Mitochondrial hypermetabolism precedes impaired autophagy and synaptic disorganization in App knock-in Alzheimer mouse fashions

Animals and moral permits

Homozygous AppNL-F/NL-F (denoted herein AppNL-F) and AppNL-G-F/NL-G-F (denoted herein AppNL-G-F) [14] and Appwt/wt (WT) (all on C57BL/6 J background) have been housed at Karolinska Institutet animal facility below circumstances of managed temperature (22–23 °C) and a 12-h mild/12-h darkish cycle. Meals and water have been obtainable advert libitum. All experimental procedures have been carried out in accordance with the rules of the Institutional Animal Care and Use of Committee and the European Group directive (2010/63/EU) and authorized by Linköping Animal Moral Committee (ID 407) and Stockholm Animal Moral Committee (15758-2019). For this examine two-, six-, 10- to 12-, 15- to 18- and 22- to 24-month-old females. Variety of animals used for every experiment is described within the respective technique part and determine legends.

RNA isolation and sequencing

RNA was extracted from dissected hippocampal mind tissue (n = 3 females/genotype and age) preserved in RNAlater (AM7020, Thermo Scientific) utilizing RNeasy Lipid Tissue Mini Equipment (74804, Qiagen) in keeping with producer’s directions. RNA high quality (RNA integrity quantity, RIN) and amount was measured in a Bioanalyzer 2100 (Agilent) utilizing the Agilent RNA 6000 Nano Equipment (half quantity 5067-1511). NEBNext Extremely II Directional RNA Library Prep Equipment for Illumina (E7760S, New England Biolabs) was used to arrange the sequencing libraries, beginning with 200 ng of complete RNA. Briefly, mRNA was remoted and fragmented utilizing the NEBNext poly(A) mRNA magnetic isolation module (E7490S, New England Biolabs). The primary and second strands of cDNA have been synthesized and purified utilizing AmPure XP beads (A63880, Beckman Coulter). Adaptor ligation and dimension choice have been carried out in keeping with the producer’s protocol. Adaptor ligated cDNA was PCR enriched to include an Illumina suitable index sequence (NEBNext Multiplex Oligos for Illumina, Twin Index Primers Set1, E7600S, New England Biolabs). The libraries have been purified utilizing AmPure XP beads, the scale distribution of the libraries was analyzed with the Bioanalyzer 2100 utilizing the Agilent Excessive Sensitivity DNA Equipment (half quantity 5067-4626). Quantification of the libraries was carried out with the Qubit® 2.0 Fluorometer (ThermoFisher Scientific) and QubitTM dsDNA HS Assay Kits (Q32851, Invitrogen). Lastly, all 30 libraries have been pooled and diluted to three.5 nM for sequencing on one lane of a Hiseq 3000 sequencer (Illumina), utilizing a single learn 50 bp and twin listed sequencing technique. On common the reads have been 41.7 M to 49 M per pattern.

Sequence processing and differential gene expression evaluation

All uncooked sequence reads obtainable in FastQ format have been mapped to the mouse genome (mm10) utilizing Tophat2 with Bowtie2 possibility [16, 17], the place adaptor sequences have been eliminated utilizing trim galore earlier than learn mapping. BAM recordsdata containing the alignment outcomes have been sorted in keeping with the mapping positions. Uncooked learn counts for every gene have been calculated utilizing featureCounts from Subread package deal [18]. DEseq2 was used to carry out the evaluation of differential gene expression, the place genes with uncooked counts have been used as enter [19]. The differentially expressed genes (DEGs) have been recognized by adjusted p-value for a number of testing utilizing Benjamini-Hochberg correction with False Discovery Fee (FDR) values lower than 0.1.

Gene ontology enrichment evaluation

The considerably DEGs have been utilized to Gene ontology (GO) enrichment evaluation utilizing on-line software program AmiGO web site (, and the numerous enrichment GO phrases was recognized utilizing Fisher’s Precise check with Bonferroni corrected p-values ≤ 0.05. Identification of genes concerned in mitochondrial operate, neuroinflammation and autophagy was carried out by utilizing free on-line databases and software program together with Uniprot (, DAVID v6.8 ( and PANTHER v13.1 ( Genes included inside the GO phrases (in all ontologies: organic processes, molecular operate, mobile element) containing the phrase “autophag” have been thought of genes concerned within the autophagosomal-lysosomal system, these containing the phrase “inflamm” have been thought of inflammation-related genes, these containing the phrase “mitochondri” have been thought of to be genes associated to mitochondrial processes and used for additional enrichment evaluation.

Pathway evaluation

Gene Set Enrichment Evaluation (GSEA) [20] was utilized to carry out pathway evaluation utilizing the KEGG pathways dataset. First, genes have been ranked decreasingly in keeping with the Log 2 Fold Change (Log2FC) of expression. For every question pathway, if gene i is a member of the pathway, it’s outlined as

$${X}_{i}, = , -sqrt{frac{G}{N-G}}$$

If gene i is just not a member of the pathway, it’s outlined as

$${X}_{i}, = , sqrt{frac{N-G}{G}}$$

the place N signifies the whole variety of genes and G signifies the variety of genes within the question pathway. Subsequent, a max working sum throughout all N genes Most Estimate Rating (MES) is calculated as

$${MES}=mathop{max }limits_{1le jle N}mathop{sum }limits_{i=1}^{j}{X}_{i}$$

The permutation check was carried out 1000 instances to evaluate the importance of MES values, the place the null speculation is that the pathway is just not enriched in rating. If the question pathway with a nominal p-value lower than 0.05 and adjusted p-value for a number of testing utilizing Benjamini-Hochberg correction with FDR values lower than 0.1, the null speculation could be rejected, and the question pathway could be thought of as considerably enriched. MES worth represents the expression course of a pathway, the place a constructive MES worth signifies up-enrichment (up-regulation) whereas a unfavorable MES worth signifies down-enrichment (down-regulation) of a pathway.

Unsupervised genome-wide clustering

The uncooked learn matrix distributed column-wisely with samples names and row-wisely with gene names, was normalized utilizing between-samples normalization technique carried out in DESeq2 package deal [19]. Unsupervised genome-wide clustering was then carried out utilizing t-SNE plot with the R package deal Rtsne [21]. The t-SNE plot is predicated on the 50 most variant dimensions of the preliminary PCA plot, whereby genes with duplicate reads are filtered out. The Pace/accuracy trade-off was set as 0.0 to precise t-SNE distance matrix. The perplexity is adjusted accordingly for the optimum clusters form. Plots displaying all samples are based mostly on the t-SNE area parameters V1 and V2.

cDNA reverse transcription (RT) and qPCR

100 ng of RNA was reverse transcribed utilizing the Excessive-Capability cDNA Reverse Transcription Equipment (Thermo Fisher Sci., Cat. no: 4374966) in keeping with producer’s directions. The TaqMan Quick Superior Grasp Combine (Thermo Fisher Sci., Cat. no: 4444557) was used to carry out the qPCR utilizing the next TaqMan mouse gene expression assays (FAM) (Thermo Fisher Sci., Cat. no: 4331182): Mm04225236_g1 for Atp8; Mm00432648_m1 for Cox8b; Mm00444593_m1 for Ndufa1; Mm01352366_m1 for Sdha; Mm01615741_gH for Uqcrb; Mm01183349_m1 for Clec7a; Mm00441259_g1 for Ccl3; Mm04209424_g1 for Trem2; Mm00437893_g1 for C4b; Mm01312230_m1 for Chrna4; Mm00495267_m1 for Lamp2; Mm00523599_g1 for Rab7b; Mm01310727 for Rubcnl; Mm01274264_m1 for Trim30a; Mn00450314_m1 for Vamp8. Experiments have been carried out within the 7500 Quick Actual-Time PCR System (Utilized Biosystems) and every pattern was run in triplicates. Gene expression was normalized to β3-tubulin mRNA (Mm00727586_s1).

Mind homogenates

Proper hippocampi from the identical people used for RNA-seq experiments (n = 3/genotype and age) have been minimize in items whereas saved chilly on ice and homogenized with a 2 cm3 glass-Teflon homogenizer in RIPA buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 0.5% DOC, 0.1% SDS; pH = 7.5) supplemented with 1:100 protease inhibitors (G-Biosciences, Cat. no: 786-433) and 1:100 phosphatase inhibitors (Sigma–Aldrich, Cat. no: P0044) in a proportion of 1:15 (mg tissue/µl buffer). The homogenates have been then sonicated for 15 s on ice and centrifuged at 20,800 g for 30 min, at 4 °C, to take away cell particles. Supernatant was collected and protein focus decided by Pierce™ BCA Protein Assay Equipment (Thermo Fisher Sci., Cat. no: 23225).

Crude synaptosomal fractions

Crude synaptosomal fractions have been remoted from dissected left hippocampal mind tissues (n = 4/genotype and age) by homogenizing the tissue with a 2 cm3 glass-Teflon homogenizer at 800 rpm in lysis buffer (0.32 M sucrose, 5 mM Hepes, and 10 ml ddH 2 O) supplemented with 1:100 diluted protease inhibitors (G-Biosciences, Cat. no: 786-433) and 1:100 diluted phosphatase inhibitors (Sigma–Aldrich, Cat. no: P0044) in a proportion of 1:10 (mg tissue/µl buffer). The homogenates have been centrifuged at 1000 g at 4 °C for 10 min to take away nuclei and particles, and the supernatant was centrifuged once more at 12,000 g at 4 °C for 20 min. Supernatant composed of the sunshine membrane fraction and soluble proteins (S2) was transferred to a brand new tube, and the pellet containing crude synaptosomes and mitochondria (P2) was resuspended in 75 μl RIPA buffer supplemented with 1:100 diluted protease inhibitors and 1:100 diluted phosphatase inhibitors. The protein focus was decided by Pierce™ BCA Protein Assay Equipment (Thermo Fisher Sci., Cat. no: 23225).

Western blotting

15 to twenty µg of protein have been loaded and separated by 4–12% Bis-Tris gels (Novex, Cat. no: NP0336BOX) or 4–20% Tris-Glycine prolonged gels (BIO-RAD, Cat. no: 4561095) and transferred to nitrocellulose or methanol activated PVDF membranes. Membranes have been blocked in 5% BSA TBS-T or 5% skim milk TBS-T and incubated in a single day at 4 °C with the next main antibodies diluted in 5% BSA or 5% skim milk dissolved in TBS-T: pSer293-PDH (1:1000) (Merck Millipore, Cat. no. ABS204), PDH (1:1000) (Santa Cruz, Cat. no. sc-377092), IDE (1:900) (Abcam, Cat. no. ab32216), LC3 (1:500) (MBL, Cat. no. PM036), pSer403-p62/SQSTM1 (1:500) (Merck Millipore, Cat. no. MABC186-I), p62/SQSTM1 (1:1000) (Cell Signaling, Cat. no. 5114), Synaptophysin (1:500) (Merck Millipore, Cat. no. MAB5258), PSD95 (1:1000) (Cell Signaling, Cat. no. 3409), ULK1 (1:500) (Cell signaling, Cat. no. 8054), pSer555-ULK1 (1:500) (Cell Signaling, Cat. no. 5869), pSer757-ULK1 (1:500) (Cell Signaling, Cat. no. 14202), β3 tubulin (1:2000) (Santa Cruz, Cat. no. sc-80016). After washing with TBS-T for thrice, membranes have been incubated with fluorescent secondary antibodies (1:10,000–20,000) at room temperature for 1 h. Blots have been developed utilizing Odyssey CLx (LI-COR) and the ensuing band intensities have been quantified by utilizing Picture Studio Lite (LI-COR). n = 4/genotype.

Immunofluorescence staining

Brains have been dissected from WT and AppNL-G-F mice (n = 4/genotype) after perfusion with PBS below anesthetization by isoflurane, and stuck in 10% formalin answer (Merck Millipore, Cat. no: HT501128). Brains have been dehydrated and the paraffin-embedded brains have been sliced into 4 μm sections by a microtome (Microm HM 360). For triple staining, after deparaffinization and antigen retrieval, sections have been stained utilizing Opal 4-Colour IHC package (Akoya, NEL810001KT) in keeping with producer’s directions with the next main antibodies: LC3A and B (1:2000) (Novus, Cat. no. NB100-2220), synaptophysin (1:400) (Synaptic Programs, Cat. no. 101 002). After LC3 and synaptophysin staining, sections have been incubated in a single day at 4 °C with anti-Aβ antibody 82E1 (1:1000) (IBL, Cat. no. 10323) diluted in PerkinElmer antibody diluent/blocking buffer, adopted by incubation with anti-mouse IgG antibody conjugated with Alexa Fluor 647 (1:200) (Thermo Fisher Scientific, Cat. no. A-21235) diluted in PerkinElmer antibody diluent/blocking buffer at room temperature for 1 h. For double staining, after deparaffinization and antigen retrieval, sections have been blocked with 5% regular goat serum in PBS-T. Sections have been first incubated in a single day at 4 °C with the first antibody in opposition to LC3A and B (1:2000) (Novus, Cat. no. NB100-2220 ) diluted in 5% regular goat serum in PBS-T. After washing, sections have been incubated with biotinylated anti-rabbit IgG antibody (1:200) (Vector, Cat. no. BA-1000) diluted in 5% regular goat serum in PBS-T for 1 h at room temperature. The indicators have been amplified by TSA Fluorescein system (Akoya Biosciences, Cat. no. NEL701A001KT) in keeping with the producer’s directions. Sections have been then incubated in a single day at 4 °C with anti-Aβ antibody 82E1 (1:1000) (IBL, Cat. no. 10323) diluted in 5% regular goat serum in PBS-T, adopted by incubation with Alexa Fluor 546 conjugated anti-mouse IgG antibody (1:200) (Thermo Fisher Scientific, Cat. no. A-11030) diluted in 5% regular goat serum in PBS-T for 1 h at room temperature. Nuclei have been counterstained with Hoechst 33342 (1:1000). The photographs have been acquired utilizing Zeiss LSM980 and Nikon fluorescence microscope (Nikon, eclipse E800).

Isolation of purposeful mitochondria

Hippocampi have been dissected from mouse brains (n = 5–6/genotype and age) and washed as soon as in ice-cold PBS. Mitochondria have been remoted utilizing discontinuous Percoll density gradient centrifugation as beforehand described [22]. Briefly, hippocampi have been transferred to an 8 ml Dounce tissue grinder of the Potter-Elvehjem PTFE and homogenized roughly 6 instances in ice-cold mitochondria isolation buffer (225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM HEPES–KOH, pH 7.2) supplemented with 1 mg/ml fatty acid-free BSA. The ultimate homogenate was centrifuged at 1100 g at 4 °C for two min. The supernatant was collected and blended with contemporary 80% Percoll (GE Healthcare, Cat. no. 17-5445-02) ready in mitochondrial dilution buffer (1000 mM sucrose, 50 mM HEPES–KOH, 10 mM EGTA, pH 7.0), to acquire a 5% Percoll answer, which was additional rigorously layered on the highest of the contemporary 10% Percoll. The mitochondrial fraction was pelleted by centrifugation at 18,500 g at 4 °C for 10 min. The pellet was then resuspended in 1 ml of mitochondria washing buffer (250 mM sucrose, 5 mM HEPES–KOH, 0.1 mM EGTA, pH 7.2) and centrifuged at 10,000 g at 4 °C for five min. Mitochondrial pellet was once more resuspended in a small quantity of ice chilly mitochondria washing buffer to attain a excessive focus of mitochondria that was saved on ice for additional evaluation for a most of three h. Protein content material of remoted mitochondria was quantified utilizing Pierce™ BCA Protein Assay Equipment.

Oxygen consumption fee (OCR) analysis

OCR was evaluated utilizing the Seahorse Analyzer (Agilent) (n = 5–8/genotype and age). This assay is predicated on the fluorometric detection of oxygen (O 2 ) and H+ ranges in actual time through strong state probes in a sensor cartridge which is additional outfitted with 4 reagent supply chambers for injecting compounds through the assay. 2.5 µg of remoted mitochondria diluted in mitochondrial assay answer (MAS; 70 mM sucrose, 220 mM mannitol, 10 mM Okay 2 HPO 4 , 5 mM MgCl 2 , 1 mM EGTA, 2 mM HEPES–KOH) supplemented with 0.2% (w/v) fatty acid-free BSA have been seeded in poly(ethyleneimine)-coated (1:15,000; Sigma–Aldrich, 03880) XFe96 seahorse plates by centrifugation at 2000 g for 18 min at 4 °C. For respiratory coupling, MAS was additional supplemented with 10 mM succinate plus 2 μM rotenone to make sure state II respiration, adopted by the injection of 4 mM ADP to induce state III, which was then inhibited by addition of three.2 μM oligomycin. The addition of 4 μM uncoupler FCCP (state IIIu) displays the maximal respiratory chain exercise in addition to the maximal substrate oxidation fee. Lastly, 4 μM antimycin A was added to completely block the respiratory chain and the residual OCR. To find out electron movement exercise, i.e., sequential dedication of complexes I-IV-dependent respiration, MAS was supplemented with 10 mM pyruvate, 2 mM malate and 4 µM FCCP. Sequential injection of rotenone (2 μM; complicated I inhibitor), succinate (10 mM; complicated II substrate), antimycin A (4 μM; complicated III inhibitor) and ascorbate/TMPD (N,N,N′,N′-tetramethyl p-phenylenediamine) (10 mM/100 μM; electron donors to cytochrome C/complicated IV) have been carried out to judge particular person mitochondrial complexes exercise [22, 23]. Information was normalized to residual non-mitochondrial respiration.

Mitochondrial ATP ranges

ATP ranges have been quantified by luminescence utilizing the CellTiter-Glo® Luminescent Assay (Promega, Cat. no. G7571) (n = 5–6/genotype and age). This assay depends on the properties of a thermostable luciferase that in presence of ATP mono-oxygenates luciferin and generates a steady glow-type luminescent sign. 10 µg of remoted mitochondria have been diluted in PBS to achieve 100 µL of ultimate quantity. An ATP customary curve was carried out utilizing a serial ten-fold dilution of ATP in PBS (1 µM to 10 nM). 100 µL of CellTiter-Glo® Buffer have been added to each samples and customary curve and the plate was agitated for two min and incubated for 10 min at room temperature earlier than luminescence measurement utilizing the CLARIOstar Plus plate reader (BMG Labtech). Information have been analyzed utilizing the MARS Information Evaluation Software program.

Calcium retention capability

Calcium (Ca2+) uptake by remoted mitochondria was measured utilizing the Ca2+ delicate probe Calcium Inexperienced-5N (n = 5–6/genotype and age). Calcium Inexperienced-5N is a visual light-excitable Ca2+ indicator that doesn’t permeate the mitochondrial membrane and, due to this fact, reveals elevated fluorescence depth upon extra-mitochondrial Ca2+ binding. Briefly, 10 μg of mitochondria have been incubated in Okay+-based buffer (125 mM KCl, 0.5 mM MgCl 2 , 3 mM KH 2 PO 4 , 10 mM HEPES, 0.01 mM EGTA, 3 mM succinate, 3 mM glutamate, 0.1 mM ADP-Okay, pH 7.4) supplemented with 1 μM oligomycin and 150 nM Calcium Inexperienced-5N (Thermo Fisher Sci., Cat. no: C3737) [22, 24]. Mitochondria have been then allotted in a 96-multiwell plate and fluorescence was measured within the microplate reader Fluostar Galaxy (LabVision) by excitation at 506 nm and emission at 523 nm. After a baseline of three min, pulses of 10 μM CaCl 2 have been added to mitochondria in 3 min intervals. A lower within the exterior Ca2+ focus signifies mitochondrial Ca2+ uptake. Mitochondrial Ca2+ retention capability was calculated by the realm below the curve after CaCl 2 pulses, which signifies the quantity of extramitochondrial Ca2+ taken up by mitochondria.

Measurement of hydrogen peroxide (H 2 O 2 ) manufacturing

Ranges of H 2 O 2 have been evaluated utilizing the Amplex Purple reagent as beforehand described by us [22] (n = 5–6/genotype and age). This assay is predicated on the oxidation of 10-acetyl-3,7-dihydroxypenoxazine, which is catalyzed by horseradish peroxidase (HRP) within the presence of H 2 O 2 to supply a crimson fluorescent oxidation product, resorufin. 5 µg of remoted mitochondria have been resuspended in response buffer (100 mM sucrose, 100 mM KCl, 2 mM KH 2 PO 4 , 0.01 mM EGTA, 5 mM HEPES–KOH, pH 7.4) supplemented with 3 mM succinate, 3 mM glutamate, 0.1 mM ADP plus 10 μM Amplex Purple and 0.5 items/ mL HRP. Fluorescence depth was evaluated for 20 min (30 sec interval) within the CLARIOstar Plus plate reader (BMG Labtech) utilizing 571 nm excitation and 585 nm emission (37 °C). 2 μM Antimycin A was used as constructive management. H 2 O 2 manufacturing was quantified utilizing the slope of the curves.

Transmission electron microscopy

Ten- to twelve-month-old AppNL-G-F and 22- to 24-month-old AppNL-F mice have been anaesthetized and perfused by means of intracardial perfusion with 2% glutaraldehyde and 1% formaldehyde in 0.1 M phosphate buffer (n = 4/genotype). Hemispheres have been minimize in a mind slicer matrix and coronal slices collected for sectioning. Leica Ultracut UCT or EM UC7 was used to create ultrathin sections, with uranyl acetate and lead citrate used as contrasting brokers. For mitochondria and endoplasmic reticulum (ER) evaluation, sections have been examined at 100 kV utilizing a Tecnai 12 BioTWIN transmission electron microscope. Pictures have been acquired from the hippocampus Cornu Ammonis space 1 (CA1) at a main magnification of 30,000×. Seven completely different cells have been snapped per mind and 30–40 synapses have been analyzed per situation. A synapse was thought of when the presence of each synaptic vesicles and post-synaptic density have been detected. Synaptic vesicles have been counted utilizing the cell counter plugin from ImageJ. ER side ratio was quantified by dividing the foremost axis of the ER profile to the minor axis. For the remark of autophagic vacuole accumulation, photos have been acquired from the hippocampus CA1 at a main magnification of 16,500× or 26,500×.

CSF sampling and proximity extension assay (PEA)

Mice have been anesthetized with 1.5% isoflurane and thereafter positioned in a stereotactic instrument with 120–130° head to physique angle (n = 5/genotype). A sagittal incision was made posterior to the occipital crest. The dura mater was uncovered by a mild blunt dissection of subcutaneous tissue and muscle tissue below dissection microscope and cleaned by cotton swabs soaked in PBS to take away any blood contamination. The dura mater was punctured with a 27-gauge needle, avoiding seen blood vessels, and CSF was subsequently collected in a glass capillary tube. The collected CSF was inspected below the microscope and discarded in case of detected blood contamination. Samples have been collected in low-affinity polypropylene tubes, snap-frozen in liquid nitrogen and saved at −80 °C till additional evaluation. Ranges of 92 proteins in CSF have been analyzed utilizing Goal 96 Mouse Exploratory Panel with PEA know-how, in keeping with producer’s directions (Olink Proteomics). This twin recognition method is predicated on matched pairs of antibodies labeled with complementary DNA oligonucleotide tags, which creates a singular DNA barcode for every antibody pair that’s amplified by qPCR.

Statistical evaluation

Statistical evaluation was executed by utilizing GraphPad Prism model 9 and information have been introduced as imply ± SEM. n refers back to the variety of distinctive particular person organic samples. Since all samples included on this report have been derived from animals, the variety of animals used have been determined based mostly on a compromise of fulfilling the three R suggestion of lowering the variety of animals used and our earlier expertise of the experimental variation noticed within the utilized methods. No energy evaluation was carried out. No blinding nor randomization of samples have been carried out. Non-parametric Kruskal-Wallis assessments adopted by Dunn’s a number of comparability check or unpaired t-test (two tailed) have been carried out as indicated within the particular person determine legends the place p-values are reported. Impact dimension ± SEM is given in determine legends for statistically important values. Outliers have been detected utilizing ROUT technique (Q = 1%).

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