Fluorescent biosensors for dynamic monitoring of paracrine signaling in extracellular matrix

DESCRIPTION provided by applicant Inflammation has both beneficial and harmful effects in chronic neurodegenerative diseases such as Alzheimerandapos s disease AD and Parkinsonandapos s disease PD and paracrine signaling is central to the inflammatory response Rapid advances in in vitro models that incorporate different neural cell types in the context of the brainandapos s uniqu extracellular matrix ECM are helping to define the paracrine signaling pathways that contribute to neuroinflammatory disease pathologies However to be useful for drug discovery these models must be amenable to analysis on automated high throughput screening HTS platforms Unfortunately dynamic non invasive detection of soluble molecules in ECM is not feasible with the current immunodetection methods used for automated high content analysis HCA which typically involve multiple components and wash steps To overcome this technical gap we propose to develop fluorescent andquot single reagentandquot paracrine biosensors based on aptamers that transduce signals intramolecularly to quantum dots Unlike antibodies aptamers can be readily engineered to incorporate ligand dependent structural switches that can be harnessed for Forster resonance energy transfer FRET signals And quantum dots have distinct advantages over organic fluors including greater brightness resistance to photobleaching and multiplexing capability As a first step we are proposing to develop a fluorescent biosensor using an existing aptamer to TNF a key inflammatory signal molecule Proof of concept for the TNF andapos AptaFluorandapos will be demonstrated in BellBrookandapos s iuvo r Microchannel Array Plates an alternative to multiwell plates which enable dynamic imaging of ECM biology on automated HCA platforms Phase I studies will lay the technical foundation for development of AptaFluor biosensors for several inflammatory cytokines and their incorporation into mutlitplex assays in Phase II AptaFluors in combination with BellBrookandapos s iuvo microchannel plates would be an extremely powerful platform for probing cell cell signaling in ECM as it would allow non invasive spatially resolved detection of soluble signaling molecules in real time using existing high content analysis platforms PUBLIC HEALTH RELEVANCE Harmful brain inflammation is a hallmark of neurodegenerative diseases such as Alzehimerandapos s and Parkinsons diseases and inflammation is controlled largely by signals sent from one cell type to another To accelerate discovery of more effective therapies for these disabling diseases we are proposing to develop biosensors for detecting the inflammatory signals in the context of the gelatinous matrix that supports cells in the brain