TREM2 splice isoforms generate soluble TREM2 species that disrupt long-term potentiation

TREM2 expression analysis using AMP-AD datasets

The generation of RNA-Seq data in the Accelerating Medicines Partnership for Alzheimer’s Disease Consortium (AMP-AD) and demographic information has been previously described in detail [33]. Briefly, RNA-Seq data were downloaded from the (AMP-AD) through the Synapse database (https://www.synapse.org/): the Mayo Clinic Brain Bank (Mayo Clinic) [34], the Mount Sinai Medical Center Brain Bank (MSBB) [35], and the Religious Orders Study and Memory and Aging Project (ROS/MAP) cohorts [36].

In the Mayo Clinic, RNA-Seq data were generated from the temporal cortex and cerebellum. In the MSBB, RNA-Seq data were generated from the parahippocampal gyrus, inferior frontal gyrus, superior temporal gyrus, and frontal pole. In ROSMAP, RNA-Seq data were generated from the dorsolateral prefrontal cortices. The procedures for sample collection, post-mortem sample descriptions, tissue and RNA preparation methods, library preparation and sequencing methods, and sample quality controls were previously described in detail [34,35,36,37,38,39]. We converted each mapped BAM file into a FASTQ file using samtools v.1.9 and then re-mapped the converted FASTQ files onto the hg19 human reference genome using STAR aligner v.2.5, as previously described in detail [40]. Using the processed RNA-Seq data, we identified TREM2 splice transcripts and calculated their expression levels. We used the software tool RSEM to accurately estimate the TREM2 transcripts expressions from RNA-Seq [41]. RSEM generates three different TREM2 transcript sequence references, and RNA-Seq reads are mapped to them. After the alignment of reads, RSEM uses a statistical model to accurately calculate transcript abundances by estimating a maximum likelihood (ML) based on expectation-maximization (EM) algorithm. Additionally, by utilizing paired-end reads to classify the different isoforms, RSEM improves the estimation of the relative isoform levels within single genes. Based on RSEM’s statistical model and additional benefits, it accurately estimates transcript abundances from reads mapped to distinct and shared regions among the three isoforms. Differential expression analysis of the TREM2 splice transcripts between cognitively normal controls and AD patients was done using a generalized linear regression model [33]. The regression was performed with the “glm” function of the stats package in R (version 3.6.1). Age and sex were used as covariates. We used the false discovery rate (FDR) to correct for multiple testing.

Mouse models

5xFAD mice were obtained from the Jackson Laboratory (Stock #34840-JAX) and express five human familial Alzheimer’s disease mutations driven by the mouse Thy1 promoter (APPSwFlLon, PSEN1*M146L*L286V]6799Vas) [42]. The APPPS1–21 mouse model expresses the Swedish APP mutation (KM670/671NL) and the L166P mutation in PSEN1 driven under the Thy-1 promoter [43]. Cortical RNA from the transgenic mouse model that harbors the human TREM2 BAC (control B6hTREM2 and 5xFADhTREM2) [35] was kindly provided by Dr. Daniel Lee and Dr. William Yang (Semel Institute, UCLA). To collect brain tissue, mice were anesthetized with 1.2% 2,2,2-tribromoethanol (Avertin), perfused with ice-cold phosphate-buffered saline (PBS), and the brain removed. Brain tissue was used for RNA extraction. All animals were maintained, and experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Indiana University School of Medicine.

Human samples

Postmortem brain tissue (middle frontal gyrus) from control and AD subjects were provided in the form of frozen blocks by the Brain Resource Center at Johns Hopkins. AD samples were examined at the Division of Neuropathology at John Hopkins University and consisted of pathologically severe AD, stages V–VI (Additional file 1: Table S1). The brains were kept at − 80 °C until used.

RNA extraction and quantitative real-time PCR

Homogenates from 20 mg of human postmortem tissue were used for RNA isolation (Qiagen #74104); 200 ng of RNA was converted to cDNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems 4368814). Murine tissue was homogenized in buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Sigma Aldrich, P8340) and mixed in an equal volume of RNA-Bee (Amsbio, CS-104B). RNA was isolated using phenol-chloroform extraction and a Purelink RNA Mini Kit (Life Technologies, 12183020) with an on-column DNAse Purelink Kit (Life Technologies, 12183025); 500–1000 ng of RNA was converted to cDNA with the High Capacity RNA-to-cDNA kit (Applied Biosystems, 4388950). qPCR was performed on StepOne Real-Time PCR System with PowerUp™ SYBR™ (Thermo Fisher Scientific A25742). The mRNA levels of mouse Trem2 and human TREM2 were normalized to Gapdh and GAPDH, respectively, and expressed as fold changes relative to controls, using the ΔΔCt method. Both mouse and human TREM2 transcripts sequences were retrieved from NCBI, and specific nucleotide sequences for each transcript were identified by analyzing the sites of splicing events (exon skipping, frameshift, and sequence insertions). These unique sequences were used to design the primers that amplify specific amplicons for each transcript (Fig. 1B). The primer sequences are available in Additional file 1: Table S2. The qPCR reactions were performed in a StepOnePlus™ Real-Time PCR (Applied Biosystems) with 40 amplification cycles with each cycle consisting of 95°C for 15 s followed by 1 min at 60 °C for all isoforms, except for the human TREM2230, for which we used a temperature of 68 °C.

Fig. 1figure 1

TREM2 isoforms expression increases in Alzheimer’s disease. A Expression analysis of TREM2 isoforms (TREM2230, TREM2222, and TREM2219) in different brain regions in control subjects (Control) and patients with late-onset Alzheimer’s disease (AD) using the AMP-AD dataset. B Design of qPCR primers to specifically detect TREM2 isoforms. C qPCR expression analysis of TREM2 isoforms in middle frontal gyrus tissue of control subjects (C) and patients with Alzheimer’s disease (AD). Statistical analysis was performed by the Student t-test for TREM2230 and Mann-Whitney test for TREM2222 and TREM2219. D qPCR expression analysis of TREM2 isoforms in brain cortical tissue of 7-month-old control and 5xFAD mice expressing human TREM2 gene (B6hT2and 5xFADhT2). Animals from both sexes were analyzed together (3 males and 1 female B6hT2; 3 males and 3 females 5xFADhT2). Statistical analysis was performed by the Student t-test. Data are expressed as mean values ± SEM (**P < 0.01; ***P < 0.001)

Plasmid design, cell culture, and transfection

Custom TREM2 plasmids were generated by GeneArt Gene Synthesis (Thermo Fisher Scientific) in the pcDNA3.1(+) backbone. The plasmids encoded for human and mouse TREM2 isoforms harboring a N-terminus HA tag and a C-terminus FLAG tag connected with short linker sequences (Additional file 1: Table S3). The human embryonic kidney 293T (HEK-293T) and human microglia cell line (HMC3) were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) high glucose, with GlutaMAX™ (Gibco, 10566024), supplemented with 10% fetal bovine serum (FBS) (Gibco, 16000) and 1% penicillin-streptomycin (Gibco, 15140). TREM2 plasmids were transfected into both cell types using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) according to the manufacturer’s protocol. HMC3 cells were transfected for 24 h and used for immunocytochemistry. HEK-293T transfected for 24 h were used for RNA extraction using the DNAse Purelink Kit, followed by cDNA conversion and qPCR as described above. HEK-293T transfected for 48 h were maintained with media without FBS after transfection, and protein extracts were collected from cells and media.

Immunocytochemistry

Cells were washed with PBS and fixed with paraformaldehyde 4% in PBS for 15 min at room temperature. Subsequentially, cells were washed with PBS and incubated with blocking buffer (5% BSA, 0.1% Triton, and 0.1% Tween-20) for 1 h at room temperature, followed by overnight incubation with primary antibody for HA 1:500 in blocking buffer (Cell signaling, C29F4) at 4 °C. Afterwards, cells were washed with PBS and incubated with secondary antibody (Alexa Fluor™ donkey anti-rabbit) 1:1000 and DAPI (1 μg/ml) in blocking buffer for 1 h at room temperature. Cells were washed with Tween 0.01% in PBS and mounted with ProLong™ Gold Antifade Mountant (Thermo P36930). Images were taken using the Nikon AR1 confocal microscope.

Protein extraction

Cells were lysed using a lysis buffer containing 1% Triton-X 100, 50 mM Tris-HCl pH = 8, and 150 mM NaCl, sonicated followed by centrifugation at 12.000g for 15 min at 4 °C. Human brain tissue was mechanically homogenized with a glass tissue homogenizer (Dounce) in Tris-buffered solution (TBS—150 mM NaCl, 50 mM Tris-HCl, pH 7.6) or RIPA (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA), followed by sonication and centrifugation for 10 min, and the supernatant (soluble fraction) was collected. All lysis buffers were supplemented with protease and phosphatase inhibitors. Protein concentration was determined with a BCA kit (Thermo Scientific).

We used trichloroacetic acid (TCA) to precipitate proteins from transfected HEK-293T-conditioned media without FBS. Conditioned media was filtered through a 0.2-μm pore size filter and mixed with TCA 20% in the ratio of 50:50 (volume), followed by incubation on ice for 1 h. After that, samples were centrifuged at 4 °C for 5 min at 10.000g, and the supernatant was discarded. The pellets were washed with ice-cold acetone followed by centrifugation at 4 °C for 5 min at 10,000g. The washing step was performed 3 times, the pellet was dried at room temperature, and then resuspended in the western blot loading buffer.

Western blot

Protein extracts were heated for 5 min at 95 °C, loaded into 4–12% Bis-Tris gels (Life Technologies), and run at 150V. Proteins were transferred into immobilon-P PVDF membranes at 400 mA, blocked in 5% milk in TBS-Tween 0.1% (TBST), and incubated with primary antibodies overnight at 4 °C. All secondary HRP-conjugated antibodies were incubated for 1 h at room temperature. The following primary antibodies were used: Anti-HA-Tag (Cell Signaling, C29F4), Anti-FLAG® (Sigma-Aldrich, F1804), Anti-DYKDDDDK Tag (Cell Signaling, D6W5B), anti-β-actin (Santa Cruz, sc-47778), anti-α-tubulin (Licor, 926-42213), anti-vinculin (Sigma-Aldrich, V9131), anti-hTREM2 (R&D AF1828), anti-TREM2222 (Ab222), and anti-TREM2219 (Ab219). Ab222 and Ab219 are custom monospecific antibodies generated by Pacific Immunology. The antigen for Ab222 is CSLAWTEARDTSTQ, and for Ab219 is RAERHVKEDDGRKSPGEVPPGTS-Cys. Rabbits were immunized, and their serum was used to purify antibodies by affinity purification against the above mentioned protein sequences. This process allows the isolation of highly specific antibodies that approach the specificity of monoclonal antibodies and provide the superior affinity of polyclonal antibodies (https://www.pacificimmunology.com). All antibodies were diluted in 5% milk in TBST, except for anti-vinculin which was dissolved in 5% bovine serum albumin (BSA) in TBST.

sTREM2 production, purification, and quantification

HEK-293T cells were transfected with TREM2 plasmids as described above and maintained in FBS-free media for 48 h. Conditioned media were collected, filtered with a 0.2-μm pore size filter, and concentrated using Pierce Protein Concentrators (Thermo Fisher Scientific, 88535). Afterwards, we purified sTREM2 from concentrated conditioned media using the HA-tagged Protein Purification Kit (MBL International, 3320) and following the manufacturer’s guidelines. This kit allows HA-tagged protein to be purified based on the use of HA beads and competitive elution. Proteins were stored in PBS and glycerol (50:50) at − 20 °C. Soluble TREM2 species were visualized by silver staining using the Pierce™ Silver Stain Kit (Thermo Fisher Scientific, 24612) after electrophoresis in 4–12% Bis-Tris gel in denaturing and reducing conditions. Soluble TREM2 proteins were quantified by ELISA, similar to what has been previously described [44]. Briefly, F8 Maxisorp Nunc-Immuno Module (Thermo Fisher, 468667) wells were coated with 2 μg/ml of the TREM2 capture antibody (R&D Systems, MAB17291) in 0.05 M carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C and blocked with 3% BSA in PBS with 0.05% Tween (PBST) for 1 h at room temperature (RT). Wells were washed 4 times with PBST and incubated with samples diluted in 0.5% BSA in PBST for 2 h at RT, followed by incubation with 0.25 μg/ml of the human TREM2 biotinylated detection antibody (R&D Systems, BAF1828) for 1 h at RT. After washing, samples were incubated with HRP-conjugated streptavidin (PerkinElmer, NEL750001EA, 1:10.000). The samples were washed and incubated with the chromogenic substrate TMB (3,3′,5,5′-tetramethylbenzidine) using Pierce TMB Substrate kit (Thermo Fisher Scientific, 34021). Upon optimal color development, reactions were stopped using 1 N HCL, and the optical density (O.D.) of the wells was read at 450 nM using the Epoch2 microplate reader (BioTek). The first batch of soluble TREM2 protein was quantified by Coomassie Brilliant Blue staining after electrophoresis in a 4–12% Bis-Tris gel with a standard curve of bovine serum albumin. These proteins were then used as standards for the quantification of the next batch of soluble TREM2 protein by ELISA. Subsequently, all quantifications were done by ELISA, and within each batch, a fraction of protein was stored to be used as ELISA standards for the following batches. We controlled for the presence of endotoxins using samples from different batches. To specifically detect TREM2222 TREM2219 by ELISA, we used the same protocol described above using different capture antibodies to coat the wells, Ab222 at 4 μg /ml and Ab219 at 2 μg/ml. The generation of ELISA standard curves and their interpolation was done using the BioTek Gen5 Analysis software.

Electrophysiology

For these experiments, 4-month-old male mice were anesthetized and transcardially perfused with cold artificial cerebral spinal fluid (aCSF) (124 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 26 mM NaHCO3, 1.2 mM NaH2PO4, 10 mM glucose, 2 mM CaCl2) bubbled with 95% O2 and 5% CO2. The coronal slices (280 μm) were cut in an ice-cold sucrose-based solution using a vibratome Leica VT1200S. The slices were stored for 60 min in oxygenated aCSF at 30 °C and then kept at room temperature. Prior to recording slices were incubated for 60 min at room temperature in either a control-protein or active-protein solutions. The incubation system we used is like the one described by Hupp et al. [45]. We used 12 well-standard culture plates with a strainer basket inside. 95% O2 and 5% CO2 were bubbled through a fine tube placed between the basket and the wall of the plate without mechanically disturbing the slices. The volume of each well was 3 ml of aCSF in which the protein was dissolved to a concentration of 15 ng/ml. Heat-inactivated proteins (100 °C for 1 h) were used as controls.

The recordings were performed at 30–32 °C in a chamber that was perfused with oxygenated aCSF at a rate of about 2 mL/min. In some experiments, picrotoxin (50 μM) was added to the medium to block GABAA receptors. Field excitatory postsynaptic potentials (fEPSPs) were recorded using a Multiclamp 700B amplifier and Clampex software (Molecular Devices). Signals were low-pass filtered at 2 kHz and digitized at 50 kHz. Tungsten stereotrodes (~ 1 MΩ) were used to stimulate the Schaffer collaterals in the hippocampus CA1 region. Stimulation parameters were adjusted using a constant current isolated stimulator (Digitimer). An input-output curve was obtained, and then using the stimulation strength that produced about 50% of the maximum intensity, a stable baseline was recorded for 10 min stimulating at 0.05 Hz. Long-term potentiation (LTP) was induced by applying 4 100-Hz trains of 100 ms duration every 20 s. Changes in the slope of the response (mV/ms) fEPSPs were recorded for 60 min post-induction monitoring at 0.05 Hz. Data were expressed as a percentage of change with respect to the average baseline.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 8 for Windows (GraphPad Software www.graphpad.com). Data were first analyzed for normality followed by statistical tests. The tests used were Student’s t-test or Mann-Whitney test.

留言 (0)

沒有登入
gif