Species: Mouse
Genes: TREM2, APP, PSEN1
Mutations: APP K670_M671delinsNL (Swedish), APP I716V (Florida), APP V717I (London), PSEN1 M146L (A>C), PSEN1 L286V
Modification: TREM2: Virus; APP: Transgenic; PSEN1: Transgenic
Disease Relevance: Alzheimer's Disease
Strain Name: N/A
Genetic Background: 5xFAD (C57BL6)
Availability: 5xFAD mice are available from The Jackson Laboratory, JAX MMRRC Stock# 034848. Research with 5xFAD is available from Scantox Neuro.

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.

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. The 5xFAD line carries human APP and PSEN1 transgenes with a total of five AD-linked mutations; mice begin depositing amyloid plaques by 2 months of age and show age-dependent gliosis, deficits in synaptic plasticity, and cognitive impairment. AAV-mediated over expression 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 (Zhong et al., 2019; 05 Apr 2019 news).

There are a few caveats associated with this model. Endogenous sTREM2 generated by protease cleavage encompasses amino acids 1-157, not amino acids 1-171 and, of course, lacks the EGFP and FLAG sequences. Endogenous sTREM2 is produced from TREM2 located on the surface of microglial cells, while AAV-derived sTREM2 apparently was made by neurons. Additionally, the levels of transgenic sTREM2 appear to be extremely high relative to the amount of endogenous sTREM2 found in the brains of 5xFAD mice.

The description below refers to mice injected with AAV as neonates and examined at 6 to 7 months of age.

Expression of sTREM2 | Neuropathology | Long-term potentiation | Behavior | Modification details | Related Models

Expression of sTREM2

sTREM2 appears to be over expressed in the brains of 7-month-old 5xFAD mice who received injections of AAV-sTREM2 as neonates, although the assay used by Zhong and colleagues did not permit the direct comparison of levels of transgenic human sTREM2 and endogenous mouse sTREM2.

Using an ELISA that detects human TREM2, approximately 400 ng sTREM2/mg protein were measured in lysates of AAV-sTREM2 5xFAD brains prepared using a detergent-free buffer (Tris-buffered saline, or TBS) expected to extract soluble proteins. Mouse TREM2 was not measured in this assay. However, in a later study conducted by Guojun Bu and colleagues (Qiao et al., 2023), endogenous mouse TREM2 in TBS lysates of wild-type brains was quantified from western blots and found to be approximately 0.4 ng/mg protein, three orders of magnitude less than the human sTREM2 in the AAV model. Even considering that the ELISA may be a more sensitive assay and that levels of total TREM2 have been shown to be elevated in the brains of 5xFAD mice compared with wild-type mice (Wang et al., 2020), it is likely that the levels of sTREM2 in AAV-sTREM2 5xFAD brains are supraphysiological.

In AAV-sTREM2 5xFAD mice, immunoreactivity for human sTREM2 was found primarily in neurons. To confirm that neuronally expressed sTREM2 could be released into the extracellular space, primary neurons were transduced with the AAV-sTREM2 vector, and sTREM2 was detected in the culture medium. Thus, although the AAV-sTREM2 5xFAD model does not replicate the physiological process of sTREM2 generation from microglia, it can be used to study the effects of soluble TREM2 fragments, albeit at supraphysiological concentrations.

Neuropathology

Over expression of sTREM2 decreased the amyloid plaque burdens in the hippocampus and cortex by about half, comparing AAV-sTREM2 5xFAD mice with 5xFAD mice who were injected with a control virus (AAV carrying EGFP cDNA). The number of plaque-associated microglia was increased in AAV-sTREM2 5xFAD mice, compared with controls.

Long-term potentiation

Long-term potentiation (LTP) at Shaeffer collateral-CA1 synapses is impaired in 5xFAD mice. AAV-mediated over expression of sTREM2 rescued LTP in AAV-sTREM2 5xFAD mice. This beneficial effect of sTREM2 on LTP is congruent with the finding of enhanced LTP in TREM2-H157Y knock-in mice, in which the mutation slightly increases the generation of endogenous sTREM2 (Qiao et al., 2023).

Behavior

5xFAD mice show impaired learning and memory in the Morris water maze. When tested 6 months after injection of AAV-sTREM2, AAV-sTREM2 5xFAD mice performed as well as non-transgenic controls.

Modification details

Adeno-associated viruses (AAV2/8) expressing EGFP- and FLAG-tagged human TREM2 (amino acids 1-171) under the control of the CAG promoter were injected bilaterally into the lateral ventricles of neonatal 5xFAD mice. Control mice received injections of AAV expressing only EGFP.

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-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 early, but not late, stages of plaque deposition.

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 AAV-sTREM2 5xFAD model employed AAV-mediated over expression of an extracellular fragment of TREM2 in the brains of 5xFAD mice to study the effects of chronically elevated soluble TREM2 (sTREM2) in the context of amyloidosis.

Another approach to investigating the role of sTREM2 is the direct application of the protein to animals or cells, but these studies generally look at short-term effects. sTREM2-Fc is a recombinant, chimeric protein intended to mimic sTREM2, consisting of the human TREM2 extracellular domain (amino acids 1-171) fused to human IgG-Fc to aid in purification of the recombinant protein (Zhong et al., 2017). Guojun Bu, Xiao-Fen Chen and colleagues injected sTREM2-Fc into the right hippocampi of 7-month-old 5xFAD mice; the left hippocampi were injected with buffer (Zhong et al., 2019). One day post-injection, sTREM2-Fc was found throughout the right hippocampus, but the injected protein was barely visible via immunohistochemistry at three-days post-injection. When examined one week after injection, the hippocampi injected with sTREM2-Fc had reduced plaque loads compared with the hippocampi injected with buffer. This finding is congruent with the decreased plaque loads seen in AAV-sTREM2 5xFAD mice. Exposure to sTREM2-Fc also resulted in an increased number of plaque-associated microglia—like the finding in AAV-sTREM2 5xFAD—increased microglial phagocytosis of Aβ, and increased expression of inflammatory cytokines. sTREM2-Fc did not appear to affect astrogliosis, as assessed by GFAP immunoreactivity. Neuritic pathology was ameliorated by exposure to sTREM2-Fc.

The protease-cleavage site in TREM2 had not yet been identified at the time Bu, Chen and colleagues began the study described above (i.e., it was not known that the physiological form of sTREM2 ended at amino acid 157). These investigators subsequently made a chimeric protein consisting of amino acids 1-157 of human TREM2 fused to IgG-Fc. One week following injection of sTREM2(1-157)-Fc into the right hippocampi of 5xFAD mice, a reduced plaque load and an increased number of plaque-associated microglia were seen in the right side, compared with the left hippocampi, which received buffer injections.

A concern with these experiments is the amount of recombinant sTREM2-Fc injected into the mouse brains. Bu and colleagues immunoprecipitated TREM2 from the brains of 5xFAD mice and measured the amount of sTREM2 by mass spectrometry, finding around 15 fmol sTREM2/mg brain tissue (Qiao et al., 2023). They then retrospectively estimated the concentration of sTREM2-Fc in the hippocampi of the mice in their 2017 study, in which they injected sTREM2-Fc into the hippocampi of wild-type mice (Zhong et al., 2017). Assuming that the injected sTREM2-Fc spread throughout the entire hippocampus but did not spread much beyond this structure, they calculated that the concentration of sTREM2-Fc was about 3.3 pmol sTREM2-Fc/mg brain tissue—more than 200-fold that of endogenous sTREM2 in 5xFAD. (It should be noted that this comparison also depends upon the assumption that there was nearly complete recovery of endogenous sTREM2 from brain lysates.) Twice the amount of sTREM2-Fc was injected into the hippocampi of the 5xFAD mice in the 2019 study (Zhong et al., 2019) as was injected into the wild-type mice in the 2017 study.

When applied to hippocampal slices from 5xFAD mice, sTREM2-Fc (50 nM) rescued deficits in long-term potentiation, as did AAV-mediated over expression of sTREM2 in 5xFAD brains (Zhong et al., 2019).

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References

Research Models Citations

1. 5xFAD (C57BL6)
2. Trem2-H157Y knock-in
3. Trem2-H157Y x 5xFAD
4. TREM2-sol
5. Trem2 KO (KOMP)
6. TREM2-IPD
7. Trem2-IPDxAPP23xPS45
8. APP23
9. AAV-sTREM2 PS19
10. Tau P301S (Line PS19)

News Citations

1. Cut Loose, Soluble TREM2 Beckons Microglia to Mop Up Plaques 5 Apr 2019

Mutations Citations

1. APP K670_M671delinsNL (Swedish)
2. PSEN1 G384A

Paper Citations

1. Zhong L, Xu Y, Zhuo R, Wang T, Wang K, Huang R, Wang D, Gao Y, Zhu Y, Sheng X, Chen K, Wang N, Zhu L, Can D, Marten Y, Shinohara M, Liu CC, Du D, Sun H, Wen L, Xu H, Bu G, Chen XF. Soluble TREM2 ameliorates pathological phenotypes by modulating microglial functions in an Alzheimer's disease model. Nat Commun. 2019 Mar 25;10(1):1365. PubMed.
2. 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.
3. Wang Y, Lin Y, Wang L, Zhan H, Luo X, Zeng Y, Wu W, Zhang X, Wang F. TREM2 ameliorates neuroinflammatory response and cognitive impairment via PI3K/AKT/FoxO3a signaling pathway in Alzheimer's disease mice. Aging (Albany NY). 2020 Oct 16;12(20):20862-20879. PubMed.
4. 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.
5. 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.
6. Zhong L, Chen XF, Wang T, Wang Z, Liao C, Wang Z, Huang R, Wang D, Li X, Wu L, Jia L, Zheng H, Painter M, Atagi Y, Liu CC, Zhang YW, Fryer JD, Xu H, Bu G. Soluble TREM2 induces inflammatory responses and enhances microglial survival. J Exp Med. 2017 Mar 6;214(3):597-607. Epub 2017 Feb 16 PubMed.
7. Zhong L, Xu Y, Zhuo R, Wang T, Wang K, Huang R, Wang D, Gao Y, Zhu Y, Sheng X, Chen K, Wang N, Zhu L, Can D, Marten Y, Shinohara M, Liu CC, Du D, Sun H, Wen L, Xu H, Bu G, Chen XF. Soluble TREM2 ameliorates pathological phenotypes by modulating microglial functions in an Alzheimer's disease model. Nat Commun. 2019 Mar 25;10(1):1365. PubMed.
8. 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.

External Citations

1. The Jackson Laboratory, JAX MMRRC Stock# 034848
2. Scantox Neuro

Further Reading

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