Study Design
This study examined the effect of tau expression on nSMase2 enzymatic activity and ceramide production as well as the therapeutic efficacy of the nSMase2 inhibitor PDDC in mouse models of AD. We utilized two distinct AD mouse models: PS19 transgenic mice and an AAV-hTau(P301L/S320F) propagation model. Animals were randomly assigned to either vehicle or drug groups with an equal number of male and female in each. All data were acquired in a blinded manner with a number assigned to each animal unrelated to their treatment status. Where possible, the experimenter dosing the animals was different from the experimenter carrying out the data acquisition and statistical analysis to maintain the blind. Sample sizes were determined using power calculations based on previously observed statistically significant differences to generate at least 90% power with fve extra animals per group to account for any premature animal losses.
Primary neuronal cell culture
Primary hippocampal neurons were isolated from day 18 embryos of Sprague-Dawley rats acquired from Jackson Laboratories (Bar Harbor, ME), as previously described(29–31). Briefly, hippocampal tissue was dissected, gently titurated with trypsinization in calcium-free Hank’s balanced salt solution (HBSS, calcium, magnesium, and phenol red free; Corning Inc., Manassass, VA) and cells were resuspended in Neurobasal media (Gibco, Waltham, MA) supplemented with B27 (Gibco), 1% antibiotic/antimitotic solution (Gibco), 10% FBS (Sigma, St. Louis, MO), Hepes (4.8mM, Sigma) and L-glutamine (1.2mM, Sigma). For imaging studies, cells were plated on 12mm glass coverslips coated with polyethyleneimine (PEI, Sigma) at a density of 70,000cells/well. For nSMase2 activity and ceramide analyses, cells were directly plated on PEI coated 12-well cell culture plates at a density of 700,000cells/well. After 4 days in vitro (DIV), cells were transduced with either pAAV1-CAG-GFP (Addgene, Watertown, MA) or an AAV1-serotype viral particles packaged with the hTau vector CBA-hTau24(P301L)(S320F)-WPRE (kindly provided by the Chakrabarty lab (University of Florida, Gainesville, FL))(32). Cells were transduced at a multiplicity of infection (MOI) of 50,000. At 8 DIV, cells were harvested for nSMase2 activity and ceramide assessments or prepped for imaging. Cells for nSMase2 activity were washed twice with ice-cold DMEM/F12 containing HEPES and without phenol red (ThermoFisher) and incubated with mammalian protein extraction reagent (ThermoFisher) containing 1X HALT protease inhibitor without EDTA (ThermoFisher), with shaking, for 10min at 4°C and for another 5min at RT. Final cell detachment was carried out using cell scrapers and sonicated (three 15s pulses on ice). The resulting lysates were assayed for both nSMase2 activity and total protein content as detailed below. Cells harvested for ceramide assessments were washed 3x on ice with 1X PBS prior to gently scraping with a cell scraper, centrifugation at 300×g for 5min at 4°C, freezing the pellet in liquid nitrogen, and storing at -80°C. Cells for imaging were fixed with ice cold 4% paraformaldehyde (PFA, Electron Microscopy Sciences) for 10min and washed 3x in 1X PBS followed by immediate staining.
Animal studies and PDDC dosing
All animal care and experimental procedures complied with the National Institutes of Health guidelines on animal care and were approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Mice were housed in a temperature and humidity-controlled environment under a 14‐h light, 10-h dark cycle. Food and water were available ad libitum. Animals acclimated to the facility for at least 7 days after arrival, prior to any experimentation. PDDC was synthesized in our laboratory as previously described(18, 19), before being formulated into an OpenStandard Diet (15 kcal% mouse chow) at an approximate 100mg/kg daily dose and treated as previously described(33). PS19 breeder mice were purchased from Jackson Laboratories (Bar Harbor, ME; strain #008169) and bred in-house to generate appropriately sized litters. Non-carrier littermates were deemed WT controls. Dosing for PS19 animals was initiated at 4 months of age, prior to overt pathology and symptom onset(34), and continued until the animals were 9 months of age. For the AAV-hTau mice, C57BL6/J mice were purchased from Jackson Labs (strain #000664) and stereotaxically injected at 10 weeks of age and then dosed for 6 weeks. Weekly body weights were measured for all studies. Equal male and female animals were enrolled. No significant differences between males and females were observed so the groups were combined.
At the end of the study, animals were euthanized using an overdose of isoflurane. The chest cavity was opened up and blood was collected via cardiac puncture into cold EDTA-coated BD microtainers (Franklin Lakes, NJ). For histological assessments, the animals were cardiac perfused with 1X ice cold PBS followed by 2% paraformaldehyde (PFA; Electron Microscopy Sciences). For all other experiments, mice were cardiac perfused with PBS only. Tissue was collected following perfusion and the assays were performed as described below.
PDDC in vivo pharmacokinetics (PK) and bioanalysis
12 mice (6 male/6 female) were enrolled in a PK study with n = 3/time point and the brain and plasma drug levels were quantified at 4 timepoints throughout the 24h day (00:00, 07:00, 12:00, and 19:00). Times were chosen based on the 14h light-10h dark cycle where 7:00 is when the lights come on and 19:00 was 2h before lights went off. Plasma was isolated from fresh whole blood by centrifugation at 500×g for 15min and stored at − 80°C until LC/MS/MS bioanalysis. Whole brains were harvested following blood collection and cut into hemispheres before freezing in liquid nitrogen and stored at − 80°C.
The bioanalysis was carried out as previously described(18, 33). Briefly, protein precipitation using acetonitrile (Sigma-Aldrich, St. Louis, MO)(100% v/v) containing the internal standard (losartan 500nM; Tocris, Minneapolis, MN) was used to extract PDDC standards and samples from plasma and brain prior to vortexing and centrifugation at 10,000×g. The supernatant was diluted 1:1 with water and then analyzed via LC/MS/MS. Plasma (nmol/ml) and tissue (nmol/g) concentrations were determined and plots of mean plasma concentration versus time were constructed. Phoenix WinNonlin version 7.0 (Certara USA, Inc., Princeton, NJ) was used to quantify exposures (AUC0–t) using non-compartmental analysis modules.
nSMase2 enzymatic activity assay
nSMase2 enzymatic activity was assessed as previously described(15, 33, 35, 36). nSMase2 activity measurements were initiated upon the addition of sphingomyelin (SM) and coupling enzymes, in the Amplex Red system (25 µl), and SM hydrolysis carried out in total reaction volumes of 50 µl in 384-well microplates for 3h at 37°C. At the end of the reaction period, the relative fluorescence units were measured at Ex 530nm, Em 590nm. Total protein measurements were carried out as per manufacturer’s instructions using PierceTM 660nm Protein Assay Reagent (ThermoFisher) and data presented as RFU/mg/h.
Brain lipid extraction and LC-ESI-MS/MS ceramide quantification
Lipid extractions of hippocampal cells and brain tissue from a single hemisphere (for PS19) or micro-dissected hippocampi and cortex (for AAV-hTau) were carried out using a modified Bligh & Dyer method(37) as previously described(38). Tissue samples were weighed and homogenized in water (10×) before adding 3× methanol containing a 1.3µg/mL internal standard of ceramide (d18:1/12:0)(Avanti Polar Lipids, Alabaster, AL, USA))(39) followed by an addition of 4× chloroform. Organic layers containing crude lipid extracts were collected following clear phase separation, before being dried in a nitrogen evaporator (Organomation, Berlin, MA, USA) and stored at − 80°C. Prior to analysis, pure methanol was used to resuspend the dried extracts. A Shimadzu ultra-fast liquid chromatography (UFLC) system (Shimadzu, Nakagyo-ku, Kyoto, Japan) coupled to a hybrid triple quadrupole LIT (linear ion trap) mass spectrometer 4000 QTRAP system equipped with Turbo Ion Spray (SCIEX, Foster City, CA, USA) with an ULTRA HPLC In-Line Filter (0.5µm Depth Filter×0.004 in ID)(Phenomenex, Torrance, CA, USA) was used to separate ceramides on a C18 reverse-phase column (2.6µm, 50×2.1mm). The lipids were ionized using positive electrospray ionization (ESI, +ve) and individual ceramide species were detected by multiple reaction monitoring (MRM) with instrument conditions and HPLC parameters previously described(40). Quality control (QC) samples were injected in every 10 injections. Eight-point calibration curves (0.1–1000ng/mL) were constructed by plotting area under the curve (AUC) for each ceramide calibration standard d18:1/C16:0, d18:1/C18:0, d18:1/C20:0, d18:1/C22:0, d18:1/C24:0 (Avanti polar lipids, Alabaster, AL, USA) with correlation coefficients > 0.999. Identified ceramide concentrations were calculated by fitting them to these standard curves based on acyl chain length. Instrument control and data acquisition were performed by using Analyst (version 1.4.2, SCIEX Inc. Thornhill, ON, Canada) and data analysis were completed using MultiQuant software (version 2.0, SCIEX).
Hippocampal tau isolation and western blotting
Left and right hippocampus were micro-dissected from whole brains following PBS perfusion, weighed, and then snap frozen on dry ice. Hippocampus tissue was mechanically homogenized in ten volumes of ice cold 1X RIPA buffer (Thermo Fisher) with Pierce Protease and Phosphatase Inhibitor Mini Tablets (Thermo Fisher Scientific, Waltham, MA) followed by brief sonication. The lysate was centrifuged for 15min at 10,000×g at 4°C and the supernatant was collected, frozen on dry ice, and stored at -80°C. Sarkosyl soluble and insoluble isolation was carried out based on a modified version of Sahara and Kimura (2018)(41). Briefly, hippocampus tissue was mechanically homogenized in ten volumes of ice cold 2X TBS buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktail prior to being centrifuged at 27,000×g at 4°C for 20min. The pellet was resuspended in five volumes of ice-cold high salt/sucrose buffer and centrifuged at 27,000×g at 4°C for 20 min. The supernatant was then adjusted to 1% sarkosyl and incubated on a shaker for 1h at 37°C before ultracentrifugation at 150,000×g at 4°C for 1h. The sarkosyl soluble supernatant was removed and frozen on dry ice to be stored at -80°C. The sarkosyl insoluble pellet was resuspended in 0.5 volume of 1X TE buffer (Thermo Fisher Scientific) and frozen on dry ice to be stored at -80°C. All western blots were run in a similar manner. Equal volumes of samples were loaded onto a NuPAGE 4–12% bis-tris protein gel (Invitrogen) and transferred on to an PVDF membrane using an iBlot2 Gel Transfer Device (Life Technologies). Total protein stain was performed for loading controls using Revert 700 total protein stain (LI-COR). An HRP-conjugated GAPDH antibody was also used as a loading control where applicable. Blots were blocked with EveryBlot blocking buffer (Bio-Rad) and stained overnight at 4°C for total tau (Tau 46; Santa Cruz Biotechnology, #sc-32274) and Thr181 phosphotau (D9F4G; Cell Signaling Technologies, #12885S). Appropriate HRP-conjugated secondary antibodies were used. Blots were incubated briefly with Clarity ECL substrate (Bio-Rad). All blots were imaged using the Bio-Rad ChemiDoc MP imager. Analysis was done using raw TIFF files in ImageJ. Mean pixel intensity was measured for each band and normalized to total protein or GAPDG intensity levels. To compare across blots, each blot was also normalized to the average value of the vehicle intensity.
Immunofluorescence staining
Fixed primary neuronal cells were permeabilized using 0.1% Triton X-100 in 1X PBS (0.1% PBST) for 10 min at room temperature (RT) before blocking with 5% normal goat serum in 0.1% PBST for 1h at RT. Primary antibody to phospho tau Thr181 (pThr181-Tau, Cell Signaling Technology) was incubated overnight at 4°C followed by the appropriate secondary antibody for 1h at RT. Neurons were stained using Alexa Fluor® 647 conjugated Anti-NeuN antibody (Abcam, #ab190565) for 2h at RT. Nuclei were then stained with Hoechst 33342 (Invitrogen, #H3570) before mounting with ProLong Glass antifade mountant (Invitrogen).
Brain tissues were prepped for immunofluorescence staining as previously described(15, 42). Briefly, following PFA perfusion, whole brains were dissected out and post-fixed overnight at 4°C in 2% PFA before being transferred to 15% then 30% sucrose, each overnight at 4°C. Brains were then frozen in TissueTek O.C.T. (Sakura FineTek USA, Inc., Torrence, CA, USA) and sectioned on a cryostat (Microm HM 505E, International Medical Equipment, MI, USA) at 20µm thickness. Sections were permeabilized and blocked followed by primary antibody (pThr181-Tau, Cell Signaling Technology; GFAP, Abcam, #ab4674; Iba1, Fujifilm Wako Chemicals, #019-19741; Synaptophysin, SinoBiological, #100298-T40) incubation overnight at 4°C. Sections were then stained with appropriate secondary antibodies for 1h at room temperature. Neurons were then stained with Alexa Fluor® 647-conjugated Anti-NeuN antibody (Abcam, #ab190565) for 2h at RT. Nuclei were stained with Hoechst 33342 (Invitrogen, #H3570) before coverslipping with ProLong Glass antifade mountant. All slides grouped together for MFI assessments were stained within the same batch of slides to minimize possible differences in antibody amounts and incubation times.
All images were taken on a LSM 800 confocal microscope (Zeiss) using identical imaging parameters for all images acquired. Images were acquired by focusing to the center of the section where the signal of interest was at a maximal intensity, with the brightest slide used to set the imaging parameters. Both hippocampi from 3–5 sections were imaged per animal and 3–6 images were acquired per hippocampal section (CA1, CA3, and DG, where applicable). Values from the images of both hippocampi per section were averaged and reported. All image analysis was done using Zen Blue imaging software (Zeiss).
Single cell mean fluorescence intensity (MFI) quantification
Single cell MFI was determined using images stained with pThr181-Tau, NeuN, and Hoechst 33342 and imaged with a 40X objective. Tau positive neurons were determined based on triple staining of tau, NeuN, and nuclei in order to ensure cells counted were imaged at a similar level and differences in intensity were not due to different imaging planes. Cells deemed “tau+” then had their cell bodies intricately traced, stopping at the axonal hilus as it was not possible to include axons and dendrites in the tracing. The MFI of the tau signal was recorded for each cell and an average of each individual cell per section was determined and recorded.
Pyramidal and granular cell layer thickness
Cell layer thickness was determined using images stained with NeuN and Hoechst 33342 with a 40X objective as previously reported(43). The thickness of the NeuN cell layer in the CA1 and DG regions was determined by drawing a line perpendicular to the cell layer at three points along the layer in each image, taking the thickest, thinnest and middle of the section. The three values were then averaged and the values for each section were then averaged and reported as a replicate. The CA3 region was not evaluated as it was more subject to slight variations in the plane of section and was highly variable.
Synaptophysin fluorescence intensity quantification
To quantify synaptic loss previously observed in PS19 mice(34), we measured the MFI intensity of synaptophysin staining in the Mossy fiber layer of the CA3. Images were acquired with identical parameters using a 20X objective and three images per hippocampus were taken from three sections per mouse. The Mossy fiber layer was traced and the MFI was recorded from each image. The values of all images per section were averaged and reported as a replicate.
Iba1 and GFAP intensity quantification
Sections were stained with Iba1, GFAP, and Hoechst 33342 and imaged with a 20X objective with identical parameters. 8 images per section from 3 sections per animal were obtained with identical parameters in the stratum radiatum, stratum moleculaire, and hilus regions around the CA1, CA3 and dentate gyrus regions. The MFI of the entire field of view was recorded and the average of all images per section was reported as a replicate.
Plasma nEV isolation, quantification and characterization
Mouse plasma nEV isolation was carried out as previously described(44). Briefly, plasma samples were defibrinated with Thrombin (System Biosciences, Mountainview, CA, USA) for 30min at RT and total EVs isolated via size exclusion chromatography (SmartSEC, System Biosciences). NEVs were isolated from total EVs via immunocapture against L1CAM/CD171 (clone 5G3). Protease and phosphatase inhibitors were included in multiple steps. Intact EVs were used for determination of particle concentration and diameter using nanoparticle tracking analysis (NTA)(Nanosight NS500; Malvern, Amesbury, UK). To confirm the isolation of bona fide EVs via L1CAM immunocapture, 30µL of intact L1CAM + nEVs were subjected to ExoView™ for the fluorescent detection of canonical EV markers CD9, CD63 and CD81 (NanoView Biosciences, #EV-TETRA-M2). Additionally, nEVs were lysed with protein extraction solution and the protein concentration was determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The pThr181-tau content was quantified in duplicate using the Human Tau pT181 ProQuantum Immunoassay Kit as per the manufacturer’s protocol (Invitrogen) at a dilution of 1:4. Samples were read on a qPCR equipment (StepOnePlus, Applied Byosystems). All Ct values were below 35. The LOD was found to be 0.0847pg/mL. To avoid excluding WT samples from the analysis, since they lack human tau, samples read below the limit of detection were set at a value of ½ LOD.
Nanoscale multiplex flow cytometry analysis (FCA) was carried out as previously described with slight modifications(45). Intact L1CAM + nEVs or total EVs isolated via SEC were diluted to with 1X-PBS and incubated with an equal volume of 40 µM blue succinimidyl ester (BSE; Thermo Fisher Scientific, #C34568) for 90min at 37°C. Excess BSE was removed via ultrafiltration (100kDa cutoff; Millipore Sigma, #C7719) bringing the final retentate volume to 1mL with 1X-PBS incubated with 100µL of Capto Core 400 beads (Cytiva, #17372401) for 30mins at RT with gentle rotation mixing. The EV supernatant was then incubated with a mouse-specific Fc receptor blocker reagent (Miltenyi, #130-092-575) for 30mins at RT with gentle rotation and labeled with fluorescent antibodies (each at 0.2ng/µL; PE-anti-Syntenin-1, Abcam, #210837; PE-anti-pSer262-Tau, Thermo Fisher Scientific, #44-750G; APC-anti-β-III-tubulin, Biolegend, #801219; APC-Iba-1, Abcam, #5076) in 0.05% tween-20 for partial membrane permeabilization. Labeled EVs were detected with a CytoFLEX LX flow cytometer (Beckman Coulter) using 405nm fluorescence triggering and analyzed with CytExpert software v2.3.0.84 (Beckman Coulter). For fluorescence detection, we used a 660/10 bandpass filter for APC, and 585/42 for PE, with gain voltage not exceeding 1500V. The instrument was aligned using FITC-tagged beads with sizes ranging from 100 to 1300nm (100nm beads, #834, Bangs Laboratories; 130–1300nm beads, #NFPPS-52-4K and #NFPPS-0152-5, Spherotech). Samples were diluted with 1X-PBS to control the abort rate below 1% without exceeding 200 events/second rate to avoid coincident detection of events and analyzed for 3mins.
AAV-hTau(P301L/S320F) stereotaxic injection model
The rapid tau propagation model was performed as previously described(15). The same AAV-hTau vector used in the cell culture experiments was used for the animal injections. A modified stereotaxic surgical method was used(16, 32), where mice had the AAV vector injected into their left dorsal hippocampus near the CA3 region using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA) and a pulled glass capillary needle (tip diameter < 50µm) at the coordinates AP-2.3, ML-2.1, and DV-2.2. 5×109 viral particles in < 250nL PBS were injected using a digital nanoinjector (Stoelting) attached to a mineral oil-filled 5uL gas-tight syringe (Hamilton) over 5min and the syringe was left in place for an additional 5min. Afterwards, the syringe was removed and the incision closed with cyanoacrylate glue (Vetbond, 3M) and the mouse provided ketoprofen analgesia and monitored for distress over 48h. Mice were given 2 days of recovery prior to treatment initiation and were treated for 6 weeks.
Contralateral tau mean fluorescence intensity quantification
Sections from the AAV-hTau mice were analyzed as previously described(15). Following staining for NeuN, phosphor tau, and Hoechst 33342, 2 images each from both the left and right dorsal dentate gyrus were acquired on an LSM 800 confocal microscope (Zeiss). The two images per section were averaged and each section was treated as a replicate. The ratio of the contralateral to ipsilateral MFI per section was calculated to account for injection variability. Animals with improper injection sites were excluded from analysis.
Statistical Analysis
All statistical analysis was done using GraphPad Prism 9 (GraphPad Software, LLC, San Diego, CA, USA). Comparisons made between two normally distributed groups utilized a two-tailed, unpaired student’s t-test. Comparisons made between two non-parametric groups utilized a Mann-Whitney U test. Comparisons made between three or more groups utilized a one-way ANOVA with Tukey’s multiple comparison. Results were considered statistically significant when p < 0.05.