# LABORATORY OF BIOPHYSICAL METHODS

| Head: Prof. Danek ELBAUM, Ph.D. E-mail: elbaum@nencki.gov.pl Staff: Maria BRZYSKA, Ph.D. Ph.D. student: Małgorzata PERGOL, M.Sc.

The main area of interest is focused on understanding etiology of Alzheimer disease.

More specifically, we study the mechanism of beta-amyloid toxicity on the macromolecular

and cellular level. We are interested how beta-amyloid induces free radical generation thus renders the aggregated protein toxic to neurons and other peripheral cells. The other area of

our interest is aimed to study Ca(2+) homeostasis abnormality observed in cells taken from

Alzheimer patients. Two aspects of the research are exiting to us: (1) understanding the basic mechanisms of the amyloid toxicity, (2) to define the observed differences between normal and the pathological fibroblasts as a potential diagnostic marker.

Beta-amyloid peptides are proteolytic fragments derived from amyloid precursor protein

(APP), a transmembrane glycoprotein. Amyloid fragments form the core of Alzheimer disease (AD) senile plaques located predominantly in the cerebral cortex and are implicated as a major determinant of the pathology. Beta-amyloid formation requires multiple abnormal hydrolytic events or proteolytic cleavages of APP. An increasing number of studies show that

oxidative reactions occur in AD. There is a correlation between areas with elevated levels of oxidation biomarkers and density of Beta-amylod cortical deposits. It suggests that Beta-amyloid may be a molecular link between oxidative stress and AD. Moreover the studies revealed that Beta-amyloid aggregates induce the accumulation of hydrogen peroxide in neurons.

Our studies implicated a novel biochemical property of Beta-amyloid: the ability to cleave

esters and peptide bonds ( Elbaum, et.al., 2000, BBRC, 267, 733-738) and more recently, the propensity to hydrolyze and oxidize as well ( Brzyska, et.al., 2001, Eur.J. Biochem., 268, 3443-3454). The results indicate that the amyloid proteolytic, hydrolytic and oxidative properties are distinct feature of the peptide and require different mechanism to occur, but three of them could contribute to its toxicity. The reported properties could be responsible for early pathogenesis of AD since there is emerging evidence that the non-plaque amyloid is elevated in AD patients.

Our methodology has been confined to determination fluorescence emission and absorption spectroscopy using several probes capable to report the mechanisms of the above reactions.

The sensitivity of the techniques have allowed us to follow small changes using a nano-Molar

concentrations range. The changes of the protein conformation have been followed with Circular Dichroism. The protein-protein interactions have been studied using Fluorescence

Resonance Energy Transfers.

On the cellular level, we analyzed the operation of K(+) channels in human Alzheimer

donor’s fibroblasts by determination Ca (2+) influx in response to chemically induced

membrane depolarization. The results were compared to aged normal and young donors.

Brzyska, et.al., 2002, Neuro. Res. Comm., 30, 51-61). The results supported the earlier findings that calcium responses were altered in AD cells. From a clinical perspective,

especially the experiments on peripheral cells, such as skin fibroblasts seem to be very valuable because they offer the possibility of early diagnosis.

Selected publications:

1. Elbaum, D., Brzyska, M., Bacia, A., Alkon, D. „ Implication of Novel Biochemical Property of Beta-amyloid”, Biophysical Research Communication, 267, 733-738, ( 2000)

2. Brzyska, M., Bacia, A., Elbaum, D., „ Oxidative and hydrolytic properties of Beta-amyloid”, Eur. J. Biochem., 268, 1-13, ( 2001).

3. Brzyska,M., Michalski,A., Elbaum, D., „ Tetraethylammonium induced changes in intracellular calcium in Alzheimer donor fibroblasts”, Neuroscience Research Communication, accepted for publication, 30, 1, (2002).

4. Chacinska, A., Wozny, W., Boguta, M., Misicka, A., Brzyska, M., Elbaum, D., “ Effect of Beta-Amyloid on proliferation and morphology of yeast Saccharomyces cerevisiae” , LIPS, accepted for publication,