Inflammation has both beneficial and harmful effects in chronic neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson'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 brain'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, single-reagent 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? 'AptaFluor' will be demonstrated in BellBrook'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 BellBrook'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.
Harmful brain inflammation is a hallmark of neurodegenerative diseases such as Alzehimer'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.
