Alzheimer's disease, ischemic etiology, amyloid, secretases, tau protein, alpha-synuclein, apolipoproteins, expresion of genes

1. Research project objectives/ Research hypothesis

We will look for evidence linking brain ischemia to Alzheimer's disease development. We will study 16 genes (e.g. 2 for amyloid precursor protein, presenilins 1 and 2, τ-protein, apolipoproteins A1, E and J, α-synuclein, β-secretase, alpha secretase, receptors RAGE and LRP1, caspase 3, BECN1 and BNIP3,) expression in brain after ischemia (8 areas) and in fractions of all cellular elements of blood (10 types of cells). Next we will study step by step Alzheimer’s proteins levels (16 proteins) in serum during the development of Alzheimer’s disease. We will try finally establish ischemic etiology as trigger of Alzheimer’s disease and the role of ischemic regulation in the development full-blown Alzheimer’s disease. Moreover, we will consider how ischemia and ischemic expression of Alzheimer’s genes and proteins are important in Alzheimer’s disease etiology. Additionally, we will try to understand the role of ischemic signaling to Alzheimer’s genes and proteins in blood as biomarkers during clinical progression

and maturation of ischemic brain injury with association of Alzheimer’s disease development. Thus, we will present step by step body of evidence, which will support the crucial responsibility of ischemia including ischemic signaling to Alzheimer-associated genes expression in brain and fractions of blood cells and Alzheimer’s proteins in serum in the development in Alzheimer’s disease neuronal death and judgment of Alzheimer’s disease state. These findings will likely lead to the development of the much needed novel etiology and treatment of Alzheimer’s disease and finally diagnostic biomarkers, which will diagnose this incurable disease finally ante mortem.

2. Research project methodology

We have working a unique rat model of complete brain ischemia with a survival after ischemia up to 2 years (in humans circa 80 years). Rats will be anesthetized and brain ischemia due to cardiac arrest will be produced using a method established in the laboratory. Briefly, a blunt tipped, steel hook bent at a 90o angle will be inserted at the right parasternal line across the third intercostal space into the mediastinum. The tip of the hook will be positioned under the cardiac vessel bundle and lifted simultaneously with the compression on the sternum with two fingers of the other hand. Vessel occlusion will be discontinued after 3.5 min and the animals remain in this condition until the onset of resuscitation. Resuscitation will be carried out by external heart massage and artificial ventilation. Brain ischemia will be produced for 10 min with survival time 2, 7 and 30 days, and 6, 12, 18 and 24 months. Experimental and control used rats - 200. Pieces of ischemic brains (8 parts; hippocampus CA1 and CA3 area, thalamus, cerebellar cortex, temporal, parietal, frontal and occiputal cortex) and blood cells (10 types of cells) will be used for studies Alzheimer’s genes. Additionally, we will study Alzheimer’s proteins (16 proteons) in serum. Cells from blood will be sorted using a MoFlo XDP fluorescence activated cell sorter using full software and FlowSight multi-color flow cytometer. The Thermo Scientific NanoDrop and Agilent 2100 Bioanalyzer will be used to automated qualitative and quantitative analysis of RNA. The particular genes in brain and blood cells samples will be detected using the Rat Alzheimer’s Disease RT Profiler PCR Array profiles to detection the expression of 16 genes, important in Alzheimer’s disease. The particular protein (16) in serum, will be detected on the automatic Elisa Analyzer, microtiter, ETI Max 3000.

3. Expected impact of the research project on the development of science, civilization and society

Studies which we started as first propose ischemic etiology of Alzheimer’s disease and this proposal actually is new, when amyloid theory disappointed. Until now nobody investigated Alzheimer’s genes expression (16 genes) in brain and blood cells, step by step for long-term following brain ischemia. Additionally, nobody investigated Alzheimer’s proteins step by step in serum for long time following brain ischemia with association with structural and ultrastructural changes in ischemic brain. Looking for above data together step by step it will be possible to elaborate biomarkers for early final ante mortem diagnosis of Alzheimer’s disease on which now medical doctors and their patients are waiting. Our group will markedly improve our understanding of ischemic etiology/mechanisms in Alzheimer-type dementia development and will propose new final ante mortem diagnostic biomarkers, which will identify asymptomatic stages of disease important in prevention and treatment. This team grant offers considerable opportunity to provide insight into mechanisms responsible for comorbidities of Alzheimer’s disease and progression of dementia. Exploring the relation between brain ischemia and Alzheimer's type cognitive decline is highly important from clinical point of view and has the potential clinical application in final ante mortem diagnosis, prevention and therapy late effects

of dementia. The design and studies of the project follow a clear stepwise approach with the goal to translate the data of the experimental studies into the clinical setting. The presented approach shall provide step by step maximum preclinical evidence before any application of therapy undergoes further testing in the patient. It will be next element, which will support etiological role of ischemic factors in Alzheimer's disease etiology. The work on animals is justified by its potential benefits to the community, since the primary objectives of proposal are to provide a clear understanding step by step mechanisms induced Alzheimer’s dysfunction and biomarkers for early final ante mortem diagnosis and to provide a strong and coherent scientific basis for rational and low cost-effective quality of care in Alzheimer’s disease patients.