Species: Mouse
Genes: Trem2
Modification: Trem2: Knock-In
Disease Relevance: Alzheimer's Disease
Strain Name: B6-Trem2em2Npa
Genetic Background: C57BL/6J
Availability: Available from Novartis Pharma AG under an MTA. Contact Ivan Galimberti (ivan.galimberti@novartis.com) or Derya Shimshek (derya.shimshek@novartis.com).

Summary

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a transmembrane receptor found on microglia, where it modulates cell activity and survival. In addition to membrane-associated TREM2, there are soluble forms of the protein—generated by protease cleavage of the extracellular domain or expression of alternative transcripts that lack a transmembrane domain.

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). A rare variant in TREM2, H157Y, increases TREM2 shedding (Schlepckow et al., 2017; Thornton et al., 2017) and has been associated with an increased risk of Alzheimer’s disease (Jiang et al., 2016; Song et al., 2017).

In this model, 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 (Dhandapani et al. 2022). In the cuprizone model of demyelination, the mutation resulted in persistent neuroinflammation during the recovery phase (Beckmann et al., 2023).

The description below refers to animals homozygous for the IPD mutation.

Mutation of the ADAM10/17 recognition site decreased TREM2 shedding from the cell surface, although the magnitude of the decrease appeared to depend on experimental conditions. Levels of sTREM2 generated by TREM2-IPD mice were approximately half those produced by wild-type animals, as measured in the media of brain slices from 6-wek-old female mice (Dhandapani et al. 2022). The difference between genotypes was not as pronounced when TREM2—presumably sTREM2—was measured in detergent-free extracts of forebrains from 2- to 3-month-old female mice: Under these conditions, levels of sTREM2 in TREM2-IPD mice were about 90 percent those of wild-type mice (Beckmann et al., 2023). (It should be noted that the assays used to measure sTREM2 are not expected to distinguish between soluble fragments generated by protease cleavage and soluble isoforms generated by alternative splicing.)

Cell-surface TREM2, measured on bone-marrow-derived macrophages (BMDMs) cultured from adult mice, was increased in TREM2-IPD, compared with wild-type—approximately two-fold in one study (Dhandapani et al. 2022) and greater than five-fold in a second study (Beckmann et al., 2023). These increases in cell-surface TREM2 were accompanied by greater survival of TREM2-IPD BMDMs when macrophage colony-stimulating factor was removed from the culture medium.

Microglia cultured from neonatal TREM2-IPD mice showed increased phagocytic capacity. Compared with microglia from wild-type mice, TREM2-IPD microglia showed increased uptake of pHrodo-myelin (myelin tagged with a pH-sensitive dye) (Beckmann et al., 2023) and of fluorescently labeled bacterial particles, LDL, and tau “seeds” derived from the brains of transgenic mice expressing human tau with the P301S mutation linked to frontotemporal dementia (Dhandapani et al. 2022).

Transcriptomic analyses were performed on cortical samples from 3- and 7-month-old mice, using single-cell RNA sequencing (Dhandapani et al. 2022). When cells were assigned to cell-type clusters based on the expression of marker genes (e.g., cells expressing P2ry12, Tmem119, C1qa, C1qb were identified as microglia), a greater proportion of cells were identified as microglia in TREM2-IPD samples, compared with wild-type samples. This increase in microglial abundance was confirmed by immunohistochemistry: Three-month-old TREM2-IPD mice had more Tmem119-positive microglia and a greater percentage of proliferating microglia, shown by BrdU staining, than did wild-type mice. Further analysis of microglial expression patterns showed a shift from 3 months to 7 months in wild-type mice, which the investigators characterized as “early” and “late” homeostatic profiles, respectively. Microglial expression patterns in TREM2-IPD mice did not change appreciably between 3 and 7 months and resembled the “late homeostatic” profiles of 7-month wild-type mice.

Pathological contexts

TREM2-IPD mice have been used to study the effects of reduced TREM2 cleavage in two pathological contexts: amyloidosis (Dhandapani et al. 2022) and demyelination (Beckmann et al., 2023).

As described here, the Trem2 IPD mutation exacerbated plaque pathology at an early—but not late—stage of plaque deposition in mice expressing human APP and PSEN1 transgenes with AD-linked mutations.

In the second model of neuropathology, demyelination was induced by feeding mice the copper chelator cuprizone. The results described here refer to the external capsule, as this white-matter tract was particularly affected. Five-week administration of cuprizone caused similar degrees of demyelination and loss of oligodendrocytes in TREM2-IPD mice and wild-type mice. (Measures of myelination and oligodendrocytes did not differ between the two genotypes under control conditions.) Microgliosis, astrogliosis, and increased staining for the lysosomal marker LAMP1 developed similarly in the two genotypes during cuprizone administration. There was no evidence of accumulation of myelin debris or axonal damage. Four weeks after the discontinuation of cuprizone, oligodendrocytes had partially recovered in both genotypes, but remyelination had not yet occurred. Microglial numbers returned towards baseline in both genotypes, but recovery was greater in the wild-type mice. While GFAP and LAMP1 immunoreactivity returned to normal in wild-type mice, these markers remained elevated in TREM2-IPD mice. These observations suggest that there was limited resolution of neuroinflammation in TREM2-IPD mice during the 4-week recovery period following cuprizone intoxication.

Modification details

CRISPR/Cas9 gene editing was used to disrupt the ADAM10/17 recognition site on TREM2, changing histidine-157 to isoleucine (I), serine-158 to proline (P), and threonine-159 to aspartate (D).

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-H157Y x 5xFAD. Trem2-H157Y mice were intercrossed with 5xFAD mice, a model of aggressive amyloidosis. Levels of Trem2 mRNA and TREM2 protein—both full-length, membrane-associated TREM2 and sTREM2—were lower in 5xFAD mice homozygous for Trem2-H157Y, compared with 5xFAD mice expressing wild-type Trem2. This difference may reflect the lower number of microglia in the brains of the Trem2 mutation carriers. However, the ratio of sTREM2 to full-length TREM2 was higher in the Trem2 mutation carriers, consistent with increased shedding of TREM2-H157Y.

Compared with 5xFAD mice homozygous for wild-type Trem2, mice homozygous for the H157Y variant showed age-dependent decreases in plaque burdens, microgliosis and plaque-associated neuritic damage. At 8.5 months, the only timepoint reported to date, expression of neuroinflammation-related genes was downregulated in the H157Y mutation carriers.

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-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

Complementary Models

The TREM2-IPD model employs a genetic manipulation to disrupt the ADAM cleavage site on TREM2—reducing the generation of sTREM2 and increasing signaling-competent TREM2 on the cell surface. A team led by Christian Haass in Munich and Joseph Lewcock at Denali used an alternate strategy to block ADAM cleavage of TREM2, employing a monoclonal antibody (4D9) that binds near the cleavage site and impedes access of the enzyme to the receptor (Schlepckow et al., 2020; see 10 Mar 2020 news). Like the IPD mutation, antibody 4D9 reduces levels of sTREM2 and increases cell-surface TREM2, but the models differ in an important aspect: Antibody 4D9 activates TREM2 signaling, likely through cross-linking cell-surface TREM2.

The IPD mutation and 4D9 treatment had similar effects on the physiology of myeloid cells in vitro. Both manipulations increased the survival of bone-marrow-derived macrophages after withdrawal of macrophage colony-stimulating factor, and both increased phagocytic activity of microglia.

The effects of 4D9 treatment and of the IPD mutation have also been studied in mouse models of amyloidosis. As the effects of these manipulations were studied in two different mouse models, direct comparisons are difficult. Nonetheless, both manipulations appeared to accelerate the transition of microglia from a homeostatic to a disease-associated phenotype. In Trem2-IPDxAPP23xPS45 mice, the IPD mutation led to higher plaque burdens and more severe plaque-associated pathology at an early stage of plaque deposition, but these effects disappeared as the mice aged (Dhandapani et al. 2022). Administration of the antibody over a 10-day period to 6-month-old APP NL-G-F knock-in mice—a late stage of plaque deposition in this line—led to decreased cortical plaque loads.

While the agonist (i.e., TREM2-activating) effect of 4D9 limits its usefulness as a tool to study the consequences of reduced TREM2 shedding, agonist antibodies are being investigated as potential therapeutics for AD (Price et al., 2020; Wang et al., 2020; 26 Jun 2020 news), and at least one such antibody is in clinical trials.

Reduced generation of sTREM2—in this case, by HEK293 cells overexpressing TREM2 and its adaptor DAP12—has also been achieved using single-chain variable fragments (scFvs) that bind the extracellular domain of TREM2 (Szykowska et al., 2021; 29 Jul 2021 news). However, in this case, decreased production of sTREM2 was likely caused by internalization of the receptors rather than blockage of the ADAM cleavage site.

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References

News Citations

1. TREM2 Cleavage Site Pinpointed: A Gateway to New Therapies? 31 Aug 2017
2. In Mice, Activating TREM2 Tempers Plaque Toxicity, not Load 26 Jun 2020
3. New Ways to Target TREM2 Beg the Question: Up or Down? 29 Jul 2021

Research Models Citations

1. Trem2-IPDxAPP23xPS45
2. Trem2-H157Y knock-in
3. Trem2-H157Y x 5xFAD
4. 5xFAD (C57BL6)
5. TREM2-sol
6. Trem2 KO (KOMP)
7. APP23
8. AAV-sTREM2 5xFAD
9. AAV-sTREM2 PS19
10. APP NL-G-F Knock-in

Mutations Citations

1. APP K670_M671delinsNL (Swedish)
2. PSEN1 G384A

Therapeutics Citations

1. AL002

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. 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.
5. 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.
6. Dhandapani R, Neri M, Bernhard M, Brzak I, Schweizer T, Rudin S, Joller S, Berth R, Kernen J, Neuhaus A, Waldt A, Cuttat R, Naumann U, Keller CG, Roma G, Feuerbach D, Shimshek DR, Neumann U, Gasparini F, Galimberti I. Sustained Trem2 stabilization accelerates microglia heterogeneity and Aβ pathology in a mouse model of Alzheimer's disease. Cell Rep. 2022 May 31;39(9):110883. PubMed.
7. Beckmann N, Neuhaus A, Zurbruegg S, Volkmer P, Patino C, Joller S, Feuerbach D, Doelemeyer A, Schweizer T, Rudin S, Neumann U, Berth R, Frieauff W, Gasparini F, Shimshek DR. Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model. J Neuroinflammation. 2023 Feb 8;20(1):29. PubMed.
8. 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.
9. 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.
10. Schlepckow K, Monroe KM, Kleinberger G, Cantuti-Castelvetri L, Parhizkar S, Xia D, Willem M, Werner G, Pettkus N, Brunner B, Sülzen A, Nuscher B, Hampel H, Xiang X, Feederle R, Tahirovic S, Park JI, Prorok R, Mahon C, Liang CC, Shi J, Kim DJ, Sabelström H, Huang F, Di Paolo G, Simons M, Lewcock JW, Haass C. Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region. EMBO Mol Med. 2020 Apr 7;12(4):e11227. Epub 2020 Mar 10 PubMed.
11. Dhandapani R, Neri M, Bernhard M, Brzak I, Schweizer T, Rudin S, Joller S, Berth R, Kernen J, Neuhaus A, Waldt A, Cuttat R, Naumann U, Keller CG, Roma G, Feuerbach D, Shimshek DR, Neumann U, Gasparini F, Galimberti I. Sustained Trem2 stabilization accelerates microglia heterogeneity and Aβ pathology in a mouse model of Alzheimer's disease. Cell Rep. 2022 May 31;39(9):110883. PubMed.
12. Price BR, Sudduth TL, Weekman EM, Johnson S, Hawthorne D, Woolums A, Wilcock DM. Therapeutic Trem2 activation ameliorates amyloid-beta deposition and improves cognition in the 5XFAD model of amyloid deposition. J Neuroinflammation. 2020 Aug 14;17(1):238. PubMed.
13. Wang S, Mustafa M, Yuede CM, Salazar SV, Kong P, Long H, Ward M, Siddiqui O, Paul R, Gilfillan S, Ibrahim A, Rhinn H, Tassi I, Rosenthal A, Schwabe T, Colonna M. Anti-human TREM2 induces microglia proliferation and reduces pathology in an Alzheimer's disease model. J Exp Med. 2020 Sep 7;217(9) PubMed.
14. Szykowska A, Chen Y, Smith TB, Preger C, Yang J, Qian D, Mukhopadhyay SM, Wigren E, Neame SJ, Gräslund S, Persson H, Atkinson PJ, Di Daniel E, Mead E, Wang J, Davis JB, Burgess-Brown NA, Bullock AN. Selection and structural characterization of anti-TREM2 scFvs that reduce levels of shed ectodomain. Structure. 2021 Nov 4;29(11):1241-1252.e5. Epub 2021 Jul 6 PubMed.

Other Citations

1. 10 Mar 2020 news

Further Reading