All experiments performed in this study comply with all relevant ethical regulations. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at St Jude Children’s Research Hospital.
Statistics and reproducibility. Sample sizes were not predetermined using statistical methods; however, they closely resembled those reported in previous publications68-70. While the assumption of normal data distribution was made, formal testing for normality was not conducted. Statistical methods were subsequently employed to estimate p-values, and the analysis of false discovery rate was conducted. No exclusions of animals occurred in the study, and the experiments were not randomized. Animals were utilized whenever available without randomization. The investigators were blinded to genotype information during the quantitation of immunostaining for plaque and microglia.
Human brain samples. Human postmortem brain tissue samples (frontal gyrus) were provided by the Brain and Body Donation Program at Banner Sun Health Research Institute. Clinical and pathological diagnoses were based on the established criteria67.
Mouse model. All mice (C57BL/6J) were housed under a 12-h light: 12-h dark cycle at 22-25 °C and 40%-60% humidity. Mdk knockoutmice were generated using CRISPR-Cas9 technology to partially delete coding region in the genome to cause frame shift mutation. Briefly, dual gRNAs were designed with in silico off-target analysis to determine highly unique spacer sequence with at least 3bp of mismatch to any other site in the mouse genome. In addition, we also consider the targeted region covered among all isoforms, and select CAGE257.MDK.g2 (5’- UGCGGCAUGGGCUUCCGCGA- 3’) and g14 (5’-GCACCUUGCAAUGGACGCGC-3’). Precomplexed ribonucleic proteins (RNPs) consisting of 25 ng/µl of each sgRNA (Synthego) and 50 ng/ µl SpCas9 mRNA (Trilink) were injected into the cytoplasm of C57BL/6J fertilized zygotes and transferred to CD-1 psuedopregnant fosters. Resulting animals were genotyped by targeted next generation sequencing using primers CAGE257.F: 5’-GTGAGGCAGGCCGTGTGACCAAGTG-3’ and CAGE257.R:5’- TGCAGTCGGCTGATGGGAGAGTGGC-3’ and analyzed using CRIS.py as previously described68,69 Founder animals with out-of-frame mutation were backcrossed to C57BL/6J mice for two generations. A founder carrying a 23bp deletion in exon 2 or 3 of all isoforms was selected for the study. FAD mice (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax) were obtained from Jackson Laboratory (MMRRC_034848-JAX) and underwent backcrossing with C57BL/6J for 10 generations. FAD/Mdk-/- (FAD/KO) mice were generated by crossing with FAD/Mdk+/- with Mdk+/-mice. In all mouse experiments, same ratio of male and female animals was utilized.
Western blotting. Protein lysates underwent separation via 4-20% Tris-Glycine gel and transfer to 0.22 µm nitrocellulose membranes. Blots underwent a 1-h blocking with 3% BSA for MDK or 5% skim milk for other targets. The following primary antibodies were incubated overnight at 4°C: anti-mouse MDK (1:1000, sheep polyclonal, R&D, #AF-7769), anti-Aβ (1:1000, mouse monoclonal, clone 82E1, IBL, #10323), and anti-β-tubulin (1:5000, rat monoclonal, clone YOL1/34, abcam, #ab6161). After three 5-minute washes, blots were exposed to secondary antibodies conjugated with HRP (1:40,000, Jackson ImmunoResearch) for 1 h at room temperature, followed by another three 5-minute washes. Chemiluminescence was developed by using either the SuperSignal West Pico PLUS or Femto substrate (Thermo Fisher Scientific) and subsequent detected by ChemiDoc system (Bio-Rad).
ELISA analysis. Aβ40 and Aβ42 levels in mouse brain lysate were measured using Meso Scale Diagnostics (MSD) ELISA plate (V-PLEX Aβ Peptide Panel 1 (6E10) Kit)70. Briefly, the 96-well plate coated with Aβ40 and Aβ42 antibodies were blocked with blocker diluent for 1 h at room temperature to reduce non-specific protein binding. The plate was washed 3 times with 150 µl /well MSD wash buffer. Then 25 µl of detection antibody followed by 25 µl of diluted standards or mouse brain lysate were loaded and incubated for 2 h. The plate was washed 3 times with 150 µl /well wash buffer. Finally, signals were developed by adding 150 µl of 2x read buffer followed with immediately read on the MESO SECTOR S 600 (MSD).
Immunohistochemistry. The staining was modified from previously report12. Briefly, brain tissue samples were fixed with 10% formalin buffer, and embedded in paraffin. 5-10 µm sections were deparaffinized, rehydrated and rinsed with water. Antigen retrieval was performed with 10 mM citric buffer (pH 6.0) with boiled water bath for 20 min and cooled down to room temperature. Endogenous peroxidase activity is blocked by 1% H2O2 in PBS buffer for 10 min. After rinsed by PBS, sections were then blocked with 5% donkey serum in PBS with 0.3% Triton X-100 (PBST) for 30 min at room temperature followed with primary antibodies diluted with PBST plus 2% BSA and 0.2% skim milk for overnight at 4°C: anti-human MDK, (goat polyclonal, 1:200, R&D, #AF-258-SP), anti-mouse MDK (1:200, sheep polyclonal, R&D, #AF-7769), anti-Aβ (1:100, clone 82E1, IBL, #10323), anti-IBA1 (1:400, rabbit polyclonal, Fujifilm Wako, #019-19741). After washing with PBS, sections were then incubated with secondary antibodies (1:500, Jackson ImmunoResearch) for 1 hour at room temperature. For immunofluorescence staining of Aβ and IBA1, we used Alexa Fluor 488 and Cy3-conjugated secondary antibodies, respectively. For MDK, we went with HRP polymer-conjugated secondary antibodies (Vector Laboratories, # MP-7405). Signal was amplified using TSA-Cy5 (Akoya Biosciences). For chromogenic immunodetection, biotin-conjugated secondary antibodies were used followed with ABC reaction by Vectastain ABC kit (Vector Laboratories, #PK-4000) and developed by 3,3-diaminobenzidine solution (Vector Laboratories, #SK-4100). Images were captured by Zeiss Axioscan.Z1 and Zeiss LSM 780 confocal microscopy.
Quantitation of Aβ plaques and microglia. Brain tissue serially sectioned sagittally at 10 μm thickness. Three slides, each representing every 30th section, were utilized for immunohistochemistry followed with imaging captured with Zeiss Axioscan.Z1. QuPath, an open-source software71 was carried out for quantitation of density and area of Aβ plaques and microglia in an investigator blinded manner.
MDK protein purification. The MDK construct (22-143 aa) was over-expressed in BL21 (DE3) Rosetta 2 cells. A single colony was employed to cultivate a starter culture overnight. Subsequently, 10 ml of the starter culture was utilized to inoculate 1L of 2XYT media, which contained 50 μg/ml Kanamycin and 35 μg/ml Chloramphenicol. The inoculation took place at 37°C, with agitation at 220 rpm, until the cell density reached an OD600 nm of approximately 0.8. The induction of protein over-expression was initiated by adding 0.5 mM Isopropyl β-D-thiogalactopyranoside (IPTG), and the culture was further incubated at 18°C for 16 h. The cells were harvested by centrifugation at 5000 rpm for 15 min at 4 °C and the cell pellet was frozen in - 80°C until used. The cell pellet was resuspended in lysis buffer (20mM HEPES, pH 7.4, 500mM NaCl, 5% glycerol), supplemented with an EDTA-free complete protease inhibitor cocktail tablet (Roche). Disruption was achieved using a cell disruptor (Constant Systems Ltd.) at 20.0 kPSI pressure in 2 rounds of lysis. The resulting lysate was cleared by centrifugation at 20,000 rpm for 30 min at 4 °C and subsequently applied to a gravity column following a 30-minute incubation with pre-equilibrated Ni-NTA resin at 4 °C. The column was then washed by 10 CV of wash buffer A (20mM HEPES pH 7.4, 500mM NaCl, 12.5 mM Imidazole and 5% glycerol), followed by 10 CV of wash buffer B (20mM HEPES pH 7.4, 500mM NaCl, 50 mM Imidazole and 5% glycerol). The protein was eluted with 20mM HEPES pH 7.4, 500mM NaCl, 250 mM Imidazole and 5% glycerol. The eluted fractions were analyzed by loading onto a 4-20% SDS-PAGE gel. Subsequently, the elution fractions were combined, and the His-tag was cleaved from the protein using tobacco etch virus (TEV) protease overnight at 4 °C. The cleaved protein was then further purified by size exclusion chromatography using a Superdex 75 16/600 column (Cytiva) equilibrated with 50 mM Tris-HCl, pH 7.5, 500mM NaCl, and 5% glycerol. Finally, the protein fractions from the column were pooled, concentrated to the desired concentration, flash-frozen in liquid nitrogen, and stored at -80 °C for subsequent experiments.
ThT fluorescence assay. Aβ40 and Aβ42 (rPeptide, #A-1153-1, and #A-1163-2) with a purity of > 97%. Each peptide vial was suspended in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), incubated for an hour to disperse any preexisting aggregates. Further these samples were aliquoted and carefully dried in nitrogen stream and stored in -80°C. Aliquots of purified Aβ species were dissolved in 10 mM NaOH to a concentration of 2 mg/ml followed by 10 min cooling and sonication in an ice water bath for 1 min. The concentration of the final monomeric sample was quantified by nanodrop at 280 nm. Purified MDK was dialyzed overnight in 50 mM Tris-HCl buffer, pH 7.5 and final concentration was determined by absorbance measurements at 280 nm Further, the freshly dissolved Aβ peptides were diluted to 5 µM in absence and presence of different concentrations of purified MDK containing 50 mM Tris-HCl, pH 7.5 buffer containing 20 µM Thioflavin. 100 µl of solution was added into 96-well half area, solid bottom, clear, sterile, microtiter plates and sealed with sealing tape to prevent evaporation. All kinetic experiments were performed at 37˚C under quotient conditions at every 5 min in a Clariostar plate reader (BMG Labtech) using an excitation and emission wavelengths of 440 and 480 nm respectively. All these assays were performed in three replicates. Aggregation kinetics of MDK at different concentrations in the same buffer with fixed ThT concentrations were also recorded.
NMR HSQC Spectroscopy. The 15N-labeled Aβ40 and Aβ42 peptides (rPeptide, #A-1101-2 and #A-1102-2) were dissolved in 10mM NaOH to a concentration of 2 mg/ml, followed by 10 min cooling and 1 min sonication in an ice water bath. After aliquoting on ice, the samples were stored in a -80°C freezer. Subsequently, 10 µM of the 15N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 µM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively. The NMR spectra of all these samples including 10 µM monomeric Aβ species were acquired on Bruker Avance 600 MHz or 800 MHz at 278K. The spectrometers are equipped with a triple-resonance cryoprobe, and the data were processed in NMR Pipe72. and analyzed using NMRFAM-SPARKY73. The backbone resonance assignment of Aβ40 was taken from BMRB (ID: 17795). Binding of MDK to the 15N-labeled Aβ40 and Aβ42 was investigated using two-dimensional (2D) [1H-15N] so fast HMQC74 spectrum recorded either with 32 scans or 128 scans with an interscan delay of 0.2 s. The intensity of the peaks from different spectra were quantified, normalized with respect to the highest peak and used in analyzing the data.
Circular dichroism spectroscopy. Aβ aggregated triplicate samples in the absence and presence of MDK were collected directly from the 96-well plates. CD spectra of monomeric and fibrillated Aβ species (in the absence and presence of MDK) were recorded on a JASCO-1500 Spectrophotometer at a wavelength of 195 - 250 nm with a step size of 0.2 nm, 2 nm bandwidth, and a scan speed of 50 nm/min. CD spectra of MDK alone before and after incubation were also recorded. For each sample, the average of five scans was recorded and background correction was done by subtraction of corresponding buffer spectra. Spectra of native and aggregated MDK were collected with the same parameters at a wavelength of 200 to 250 nm.
Negative stain transmission electron microscopy. Aβ species alone and reactions involving Aβ together with 10 µM of MDK samples from the kinetic experiments were collected at the end point of the ThT experiments. 400-mesh copper grids (CF400-Cu grids, Electron Microscopy Sciences) were plasma cleaned with an Ar/O2 gas mixture for 10s using Solarus plasma cleaner (Gatan), followed by 5 µl of sample. The samples were allowed to adsorb for 1 min before blotting away the excess liquid, followed by rinsing using Milli Q water and subsequent staining using three successive applications of 2% uranyl acetate. The last round of stain application was allowed to sit for a min before blotting away the excess stain. The grids were air-dried prior to imaging using a 120 kV Talos L120C TEM (Thermo Fisher Scientific) equipped with a CETA detector (TFS).
Immunogold negative-stain electron microscopy. Extraction of Aβ filaments was performed as in the previous report75. Aβ filaments were deposited on glow-discharged 400 mesh formvar/carbon coated copper grids (EM Sciences CF400-Cu) for 40 s. Subsequently, the grids were blocked for 10 min with 1% BSA in PBS and incubated with anti-MDK (1:50, goat polyclonal, Bio-Techne, #AF-258-SP). After rinsing with blocking buffer, the grids were incubated with anti-goat IgG conjugated with 10 nm gold particle (1:20, Sigma), followed with wash and stained with 2% uranyl acetate for 1 min. Images were acquired at 11,000x with a Gatan Orius SC200B CCD detector on a Tecnai G2 Spirit at 120 kV.
Detergent extraction for insoluble proteome. The analysis procedure was adapted from a previously published method19. Mouse cortices were extracted with Lysis Buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 1 mM DTT, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% Sarkosyl, 10% glycerol, and 1x Sigma cOmplete Protease Inhibitor Cocktail). Protein lysates were centrifuged at 3000 x g for 5 min at 4°C. The resulting supernatant was subjected to a second centrifugation at 130,000 x g for 1 h at 4°C. The resulting pellet was washed with 50 mM HEPES containing 0.5% sodium deoxycholate and centrifuged at 17,200 x g for 30 min at 4°C. The insoluble pellet was subsequently resuspended in 8 M Urea with 0.5% sodium deoxycholate for proteome profiling.
Mass spectrometry-based proteomics. We used an optimized protocol of TMT-LC/LC-MS/MS for deep proteome profiling44,45. Protein samples were lysed by homogenization in the lysis buffer (50 mM HEPES, pH 8.5, 8 M urea, and 0.5% sodium deoxycholate), and their concentrations were measured by the BCA assay (Thermo Fisher Scientific, #23227) and confirmed by Coomassie-stained short SDS gels 76. Quantified protein samples (~0.1 mg per TMT channel) were digested with Lys-C (1:100 w/w, Wako, distributor, #121-05063) for 2 h at 21 ºC, followed by dilution to decrease urea to 2 M and trypsin digestion (1:50 w/w, Promega, #V5113) overnight at 21 ºC. Cys residues were reduced and alkylated by iodoacetamide. The proteolysis was terminated by adding trifluoroacetic acid to 1%. The resulting peptides were desalted with the Sep-Pak C18 cartridges (Waters), TMT-labeled (Thermo Fisher Scientific, #A34808 and A44520), and pooled equally. The pooled peptides were resolved by basic pH reverse phase LC on an XBridge C18 column (3.5 μm beads, 4.6 mm x 25 cm, Waters; buffer A: 10 mM ammonium formate, pH 8.0; buffer B: 95% acetonitrile, 10 mM ammonium formate, pH 8.0, ~2 h gradient, 40-80 concatenated fractions collected)77. Each fraction was analyzed by acidic pH LC-MS/MS (75 µm x ~20 cm, 1.9 µm C18 resin from Dr. Maisch GmbH, buffer A: 0.2% formic acid, 5% DMSO; buffer B: buffer A plus 65% acetonitrile, ~1.5 h gradient). The settings of Q Exactive HF Orbitrap MS (Thermo Fisher Scientific) included the MS1 scan (~410-1600 m/z, 60,000 resolution, 1 x 106 AGC and 50 ms maximal ion time) and 20 data-dependent MS2 scans (fixed first mass of 120 m/z, 60,000 resolution, 1 x 105 AGC, ~110 ms maximal ion time, HCD, 32-35% normalized collision energy, ~1.0 m/z isolation window with 0.3 m/z offset, and ~15 s dynamic exclusion)45.
The raw MS data were searched against protein database by the JUMP software (v1.13.4)78, which utilizes both pattern matching and de novo tag scoring to improve the sensitivity and specificity. A composite target/decoy database was used to evaluate FDR in peptide identification79,80. The protein target database combined downloaded Swiss-Prot, TrEMBL, and UCSC databases (human: 83,955 entries; mouse: 59,423 entries). Search parameters included precursor/product ion mass tolerance (± 10 ppm), full trypticity, static mass shift (TMT tags of 229.16293 and Cys carbamidomethylation of 57.02146 on cysteine), dynamic mass shift (Met oxidation of 15.99491), two maximal miscleavage sites, and three maximal modification sites. Peptide-spectrum matches (PSMs) were filtered by matching scores and mass accuracy to limit protein FDR below 1%.
Proteins were quantified from TMT reporter ions based on our published method46. Briefly, TMT reporter ion intensities were extracted for each PSM, corrected by isotopic distribution of TMT reagents, filtered to remove poor PSMs (e.g., minimum intensity of 1,000), and adjusted to alleviate sample pooling bias. The relative protein intensities were averaged from all assigned PSMs after removing outliers (e.g., Dixon’s Q-test or generalized extreme Studentized deviate test). Finally, the absolute protein intensities were derived by multiplying the relative intensities by the grand mean of top three abundant PSMs.
Pathway enrichment by KEGG and gene ontology databases. Pathway enrichment analysis was carried out by the JUMPn software (v1.13.0)81 to identify the biological functions of dysregulated genes/proteins in a given dataset. The analysis was performed using Fisher’s exact test against the Gene Ontology (GO) biological process, molecular function, and cellular component annotations, and KEGG pathway database, separately. The homologous genes between human and mouse were used as the background. The p values derived from Fisher’s exact test were further adjusted into FDR using the Benjamini-Hochberg procedure for multiple testing. Enriched pathways with FDR values < 0.05 were considered statistically significant.
Protein-protein interaction (PPI) network analysis. The analysis was performed based on our previously published protocol82 with modifications. DE genes/proteins were superimposed onto a composite PPI database by combining STRING (v11)83, BioPlex (v3.0)84, and InWeb_IM (v2016_09_12)85. The BioPlex database was developed by the method of affinity purification and mass spectrometry, whereas the STRING and InWeb contain information from various sources. Due to this heterogeneity of PPI interactions, the STRING and InWeb databases were further filtered by the edge score to ensure high quality, with the following rules: (i) only edges with evidence of physical interactions (e.g. through co-IP or yeast two-hybrid) were considered; (ii) edges of high confidence, as filtered by the edge score, with the cutoff determined by best fitting the log-log degree distribution using the scale free criteria86. The finally accepted STRING and InWeb databases were combined with BioPlex and the inhibitory postsynaptic density interactome87 to construct a composite PPI database, which includes 20,485 proteins and 1,152,607 PPI connections. PPI modules were then defined by a three-step procedure: (i) extracting a subnetwork by retaining PPI between two proteins if both were from the DE protein list; (ii) calculating a topologically overlapping measure (TOM)88 between each pair of proteins for the resulting PPI subnetwork, and (iii) dividing this network into individual modules based on the TOM clustering using the hybrid dynamic tree-cutting method48. The biological functions of each PPI module were further obtained using the proteins in each module as the input to perform the pathway enrichment analysis as described above.
Quantitation and statistical analysis. In small-scale analyses, two-tailed unpaired Student’s t-test was used for two-sample comparisons (Graphpad Prism 7.0.5). Proteomics analysis for differentially expressed proteins primarily utilized the limma R package (v3.48.3) 89 in several steps: (i) obtain the protein quantification data from MS as described above, (ii) perform a log transformation of the data90, (iii) calculate the p values by moderated t-test, and FDR values by the Benjamini-Hochberg procedure, using limma R package, (iv) calculate the mean for each protein under different conditions and derive the log2(fold change), (v) fit the log2(fold change) data of all proteins to a Gaussian distribution to generate a 'global' standard deviation value. Statistically significant changes were typically determined using an P value cutoff of 0.05 and a log2 (fold change) cutoff equivalent to two standard deviations.