Phospholipase D3 drives microglial response to amyloid pathology in Alzheimer’s disease

Abstract

AbstractBackgroundAmyloid‐beta (Aβ) plaque formation is a well‐established hallmark for Alzheimer’s disease (AD). However, the processes behind plaque formation are not understood. Previous work from our group identified rare coding variants for PLD3, with one variant in particular, p.A442A, disrupting a splicing enhancer binding site within the mRNA transcript. In this study, we present findings that PLD3 regulates Aβ clearance in the brain through both cell autonomous and non‐autonomous means.MethodTo replicate the splicing effect observed in patient brain samples in vitro, PLD3 p.A442A expressing induced pluripotent stem cells (iPSCs) were differentiated into neurons or microglia and evaluated for extracellular Aβ and Aβ uptake, respectively. In an APP/PSEN1xPld3KO mouse model, Aβ dynamics were assessed with in vivo microdialysis and plaque pathology was assessed with immunohistochemistry. Frozen cortical tissue from these mice was analyzed through bulk RNAseq to identify transcriptomic and molecular changes that are attributed to Pld3 deficiency. PLD3 expression was assessed in an independent single‐cell RNAseq (scRNAseq) dataset from iPSC‐derived microglia to assess a potential cell‐autonomous role for PLD3 in microglia.ResultTranscriptomics from human samples reveal a significant decrease of PLD3 expression in AD brains versus controls, particularly in the brain regions most susceptible to amyloid pathology. Similar to PLD3‐variant brains, iPSC‐derived neurons carrying PLD3 p.A442A exhibited alternative splicing and displayed elevated Aβ levels in the conditioned media. Loss of Pld3 in APP/PSEN1 mice led to increased interstitial levels of Aβ and reduced Aβ clearance. Immunohistochemistry revealed an increase in diffuse, nonfibrillar plaque area and reduced microglial recruitment in APP/PSEN1xPld3KO. Transcriptomics data from brain tissue in these mice revealed altered expression of genes that define the disease associated microglia signatures. To begin to understand the role of PLD3 in microglia, we analyzed scRNAseq from CRISPRi screen in iPSC‐derived microglia. We found that PLD3 expression is enriched in interferon‐associated microglia. Finally, we demonstrate that iPSC‐derived microglia carrying PLD3 p.A442A exhibit reduced internalization of Aβ and myelin, suggesting a defect in phagocytic capacity.ConclusionTogether, we show that PLD3 plays a critical role in the brain by facilitating the microglial response to Aβ plaque pathology, modulating plaque composition.