Aducanumab anti-amyloid immunotherapy induces sustained microglial and immune alterations

Abstract

Aducanumab, an anti-amyloid immunotherapy for Alzheimer's disease, efficiently reduces Aβ, though its plaque clearance mechanisms, long-term effects, and effects of discontinuation are not fully understood. We assessed the effect of aducanumab treatment and withdrawal on Aβ, neuritic dystrophy, astrocytes, and microglia in the APP/PS1 amyloid mouse model. We found that reductions in amyloid and neuritic dystrophy during acute treatment were accompanied by microglial and astrocytic activation, and microglial recruitment to plaques and adoption of an aducanumab-specific pro-phagocytic and pro-degradation transcriptomic signature, indicating a role for microglia in aducanumab-mediated Aβ clearance. Reductions in Aβ and dystrophy were sustained 15 but not 30 wk after discontinuation, and reaccumulation of plaques coincided with loss of the microglial aducanumab signature and failure of microglia to reactivate. This suggests that despite the initial benefit from treatment, microglia are unable to respond later to restrain plaque reaccumulation, making further studies on the effect of amyloid-directed immunotherapy withdrawal crucial for assessing long-term safety and efficacy.

Figure 1.

Aducanumab modestly reduces Aβ and neuritic dystrophy in APP/PS1 mice during acute treatment phases. (A) Timeline of the acute treatment phase of the study. Male and female 10- to 10.5-mo-old APP/PS1 mice received either two or four doses of aducanumab or IgG control (40 mg/kg, IP injection) and were harvested for analysis 1 wk after the final dose. (B–D) Representative images of (B) total Aβ marked by MOAB-2, (C) X34⁺ fibrillar plaques, and (D) neuritic dystrophy marked by LAMP1, in male mice treated with four doses of aducanumab (Adu) or IgG control. (E and F) Quantification of percent of the tissue covered by MOAB-2⁺ (E) and LAMP1⁺ staining in the cortex (F), following two doses of aducanumab. (G and H) Quantification of percent of the tissue covered by MOAB-2⁺ (G) and LAMP1⁺ staining in the hippocampus (H), following two doses of aducanumab. (I and J) Quantification of the percent of tissue covered by MOAB-2⁺ (I) and LAMP1 staining in the cortex (J), following four doses of aducanumab. (K and L) Quantification of percent of tissue covered by MOAB-2⁺ (K) and LAMP1 staining in the hippocampus (L), following four doses of aducanumab. (M) LAMP1⁺ neuritic dystrophy surrounding amyloid plaques in IgG control and aducanumab-treated mice. Scale bars in B–D = 500 µm for whole tissue view, 100 µm for insets; scale bars in M = 100 µm. Data in E–L expressed as fold change relative to IgG control of the same sex. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (E; F; G females; H females; I; J males; K males; L females), Welch’s t test for normally distributed samples with differences in variances (J females; K females; L males), Mann–Whitney test for non-normally distributed samples (G males; H males), with males and females analyzed separately. N = 6–9 mice/sex/treatment.

Figure S1.

Aducanumab modestly reduces total Aβ after two and four doses. (A–H) Quantification of plaques and neuritic dystrophy in the cortex after two weekly doses of IgG or aducanumab (Adu). (A) Percent of the tissue covered by X34⁺ staining (A), number of X34⁺ plaques per area (B), average X34⁺ plaque size (C), percent of X34⁺ fibrillar plaques (D), number of MOAB-2⁺ plaques per area (E), average MOAB-2⁺ plaque size (F), number of LAMP1⁺ inclusions per area (G), average LAMP1⁺ inclusion size (H). (I–P) The same as A–H, but in the hippocampus, after two weekly doses of IgG or aducanumab. (Q–X) The same as A–H, but in the cortex, after four weekly doses of IgG or aducanumab. (Y–FF) The same as A–H, but in the hippocampus, after four weekly doses of IgG or aducanumab. (GG) Staining of X34⁺ plaques with Cy3 mouse IgG secondary antibody to detect aducanumab in the acute treatment phase. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (A; B females; C; D; E females; F; G; H females; I; J males; K males; L; M; N males; O males; P females; Q; R males; S females; T females; U–Z; AA females; BB females; CC; DD; EE males; FF), Welch’s t test for normally distributed samples with differences in variances (E males; K females; N females; P males; BB males; EE females), Mann–Whitney test for non-normally distributed samples (B males; H males; J females; O females; R females; S males; T males; AA males), with males and females analyzed separately. N = 6–9 mice/sex/treatment.

Figure S2.

Aducanumab increases microglial activation acutely. (A) Diagram showing dissociation methods used for flow cytometry and scRNAseq. (B) Flow cytometry gating strategy used for microglial analysis. Singlet gating axes—FSC-A: 0, 50K, 100K, 150K, 200K, 250K; FSC-H: 0, 50K, 100K, 150K, 200K. Live cell gating axes—UV Blue Zombie UV: −103, 0, 103, 104; SSC-A: 50K, 100K, 150K, 200K. CD11B⁺TMEM119⁺ gating axes—BV510 CD11B: −103, 0, 103, 104; PE TMEM119: −103, 0, 103, 104. CD11C⁺ gating axes—FITC CD11C: −103, 0, 103; SSC-A: 0, 50K, 100K. CD45⁺ gating axes—APC CD45: −103, 0, 103; SSC-A: 0, 50K, 100K. (C) RT-qPCR relative expression of microglial activation genes after two doses of aducanumab (Adu). (D) The same as C, but after four doses of aducanumab. All data in this figure expressed as fold change relative to IgG control within a single sex. (E) Metascape gene ontology heatmap generated from all genes significantly (P < 0.05) upregulated in aducanumab-treated mice. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (C: Trem2, Lpl males, Lyz2 males, Spp1 females, H2-K1, B2m males, Ctsh, Ly86 males, Cd81 females, Unc93b1 males, Ifngr1 males, Sirpa, Ctsz males, Lgmn, H2-D1 males; D: Trem2 males, Lpl, Lyz2, Itgax females, Spp1 males, Ctsh, Ctsz, Ly86, Cd81, Unc93b1, Sirpa), Welch’s t test for normally distributed samples with differences in variances (C: Lpl females, Lyz2 females, Ccl4 females, B2m females, Itgax females, Ly86 females, Unc93b1 females, Ctsz females; D: Spp1 females), Mann–Whitney test for non-normally distributed samples (C: Cd74, Spp1 males, Ccl4 males, Itgax males, Cd81 males, Ifngr1 females, H2-D1 females; D: Trem2 females, Itgax males, Ccl4), with males and females analyzed separately. N = 6–11 mice/sex/treatment.

Figure 2.

Aducanumab increases microglial activation, upregulates pro-clearance genes, and recruits microglia and astrocytes to plaques during acute treatment phases. (A–D) Flow cytometry data showing (A) percent of activated CD11C⁺ microglia, (B) mean fluorescence intensity of CD11C within the CD11C⁺ microglia population, (C) percent of activated CD45⁺ microglia, and (D) mean fluorescence intensity of CD45⁺ within the CD45⁺ microglia population, following four 40 mg/kg doses of aducanumab (Adu) or IgG control. Microglia were identified as CD11B⁺ TMEM119⁺ live cells. (E) UMAP projection of microglia subcluster from scRNAseq analysis, following four doses of aducanumab (N = 2 male mice/treatment). (F) Percent of homeostatic and activated cells in the microglia subcluster in aducanumab- or IgG-treated mice. (G) Violin plots of canonical homeostatic and activated microglial genes used to identify homeostatic and activated microglial subclusters. (H) Heatmap showing the top 30 most highly upregulated genes in aducanumab versus IgG control mice, with P_val_adj < 0.05. (I) RT-qPCR data from bulk hemiforebrain tissue showing relative expression of genes involved in immune system regulation, antigen processing, and lysosomal degradation, including validation of targets from the scRNAseq experiment. (J) Representative images of X34⁺ plaques and LGALS3⁺ and IBA1⁺ microglia following four doses of IgG control or aducanumab. (K) Quantification of total mean intensity of LGALS3⁺ microglia after four doses, related to J. (L) Quantification from Sholl analysis of mean intensity of LGALS3⁺ microglia 5 µm from plaques after four doses, related to J. (M) Representative images of X34⁺ plaques and GFAP⁺ astrocytes following two doses. (N) Quantification of total GFAP⁺ astrocyte coverage after two doses, related to M. (O) Quantification from Sholl analysis of GFAP⁺ astrocyte coverage 15 µm from plaques after two doses, related to L. Scale bars in J = 20 µm; scale bars in M = 100 µm. A–D and I expressed as fold change relative to IgG control of the same sex. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (A females; B females; C; D; I: Ifngr1, Lgmn, B2m, Cd74 males, H2-D1, H2-K1 females; K females; N), Welch’s t test for normally distributed samples with differences in variances (I: Cd74 females; L females; O females), Mann–Whitney test for non-normally distributed samples (A males; B males; I: H2-K1 males; K males; L males; O males), with males and females analyzed separately. N = 5–9 mice/sex/treatment.

Figure 3.

Bulk RNAseq reveals the global acute aducanumab signature is dominated by immune genes. (A and B) Volcano plots showing DEGs from bulk RNAseq of bulk hemiforebrain tissue following (A) two or (B) four 40 mg/kg doses of aducanumab (Adu) or IgG control. Gene names displayed are the top 15 upregulated and downregulated genes. (C and D) Metascape (C) gene ontology and (D) protein–protein interaction analysis of all genes upregulated in aducanumab treatment at weeks 3 and 5. (E) Venn diagram showing genes differentially expressed at either week 3 only (early response), week 5 only (intermediate response), or at both weeks (shared acute response). (F and G) Metascape (F) gene ontology and (G) protein–protein interaction analysis of all genes downregulated in aducanumab treatment at week 5. (H) Heatmap of selected canonical microglial activation genes curated by Chen and Colonna (2021) from multiple studies of microglial activation, showing differential expression of these genes after two doses of aducanumab (week 3 aducanumab versus week 3 IgG), four doses of aducanumab (week 5 aducanumab versus week 5 IgG), and aged amyloid only (week 34 IgG versus week 3 IgG, or 31 wk of aging in APP/PS1 mice). (I) Venn diagram showing the overlap of genes differentially expressed during aducanumab treatment with genes from the Chen and Colonna list. N = 4 mice/sex/treatment/time point, significance at adjusted P val < 0.1 with FDR correction (statistics computed with analysis in edgeR; see Materials and methods for additional details). For heatmaps, *P_val_adj < 0.1, **P_val_adj < 0.05; ***P_val_adj < 0.01.

Figure 4.

Reduction of amyloid load is sustained 15 but not 30 wk after cessation of aducanumab treatment. (A) Timeline of the withdrawal phase of the study. 10- to 10.5-mo-old male and female APP/PS1 mice received four doses of aducanumab or IgG control (40 mg/kg, IP injection) and were harvested either 15 or 30 wk after the final dose. (B–D) Representative images of (B) total Aβ marked by MOAB-2, (C) X34 fibrillar plaques, and (D) neuritic dystrophy marked by LAMP1, in male mice treated with four doses of aducanumab or IgG control, and harvested 15 wk after final treatment. (E and F) Quantification of the percent of the tissue covered by MOAB-2⁺ (E) and LAMP1⁺ staining in the cortex (F), following a 15-wk washout period. (G and H) Quantification of percent of the tissue covered by MOAB-2⁺ (G) and LAMP1⁺ staining in the hippocampus (H), following a 15-wk washout period. (I and J) Quantification of percent of tissue covered by MOAB-2⁺ (I) and LAMP1⁺ (J) staining in the cortex, following a 30-wk washout period. (K and L) Quantification of percent of tissue covered by MOAB-2⁺ (K) and LAMP1⁺ (L) staining in the hippocampus, following a 30-wk washout period. (M) LAMP1⁺ neuritic dystrophy surrounding amyloid plaques in following a 15-wk washout period. Scale bars in B–D = 500 µm for whole tissue view, 100 µm for insets; scale bars in M = 100 µm. Data in E–L expressed as fold change relative to IgG control of the same sex. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (E females; F; G; I females; J; K males; L), Welch’s t test for normally distributed samples with differences in variances (H; K females), Mann–Whitney test for non-normally distributed samples (E males; I males), with males and females analyzed separately. N = 4–11 mice/sex/treatment.

Figure S3.

Reduction in amyloid and neuritic dystrophy is sustained 15 but not 30 wk after aducanumab treatment. (A–H) Quantification of plaque and neuritic dystrophy in the cortex 15 wk after four weekly doses of IgG or aducanumab (Adu). Percent of the tissue covered by X34⁺ staining (A), number of X34⁺ plaques per area (B), average X34⁺ plaque size (C), percent of X34⁺ fibrillar plaques (D), number of MOAB-2⁺ plaques per area (E), average MOAB-2⁺ plaque size (F), number of LAMP1⁺ inclusions per area (G), average LAMP1⁺ inclusion size (H). (I–P) The same as A–H, but in the hippocampus, 15 wk after four weekly doses of IgG or aducanumab. (Q and X) The same as A–H, but in the cortex, 30 wk after four weekly doses of IgG or aducanumab. (Y–FF) The same as A–H, but in the hippocampus, 30 wk after four weekly doses of IgG or aducanumab. (GG) Staining of X34⁺ plaques and Cy3 mouse IgG secondary antibody to detect aducanumab in the washout phase. (HH) CD11C and CD45 mean fluorescence intensity from flow cytometry data from week 19. (II) CD11C and CD45 mean fluorescence intensity from flow cytometry data from week 34. Data in A–II expressed as fold change relative to IgG control within a single sex. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (A–D; E females; F–G; H females; I females; K–L; M males; N females; O females; Q females; R and S; T females; U–X; Y females; Z females; AA males; BB females; CC; DD males; EE males; FF females; HH: CD11C MFI, CD45 males; II), Welch’s t test for normally distributed samples with differences in variances (AA females; DD females; EE females), Mann–Whitney test for non-normally distributed samples (E males; H males; I males; J; M females; N males; O males; P; Q males; T males; Y males; Z males; BB males; FF males; HH: CD45 females), with males and females analyzed separately. N = 4–11 mice/sex/treatment.

Figure 5.

Microglial activation is blunted up to 30 wk after aducanumab treatment. (A and B) Flow cytometry data showing percent of activated (A) CD11C⁺ and (B) CD45⁺ microglia 15 wk after four doses of 40 mg/kg IgG control or aducanumab (Adu). (C and D) Percent of activated (C) CD11C⁺ and (D) CD45⁺ microglia 30 wk after four doses of 40 mg/kg IgG control or aducanumab. Microglia were identified as CD11B⁺ TMEM119⁺ live cells. (E and F) RT-qPCR from bulk hemiforebrain tissue showing relative expression of microglial activation genes (E) 15 wk or (F) 30 wk after four doses of IgG control or aducanumab. (G) Representative images of X34⁺ plaques and IBA1⁺ and LGALS3⁺ microglia 15 wk after four doses of IgG control or aducanumab. (H) Quantification of total mean intensity of LGALS3⁺ microglia 15 wk after four doses, related to G. (I) Quantification from Sholl analysis of mean intensity of LGALS3⁺ microglia 5 µm from plaques, 15 wk after four doses related to G. (J) Representative images of X34⁺ plaques and GFAP⁺ astrocytes 15 wk after four doses. (K) Quantification of total GFAP⁺ astrocyte coverage 15 wk after four doses, related to J. (L) Quantification from Sholl analysis of GFAP⁺ astrocyte coverage 15 µm from plaques 15 wk after four doses, related to J. Scale bars in G = 100 µm. Scale bars in J = 20 µm. A–F expressed as fold change relative to IgG control of the same sex. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (A females; B females; C females; D females; E: Trem2 females, H2-D1, Cst7 females; F: Trem2 females, H2-D1 males, Cst7, Ccl4 females; H males; I males; K; L females), Welch’s t test for normally distributed samples with differences in variances (E: Ccl4; F: Ccl4), Mann–Whitney test for non-normally distributed samples (A males; B males; C males; D males; E: Trem2 males, Cst7 males; F: Trem2 males; H females; I females; L males), with males and females analyzed separately. N = 4–11 mice/sex/treatment.

Figure 6.

Immune pathways activated during acute aducanumab treatment are blunted following withdrawal. (A and B) Volcano plots showing DEGs from bulk RNAseq of bulk hemiforebrain tissue after a (A) 15- or (B) 30-wk washout period following four 40 mg/kg doses of aducanumab (Adu) or IgG control. Gene names displayed are the top 15 upregulated and downregulated genes. (C and D) Metascape (C) gene ontology and (D) protein–protein interaction analysis of all genes downregulated with aducanumab treatment at both washout points. (E) Venn diagram showing genes differentially expressed at either week 19 only (early washout), week 34 only (late washout), or at both weeks (shared washout response). (F) Heatmap of selected canonical microglial activation genes curated by Chen and Colonna (2021) from multiple studies of microglial activation, showing differential expression of these genes after two doses of aducanumab (week 3 aducanumab versus week 3 IgG), four doses of aducanumab (week 5 aducanumab versus week 5 IgG), 15-wk washout (week 19 aducanumab versus week 19 IgG), and 30-wk washout (week 34 aducanumab versus week 34 IgG). N = 3–4 mice/sex/treatment/time point, significance at adjusted P val < 0.1 with FDR correction (statistics computed automatically with analysis in edgeR; see Materials and methods for additional details). For heatmaps, *P_val_adj <0.1, **P_val_adj < 0.05; ***P_val_adj < 0.01.

Figure S4.

Females respond earlier and more strongly to aducanumab, and display greater immune deactivation at washout. (A and D) Volcano plots showing DEGs from bulk RNAseq of bulk hemiforebrain tissue in (A) females after two doses of 40 mg/kg aducanumab (Adu) or IgG control (week 3), (B) females after four doses (week 5), (C) males after four doses (week 5), (D) females after 30-wk washout period following four doses (week 34). Gene names displayed are the top 15 upregulated and downregulated genes. (E–F) Heatmap of selected canonical microglial activation genes curated by Chen and Colonna (2021) from multiple studies of microglial activation, showing differential expression of these genes after two doses of aducanumab (week 3 aducanumab versus week 3 IgG), four doses of aducanumab (week 5 aducanumab versus week 5 IgG), 15-wk washout (week 19 aducanumab versus week 19 IgG), and 30-wk washout (week 34 aducanumab versus week 34 IgG) in (E) males and (F) females. N = 3–4 mice/sex/treatment/time point, significance at adjusted P val < 0.1 with FDR correction (statistics computed automatically with analysis in edgeR; see Materials and methods for additional details). For heatmaps, *P_val_adj < 0.1, **P_val_adj < 0.05; ***P_val_adj < 0.01.

Figure S5.

Microglial and astrocytic recruitment to plaques during acute and washout phase. (A) Representative image of staining of X34⁺ plaques and P2RY12⁺ microglia following two doses of IgG control or aducanumab. Scale bar = 50 µm. (B and C) Representative image of staining of X34⁺ plaques and LGALS3⁺ microglia during (B) acute treatment phase after four doses, and (C) washout phase, 15 wk after four doses. Scale bar = 100 µm. (D) Representative image of staining of activated CD68⁺ microglia around X34⁺ plaques following two doses of IgG or aducanumab. Scale bar = 20 µm. (E) Quantification of total mean intensity of LGALS3⁺ microglia after two doses. (F) Quantification from Sholl analysis of intensity of LGALS3⁺ microglia 5 µm from plaques after two doses. (G) Quantification of total mean intensity of LGALS3⁺ microglia at 30 wk washout. (H) Quantification from Sholl analysis of intensity of LGALS3⁺ microglia 5 µm from plaques at 30 wk washout. (I) Quantification of total mean intensity of CD68⁺ microglia after two doses. (J) Quantification from Sholl analysis of intensity of CD68⁺ microglia 5 µm from plaques after two doses. (K) Quantification of total mean intensity of CD68⁺ microglia after four doses. (L) Quantification from Sholl analysis of intensity of CD68⁺ microglia 5 µm from plaques after four doses. (M) Quantification of total mean intensity of CD68⁺ microglia at 15 wk washout. (N) Quantification from Sholl analysis of intensity of CD68⁺ microglia 5 µm from plaques at 15 wk washout. (O) Quantification of total mean intensity of CD68⁺ microglia at 30 wk washout. (P) Quantification from Sholl analysis of intensity of CD68⁺ microglia 5 µm from plaques at 30 wk washout. (Q) Quantification of total coverage of GFAP⁺ astrocytes after four doses. (R) Quantification from Sholl analysis of coverage of GFAP⁺ astrocytes 15 µm from plaques after four doses. (S) Quantification of total coverage of GFAP⁺ astrocytes at 30 wk washout. (T) Quantification from Sholl analysis of coverage of GFAP⁺ astrocytes 15 µm from plaques at 30 wk washout. *P < 0.05; **P < 0.01, Student’s t test for normally distributed samples with no significant difference in variance (F females; G and H; I females; J–M; N males; O; P females; Q; R females; S; T males), Welch’s t test for normally distributed samples with differences in variances (F males; N females; P males; R males; T females), Mann–Whitney test for non-normally distributed samples (E; I males), with males and females analyzed separately. N = 4–11 mice/sex/treatment.