19-23 DIV, neurons were imaged with a 100X objective on a Nikon Eclipse Ti Spinning Disk Confocal Microscope using a 488 laser. A 10 μm Z stack at 0.1 μm intervals was obtained every 15 min over 6 hours from multiple fixed locations per 8-well dish with an automated stage. 500 nM Aß oligomer or F12 vehicle control were added after one hour of imaging and additional images collected over 5 hours. In some conditions, 50 nM Z or drug vehicle control were added immediately before Aß or vehicle. Spine number in consecutive images for specific dendritic segments was measured using ImageJ software without knowledge of drug or genotype. For each condition, at least 4 segments with 30 spines at time zero were assessed. Hippocampal neurons of various genotype were obtained from E17-19 mouse embryos (). After hippocampal digestion with papain (37°C; 5% COfor 30 min), the neurons were transfected with myristoyl-GFP expression vector by Amaxa Nucleofector. Cells were plated at 100,000 cells per well on poly-D-lysine-coated glass 8 well plates (Lab-Tek Chambered Coverslip 155411). The culture medium was Neurobasal A supplemented with 1X penicillin/streptomycin, 1 mM Na-pyruvate, 2 mM GlutaMax, and B27 supplement with weekly replenishment. After 19-23 DIV, neurons were imaged with a 100X objective on a Nikon Eclipse Ti Spinning Disk Confocal Microscope using a 488 laser. A 10 μm Z stack at 0.1 μm intervals was obtained every 15 min over 6 hours from multiple fixed locations per 8-well dish with an automated stage. 500 nM Aß oligomer or F12 vehicle control were added after one hour of imaging and additional images collected over 5 hours. In some conditions, 50 nM Z or drug vehicle control were added immediately before Aß or vehicle. Spine number in consecutive images for specific dendritic segments was measured using ImageJ software without knowledge of drug or genotype. For each condition, at least 4 segments with 30 spines at time zero were assessed.

LDH release Cell toxicity was quantitatively assessed by the measurement of Lactose dehydrogenase (LDH) activity in the medium. LDH activity in the culture medium was measured by Cytotoxicity Detection Kit (Roche) according to manufacturer’s procedure. In brief, 60 μL of supernatant from each well was transferred to a 96 well plate and 60 μL of reconstituted substrate solution was added to each well and then the plates were incubated for 30 min. Total LDH release was achieved by adding 2% Triton X-100 solution to untreated control cells. The absorbance of the samples was measured at 490 nm using a VictorX3 Multilabel Plate Reader (PerkinElmer). The values were expressed as a percent of the total LDH release.

Animal Treatment For Morris water maze, mice were randomly assigned to treatment groups and the experimenter was unaware of both genotype and treatment group. Groups were balanced for age, sex, and weight. Mice used were 12-14 months of age at experiment initiation. During treatment, the experimenter was blinded to genotype. For drug treated mice, Poly(4-styrenesulfonic acid co-maleic acid) was administered by twice daily oral gavage of 5.0 mg and 15.0 mg Poly(4-styrenesulfonic acid co-maleic acid) per kg body weight in a vehicle of 0.5% w/v hydroxypropyl methylcellulose and 0.1% w/v polysorbate 80 to APP/PS1 and WT mice, respectively. Vehicle treated animals were gavaged twice daily with vehicle. 500 mg ceftazidime as Fortaz was dissolved per manufacturer’s instructions in 1.5 mL sterile milliQ H 2 O to obtain 333 mg/ml in sodium carbonate solution. Animals treated peripherally with fresh ceftazidime were injected directly after dissolution intraperitoneally with 100 mg ceftazidime per kg body weight in a vehicle of PBS. Animals treated centrally with aged ceftazidime were fitted with an intracerebroventricular cannula (Alzet brain infusion kit 0008663) and subcutaneous osmotic minipump (Alzet model 1004) loaded with aged ceftazidime diluted in PBS. All animals were treated for 4 weeks prior to the Morris water maze and thoughout assessment.

Behavioral Testing Morris, 1984 Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. Smith et al., 2018 Smith L.M.

Zhu R.

Strittmatter S.M. Disease-modifying benefit of Fyn blockade persists after washout in mouse Alzheimer’s model. Morris water maze was performed as previously described (). Thoughout experimentation, the experimenter was blinded to treatment group, and genotype. Each animal was handled by the experimenter for five minutes each day for thee consecutive days preceding the initiation of behavioral experiments to minimize animal stress on testing days. The testing pool was ∼1 m in diameter with four unique spatial cues placed evenly around the perimeter. For learning swims, a clear plastic platform was submerged 1 cm below the surface of the water and fixed to the bottom of the pool in the target quadrant. For each swim a mouse was placed in the water facing the pool wall opposite the target quadrant in one of four positions. The sequence of the entry positions was changed for each of the six trial blocks. For a single trial block, each animal was swum four times. For each swim mice were given 60 s to locate the hidden platform. Mice were given a 60 s rest interval with access to a heating lamp between swims. Trial blocks were initiated every 12 hours over thee consecutive days for a total of 6 trial blocks. If a mouse failed to locate the hidden platform in the allotted time during trial blocks one or two, the mouse was gently guided to the platform and placed there for 15 s. The reverse swim began the day after completion of the forward swims and followed the same protocol with the hidden platform placed in the quadrant opposite that of the forward swims. Twenty-four hours after the last learning trial block of the reverse swim, the platform was removed from the pool for the probe trial. During the probe trial, each animal was placed in the pool once and allowed to freely swim for 60 s. For all swims animals were tracked using SMART 3.0 software (Panlab, S.L. - Harvard Apparatus, Inc, Holliston, MA) with a JVC Everio G-series camcorder (Yokohama, Japan). To account for differences in visual acuity, a marker that extended above the surface of the water was placed on the platform and mice we placed in the water facing the wall opposite the platform. Latency to reach the visible platform was recorded and any animals that did not reach the platform within two standard deviations of the mean were excluded from analysis. For analysis of the learning swims, a single mouse’s latency to find the platform was averaged across four swims to generate a trial average.

Fluorescence Recovery After Photobleaching (FRAP) COS-7 green monkey kidney cells (ATCC® CRL-1651) were passaged in high-glucose DMEM (ThermoFisher, 11965092) supplemented with sodium pyruvate, 10% Fetal Bovine Serum and Pen/Strep antibiotic mix. Trypsinized cells were seeded in 8-well chambered sterile coverglass slides (ThermoFisher, 155411) at 10000 cells/well in 250 μl of complete growth medium and cultured overnight. The following day, the cells were transfected with a total of 200 ng DNA/well using Lipofectamine 3000 lipid transfection reagent (ThermoFisher, L3000015) according to manufacturer’s protocol. For fluorescent labeling, cells expressing SNAP-PrP were incubated with 500 nM SNAP-Surface Alexa Fluor647 in complete medium for 30 min at 37°C. Cells were washed twice with PBS supplemented with calcium and magnesium (Sigma, D8662) to remove the excess labeling fluorophores and then incubated for 15 min in PBSCa,Mg with 1 μM PSCMA or PBSCa,Mg alone as a control. Aβo or PBS (vehicle) was subsequently applied to 1 μM final concentration for 1 h at 37°C and cells were then imaged at room temperature. All FRAP experiments were performed on UltraVIEW VoX (Perkin Elmer) SDC microscope equipped with PhotoKinesis FRAP unit using 60x oil immersion objective. Images were collected every second for 7 s. before bleaching to measure the baseline fluorescence. Following the bleaching cycle, the imaging was performed with 1 s intervals for the first 30 s and with 4 s intervals for an additional 220 s. 640 nm laser was used for selective photobleaching of SNAP-PrP conjugated with Alexa 647. All the imaging was performed in the apical membrane of the cells and at least thee 2x2 μm areas per cell were bleached to average the intrinsic variability in the protein mobility between the regions of the plasma membrane. Quantitation of fluorescence recovery was performed in Volocity software (PerkinElmer).

Immunohistology Mice were euthanized by CO2 asphyxiation, perfused with cold PBS and brains were dissected and post-fixed in 4% paraformaldehyde for 72 h at 4°C. Brains were sliced into 40 μm coronal brain sections using a Leica WT1000S vibratome. Sections were permeabilized in PBS + 0.1% Triton X-100 for 15 min. All slices underwent an antigen retrieval step prior to exposure to primary antibody by incubating slices in 1x Reveal Decloaker buffer (RV1000M, Biocare Medical) for 15 min at 90C in an oven. After antigen retrieval, sections were blocked in 10% normal horse serum (Jackson ImmunoResarch Laboratories) in PBS for 1 hour at room temperature and then incubated with primary antibodies for 24 hours at 4C. The following primary antibodies were used: anti-GFAP (glial fibrillary acidic protein; Abcam ab4674; 1:500), anti-Iba1 (ionized calcium-binding adaptor molecule 1; Wako 019-19741; 1:250), anti-PSD95 (postsynaptic density protein 95; Invitrogen 51-6900; 1:250), and anti-SV2a (synaptic vesicle glycoprotein 2A; Abcam 32942; 1:250). Sections were washed 3 times in PBS and incubated with secondary antibodies (donkey anti-rabbit or donkey anti-chicken fluorescent antibodies; Invitrogen Alexa Fluor; 1:500) for 1 hour at room temperature. After 3 washes in PBS, the sections were mounted onto glass slides (Superfrost Plus, Fisher Scientific) and coverslipped with Vectashield (Vector Laboratories H-1200) antifade aqueous mounting medium.

Imaging and analysis of immunohistochemistry Gimbel et al., 2010 Gimbel D.A.

Nygaard H.B.

Coffey E.E.

Gunther E.C.

Laurén J.

Gimbel Z.A.

Strittmatter S.M. Memory impairment in transgenic Alzheimer mice requires cellular prion protein. For imaging of synapse density stained by anti-SV2a and anti-PSD95 antibodies, a Zeiss 800 confocal microscope with a 63X 1.4 NA oil-immersion lens was used. The area occupied by immunoreactive synaptic puncta from the molecular layer of the dentate gyrus was measured as described previously (). For imaging and analysis of the tissue stained for Iba1 and GFAP, a Zeiss 800 confocal microscope with a 20X 0.3 NA air-objective lens was used and a full tiled z stack of the hippocampus was taken. β-amyloid plaque load was imaged on a Zeiss AxioImager Z1 fluorescent microscope with a 4X air-objective lens. ImageJ software was used for quantification of image area containing tissue only, excluding image area not containing tissue. Statistical analysis was based on separate mice.

Thioflavin S Aß plaque staining 60 μm sections were incubated in pre-heated 10 mM sodium citrate with 0.05% Tween 20, pH 6 at 95°C for 1 hour, rinsed twice with PBST and blocked with 10% donkey serum (Jackson ImmunoResearch 017-000-121) for 1 hour. Next, sections were incubated in 0.1% Thioflavin S (Sigma T1892) in 70% ethanol at room temperature for 15 min, washed twice with 70% ethanol, then twice with distilled water. Images of cortical Thioflavin S staining were collected from thee slices for each animal and quantified using ImageJ. Thee values for a single animal were averaged and graphed as a single data point per animal.

Mouse pharmacokinetic (PK) study 2 O and purified using Oasis® WAX cartridges (Waters, 186002489). Eluted extracted PSCMA was assayed for Aßo/PrPC inhibitory activity by PrP-ELISA or PLISA ( Kostylev et al., 2015 Kostylev M.A.

Kaufman A.C.

Nygaard H.B.

Patel P.

Haas L.T.

Gunther E.C.

Vortmeyer A.

Strittmatter S.M. Prion-protein-interacting amyloid-β oligomers of high molecular weight are tightly correlated with memory impairment in multiple Alzheimer mouse models. Brain penetration of 20 kDa PSCMA (Sigma, 434566) was characterized in six male C57BL/6 mice. Mice received drug at 40 mg/kg as a solution in 95% PEG400/5% Solutol (dose volume = 5 mL/kg) by oral gavage. After 10 days of treatment, mice were euthanized by CO2 asphyxiation, perfused with ice-cold PBS for 60 s, and brains were rapidly dissected. Whole brains were Dounce homogenized in 1:10 w:v brain:PBS, followed by polyanion extraction using TRIzol reagent (Invitrogen, 15596026). 1 mL trizol per 100 mg tissue was added, vortexed, 0.2 mL chloroform per ml TRIzol added, vortexed, centrifuged 15 min at 12,000 x g, aqueous phase transferred to a new tube, 0.5 mL isopropanol added per ml TRIzol used for lysis, incubated 15 min, centrifuged at 12,000 x g, supernatant retrieved leaving RNA pellet, supernatant speed vacuumed overnight, resuspended in HO and purified using Oasis® WAX cartridges (Waters, 186002489). Eluted extracted PSCMA was assayed for Aßo/PrPinhibitory activity by PrP-ELISA or PLISA () using similarly Dounce homogenized brain, spiked with varying concentrations of PSCMA followed by extraction, to establish a standard curve.

Toxicological Assessment of PSCMA For the survival study, WT mice were orally gavaged with 120 mg/kg, 40 mg/kg, 2.4 mg/kg, or vehicle twice daily for 10 days. For the tissue sampling study, groups of 12-month-old WT mice balanced for sex and weight were orally gavaged with 15 mg/kg, 3 mg/kg, or vehicle twice daily for 1 week. All group received the same volume of vehicle per gram of body weight. After treatment, animals were sacrificed and whole blood was collected via cardiac puncture using a 21 gauge needle, and aliquoted into a plasma separator tube (BD Microtainer 365985) and a K 2 EDTA tube (BD Microtainer 365974). Plasma separator tubes were centrifuged at 6,000 x g for 90 s. Following blood collection, heart, liver, spleen, and left kidney were removed and weighed. Blood samples were sent to ANTECH Diagnostics for complete blood counts and chemistry panels.