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Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease
Published on: December 26, 2016
The CD33 short isoform is a gain-of-function variant that enhances Aβ1-42 phagocytosis in microglia
Abhishek Bhattacherjee1, Jaesoo Jung1, Sameera Zia2
1Department of Chemistry, University of Alberta, 11227 Saskatchewan Dr., Gunning Lemieux Chemistry Centre E5-18A, Edmonton, T6G 2G2, Canada.
Background:
CD33 is genetically linked to Alzheimer's disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant.
Methods:
We have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently.
Results:
In both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m+ microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding.
Conclusions:
These results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.
Insights
The short isoform of CD33 (hCD33m) enhances microglial phagocytosis, acting as a gain-of-function variant. This finding provides insights into Alzheimer's disease (AD) protective mechanisms.
Area of Science:
- Neuroscience
- Immunology
- Genetics
Background:
- CD33 genetic variations are linked to Alzheimer's disease (AD) susceptibility.
- The role of the short isoform of human CD33 (hCD33m), associated with AD protection, remains unclear.
- This study investigates whether hCD33m is a loss-of-function or gain-of-function variant.
Purpose of the Study:
- To determine the functional role of the hCD33m isoform.
- To elucidate the mechanism by which hCD33m influences microglial function.
- To explore therapeutic strategies targeting the AD-protective CD33 allele.
Main Methods:
- Development of transgenic mouse models expressing hCD33m in microglia.
- Utilized CRISPR/Cas9 gene editing in U937 cells to study hCD33 variants.
- Performed phagocytosis assays and single-cell RNA sequencing (scRNAseq) on microglia.
Main Results:
- hCD33m was found to enhance phagocytosis in both primary microglia and U937 cells.
- scRNAseq revealed hCD33m+ microglia exhibit upregulated immediate early gene expression.
- A novel antibody confirmed hCD33m's intracellular localization and gain-of-function role dependent on signaling motifs.
Conclusions:
- hCD33m functions as a gain-of-function isoform, enhancing microglial phagocytosis.
- This gain-of-function contrasts with the inhibitory role of the long isoform (hCD33M).
- Findings offer a basis for developing therapeutics that leverage the AD-protective CD33 allele.
