Development of an optogenetic TrkB.T1 probe.

Abstract

Astrocytes are the most abundant cell type in the human brain and are involved in regulating synaptic transmission and synapse formation. Astrocytes have a unique star-like morphology containing multi-branched processes that reach sub-diffraction limited scales. Fine processes protruding from astrocytes are known to interact directly with synapses to form the canonical tripartite synapse. These Perisynaptic Astrocytic Processes (PAPs) can release and uptake various neuroactive molecules to regulate synaptic function. Astrocyte morphology and PAP interactions are dynamic and vary throughout different brain regions and in diseased states, suggesting that astrocyte branching dynamics are important for regulating brain function. Despite this, no tools have been developed to directly probe astrocyte morphology for in vivo studies. Previously, signaling via a truncated receptor tyrosine kinase, TrkB.T1, has been found to regulate astrocyte morphology. TrkB.T1 is a transmembrane receptor highly expressed in astrocytes. Binding of Brain Derived Nerve Growth Factor (BDNF) to the extracellular receptor of TrkB.T1 results in the dimerization of its 23 amino acid intracellular domain. This dimerization activates Phospholipase C (PLC) calcium signaling and inhibits RhoGTPases via the release of the RhoGTPase inhibitor RhoGDI1 into the cytosol. Application of BDNF to astrocytes in culture and brain slices has been shown to increased astrocyte branching and promote synapse formation. In this work, we develop Cry2 and iLID based optogenetic TrkB.T1 probes (OptoT1s). OptoT1s allows for light induced dimerization of the intracellular TrkB.T1 domain at the cell membrane, which should subsequently activate downstream TrkB.T1 signaling pathways. We are currently characterizing the ability of these OptoT1 probes to induce PLC-dependent calcium transients and RhoGTPase inhibition. The development and optimization of this optogenetic tool will allow for in vivo spatiotemporal control of astrocyte branching that can help us investigate the role of astrocyte morphology on brain function and diseases.