An attempt to elucidate the role of clusterin and lipid rafts in Alzheimer's disease etiology

1. Research project objectives/ Research hypothesis

Accumulation of amyloid fibrils is one of the pathological hallmarks of Alzheimer’s disease (AD). Fibrils form senile plaques (SP) composed of the beta-amyloid peptide (Ab a peptide fragment processed sequentially from amyloid precursor protein, APP). Hypercholesterolemia is known to be a major risk in AD as recent studies revealed that elevated level of LDL predisposes to such

neuropathy. The relationship between the blood cholesterol (CHOL) level, proinflammatory cytokines, clusterin (CLU) expression and AD have not been completely explained. LR nanodomains are essential sites for CHOL deposition and constitutive expression of b-secretase and presenilin 1. The role played by CLU in CHOL redistribution is apparently ambiguous. In this project, LR and CLU will be modulated in PC12 neural cells by CHOL chelators/statins and silencing/profection, respectively, in order to determine the susceptibility of PC12 cells to proinflammatory cytokines. The position of CLU in this process will be established either.

Alzheimer’s disease is a complex of disturbances that show a definite but limited familial component. Several genes and their mutations have been associated with AD, such as amyloid beta precursor protein (AβPP), apolipoprotein E (ApoE), presenilins (2). However, the epigenetic regulation of other, undefined genes could explain the sporadic AD cases. One of the known risk

factors of AD development is hypercholesterolemia. It is apparent, that the elevated level of serum cholesterol fraction may influence the brain function. Recent studies revealed that people with total cholesterol levels between 249 and 500 mg/dl were one-and-a-half times more likely to develop Alzheimer’s disease in elderly than those with cholesterol levels of less than 198 mg/dl. People with total cholesterol levels of 221 to 248 mg/dl were more than one-and-a-quarter times more likely to develop AD. A total cholesterol level below 200 mg/dl is generally considered healthy (3). Previously, similar effect of diet-induced hypercholesterolemia on AD incidence was reported in transgenic mouse model of AD (4). Biochemical analysis showed that, compared to control, the hypercholesterolemic mice had significantly decreased sAPPalpha and increased levels of C-terminal fragments (beta), suggesting alterations in amyloid precursor processing in response to high dietary cholesterol (4). How the blood cholesterol can affect the APP processing in not fully understood. The possible mechanisms which could explain cholesterol-AD relationship is the ability of clusterin to bind lipids, including cholesterol and lipid rafts with constitutive expression of beta/gamma secretase. It was demonstrated that CLU forms are linked with ApoA-I lipoproteins and with lipids, where lipids were composed of 54% total cholesterol, 42% phospholipids and 4% triglycerides (mol/mol). The molar ratio between unesterified and esterified CHOL amounted to 0.58. Thus, the CLU complex is a lipoprotein complex of unusual composition in that it has very high protein content and is rich in free CHOL (5). More importantly, CLU levels could be elevated in response to atherogenic diet, containing oxidized LDL and lipids (5). The next cholesterol transporter, which is also relevant to AD is apolipoprotein E (ApoE). The CLU or ApoE might be transported individually or in complexes through the blood-brain barrier (BBB) by megalin/gp330 receptor (6). Megalin/gp330 receptor is a member of the low density lipoprotein (LDL) receptor family, expressed in various tissues, including brain, reproductive organs, epithelia of tracheal, mammary, kidneys and other organs (7). It is possible that BBB-localized megalin/gp330 receptor promotes ApoA-I-CLU-cholesterol transit, or CLU-cholesterol, and ApoE-cholesterol complexes (8). Our assumption is supported by the results reported by Assemat et al. (9), who found that megalin is capable to endocytose dietary sterols such as CHOL, and this function is important both in adult and during embryogenesis. Thus, the relationship between serum and brain CHOL seems to be functional. The hypothesis is illustrated on Figure below. This project is intended to shed light on uncertainty, how cholesterol is linked to clusterin and LR functions. It is a rather new idea established on the foundations that recapitulate our studies performed on neoplastic cells.

2. Research methodology

We are well acquainted with the molecular biology. During last decade we adopted and developed new techniques essential for determination of gene expression (qRT PCR), protein expression levels (WB), immunoprecipitation, cell phenotyping, cytometry, confocal microscopy imaging, transmission electron microscopy imaging with postembedement immunocytochemistry, scanning confocal microscopy, scanning ultrastructural imaging of cell morphology, scanning electron microscopy imaging of surface proteins etc. Additionally, for efficient biochemical examination (kinetics) the multiplate reader with monochromators is needed. The set-up of each is as follows:

Task 1 – Cell culture and transfection approaches. Comparative studies on PC12 cells (wt PC12 vs. AbetaPPsw PC12). The effect of death receptor ligands (TNF-alpha, TRAIL) and reversible CHOL chelation (MbetaCD or nystatin) on cell viability (determination of dose-response and time-course curves). As reported previously (15) the expression levels of proteins involved in PI3-K/AKT/GSK-3beta signaling are crucial for PC12 cell survival. Thus immunobloting of AKT in the DRM fraction isolated with Brij 98 as well as pro- and antiapoptotic proteins, transcription factors, upstream regulators in cytosolic fraction will be performed. WB will be used to determine nCLU and immunopreciptation of sCLU to find its partners.

Task 2 – Ultrastructural studies, cell surface antigens imaging with Quantomix system (TNF-R1, TRAIL-R1 and TRAIL-R2). Transmission and scanning electron microscopy of control and treated cells. Morphological criteria of autophagy and apoptosis will be evaluated by TEM. Postembedment immunocytochemistry technique to co-localize APP, beta-secretase, presenilin1,

Abeta and senile plaques.

Task 3 - Molecular examination of TNF-alpha and TRAIL signal transduction pathways in PC12 cells, identification of cellular proteins involved in PC12 cell death. Modulation of CHOL- and CER-rafts with acidic sphingomyelinase inhibitor (imipramine). Biochemical studies with HTRF technology of enzyme activities by Förster Resonance Energy Transfer (FRET) and Tag-lite

technology from Cisbio (Cisbio Bioassays, France) established to use modern VIS/UV/FL multiplate reader (Tecan M1000, Bern, Switzerland).

Department of Cell Ultrastructure, Mossakowski Medical Research Centre PAS in Warsaw is equipped with cell culture unit, molecular biology lab, transmission and scanning electron microscopes (TEM, SEM), and fluorescent microscope. In order to conduct biochemical analysis of enzyme activities by HTRF (FRET) and Tag-lite technology from Cisbio (Cisbio Bioassays,

France) the access to modern VIS/UV/FL multiplate reader with monochromators is essential (Tecan M1000, Bern, Switzerland).

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

The main form of broadening the results are publications (original and review papers) in high rank and leading journals in human medicine. Lectures and oral presentations are considered as an important route to address the research to specialists in the field. Thus, meetings and workshops would be an excellent opportunity for multimedia presentations and direct dialogue with AD experts. Invited reviews and monographs would be of special interest for the group leader. At  least 2 candidates will be recruited to carry PhD study within the team. They will be thought the methodology and will perform the tasks of the project. Additional benefit would be the implementation of a modern HTFR technique to study LR without detergent application to isolate DRM fraction. This method is the most valuable as the use of detergents led to questioning of the existence of rafts in the past.