Proteomic analysis of changes in brain and liver mitochondria in apoE-knockout mice – influence of metformin – an AMP-activated protein kinase (AMPK) activator

Apolipoprotein E-deficient mice (apoE-knockout mice) are one of the most widely used animal model in biomedicine. These animals develop atherosclerosis with non-alcoholic fatty liver disease and neurodegenerative changes similar to those observed in Alzheimer;s disease. Importantly, recent studies underline crucial role of mitochondrial dysfunction in the development of atherosclerotic vascular changes, liver steatosis and neurodegeneration. Proteomic methodology – two-dimensional electrophoresis coupled with tandem mass spectrometry (2DE-LC-MS/MS) – offers a unique capability to perform simultaneous analysis in expression of hundreds of proteins in complex tissue/cell/organelle samples. The aim of the study is to investigate changes in brain and liver mitochondria in apoE – knockout mice and to analyze the mitochondrial action of metformin – an activator of AMP-activated protein kinase (AMPK) - widely used antidiabetic drug with putative antiatherosclerotic and neuroprotective action. Results of our study may shed a new light on the mechanisms of involvement of mitochondria in the pathogenesis of organ damage in atherosclerosis and amyloid- β-related neurodegeneration.

Despite remarkable progress in modern medicine, atherosclerosis and atherosclerosis-related organ injury are one of the major cause of morbidity in Western countries. Organ injury develops in time-dependent manner, reaping its outcome especially in the elderly population. What is more, Alzheimer’s disease is the most common form of neurodegeneration, affecting 5% of older people above the age of 65 and up to 40% of the society over 85 years of age, exhibiting a profound decline in daily activities and dementia. In spite of a large effort in investigating the etiology and pathogenesis of those diseases, the precise mechanisms responsible for initiation and progression of both are not fully known. Recent literature highlights the potential key role of mitochondria and their dysfunction as a common ground for vascular and organ pathology in atherosclerosis, including liver steatosis as well as for b-amyloid-mediated brain neurodegeneration. It is also widely recognized that 5’ AMP-activated protein kinase (AMPK) is the one of the major regulator of cellular metabolism and function of mitochondria.

Our project is aimed to identify the protein targets of possible importance for pathogenesis of non-alcoholic fatty liver disease and neurodegeneration in mice. In this regard our results should open new ways for further functional and molecular experiments on animals as well as guide future studies on patients. We believe, that it may broaden the understanding of the nature of the processes responsible for dysfunction of mitochondria and possibly indicate the mechanisms of its compensation. Reversal of the unfavorable protein changes in mitochondria could represent a new pharmacological approach to treatment of fatty liver disease and neurodegeneration.

Experimental protocol will be based on comparison of the protein maps of brain and liver mitochondria between C57BL/6J (wild type), apoE^-/- mice, and apoE^-/- mice treated with metformin. For identification of proteins the methods of differential proteomics will be used: 2-dimensional electrophoresis (2DE) and mass spectrometry (MS). The study outcome will consist of list of mitochondrial proteins with changed expression in apoE^-/- mice vs. wild type mice, and the list of proteins in mitochondria of apoE^-/- mice affected by use of metformin. Then, selected results obtained from 2DE-LC-MS/MS experiments will be validated by molecular biology techniques (Western blot, RT-PCR). Finally, mitoproteome changes at molecular level will be correlated with histological assessment of brain and liver tissue. Our preliminary results, regarding changes in liver mitochondria in apoE^-/- mice, obtained according to such workflow have been published last month (Suski et al.. J Proteomics 2011 May 16;74(6):887-93).

Our results of proteomic investigation of atherosclerosis development-related liver mitochondria proteome changes in apoE-knockout mice have been published and delivered preliminary protein targets for following detailed studies. Differential proteomics have not been used before to reveal protein expression changes in the brain in this experimental model of Alzheimer’s disease. Proteomic examination of the effect of metformin on brain and liver mitochondria represent a novel and original approach to study possible simultaneous alterations of hundreds of proteins, related to AMP-activated protein kinase (AMPK) activation.

Our project is aimed to identify the protein targets of possible importance for pathogenesis of non-alcoholic fatty liver disease and neurodegeneration in mice. In this regard our results should open new ways for further functional and molecular experiments on animals as well as guide future studies on patients. We believe, that it may broaden the understanding of the nature of the processes responsible for dysfunction of mitochondria and possibly indicate the mechanisms of its compensation. Reversal of the unfavorable protein changes in mitochondria could represent a new pharmacological approach to treatment of fatty liver disease and neurodegeneration.