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. 2018 Sep 6;13(1):49.
doi: 10.1186/s13024-018-0280-6.

The Trem2 R47H Alzheimer's Risk Variant Impairs Splicing and Reduces Trem2 mRNA and Protein in Mice but Not in Humans

Affiliations
Free PMC article

The Trem2 R47H Alzheimer's Risk Variant Impairs Splicing and Reduces Trem2 mRNA and Protein in Mice but Not in Humans

Xianyuan Xiang et al. Mol Neurodegener. .
Free PMC article

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.

Keywords: Alzheimer’s disease; Human microglia; Microglia; Neurodegeneration; Pre-mRNA splicing; TREM2.

Conflict of interest statement

Ethics approval

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

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

Competing interests

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.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Trem2 mRNA and protein are reduced in a novel Trem2 R47H knock-in mouse model. a and b Evolutionary conservation of TREM2 at the DNA (a) and protein (b) level. c Strategy to generate Trem2 R47H knock-in (R47H ki) mice indicating the protospacer region (green), protospacer adjacent region (PAM, purple), and the introduced nucleotide changes (orange or red). The restriction site for SalI is underlined. d Trem2 and Tyrobp mRNA levels in brains from R47H ki mice. TaqMan probes for the exon 4/5 boundary were used to detect total Trem2 mRNA. TaqMan probes for the Trem2 exon 3/4 boundary were used for isoform discrimination. (N = 3, +/-SEM, one way ANOVA, Bonferroni-corrected pair-wise post hoc tests, total Trem2 WT vs. Het p = 0.0002, WT vs. Hom p < 0.0001; Trem2 isoform 1 WT vs. Het p = 0.0029, WT vs. Hom p < 0.0001; Trem2 isoform 2 WT vs. Het p = 0.0031, WT vs. Hom p = 0.0002. n.s. Non-significant). e Allele specific Trem2 mRNA expression in heterozygous R47H ki mice. Customized probes were against Trem2 R47H and its neighbor region (see also Methods). (N = 3, +/-SEM, unpaired t test, p < 0.0001). f Trem2 protein expression in microglia isolated from Trem2 wt or R47H ki mice. (N = 3,+/-SEM, one way ANOVA, p < 0.0001, Bonferroni-corrected pair-wise post hoc tests, WT vs. Het p = 0.0005, WT vs. Hom p < 0.0001). g Trem2 and Tyrobp mRNA levels in bone marrow derived macrophages (BMDM) isolated from Trem2 wt and R47H ki mice. (N = 3, +/-SEM, one way ANOVA, Bonferroni-corrected pair-wise post hoc tests, Trem2 WT vs. Het p = 0.0002, WT vs. Hom p < 0.0001; Trem2 isoform 1 WT vs. Het p < 0.0001, WT vs. Hom p < 0.0001; Trem2 isoform 2 WT vs. Het p = 0.0008, WT vs. Hom p < 0.0001. n.s. Non-significant.) h Expression levels of membrane bound and soluble Trem2 (sTrem2) protein in BMDM isolated from Trem2 wt or R47H ki mice. (N = 3, +/-SEM, one way ANOVA, Trem2 p = 0.0003, Bonferroni-corrected pair-wise post hoc tests, WT vs. Het p = 0.0026, WT vs. Hom p = 0.0002, sTrem2 p = 0.0007, WT vs. Het p = 0.0469, WT vs. Hom p = 0.0005)
Fig. 2
Fig. 2
Trem2 haploinsufficiency in an independent R47H knock-in mouse model provided by Jackson laboratories. a DNA sequence comparison of in-house made Trem2 R47H ki mice (R47H ki mice) and Jax Trem2 R47H ki mice (Jax R47H ki mice). b Trem2 and Tyrobp mRNA levels in brains of wt or Jax R47H ki mice. (N = 3, +/-SEM, unpaired t test, Total Trem2 p = 0.0005; Trem2 isoform1 p = 0.0002; Trem2 isoform2 p = 0.0001. n.s. Non-significant.). c Expression levels of membrane bound and soluble Trem2 (sTrem2) protein in bone marrow derived macrophages (BMDM) isolated from Trem2 wt or Jax R47H ki mice (N = 3, +/-SEM, unpaired t test, Trem2 p = 0.0016, sTrem2 p = 0.0433)
Fig. 3
Fig. 3
Aberrant splicing of exon1/2 in two independent Trem2 R47H knock-in mice. a Schematic representation of exon/intron boundaries of Trem2 and the strategy used to investigate exon 1/2 splicing. b RT-PCR mediated amplification of splicing products generated by R47H ki mice. c RT-PCR mediated amplification of splicing products generated by Jax R47H ki mice. d DNA and amino acid sequence of the two splice products identified. Fwd: Forward; Rev: Reverse
Fig. 4
Fig. 4
Aberrant splicing of Trem2 variants containing the R47H mutation with and without additional mutations used to create three different R47H ki mice. a The minigene construct used to investigate exon1/2 splicing of the Trem2 variants shown in (b). b Sequence alignment of Trem2 variants investigated for aberrant splicing. c Exon 1/2 splicing of mouse Trem2 variants described in (b). d Exon 1/2 splicing of human TREM2 variants described in (b). Note that only mouse transcripts undergo aberrant splicing. EV: empty vector
Fig. 5
Fig. 5
Normal exon 1/2 splicing of human TREM2 pre-mRNA encoding the R47H variant. a Normal splicing of human TREM2 upon ectopic expression of the human wt or R47H mutant TREM2 locus in Trem2−/− mice. b Normal exon 1/2 splicing of Trem2 in human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMG) with the wt TREM2 allele or heterozygous for the TREM2 R47H variant. c No aberrant splicing of the R47H variant in an AD case carrying one R47H mutant allele. d No reduction of total TREM2 mRNA in iMG with one R47H allele. (N = 4, +/-SEM, unpaired t test, non-significant.) e Allele specific qPCR demonstrates that the expression of the R47H allele is comparable to the wt allele in iMG. (N = 7, +/-SEM) f Allele specific qPCR demonstrates that the expression of the R47H allele is comparable to wt allele in human brains derived from R47H carriers. (N = 2). Customized probes were against Trem2 R47H and its neighbor region (see also Methods)

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References

    1. Sims R, Van Der Lee SJ, Naj AC, Bellenguez C, Badarinarayan N, Jakobsdottir J, et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat Genet. [Internet] 2017;49:1373–84. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28714976. - PMC - PubMed
    1. Jiang T, Tan L, Chen Q, Tan MS, Zhou JS, Zhu XC, et al. A rare coding variant in TREM2 increases risk for Alzheimer’s disease in Han Chinese. Neurobiol Aging [Internet]. Elsevier Inc. 2016;42:17.e1–217.e3. Available from: 10.1016/j.neurobiolaging.2016.02.023. - PubMed
    1. Jonsson T, Stefansson H, Steinberg S, Jonsdottir I, Jonsson PV, Snaedal J, et al. Variant of TREM2 associated with the risk of Alzheimer’s disease. N Engl J Med. [Internet]. 2013;368:107–16. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23150908. - PMC - PubMed
    1. Guerreiro R, Wojtas A, Bras J, Carrasquillo M, Rogaeva E, Majounie E, et al. TREM2 variants in Alzheimer’s disease. N Engl J Med [Internet]. 2013;368:117–27. Available from: http://www.nejm.org/doi/abs/10.1056/NEJMoa1211851. - DOI - PMC - PubMed
    1. Chan G, White CC, Winn P a, Cimpean M, Replogle JM, Glick LR, et al. CD33 modulates TREM2: convergence of Alzheimer loci. Nat. Neurosci. [Internet]. 2015;2015. Available from: 10.1038/nn.4126. - PMC - PubMed

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