Abstract
Amyloid plaques are crucial for the pathogenesis of Alzheimer disease (AD). Phagocytosis of fibrillar β-amyloid (Aβ) by activated microglia is essential for Aβ clearance in Alzheimer disease. However, the mechanism underlying Aβ clearance in the microglia remains unclear. In this study, we performed stable isotope labeling of amino acids in cultured cells for quantitative proteomics analysis to determine the changes in protein expression in BV2 microglia treated with or without Aβ. Among 2742 proteins identified, six were significantly up-regulated and seven were down-regulated by Aβ treatment. Bioinformatic analysis revealed strong over-representation of membrane proteins, including lipoprotein lipase (LPL), among proteins regulated by the Aβ stimulus. We verified that LPL expression increased at both mRNA and protein levels in response to Aβ treatment in BV2 microglia and primary microglial cells. Silencing of LPL reduced microglial phagocytosis of Aβ, but did not affect degradation of internalized Aβ. Importantly, we found that enhanced cyclin-dependent kinase 5 (CDK5) activity by increasing p35-to-p25 conversion contributed to LPL up-regulation and promoted Aβ phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and Aβ internalization. Furthermore, Aβ plaques was increased with reducing p25 and LPL level in APP/PS1 mouse brains, suggesting that CDK5/p25 signaling plays a crucial role in microglial phagocytosis of Aβ. In summary, our findings reveal a potential role of the CDK5/p25-LPL signaling pathway in Aβ phagocytosis by microglia and provide a new insight into the molecular pathogenesis of Alzheimer disease.
Highlights
From the ‡State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China
A Proteomic Screen to Detect Changes in BV2 Cell Phagocytosis of fA—Considering that BV2 cells could be activated and phagocytose fA, we investigated the proteins that might be implicated in microglial phagocytosis of fA by performing SILAC quantitation and high resolution nanoLC-MS/MS analysis (Fig. 2A)
We investigated the changes of proteins in BV2 microglia treated with fA using the SILAC approach and identified six up-regulated and seven down-regulated proteins in A-activated BV2 microglia
Summary
Preparation of fA and Electron Microscopy—Human A1–42 (AnaSpec, San Jose, CA) was dissolved in 1 mM PBS and incubated at 37 °C for 1 week before use. Data Analysis—Raw mass spectra files were processed with MaxQuant software [27, 28] (version 1.3.0.5), that performs peak list generation, SILAC and extracted ion current-based quantitation, false positive rate determination based on search engine results, peptide to protein group assembly, and data filtration and presentation. BV2 cells or primary microglia were seeded in 12-well culture plates and incubated in the absence or presence of 2 M A1–42 for 24 h in serum-free DMEM. CDK5 Kinase Assay—BV2 cells were treated with 2 M fAfor 24 h, lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1% PI, 1 mM Na3VO4, and 1 mM NaF on ice, placed on a roller at 4 °C for 30 min, and centrifuged at 14,000 ϫ g for 15 min. The significance level was set at p Ͻ 0.05
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