Laboratory techniques for the analysis of synaptic ADAM10 expression in Alzheimer's Disease

• Grant number: 13/19521-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: May 01, 2014
• End date: July 31, 2014
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Márcia Regina Cominetti
• Grantee: Patricia Regina Manzine Moralles
• Supervisor: Monica M. G. DiLuca
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Institution abroad: Università degli Studi di Milano, Italy
• Associated to the scholarship: 12/08654-7 - Blood biomarkers for Alzheimer's Disease: evaluation of ADAM10 gene expression and micro-RNAs, BP.DR

Abstract

The amyloid precursor protein (APP) has been the focus of intense research in recent years because of its association with the Alzheimer´s disease (AD) pathogenesis. In non-amyloidogenic pathway, APP is cleaved by A Disintegrin And Metallopeptidase 10 (ADAM10), an ±-secretase, between lysine16 and leucine17 in the middle of ß-amyloid (Aß) region, thus releasing sAPP± - an structure with neurotropic and neuroprotective functions - retaining C83 residue in the membrane. The following cleavage of C83 by ³-secretase releases the p3 - which is supposed to be beneficial, and is not found in amyloid plaques - and starts at position A²17 (A²17-40 and A²17-42), thereby inhibiting amyloidogenic Aß production. The Laboratory of Pharmacology of Neurodegeneration (LPN) from the Department of Pharmacological Sciences - UNIVERSITÀ DEGLI STUDI DI MILANO (UNIMI) operates in the investigation of the expression of synaptic proteins in neurons, including ADAM10, and the mechanisms related to the modulation of its expression from other carrier proteins. Two lines of thought drive the studies on the ADAM10 expression in neurons; both related to the intraneuronal trafficking of this protein. The first line is related to the study of a Synapse-associated protein 97 (SAP97), which in association with ADAM10 favors its cytoplasmic trafficking toward the membrane, thus improving APP proteolytic capacity, as ±-secretase. The second line is focused on aspects of ADAM10 membrane endocytosis held by the clathrin adaptor protein (AP2). This combination promotes the internalization of ADAM10, and therefore influences its expression and activity as ±-secretase. For the development of these studies, laboratory techniques for protein analysis such as: western blotting, immunoprecipitation, bi-dimensional gel electrophoresis, etc.; in vitro and in vivo techniques for protein phosphorylation analysis; subcellular fractionation; cell and slice cultures; transfection; confocal laser scan imaging; molecular biology: PCR, RT-qPCR, cloning; treating neuronal culture, co-immunoprecipitation, co-location, action potentiation/ long-term depression (LTP and LTD respectively) in neuronal plasticity and genetic sequencing of the cytoplasmic tail of ADAM10 (ADAM10 Ct) were conducted. Since UNIMI stands out as a center of excellence in the study of platelet and neuronal ADAM10, this stage will represent an important collaboration to establish a new research area in our laboratory (LABEN), directed to the study of neuronal ADAM10 using some of the techniques that will be the aim of this stage. The main aim is to incorporate theoretical and practical knowledge about laboratory techniques performed by LPN-UNIMI for the study of ADAM10 neuronal expression in order to standardize and to implement such techniques in LABEN. Contact with totally new laboratory techniques will allow further improvement in the forms of analysis and evaluation of biological materials, especially peripheral blood and neurons. This stage will provide the frame for development of future studies in our research group, which may use new laboratory methodologies in order to deepen and assess other biological materials from control subjects, mild cognitive impairment (MCI), and AD patients. It is expected that the findings obtained until date, regarding the ADAM10 gene expression in AD elderly, related to a doctoral project (Process No. 2012/08654-7) could be compared with researches that are under development by LPN-UNIMI, allowing the establishment of a collaborative study. During the three-month period, it is intended to follow and learn laboratory techniques for the evaluation of ADAM10 neuronal expression: genetic sequencing of different ADAM10 domains, neuronal culture treatment (LTP and LTD), crosslinking assays, surface biotinylations, internalization assays, co-location assays, co-immunoprecipitation assays. (AU)