Reactive or transgenic increase in microglial TYROBP reveals a TREM2-independent TYROBP-APOE link in wild-type and Alzheimer's-related mice

Abstract

Introduction: Microglial TYROBP (DAP12) is a network hub and driver in sporadic late-onset Alzheimer's disease (AD). TYROBP is a cytoplasmic adaptor for TREM2 and other receptors, but little is known about its roles and actions in AD. Herein, we demonstrate that endogenous Tyrobp transcription is specifically increased in recruited microglia.

Methods: Using a novel transgenic mouse overexpressing TYROBP in microglia, we observed a decrease of the amyloid burden and an increase of TAU phosphorylation stoichiometry when crossed with APP/PSEN1 or MAPT^P301S mice, respectively. Characterization of these mice revealed Tyrobp-related modulation of apolipoprotein E (Apoe) transcription. We also showed that Tyrobp and Apoe mRNAs were increased in Trem2-null microglia recruited around either amyloid beta deposits or a cortical stab injury. Conversely, microglial Apoe transcription was dramatically diminished when Tyrobp was absent.

Conclusions: Our results provide evidence that TYROBP-APOE signaling does not require TREM2 and could be an initiating step in establishment of the disease-associated microglia (DAM) phenotype.

Keywords: APP/PSEN1; Alzheimer's disease; DAM; Dap12; PS19; RNAscope; Trem2; Tyrobp; amyloid; apolipoprotein E; microglia; tauopathy.

FIGURE 1

Tyrobp mRNA is increased in recruited microglia. A, Dual RNA fluorescent in situ hybridization (RNAscope) and immunohistochemistry for Tyrobp mRNA (red) and IBA1 protein (green), respectively, in wild‐type (WT) mice (DAPI in blue). Scale bar = 50 μm. B‐C, Dual RNAin situ hybridization and immunohistochemistry for Tyrobp (red), IBA1 (green), and amyloid beta (Aβ; antibody 6E10; blue) in APP/PSEN1 (B) and 5xFAD (C) mice. Scale bar = 200 μm. D, Left panel: representative image of immunohistochemistry with antibody pT205 in the piriform cortex of MAPT^P301S (PS19) mice. Scale bar = 200 μm. Right panels: dual RNAin situ hybridization and immunohistochemistry for Tyrobp (red), IBA1 (green), and p‐TAU (antibody AT8; blue) in the piriform cortex of MAPT^P301S mice. Scale bars = 200 and 50 μm. E, Co‐immunohistochemistry for TYROBP (green) and human Aβ (antibody 6E10; red) in APP/PSEN1 mice (DAPI in blue). Scale bar = 50 μm. F, Real‐time quantitative polymerase chain reaction analyses of Tyrobp and TNFα mRNAs in WT primary microglia with and without lipopolysaccharide. Mice were either 4 (A) or 8 (B‐E) months of age and were all WT for Tyrobp. White and orange arrows indicate examples of non‐recruited and recruited microglia, respectively. Slice thickness = 10 μm

FIGURE 2

Generation of Iba1^Tyrobp mice. A, Hippocampi from 4‐month‐old Tyrobp^−/−, wild‐type (WT), and Iba1^Tyrobp mice were assayed for Tyrobp and Gfp mRNAs by real‐time quantitative polymerase chain reaction (n = 3–4 mice per group). B, Representative western blot and quantification of TYROBP and GAPDH in the cortex of the same groups used in (A) (n = 2–6 mice per group). C, Dual RNA fluorescent in situ hybridization and immunohistochemistry for Tyrobp mRNA (red) and IBA1 (green), respectively, (DAPI in blue) in Tyrobp ^−/−, WT, and Iba1^Tyrobp mice. Scale bar = 50 μm and slice thickness = 10 μm. D, Quantification of Tyrobp mRNA intensity from the experiment described in (C). n = 4, 17, and 17 slices per group (from N = 1 mouse per genotype) for Tyrobp^−/−, WT, and Iba1^Tyrobp mice, respectively. E, Volcano plot representation of the whole hippocampal DEGs in Iba1^Tyrobp versus WT mice (n = four 4‐month‐old males per genotype). Error bars represent means ± standard error of the mean. Statistical analyses were performed using a Student t‐test (A) or a one‐way analysis of variance followed by a Tukey's post hoc test (B, D), *P < .05, **P < .01, ****P < .0001. na, not applicable; ns, non‐significant

FIGURE 3

Transgene‐derived Tyrobp upregulation decreases amyloid plaque load in APP/PSEN1 mice. A, Representative images of thioflavine‐S (ThioS) staining in APP/PSEN1 and APP/PSEN1;Iba1^Tyrobp mice at 4 months of age. Scale bar = 500 μm. B, Quantification of the number of ThioS‐positive plaques per hemibrain in APP/PSEN1 and APP/PSEN1;Iba1^Tyrobp mice at 4 months of age. N = 4–5 mice per genotype and sex with three slices per animal. C, Human amyloid beta (Aβ)42 and Aβ40 concentrations measured by enzyme‐linked immunosorbent assay in the cortices of the same groups described in (B). D, Representative images of double‐label immunohistochemistry with anti‐IBA1 and anti‐6E10 antibodies in APP/PSEN1 and APP/PSEN1;Iba1^Tyrobp mice at 4 months of age. Scale bar = 100 μm. E, Quantification of the number of plaque‐associated microglia in the four groups described in (B). N = 10–24 plaques from 4–5 mice per group. F, Real‐time quantitative polymerase chain reaction analyses of microglial gene mRNAs in the hippocampi of APP/PSEN1 and APP/PSEN1;Iba1^Tyrobp mice at 4 months of age. N = 7–9 mice per group, females and males were pooled. G, Representative images of ThioS staining in male APP/PSEN1 and APP/PSEN1;Iba1^Tyrobp mice at 8 months of age. Scale bar = 500 μm. H, Quantification of the ThioS immunoreactive area in male APP/PSEN1 and APP/PSEN1;Iba1^Tyrobp mice at 8 months of age (somatomotor and visual areas of the cortex, and hippocampus were quantified). N = 3–4 mice per group. Error bars represent means ± standard error of the mean. Statistical analyses were performed using a two‐way analysis of variance followed by a Sidak post hoc test for (B and E), a Kruskal‐Wallis test for (C), a Student t‐test for (F) and a Mann‐Whitney test for (H), *P < .05, **P < .01, ***P < .001

FIGURE 4

Transgene‐induced Tyrobp upregulation increases apparent stoichiometry of TAU phosphorylation and microglial activation in 4‐month‐old MAPT^P301S mice. A, Western blot analyses of phosphorylated TAU on S202 or T205 epitopes (AT8 and pT205 antibodies) and total human TAU (HT7 antibody) in cortical homogenates from wild‐type, MAPT^P301S (PS19), and MAPT^P301S;Iba1^Tyrobp mice at 4 months‐old. n = 4–9 mice per group. B, Densitometric analyses of western blots presented in (A) standardized to GAPDH or HT7. C, Representative images of DAB‐immunohistochemistry with antibody pT205 in 4‐month‐old MAPT^P301S and MAPT^P301S;Iba1^Tyrobp mice. Scale bar = 200 μm. D, Left panel: representative images of anti‐IBA1 immunohistochemistry on the same groups described in (C). Scale bar = 200 μm. Additional representative pictures are presented in Figure S2 in supporting information. Right panel: western blot‐AT8/GAPDH quantification plotted against anti‐IBA1 immunoreactivity in the cortex. Linear regression with trend line (red line) and 95% confidence intervals (black lines) are indicated. E, Representative images of double‐label immunofluorescence with anti‐IBA1 and anti‐CD68 antibodies in the piriform cortex on the same groups described in (C). Scale bar = 500 μm. F, Real‐time quantitative polymerase chain reaction analyses of microglial gene mRNAs in the hippocampus of MAPT^P301S and MAPT^P301S;Iba1^Tyrobp mice at 4 months of age. N = 7–11 per group. Error bars represent means ± standard error of the mean. Statistical analyses were performed using a one‐way analysis of variance followed by a Tukey's post hoc test for (B) or a Student t‐test for (B) when *t is indicated and (F), *P < .05, **P < .01, ***P < .001, ****P < .0001

FIGURE 5

Increases of Tyrobp and Apoe mRNAs in microglia recruited to a site of stab injury are Trem2‐independent. A, Stab‐injured wild‐type (WT) mice were sacrificed 3 days after injury and dual RNA fluorescent in situ hybridization and immunohistochemistry for Apoe mRNA (red) and anti‐IBA1 (green), respectively, was performed. The injured ipsilateral area (red dotted line) is shown on the top row and the uninjured contralateral area is shown on the bottom row. Scale bar = 200 μm. B, Dual RNA fluorescent in situ hybridization and immunohistochemistry for Apoe (green) and GFAP (red) in non‐injured WT mice. C‐D, The same stab injury protocol was used in Tyrobp^−/− (C) and Trem2^−/− (D) mice. Anti‐IBA1 staining and DAPI staining are shown in green and blue, respectively. Top row: Trem2 mRNA (red); middle row: Tyrobp mRNA (red); bottom row: Apoe mRNA (red). Mice were 4 months of age, and slice thickness = 10 μm. The red asterisk indicates the injured side. White and orange arrows indicate examples of non‐recruited and recruited microglia, respectively

FIGURE 6

Increases in Tyrobp and Apoe mRNAs in amyloid plaque‐associated microglia are Trem2‐independent. A, Dual RNA fluorescent in situ hybridization and immunohistochemistry for Tyrobp mRNA (red), anti‐IBA1 (green), and human amyloid beta (Aβ; 6E10 antibody; blue) in TgCRND8 mice on wild‐type (WT; top row) or Trem2 ^−/− (bottom row) background. Scale bar = 200 or 50 μm. B, Dual RNA fluorescent in situ hybridization and immunohistochemistry for Apoe mRNA (red), anti‐IBA1 (green), and human amyloid (6E10 antibody; blue) in the same mice as in (A). Scale bar = 200 or 50 μm. C, Dual RNA fluorescent in situ hybridization and immunohistochemistry for Tyrobp mRNA (green), Apoe mRNA (red), and 6E10 (blue) in APP/PSEN1 mice. Scale bar = 50 μm. D, Dual RNA fluorescent in situ hybridization and immunohistochemistry for Apoe mRNA (red), anti‐IBA1 (green), and human Aβ (6E10 antibody; blue) in APP/PSEN1 mice on a WT (top row) or Tyrobp‐null (bottom row) background. Scale bar = 50 μm. Right panel: quantification of the number of plaque‐associated microglia with upregulated Apoe mRNA in the same mice as in (D). N = 2–3 mice per group (A‐D)

FIGURE 7

Proposed ligand‐induced Tyrobp signaling in recruited microglia. Left panel, in response to penetrating stab injury or accumulation of amyloid beta (Aβ) deposits or misfolded TAU, Tyrobp transcription is upregulated in microglia, thereby marking these cells as “recruited microglia.” Right panel, we observed that both microglial recruitment and Tyrobp upregulation occur in the absence of TREM2, indicating the existence of “sensing” receptors. Multiple alternative signaling pathways can be considered: Ligand signaling is initiated by APOE, Aβ, debris, or other ligands at sensing receptors and leads to phosphorylation of the tyrosine residues in the cytoplasmic ITAM of TYROBP by SRC kinases and the recruitment of SYK. In turn, SYK signaling leads to upregulated transcription of Tyrobp and Apoe. This series of events forms the basis for the phenotypic switch from homeostatic microglia to DAM. In mice lacking TREM2, microglial recruitment is retained, and transcription of both Tyrobp and Apoe is induced. Because these are constitutive TREM2 knockout mice, we are unable to exclude the possibility that some unknown sensor developed as compensation for the absence of TREM2. Another possibility is the existence of unidentified sensing receptor(s) that can upregulate TYROBP and APOE through a mechanism that does not require formation of complexes with TYROBP itself. APOE, apolipoprotein E; DAM, disease‐associated microglia; ITAM, immunoreceptor tyrosine‐based activation motif; SYK, spleen tyrosine kinase; TREM2, triggering receptor expressed on myeloid cells‐2; TYROBP, tyrosine kinase binding protein