Animals and experimental design
All mice were housed 1-5 per cage (503.22 usable cm2) in a room at 23°C ± 2°C, under a 14/10-hour light/dark cycle beginning at 6:00 AM, with ad libitum access to water and chow. The APPNLh/NLh x PS1P264L/P264L double knock-in (KI) mouse model expresses humanized amyloid precursor protein with the Swedish mutation (K670N/M671L), along with a P264L point mutation in the mouse presenilin 1 gene . The homozygous KI mice were maintained on a combined CD-1/129 background, with wildtype (WT) controls derived separately from matings of heterozygous KI animals. The experiment was carried out in a 2 x 2 diet by genotype design, where half the mice of each genotype were randomized to receive 8 weeks of control (Envigo, #TD.01636) or HHcy diet (Envigo, #TD.97345), beginning between 52 and 54 weeks of age (M = 53.4 weeks, SD = 0.36). The HHcy diet was deficient in vitamins B6, B9, and B12 and supplemented with excess methionine, while the control diet was nutritionally matched with normal methionine and B vitamin levels . Three mice died during the study and were excluded from all analyses: one KI female receiving HHcy diet, one WT male receiving control diet, and one WT male receiving HHcy diet. The final numbers of mice for each group were 8 WT on control diet (5 female), 10 WT on HHcy diet (5 female), 11 KI on control diet (4 female), and 10 KI on HHcy diet (8 female). The experiment was performed in compliance with the Institutional Animal Care and Use Committee of the University of Kentucky.
Tissue collection and blood analyses
Mice were deeply anesthetized with 5% isoflurane, and arterial blood collected from the left ventricle and placed into EDTA-plasma tubes (Greiner Bio-One, #454428) for separation of plasma by centrifugation (2000xg for 20 minutes at room temperature) followed by storage at -80°C. Plasma samples from a subset of 5 mice per group were diluted 1:5 in ARCHITECT Multi-Assay Manual Diluent (Abbott Laboratories, Chicago, IL, USA) and delivered to the University of Kentucky Clinical Laboratory for Hcy measurement on an ARCHITECT i2000SR analyzer (Abbott Laboratories). An additional subset of 7 WT mice (4 on HHcy and 3 on control diet) had whole blood taken for hematologic analysis using i-STAT CG8+ cartridges (Abbott Laboratories) according to manufacturer’s instructions. All mice subsequently underwent transcardial perfusion with 50 ml ice-cold phosphate buffered saline (PBS) at a flow rate of 10 ml/min before decapitation and brain removal and dissection. The right hemisphere was post-fixed in 4% paraformaldehyde for 24 hours at 4°C and cryo-protected in 30% sucrose for at least 48 hours at 4°C. Samples were subsequently cut into 30 mm sections with a sliding microtome and stored in cryoprotectant solution at -20°C prior to immunofluorescent staining. The hippocampus and overlying cortex were dissected from the left hemisphere, flash frozen in liquid nitrogen, and stored at -80°C until processing for biochemical endpoints.
Staining was performed on free-floating sections spaced approximately 300 microns apart through the dorsal hippocampus and overlying cortex, for a total of 6-8 sections per animal. Blocking was performed with 10% normal goat serum (Lampire Biological Laboratories, #7332500) and 0.2% Triton X-100 in PBS. All antibodies were diluted in PBS with 3% normal goat serum and 0.2% Triton X-100. Sections were incubated overnight at 4°C with rabbit anti-P2ry12 (1:500, Anaspec #AS-55043A) and mouse anti-Ab 6E10 conjugated to Alexa 647 (1:200, BioLegend #803021). Samples were subsequently incubated at room temperature for 2 hours in 1:500 secondary antibody solution with Alexa 488 goat anti-rabbit (Invitrogen, #A-11034). Sections were mounted and treated for 5 minutes with 1x TrueBlack (VWR, #10119-144) in 70% ethanol to reduce autofluorescence before drying and coverslipping in Vectashield mounting medium with DAPI (Vector Laboratories, #H-1200). Slides were imaged on a Zeiss Axio Scan Z1 digital slide scanner at 20x magnification.
The dorsal hippocampus and overlying cortex were manually outlined in the HALO analysis suite (Indica Labs, version 2.3.2089.34) by an investigator blinded to experimental groups. The algorithm minimum intensity settings for all analyses were thresholded based upon negative control (no primary antibody) samples. For cortical and hippocampal analyses of P2ry12 staining, and P2ry12 and 6E10 co-localization, the positive pixel algorithm (Area Quantification FL v1.2) was applied to the traced region across all sections per animal to give a single value of total percent area stained per region per mouse. For cortical and hippocampal analyses of 6E10 staining, the object counter algorithm (Object Colocalization FL v1.0) was applied to the traced region across all sections to give a single average count per square mm of tissue per region. For the spatial analysis of microglia around amyloid plaques, 1-3 individual plaques were manually circled per section within the outlined cortex region, totaling 8-10 plaques per animal. A series of three concentric rings, each 30 microns in width, were drawn around the centered plaque and the positive pixel algorithm was applied within each ring (inner, middle, and outer). Plaques were chosen such that the ring analysis regions were non-overlapping between plaques.
MesoScale Discovery (MSD) multiplex ELISA
Dissected hippocampal and cortical tissue pieces were homogenized using an Omni Bead Ruptor 24 (Omni International) at a 1:20 weight to volume ratio in lysis buffer: PBS with 1 mM PMSF, 0.5 mM EDTA and 0.2X Halt Protease Inhibitor Cocktail (Thermo Scientific, #87786). Homogenates were centrifuged at 12,000xg for 20 minutes at 4°C. Supernatants were collected for cytokine measurement using MSD custom V-Plex ELISA kits according to manufacturer instruction with minor modifications. Briefly, 50 mL of supernatant were loaded per well of MSD plate, and the sample was incubated overnight at 4C in an Eppendorf MixMate at 1000 rpm. Cytokine levels were normalized to the total milligrams (mg) of protein loaded in the sample as determined by BCA Protein Assay (ThermoFisher #23225).
Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)
RNA was isolated from whole cortex using the RNeasy Plus Mini Kit (Qiagen, #74136) according to manufacturer’s instructions. Tissue was weighed and homogenized in an appropriate volume of buffer, and genomic DNA removed with the gDNA eliminator column. The samples were mixed with 70% ethanol, run through the RNeasy column, washed, and eluted in RNase-free water. Quantity and quality of RNA and 260/280 absorbance ratios were assessed using a NanoDrop spectrophotometer (ThermoFisher Scientific). Reverse transcription was performed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, #4368814) according to manufacturer’s protocol. Real-time PCR was performed on a ViiA 7 Real-Time PCR System (Applied Biosystems) using individual TaqMan® probes for P2ry12, Clec7a, and Itgax and custom TaqMan®Array Cards (ThermoFisher Scientific, #4342253) with TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, #4444557). See Table 1 for a complete list of genes and probes included on the array. Established microglial markers affected by amyloid pathology were chosen based on recent studies [17, 18]. Relative gene expression was calculated using the 2-∆∆CT method and log2 normalized. HPRT and 18S were used as housekeeping genes for the individual probes and the custom array, respectively.
Statistical analysis, figure generation, and reporting
Analyses and figure generation were performed with JMP Pro 14 (SAS) and Prism 8.3.0 (GraphPad). Two-way analysis of variance (ANOVA) with Sidak’s or Dunnett’s post-hoc testing and student’s t-tests were performed in Prism as indicated in the figure legends or text. Three group comparisons were made with the gene expression data: WT HHcy versus WT control, KI control vs WT control, and KI HHcy versus KI control. To generate the heatmap, group scores were averaged and transformed into z-scores using JMP, then the variable clustering script was run to group genes by similarity of expression pattern prior to visualization in Prism. Where reported in the text, group means are followed by the standard deviation (SD) in parenthesis.