The Alzheimer's disease risk factor INPP5D restricts neuroprotective microglial responses in amyloid beta-mediated pathology

Abstract

Introduction: Mutations in INPP5D, which encodes for the SH2-domain-containing inositol phosphatase SHIP-1, have recently been linked to an increased risk of developing late-onset Alzheimer's disease. While INPP5D expression is almost exclusively restricted to microglia in the brain, little is known regarding how SHIP-1 affects neurobiology or neurodegenerative disease pathogenesis.

Methods: We generated and investigated 5xFAD Inpp5d^fl/fl Cx3cr1^Ert2Cre mice to ascertain the function of microglial SHIP-1 signaling in response to amyloid beta (Aβ)-mediated pathology.

Results: SHIP-1 deletion in microglia led to substantially enhanced recruitment of microglia to Aβ plaques, altered microglial gene expression, and marked improvements in neuronal health. Further, SHIP-1 loss enhanced microglial plaque containment and Aβ engulfment when compared to microglia from Cre-negative 5xFAD Inpp5d^fl/fl littermate controls.

Discussion: These results define SHIP-1 as a pivotal regulator of microglial responses during Aβ-driven neurological disease and suggest that targeting SHIP-1 may offer a promising strategy to treat Alzheimer's disease.

Highlights: Inpp5d deficiency in microglia increases plaque-associated microglia numbers. Loss of Inpp5d induces activation and phagocytosis transcriptional pathways. Plaque encapsulation and engulfment by microglia are enhanced with Inpp5d deletion. Genetic ablation of Inpp5d protects against plaque-induced neuronal dystrophy.

Keywords: Alzheimer's disease; INPP5D; SHIP-1; amyloid beta; amyloidosis; disease-associated microglia; microglia; neurodegenerative disease; neuroimmunology.

Figure 1.. SHIP-1-deficiency leads to increased mobilization of microglia to Aβ plaques in 5xFAD mice.

5xFAD Inpp5d^fl/flCx3cr1^ERT2Cre (5xFAD Inpp5d^ΔMG mice) and Cre-negative 5xFAD Inpp5d^fl/fl littermate controls (5xFAD mice) received tamoxifen food for 2 weeks beginning at 3 weeks of age and then were returned to regular food for the remainder of the experiment. Mice were harvested at 5 months of age to evaluate microgliosis, microglial association with Aβ plaques, and Aβ plaque burden. (A) Representative immunofluorescence staining of IBA1 (green) and Aβ plaques (ThioS, magenta) in the cortex; zoomed in view of a single plaque (inset 1 and 3) and zoomed in view of a single z-plane from a single plaque (inset 2 and 4) showing the number of IBA1+ microglia interacting with the plaque. (B) Enumeration of the number of IBA1+ cells per field of view (FOV). (C) Quantification of total IBA1 staining volume per FOV. (D) Quantification of microglia size by total IBA1 staining volume per IBA1+ microglia. (E) Quantification of the average number of IBA1+ microglia per ThioS+ Aβ plaque. (F) Quantification of the number of IBA1+ microglia within a 15 μm and 30 μm radius of ThioS+ Aβ plaques. (G) Representative immunofluorescence staining of IBA1 (green), Ki67 (blue), and Aβ plaques (ThioS, magenta) in the cortex. Arrows denote Ki67+ IBA1+ cells. (H) Enumeration of the number of Ki67+ IBA1+ cells per FOV. (I) Representative immunofluorescence staining of IBA1 (green), CLEC7A (grey), and Aβ plaques (ThioS, magenta) in the cortex. (J) Quantification of the percentage of IBA1+ CLEC7A+ staining volume per ThioS+ Aβ plaque. Percent IBA1+ CLEC7A+ staining volume was calculated by dividing the total IBA1+ CLEC7A+ staining volume by the total IBA1 staining volume. (K) Representative immunofluorescence staining of Aβ (anti-Aβ D54D2, red) and DAPI (blue) performed on sagittal brain sections. (L) Quantification of the percent area covered by Aβ across whole sagittal brain sections. For (B-E), (H), and (J), each point represents an individual mouse averaged from 6 images across 3 matching brain sections per mouse. For (F), each point represents an individual mouse with an average of 50 plaques from 3 matching brain sections per mouse. For (L), each point represents an individual mouse averaged from 3 matching brain sections per mouse. Statistical significance between experimental groups was calculated by an unpaired Student’s t-test. Error bars represent mean ± SEM.

Figure 2.. snRNAseq differentiates major brain cell types and reveals enhanced microgliosis following SHIP-1 deletion in 5xFAD mice.

snRNAseq of cortices from 5xFAD Inpp5d^ΔMG mice and 5xFAD littermate controls harvested at 5 months of age. (A) UMAP rendering of 20,818 nuclei from 5xFAD Inpp5d^ΔMG and 5xFAD samples showing 17 distinct clusters numbered 0–16. The cell type identity was determined by the expression of cell type-specific markers. (B) Gene expression heatmap depicting signature genes used to identify each cluster in (A). (C) Donut chart depicting the frequency of nuclei recovered per cluster across both genotypes. (D) Bar chart showing the relative nuclei distribution across all clusters in each genotype. (E) Relative frequency of nuclei across all clusters normalized to the number of 5xFAD nuclei per cluster (dashed horizontal line). Cluster 5 (microglia) was highly increased in 5xFAD Inpp5d^ΔMG mice.

Figure 3.. SHIP-1 deletion in 5xFAD mice induces a unique transcriptional shift in microglia.

Microglia cluster analysis following snRNAseq of cortices from 5xFAD Inpp5d^ΔMG mice and 5xFAD littermate controls harvested at 5 months of age. (A) UMAP rendering of 20,818 nuclei highlighting the microglia cluster (cyan) from Figure 2A expressing microglia signature genes, including Cx3cr1, P2ry12, Hexb, Tmem119, Csf1r, C1qa, Lrmda, and Dock8. (B) Volcano plot depicting DEGs (log₂(fold-change) >0.25 or <−0.25 and Bonferroni adjusted P value <.00001) identified between 5xFAD Inpp5d^ΔMG and 5xFAD microglia. (C) Pathway analysis of DEGs (log₂(fold-change) >0 and Bonferroni adjusted P value <.05) that were upregulated in 5xFAD Inpp5d^ΔMG microglia when compared to 5xFAD microglia. (D) Pathway analysis of DEGs (log₂(fold-change) <0 and Bonferroni adjusted P value <.05) that were downregulated in 5xFAD Inpp5d^ΔMG mice when compared to 5xFAD microglia. (E) UMAP rendering of 1,573 microglia nuclei (from Cluster 5) following re-integration and re-clustering samples showing 4 distinct subclusters numbered 0–3. (F) UMAP rendering of microglia nuclei in (E) split by genotype (5xFAD Inpp5d^ΔMG: 1,264 nuclei, 5xFAD: 309 nuclei). Subcluster 0 shows an enrichment in 5xFAD Inpp5d^ΔMG microglia. (G) Bar chart showing the raw frequency of nuclei recovered across all subclusters in each genotype. Subcluster 0 is increased in 5xFAD Inpp5d^ΔMG microglia. (H) DotPlot colored to show the relative average expression of homeostatic and disease-associated microglia genes. The size of each dot corresponds to the percent of nuclei in each subcluster expressing the gene. (I) Pathway analyses of downregulated DEGs (log₂(fold-change) <0 and Bonferroni adjusted P value <.05) enriched in microglia Subcluster 0 versus all other subclusters. For (C), (D), and (I), P values are shown and an asterisk denotes global significance (adjusted P value <.05).

Figure 4.. Loss of SHIP-1 in microglia increases CD68 expression and engulfment of Aβ in 5xFAD mice.

5xFAD Inpp5d^ΔMG mice and 5xFAD littermate controls were harvested at 5 months of age to evaluate IBA1+ microglial CD68 expression and engulfment of Aβ. (A) Representative immunofluorescence staining and Imaris three-dimensional rendering of IBA1 (green), Aβ plaques (ThioS, magenta), and CD68 (blue) in the cortex. The right panel displays the amount of Aβ that localizes with CD68 (orange) in three-dimensional space. (B) Quantification of the total CD68 staining volume per IBA1+ microglia. (C) Quantification of the total CD68 staining volume per ThioS+ Aβ plaque. (D) Quantification of the percentage of Aβ plaque volume that is engulfed within CD68 staining. Each point represents an individual mouse with an average of 50 plaques from 3 matching brain sections per mouse. Statistical significance between experimental groups was calculated by an unpaired Student’s t-test. Error bars represent mean ± SEM.

Figure 5.. Deletion of SHIP-1 enhances microglial encapsulation of Aβ plaques and protects against neuronal dystrophy.

5xFAD Inpp5d^ΔMG and 5xFAD mice were harvested at 5 months of age to evaluate IBA1+ microglia containment and encapsulation of Aβ plaques and neuronal health. (A) Representative immunofluorescence staining and Imaris three-dimensional rendering of IBA1 (green) and ThioS (magenta) in the cortex. The right panel depicts the amount of Aβ that is completely encapsulated by IBA1+ microglia (orange) in three-dimensional space. (B) Representative orthogonal view depicting IBA1+ microglia (green) surrounding Aβ plaques (ThioS, magenta) in the cortex. (C) Quantification of the percentage of the total Aβ plaque volume completely encapsulated by IBA1 staining in the cortex and dentate gyrus of the hippocampus. (D) The formation of dystrophic neurites surrounding plaques in the cortex was determined by staining for APP (yellow) and Aβ (ThioS, magenta). Representative immunofluorescence staining of APP puncta and Aβ plaques in the cortex. (E, F) Quantification of APP+ puncta found within a 10 μm and 20 μm radius of ThioS+ Aβ plaques in the (E) cortex and (F) dentate gyrus of the hippocampus. For cortical analyses, each point represents an individual mouse with an average of 50 plaques from 3 matching brain sections per mouse. For hippocampal analyses, each point represents an individual mouse with an average of 25 plaques from 3 matching brain sections per mouse. Statistical significance between experimental groups was calculated by an unpaired Student’s t-test. Error bars represent mean ± SEM.