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

Authors

• Kelli Cannon

Advisor(s)

Mark Bolding

Committee Member(s)

Lynn Dobrunz

Jeffrey Anker

Lawrence Sincich

Bradley Yoder

Alecia Gross

Document Type

Dissertation

Date of Award

2023

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.

ProQuest Publication Number

Document on ProQuest

ISBN

9798381376005

Comments

etdadmin_upload_1032112

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