PLCG2 is associated with the inflammatory response and is induced by amyloid plaques in Alzheimer’s disease

Abstract

Background Alzheimer’s disease (AD) is characterized by robust microgliosis and phenotypic changes that accompany disease pathogenesis. Accumulating evidence from genetic studies suggests the importance of phospholipase C γ 2 (PLCG2) in late-onset AD (LOAD) pathophysiology. However, the role of PLCG2 in AD is still poorly understood. Methods Using bulk RNA-Seq (N=1249) data from the Accelerating Medicines Partnership-Alzheimer’s Disease Consortium (AMP-AD), we investigated whether PLCG2 expression increased in the brains of LOAD patients. We also evaluated the relationship between PLCG2 expression levels, amyloid plaque density, and expression levels of microglia specific markers (AIF1 and TMEM119). Finally, we investigated the longitudinal changes of PLCG2 expression in the 5xFAD mouse model of AD. To further understand the role of PLCG2 in different signaling pathways, differential gene expression and co-expression network analyses were performed using bulk RNA-Seq and microglial single-cell RNA-Seq data. To substantiate the human analyses, we performed differential gene expression analysis on wild-type (WT) and inactivated Plcg2 mice and used immunostaining to determine if the differentially expressed genes/pathways were altered by microglial cell coverage or morphology. Results We observed significant upregulation of PLCG2 expression in three brain regions of LOAD patients and significant positive correlation of PLCG2 expression with amyloid plaque density. These findings in the human brain were validated in the 5xFAD amyloid mouse model, which showed disease progression-dependent increases in Plcg2 expression associated with amyloid pathology. Of note, increased Plcg2 expression levels in 5xFAD mice were abolished by reducing microglia. Furthermore, using bulk RNA-Seq data, we performed differential expression analysis by comparing cognitively normal older adults (CN) with 75th percentile (high) and 25th percentile (low) PLCG2 gene expression levels to identify pathways related to inflammation and the inflammatory response. The findings in the human brain were validated by differential expression analyses between WT and plcg2 inactivated mice. PLCG2 co-expression network analysis of microglial single-cell RNA-Seq data identified pathways related to the inflammatory response including regulation of I-kappaB/NF-kappa B signaling and response to lipopolysaccharide. Conclusions Our results provide further evidence that PLCG2 plays an important role in AD pathophysiology and may be a potential target for microglia-targeted AD therapies.

Biomarker Genes Discovery of Alzheimer’s Disease by Multi-Omics-Based Gene Regulatory Network Construction of Microglia

“…The data were normalized using the Sctransform() function in the Seurat package, instead of using the traditional NormalizeData(), ScaleData() and FindVariableFeatures() preprocessing functions. In total, 6000 highly variable genes (HVGs) were selected [ 16 ].…”

“…The batch processing of the normalized data was investigated using PCA and Harmony algorithms to facilitate the clustering analysis. Ultimately, these important steps were implemented through the RunPCA() and RunHarmony() functions in the Seurat and Harmony packages [ 16 ], and the first 20 pc were selected as input.…”

“…The resolution was chosen to be 0.5. In total, 60,137 cell nuclei were clustered, based on 6000 HVGs [ 16 ].…”

PLCγ2 impacts microglia-related effectors revealing variants and pathways important in Alzheimer’s disease

“…Expression levels of microglia-specific marker genes were significantly associated with PLCγ2 expression levels in the frontal pole, superior temporal gyrus, parahippocampal gyrus, and inferior prefrontal gyrus. Notably, expression levels of PLCγ2 were also shown to correlate significantly with mean amyloid plaque density in the parahippocampal gyrus, superior temporal gyrus, and prefrontal subcortex ( Tsai et al, 2022 ).…”

“…Andreone and colleagues have shown that PLCγ2 may affect the survival, proliferation, phagocytosis, and migration of microglia by regulating intracellular calcium release, to help microglia maintain their normal immune response ( Andreone et al, 2020 ). Cellular and animal studies targeting PLCG2 variants have revealed their impact on microglia under AD conditions, including the association of PLCγ2 with two pathological markers of AD, Aβ and p-tau, and the relationship with many AD-related effectors such as TREM2, TLR4, and others ( Shen et al, 2008 ; Matarin et al, 2015 ; Sierksma et al, 2020 ; Tsai et al, 2022 ).…”