Deletion of Abi3/Gngt2 influences age-progressive amyloid β and tau pathologies in distinctive ways

ABI3 is expressed in microglia and neurons in mice and humans

Recent reports have confirmed ABI3 as an AD risk gene [6,7,8]. ABI3 has been reported to be a microglia-specific gene using single-cell RNAseq [12, 20]. Bulk RNAseq data reveals increased levels of ABI3 RNA relative to control cohorts in both the temporal cortex and cerebellum of human patients and in mouse models of amyloid and tau pathologies [6]. In AD brains, ABI3 expression is significantly upregulated relative to controls (p = 4.47E-03) [6]. We further confirmed this in aging cohorts of APP TgCRND8 (TG) mice relative to nontransgenic (NonTG) age-matched mice (Fig. 1a; Additional File 2: Table S2). It is notable that increasing plaque burden, and not necessarily age, seems to be associated with increased Abi3 expression (Fig. 1a). In MAPT transgenic rTg4510 mice, the RNA levels of Abi3 increased in 4.5-month-old tau expressing mice but reduced to levels comparable to nonTG littermates at 6 months (Fig. 1b; Additional File 3: Table S3).

Fig. 1figure 1

ABI3 RNA is expressed in microglia and neurons in mice and humans. a, b Abi3 RNA levels (FKPM) plotted across different ages in APP CRND8 mice (a) and MAPT rTg4510 mice (b).

Source data obtained from Mayo RNAseq study (AD Knowledge Portal: https://adknowledgeportal.org). n = 9–14 (a) and n = 6 per genotype/age. b. One-way ANOVA, ****p < 0.0001; **p < 0.01; *p < 0.05. cf In situ hybridization was done to detect ABI3 (Fast Red; red color) RNA on human and mouse paraffin-embedded brain sections immunostained with anti Iba-1 antibody (brown color). Arrowheads indicate Iba-1 (microglia) associated in situ signal and arrows indicate in situ signal in non-Iba-1 cells. n = 3 (human AD cases, 6-month-old TG-Abi3-Gngt2−/− mice and 6-month-old TG-Abi3-Gngt2+/+ mice) and n = 1 (18-month-old TgCRND8 mice). Representative of two independent experimental replicates. Two separate images are shown from each cohort, indicated as c1-c2, d1-d2, e1-e2, f1-f2. Additional representative images are available in Additional File 4 Fig. S1. NonTG, nontransgenic; TG, transgenic

To provide cellular localization information complementary to this quantitative bulk RNAseq data, we used RNAscope to localize Abi3 expression in mouse and human tissues (Fig. 1c-f, Additional File 4: Fig. S1). In AD patients, we observed Abi3 transcript in Iba1-immunopositive microglia as well as in Iba-1 nonreactive cells in the grey matter, which are presumed to be neurons based on size and location (Fig. 1c1, c2; Additional File 4: Fig. S1a-f). Specificity for the RNA in situ hybridization signal was confirmed by using brain tissue from APP mice completely lacking the Abi3 locus (Fig. 1d1, d2; Additional File 4: Fig. S1g-l). In 6-month-old and 18-month-old APP TgCRND8 mice, we observed Abi3 + microglia in the cortex and in the white matter tracts (Fig. 1e1, f1, f2; Additional File 4: Fig. S1m-x). Similar to human cases, we noted Abi3 + cells in the neuronal layers in the cortex and hippocampus in these mice (Fig. 1e2, f2). Overall, RNAscope confirmed the presence of Abi3 RNA in both microglial and non-microglial cells in AD and an APP mouse model.

ABI3 and GNGT2 genes are co-regulated in APP mouse models and in AD

To elucidate how Abi3-specific immune signaling contributes to the neurodegenerative cascade in AD, we obtained the Abi3−/− mice from Jax Labs (Stock #028180). While using bulk RNAseq to characterize these mice, we serendipitously discovered that the levels of another gene, Gngt2, were dramatically reduced to the same extent as Abi3 (Additional File 5: Fig. S2a). We surveyed the gene maps and found that mouse Abi3 locus overlaps with two other genes on chromosome 11—microglia-specific G protein gamma transducing activity polypeptide 2 (Gngt2) and Phospho-1 (Additional File 5: Fig. S2b). We also confirmed that in humans, the arrangement of the ABI3, GNGT2 and PHOSPHO-1 genes is conserved, albeit being on chromosome 17. Using data from UCSC genome browser, we discovered that the Velocigene targeted deletion in the Abi3 locus knocked out both Abi3 and Gngt2 (Additional File 5: Fig. S2b). In subsequent sections, we will refer to these mice as Abi3-Gngt2−/−. Notably, we did not find any changes in Phospho-1 transcript levels in these mice.

Given that Abi3 and Gngt2 genes are overlapping, we hypothesized that their expression could be correlated. Using RNAseq data from the AMP-AD consortium, we investigated the concordance between the expression patterns of Abi3 and Gngt2. We found that ABI3 and GNGT2 genes were co-regulated in three distinct AD patient cohorts: temporal cortex samples of Mayo Clinic AD cohort (Mayo TCX: ρ = 0.644, p = 2.2e − 16) (Fig. 2a), cerebellar samples of Mayo Clinic AD cohort (Mayo CER: ρ = 0.556, p = 2.2e − 16) (Fig. 2b) and prefrontal cortex samples of Religious Orders Study and Rush Memory and Aging Project AD cohort (ROSMAP; ρ = 0.328, p = 2.2e − 16) (Fig. 2c). We also confirmed that Abi3 and Gngt2 genes are co-regulated in the APP transgenic TgCRND8 mice (Fig. 2d, ρ = 0.625, p = 7.31 − 06) and in MAPT transgenic rTg4510 mice (Fig. 2e, ρ = 0.554, p = 0.018). The nonTG littermates of these mice showed no correlation between Abi3 and Gngt2 expression. In addition, we investigated 96 additional mouse transcriptomic datasets [33, 34] and in 26 of these cohorts, we found that Abi3 and Gngt2 were both differentially regulated. Among these 26 cohorts, Abi3 and Gngt2 expression changes were concordant in 24 studies (Additional File 6: Table S4), showing that these genes are consistently co-regulated across different mouse models and experimental cohorts. Collectively, these analyses show that expression of ABI3 and GNGT2 genes are in a tight co-expression network in AD and AD mouse models.

Fig. 2figure 2

ABI3 and GNGT2 genes are in a co-regulatory expression network. ac Graphs showing co-regulation of ABI3 and GNGT2 RNA from AD patients. Data from Mayo AD cohorts (temporal cortex, TCX and cerebellum, CER) and ROSMAP AD cohorts obtained from AD Knowledge Portal (https://adknowledgeportal.org). d, e Graphs showing co-regulation of Abi3 and Gngt2 RNA from APP TgCRND8 and MAPT rTg4510 mice obtained from AD Knowledge Portal (https://adknowledgeportal.org). f Table depicting the correlation coefficient (ρ) and p-values adjusted for multiple testing for cohorts depicted in ae. Tg, transgenic for APP (d) or MAPT/tTA (e); nonTg, nontransgenic background strain matched mice (d, e). Each datapoint indicates individual sample (ae). x and y axes denote FPKM values of ABI3 and GNGT2 respectively

Loss of Abi3-Gngt2 induces reactive gliosis and a glial gene signature typically associated with AD

We evaluated baseline gliosis in the parental Abi3-Gngt2−/− line. Using Iba-1 immunohistochemistry, we found that at 3 months of age, the heterozygous Abi3-Gngt2+/− mice showed reduced hippocampal microgliosis compared to wild type Abi3-Gngt2+/+ mice (p < 0.01 in hippocampus) and Abi3-Gngt2−/− mice (ns association) (Fig. 3a–c). At 6 months of age, we noticed an interesting gene-dosage-dependent dichotomy in microgliosis in the Abi3-Gngt2 lines. The heterozygous Abi3-Gngt2+/− mice showed higher Iba-1 reactivity relative to WT Abi3-Gngt2+/+ mice (p < 0.05 in cortex) and Abi3-Gngt2−/− mice (p < 0.01 in cortex and p < 0.05 in hippocampus) (Fig. 3d–f). There were no significant differences in microgliosis between the WT Abi3-Gngt2+/+ and Abi3-Gngt2−/− mice at this age (Fig. 3d–f).

Fig. 3figure 3

Immune activation in Abi3-Gngt2−/− mice. Representative images of Iba-1 reactive microglia (af) and GFAP-reactive astrocyte (gl) in 3-month-old or 6-month-old mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 genes. Quantitation of the Iba-1 or GFAP staining from cortex or hippocampus is provided in corresponding panels on the right side. N = 6 mice (af), 7 mice (gl). Scale bar, 50 µm. Clear symbols denote female mice and filled symbols denote male mice. Data represents mean ± sem. One-way ANOVA; ***p < 0.001; **p < 0.01; *p < 0.05. KO, knockout; Het, heterozygous; WT, wild type

At 3 months of age, GFAP-reactive gliosis levels in Abi3-Gngt2−/− mice showed increased trend relative to WT Abi3-Gngt2+/+ and heterozygous Abi3-Gngt2+/− mice (p < 0.05 in cortex) (Fig. 3g–i). At 6 months of age, the astrocyte burden continued to remain elevated in the Abi3-Gngt2−/− mice relative to WT Abi3-Gngt2+/+mice (p < 0.05 in cortex and hippocampus) and heterozygous Abi3-Gngt2+/− mice (p < 0.05 in hippocampus) (Fig. 3j–l). This shows that complete loss of function of the Abi3 locus results in early astrocytosis that progresses as the mice age.

We performed bulk RNAseq from the forebrains of 3-month-old Abi3-Gngt2 mice (Fig. 4; Additional File 7: Fig. S3). Relative to WT mice, the upregulated RNAs in the Abi3-Gngt2−/− mice were predominantly microglia-specific, such as C-type lectin domain family 7 member A (Clec7a/Dectin 1), Macrophage Expressed 1 (Mpeg1), Natural resistance-associated macrophage protein 1 (Slc11a1/Nramp1), Lymphocyte Antigen 86 (Ly86) and Olfactomedin-like 3 (Olfm13) (Fig. 4a,b). Downregulated RNAs included Abi3, Gngt2, G Protein-Coupled Receptor 179 (Gpr179), and TNF Receptor Superfamily Member 1B (Tnfrsf1b) (Fig. 4a,b). Analysis of the heterozygous Abi3-Gngt2+/− mice relative to WT Abi3-Gngt2+/+ mice revealed only one significantly altered (downregulated) gene—Protocadherin Gamma Subfamily A 5 (Pcdha5)—that is involved in establishing and maintaining intercellular connections in the brain (Additional File 7: Fig. S3a). Pathway analysis of the differentially expressed gene sets in Abi3-Gngt2−/− mice relative to WT mice revealed the involvement of immune pathways, such as granulocyte (GO:0071621) and leukocyte chemotaxis (GO:0030595), proliferation of mononuclear leukocytes (GO:0032943), and leukocyte-mediated immunity (GO:0002443) (Fig. 4c). The cell types most affected in Abi3-Gngt2−/− mice were microglia which showed increase in these mice relative to WT and heterozygous Abi3-Gngt2+/− mice (p < 0.05) (Fig. 4d). There was a specific reduction in neuronal gene counts in Abi3-Gngt2−/− mice (p < 0.01 relative to WT mice) and in heterozygous mice (p < 0.001 relative to both WT and Abi3-Gngt2−/− mice) (Fig. 4d). No significant changes in astrocyte or oligodendrocyte-specific gene counts were seen among the three groups (Fig. 4d). Surprisingly, the Abi3-Gngt2−/− mice showed induction of the amyloid/AD-associated PIG network, even in the absence of Aβ (p < 0.01 relative to both WT and heterozygous mice) (Fig. 4e). We also observed suggestive upregulated trends in the DAM, MGnD, ARM, and A1 co-expression networks in the Abi3-Gngt2−/− mice (Fig. 4e). Weighted gene co-expression network analysis (WGCNA) identified several gene modules correlating the Abi3-Gngt2 genotype with the gliosis phenotype (Fig. 4f). The hub genes of selected modules that specifically correlated with the Abi3-Gngt2−/− genotype and gliosis include Unc93b1 (antiquewhite2) and immunoglobulin kappa variable 10–96 (coral2) (Additional File 7: Fig. S3b-d). The antiquewhite2 module is especially relevant to AD pathophysiology as the module members, Ctss, Siglech, Csf3r, Ly86, and C1qc, have been reported in both mouse models and humans (Additional File 7: Fig. S3b) [35]. These genes are also reported to be associated with pathologic signatures in AD, most notably DAM and MGnD [24, 25] and PIG [21] (Additional File 7: Fig. S3e). It was also highly associated with several immune and autoimmune conditions such as Staphylococcus infection and primary immunodeficiency as well as neurodegenerative diseases such as prion disease (Additional File 7: Fig. S3f). Overall, RNAseq data shows an inflammatory gliosis profile corresponding to AD-typical gene expression patterns in Abi3-Gngt2−/− mice, even in the absence of Aβ.

Fig. 4figure 4

Unbiased transcriptomic analysis of Abi3-Gngt2−/− mice reveal upregulation of immune pathways and disease-related gene expression patterns. ac Volcano plot (a), list of top 5 upregulated and top 5 downregulated genes (based on fold change; orange, upregulated genes, blue, downregulated genes) (b) and GO pathways based on enriched genes (c) in 3-month-old Abi3-Gngt2−/− mice relative to Abi3-Gngt2+/+ mice. Orange dots, upregulated genes; blue dots, downregulated genes (a). FC, fold change; DEG, differentially expressed genes; padj, p-values adjusted for multiple comparison. d Cell type population analyses indicating changes in microglia, astrocytes, neurons, and oligodendrocyte populations in 3-month-old mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 locus. One-way ANOVA, ***p < 0.001, **p < 0.01, *p < 0.05. e Gene expression signatures for microglia or astrocyte functional subtypes in 3-month-old mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 locus. These cell-type-specific signatures were identified in previous studies [21,22,23,24,25,26,27,28]. One-way ANOVA, **p < 0.01. f WGCNA gene co-expression modules correlating with specific experimental traits (Iba-1 burden, GFAP burden, and Abi3-Gngt2−/− genotype) shown. Correlation of modules to different experimental traits is colored in a heatmap (red, positive correlation; blue, negative correlation). Modules with p-values < 0.05 and correlation value < -0.5 or > 0.5 are indicated in colored tiles (see scale on right). Cell-type-specific gene lists were used to identify genes with significant overlap (odds ratio, see scale on right) within the modules. The heatmap is colored by the value of the odds ratio; higher the odds ratio of association, warmer the color. Grey squares indicate non-significant (p > 0.05, odds ratio < 2) overlaps in the gene lists. All p-values adjusted for multiple comparisons (padj). n = 4 mice (2 males, 2 females) per Abi3-Gngt2 genotype except 1 outlier removed in d, e

Reduction in Aβ levels in APP mice lacking Abi3-Gngt2

We then examined how complete insufficiency or haploinsufficiency of the Abi3 locus alters Aβ plaque pathology in APP transgenic CRND8 mice. Transgenic APP mice (referred as “TG”) that are wild type (+ / +), heterozygous (+ /−), or knocked out (−/−) for Abi3-Gngt2 genes were aged to 3 and 6 months and the Aβ levels assessed by immunohistochemistry and biochemical analysis (Fig. 5, Additional File 8: Fig. S4). There was no change in APP expression levels in these bigenic TG-Abi3-Gngt2 colonies (Additional File 8: Fig. S4a-b). At 3 months, both heterozygous Abi3-Gngt2+/− (p < 0.05) and Abi3-Gngt2−/− (p < 0.01) mice showed reduced Aβ plaques relative to APP transgenic mice wild type for Abi3-Gngt2 (Fig. 5a,b). Concurrently, there was a non-significant reduction in the number of Thioflavin S cored plaques in the TG-Abi3-Gngt2−/− mice (Fig. 5c,d). Biochemical analysis showed significant reduction of FA associated insoluble Aβ42 and Aβ40 levels in both TG-Abi3-Gngt2+/− (Aβ42: p < 0.01; Aβ40: p < 0.05) and TG-Abi3-Gngt2−/− (Aβ42: p < 0.001; Aβ40: p < 0.05) mice relative to TG-Abi3-Gngt2+/+ mice (Fig. 5e,f). In the SDS detergent-soluble fraction, both Aβ42 and Aβ40 values were reduced in TG-Abi3-Gngt2−/− mice (p < 0.05) while only Aβ42 was significantly reduced in TG-Abi3-Gngt2+/− mice (p < 0.05) relative to TG-Abi3-Gngt2+/+ mice (Fig. 5g,h). We did not observe major changes in ubiquitin labeling of the Aβ plaques across all the Abi3 genotypes (Additional File 8: Fig. S4c).

Fig. 5figure 5

Loss of Abi3-Gngt2 expression ameliorates Aβ in a gene-dosage manner. a, b Representative immunohistochemical images of total Aβ plaque burden and quantification in 3-month-old APP TG mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 locus. c, d Representative images of thioflavin S-stained cored Aβ plaques and quantitation in 3-month-old APP TG mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 locus. eh Biochemical levels of formic acid (FA) solubilized and detergent (SDS) soluble Aβ42 and Aβ40 in 3-month-old APP TG mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 locus. i, j Representative immunohistochemical images of total Aβ plaque burden and quantification in 6-month-old APP transgenic mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 locus. k, l Representative images of thioflavin S-stained cored plaques and quantitation in 6-month-old APP TG mice with WT (+ / +), heterozygous (+ /−), or KO (−/−) of Abi3-Gngt2 locus. mp Biochemical levels of FA solubilized and SDS soluble Aβ42 and Aβ40 in 6-month-old APP TG mice with WT (+ / +), heterozygous (+ /−), or KO (−/.−) of Abi3-Gngt2 locus. N = 6 mice (ah), 7 mice (i, j, m-p), 6 mice (k, l). Scale bar, 50 µm (a, i); 100 µm (ck). Clear symbols denote female mice and filled symbols denote male mice (except panel d, k). n = 3 females, 3 males (3 months) and n = 3 females, 4 males (6 months). Data represents mean ± sem. One-way ANOVA; ***p < 0.001; **p < 0.01; *p < 0.05

At 6 months of age, complete deletion of Abi3-Gngt2 resulted in reduced total Aβ plaque burden (Fig. 5i,j, p < 0.05) as well as number of cored plaques (Fig. 5k,l, p < 0.01) in TG-Abi3-Gngt2−/− mice relative to TG-Abi3-Gngt2+/+ mice. Notably, though the immunohistochemical plaque burden was similar between TG-Abi3-Gngt2+/− and TG-Abi3-Gngt2+/+ mice (Fig. 5i,j), the former group showed lower thioflavin S (ThioS) plaque number compared to the latter group (Fig. 5k,l, p < 0.05). The FA and SDS level of Aβ was mostly equivalent among all the groups except for reduction in SDS-associated Aβ40 in TG-Abi3-Gngt2−/− mice relative to TG-Abi3-Gngt2+/+ mice (Fig. 5m–p, p < 0.05). The patterns of ubiquitin staining around plaques appeared unchanged across the Abi3 genotypes (Additional File 8: Fig. S4d).

We generated an additional cohort of 9-month-old TG-Abi3-Gngt2+/− mice for neuropathological comparisons with age-matched TG-Abi3-Gngt2+/+ mice. We found no changes in Aβ burden, astrocytosis (GFAP), microgliosis (Iba-1 and cd11b), or ubiquitin staining patterns between these two cohorts of mice at this age (Additional File 8: Fig. S4e-m).

Alterations in gliosis, especially astrocyte dysfunction, impacts synaptic health [36]. To survey how amyloid plaques and gliosis in this model affect neuronal health, we evaluate several pre- and post-synaptic proteins at 3 months of age in the TG mice. Consistent with the reduction in Aβ levels, we saw improved synaptic function as exemplified by increased synaptophysin level in both TG-Abi3-Gngt2+/− mice (p < 0.05) and TG-Abi3-Gngt2−/− mice (P < 0.01) (Additional File 9: Fig. S5a-b). We observed an insignificant trend in increased PSD95 levels in TG-Abi3-Gngt2−/− mice while levels of synaptogyrin3 and spinophilin were unaltered in the three genotypes rested (Additional File 9: Fig. S5c-h). There was a reduction in vGlut1 (p < 0.01 in TG-Abi3-Gngt2+/−; p < 0.05 in TG-Abi3-Gngt2−/−), GluR1 (p < 0.05 in TG-Abi3-Gngt2+/−), and GluR2 levels (trend in TG-Abi3-Gngt2−/−) compared to TG-Abi3-Gngt2+/+ mice, signifying dysfunctional glutamatergic signaling (Additional File 9: Fig. S5i-n).

Gene dose-dependent regulation of inflammatory profile in TG-Abi3-Gngt2 -/- mice

Aβ levels are generally well-correlated with immune activation indicated by microgliosis and astrocytosis [3]. Thus, we predicted lower burden of microglia (Iba-1 immunoreactivity) and astrocytes (GFAP immunoreactivity) in TG-Abi3-Gngt2−/− mice because these mice showed robust Aβ reduction at 3 months and 6 months of age. Surprisingly, we found that at both ages, the TG-Abi3-Gngt2−/− mice showed similar levels of Iba-1 reactive microgliosis compared to TG-Abi3-Gngt2+/+ mice (3 months: Fig. 6a–c; 6 months: Fig. 6d–f). The heterozygous TG-Abi3-Gngt2+/− mice showed decreased cortical microglia at 3 months compared to TG-Abi3-Gngt2−/− mice (Fig. 6a–c; p < 0.05), but this normalized to equivalent levels by 6 months of age (Fig. 6d–f). The patterns of astrocyte burden reflected a differential scenario across the three Abi3 genotypes. At 3 months of age, the TG-Abi3-Gngt2+/− mice had significantly lower astrocytosis compared to both TG-Abi3-Gngt2+/+ (p < 0.01 in cortex; p < 0.001 in hippocampus) and TG-Abi3-Gngt2−/− mice (p < 0.001 in cortex; p < 0.01 in hippocampus) (Fig. 6g–i). At 6 months of age, these differences normalized in the cortex but hippocampal GFAP burden in TG-Abi3-Gngt2+/− mice was higher than both TG-Abi3-Gngt2+/+ or TG-Abi3-Gngt2−/− mice (Fig. 6j–l). There was no significant difference in astrocytosis burden between the TG-Abi3-Gngt2+/+ or TG-Abi3-Gngt2−/− mice in 3-month or 6-month-old cohorts (Fig. 6g–i, j–l), indicating that reduction of Aβ did not ameliorate the existing astrocytic phenotype inherent in the Abi3-Gngt2 line. Overall, the immunohistochemistry data suggests a biphasic age-dependent response of the astrocytes and microglia in APP mice in relation to Abi3-Gngt2 gene dosage.

Fig. 6figure 6

Abi3-Gngt2 regulates gliosis in APP mice. Representative images of Iba-1 reactive microglia (af) and GFAP-reactive astrocyte (g-l) in 3-month-old or 6-month-old APP transgenic mice with WT (+ / +), heterozygous (+ /−), or KO (−/.−) of Abi3-Gngt2 locus. Quantitation of the Iba-1 or GFAP staining from cortex or hippocampus is provided in corresponding panels on the right side. Scale bar, 50 µm. Clear symbols denote female mice and filled symbols denote male mice. n = 3 females, 3 males (3 months) and n = 3 females, 4 males (6 months). Data represents mean ± sem. One-way ANOVA; ***p < 0.001; **p < 0.01; *p < 0.05

We performed bulk RNAseq from forebrains of 3-month-old TG-Abi3-Gngt2 mice (Fig. 7, Additional File 10: Fig. S6). Relative to the TG-Abi3-Gngt2+/+ mice, the TG-Abi3-Gngt2−/− mice showed lower Abi3 and Gngt2 as expected (Fig. 7a,b). Other genes that were downregulated in the TG-Abi3-Gngt2−/− mice are Adgrf3 (Adhesion G Protein-Coupled Receptor F3), S100a8, and S100a9 (S100 Calcium-Binding Protein members A8 and A9). Among the genes that were upregulated in these mice were host defense proteins such as BPI Fold Containing Family B Member 4 (Bpifb4), Cxcr4, and Dermokine (Dmkn), as well as Aklr1c13 (Aldo–keto reductase family 1 member C13) and Fndc9 (Fibronectin Type III Domain Containing 9) (Fig. 7a,b). Relative to TG-Abi3-Gngt2+/+ mice, the molecular pathways represented by gene expression changes in TG-Abi3-Gngt2−/− mice include syncytium formation, cellular fusion, calcium mediated signaling, and extracellular matrix organization (Fig. 7c), recapitulating expected functional properties of the ABI family members [37]. Additional pathways that were enriched were gliogenesis (GO:0014015) and response to LPS (GO:0034189), consistent with altered glial homeostasis. We did not observe any significant gene expression changes in TG-Abi3-Gngt2+/− mice relative to TG-Abi3-Gngt2+/+mice. In the TG-Abi3-Gngt2−/− mice, most of the gene expression changes were indicative of increased microglial (p < 0.05 relative to TG-Abi3-Gngt2+/−) and astrocytic involvement (p < 0.01 relative to TG-Abi3-Gngt2+/−; p < 0.05 relative to TG-Abi3-Gngt2+/+), with no changes observed in neuronal and oligodendrocyte-specific gene expression (Fig. 7d). Surprisingly, we found that in spite of reduced Aβ plaques, TG-Abi3-Gngt2−/− mice showed elevated gene signatures typically identified in AD tissues or preclinical models of AD. These mice showed upregulated PIG (p < 0.05 relative to TG-Abi3-Gngt2+/−; p < 0.05 relative to TG-Abi3-Gngt2+/+) [21], DAM (p < 0.05 relative to TG-Abi3-Gngt2+/−) [24], MGnD (p < 0.05 relative to TG-Abi3-Gngt2+/−) [25], and A1 astrocyte (p < 0.05 relative to TG-Abi3-Gngt2+/+) [38] gene profile signatures (Fig. 7e). We did not detect any selective induction of either the ARM [26] or A2 astrocyte [38] phenotypes in the three TG-Abi3-Gngt2 genotypes (Fig. 7e). Notably, most of these modules (PIG, DAM, and MGnD) are driven by Apoe, Tyrobp, and Trem2 while the ARM signature is driven by specialized microglial subgroups overexpressing MHC II genes. WGCNA identified several gene co-expression modules that allowed us to correlate neuropathological traits to cell types (Fig. 7f), KEGG pathways (Additional File 10: Fig. S6a), module hub genes (Additional File 10: Fig. S6b-e), and glial signatures correlating with the modules (Additional File 10: Fig. S6f). Modules that were positively correlated with the Abi3-Gngt2 genotype but were negatively correlated with Aβ biochemical levels or Aβ plaque burden included honeydew1 and plum3. These gene modules were primarily driven by microglia- and astrocyte-specific genes, respectively. We identified hub genes that regulate these different gene network modules (Additional File 10: Fig. S6b-e). Notably, Chil1/CHI3L1/YKL-40 (Chitinase-like 1) that is the top hub gene in honeydew1 is related to inflammation and AD pathophysiology [39]. The top hub gene in the plum3 module—Interferon Regulatory Factor 8 (Irf8)—corresponds to interferon signaling that has recently been identified to be upregulated in microglia from human AD [40]. This module is especially enriched for immune function, with the cd37 hub gene related to a Tyrobp-regulated microglial module in AD [41] and Lat2 identified as a core transcriptional signature of AD microglia [

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