BILIVERDIN REDUCTASE A DEFICIENCY ENHANCES THE PERFORMANCE OF BPHP-BASED OPTOGENETIC TOOLS AND IMAGING REPORTERS IN PRIMARY NEURONS AND<EM> IN VIVO</EM>

Near-infrared (NIR) optogenetic tools and fluorescent proteins engineered from bacterial phytochrome photoreceptors (BphPs) enable deeper tissue modulation than visible-light systems. Yet these proteins critically depend on the availability of endogenous chromophore biliverdin IXa, which limits the formation of their functional holoforms and defines their performance in vivo. We demonstrated improved activation of the BphP-based optogenetic system iLight in biliverdin reductase A knock-out (Blvra^−/−) primary neurons and in vivo.
Optogenetic system performance was quantified by measuring iLight-induced transcriptional activation of a NIR luciferase reporter (AkaLuc) and was defined as the fold increase in bioluminescence signal in illuminated samples relative to dark controls.
Primary Blvra^−/− neurons showed prominent enhancement of iLight optogenetic performance, with light-to-dark luciferase signal ratios increasing from ~4.5-fold in wild-type neurons to nearly 100-fold. Exogenous biliverdin improved iLight activation in wild-type neurons, but had no effect in Blvra^−/− neurons, indicating that endogenous biliverdin levels in Blvra-deficient neurons are sufficient for ‘saturation’ of optogenetic system.
To assess the impact of biliverdin availability on near-infrared imaging reporters, we examined the fluorescence of the BphP-derived protein miRFP720. Following AAV-mediated expression and quantitative measurements, Blvra^−/− neurons exhibited approximately a 2-fold increase in miRFP720 fluorescence intensity compared with wild-type cells. Such enhancement was also observed in non-neuronal cells, indicating that Blvra deletion improves chromophore availability and brightness of BphP-derived reporters. Our findings demonstrate that biliverdin reductase deficiency substantially boosts the performance of BphP-based optogenetic and reporter systems in the brain, improving their suitability for deep-tissue and in vivo manipulations.