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Mass spectrometry has become a prominent technique in biological research for the identification, characterization, and quantification of proteins. In WP1 mass spectrometry methods will be implemented to identify and quantify peptides derived from P. g in different tissue samples (blood, brain, gingivia, cerebral fluid).
The pgQC-inhibitors will be characterized in vitro and selected compounds will be tested in vivo using mouse model. The effect of pgQC-inhibitors treatment will be evaluated in behavioral studies.
WP 2 will be executed by the Molecular Biotechnology group headed by Holger Cynis at the Fraunhofer Institute for Cell Therapy and Immunology (IZI-MWT) in Halle, Germany. The group is responsible for general preclinical testing of pgQC inhibitors in vitro and in vivo P.g.
Detection of the bacteria within the CNS using currently available culture- or PCR-based methods requires highly invasive and dangerous procedure, brain biopsy. Therefore, a set of chemical tools for identification, visualization and quantification of gingipains will be developed by Partner 3. First, a highly specific and ultrasensitive diagnostic probe based in the DIANA technology to detect gingipains will be constructed. Secondly, specific polymer conjugates decorated by gingipain inhibitors enabling imaging and potentially treatment of P.g-induced inflammation will be developed. These tools will be designed based on the structures of known specific inhibitors of gingipain.
To induce periodontitis in mice the P.g infection oral gavage model will be used. The alveolar bone loss in response to P.g infection will be determined using a microcomputed tomography (µCT) scanner. The dissemination of P. gingivalis to CNS will be analyzed in post-in vivo obtained brain tissues using qRT-PCR method. Additional analysis will also be performed to show the presence of anionic LPS derived from P. gingivalis (A-LPS) and gingipain antigen in brain structures using the methods of immunohistochemistry, immunofluorescence and confocal microscopy. The effect of P.g infection on neurodegenerative changes will be determined based on phenotypic characterization of glial cell (microglia and astrocytes) and accumulation of β-amyloid deposits.
Capacity of pgQC-inhibitors to prevent decrease of the cognitive function in mice infected with P.g will be tested in WP5. Additionally, state-of-the-art Hyperion Imaging System will be used to set up a novel approach to analyze brain tissue. It combines a precisely directed laser to collect biological samples stained with metal-tagged antibodies and analyze them using CyTOF technology allowing for simultaneous interrogation a wide panel of protein markers. The mice brain tissues will be analyzed at sub-cellular resolution while preserving the information on tissue architecture and cellular morphology to uncover new biomarker correlations and cell interactions. Acquired knowledge will help to construct a novel model of the etiopathogenesis of AD based on the effect of P.g infection and might open an opportunity to develop new biomarkers and diagnostic methods.