The R47H mutation in the TREM2 gene triples a person’s risk of Alzheimer’s disease, and for a while researchers thought they had discovered why. Using CRISPR to introduce the mutation in mice, two independent groups reported that it dramatically diminished TREM2 expression. A third model, described September 10 in Molecular Neurodegeneration, confirmed this and uncovered the mechanism: The R47H mutation bungles splicing of the mouse gene, leading to a stunted transcript destined for proteolysis. The rub? This only happens in the mouse version of the gene. The researchers, led by Christian Haass at the German Center for Neurodegenerative Diseases in Munich, reported that in people with the R47H mutation, TREM2 splicing and expression were completely normal.
- R47H mutation in TREM2 dampens expression of the mouse gene.
- Suppression linked to alternative splice site.
- CRISPR-induced silent mutations exacerbate the effect.
The results point to an inconvenient truth: R47H-knock-in models cannot provide reliable insight into how the mutation alters TREM2 function in human carriers, the researchers contend. “This is just bad luck,” Haass told Alzforum. Mouse models that use a humanized version of the gene will be needed to study the biology of the mutation, he suggested.
In a joint comment to Alzforum, the researchers who had generated two other R47H-TREM2 models weren’t so quick to write them off completely. They pointed out that homozygous animals do express a significant amount of the correctly spliced gene, meaning that they may yet yield some information about how the mutation affects TREM2 function.
In the six years since researchers linked the R47H-TREM2 variant to AD, mounting evidence from cell culture models has indicated that the mutation stifles the microglial receptor’s interactions with ligands, such as phospholipids, ApoE, and Aβ (Nov 2012 news; Oct 2015 news; Kober et al., 2016). TREM2-deficient mouse models suggest the receptor is crucial for activating microglia and containing Aβ plaques (Feb 2015 news; May 2016 news; Jun 2017 news; Jul 2018 conference news).
To learn how R47H affects TREM2 function in vivo, several groups have developed mouse models expressing the variant. Researchers led by Marco Colonna at Washington University in St. Louis used a bacterial artificial chromosome (BAC) to express the human gene, with or without the mutation, in mice lacking their own TREM2. The R47H-TREM2 bound poorly to ligands, and in mice crossed to 5xFAD mice, the mutated receptor failed to rally microglia to surround and contain plaques (TREM2, humanized (R47H) X 5XFAD mouse model; Jan 2018 news). However, the BAC model expressed eight copies of the human transgene, making it difficult to distinguish between effects of R47H and effects of overexpression.
To avoid the latter, four groups used CRISPR-Cas9 to introduce R47H directly into endogenous mouse TREM2. They were led by Bruce Lamb and Gary Landreth at Indiana University School of Medicine in Indianapolis (Trem2 R47H KI (Lamb/Landreth) mouse model; Cheng-Hathaway et al., 2018); the MODEL-AD consortium at the Jackson Laboratory in Bar Harbor, Maine (Trem2 R47H KI (JAX)); Shilpa Shambashivan and colleagues at Amgen (Cheng et al., 2018); and now Haass’ group.
In addition to the G-to-A point mutation required to generate R47H, the Munich group introduced three silent mutations: GA-to-TC mutations to create a restriction site to ease genotyping; and a G-to-A mutation to boost CRISPR gene editing efficiency. First author Xianyuan Xiang found that heterozygous R47H-TREM2 mice had about 40 percent less TREM2 mRNA in their brains than wild-type, while homozygous R47H-TREM2 mice had about 70 percent less. In the heterozygotes, only the mutant copy was reduced. Similarly, microglia from the mutant mice expressed less TREM2 RNA and made less TREM2 protein than cells from wild-type mice. Xiang came to similar conclusions when she measured TREM2 expression in the R47H-TREM2 mice generated at Jackson Labs.
Analysis of TREM2 RNA transcripts pointed to splicing gone awry. Using RT-PCR, Xiang found that the R47H mutation, which lies in the second exon, introduced a new splice site. This resulted in removal of 119 base pairs at the 5' end of exon 2, introducing a premature stop codon that created a stunted transcript. The researchers speculated that this short transcript would make fodder for nonsense-mediated decay, a cellular surveillance mechanism that gobbles up transcripts with premature stop codons. In support of this idea, RT-PCR amplified a only a small amount of the short TREM2 transcript, even in the R47H TREM2 homozygotes. The researchers did detect traces of the correctly spliced transcript as well, suggesting some residual normal splicing still occurred. They also analyzed splicing patterns in the R47H-TREM2 mice generated at Jackson Lab, and found both transcripts in those mice as well, although the Jackson Lab mice appeared to have a greater proportion of the correctly spliced transcript than the Haass mice did.
In a joint comment to Alzforum, Landreth, Lamb, and Michael Sasner of Jackson Labs confirmed that the splicing defects occurred in their two R47H-TREM2 models (see full comment below). Amgen did not respond to requests for comment about its model.
Itching to see whether R47H TREM2 expression in people also fell prey to splicing antics, Xiang and colleagues expressed human and mouse TREM2 genes in human embryonic kidney (HEK) 293 cells. The R47H mutation triggered the same splicing defects in mouse TREM2 as it had in the CRISPR mice. Additional silent mutations introduced to make CRISPR more efficient exacerbated the problem. In stark contrast, neither R47H nor the silent mutations affected splicing of the human TREM2 gene. Moreover, TREM2 was spliced normally in induced microglia derived from R47H-TREM2 mutation carriers, and also in a postmortem brain sample from an AD patient with the R47H TREM2 mutation. Xiang also found that human TREM2 was spliced normally in Colonna’s BAC model.
Haass told Alzforum that to his mind, these findings render all the R47H-TREM2 CRISPR models unsuitable for studying human TREM2 biology. Rather than modeling functional effects of the R47H variant, the mice more closely model TREM2 deficiency, he said. Moving forward, researchers will need to employ different strategies, such as generating knock-in mice that express human TREM2 variants under control of the endogenous mouse promoter, he said.
Lamb, Landreth, and the MODEL-AD consortium scientists acknowledge that better models are needed. Even so, they think the R47H-TREM2 CRISPR models have some use. They noted that although the majority of the mutant TREM2 transcript is mis-spliced, their mice still produce some full-length mutant transcript. They are comparing whole-brain transcriptomes of TREM2-deficient mice to those of R47H CRISPR mice; this should provide some insight into specific effects of the R47H allele, they wrote.
The MODEL-AD consortium is considering strategies to generate TREM2 R47H models that splice the transcript normally, and seeks ideas from the scientific community on how to make that happen.—Jessica Shugart
- Enter the New Alzheimer’s Gene: TREM2 Variant Triples Risk
- Alzheimer’s Risk Genes Interact in Immune Cells
- TREM2 Buoys Microglial Disaster Relief Efforts in AD and Stroke
- Barrier Function: TREM2 Helps Microglia to Compact Amyloid Plaques
- Hot DAM: Specific Microglia Engulf Plaques
- TREM2: Diehard Microglial Supporter, Consequences Be DAMed
- New Mouse Models Reveal Unexpected Property of TREM2
Research Models Citations
- TREM2, humanized (R47H) X 5XFAD
- Trem2 R47H KI (Lamb/Landreth)
- Trem2 R47H KI (JAX)
- Trem2 R47H KI (Haass)
- Kober DL, Alexander-Brett JM, Karch CM, Cruchaga C, Colonna M, Holtzman MJ, Brett TJ. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms. Elife. 2016 Dec 20;5 PubMed.
- Cheng-Hathaway PJ, Reed-Geaghan EG, Jay TR, Casali BT, Bemiller SM, Puntambekar SS, von Saucken VE, Williams RY, Karlo JC, Moutinho M, Xu G, Ransohoff RM, Lamb BT, Landreth GE. The Trem2 R47H variant confers loss-of-function-like phenotypes in Alzheimer's disease. Mol Neurodegener. 2018 Jun 1;13(1):29. PubMed.
- Cheng Q, Danao J, Talreja S, Wen P, Yin J, Sun N, Li CM, Chui D, Tran D, Koirala S, Chen H, Foltz IN, Wang S, Sambashivan S. TREM2-activating antibodies abrogate the negative pleiotropic effects of the Alzheimer's disease variant Trem2 R47H on murine myeloid cell function. J Biol Chem. 2018 Aug 10;293(32):12620-12633. Epub 2018 Mar 29 PubMed.
- Xiang X, Piers TM, Wefers B, Zhu K, Mallach A, Brunner B, Kleinberger G, Song W, Colonna M, Herms J, Wurst W, Pocock JM, Haass C. The Trem2 R47H Alzheimer's risk variant impairs splicing and reduces Trem2 mRNA and protein in mice but not in humans. Mol Neurodegener. 2018 Sep 6;13(1):49. PubMed.