Towards Minimally Invasive Genetically Targeted Control Of Neural Activity Using X-Rays

Authors

Kelli Cannon, University of Alabama at Birmingham

Advisory Committee Chair

Mark Bolding

Advisory Committee Members

Lynn Dobrunz

Jeffrey Anker

Lawrence Sincich

Bradley Yoder

Alecia Gross

Document Type

Dissertation

Date of Award

2023

Degree Name by School

Doctor of Philosophy (PhD) School of Optometry

Abstract

X-genetics aims to replace visible light stimulation in optogenetics with X-radiation, offering improved bone penetration, minimal scattering, and enhanced targeting capabilities. This eliminates the need for surgical optic fiber implantation, reducing invasiveness and minimizing off-target effects. Discovering a receptor protein sensitive to X-rays or their byproducts is pivotal for advancing X-genetics.Two genetically encoded X-ray receptors (GEXRs) were investigated for X-genetics application: rhodopsin, historically associated with X-ray responses, and LITE-1, an unexpected candidate. Rhodopsin, when expressed in human embryonic kidney (HEK293) cells, demonstrated robust cAMP decreases upon visible light stimulation but remained unresponsive to moderate and high-dose X-rays, suggesting it may not be suitable as a GEXR. LITE-1, initially explored for UV sensitivity, surprisingly exhibited X-ray sensitivity in wild type C. elegans, as indicated by a short latency X-ray avoidance response absent in lite-1 mutants. Ectopic LITE-1 expression in C. elegans muscle cells mediated X-ray-induced muscle contraction and paralysis responses, demonstrating the receptor’s capability to confer X-ray sensitivity to otherwise insensitive cells. To assess LITE-1's potential as an X-ray receptor in mammalian systems, lite-1::1D4 was cloned into a mammalian expression vector with which HEK293 cells were transfected. While robustLITE-1 expression localized to the plasma membrane was observed, cAMP luminescence-based GsO assays failed to detect g-protein activation in response to UV or X-ray stimulation. Immunolabeling of membrane-intact cells failed to label LITE-1, suggesting improper expression in HEK293 cells. These findings help identify GEXRs for X-genetics. Rhodopsin's inability to confer X-ray sensitivity to HEK293 cells challenges its role as an X-ray receptor. On the other hand, LITE-1 successfully imparts X-ray sensitivity in C. elegans but fails to do so in HEK293 cells, likely due to membrane trafficking issues. These results eliminate one potential GEXR while introducing a new GEXR with promise in C. elegans. Further research in mammalian neurons is necessary to fully explore LITE-1's potential for X-genetics in mammals.

Recommended Citation

Cannon, Kelli, "Towards Minimally Invasive Genetically Targeted Control Of Neural Activity Using X-Rays" (2023). All ETDs from UAB. 3484.
https://digitalcommons.library.uab.edu/etd-collection/3484