Species: Mouse
Genes: Trem2, APP, PSEN1
Mutations: TREM2 H157Y, APP K670_M671delinsNL (Swedish), APP I716V (Florida), APP V717I (London), PSEN1 M146L (A>C), PSEN1 L286V
Modification: Trem2: Knock-In; APP: Transgenic; PSEN1: Transgenic
Disease Relevance: Alzheimer's Disease
Strain Name: N/A
Genetic Background: C57Bl/6J
Availability: Trem2-H157Y knock-in mice are available through Na Zhao (zhao.na@mayo.edu). 5xFAD mice are available from The Jackson Laboratory, JAX MMRRC Stock# 034848.

Summary

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a transmembrane receptor found on microglia, where it modulates cell activity and survival. TREM2 is cleaved by ADAM proteases after amino acid 157 to release a soluble fragment, sTREM2, whose function is still being elucidated (Feuerbach et al., 2017; Schlepckow et al., 2017; Thornton et al., 2017; 31 Aug 2017 news). H157Y is a rare TREM2 variant that was shown to increase the generation of sTREM2 (Schlepckow et al., 2017; Thornton et al., 2017; Qiao et al., 2023). The variant has been associated with an increased risk for Alzheimer’s disease (Jiang et al., 2016; Song et al., 2017).

To study the effects of the H157Y variant in the context of AD-related pathology, Trem2-H157Y knock-in mice were crossed with 5xFAD mice (Qiao et al., 2023). 5xFAD mice carry transgenes for human APP and PSEN1 with a total of five AD-linked mutations, deposit amyloid plaques and exhibit gliosis as early as 2 months of age, and are one of the most popular transgenic models of amyloidosis in use today. Perhaps surprisingly for a variant associated with increased AD risk, Trem2 H157Y accelerated Aβ42 clearance from the interstitial fluid and decreased plaque pathology, microgliosis and plaque-associated neuritic damage in 5xFAD mice. The variant also led to downregulation of DAM (disease-associated microglia) genes, microglial immune-related genes, and genes encoding inflammatory cytokines.

TREM2 levels and activity | Aβ accumulation and plaque pathology | Gliosis | Dystrophic neurites | Synaptic markers | Transcriptomics | Modification details | Related Models

TREM2 levels and activity

Levels of Trem2 mRNA and TREM2 protein—both full-length, membrane-associated TREM2 and sTREM2—were lower in the cortices of 8.5-month-old 5xFAD mice homozygous for the H157Y variant (5xFAD; Trem2^H157Y/H157) than in 5xFAD mice homozygous for wild-type Trem2 (5xFAD; Trem2^+/+). This difference may reflect the lower number of microglia in the brains of 5xFAD; Trem2^H157Y/H157Y mice, as discussed below. The sTREM2/full-length-TREM2 ratio was higher in 5xFAD; Trem2^H157Y/H157Y mice, consistent with increased sTREM2 shedding in H157Y carriers. Levels of serum sTREM2 were higher in homozygous carriers of the H157Y mutation.

TREM2 signaling, assessed as the ratio of phosphorylated (activated) SYK to total SYK, was lower in microglia from 5xFAD; Trem2^H157Y/H157Y mice than microglia from 5xFAD; Trem2^+/+.

Levels of Trem2 mRNA in heterozygous carriers of the Trem2 mutation were intermediate between homozygous carriers of the mutation and mice homozygous for the wild-type allele but did not significantly differ from the latter. TREM2 shedding (sTREM2/full-length-TREM2) and levels of sTREM2 in serum were also increased in H157Y heterozygotes, compared with mice homozygous for the wild-type allele, and did not differ from homozygous carriers of the mutation. TREM2 signaling was not reported for 5xFAD; Trem2^+/H157 mice.

Aβ accumulation and plaque pathology

The H157Y variant had age-dependent effects on Aβ accumulation and plaque pathology.

At 4 months of age, the genotypes did not differ with regard to cortical or hippocampal plaque burden (percent area occupied by plaques), density, or size—regardless of whether plaques were visualized using Aβ immunohistochemistry or staining with X-34, which labels fibrillar Aβ.

Levels of Aβ40 and Aβ42 in 4-month-old mice, assessed biochemically, also did not differ among the genotypes: Tissue was sequentially solubilized in Tris-buffered saline (TBS), TBS with the detergent Triton X-100 (TBS-T), and guanidine hydrochloride to extract soluble, membrane-associated, and insoluble proteins, respectively. Levels of Aβ40 and Aβ42, measured by ELISA, were similar among the genotypes in all three fractions.

At 8.5 months of age, plaque burdens and densities, visualized using anti-Aβ immunohistochemistry, were lower in the cortices and hippocampi of 5xFAD; Trem2^H157Y/H157 mice, compared with 5xFAD; Trem2^+/+ mice. However, the genotypes did not differ when plaques were stained with X-34. Biochemical findings were consistent with the histology: Levels of insoluble Aβ40 and Aβ42 were lower in 5xFAD; Trem2^H157Y/H157 mice, compared with 5xFAD; Trem2^+/+ mice. While the amounts of Aβ40 and Aβ42 in the TBS and TBS-T fractions did not differ between the genotypes, levels of Aβ42 oligomers in these two fractions were lower in the H157Y homozygotes than in the Trem2 wild-type mice.

For the most part, measures of plaque pathology did not differ between 8.5-month-old H157Y heterozygotes and the other two genotypes (5xFAD; Trem2^H157Y/H157 and 5xFAD; Trem2^+/+), but cortical plaque density was greater in heterozygotes than in H157Y homozygotes. Levels of insoluble Aβ40 and Aβ42 did not differ between H157Y heterozygotes and the other two genotypes.

Although the H157Y mutation did not affect the levels of Aβ42 in brain homogenates from young mice, the mutation accelerated Aβ42 clearance from the interstitial fluid (ISF). Microdialysis was used to collect Aβ42 from the ISF of 3-month-old awake, freely moving mice who had been treated with a γ-secretase inhibitor to halt further production of Aβ. The half-life of the peptide was shortened by 50 percent in 5xFAD; Trem2^H157Y/H157, compared with 5xFAD; Trem2^+/+. (Aβ42 clearance was not measured in H157Y heterozygotes.)

Gliosis

The effects of the H157Y variant on gliosis were also age-dependent.

At 4 months, neither microgliosis (percent coverage Iba1 immunoreactivity) nor astrogliosis (percent coverage GFAP immunoreactivity) differed between genotypes.

However, by 8.5 months, both microgliosis and astrogliosis were reduced in homozygous carriers of the H157Y variant, compared with mice homozygous for wild-type Trem2. 5xFAD; Trem2^H157Y/H157 showed decreased immunoreactivity for the microglial marker Iba1 and the phagocytic marker CD68 in hippocampus and cortex. The H157Y homozygotes also showed less immunoreactivity for TREM2, consistent with the biochemical findings mentioned above. There was a positive correlation between the TREM2- and Iba1-immunoreactive areas, supporting the idea that the reduction in TREM2 in the mutation carriers is due to their having fewer microglia than their Trem2^+/+ counterparts.

There were fewer microglia in the area immediately surrounding plaques in the 5xFAD; Trem2^H157Y/H157 mice, but this reduction in plaque-associated microglia likely reflected the general decrease in microgliosis in the mutation carriers, rather than a specific effect on microglia-plaque interactions.

Microgliosis in H157Y heterozygotes did not differ from that seen in homozygous carriers of the wild-type allele, but was exacerbated compared with H157Y homozygotes (areas of Iba1 and CD68 immunoreactivity in the hippocampus were greater in Trem2^+/H157 mice than in Trem2^H157Y/H157 mice).

Astrogliosis was reduced in the cortices and hippocampi of 5xFAD; Trem2^H157Y/H157, compared with 5xFAD; Trem2^+/+. Additionally, there was less astrogliosis in the hippocampi of heterozygous carriers of the H157Y variant, compared with mice homozygous for wild-type Trem2.

Dystrophic neurites

At 8.5 months, neuritic pathology—assessed as the percent area stained using an antibody directed against the C-terminal of APP—was reduced in the hippocampi of  5xFAD; Trem2^H157Y/H157, compared with 5xFAD; Trem2^+/+ mice. This finding was supported using a second marker of dystrophic neurites, LAMP1.

Neuritic pathology in H157Y heterozygotes did not differ from that in homozygous carriers of the wild-type Trem2 allele, assessed using either of the two markers. Compared with H157Y homozygotes, the heterozygotes showed greater immunoreactivity for APP, but the two genotypes did not differ with regard to LAMP1.

Synaptic markers

Levels of the presynaptic marker synaptophysin and the postsynaptic marker PSD95, measured in western blots of cortical lysates from 8.5-month-old mice, did not differ between genotypes.

Transcriptomics

Bulk RNA sequencing revealed 183 differentially expressed genes in the cortices of 8.5-month-old 5xFAD; Trem2^H157Y/H157, compared with 5xFAD; Trem2^+/+ mice, 177 of which were downregulated in the Trem2 mutation carriers. The H157Y variant led to downregulation of DAM (disease-associated microglia) genes, microglial immune-related genes, and genes encoding inflammatory cytokines. Several genes expressed by astrocytes were also among those downregulated in 5xFAD; Trem2^H157Y/H157 animals.

Modification details

CRISPR/Cas9 gene editing was used to introduce the H157Y mutation into the mouse Trem2 gene. The resulting Trem2-H157Y knock-in mice were then intercrossed with 5xFAD mice.

Related Models

The following models have been used to manipulate levels of soluble TREM2, through genetic alteration of the ADAM protease cleavage site or AAV-mediated expression of an extracellular fragment of TREM2.

Trem2-H157Y knock-in. CRISPR/Cas9 gene editing was used to introduce the H157Y mutation into the mouse Trem2 gene. This variant first gained attention when it was found to associate with an increased risk for Alzheimer’s disease in a Han Chinese cohort (Jiang et al., 2016). Elevated levels of sTREM2 were found in mice homozygous for the variant. The H157Y mutation did not affect microglial density or morphology, performance on a battery of behavioral tests, or levels of synaptic markers, but enhanced hippocampal synaptic plasticity. Perhaps surprisingly, when Trem2-H157Y mice were crossed with 5xFAD mice, the Trem2 variant decreased amyloid pathology, microgliosis, and plaque-associated neuritic damage.

Trem2-sol. In an attempt to create a model that generates only sTREM2—but no full-length, signaling-competent receptor—CRISPR/Cas9 gene editing was used to introduce a stop codon after H157 of murine Trem2. These mice, called “TREM2-sol,” were found to express very low levels of sTREM2 and Trem2 mRNA. Nonetheless, differences between TREM2-sol and Trem2 knockout mice (Trem2-KO) were observed, including prolonged microglial responses to injury, increased vulnerability of bone marrow-derived macrophages to growth factor deprivation, and preservation of endo-lysosomal function in TREM2-sol compared with Trem2-KO mice.

Trem2-IPD. CRISPR/Cas9 gene editing was used to disrupt the ADAM10/17 recognition site on mouse TREM2, changing histidine-157 to isoleucine (I), serine-158 to proline (P), and threonine-159 to aspartate (D)—leading to both a reduction in sTREM2 and an increase in cell-surface TREM2. The IPD mutation accelerated microglial maturation and increased microglial phagocytic activity. In the cuprizone model of demyelination, the mutation resulted in persistent neuroinflammation during the recovery phase.

Trem2-IPDxAPP23xPS45. To study the effects of reducing TREM2 cleavage in the context of amyloid pathology, Trem2-IPD mice were intercrossed with APP23 and PS45 mice (Herzig et al., 2004), which carry transgenes for human APP with the AD-linked Swedish mutation and PSEN1 with the AD-linked G384A mutation, respectively. Compared with APP23xPS45 mice carrying wild-type Trem2, Trem2-IPDxAPP23xPS45 mice had higher plaque burdens and more severe plaque-associated pathology at an early—but not late—stage of plaque deposition.

AAV-sTREM2 5xFAD. AAV-sTREM2 5xFAD mice were created to study the long-term effects of soluble TREM2 (sTREM2) in the context of amyloidosis. To generate this model, AAV carrying cDNA encoding EGFP- and FLAG-tagged sTREM2 (amino acids 1-171 of human TREM2) was injected into the brains of neonatal 5xFAD mice. Overexpression of sTREM2 led to decreased plaque burdens, increased the number of plaque-associated microglia, and rescued hippocampal long-term potentiation and performance in the Morris water maze in 5xFAD mice aged 6 to 7 months.

AAV-sTREM2 PS19. AAV-sTREM2 PS19 mice were created to study the long-term effects of soluble TREM2 (sTREM2) in the context of tauopathy. To generate this model, AAV carrying cDNA encoding EGFP- and FLAG-tagged sTREM2 (amino acids 1-171 of human TREM2) was injected into the brains of 3-month-old PS19 mice. AAV-mediated expression of sTREM2 protected against hippocampal synapse loss, improved performance in the Morris water maze and Y-maze, enhanced long-term potentiation, and reduced levels of p-tau202 and p-tau396 in PS19 mice studied at 7 months of age.

Phenotype Characterization

Q&A with Model Creator

Q&A with Wenhui Qiao and Na Zhao.

What would you say are the unique advantages of this model? This model is the first animal model to feature a knock-in of the Trem2 H157Y mutation, enabling the study of how this mutation influences amyloid-β development. The phenotypes derived from these mice were validated across two different founder lines. Both heterozygous and homozygous littermates were included and characterized in terms of their functions in impacting amyloid burden.

What do you think this model is best used for? This model can be used to understand Trem2's overall function, particularly how increased soluble TREM2 due to the H157Y mutation influences microglial functions under various pathological conditions.

COMMENTS / QUESTIONS

No Available Comments

Make a comment or submit a question

To make a comment you must login or register.

References

News Citations

1. TREM2 Cleavage Site Pinpointed: A Gateway to New Therapies? 31 Aug 2017

Research Models Citations

1. Trem2-H157Y knock-in
2. TREM2-sol
3. Trem2 KO (KOMP)
4. TREM2-IPD
5. Trem2-IPDxAPP23xPS45
6. APP23
7. AAV-sTREM2 5xFAD
8. AAV-sTREM2 PS19
9. Tau P301S (Line PS19)

Mutations Citations

1. APP K670_M671delinsNL (Swedish)
2. PSEN1 G384A

Paper Citations

1. Feuerbach D, Schindler P, Barske C, Joller S, Beng-Louka E, Worringer KA, Kommineni S, Kaykas A, Ho DJ, Ye C, Welzenbach K, Elain G, Klein L, Brzak I, Mir AK, Farady CJ, Aichholz R, Popp S, George N, Neumann U. ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157. Neurosci Lett. 2017 Nov 1;660:109-114. Epub 2017 Sep 18 PubMed.
2. Schlepckow K, Kleinberger G, Fukumori A, Feederle R, Lichtenthaler SF, Steiner H, Haass C. An Alzheimer-associated TREM2 variant occurs at the ADAM cleavage site and affects shedding and phagocytic function. EMBO Mol Med. 2017 Oct;9(10):1356-1365. PubMed.
3. Thornton P, Sevalle J, Deery MJ, Fraser G, Zhou Y, Ståhl S, Franssen EH, Dodd RB, Qamar S, Gomez Perez-Nievas B, Nicol LS, Eketjäll S, Revell J, Jones C, Billinton A, St George-Hyslop PH, Chessell I, Crowther DC. TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant. EMBO Mol Med. 2017 Oct;9(10):1366-1378. PubMed.
4. Qiao W, Chen Y, Zhong J, Madden BJ, Charlesworth CM, Martens YA, Liu CC, Knight J, Ikezu TC, Aishe K, Zhu Y, Meneses A, Rosenberg CL, Kuchenbecker LA, Vanmaele LK, Li F, Chen K, Shue F, Dacquel MV, Fryer J, Pandey A, Zhao N, Bu G. Trem2 H157Y increases soluble TREM2 production and reduces amyloid pathology. Mol Neurodegener. 2023 Jan 31;18(1):8. PubMed. Correction.
5. Jiang T, Hou JK, Gao Q, Yu JT, Zhou JS, Zhao HD, Zhang YD. TREM2 p.H157Y Variant and the Risk of Alzheimer's Disease: A Meta-Analysis Involving 14,510 Subjects. Curr Neurovasc Res. 2016;13(4):318-320. PubMed.
6. Song W, Hooli B, Mullin K, Jin SC, Cella M, Ulland TK, Wang Y, Tanzi RE, Colonna M. Alzheimer's disease-associated TREM2 variants exhibit either decreased or increased ligand-dependent activation. Alzheimers Dement. 2017 Apr;13(4):381-387. Epub 2016 Aug 9 PubMed.
7. Jiang T, Tan L, Chen Q, Tan MS, Zhou JS, Zhu XC, Lu H, Wang HF, Zhang YD, Yu JT. A rare coding variant in TREM2 increases risk for Alzheimer's disease in Han Chinese. Neurobiol Aging. 2016 Jun;42:217.e1-3. Epub 2016 Mar 3 PubMed.
8. Herzig MC, Winkler DT, Burgermeister P, Pfeifer M, Kohler E, Schmidt SD, Danner S, Abramowski D, Stürchler-Pierrat C, Bürki K, van Duinen SG, Maat-Schieman ML, Staufenbiel M, Mathews PM, Jucker M. Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis. Nat Neurosci. 2004 Sep;7(9):954-60. PubMed.

External Citations

1. The Jackson Laboratory, JAX MMRRC Stock# 034848

Further Reading

No Available Further Reading