Proteomic analysis of inflammatory protein expression patterns in cell culture and transgenic animal models for Alzheimer's disease

Abstract

Dementia is a syndrome characterized by failure of recent memory and other cognitive functions that is usually insidious in onset but steadily progresses with age. Alzheimer’s disease (AD) is the most common form of senile dementia. It is neuropathologically characterized by extracellular and perivascular aggregation of amyloid β (Aβ) peptide, by the generation of intracellular neurofibrillary tangles due to a hyperphosphorylation of tau protein and by an increased rate of neuronal degeneration. The degenerative process starts 20-30 years before the clinical onset of the disease. Clinical diagnosis of AD is difficult but possible, but can only be confirmed by biopsy or autopsy. At present, no biological marker exists for early diagnosis of AD during life. Therefore, identification of biomarkers for AD would be of great value for clinical diagnosis of incipient AD. Recent studies have proven the involvement of inflammatory processes in the neurodegenerative events in AD. Inflammation may not be the first event in the progression of the disease, but it involves activation of glia cells including microglia and astrocytes and subsequent release of proinflammatory mediators. Cytokines released such as IL-1, TNF-α and IL-6 are the main proinflammatory cytokines that can modulate inflammatory responses as well as glial proliferation and activation. Oxidative stress triggered by inflammatory processes causes changes in proteins such as tyrosine nitration or lipid peroxidation. Aβ deposits, tau hyperphosphorylation, inflammation and oxidative stress may finally lead to changes in synaptic connectivity and efficacy including perturbation of long-term potentiation (LTP), important in the formation of memory. Proteomic technology used in these studies is a recent technology which is a two step process: separation of proteins and their subsequent analysis by mass spectrometry. Moreover, this technology can provide new information concerning the expression level, post-translational modification of specific proteins as well as their conformational changes during disease progression. In our study, this technology was modified and improved, e.g by the miniaturization of the complete process. Proteomic technology was also used in parallel with other methods such as chromatography in order to increase the sensitivity of detection by mass spectrometry. This study aimed: 1) To establish that cytokine treatment of human microglia cells is an efficient method to study certain aspects of AD pathogenesis. For this analysis, a map of protein expression in normal and in treated microglia cells was made. 2) To map protein expression in APP/PS2 transgenic mice, a model for human AD, in order to compare human AD brain with murine models. 3) To identify highly nitrated proteins in brains of transgenic animals. Several proteins were found to be modified after injury. 4) To provide evidence for instability of synapses in AD brains. To start with this study, the technologies used to map mouse brain cytosolic proteins were improved. 5) To isolate synaptosomal membranes from the whole brain and to analyse it by massspectrometry. For mapping synaptic membrane protein expression in controls or transgenic mouse models, the technology was miniaturizated and optimized. This study is still in progress.