Step‐function luminopsins for bimodal prolonged neuromodulation

Abstract

Although molecular tools for controlling neuronal activity by light have vastly expanded, there are still unmet needs which require development and refinement. For example, light delivery into the brain is still a major practical challenge that hinders potential translation of optogenetics in human patients. In addition, it would be advantageous to manipulate neuronal activity acutely and precisely as well as chronically and non‐invasively, using the same genetic construct in animal models. We have previously addressed these challenges by employing bioluminescence and have created a new line of opto‐chemogenetic probes termed luminopsins by fusing light‐sensing opsins with light‐emitting luciferases. In this report, we incorporated Chlamydomonas channelrhodopsin 2 with step‐function mutations as the opsin moiety in the new luminopsin fusion protein termed step‐function luminopsin (SFLMO). Bioluminescence‐induced photocurrent lasted longer than the bioluminescence signal due to very slow deactivation of the mutated channel. In addition, bioluminescence was able to activate most of the channels on the cell surface due to the extremely high light sensitivity of the channel. This efficient channel activation was partly mediated by radiationless bioluminescence resonance energy transfer due to the proximity of luciferase and opsin. To test the utility of SFLMOs in vivo, we transduced the substantia nigra unilaterally via a viral vector in male rats. Injection of the luciferase substrate as well as conventional photostimulation via fiber optics elicited circling behaviors. Thus, SFLMOs expand the current approaches for manipulation of neuronal activity in the brain and add more versatility and practicality to optogenetics in freely behaving animals.

The BioLuminescent‐OptoGenetic in vivo response to coelenterazine is proportional, sensitive, and specific in neocortex

“…BioLuminescent OptoGenetics (“BL‐OG”) is an emerging dual strategy that provides both optogenetic and chemogenetic capabilities. In this approach, binding of an oxidative enzyme (luciferase) to a small light‐emitting molecule (luciferin) drives bioluminescence that, in turn, regulates a neighboring opsin (Berglund, Birkner, Augustine, & Hochgeschwender, ; Berglund, Clissold, et al, ; Berglund, Fernandez, Gutekunst, Hochgeschwender, & Gross, ; Berglund, Tung, et al, ; Birkner, Berglund, Klein, Augustine, & Hochgeschwender, ; Park et al, ; Prakash, Medendorp, & Hochgeschwender, ; Tung et al, ; Tung, Shiu, Ding, & Gross, ; Zenchak et al, ). To ensure proximity of the bioluminescent reaction to the recipient opsin, the luminopsin (LMO) construct was invented, in which a luciferase is linked to the optogenetic element by a short 15 amino acids linker (Berglund, Tung, et al, ).…”

The BioLuminescent-OptoGenetic in vivo Response to Coelenterazine is Proportional, Sensitive and Specific in Neocortex

“…BioLuminescent OptoGenetics ('BL-OG') is an emerging dual strategy that provides both optogenetic and chemogenetic capabilities. In this approach, binding of an oxidative enzyme (luciferase) to a small light-emitting molecule (luciferin) drives bioluminescence that, in turn, regulates a neighboring opsin (Berglund et al, 2013;Birkner et al, 2014;Tung et al, 2015;Berglund et al, 2016b;Berglund et al, 2016a;Park et al, 2017;Prakash et al, 2018;Tung et al, 2018;Zenchak et al, 2018;Berglund et al, 2019). To ensure proximity of the bioluminescent reaction to the recipient opsin, the luminopsin (LMO) construct was invented, in which a luciferase is linked to the optogenetic element by a short 15 amino acids linker (Berglund et al, 2016a).…”

Opto‐chemogenetics with luminopsins: A novel avenue for targeted control of neuronal activity

“…The channel is tagged with enhanced yellow fluorescent protein (EYFP) at the C‐terminus for convenient identification of LMO‐expressing cells. Park et al () and Berglund et al () combined improved variants of GLuc and ChR, respectively, to generate new LMOs. The latter article further demonstrated that the chromophore, all trans ‐retinal (ATR), within ChR acts as the acceptor for BRET from GLuc.…”

“…The same molecule can be activated by external light, similar to conventional optogenetics (right). Prakash et al () and Berglund et al () capitalized on this unique property of bimodal activation in vitro and in vivo…”

“…To improve the efficacy of LMOs, Berglund, Fernandez, Gutekunst, Hochgeschwender, and Gross () took a complementary approach as a workaround for inherently dim bioluminescence. They replaced the opsin moiety with channelrhodopsin‐2 variants of superior light sensitivity, step‐function opsins (SFOs).…”