Developing a protein biotinylation system to unravel the astrocyte specific secretome

Abstract

Neurons communicate at specialist points of contact called synapses that can be excitatory or inhibitory depending on the neurotransmitter used and the effect they elicit. Correct synapse formation and maturation is essential for maintaining the excitation/inhibition balance in the brain, with aberrant synapse formation linked to a range of neurodevelopment disorders. Astrocytes are closely associated with synapses and have been shown to regulate synaptic formation and function, both through cell surface adhesion molecules and secreted proteins. However, many of these factors are yet to be identified, especially the ones involved in inhibitory synapse assembly and maturation. I aim to develop a system that will allow the host group to identify astrocyte secreted factors that could be involved in inhibitory or excitatory synapse formation. To do this, TurboID, a promiscuous biotin ligase, can be directed to the ER of primary astrocytes, to biotinylate proteins that will then be secreted into the medium. After an easy one-step purification, rather than the labor intensive chromatographic approaches used to date, biotinylated proteins in the medium can be identified by mass spectrometry. From the determined astrocyte secretome, candidate proteins can be selected to test for synaptogenic potential. I have tested two constructs expressing TurboID linked to ER retention sequences - KDEL and Sec61b. In commonly used cell lines, I validated the constructs both by immunofluorescence and Western Blot analysis. In transfected cells, I observed colocalization of both constructs with the ER resident protein calnexin. Additionally, I confirmed the presence of biotinylated proteins inside transfected cells and in their conditioned media. Moreover, in an astrocyte-like cell line, by increasing biotin incubation and chase times, I maximized the amount of biotinylated proteins that could be collected from conditioned medium. Lastly, I used lentiviral vectors expressing the same constructs (TurboID-KDEL and Sec61b-TurboID) to transduce mouse primary astrocytes. Preliminary results show that TurboID is active in these transduced cells. This work paves the way for this system to be used to obtain the secretome of mouse astrocytes, allowing the host group to choose candidate proteins and study their involvement in inhibitory and excitatory synapse formation. Keywords: astrocytes, secretome, proximity biotinylation, synapse formation, TurboID