Genetic inactivation of Alzheimer’s disease risk gene CD33 in human macrophages causes changes in gene signalling and cell function

Pu, Shuang (2025) Genetic inactivation of Alzheimer’s disease risk gene CD33 in human macrophages causes changes in gene signalling and cell function. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Late-onset sporadic AD(LOAD), the most common form of dementia, is influenced by multiple risk genes identified by Genome-Wide Association Studies (GWAS). Among these, the sialic acid receptor CD33 has emerged as a significant factor due to its strong negative correlation with phagocytic capabilities, which is essential for the clearance of AD hallmark—amyloid β(Aβ). Current understanding of CD33 in Aβ plaque clearance and AD pathology is insufficient. Thus, in this project, the human U937 monocyte-derived macrophage model was developed to investigate CD33 signaling pathway in Aβ plaque clearance and its implications for LOAD.CRISPR/Cas9 technology was used to generate human CD33KO-U937 monocytes. Both CD33 wild type (CD33WT) and knockout (CD33KO) U937 monocytes were differentiated into macrophages using PMA treatment and total RNA was extracted for RNA deep sequencing. Key gene expression changes were validated by RT-qPCR and Western blot assays. Phagocytosis assays using fluorescent beads and pre- aggregated Aβ40 and Aβ42, were analyzed by flow cytometry and confocal microscopy to assess the impact of CD33 on phagocytic capacity. Holomonitor and scanning electron microscopy were applied to examine changes in macrophage mobility and morphology following CD33 knockout. The results demonstrated significant changes in multiple signaling pathways due to CD33 knockout, impacting immune response, cell proliferation, differentiation, apoptosis, autophagy, phagocytosis, and cell migration. Key findings, validated by RT-qPCR, Western blot, and fluorescence confocal microscopy indicated that CD33 knockout :(i) enhanced Aβ clearance through phagocytosis and autophagy-mediated Aβ degradation;(ii)resulted in dramatic increase of phagocytosis related gene MSR1 expression;(iii)elevated LAMP2 and LC3b expression, indicating enhanced lysosomal degradation; (iv)inhibited both JAK/STAT3 signaling, and PDK1/PI3K/AKT signalling, then promoted autophagy activity in macrophages;(v)exhibited increase in IL-1β, TNF-α, and NOX2, accompanied by increased anti-inflammatory IL-1RN expression to counterbalance pro-inflammatory effects;(vi) enhanced cell motility resulting from elevated chemokines (CCL2, CCL3, CCL7) to boost Aβ clearance. In summary, this thesis provides insights into the signalling changes related to Aβ clearance in CD33KO-U937 monocyte-derived macrophage model, and sheds new light on possible future therapeutic strategies for Alzheimer’s disease.