Animals
We used 5XFAD mice (Tg6799; Jackson Laboratory, Stock #006554) that express both mutant human amyloid precursor protein (APP) with the Swedish mutation (K670N, M671L), the Florida mutation (I716V), and the London mutation (V717I) and mutant human presenilin1 (PS1) with M146L and L286V mutations under transcriptional control of mouse Thy-1 promoter. All experiments were performed using female 5XFAD mice. 8-week-old C57BL6 male mice were used for virus injection. All animal experiments and management procedures were performed as outlined in the guidelines of the Institutional Animal Care and Use Committee of Seoul National University.
Real time q-PCR
Total RNA was prepared by using a RNeasy Plus Mini kit (QIAZEN). cDNA was synthesized from 100-200 ng/μl of total RNA by using a Maxime RT PreMix kit (iNtRON Biotechnology, Korea). The real time q-PCR was carried out using KAPA SYBR FAST ABI Prism qPCR kit (KAPA Systems). mRNA levels of interested genes were normalized with 18S ribosomal RNA (18S rRNA) levels as a housekeeping gene. PCR primers used in the study were listed in Additional file 1: Table S1.
Real-time oxygen consumption rate measurement
Oxygen consumption rate (OCR) was measured using a Seahorse XF24 analyzer (Seahorse Bioscience). 4 × 104 cells were placed on XF24 cell culture microplates and cultured. The cartridge plate was reacted with XF Calibrant buffer for one day (37 °C, CO2 free); Analytical medium (1 mM pyruvate, 4 mM glutamine and 25 mM glucose added to the XF basal medium) was prepared immediately before the analysis.
Immunohistochemistry (IHC)
After anesthetizing the mice with cold PBS, brains were extracted and reacted at 4 °C for 24 hrs in 4 % PFA solution. Then, it was reacted at 4 ℃ for 72 hrs in a 30 % sucrose solution. Brain tissue was cut to 30 μm thickness using Cryostat (Leica) and used in the experiment. Brain sections with hippocampal sections were washed with PBS and then reacted with a solution containing BSA, 0.3 % Triton X-100 and 5% Horse serum to reduce nonspecific responses and enhance antibody permeability. Subsequently, the primary antibody for the target protein was reacted at 4 °C for one day. The proportion of primary antibodies used is as follows; Iba-1 (1:1,000, Wako), 4G8 (1:500). After the reaction with the primary antibody, the IgG secondary antibody (1:500, Invitrogen) conjugated with Alexa-488, 594 or 647 was used at room temperature for 1 hr. DAPI (0.4 μg/ml) was stained for 10 min for the purpose of staining confirmation. Stained brain sections were taken using an LSM 700 confocal laser scanning microscope (Carl Zeiss) and images were analyzed via ImageJ software.
Behavioral analysis experiments
1) Y-maze test
Mice in the control and experimental groups were subjected to spatial adaptation and habituation in a Y-shaped maze one day before the test. In the next day, the Y labyrinth was freely explored for 8 min under the same experimental conditions. The results of the test were analyzed by measuring the percentage of the total number of entrances into the maze, alternating with three different types of mazes (entry without overlapping like ABC).
2) Novel object recognition (NOR) test
Mice injected with LV-scramble siRNA or LV-Kdm5a siRNA were habituated to an empty rectangular box for 10 min on day 1. On day 2, two identical objects were placed at the corner of the box, 5 cm away from the same wall, and then individual mice were allowed to explore freely in the box for 10 min. After 4 hrs, one familiar object was replaced with a novel object, and we then measured the time exploring each object, respectively for 8 min. Recognition memory was assessed by the discrimination index, which was analyzed by the percentage of the total time to the exploration time for a novel object during the test session.
3) Passive avoidance task (PAT)
Mice injected with LV-GFP or LV-mitoAβ were allowed to explore in the chamber which is divided into a lighted and dark compartment with a gate. In the acquisition trial, each mouse experienced an electronic shock (0.7 mA, 3 s) only in the dark compartment. On the following day, mice were placed back and explored in the chamber for 300 s. Learning and memory were evaluated by the measurement of the latency time to cross between the compartments.
Transmission Electron Microscope (TEM) Shooting
Hippocampal tissue and HT22 cells expressing mitoAβ were fixed for one day in a mixture of cold 2.5 % glutaraldehyde diluted in 0.1 M phosphate buffer (pH 7.2) and 2 % paraformaldehyde diluted in 0.1 M phosphate or cacodylate buffer (pH 7.2). After the reaction, the tissue and cells were immersed using only epoxy resin, and the samples were loaded into capsules and polymerized for 12 hrs at 38 °C and 48 hrs at 60 °C. Ultramicrotome (RMC MT-XL; RMC Products) was used to cut into thin sections and collected in a copper mold. Thin sections were cut at 65 nm, stained with 4 % uranyl acetate and 4 % lead citrate, and photographed by transmission electron microscopy (JEM-1400; Japan) at 80 Kv.
Mitochondrial ROS Measurement
Mitochondrial-derived reactive oxygen species (ROS) were visualized using MitoSOX Red Mitochondrial Superoxide Indicator (M36008, Invitrogen). The dye is mixed in the cell culture medium at a final concentration of 5 μM. The reaction was carried out at 37 °C for 10 min and then washed with PBS. Fluorescence signals were imaged using fluorescence microscopy (EVOS FL Auto2, Invitrogen).
Mitochondrial membrane potential measurement
The cell culture medium is removed, and TMRM (Invitrogen) is mixed and treated with the cells so that the final concentration is 100 nM in the cell culture medium. After reacting for 30 min at 37 ℃ was photographed using a fluorescence microscope (Olympus).
α-ketoglutarate assay
The amount of α-ketoglutarate was measured using the α-ketoglutarate assay kit (Abcam, ab83431) following the manufacturer’s instructions. ReN CX cells expressing GFP or mitoAβ were homogenized with 500 μl Alpha KG assay buffer by pipetting. Cell lysates were centrifuged at 13,000 x g for 5 min, and the resulting supernatant was used for further procedures. To remove proteins, perchloric acid (PCA) solution was added to the homogenate solution (final concentration, 1 M), and the solution was centrifuged at 13,000 x g for 2 min. 2 M KOH solution (34 % of sample volume) was added to precipitate remained PCA, and neutralized sample was centrifuged at 13,000 x g for 15 min. 50 μl of α-ketoglutarate standard or cell lysates were mixed with the reaction mixture including probe and converting enzyme. After incubation at 37 ℃ for 30 min, the results were measured on a PowerWave XS microplate reader (BioTek) at OD570 nm.
Mitochondria fractionation
In order to separate the cytoplasm and mitochondria, the cells were placed in a solution containing 20 mM HEPES, pH 7.5, 250 mM sucrose, 20 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, using a dounce homogenizer. Cell lysates were centrifuged at 600 x g for 10 min to remove undissolved cell debris and nuclei. The supernatant was again centrifuged at 7,000 x g for 10 min at 4 °C, and the resulting pellet was collected as a mitochondria concentrated fraction.
Immunocytochemistry
Cells washed with PBS are fixed in PFA solution for 15 min in 4 % PFA solution. After washing sufficiently with PBS, the antibody was reacted with a solution containing 0.3 % Triton X-100 for 1 hr to improve the accessibility of the antibody. The primary antibody, KDM5A or GFP antibody was added and reacted at 4 °C for one day. After washing with PBS the next day, the cells were reacted with a fluorescent-linked secondary antibody (1:500, Invitrogen) at room temperature for 1 hr. DAPI was stained for 10 min for the purpose of staining confirmation. Labeled cells were transferred to glass slides and photographed with confocal microscopy (LSM700, ZEISS).
Construction of pLentiSyn1.41g-mitoAβ
To prepare the pLentiSyn1.41g-mitoAβ construct, a 222-bp of mitoAβ gene was amplified from pcDNA-mitoAβ by PCR using the following primers: mitoAβ-EcoR V-F, 5‘-CGGATATCATGTCCGTCCTGACGCCGCT-3’ and mitoAβ-EcoR V-R, 5‘-GCGATATC CTACGCTATGACAACACCGC-3’. The resulting PCR fragment was digested with EcoR V and ligated into a lentiviral vector pLentiSyn1.41g under the control of the murine cytomegalovirus immediate early gene promoter. The pLentiSyn1.41g encodes GFP which is expressed by human synapsin 1 promoter.
roduction of pLentiSyn1.41g-mitoAβ
Control and mitoAβ lentivirus were produced as previously described1. Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone Laboratories Inc., Logan, UT, USA) with 10 % fetal bovine serum (Gibco-BRL, Invitrogen, Grand Island, NY, USA) and maintained in a 5 % CO2 incubator at 37 °C. The lentivirus particles were produced by co-transfection of HEK 293T cells with the following three plasmids using Lipofectamine Plus (Invitrogen): VSV-G, gag-pol, and pLentiSyn1.41g-mitoAβ. At 48 hrs post-transfection, culture supernatants containing virus particles were collected and clarified with a 0.45 μm membrane filter (Nalgene, USA) and stored in a -70 °C deep-freezer immediately. Titers were determined with p24 enzyme-linked immunosorbent assays (Perkin-Elmer Life Science) or western blot analyses using a monoclonal anti-p24 antibody (obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health, Bethesda, MD, USA). In our routine preparation, the titers were ≒ 107 transduction units (TU) per mL without further concentration. For stereotaxic injection, the lentivirus particles were concentrated by ultracentrifugation on a 20 % sucrose cushion (2 hrs at 50,000 × g) at 4 °C.
Stereotaxic injection
8-week-old C57BL/6 male mice were anesthetized with zolazepam and tiletamine (30mg/kg; Virbac), mixed with xylazine (10mg/ml; Bayer Korea), and then fixed in a stereotaxic instrument (Neurostar, Germany). Coordinate for the injection was set at the AP axis -2.0 mm, the ML axis 1.3 mm and the DV axis 2.1 mm from bregma of the skull. A total volume of 2 μl (1×107 titer of lentivirus) was injected into the dorsal DG of the hippocampus, using an automatic microinjector, at a rate of 0.2 μl/min. After reaching the desired coordinates, a Hamilton syringe was left in place for 10 min to prevent the backflow of the virus liquid.
Hippocampal neural stem cell culture
Hippocampal neural stem (HCN) cells derived from adult rat were cultured at 37℃ and 5% CO2 on dishes coated with 10 mg/mL poly-L-ornithine (Sigma-Aldrich, P3655) and 5 mg/mL mouse laminin (Corning, 354232), and maintained on Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Invitrogen, 12400-024) containing 5 mg/L insulin (Sigma-Aldrich, I6634), 100 mg/L apo-transferrin (Prospec, PRO-325), penicillin (100 U/L)/streptomycin (100 mg/L) (Hyclone, SV30010), 16 mg/L putrescin (Sigma-Aldrich, P5780), 30 nM sodium selenite (Sigma-Aldrich, S1382), 20 nM progesterone (Sigma-Aldrich, P0130), and 20 ng/ml basic fibroblast growth factor (bFGF) (Peprotech, #100-18B) [19]. For differentiation, HCN cells were plated onto coated dishes at a density of ~ 0.5×105/cm2, and 24 hrs later, culture medium was changed with media containing 1 µM retinoic acid (Enzo, BML-GR100), 5 µM forskolin (Enzo, BML-CN100), and 0.1% fetal bovine serum (Tissue Culture Biologicals, 101) [20]. The medium was changed half every 2 days until experiment.
Chromatin immunoprecipitation (ChIP)
ReN CX cells or differentiated cells (after 5 days) were fixed with 1 % formaldehyde at room temperature for 10 min. After cross-linking reactions, the fixation was stopped by adding 1 M glycine solution to be at a final concentration of 125 mM. The solution was centrifuged at 3,000 rpm for 5 min, and the resulting pellet was then washed 3 times with ice-cold phosphate-buffered saline (PBS). Cell pellets were suspended with lysis buffer (sc-45000, Santa Cruz) and centrifuged at 3,000 rpm for 5 min. Crude nuclear extract was re-suspended with high salt lysis buffer (sc-45001, Santa Cruz) and sonicated 15 times by 30 s burst, each followed by 1 min incubation on ice. The sample was centrifuged at 14,000 rpm for 10 min, and then the supernatants containing chromatin were diluted 1:10 with the dilution buffer containing 0.01 % SDS, 1.1 % Tritonx-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl, and proteinase inhibitor cocktail. For preclearing the chromatin samples, 50 μl Protein A/G PLUS-Agarose beads (sc-2003, Santa Cruz) were added to the samples and incubated at 4 ℃ for 30 min. To precipitate agarose beads, the chromatin solution was centrifuged at 12,000 rpm for 20 s. 5 – 10 μg antibodies were added in the precleared chromatin solution and incubated overnight at 4 ℃. On the following day, 50 μl Protein A/G PLUS-Agarose beads were added and incubated at 4 ℃ for 2 hrs. To collect the beads, the solution was centrifuged at 12,000 rpm for 20 s. After the beads were washed with high salt lysis buffer and wash buffer (sc-45002, Santa Cruz), the beads were suspended with 500 μl of elution buffer with 20 μl of 5 M NaCl. Cross-linking was reversed by heating at 65 ℃ for 4 hrs. To isolate DNA fragments, the supernatants were mixed with 500 μl phenol/choloroform solution
Library preparation and sequencing
The construction of library was performed using NEBNext® UltraTM DNA Library Prep Kit for Illumina (New England Biolabs, UK) according to the manufacturer’s instructions. Briefly, the chipped DNA was ligated with adaptors. After purification, PCR reaction was done with adaptor-ligated DNA and index primer for multiplexing sequencing. Library was purified by using magnetic beads to remove all reaction components. The size of library was assessed by Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands). High-throughput sequencing was performed as paired-end sequencing (101 bp) using HiSeq 2500 (Illumina, Inc., USA).
ChIP sequencing data analysis
The reads were trimmed and aligned using Bowtie2 (Langmead and Salzberg, 2012). Bowtie2 indices were generated from genome assembly sequence or the representative transcript sequences during the alignment of the reads to the genome or transcriptome, respectively. With the mapped reads, we used MACS2 (Model-base Analysis of ChIP-seq) for identifying the peaks representing DNA binding of KDM5A. Transcription factor binding sites enriched in the peaks were identified using HOMER (Hypergeometric Optimization of Motif EnRichment), a software suite for ChIp-Seq analysis. Gene classification was performed based on the results from functional enrichment analysis using g:Profiler (https://biit.cs.ut.ee/gprofiler/).
Protein extraction
Collected hippocampal tissues from two control (LV-GFP) and two Aβ (LV-Mito-Aβ) groups were individually pulverized using Covaris CP02 Cryoprep device (Covaris, Woburn, MA). Briefly, each tissue piece was placed in a Covaris tissue bag (Covaris, 430487), and the bag was placed in a liquid nitrogen for 30 s. The frozen tissue was subsequently pulverized at impact level 2. We then added 500 µl of lysis buffer (4%SDS in Tris-HCl pH 7.6 and phosphatase inhibitor) to the tissue powder, which were subsequently sonicated using a probe sonicator (Q55, QSONICA, Newtown, CT) at 30 W on ice for 30 s. The tissue lysate was centrifuged at 16,000 x g for 10 min, and supernatants was transferred to a siliconized low-retention tube. Protein concentration was then measured using the BCA protein assay (Pierce, Rockford, IL).
Protein digestion
All hippocampal proteins were digested by the filter aided sample preparation (FASP) method [21] with a slight modification. Briefly, hippocampal proteins were reduced with SDT buffer (4 % SDS in 0.1 M Tris-HCl pH 7.6 and 0.1 M DTT) at 37 °C for 45 min by shaking at 300 rpm followed by boiling for 10 min at 95 °C on a thermomixer (Eppendorf, Hamburg, Germany). The protein sample was centrifuged at 16,000 x g for 5 min and transferred to a YM-30 microcon filter, in which the protein sample was mixed with 200 µl of UA buffer (8 M urea in 0.1 M Tris-HCl pH 8.5). The protein samples were then centrifuged at 14,000 x g at 20 °C for 60 min to remove SDS. This step was repeated three times. After alkylation for 25 min with 100 μL of 0.05 M iodoacetamide in 8 M urea at room temperature in the dark, the filter was centrifuged at 14,000 x g for 30 min, followed by washing with 200 µl of UA buffer four times. 100 μL of 0.05 M ammonium bicarbonate was added to the filter before it was centrifuged at 14,000 x g for 30 min for buffer exchange. This step of buffer exchange was repeated twice. Subsequently, sequencing grade modified trypsin (Promega; Madison; WI) was added to protein samples on the filter at an enzyme to protein ratio of 1: 50 (w/w), and the proteins were digested overnight at 37 °C. After the first digestion, the second digestion was carried out with trypsin (1: 100 w/w) at 37 °C for 6 hrs. The concentration of hippocampal peptide samples was measured by the BCA assay.
Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) labeling and fractionation
The hippocampal peptide samples from two LV-GFP and two LV-mitoAβ injected groups (700 µg each) were labeled with 4-plex iTRAQ reagent (AB Sciex, Foster City, CA) according to the manufacturer’s instructions. The two LV-GFP groups were labeled with 114/116 iTRAQ reagents, respectively, while two LV-mitoAβ groups were labeled with 115/117 iTRAQ reagents, respectively. All labeled peptide samples were then pooled and dried by vacuum centrifugation. The pooled iTRAQ labeled peptide sample was fractionated using Agilent 1269 Infinity HPLC system (Agilent, Palo Alto, CA) as previously described [22]. The Xbridge C18 column (4.6 mm × 250 mm, 130 Å, 5 µm, Waters, Milford, MA) and a guard column (4.6 mm × 20 mm, 130 Å, 5 µm) were used for the peptide separation. The peptide fractionation was performed with a 115 min gradient at a flow rate of 500 µL/min: at 0 % solvent B [10 mM TEAB in 90 % ACN (pH 7.5)] for 10 min, from 0 % - 5 % solvent B in 10 min, from 5 % to 35 % in 60 min, from 35 % - 70 % in 15 min, at 70 % solvent B for 10 min, from 70 % - 0 % solvent B in 10 min, and at 0 % solvent over 15 min. A total of 96 fractions were collected in every 1 min from 15 min to 110 min, and they were non-contiguously concatenated into 24 fractions (i.e., #1-#25-#49-#73, #2-#26-#50-#74, …, #24-#48-#72-#96). The resultant 24 fractions were dried by vacuum centrifugation. 10 µg of each fraction was used for global proteome profiling, and the remaining samples were used for phosphopeptide enrichment by immobilized-metal affinity chromatography (IMAC) [22]. For IMAC experiment, the 24 fractions were further concatenated into 12 fractions by pooling two adjacent fractions (i.e., #1-#2, #3-#4, …, #23-#24) before drying and storing at -80 °C.
Phosphopeptide enrichment
For phosphopeptide enrichment, 1.5 mL of Ni-NTA bead slurry (36113, Qiagen, Valencia, CA) was washed three times with 1.2 mL of deionized water (DIW), and Ni2+ ions were removed from NTA bead by adding 1.2 mL of 100 mM EDTA (pH 8.0) and mixing for 30 min on an end-over-end rotator (SB3, Stuart). After removal of the EDTA solution, NTA beads were washed three times with 1.2 mL of DIW and then reacted with 1.2 mL of 10 mM aqueous FeCl3 solution for 30 min on an end-over-end rotation. The Fe3+-NTA beads were washed with 1.2 mL of DIW three times and resuspended in 1.2 mL of 1:1:1 ACN/MeOH/0.01 % acetic acid for aliquoting into 12 microcentrifuge tubes and each of the Fe3+-NTA beads was washed with 400 µL of binding buffer (80 % ACN/0.1% TFA). Each of the 12 fraction samples was then resuspended in 500 µL of binding buffer and individually transferred to a tube of the aliquoted Fe3+-NTA beads. The binding reactions of the 12 aliquoted peptide samples were performed in parallel for 30 min on an end-over-end rotation. After the binding reaction, the beads were washed with 500 µL of binding buffer for four times. The bound phosphopeptides were eluted by incubating in 125 µL of 1:1 ACN/2.5 % ammonia in 2 mM phosphate buffer (pH 10) for 1.5 min. The eluted phosphopeptides were acidified with 10 % TFA to pH 3.5 - 4.0 immediately before drying.
LC-MS/MS experiments
The 24 fraction global peptide and 12 fraction phosphopeptide samples from hippocampal tissues were individually analyzed using a proteome profiling platform (at the Center for Proteogenomic Research, kore) that consists of a dual-online UPLC system (Waters, Milford, MA) [23] online coupled with Q-Exactive orbitrap mass spectrometer (ThermoScientific, Bremen, Germany). The dual-online UPLC system was equipped with two analytical columns (75 μm i.d. x 360 μm o.d, 100 cm) and two solid phase extraction (SPE) columns (150 μm i.d. x 360 μm o.d, 3 cm), and all columns were manufactured by slurry packing of C18 materials (3 μm diameter, 300 Å pore size, Jupiter, Phenomenex, Torrance, CA, USA) as previously reported [24]. The column was heated to 60 °C, and a flow rate was set to 300 nL/min [24]. The global peptides were separated by a 180 min linear gradient (1-40 % solvent B over 160 min, 40 - 80 % over 5 min, 80 % for 10 min and holding at 1 % for 5 min), and phosphopeptides were separated by a 240 min gradient (1 % to 50 % solvent B over 220 min, 50 % - 80 % solvent B over 5 min, 80 % solvent B for 10 min and holding at 1 % for 5 min). The solvent A and B were 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile, respectively. The eluted peptides from LC were ionized at 2.4kV, and the desolvation capillary temperature was kept at 250 °C. Full MS data was acquired between 400 and 2,000 m/z at the resolution of 70,000, and the automated gain control (AGC) target value was set to 1.0 x 106 with a maximum ion injection time of 20 ms. The tandem mass (MS/MS) data were obtained using a data-dependent acquisition (DDA) mode, and isolation of the most abundant top 10 ions was performed within ± 0.8 Th window. The isolated precursor ion was fragmented at a normalized collision energy (NCE) of 30 for higher energy collisional dissociation (HCD). The resolution of MS/MS data was set to 17,500, and AGC target value was to 1.0 x 106 with a maximum ion injection time of 60 ms.
LC-MS/MS data analysis
The LC-MS/MS data from global proteome and phosphoproteome profiling experiments were processed with the PE-MMR (Post-experiment monoisotopic mass refinement) method as previously described [25]. The resultant tandem mass data were searched against a mouse uniprot database (April, 2014; 51,388 entries) through MS-GF+ (v9387) search engine [26] with the search parameters: Semi tryptic, 10 ppm precursor mass tolerance, iTRAQ (+144.102063 Da) modification of lysine and N-termini and carbamidomethylation (+57.0214 Da) of cysteine as static modifications; and methionine oxidation (+15.994915 Da) as a variable modification. For phosphopeptide search, phosphorylation (+79.966) to serine/threonine/tyrosine was set as an additional variable modification. The identified phosphopeptides were further subjected to the unique mass class (UMC) filtering to localize the sites of phosphorylation as previously described [27] . The search results from the 24 MS/MS data sets of global proteome and the 12 MS/MS data sets from phosphoproteome were combined, respectively. Peptides were finally identified from the peptide spectrum matches (PSMs) with the false discovery rate (FDR) less than 0.01 and used for the downstream analyses. The identified non-redundant peptides were used to obtain protein groups by a bipartite graph analysis [28]. Among the component proteins of each protein group, the protein of the highest number of associated peptides was selected as the representative of the protein group. When multiple proteins had the same number of non-redundant peptides, the protein of a higher protein sequence coverage was chosen as the representative protein. Additionally, a protein having any unique peptides was selected as a separate representative protein. For global proteome analysis, two or more sibling non-redundant peptides were required for protein group identification. In the case of the phosphoproteome analysis, all protein groups identified by the phosphorylated peptides were used.
Identification of differentially expressed peptides and proteins
The intensities of the iTRAQ reporter ions for hippocampus samples from two LV-GFP and two LV-Mito-Aβ injected mice were normalized using the quantile normalization method [29]. Using the normalized intensities of each peptide, we performed Student t-test and calculated log2-median-ratios (fold-changes) for LV-Mito-Aβ to LV-GFP groups. We next estimated the null hypothesis distributions of these values by performing random permutations of the samples and computing t-statistic values and log2-median-ratios as previously described [30]. An adjusted p value was calculated for a t-statistic value for each peptide by two sample test using the empirical distribution. Differentially expressed (DEpeptides) and phosphorylated peptides (DPpeptides) were then identified as the peptides with p < 0.05 from the two sample t-test and log2-median-ratios > 95th percentiles in its empirical null distribution (1.32- and 1.58-fold for global and phosphoproteome profiles, respectively). The DEPs were identified as the proteins with at least two unique DEpeptides showing consistent up- or down-regulation in LV-Mito-Aβ injected mice. DPpeptides with more than two spectral counts were used for the subsequent analyses, and differentially phosphorylated proteins (DPPs) were defined as the proteins containing the DPpeptides.
GOBP association analysis.
We built a network model describing the links among the 586 GOBPs enriched by the three sets of proteins affected by LV-mitoAβ: 1) 281 up- and 2) 218 down-regulated proteins and 3) 191 DPPs. For each pair of the 586 GOBPs (GOBP1 with n proteins, GOBP2 with m proteins, and k shared proteins between GOBP1 and 2), we computed the similarity score as 2k/(n+m) and connected the two GOBPs with the similarity score > 0.52 (99 % of the null distribution for the similarity score). This procedure resulted in 43 connected subnetworks, called functional modules. The importance (weighted degree) of GOBPs in each module was estimated by summing the similarity scores of its interactors in the corresponding subnetwork. The GOBPs were then ranked by their weighted degrees such that the top-ranked GOBP has the largest weighted degree (i.e., a hub-like term). Also, the key GOBPs should be highly enriched with up- and down-regulated proteins and DPPs. Thus, we evaluated the enrichment of the GOBPs in each module using EASE scores obtained from DAVID [31]. The GOBPs in each module was then ranked such that the top-ranked GOBP has the largest enrichment score. After summing the two ranks from the weighted degrees and the enrichment scores, finally, the key GOBP in each module was selected as the top-ranked one based on the summed ranks.
Statistical analysis
All values are expressed as mean ± standard error of the mean (SEM) from. The significant differences between two groups were analyzed by Student’s unpaired t test. In three or more groups the significant differences were evaluated by one-way ANOVA or two-way ANOVA test. Statistical significance is displayed as: N.S., not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.