RICERCA ITALIANA     

Chemical processes and structural modifications in neurodegeneration

Università degli Studi di Pavia

Abstract

The research proposed in this project addresses the problem of the origin of the chemical modifications and the conformational changes undergone by the proteins involved in the pathogenesis of two important neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer's disease (AD). The effects produced on the proteins by various agents, including oxidative stress, nitrative stress, redox active metal ions, and dopamine will be investigated. The nature and specific sites of protein modifications produced by reaction with dopaminoquinones (DAQ) and other enzyme- or chemically-generated species under oxidative and nitrative stress conditions will be clarified. In particular, the characterization of the different protein-DAQ adducts formed in dopamine neurons and their role in neuromelanin synthesis and neurodegeneration will be pursued. Neuronal cultures and brain tissues from monkeys will be used as in vivo models. A parallel, detailed analysis of the structural, conformational, and stability changes produced by metal ion binding to toxic amyloid beta-peptide 1-42 (Abeta 1-42) and the N-terminal peptide fragment of human prion protein (PrP 84-114), which are at the origin of AD and transmissible encephalopaties, respectively, will be carried out. The research activities to be developed can be summarized in the following main research lines.
1) Expression of human tyrosinase and other proteins, generation of protein-DAQ adducts by tyrosinase and peroxidase, and >>>

Principal Investigator

Luigi CASELLA Università degli Studi di PAVIA

Research Objectives

The aim of the proposed research project is to clarify the molecular basis of the processes that induce the misfolding and aggregation of the proteins involved in two important neurological diseases, Parkinson's disease (PD) and Alzheimer's disease (AD). The starting point is the observation from a variety of experiments that oxidative and nitrative reactions accompany the misfolding and aggregation of neuronal proteins, although it is still unclear whether these processes have a primary role in neurodegeneration or they occur as a secondary phenomenon mostly at the end-stage of the diseases. Understanding the mechanisms that induce the protein changes is thus of enormous importance to clarify the origin of neurodegenerative processes and to devise future strategies to prevent their occurrence. The research proposed here exploits the complementary expertise of the three UOs for a thorough interdisciplinary investigation of the chemical modifications undergone by several neuronal proteins and their detection in cellular and animal models. Complementary expertise is needed at this level because the competence to deal with all aspects involved cannot be present in a single research group. In fact, the high reactivity of the chemical species involved in protein modification (DAQ, ROS and RNS) requires the definition of methodologies and protocols for the analysis and identification of the sites where reactions occurs, the structural and conformational consequences of the >>>

Timescale

24 months

National and international background

In the last decade, a large body of evidence has accumulated that protein misfolding and aggregation is the most likely cause of various neurological diseases, that for this reason are often grouped together under the name of protein conformational disorders (PCDs) (Dobson, 1999; Soto, 2003). This group includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease, transmissible spongiform encephalopaties (TSEs), diabetes type II, amyotrophic lateral sclerosis, and more than 15 other less well known diseases (Martin, 1999). The hallmark feature of conformational disorders is that a particular protein folds into a different conformation, which in most cases results in its aggregation and accumulation in tissues as fibrillar deposits. These deposits have some similar morphologic, structural and staining characteristics, but different protein deposits have also distinct biochemical features, depending on their origin and their intra- or extracellular localization. In general, aggregated proteins have structures that vary from amorphous to highly ordered beta-sheet aggregates. The term amyloid, originally used to refer to the extracellular protein deposits found in AD and systemic amyloid disorders, is now generally used in a broad sense referring to protein aggregates organized in cross-beta-sheet structure, resistant to proteolytic degradation and exhibiting fibrillar appearance under electron microscopy. To date, nearly 20 such disease-causing proteins have >>>