Activated cyclin-dependent kinase 5 promotes microglial phagocytosis of fibrillar ß-amyloid by up-regulating lipoprotein lipase expression.

Amyloid plaques are crucial for the pathogenesis of Alzheimer disease (AD). Phagocytosis of fibrillar ß-amyloid (Aß) by activated microglia is essential for Aß clearance in Alzheimer disease. However, the mechanism underlying Aß 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 Aß. Among 2742 proteins identified, six were significantly up-regulated and seven were down-regulated by Aß treatment. Bioinformatic analysis revealed strong over-representation of membrane proteins, including lipoprotein lipase (LPL), among proteins regulated by the Aß stimulus. We verified that LPL expression increased at both mRNA and protein levels in response to Aß treatment in BV2 microglia and primary microglial cells. Silencing of LPL reduced microglial phagocytosis of Aß, but did not affect degradation of internalized Aß. Importantly, we found that enhanced cyclin-dependent kinase 5 (CDK5) activity by increasing p35-to-p25 conversion contributed to LPL up-regulation and promoted Aß phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and Aß internalization. Furthermore, Aß 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 Aß. In summary, our findings reveal a potential role of the CDK5/p25-LPL signaling pathway in Aß phagocytosis by microglia and provide a new insight into the molecular pathogenesis of Alzheimer disease.

Peroxiredoxin 6 Is a Crucial Factor in the Initial Step of Mitochondrial Clearance and Is Upstream of the PINK1-Parkin Pathway.

AIMS: PTEN-putative kinase 1 (PINK1)-Parkin-mediated mitophagy is crucial for the clearance of damaged mitochondria. However, the mechanisms underlying PINK1-Parkin-mediated mitophagy are not fully understood. The goal of this study is to identify new regulators and to elucidate the regulatory mechanisms of mitophagy. RESULTS: Quantitative mitochondrial proteomic analysis revealed that 63 proteins showed increased levels and 36 proteins showed decreased levels in cells subjected to carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment. Peroxiredoxin 6 (PRDX6 or Prx6), a unique member of the ubiquitous PRDX family, was recruited to depolarized mitochondria. Reactive oxygen species (ROS) generated by CCCP promoted PRDX6 accumulation and PINK1 stabilization in damaged mitochondria and induced mitophagy. In addition, depletion of PRDX6 resulted in the stabilization of PINK1, accumulation of autophagic marker, p62, translocation of Parkin to mitochondria, and lipidation of microtubule-associated protein 1 light chain 3. Furthermore, these events were blocked upon supplementation with antioxidant N-acetyl-l-cysteine or depletion of PINK1. INNOVATION: This is the first study to demonstrate that PRDX6 is the only member of the PRDX family that relocates to damaged mitochondria, where it plays a crucial role in the initial stage of mitophagy by controlling ROS homeostasis. CONCLUSION: ROS induce the recruitment of PRDX6 to mitochondria, where PRDX6 controls ROS homeostasis in the initial step of PINK1-Parkin-mediated mitophagy. Our study provides new insight into the initial regulatory mechanisms of mitophagy and reveals the protective role of PRDX6 in the clearance of damaged mitochondria.

Quantitative proteomics reveals that PEA15 regulates astroglial Aß phagocytosis in an Alzheimer's disease mouse model.

Amyloid-beta (Aß) deposition plays a crucial role in the progression of Alzheimer's disease (AD). The Aß deposited extracellularly can be phagocytosed and degraded by surrounding activated astrocytes, but the precise mechanisms underlying Aß clearance mediated by astrocytes remain unclear. In this study, we performed tandem mass tag-based quantitative proteomic analysis on the cerebral cortices of 5-month-old APP/PS1 double-transgenic mice. Among the 2668 proteins quantified, 35 proteins were upregulated and 12 were downregulated, with most of these proteins being shown here for the first time to be differently expressed in the APP/PS1 mouse. The altered proteins were involved in molecular transport, lipid metabolism, autophagy, inflammation, and oxidative stress. One specific protein, PEA15 (phosphoprotein enriched in astrocytes 15 kDa) upregulated in APP/PS1 mice, was verified to play a critical role in astrocyte-mediated Aß phagocytosis. Furthermore, PEA15 levels were determined to increase with age in APP/PS1 mice, indicating that Aß stimulated the upregulation of PEA15 in the APP/PS1 mouse. These results highlight the function of PEA15 in astrocyte-mediated Aß phagocytosis, and thus provide novel insight into the molecular mechanism underlying Aß clearance. The protein-expression profile revealed here should offer new clues to understand the pathogenesis of AD and potential therapeutic targets for AD. BIOLOGICAL SIGNIFICANCE: Activated astrocytes are known to clear the Aß deposited in the extracellular milieu, which is why they play a key role in regulating the progression of Alzheimer's disease (AD). However, the molecular mechanism underlying astrocyte-mediated Aß phagocytosis and degradation remains unclear. By performing tandem mass tag-based quantitative proteomic analysis, we identified 47 proteins that were differentially expressed in APP/PS1 double-transgenic. To our knowledge, this is the first time most of these proteins have been reported to exhibit altered expression in the mouse model of AD. Furthermore, our results indicate that one of the proteins upregulated in the APP/PS1 mouse, PEA15 (phosphoprotein enriched in astrocytes 15 kDa), regulates astroglial phagocytosis of Aß. Our findings provide new insights into the molecular mechanism underlying Aß clearance in AD. The altered profile of protein expression in APP/PS1 mice described here should offer valuable clues to understand the pathogenesis of AD and facilitate the identification of potential targets for the treatment of AD.