Our main project is proteomics to investigate apoptotic pathway in neurodegenerative diseases, e.g., Alzheimer’s disease (Figure 1). Alzheimer's disease has been identified as a protein misfolding disease due to the accumulation of abnormally folded beta amyloid proteins in the brains with AD. beta amyloid induces neuronal cell death, apoptosis, and loss of neurons, resulting in atrophy. Left panel is the brain with Alzheimer's disease, and right panel is a normal aged brain. In the Alzheimer brain, atrophy is clearly seen.β amyloid activates cPLA2, cPLA2 liberates AA from phospholipids. COX-2 is up-regulated in the brain of AD patients, and catalyzes AA to PGG₂ and PGH2. PGD₂ synthase is also upregulated in the cerebrospinal fluid, and generates PGD₂ from PGH. PGD₂ was increased in the AD brain. However, specific binding sites of PGD₂ has not yet been detected in neurons. PGD₂ can be non-enzymatically metabolized to PGJ₂, ^△12-PGJ₂ and 15d-^△12,14-PGJ₂ (15d-PGJ₂). We have found 15d-PGJ₂ as one of endogenous neurotoxins, and detected the specific binding sites of 15d-PGJ₂.
　　15d-PGJ₂ is actively transported into cytosol, bound to PPARγ, a nuclear receptor for 15d-PGJ₂ (Figure 2). PPARγis a target of drug for diabetic, e.g. Actos (pioglitazone). Activation of PPARγ induced neuroprotective effects, but did not apoptosis. 15d-PGJ₂ induced neuronal apoptosis via independent pathway from PPARγ. We predict that the specific binding site might be involved in the novel apoptotic pathway.
　　To clear the apoptotic pathway, we are challenging the identification of membrane receptor for 15d-PGJ₂ (Figure 3). Carboxy group of 15d-PGJ₂ can be biotinylated. Biotinylated 15d-PGJ₂ are incubated with neuronal plasma membranes. This analog binds to cysteine residues by Michael addition.
　　These samples are analyzed in parallel by two-dimensional (2D) electrophoresis followed by Western blotting and Sypro Ruby Staining　(Figure 4). First, proteins can be separated by their isoelectric characters. Second, they can be separated by their molecular weights. We can detect biotinylated 15d-PGJ₂–modified proteins by western blotting. Non-specific signals of biotinylated 15d-PGJ₂–modified proteins can be detected in the presence of excess 15d-PGJ₂. Specific signals are indicated by arrowheads.
　　Spots which corresponds to the specific signals are excised for a Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis (Figure 5). Spot in Figure 3 was digested in gel with trypsin, and the resulting peptides were analyzed by MALDI-TOF MS. (A) Typical mass spectrum from a representative experiment. (B) List of the monoisotopic masses of some of the peptides identified by MASCOT analysis.