Engineering new optogenetic proteins and biosensors: Tools to study glial signaling

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It is increasingly appreciated that glia, the non-neuronal cells in the brain, dynamically regulate the way individual synapses and neuronal networks function. Glia perform dichotomous roles in regulating synapse formation and elimination, synaptic plasticity, and neuronal death and dysfunction. Of central nervous system glia, microglia and astrocytes synthesize and release a variety of signaling molecules that dynamically regulate synaptic transmission and neural circuit dynamics, including pro- and anti-inflammatory cytokines, chemokines and neurotrophic factors. While our knowledge surrounding the importance of these signaling molecules in regulating brain development, synaptic plasticity and neuronal dysfunction has expanded since the advent of genome editing technologies, there remains a void in correlating spatiotemporal information on glial signaling with neuronal functional dynamics. This is mainly due to limitations in measuring local concentrations and temporal dynamics of individual signaling molecules and manipulation of heterogeneous glial populations in specific ways in the intact brain. Optogenetics, the genetic encoding of optically-controlled proteins and biosensors, has had a tremendous impact on the study of neuronal signaling dynamics in the intact brain. The combination of genetically encoded calcium indicators and voltage sensors with optically-controlled ion channels and pumps now facilitates direct measurements of neuronal activity with the ability to excite or silence individual or populations of neurons, shedding unparalleled light on brain organization and the encoding of diverse cognitive, locomoter and sensory functions. While these state-of-the-art tools have significantly impacted our understanding of neuronal signaling, complementary light-controlled proteins and biosensors are currently lacking for the study of glia. By expanding the experimental toolbox with novel glia-centric optogenetic tools, we will considerably increase our understanding of glial signaling dynamics and the impact on neuronal signaling and circuit dynamics. This Discovery Grant application aims to meet these needs by focusing on three central axes. We specifically aim to 1) create new genetically-encoded photoreleasable signaling molecules 2) engineer genetically-encoded biosensors for glial derived signaling molecules and 3) engineer new optogenetic proteins to manipulate glial phenotypes and effector functions. This proposal specifically focuses on the inflammatory cytokine interleukin-1 beta (IL-1b) due to its central role in glial signaling. Utilizing our new photocleavable protein technology we will generate an optically-controlled protease system to process and release mature IL-1b, utilize FRET-based technology and structural information for the IL-1b receptor complex to create a new IL-1b biosensor and engineer a novel optically-controlled NFkB transcription factor system to regulate gene expression downstream of IL-1b signaling.