Anti-MRC1 monoclonal antibody, clone 15-2

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that similar to 50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.

BACKGROUND AND PURPOSE Microglial phenotype and phagocytic activity are deregulated in Parkinson's disease (PD). PPAR gamma agonists are neuroprotective in experimental PD, but their role in regulating microglial phenotype and phagocytosis has been poorly investigated. We addressed it by using the PPAR gamma agonist MDG548. EXPERIMENTAL APPROACH Murine microglial cell line MMGT12 was stimulated with LPS and/or MDG548, and their effect on phagocytosis of fluorescent microspheres or necrotic neurons was investigated by flow cytometry. Cytokines and markers of microglia phenotype, such as mannose receptor C type 1; MRC1), Ym1 and CD68 were measured by elisa and fluorescent immunohistochemistry. Levels of Beclin-1, which plays a role in microglial phagocytosis, were measured by Western blotting. In the in vivo MPTP-probenecid (MPTPp) model of PD in mice, MDG548 was tested on motor impairment, nigral neurodegeneration, microglial activation and phenotype. KEY RESULTS In LPS-stimulated microglia, MDG548 increased phagocytosis of both latex beads and necrotic cells, up-regulated the expression of MRC1, CD68 and to a lesser extent IL-10, while blocking the LPS-induced increase of TNF-alpha and iNOS. MDG548 also induced Beclin-1. Chronic MPTPp treatment in mice down-regulated MRC1 and TGF-beta and up-regulated TNF-alpha and IL-1 beta immunoreactivity in activated CD11b-positive microglia, causing the death of nigral dopaminergic neurons. MDG548 arrested MPTPp-induced cell death, enhanced MRC1 and restored cytokine levels. CONCLUSIONS AND IMPLICATIONS This study adds a novel mechanism for PPAR gamma-mediated neuroprotection in PD and suggests that increasing phagocytic activity and anti-inflammatory markers may represent an effective disease-modifying approach.