PTP1B inhibition promotes microglial phagocytosis in Alzheimer's disease models by enhancing SYK signaling

Abstract

Amyloid β (Aβ) accumulation is a hallmark of Alzheimer's disease (AD). Emerging evidence suggests that impaired microglial Aβ phagocytosis is a key feature in AD, highlighting the therapeutic potential of enhancing this innate immune function. Here, we demonstrate that genetic deletion or pharmacological inhibition of protein tyrosine phosphatase 1B (PTP1B) ameliorated memory deficits and reduced Aβ burden in APP/PS1 mice. Moreover, we show that PTP1B was highly expressed in microglia, and its deficiency promoted a transcriptional shift toward immune activation and phagocytosis. Consistently, PTP1B deletion in microglia enhanced phagocytosis and energy metabolism, supported by increased AKT-mTOR signaling, a pathway essential for meeting the energy demands of activation. Mechanistically, we identified spleen tyrosine kinase (SYK), a key regulator of microglial phagocytosis, as a direct substrate of PTP1B. Inhibition of SYK showed that PTP1B modulates microglial activation in a SYK-dependent manner. These findings established PTP1B as a critical modulator of microglial activation and a potential therapeutic target for AD.

Keywords: Alzheimer’s disease; PTP1B; microglia; signal transduction; therapeutic target.

Figure 1.. Deletion or inhibition of PTP1B in APP/PS1 model improved learning and memory.

(A) Schematic of the NOR test. (B) Percentage of exploration of old or novel object in WT- PTP1B^+/+ (n=16, 7 males, 9 females), WT-PTP1B^−/−(n=18, 8 males, 10 females), APP/PS1-PTP1B^+/+ (n=20, 10 males, 11 females), APP/PS1-PTP1B^−/− (n=18, 9 males, 9 females); unpaired two-tailed t-tests. (C) Schematic of the MWM tests. (D-F) MWM test in WT-PTP1B^+/+ (n=16, 7 males, 9 females), WT-PTP1B^−/− (n=18, 8 males, 10 females), APP/PS1-PTP1B^+/+ (n=20, 10 males, 10 females), APP/PS1-PTP1B^−/− (n=20, 10 males, 11 females); (D) Escape latency to submerged platform in acquisition trials (repeated-measures two-way ANOVA with Tukey’s post hoc test; *: WT-PTP1B^+/+ vs APP/PS1-PTP1B^+/+; #: APP/PS1-PTP1B^+/+ vs APP/PS1-PTP1B^−/−); (E) Crossing frequency in the platform area in probe trial and (F) Time in target quadrant in probe trial (One-way ANOVA with Tukey’s post hoc test); the symbol legend in Fig. 1B also applies to Fig. 1D-F. (G) Percentage of exploration on old or novel object on WT-veh (n= 16, 6 males and 10 females), WT-DPM-1003 (n= 19, 9 males and 10 females), APP/PS1-veh (n= 27, 13 males and 14 females), APP/PS1-DPM-1003 (n= 24, 14 males and 10 females) unpaired two-tailed t-tests. (H-J) Morris water maze in WT-veh (n= 17, 7 males and 10 females), WT-DPM-1003 (n= 19, 9 males and 10 females), APP/PS1-veh (n= 27, 15 males and 12 females), APP/PS1-DPM-1003 (n= 27, 14 males and 13 females); (H) Escape latency to submerged platform in acquisition trials (repeated-measures two-way ANOVA, Tukey’s post hoc test was applied; *: WT-veh vs APP/PS1-veh; #: APP/PS1-veh vs APP/PS1-DPM-1003); (I) Crossing frequence in the platform area in probe trial and (J) Time in target quadrant in probe (One-way ANOVA with Tukey’s post hoc test); the symbol legend in Fig. 1G also applies to Fig. 1H-J. Data represent mean ± SEM; p > 0.05(ns), p < 0.05 (*), p < 0.05(* or #), p < 0.01 (** or ##), p < 0.001 (***or ###), p < 0.0001 (**** or ####).

Figure 2.. Deletion or inhibition of PTP1B in APP/PS1 reduced Aβ levels in APP/PS1 mice.

(A-C) Immunofluorescence and quantitation of Aβ levels in APP/PS1 mice with or without PTP1B in hippocampal region; (A) Representative images showing Thioflavin S and 6E10 staining, Scale bar =500μm; Quantitation of percent area occupied by ThioS (B) and 6E10 (C); n=8 mice/group (4 males, 4 females), with 3-4 brain slices analyzed per mouse. (D-F) Immunofluorescence and quantitation of Aβ levels in APP/PS1 mice with or without DPM-1003 treatment in hippocampal region; (D) Representative images showing Thioflavin S and 6E10 staining, Scale bar =500μm; Quantitation of percent area occupied by ThioS (E) and 6E10 (F); n=8 mice/group (4 males, 4 females), with 3-4 brain slices analyzed per mouse. (G) ELISA quantitation of Aβ_1–42 levels in the brain diethylamine (soluble) and formic acid (insoluble) fractions of cortical tissue in APP/PS1-PTP1B^+/+ (n= 15, 8 males and 7 females) and APP/PS1-PTP1B^−/− (n= 16, 7 males and 9 females); (H) ELISA quantitation of Aβ_1–42 levels in the brain diethylamine (soluble) and formic acid (insoluble) of cortical tissue in APP/PS1-veh (n= 11, 5 males and 6 females) APP/PS1-DPM-1003 (n= 9, 5 males and 4 females) Each dot represents an individual animal; data represent mean ± SEM; unpaired two-tailed t-tests; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Figure 3.. PTP1B is highly expressed in brain immune cells and limits microglial reactivity in APP/PS1 mice.

(A) Schematic of the scRNA-seq experimental design on 13-month-old female mice. (B) Ptpn1 expression across all annotated cell types in APP/PS1 mice. (C) UMAP plots of microglia subclusters split by genotype and colored according to subclusters. (D) Proportion of microglia subclusters from each genotype. (E) Volcano plot showing significant differential expressed genes in red dots (adjusted P < 0.05, log₂FC> 0.3 or log₂FC< −0.3) in microglia of APP/PS1-PTP1B^−/− versus APP/PS1; pseudobulk expression was generated by summing all counts per gene. (F) Gene ontology bar graph generated from genes significantly upregulated (adjusted P < 0.05, basemean>200, log₂FC>0.3) in APP/PS1-PTP1B^−/− mice.

Figure 4.. PTP1B deletion enhanced Aβ engulfment in APP/PS1 mice and promoted phagocytosis in vitro.

(A) Quantitation of WT and PTP1B^−/− primary microglia, treated with vehicle or AβOs, and measurement of phagocytic activity by fluorescence-activated cell sorting (FACS)-based microparticle-uptake assay (n = 9/group); repeated-measures one-way ANOVA with Tukey’s post hoc test; representative micrographs are shown in Supplementary Figure S5A. (B) Representative images of microglia (IBA1, pseudo green), Aβ plaques (6E10, pseudo grey) and lysosomal marker (CD68, pseudo red) co-stained in APP/PS1 mice with or without PTP1B; nuclei are stained with DAPI (blue); Aβ engulfed by IBA1⁺ microglia and by CD68⁺ microglia were shown in grey, scale bar=10μm. (C) Quantitation of the percent volume of Aβ engulfed by IBA1⁺ microglia, averaged across multiple images per mouse; each dot represents one mouse (n=7 mice/group); unpaired two-tailed t-tests. (D) Quantitation of the percent volume of Aβ engulfed by CD68⁺ microglia, averaged across multiple images per mouse; each dot represents one mouse (n=7 mice/group); unpaired two-tailed t-tests. Data represent mean ± SEM; p > 0.05 (ns), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Figure 5.. PTP1B deletion enhanced AβOs-induced PI3K-AKT-mTOR signaling and energy metabolism in microglia.

(A) Representative immunoblot analysis of AKT, p-AKT, mTOR, p-mTOR, HIF1α, and Actin in WT or PTP1B^−/− primary microglia treated with vehicle or AβOs; quantitation from independent biological replicates (n = 5-6) are shown in Supplementary Figure S6A. (B) Glycolysis measured by lactate secretion (n = 4 per group); repeated-measures one-way ANOVA with Tukey’s post hoc test. (C-D) WT and PTP1B^−/− primary microglia were treated with and without AβOs for 24h. After treatment, real time oxygen consumption rate (OCR) was measured following sequential addition of oligomycin, FCCP, rotenone and antimycin A as illustrated (n = 3 independent experiments); (C) OCR curve; (D) quantitation of basal respiration (repeated-measures one-way ANOVA with Bonferroni's post hoc test). Data represent mean ± SEM; p < 0.05 (*), p < 0.01 (**).

Figure 6.. PTP1B plays a critical role in regulating microglia activation upon AβOs stimulation via SYK-dependent pathway

(A) Representative immunoblot analysis of SYK, p-SYK, AKT, p-AKT, mTOR, p-mTOR, HIF1α, and Actin in WT or PTP1B^−/− microglia treated with AβOs or varying concentrations of SYK inhibitor BAY-61-3606, quantitations from independent biological replicates (n = 3-6/group) are shown in Supplementary Figure S7B. (B) Quantitation of microglia and measurement of phagocytic activity by fluorescence-activated cell sorting (FACS)-based microparticle-uptake assay (n = 7/group); repeated-measures one-way ANOVA with Tukey’s post hoc test; representative micrographs are shown in Supplementary Figure S7C. (C) Glycolysis measured by lactate secretion (n = 4/group); repeated-measures one-way ANOVA with Tukey’s post hoc test. (D) OCR measurements of primary cultured microglia with or without the presence of BAY61-3606; repeated-measures one-way ANOVA with Bonferroni's post hoc test. Data represent mean ± SEM; p > 0.05 (ns), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Figure 7.. SYK is a direct substrate of PTP1B.

(A) Representative immunoblot of HA-SYK co-immunoprecipitated with FLAG-PTP1B in CRISPR-generated PTP1B-KO HEK293T cells transiently transfected with FLAG-PTP1B (WT or D181A mutant) and HA-SYK (WT, Y525F, Y526F, YY525/526FF, or Y352F mutant) constructs, alone or in combination as indicated. Whole-cell lysates were probed for HA and FLAG; quantitation from independent biological replicates (n = 4) is shown in Supplementary Figure S8A. (B) Representative Immunoblot and quantitation of SYK and PTP1B substrate trapping assay in primary microglia stimulated with AβOs; Lysates were incubated with purified His-tagged PTP1B (WT or D181A mutant, residue 1-321 truncated form comprising the catalytic domain) in the presence or absence of pervanadate prior to immunoprecipitation; repeated-measures one-way ANOVA followed by Dunnet post hoc test, comparing each group to the control (lane 2); n=3. (C) Proposed model: In microglia, PTP1B directly dephosphorylate SYK, suppressing the downstream AKT-mTOR signaling; deletion of PTP1B prevents SYK dephosphorylation, leading to enhanced AKT-mTOR signaling, which promotes increased activation, phagocytosis, mitochondrial and glycolytic metabolism in response to Aβ. In APP/PS1 mice, PTP1B deletion facilitates Aβ clearance and improve cognitive function. Data represent mean ± SEM; p < 0.05 (*).