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Basic Science and Pathogenesis
Kanayo Satoh1, Ye Zhou1, Roger B Dodd2
1University of Toronto, Toronto, ON, Canada.
Background:
The sialic-acid binding immunoglobulin-like lectin 3 receptor (Siglec-3 / CD33) expressed on microglia, regulates immune functions relevant to Alzheimer's disease (AD). Clusterin (CLU) and apolipoprotein E (ApoE) are soluble, sialylated proteins implicated in AD pathogenesis through genetic associations and their interactions with amyloid-beta (Aβ). However, the role of these proteins as potential CD33 ligands remains unclear. This study explores whether CLU and/or ApoE bind CD33 and examines the functional impact of these interactions on Aβ uptake and amyloid plaque clearance.
Methods:
The binding of CD33 to CLU and ApoE was assessed through co-immunoprecipitation using U937 cells (endogenously expressing CD33) and HEK293 cells (expressing exogenous CD33). Quantitative bio-layer interferometry (BLI) and microscale thermophoresis determined binding affinities, focusing on the role of CD33's Arg119 sialic acid binding site. In situ proximity ligation assays (PLA) and co-immunoprecipitation from AD and control human brain lysates validated in vivo interactions. Functional assays examined Aβ uptake and amyloid plaque clearance in monocytes and U937 cells, with or without CLU treatment.
Results:
The quantitative binding assay revealed that CLU, but not ApoE, was a sialylation-dependent ligand for CD33, binding with high affinity (Kd = 28.9 ± 10.3 nM). Binding required an intact Arg119 residue and dimeric CD33 structure. PLA and co-immunoprecipitation studies demonstrated colocalization of CD33 and CLU on microglia in AD brains, especially near amyloid plaques. Functionally, sialylated CLU inhibited Aβ uptake in monocytes from CD33 "CC" risk allele carriers and reduced amyloid plaque clearance in U937 cells. Desialylated CLU showed no significant effects. Notably, CLU + Aβ oligomers induced stronger CD33 ITIM signaling than CLU alone, enhancing phosphorylation and SHP-1 recruitment.
Conclusion:
This study identifies CLU as a specific CD33 ligand and highlights its role in modulating microglial functions via CD33 ITIM signaling. Sialylated CLU inhibits Aβ uptake and amyloid plaque clearance, suggesting a potential mechanism underlying microglial dysfunction in AD. These findings underscore the therapeutic potential of targeting the CD33-CLU axis to restore microglial homeostasis and enhance amyloid clearance in AD.
Insights
Clusterin (CLU) binds CD33 (sialic-acid binding immunoglobulin-like lectin 3 receptor) in Alzheimer's disease (AD) brains. This interaction impairs Aβ uptake and plaque clearance by microglia, suggesting a therapeutic target for AD.
Area of Science:
- Neuroimmunology
- Alzheimer's Disease Pathogenesis
- Microglial Function
Background:
- Sialic-acid binding immunoglobulin-like lectin 3 receptor (CD33) on microglia regulates immune responses relevant to Alzheimer's disease (AD).
- Clusterin (CLU) and apolipoprotein E (ApoE) are implicated in AD pathogenesis but their role as CD33 ligands is unknown.
- Investigating CLU and ApoE interactions with CD33 is crucial for understanding AD mechanisms.
Purpose of the Study:
- To determine if Clusterin (CLU) and/or Apolipoprotein E (ApoE) bind to CD33.
- To examine the functional consequences of CD33-CLU/ApoE interactions on amyloid-beta (Aβ) uptake and plaque clearance.
- To elucidate the role of CD33-CLU interactions in AD pathogenesis.
Main Methods:
- Co-immunoprecipitation and quantitative bio-layer interferometry (BLI) assessed CD33 binding to CLU and ApoE.
- Binding affinities and the role of CD33's Arg119 residue were analyzed.
- In situ proximity ligation assays (PLA) and functional assays evaluated Aβ uptake and plaque clearance in relevant cell models and human brain lysates.
Main Results:
- Clusterin (CLU), but not ApoE, demonstrated sialylation-dependent high-affinity binding to CD33, requiring an intact Arg119 residue and dimeric CD33.
- In AD brains, CD33 and CLU colocalized on microglia near amyloid plaques.
- Sialylated CLU inhibited Aβ uptake in monocytes and reduced amyloid plaque clearance, while desialylated CLU had no effect; CLU + Aβ oligomers enhanced CD33 ITIM signaling.
Conclusions:
- Clusterin (CLU) is identified as a specific CD33 ligand, modulating microglial function through CD33 ITIM signaling in AD.
- Sialylated CLU impairs Aβ uptake and amyloid plaque clearance, contributing to microglial dysfunction in AD.
- Targeting the CD33-CLU axis offers potential therapeutic strategies for restoring microglial homeostasis and enhancing amyloid clearance in AD.
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