Effect of secreted frizzled-related protein 3 on tau aggregation
10.3760/cma.j.cn115354-20250324-00168
- VernacularTitle:分泌性卷曲相关蛋白3对tau蛋白聚集的影响研究
- Author:
Jiangyu WANG
1
;
Xingyu ZHANG
1
;
Zhentao ZHANG
1
Author Information
1. 武汉大学人民医院神经内科，武汉 430060
- Publication Type:Journal Article
- Keywords:
Secreted frizzled-related protein 3;
tau;
Neurodegenerative disease;
Protein aggregation;
Protein-protein interaction
- From:
Chinese Journal of Neuromedicine
2025;24(9):888-900
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effect of secreted frizzled-related protein 3 (sFRP3) on tau aggregation.Methods:(1) Twelve wild-type C57BL/6J mice and 12 P301S mutant tau transgenic (tau P301S) mice were selected; Western blotting was used to detect the sFRP3 protein expression in the brain tissues; co-immunoprecipitation experiment was conducted to verify endogenous sFRP3 binding to tau; and double immunofluorescent staining was performed to observe whether sFRP3 co-localized with phosphorylated tau. (2) Recombinant human tau (K18) and sFRP3 protein were purified, and pre-formed fibrils (PFFs) containing only K18 or mixed PFFs (containing both sFRP3 and K18) were prepared; His pull-down assay was adopted to verify exogenous purified sFRP3 binding to tau. Aggregation of these two types of PFFs was observed by thioflavin T (ThT) fluorescence,and anti-digestibility of these two types of PFFs was detected by proteinase K (PK) assay. Ultrastructure of two types of PFFs was observed under transmission electron microscope. (3) HEK293 cells stably expressing green fluorescent protein (GFP) and tau repeat domain (GFP-tau RD HEK293) were treated with the two tpyes of PFFs; intracellular tau aggregation was observed by immunofluorescence; intracellular soluble or insoluble component was evaluated by density gradient lysis. SH-SY5Y cells were treated with the two types of PFFs; cell counting kit-8 (CCK-8) assay was used to detect cell viability; Western blotting was used to detect the expressions of apoptosis-related proteins (Bcl-2-associated X protein [Bax] and cleaved caspase-3); double staining with propidium iodide (PI) and Hoechst 33342, and TUNEL were used to detect cell apoptosis. Primary neurons were treated with the two types of PFFs; density of dendritic spines was measured by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) staining; expressions of synaptic proteins, such as synapsin I and vesicle-associated membrane protein 2 (VAMP2), were detected by Western blotting and double immunofluorescent staining. Results:(1) sFRP3 expression in the brain tissues of tau P301S mice was significantly higher than that in wild-type mice (1.10±0.05 vs. 0.79±0.06, P<0.05). In the brain tissues of wild-type mice, endogenous sFRP3 could bind to tau, and in the brain tissues of tau P301S mice, sFRP3 and phosphorylated tau were co-localized. (2) Exogenous tau and sFRP3 could interact with each other. After constant temperature oscillation for approximately 180 minutes, the ThT fluorescent intensity in mixed PFFs was significantly higher than that in K18 PFFs. Remaining proteins of mixed PFFs were significantly increased compared with those of K18 PFFs after the same digestion time of 1 and 2 μg/mL PK (0.77±0.02 vs. 0.67±0.03; 0.52±0.04 vs. 0.33±0.02, P<0.05). The ultrastructure of mixed PFFs was longer and more compact than K18 PFFs. (3) Compared with K18 PFFs, mixed PFFs formed more intracellular tau aggregation points and had more insoluble components (insoluble protein and soluble protein ratio: 0.43±0.04 and 0.92±0.08), with significant differences ( P<0.05). Compared with K18 PFFs, mixed PFFs had significantly decreased SH-SY5Y cell viability (0.44±0.01 vs. 0.24±0.02), significantly up-regulated Bax and cleaved caspase-3 protein expressions (0.71±0.04 vs. 0.87±0.04; 0.60±0.06 vs. 0.88±0.03), significantly increased percentage of apoptotic cells (PI/Hoechst staining: 8.00%±0.49% vs. 18.11%±1.13%; TUNEL staining: 6.62%±0.91% vs. 14.94%±1.32%, P<0.05). Compared with the K18 PFFs group, the mixed PFFs group had significantly decreased density of dendritic spines ([5.7±0.28]/10 μm vs. [2.7±0.29]/10 μm), synapsin I and VAMP2 expressions (0.95±0.02 vs. 0.48±0.04; 0.88±0.03 vs. 0.52±0.06), and average fluorescent intensity of synapsin I and VAMP2 in the primary neurons (7.73±0.70 vs. 2.74±0.34; 6.14±0.60 vs. 2.96±0.54, P<0.05). Conclusion:sFRP3 interacts with tau to promote tau aggregation, and enhance the cytotoxic effect of tau aggregation.
