An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward P_fr → P_o transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse P_o → P_fr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm⁻¹. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.     

Optoregulated Drug Release from an Engineered Living Material: Self‐Replenishing Drug Depots for Long‐Term,Light‐Regulated Delivery

On‐demand and long‐term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self‐replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin‐free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light‐regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.     

In Vivo Electrophysiology of Peptidergic Neurons in Deep Layers of the Lumbar Spinal Cord after Optogenetic Stimulation of Hypothalamic Paraventricular Oxytocin Neurons in Rats

The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain–spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior.     

Photon Upconversion Hydrogels for 3D Optogenetics

The ability to optically induce biological responses in 3D has been dwarfed by the physical limitations of visible light penetration to trigger photochemical processes. However, many biological systems are relatively transparent to low-energy light, which does not provide sufficient energy to induce photochemistry in 3D. To overcome this challenge, hydrogels that are capable of converting red or near-IR (NIR) light into blue light within the cell-laden 3D scaffolds are developed. The upconverted light can then excite optically active proteins in cells to trigger a photochemical response. The hydrogels operate by triplet–triplet annihilation upconversion. As proof-of-principle, it is found that the hydrogels trigger an optogenetic response by red/NIR irradiation of HeLa cells that have been engineered to express the blue-light sensitive protein Cry2olig. While it is remarkable to photoinduce the clustering of Cry2olig with blanket NIR irradiation in 3D, it is also demonstrated how the hydrogels trigger clustering within a single cell with great specificity and spatiotemporal control. In principle, these hydrogels may allow for photochemical control of cell function within 3D scaffolds, which can lead to a wealth of fundamental studies and biochemical applications.     

Optical Control of Antibody Activity by Using Photocleavable Bivalent Peptide–DNA Locks

Antibody-based molecular recognition plays a central role in today's life sciences, ranging from immunoassays to molecular imaging and antibody-based therapeutics. Control over antibody activity by using external triggers such as light could further increase the specificity of antibody-based targeting. Here we present bivalent peptide–DNA ligands containing photocleavable linkers as a noncovalent approach by which to allow photoactivation of antibody activity. Light-triggered cleavage of the 3-amino-3-(2-nitrophenyl)propionic acid peptide linker converted the high-affinity bivalent peptide–DNA lock into weakly binding monovalent ligands, effectively restoring antibody targeting of cell-surface receptors. In this work, a proof of principle was provided with an anti-hemagglutinin antibody, but the molecular design of the lock is generic and applicable to any monoclonal antibody for which an epitope or mimotope of sufficient affinity is available.     

Quasi‐Continuous Wave Near‐Infrared Excitation of Upconversion Nanoparticles for Optogenetic Manipulation of C. elegans

Optogenetics is an emerging powerful tool to investigate workings of the nervous system. However, the use of low tissue penetrating visible light limits its therapeutic potential. Employing deep penetrating near‐infrared (NIR) light for optogenetics would be beneficial but it cannot be used directly. This issue can be tackled with upconversion nanoparticles (UCNs) acting as nanotransducers emitting at shorter wavelengths extending to the UV range upon NIR light excitation. Although attractive, implementation of such NIR‐optogenetics is hindered by the low UCN emission intensity that necessitates high NIR excitation intensities, resulting in overheating issues. A novel quasi‐continuous wave (quasi‐CW) excitation approach is developed that significantly enhances multiphoton emissions from UCNs, and for the first time NIR light‐triggered optogenetic manipulations are implemented in vitro and in C. elegans. The approach developed here enables the activation of channelrhodopsin‐2 with a significantly lower excitation power and UCN concentration along with negligible phototoxicity as seen with CW excitation, paving the way for therapeutic optogenetics.     

Reversible Photocontrol of Dopaminergic Transmission in Wild-Type Animals

Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions, and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D₁-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes.     

Protective Effect of Memantine on Bergmann Glia and Purkinje Cells Morphology in Optogenetic Model of Neurodegeneration in Mice

Spinocerebellar ataxias are a family of fatal inherited diseases affecting the brain. Although specific mutated proteins are different, they may have a common pathogenetic mechanism, such as insufficient glutamate clearance. This function fails in reactive glia, leading to excitotoxicity and overactivation of NMDA receptors. Therefore, NMDA receptor blockers could be considered for the management of excitotoxicity. One such drug, memantine, currently used for the treatment of Alzheimer’s disease, could potentially be used for the treatment of other forms of neurodegeneration, for example, spinocerebellar ataxias (SCA). We previously demonstrated close parallels between optogenetically induced cerebellar degeneration and SCA1. Here we induced reactive transformation of cerebellar Bergmann glia (BG) using this novel optogenetic approach and tested whether memantine could counteract changes in BG and Purkinje cell (PC) morphology and expression of the main glial glutamate transporter—excitatory amino acid transporter 1 (EAAT1). Reactive BG induced by chronic optogenetic stimulation presented increased GFAP immunoreactivity, increased thickness and decreased length of its processes. Oral memantine (~90 mg/kg/day for 4 days) prevented thickening of the processes (1.57 to 1.81 vs. 1.62 μm) and strongly antagonized light-induced reduction in their average length (186.0 to 150.8 vs. 171.9 μm). Memantine also prevented the loss of the key glial glutamate transporter EAAT1 on BG. Finally, memantine reduced the loss of PC (4.2 ± 0.2 to 3.2 ± 0.2 vs. 4.1 ± 0.3 cells per 100 μm of the PC layer). These results identify memantine as potential neuroprotective therapeutics for cerebellar ataxias.