The Trem2 R47H Alzheimer’s risk variant impairs splicing and reduces Trem2 mRNA and protein in mice but not in humans

Animal handling and animal experiments were performed in accordance to local animal laws and housed in standard cages in a specific pathogen-free facility on a 12-h light/dark cycle with ad libitum access to food and water.

Jax Trem2 R47H knock-in mice were purchased from Jackson laboratory. In-house Trem2 R47H knock-in mice were generated using CRISPR/Cas9 technology in C57BL/6 N background. Both strains were housed and bred in the same animal facility. To extract bone marrow, mice were first euthanized by CO₂ followed by cervical dislocation.

Trem2 R47H knock-in mice (R47H ki mice) were generated by CRISPR/Cas9-assisted gene targeting in zygotes as described previously [28, 29]. Briefly, pronuclear stage zygotes were obtained by mating C57BL/6 N males with superovulated C57BL/6 N females (Charles River). Embryos were then microinjected into the male pronucleus with an injection mix containing 25 ng/μl Cas9 mRNA, 12.5 ng/μl Trem2-specific sgRNA, and 25 ng/μl single-stranded oligodeoxynucleotide (ssODN). Cas9 mRNA was prepared from XbaI-linerized pCAG-Cas9v2-162A by in vitro transcription using the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Thermo Fisher Scientific, #AM1345) and purified using the MEGAclear™ Transcription Clean-Up Kit (Thermo Fisher Scientific, #AM1908). Trem2-specific sgRNA (protospacer: GAAGCACTGGGGGAGACGCA) was prepared by IVT from pBS-T7-sgTrem2 using the MEGAshortscript™ T7 Transcription Kit (Thermo Fisher Scientific, #AM1354) and purified with the MEGAclear™ Transcription Clean-Up Kit. The 130 nt ssODN targeting molecule ssTrem2 R47H (5′- GGGCATGGCCGGCCAGTCCTTGAGGGTGTCATGTACTTATGACGCCTTGAAGCACTGGGGTCGACACAAAGCCTGGTGTCGGCAGCTGGGTGAGGAGGGCCCATGCCAGCGTGTGGTGAGCACACACGGT -3′), comprising the G > A substitution (underlined) and three additional silent mutations (bold), was synthetized by Metabion. After microinjection, zygotes were cultured in KSOM medium until they were transferred into pseudopregnant CD-1 foster animals.

To identify putative off-target sites of the Trem2-specific sgRNA, the online tool CRISPOR (http://crispor.tefor.net/↗) [30] was used. Predicted sites with a CFD score > 0.5 and an MIT score > 0.6 were chosen for off-target analysis. For analysis, genomic DNA of wildtype and heterozygous mutant Trem2 R47H mice was isolated and the loci were PCR amplified with primers flanking the putative cut sites. PCR amplicons were subsequently Sanger sequenced using PCR amplification primers and the traces compared to a reference sequence.

Bone marrow derived macrophages (BMDM) were prepared as previously described [31, 32]. Briefly, the bone marrow cells were flushed out using advanced RPMI 1640 (Life Technologies). Cells were differentiated using advanced RPMI 1640 supplemented with 2 mM L-Glutamine, 10% (v/v)) fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 50 ng/ml murine M-CSF (R&D System) for 7 days in non-cell culture treated dishes.

Microglia were isolated as previously described with some modification [33]. Wild type and Trem2 R47H ki mice were perfused with cold phosphate buffered saline (PBS). Whole brain without the cerebellum was cut into small pieces and gently homogenized by mechanical dissociation in homogenization buffer (HBSS no calcium, no magnesium, with phenol red; 15 mM HEPES, 0.6% glucose). Homogenized cell suspensions were passed through a 100 μm cell strainer. Cells were pelleted at 300 g for 10 min and the supernatant discarded. To remove myelin, the cell pellets were re-suspended in 22% percoll (GE Healthcare) and centrifuged for 20 min at 900 g (acceleration 4, deceleration 0). Microglia were purified from the pellet using MACS CD11b magnetic beads according to manufacturer’s instructions. Briefly, 20 μl CD11b microbeads (Miltenyi) were incubated with cell suspension for 15 min. After incubation, 500 μl MACS buffer were added and cells were passed over a pre-rinsed Miltenyi LS column attached to a magnetic field. The column was washed three times with 500 μl MACS buffer, removed from the magnetic field and CD11b positive cells were washed out in 5 ml of MACS buffer.

To detect membrane bound Trem2, membrane fractions were collected as previously described [32]. Briefly, cells were lysed in hypotonic buffer (100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 7.4) freshly supplemented with a protease inhibitors cocktail (Sigma-Aldrich). Membrane fractions were pelleted by centrifugation for 45 min at 16,000 g at 4 °C. Membranes were lysed in STEN lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.6, 2 mM EDTA, 1% Triton-X 100) on ice for 20 min. Equal amounts of protein were mixed with Laemmli sample buffer supplemented with β mercaptoethanol followed by by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes (Amersham Hybond P 0.45 PVDF, GE Healthcare Life Science) and blocked in 10% I-BlockTM (Thermo Fisher Scientific) for 1 h. Monoclonal antibody 5F4 (dilution 1:100) [32] was used to detect Trem2.

Ethical permission for this study was obtained from the National Hospital for Neurology and Neurosurgery and the Institute of Neurology joint research ethics committee (study reference 09/H0716/64). R47H heterozygous fibroblasts were acquired with a material transfer agreement between University College London and University of California Irvine Alzheimer’s Disease Research Center (UCI ADRC; M Blurton-Jones). Fibroblast reprogramming was performed by episomal plasmid nucleofection (Lonza) as previously described [34], using plasmids obtained from Addgene (#27077, #27078 and #27080). Nucleofected cultures were transferred to Essential 8 medium (Life Technologies) after 7 days in vitro (DIV) and individual colonies were picked after 25–30 DIV. All iPSCs were maintained and routinely passaged in Essential 8 medium. Karyotype analysis was performed by The Doctors Laboratory (London, UK). Control iPSC lines used in this study are as follows: CTRL1 (kindly provided by Dr. Selina Wray, UCL Institute of Neurology); CTRL2 (SBAD03, Stembancc); CTRL3 (SFC840, Stembancc); CTRL4 (BIONi010-C, EBiSC).

Using previously described protocols, iPSC-derived microglia-like cells (iMG) were generated [35, 36].

Day 0: iPSC lines were cultured to 60% confluency in E8 medium on vitronectin-coated 6-well plates. Cells were washed with PBS (w/o Ca2+/Mg2+), followed by trypLE digestion (1 ml/well; 4 min at 37 °C). The solution was added to 4 volumes of PBS (w/o Ca2+/Mg2+), and triturated to a single cell suspension. The suspension was centrifuged for 3 min at 300 g and the cell pellet resuspended in 1 ml EB differentiation medium (Adapted from [36]; EBdiff; Essential 8, 50 ng/ml BMP-4, 50 ng/ml VEGF, 20 ng/ml SCF, and 10 μM Y-27632). Cells were counted, and resuspended in EBDiff medium to a density of 10⁵ cells/ml. To generate embryoid bodies (EBs), 100 μl of the suspension was added to 96 well ultra-low attachment round bottom tissue culture plates (Corning), centrifuged at 115 g for 3 min, and transferred to a tissue culture incubator at 37 °C with 5% CO2.

Day 2: 50 μl of EBdiff medium was added to each well.

Day 3: Dense EBs were formed and collected with a P1000 Gilson pipette into a sterile 15 ml tube, and left to settle. The spent EBdiff medium was discarded and 10 ml of myeloid differentiation medium (Mdiff; X-VIVO 15 medium (Lonza), 1X Glutamax (Life Technologies), 100 U Penicillin/Streptomycin (Life Technologies), 50 μM β-mercaptoethanol (Life Technologies), 100 ng/ml MCSF (Peprotech), and 25 ng/ml IL-3 (Cell Guidance Systems)) added. Approximately 150 embryoid bodies (1.5 × 96 well plates) were transferred to a 175 cm2 flask containing a further 20 ml of Mdiff medium.

Day 9–11: Mdiff medium (30 ml) was added, to avoid acidosis, being careful not to disturb the EBs.

Day 26: Cells were collected from the medium for myeloid marker analysis.

Day 33: Once a week, 1/2 of the Mdiff medium/flask containing budded myeloid cells was collected through a 40 μm cell strainer (Falcon, Corning), replacing the collected medium with fresh Mdiff. The myeloid cell suspension was centrifuged for 3 min at 300 g and the cell pellet resuspended in 1 ml microglial differentiation medium (Adapted from [35]; MGdiff; DMEM/F12 HEPES no phenol red, 2% ITS-G (Life Technologies), 1% N2 supplement (Life Technologies), 200 μM monothioglycerol (Sigma), 1X Glutamax, 1X NEAA (Life Technologies), 5 μg/ml Insulin (Sigma), 100 ng/ml IL34 (Peprotech), 25 ng/ml MCSF and 5 ng/ml TGFβ1 (Peprotech), filtered through a 0.22 μm syringe filter. Cells were counted and plated in MGdiff, and medium replaced every 3–4 days.

Day 46: MGdiff medium was replaced with microglial maturation medium (MGmat: MGdiff + 100 ng/ml CD200 (Generon), and 100 ng/ml CX3CL1 (Peprotech)) for 4 days to generate iMG.

Monocytes were obtained from blood through centrifugation with Histopaque (Sigma) to isolate peripheral blood mononuclear cells followed by separation and purification with CD14-conjugated magnetic beads (Miltenyi). Peripheral blood monocytes (PBM) were matured into monocyte-derived macrophages (hMacs) in X-VIVO 15 medium with 1% Glutamax, 100 U Penicillin/Streptomycin, and 100 ng/ml MCSF for 7 days.

A custom gene array based on published microglial expression data [35, 37–39] was used to confirm a microglial signature in our iMG cultures (TaqMan Array Plate 32 plus Candidate Endogenous Control Genes; Thermo Fisher Scientific). Complementary DNA was generated from iMG, iPSC-derived microglial-like cells [36], and human monocyte-derived macrophages (hMacs) RNA samples using the High-Capacity RNA-cDNA kit (Life Technologies), according to the manufacturer’s instructions. Human primary microglia cDNA was also analyzed as a control sample (ScienCell). Quantitative PCR were conducted on the Mx3000p qPCR system with MxPro qPCR software (Stratagene) using TaqMan Gene Expression Mastermix (Thermo Fisher Scientific). Heat maps were generated with the gplots [40] and d3heatmap [41] packages in R.

Microglial gene signature primer details:

Cells were fixed in 4% PFA/sucrose in PBS for 20 min at room temperature (RT), quenched with 50 mM NH₄Cl in PBS for 10 min at RT, and permebalized with 0.2% Triton X-100 in PBS for 5 min at RT. Blocking was performed with 5% normal goat serum (NGS) in PBS for 30 min. Primary antibodies were diluted in 5% NGS/PBS and incubated at RT for 2 h, followed by PBS washes and incubation with corresponding secondary antibodies for 1 h at RT in the dark, with gentle rocking. Nuclei were counterstained during mounting using Vectorshield with DAPI (4′, 6-diamidino-2-phenylindol; Vector Labs). Fluorescence microscopy was performed on a Zeiss Axioskop 2 microscope and Axiovison software (Zeiss, v4.8). Confocal microscopy was performed on a Zeiss LSM 710 confocal microscope using Zen software (Zeiss, Version 2012), and all images were processed with Image J1.51 K (https://imagej.nih.gov/ij/↗). The following antibodies were used: mouse anti-CD68 (1:100, DAKO), rabbit-anti-P2YR12 (1:200, Atlas Antibodies), mouse-anti-β-Actin (1:500, Sigma), mouse-anti-EZR (1:250, Atlas Antibodies), goat anti-rabbit Alexafluor488 (1:500, Life Technologies), goat-anti-mouse Alexafluor568 (1:500, Life Technologies).

Human and mouse Trem2 genomic fragments encoding exon 1, intron 1, exon 2, and a FLAG tag were synthesized by Integrated DNA Technologies. The following primer sets were used for vector cloning: EcoRV-Hs TREM2-Fw (GGATATCCGGGCAGCGCCTGACATGCCTG) and NotI-Hs TREM2-Rv (ATGCGGCCGCTTAGGATTACAAGGATGACGACGATAAG); HindIII-Mm Trem2 Fw (CCCAAGCTTGGGGCGCCTACCCTAGTCC) and XohvI-Mm Trem2-Rv (CCGCTCGAGCGGCTACTTGTCGTCATCG). The amplified fragments were digested by EcoRV/NotI or HindIII/XohI and the digested fragments were inserted into pcDNA3.1 (+) (Invitrogen). The mutations were introduced in the human and mouse minigene using the Quikchange™ site-directed mutagenesis kit (Agilent). The following mutants were generated:

For the cellular splicing assay 8 × 10⁵ HEK293 cells were seeded in 6-well plates with Dulbecco’s Modified Eagle Medium (Life Technologies) supplemented with GlutaMAX™, 10% (v/v) FCS, 100 U/ml penicillin, 100 μg/ml streptomycin and cultured overnight. Cells were transfected with 3 μg of plasmids to express human or mouse Trem2 minigenes using 6 μl lipofectamine 2000 according the manufacturer’s instructions (Thermo Fisher Scientific). Transfected cells were cultured in normal medium for 48 h and collected for RNA extraction using RNeasy Mini Kit (Qiagen) according the manufacturer’s instructions. The RNA was used for Reverse transcription polymerase chain reaction (RT-PCR).

1 μg of total RNA was transcribed into cDNA using SuperScript IV reverse transcriptase and oligo dT (Thermo Fisher Scientific). 2 μl of cDNA was used as template and amplified by polymerase chain reaction (PCR) with GoTaq DNA polymerase (Promega) according the manufacturer’s instructions. The reaction condition and primer sequences are listed in Table 1. The PCR products were loaded into 2% agarose gel with GelRed™ (Biotium) for DNA visualization.

RNeasy Mini Kit (Qiagen) was used for total RNA isolation according the manufacturer’s instructions. 1 μg of total RNA was transcribed into cDNA using SuperScript IV reverse transcriptase and oligo dT (Thermo Fisher Scientific). RNA levels of human and mouse Trem2 and Tyrobp were analyzed by Taqman® real-time PCR using the 7500 Fast real-time PCR system (Applied Biosystems). For endogenous controls Gusb (Mm01197698_m1, Thermo Fisher Scientific) and Hsp90ab1 (Mm00833431_g1, Thermo Fisher Scientific) or GUSB (Hs00939627_m1, Thermo Fisher Scientific) and HSP90AB1 (Hs03043878_g1, Thermo Fisher Scientific) were used. Probes that target mouse Trem2 and Tyrobp are Mm04209424_g1, Mm04209423_g1, Mm01273682_g1, Mm00449152_m1 (Thermo Fisher Scientific). Probes that target human TREM2 and TYROBP are Hs01010721_m1, Hs01003899_m1, Hs00182426_m1 (Thermo Fisher Scientific). For allele specific mRNA expression custom made primers and probes were designed. The primer pair for mouse Trem2 is: CCTTGAGGGTGTCATGTACTTAT and TCCCATTCCGCTTCTTCAG. The probes for the mouse wild-type allele and R47H allele are /5HEX/CCTT+G + C + GT + CT + CC/3IABkFQ/ (+, lock nucleic acid) and /56-FAM/CTT + T + G + T + GT + C + GA + C/3IABkFQ/, respectively (Integrated DNA Technologies). The primer pair for human TREM2 is ACAAGTTGTGCGTGCTGA and ATGACTCCATGAAGCACTGG. The probes for the human wild-type allele and the R47H allele are /5HEX/CTT + G + C + GCCT+CC/3IABkFQ/ and /56-FAM/TT + G + T + GC + CT + CC/3IABkFQ/, respectively (Integrated DNA Technologies). The probes and primers were mixed in 1:2 ratios for quantitative PCR reaction.

cDNAs were diluted 1:5 with H₂O and 9 μl of diluted cDNA together with 1 μl of primer probe mix, 10 μl of Taqman® master mix (Thermo Fisher Scientific) were used in one 20 μl reaction.

The rare TREM2 variant rs75932628-T encodes a histidine instead of arginine at position 47 (R47H) and increases the risk for AD around three-fold [3, 4]. The DNA and amino acid sequence around arginine at position 47 is highly conserved across different mammalian species (Fig. 1a and b). To study the impact of this variant on TREM2 function in microglia in vivo we generated Trem2 R47H knock-in mice (R47H ki mice) by introducing a G > A mutation using the CRISPR/Cas9 technology (Fig. 1c). Two silent mutations were additionally introduced (GA > TC) to create a SalI restriction site for genotyping (Fig. 1c). In addition a silent G > A mutation was generated to block the protospacer-adjacent motif (PAM) to allow higher gene editing efficiency (Fig. 1c). The predicted potential off-target sites were analyzed. An off-target event with a Δ10-Indel mutation was identified at an intragenic region on chromosome 11 (Additional file 1: Figure S1, putative off-target #2). The founder mouse was back-crossed to C57BL/6 N, and off-spring with positive Trem2 R47H ki but negative off-target #2 (i.e ID-7-1; ID-7-2; ID-7-4) were used for establishing the mouse line. Expression of total Trem2 mRNA as well as both Trem2 mRNA transcripts (NM_031254.3↗ and NM_001272078.1↗), encoding either membrane bound Trem2 or a truncated soluble version were validated in brain. Interestingly, total Trem2 mRNA including both Trem2 transcripts was significantly reduced in a gene dose-dependent manner, whereas mRNA expression of Tyrobp (NM_011662↗), the adaptor protein of Trem2 [42], remained unchanged (Fig. 1d). Using allele specific qPCR we confirmed that expression of the R47H allele was selectively reduced compared to the wt allele in heterozygous R47H ki mice (Fig. 1e and Additional file 2: Table S1).

To confirm decreased Trem2 expression on protein level, we purified microglia from wild-type (wt), heterozygous (het), and homozygous (hom) R47H ki mice and performed western blotting of membrane fractions. Membrane-bound Trem2 showed a gene dose-dependent reduction (Fig. 1f).

Reduced Trem2 R47H mRNA and protein expression was further confirmed in bone marrow derived macrophages (BMDM). Consistent with our findings in brain and microglia, mRNA of both Trem2 transcripts decreased in a gene dose dependent manner whereas mRNA of Tyrobp remained unchanged (Fig. 1g). Furthermore, immature and mature Trem2 as well as soluble Trem2 (sTrem2) were also reduced in a gene dose dependent manner (Fig. 1h).

To exclude that reduced Trem2 mRNA and protein expression is artificially caused by the introduction of the three silent mutations, we analyzed Trem2 R47H knock-in mice generated by Jackson laboratory (Jax R47H ki mice). In addition to the target variant R47H, these mice harbor two silent mutations (Fig. 2a). One of the silent mutations is a G > A exchange to block the PAM sequence exactly like in the mice generated within our laboratory. The second silent mutation C > A is only present in the Jax R47H ki mice (Fig. 2a). We investigated Trem2 mRNA and protein levels using homozygous Jax R47H ki mice and their wt counterparts. Again, mRNA from total Trem2 as well as from both transcripts of Trem2 decreased in brains of homozygous ki mice whereas mRNA expression of Tyrobp remained unchanged (Fig. 2b). Western blotting of membrane fractions from BMDM from the Jax R47H ki mice also confirmed a significant reduction of membrane bound and soluble Trem2 (Fig. 2c). Thus reduction of mRNA and protein levels in Trem2 R47H ki mouse was confirmed in two independent mouse models.

Next we searched for the cellular mechanism responsible for the substantial reduction of the Trem2 R47H mRNA in the two mouse models. Since nonfunctional mRNAs are rapidly removed by mRNA surveillance systems [43], we studied splicing of the first intron which separates exon 2 containing the R47H variant from exon 1 harboring the translation initiation site and upstream untranslated sequences. Using RT-PCR with primer pairs against the 5′ end of exon 1 and the 3′ end of exon 2, we compared the splicing patterns of Trem2 in R47H ki and wt mice (Fig. 3a). A single splicing product of the expected length (465 base pairs) was obtained from wt Trem2 mRNA (Fig. 3b). Surprisingly, an additional smaller splicing product (346 base pairs) was observed in Trem2 R47H heterozygous mice, which was even more abundant in Trem2 R47H homozygous mice (Fig. 3b) while at the same time the larger splicing product was reduced in a gene dose dependent manner (Fig. 3b). This suggests aberrant splicing of Trem2 pre-mRNA derived from the mutant allele. To independently confirm aberrant splicing, we investigated the Jax R47H ki mice described in Fig. 2. Again, we found an additional smaller RT-PCR product in mutant but not wt mice (Fig. 3c). Furthermore, the larger splicing product was reduced in homozygous ki mice (Fig. 3c). Thus aberrant splicing of R47H pre-mRNA occurs in two independent mouse models. DNA sequencing of the splicing products revealed correct splicing of exon 1 and exon 2 in wt mice and to a lesser extent also in the ki mice (Fig. 3d). However, DNA sequencing of the additional shorter RT-PCR product of both mutant mice revealed that 119 base pairs were deleted at the 5′ end of exon 2 (Fig. 3d and Additional file 3: Figure S2). The deletion leads to a frame shift and a premature stop codon in exon 2 (Fig. 3d), which may lead to nonsense mediated mRNA decay and thus explain the consistent reduction of the mutant mRNA in both mouse models.

To directly compare the splicing pattern of mouse and human TREM2 R47H, we expressed mouse or human TREM2 minigenes containing exon 1, intron 1, and exon 2 in human embryonic kidney 293 cells (HEK 293) (Fig. 4a). To separately investigate the disease causing TREM2 R47H variant and the silent mutations, we introduced the corresponding sequence variants either alone or together (Fig. 4b). Upon expression of the minigene encoding the mouse Trem2 sequence, we observed miss-splicing induced by the R47H variant alone (R47H) (Fig. 4c). Similar aberrant splicing was observed when the two silent mutations of the Jax R47H ki mice were expressed in addition to the R47H variant (R47H^AA), whereas introduction of the two silent mutations alone (AA) did not affect splicing (Fig. 4c). Moreover, when the three silent mutations used to generate our in-house mouse model were combined with the R47H (R47H^TCA), a striking increase of aberrant splicing was observed. When we only introduced the unique TC mutations used to generate our mouse model (TC) we also observed impaired splicing (Fig. 4c), suggesting that R47H and TC together display synergistic effects on aberrant splicing. This demonstrates that the R47H variant by itself is sufficient to induce aberrant splicing, but enhanced miss-splicing upon the addition of silent mutations implies that this genomic region is very sensitive for splicing errors induced by minor changes within the pre-mRNA sequence. Finally, we also investigated if the mutations introduced into the mouse genome by Cheng-hathaway et al. [26] affect pre-mRNA splicing of Trem2. Very similar to our R47H ki mice and those generated by the Jackson Laboratories, the mutations introduced by Cheng-hathaway and colleagues (R47H^T) caused aberrant splicing (Fig. 4c), which is consistent with the haploinsuffciency also observed in this model [26].

To prove if this is also true for human TREM2, we investigated the same TREM2 variants in human minigene constructs (Fig. 4a, b and d). Surprisingly, our results demonstrate that neither the R47H variant alone or in combination with the silent mutations used to generate the ki mouse models affected correct exon1/2 splicing (Fig. 4d). This suggests that only the mouse gene locus is vulnerable for aberrant splicing upon introduction of these sequence variants and implies that the R47H mutation does not affect splicing and mRNA levels in humans.

To provide direct evidence for this prediction, we investigated exon 1/2 splicing in humanized TREM2 mice generated by ectopic expression of the human wt or R47H mutant TREM2 locus in Trem2^−/− mice [14]. Using the same RT-PCR strategy as described above (Fig. 3a), we could only detect the correctly spliced exon 1 and 2 but no aberrantly spliced additional products (Fig. 5a).

Similarly, in human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMG) with the wt TREM2 allele or heterozygous for the TREM2 R47H variant (Additional file 4: Figure S3) we also detected only the correctly spliced exon 1 and 2 (Fig. 5b). Furthermore, no aberrant splicing was detected in AD cases carrying one R47H mutant allele (Fig. 5c). Direct sequencing demonstrated correct exon1/2 splicing in human iMG and in AD cases. Lack of aberrant splicing of human TREM2 R47H is consistent with no reduction of total TREM2 mRNA in iMG with one R47H allele (Fig. 5d). In addition, using allele specific qPCR we confirmed that the expression of the R47H allele is comparable to wt in iMG (Fig. 5e and Additional file 5: Table S2), and in human brain (Fig. 5f and Additional file 5: Table S2). Taken together, aberrant splicing of R47H mutant pre-mRNA is not observed in humans and consequently no haploinsufficiency of TREM2 could be detected.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) within the framework of the Munich Cluster for Systems Neurology (EXC 1010 SyNergy) and the Koselleck Project HA1737/16–1 (to C.H.). This work was funded in part by the German Federal Ministry of Education and Research (BMBF) through the Joint Project HIT-Tau TP2: Grant 01EK1605C to W. W. This work was supported by the Biotechnology and Biological Sciences Research Council (grant number BB/M009513/1) to A.M. and by funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115976 (for T.P to J.P). This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This work was funded by NIH grant RF1 AG051485 to M.C.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

All mice were handled according to institutional guidelines approved by the animal welfare and use committee of the government of Upper Bavaria. Ethics committee from Ludwig-Maximilians- University and University College London approved this research project using human tissue.

Consent for publication on using human tissue in this study is detailed in the Material Transfer Agreement (MTA) between organizations.

C.H. collaborates with DENALI Therapeutics and received a speaker honorarium from Novartis and Roche. The other authors declare that they have no competing interests.

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The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.