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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

Molecular Neurodegeneration 1/2018

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

Zeitschrift:
Molecular Neurodegeneration > Ausgabe 1/2018
Autoren:
Xianyuan Xiang, Thomas M. Piers, Benedikt Wefers, Kaichuan Zhu, Anna Mallach, Bettina Brunner, Gernot Kleinberger, Wilbur Song, Marco Colonna, Jochen Herms, Wolfgang Wurst, Jennifer M. Pocock, Christian Haass
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s13024-018-0280-6) contains supplementary material, which is available to authorized users.

Abstract

Background

The R47H variant of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) significantly increases the risk for late onset Alzheimer’s disease. Mouse models accurately reproducing phenotypes observed in Alzheimer’ disease patients carrying the R47H coding variant are required to understand the TREM2 related dysfunctions responsible for the enhanced risk for late onset Alzheimer’s disease.

Methods

A CRISPR/Cas9-assisted gene targeting strategy was used to generate Trem2 R47H knock-in mice. Trem2 mRNA and protein levels as well as Trem2 splicing patterns were assessed in these mice, in iPSC-derived human microglia-like cells, and in human brains from Alzheimer’s patients carrying the TREM2 R47H risk factor.

Results

Two independent Trem2 R47H knock-in mouse models show reduced Trem2 mRNA and protein production. In both mouse models Trem2 haploinsufficiency was due to atypical splicing of mouse Trem2 R47H, which introduced a premature stop codon. Cellular splicing assays using minigene constructs demonstrate that the R47H variant induced abnormal splicing only occurs in mice but not in humans. TREM2 mRNA levels and splicing patterns were both normal in iPSC-derived human microglia-like cells and patient brains with the TREM2 R47H variant.

Conclusions

The Trem2 R47H variant activates a cryptic splice site that generates miss-spliced transcripts leading to Trem2 haploinsufficiency only in mice but not in humans. Since Trem2 R47H related phenotypes are mouse specific and do not occur in humans, humanized TREM2 R47H knock-in mice should be generated to study the cellular consequences caused by the human TREM2 R47H coding variant. Currently described phenotypes of Trem2 R47H knock-in mice can therefore not be translated to humans.
Zusatzmaterial
Additional file 1: Figure S1. Off-target analysis of in-house made Trem2 R47H knock-in mice. a Sanger-sequencing chromatograms of the Trem2 on-target site and the six putative off target sites of animal Trem2 R47H ki ID-7. Mixed peaks in the Trem2 locus show the correct R47H substitution (CGC > CAC) and the three silent mutations for genotyping purposes. Mixed peaks in traces of site #2 reveal a Δ10-Indel mutation at the putative cut site, indicating a true off target event. Underlined: Protospacer; arrow head: putative cut site; green letters: PAM site on shown strand; red letters: PAM site on complementary strand; yellow: Δ10-Indel mutation. b Sanger sequencing results of Trem2 R47H positive off-springs of male ID-7, which was crossed with a C57BL/6 N female. The Δ10-Indel allele was inherited to animals ID-7-3 und ID-7-12 that were excluded from any further breedings and experiments. (PDF 201 kb)
13024_2018_280_MOESM1_ESM.pdf
Additional file 2: Table S1. Allele specific quantitative PCR for Trem2 R47H knock-in mice (PDF 92 kb)
13024_2018_280_MOESM2_ESM.pdf
Additional file 3: Figure S2. Sequence of the aberrantly spliced murine Trem2 mRNA. Trem2 gene sequence. Exon in black; intron in green; // indicates splicing sites. (PDF 93 kb)
13024_2018_280_MOESM3_ESM.pdf
Additional file 4: Figure S3. iMG differentiation and validation. a Schematic of the in vitro differentiation of iPSC-derived microglia-like cells (iMG). (i): Human iPSCs are grown in feeder free conditions with no spontaneous differentiation. Scale bar: 250 μm. (ii): Embryoid bodies are formed in the presence of 3 factors SCF, BMP4, and VEGF; Scale bar: 750 μm. (iii): Myeloid cells are generated after culturing with IL3 and MCSF growth factors for 3–4 weeks, then stained for classic myeloid/macrophage markers CD68. Scale bar: 50 μm. (iv): Further differentiation to microglia-like cells that positive for microglial markers P2RY12. Scale bar: 20 μm. (v): Addition of the two final factors (CD200 and CX3CL1) matures the iMG. Scale bar: 20 μm. b Heat map showing mRNA expression of a microglial gene signature in iMG, human monocyte-derived macrophages (hMac), human primary microglia (hMG), and iPSC samples. Clear clustering is observed between iMG and hMG. (PDF 159 kb)
13024_2018_280_MOESM4_ESM.pdf
Additional file 5: Table S2. Allele specific quantitative PCR for iMG and patient brains (PDF 104 kb)
13024_2018_280_MOESM5_ESM.pdf
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