BIOMOLECULAR TECHNOLOGIES IN THE STUDY OF CENTRAL NERVOUS SYSTEM DISEASES

The present study is focused on the analysis of three pathologies of the Central Nervous System (CNS): the autoimmune neurological pathology Hashimoto Encephalopathy (HE), the Experimental model of Multiple Sclerosis-Experimental Autoimmune Encephalomyletis (EAE) and Glioblastoma Multiforme (GBM). Specifically, the complexity of the immune response in the autoimmune diseases under investigation has been uncovered adopting a powerful tool of autoantibody investigation: 2-Dimensional PolyAcrylamide Gel Electrophoresis (2D-PAGE) coupled with immunoblotting, commonly known as 2D-immunomics. Regarding the identification of the target of IgG autoimmune response in HE, the binding of IgG present in serum and CerebroSpinal Fluid (CSF) from six patients with HE and 15 controls to human CNS white matter antigens has been studied by Immunomics approach and immunohistochemistry. It has been found that CSF IgG from HE patients specifically recognized 3 spots, which were identified as dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). DDAHI was present in two isoforms recognized respectively by five and four HE patients; immunohistochemistry with anti-DDAHI antiserum depicted endothelial cells in normal human CNS. AKRIAI was recognized by three HE CSF and this enzyme was widely distributed on neurons and endothelia by immunohistochemistry. IgG from HE CSF immunostained both neuronal and endothelial cells in mouse CNS. The presence of these autoantibodies selectively in the CSF of HE patients may have important diagnostic and pathogenetic implications, since the autoimmune response to these enzymes may lead to 3vascular and/or neuronal damage, two major mechanisms involved in the pathogenesis of HE. In the second study, the capacity of a single or multiple antigens to elicit autoantibodies targeting multiple neural autoantigens in EAE has been analysed by 2D- immunomics and ELISA. To gain this objective, EAE was induced with MBP peptide89– 104, total MBP or spinal cord homogenate. Both techniques showed anti-MBP IgG only after immunization with total MBP. In addition, 2D-immunomics revealed the presence in EAE mice of autoantibodies targeting other neural proteins, some displaying partial sequence homology with MBP. The present finding by 2D- immunomics of multiple neural proteins targeted by autoantibodies generated by a single antigen may help to explain the complex autoimmune response observed in multiple sclerosis. The third analysis, performed at the University of California Los Angeles (UCLA, U.S.A.) is based on the finding that human tumors are composed of a mixture of cancer, immune, stromal and vascular cells contributing to the heterogeneous DNA signatures and global transcriptional changes found in clinical samples. In this context, recent findings on GBM, one of the most common malignant and lethal primary brain tumor of adults, have shown that non-cancerous cellular components of the tumor microenvironment appear to play a critical role in its development and progression. The isolation of discrete cell subtypes in GBM microenvironment for identifying the cellular source of specific genomic alterations and examining their transcriptional and proteomic profiles is likely to be important for developing therapies that target vulnerable cellular subpopulations. DNA Encoded Antibody Library (DEAL) technology has been developed to sort specific cellular subtypes from solid tumor samples that are not amenable to fluorescene-activated cell sorting (FACS) because they are too small and/or highly necrotic. In particular, considering the importance of the tumor microenvironment in the etiology of GBM and the strong evidence of stromal cell involvement in the promotion of tumor progression, DEAL technology has started to be optimize to selectively capture tumor EGFR positive cells, CD31 positive lymphocytes and vascular endothelial cells, CD45 positive microglia from GBM biopsy samples, in order to evaluate the genomic and transcriptional profiles of DEAL sorted cellular subtypes, in the perspective of disposing of personalized cancer treatment. This GBM analysis by DEAL has been additionally coupled with the study of the anti-tumor properties of a novel mTOR kinase inhibitor, affecting the activity of the mammalian Target Of Rapamycin (mTOR), a protein frequently up-regulated in GBM. As further shown, the combination of multiple approach in GBM investigation, as performed in this analysis, could open up significant promises for the improvement of GBM therapeutic protocols.