Shoko Hososhima

Structural basis for Na + transport mechanism by a light-driven Na + pump

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na+ pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na+ transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na+ transport. Together with the structure-based engineering of the first light-driven K+ pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Near-infrared (NIR) up-conversion optogenetics.

Non-invasive remote control technologies designed to manipulate neural functions have been long-awaited for the comprehensive and quantitative understanding of neuronal network in the brain as well as for the therapy of neurological disorders. Recently, it has become possible for the neuronal activity to be optically manipulated using biological photo-reactive molecules such as channelrhodopsin (ChR)-2. However, ChR2 and its relatives are mostly reactive to visible light, which does not effectively penetrate through biological tissues. In contrast, near-infrared (NIR) light (650–1450 nm) penetrates deep into the tissues because biological systems are almost transparent to light within this so-called ‘imaging window’. Here we used lanthanide nanoparticles (LNPs), composed of rare-earth elements, as luminous bodies to activate ChRs since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Here, we created a new type of optogenetic system which consists of the donor LNPs and the acceptor ChRs. The NIR laser irradiation emitted visible light from LNPs, then induced the photo-reactive responses in the near-by cells that expressed ChRs. However, there remains room for large improvements in the energy efficiency of the LNP-ChR system.

Kinetic Evaluation of Photosensitivity in Bi-Stable Variants of Chimeric Channelrhodopsins

Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0–0.12 mWmm−2 of blue LED light or to 0–0.33 mWmm−2 of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.

Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca2+ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8–14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca2+-sensor, the Ca2+ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca2+ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca2+. It is suggested that the photo-activation of ChRFR facilitated the release of Ca2+ from intracellular Ca2+ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.

Near-infrared (NIR) optogenetics using up-conversion system

Non-invasive remote control technologies designed to manipulate neural functions for a comprehensive and quantitative understanding of the neuronal network in the brain as well as for the therapy of neurological disorders have long been awaited. Recently, it has become possible to optically manipulate the neuronal activity using biological photo-reactive molecules such as channelrhodopsin-2 (ChR2). However, ChR2 and its relatives are mostly reactive to visible light which does not effectively penetrate through biological tissues. In contrast, near-infrared (NIR) light penetrates deep into the tissues because biological systems are almost transparent to light within this so-called ‘imaging window’. Here we used lanthanide nanoparticles (LNPs), which are composed of rare-earth elements, as luminous bodies to activate channelrhodopsins (ChRs) since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Neuron-glioma-hybrid ND-7/23 cells were cultured with LNP(NaYF4:Sc/Yb/Er) particles (peak emission, 543 nm) and transfected to express C1V1 (peak absorbance, 539 nm), a chimera of ChR1 and VChR1. The photocurrents were generated in response to NIR laser light (976 nm) to a level comparable to that evoked by a filtered Hg lamp (530-550 nm). NIR light pulses also evoked action potentials in the cultured neurons that expressed C1V1. It is suggested that the green luminescent light emitted from LNPs effectively activated C1V1 to generate the photocurrent. With the optimization of LNPs, acceptor photo-reactive biomolecules and optics, this system could be applied to non-invasively actuate neurons deep in the brain.

General Description: Future Prospects of Optogenetics

Recent optical neuro-research methods have several advantages over conventional techniques: high resolution in space and time, together with direct measurement and manipulation of cell functions by light. The use of fluorescent proteins, bioluminescence systems, and light-sensitive proteins facilitates the optical methods in combination with genetic engineering techniques. Techniques involving the application of light-sensitive proteins are collectively termed ‘optogenetics’ because they combine optics and genetics. Light-sensitive proteins, either natural or synthetic, are termed ‘optogenetic molecular reagents’ (OMRs) or ‘optogenetic molecular tools’ if they are used as tools for optogenetics. These techniques are receiving recognition as enabling breakthroughs in neuroscience research. In this chapter, the topics and the prospects of optogenetics are discussed from three view points: the optimization of OMRs, the optimization of gene-targeting methods, and the optimization of optics.

Genetic manipulation of molecular organization and function of the calyx-type presynaptic terminal in the chick ciliary ganglion

unknown. Here, we investigated the alternative splice choice of NRXs at site 4, which regulates interaction of NRXs with NLs and other ligands. We found that depolarization of cultured neurons rapidly and reversibly altered alternative exon inclusion at site 4. This process required calcium influx via L-type voltage gated calcium cannels (L-VGCC) and calmodulin-dependent protein kinase (CaMK) activity. We identified a KH-domain RNA-binding protein that regulates exon skipping at site 4 downstream of CaMK. In splice reporter assays, this splicing factor was sufficient to drive exclusion of exon20. By contrast, knockout mice for the splicing factor exhibit loss of NRX variants lacking exon 20 at site 4, synaptic alterations and behavioral phenotypes. Thus, the calcium-dependent regulation through L-VGCC and CaMK for alternative splicing of NRXs may contribute to the specific formation of neural circuits via NRX-NL signaling during development and synaptic remodeling in response to neuronal activity in the mature nervous system.