Rational design of optogenetic tools: from bioinformatic genomic data analysis to electrophysiological validation

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Rational design of optogenetic tools: from bioinformatic genomic data analysis to electrophysiological validation

Von der Fakultät für Georessourcen und Materialtechnik der Rheinisсh -Westfälischen Technischen Hochschule Aachen

zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigte Dissertation

vorgelegt von

M. Sc. Alexey Alekseev

aus Gornyak, Russland

Berichter: Herr Univ.-Prof. Dr. Valentin Gordeliy Herr Prof. Dr. Georg Büldt

Herr Prof. Dr. Ernst Bamberg Herr Prof. Dr. Martin Engelhard

Tag der mündlichen Prüfung: 07.12.2020

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar

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Abstract

Microbial rhodopsins comprise one of the most diverse clades of light-harvesting proteins. At the beginning of the 21^st century, they found exceptional application in neuroscience with the potential for the invention of medical therapies of the new generation. Currently, a variety of channelrhodopsins were functionally described. However, the search for optogenetic tools with desired properties is still a challenge.

In the thesis, we describe a pipeline for the identification of microbial rhodopsins with potentially new properties and their structural and functional characterization. The pipeline is based on the method of bioinformatics, taking into account current knowledge of microbial rhodopsins and known x-ray structures, and patch-clamp recordings in mammalian cells and cultured neurons.

First, we identified several different clades of rhodopsins type-1, which might have unique functional properties for optogenetics. Second, we conducted a detailed structure-based bioinformatic analysis of heliorhodopsins and showed that they form a heterogeneous class of proteins. We identified several groups of heliorhodopsins that may possess different functions.

Third, we examined the electrophysiology of VirChR1, a member of viral rhodopsins group 1.

VirChR1 turned out to be ion-channel selective for monovalent cations. Besides, we showed that it could fire cultured rat hippocampal neurons. We showed that the VirChR1 is impermeable to Ca²⁺, which makes this protein a potentially perspective tool for optogenetics.

Taking into account all data, we conclude that viral rhodopsins and heliorhodopsins are widely distributed and also suggest their involvement in the global ecology.

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Abstrakt

Mikrobielle Rhodopsine umfassen eine der verschiedensten Kladen von Lichtsammelproteinen. Zu Beginn des 21. Jahrhunderts fanden sie eine außergewöhnliche Anwendung in den Neurowissenschaften mit dem Potenzial zur Erfindung medizinischer Therapien der neuen Generation. Derzeit wurde eine Vielzahl von Channelrhodopsinen funktionell beschrieben. Die Suche nach optogenetischen Werkzeugen mit gewünschten Eigenschaften ist jedoch immer noch eine Herausforderung.

In der Arbeit beschreiben wir eine Pipeline zur Identifizierung von mikrobiellen Rhodopsinen mit potenziell neuen Eigenschaften und deren struktureller und funktioneller Charakterisierung. Die Pipeline basiert auf der Methode der Bioinformatik unter Berücksichtigung des aktuellen Wissens über mikrobielle Rhodopsine und bekannte Röntgenstrukturen sowie Patch-Clamp-Aufzeichnungen in Säugetierzellen und kultivierten Neuronen.

Zunächst identifizierten wir verschiedene Rhodopsinkladen vom Typ 1, die möglicherweise einzigartige funktionelle Eigenschaften für die Optogenetik aufweisen. Zweitens führten wir eine detaillierte strukturbasierte bioinformatische Analyse von Heliorhodopsinen durch und zeigten, dass sie eine heterogene Klasse von Proteinen bilden. Wir haben mehrere Gruppen von Heliorhodopsinen identifiziert, die unterschiedliche Funktionen besitzen können. Drittens untersuchten wir die Elektrophysiologie von VirChR1, einem Mitglied der viralen Rhodopsin- Gruppe 1. VirChR1 erwies sich als ionenkanalselektiv für einwertige Kationen. Außerdem haben wir gezeigt, dass es kultivierte Hippocampus-Neuronen von Ratten abfeuern kann. Wir haben gezeigt, dass VirChR1 für Ca2 + undurchlässig ist, was dieses Protein zu einem potenziell perspektivischen Werkzeug für die Optogenetik macht.

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Unter Berücksichtigung aller Daten schließen wir, dass virale Rhodopsine und Heliorhodopsine weit verbreitet sind, und legen auch nahe, dass sie an der globalen Ökologie beteiligt sind.

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List of abbreviations

CrChR2 channelrhodopsin-2 from Chlamydomonas reinhardtii CrChR1 channelrhodopsin-1 from Chlamydomonas reinhardtii

C1C2 chimera of CrChR1 (N-terminus and 1-5 α-helices) and CrChR2 (6-7 α-helices and C-terminus) from Chlamydomonas reinhardtii

ECS extracellular constriction site ICS intracellular constriction site CCS central constriction site

HsBR bacteriorhodopsin from Halobium salinarum NpHR halorhodopsin from Natronomonas pharaonic

eNpHR3.0 halorhodopsin from Natronomonas pharaonic optimized for optogenetics ACR anion-conducting channelrhodopsin

GtACR1 anion-conducting channelrhodopsin-1 from Guillardia theta GtACR2 anion-conducting channelrhodopsin-2 from Guillardia theta NsXeR xenorhodopsin from Nanosalina

HeR heliorhodopsin SBC Schiff base cavity XeR xenorhodopsin ChR channelrhodopsin

pR proteorhodopsin

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BR bacteriorhodopsin

HR halorhodopsin

XR xanthorhodopsin

SR sensory rhodopsin

NaR sodium pumping rhodopsin of bacteria ClR chloride pumping rhodopsin of bacteria SzR schizorhodopsin

SFO, SSFO step-function opsin, stabilized step-function opsin

GECI genetically encoded calcium indicators GEVI genetically encoded voltage indicators DDM n-Dodecyl-β-D-Maltopyranoside

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1. Introduction

One of the greatest challenges is to understand how neural processes inside brain control animal behavior. Humans were trying to understand brain functions during all their history. Some new technologies like electroencephalography made a significant contribution to this field.

Nevertheless, until the 21^st century, all methods were able to monitor processes mainly on the macroscopic scale.

Optogenetics is a modern technology that made a revolution in neuroscience. In 2003 group of scientists led by Ernst Bamberg presented channelrhodopsin-2 to the scientific world and suggested that it can be a highly anticipated tool to study the brain [1]. In 2005 the suggestion was proved, and it was shown that channelrhodopsin-2 could be used to initiate an electrical signal in neurons [2]. Channelrhodopsin-2, a light-gated cation channel, can effectively depolarize neural membrane inducing action potentials. This great discovery offered principally new great opportunities for the investigation of the brain on cellular spatial resolution.

Since then, new optogenetic tools were discovered or engineered. Some of them are capable of inducing or suppressing the electrical activity of neurons on a millisecond timescale; others allow researchers to observe the activity of the whole-brain.

Currently, the most useful and powerful optogenetic tools are still microbial rhodopsins and channelrhodopsin-2, in particular. However, rhodopsins are still not perfect tools and can not ideally simulate native processes in neural cells. This and a great diversity of rhodopsins across all domains of life make expanding and optimization of rhodopsin-based optogenetic toolbox an essential and perspective scientific task.

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In my thesis, it is described how to engineer rhodopsin constructs suitable for optogenetic application in four steps:

1. Nowadays, metagenomics is developing rapidly, and it compiles large genome datasets [3]. Tons of biomaterial are collected, sorted by size, and all its DNA is sequenced. This approach provides information on new genomes. In our work, we used genomic data downloaded from public sources, including recent findings of the Tara Oceans expedition.

2. Structure-based bioinformatics allowed us to identify the most promising rhodopsins for further experimental work. We analyzed sequences and defined amino acids, which differ from those known as key determinants of rhodopsin function. Single amino acid differences can result in a protein with a new unique function.

3. One of the most critical steps is the functional validation of genes. Since we are looking for light-gated ion transporters, we used patch-clamp as the primary approach for functional characterization.

4. X-Ray structure determination gives a chance to suggest the molecular mechanism of protein function. Using structure-guided mutagenesis, we verified the hypothesis.

In the present work, we report on three major research results.

First, we conducted a bioinformatic analysis of the whole Uniprot and GenBank databases.

This analysis helped to identify rhodopsins that are not yet characterized and can potentially be of the interest of optogenetics. Currently, several of them are described experimentally by our lab. A unique phylogenetically distinct group of anion-conducting channelrhodopsins was also found and presented in this work.

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Second, we performed a structure-based bioinformatic study on heliorhodopsins clade of rhodopsins. Recently, Pushkarev et al. [4] presented a new group of rhodopsins with topologically inverted membrane orientation, spread on all domains of life. We obtained a crystal structure of 48C12 – one representative of the group and carried out a bioinformatic description of the group.

Finally, we examined electrophysiologically VirChR1 – viral cation conducting channelrhodopsin. VirChR1 is representative of viral rhodopsins group 1 described in a bioinformatic study by a group of Eugene Koonin [5]. VirChR1 turned out to be a unique calcium-sensitive Na⁺/K⁺ conducting channelrhodopsin with a potentially remarkable optogenetic applicability.

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2. Literature review

2.1 Optogenetics

Optogenetics is a biological technology that allows controlling animal behavior and reading out the current condition of an animal brain on molecular spatial and millisecond time scale.

Nobel Prize Winner of 1962, neuroscientist Francis Crick suggested in 1999 using light to control neurons with high spatial and temporal resolution [6]. At that time, neuroscientists could only define regions of the brain or groups of neurons, which are responsible for specific actions. He pointed out the necessity to fire or suppress the action of single neurons and to do it in a fast way. Francis Crick suggested using infrared light, which deeply penetrates neural tissue.

Several remarkable attempts were made in 21^st century to make Crick’s suggestions come true.

In 2002 group of scientists presented the first genetically encoded three-component system, which allowed to control group neurons by type, but not by spatial arrangement [7]. They coexpressed ten proteins that are responsible for Drosophila photo-sensing in Xenopus oocytes.

Using patch-clamp, they detected photocurrents in oocytes with a magnitude around 1 µA.

Excluding one by one the proteins that had no significant effect on photocurrents, they came up with chARGe - the combination of three proteins (arrestin-2, rhodopsin, and the alpha subunit of cognate heterotrimeric G-protein). They were sufficient to support the light-induced effect of the same magnitude. They also showed that rat hippocampal neurons with chARGe were photoactive (Fig. 1a,b). It was the first genetically encoded tool for optical stimulation of neurons.

One year later, the same group of neuroscientists presented a new way for optical stimulation of neurons [8]. This method was based on ion channels TRPV1 and P2X2 and optical uncaging

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of their agonists (capsaicin and ATP, respectively). This approach had several advantages comparing to chARGe: expression of only one heterologous gene, neuronal response stability, possibility to depolarize neurons pharmacologically, or thermally.

Figure 1. ChARGed Hippocampal neuron is firing in response to illumination. (a) Intrinsic fluorescence of EGFP shows ChARGed hippocampal neurons. (b) The membrane potential of a neuron Periods of darkness and illumination. [7].

In the 2003 year group of Ernst Bamberg and his colleagues published work on Channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) [1]. This article indicated the beginning of the revolution in optogenetics and neuroscience. The authors showed that CrChR2 could induce large photocurrents being expressed heterologously in Xenopus oocytes or in HEK293 (stationary current was 1.5 µA and 500 pA, respectively). They showed that single gene chop2 is responsible for photocurrents and that the membrane part of the corresponding protein acts as a light-sensitive ion channel (Fig. 2a). They replaced the cytoplasmic part of protein with YFP for the detection of protein distribution in animal cells.

CrChR2 was shown to be a non-selective cation channel with extremely rapid kinetics comparing to existed optogenetic tools (Fig. 2b). The authors suggested that CrChR2 was suitable for neural membrane depolarization. It took two more years to implement the first single-component optogenetic tool [2].

In 2005 teamed up German and USA scientists showed the applicability of CrChR2 in hippocampal neurons for spike firing [2]. They reported that CrChR2 combined exceptional temporal resolution with advantages of previous optogenetic approaches. First of all, the new

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techniques allowed to elicit spike sequences that mimic natural neural activity (Fig. 2c). They generated patterns of light pulses with random temporal distribution. The experiment showed that CrChR2 fired neurons in a repeatable and reliable manner according to the light pattern.

That effect had very low temporal jitter in a single neuron from trial to trial. Also, different neurons responded in the same manner to an identical light series. Second, CrChR2 enabled control of neural spikes in a wide range of physiological frequencies of firing. Scientists tested firing ability from 5 Hz up to 30 Hz and obtained expected results. Third, they showed the applicability of CrChR2 to elicit subthreshold depolarization (for example, essential for neural plasticity investigations). Finally, the authors tested whether the presence of CrChR2 in the neural membrane causes serious side-effects. Their data suggested that CrChR2 did not significantly affect the cell health or the electrical properties of its membrane.

Genetically encoded calcium indicators (GECI) are the primary optogenetic readout technique.

Synergetic use of GECI with microbial rhodopsins allows inducing and reading specific action at the same time. GECI are developing gradually in parallel with the inventing of new optogenetic approaches. The first GECI to be used for calcium imaging in living animals was invented in 1997 [9]. However, the first successful use of GECI in mammals was in 2004 [10].

In this work, scientists reported about genetically modified mice expressing GECI in smooth muscles. That allowed researchers to monitor postsynaptic signals in the muscles.

Findings in the field of microbial rhodopsins resulted in a revolution in optogenetics giving powerful tools for activating neuronal spikes. These techniques, together with GECI, pushed forward neurosciences.

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Figure 2. CrChR2 is a light-gated cation channel capable of firing neurons. (a) Voltage-clamp recording from oocytes expressing CrChR2. (b) Current-voltage relationship of stationary photocurrents for one representative oocyte. (c) Current-voltage relationship of stationary photocurrents for one representative oocyte [1], [2].

2.2 Advances in neuroscience due to optogenetics

Although there is no doubt that optogenetics is a powerful approach, the scientific achievements with the use of optogenetics are still worth mentioning.

Since 2005, neuroscientists published a bunch of papers describing how neural circuits control the behavior of animals. Most of these discoveries would not be possible through other techniques of neural modulation. Several subjectively selected publications are mentioned in this section.

2.2.1 Optogenetic curing of temporal lobe epilepsy

In 2013 it was reported that temporal lobe epilepsy (TLE) could be cured using optogenetics [11]. TLE is the most common variant of epilepsy in adults. Memory disorders and loss of memory are common in TLE. The disease also chronically affects personality and behavior.

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For example, people with TLE often suffer from Geschwind syndrome. The syndrome is characterized by hypergraphia, hyper religiosity, hyposexuality, and circumstantiality.

Current treatments of TLE have a variety of serious adverse side-effects. That happens because these therapies have no selective effect and have low temporal precision. However, temporal lobe seizures arise in the discrete brain region and can be fastly detected when started, which makes it theoretically possible to apply the high spatial-temporal precision treatment. Scientists from the University of California managed to use closed-loop optogenetics in order to suppress ictal brain activity in mice model of TLE.

Figure 3. TLE seizure detection and suppression by halorhodopsin. (a-c) Electrographic seizures detected (vertical green bars), activating amber light (589 nm) randomly for 50% of events (light: amber line, an example in b; no-light example in c). (d) Distribution of seizure durations after detection under illumination (solid amber) and no-light conditions (hashed grey). [11]

Krook-Magnuson et al. developed a method to detect epileptic seizures and fastly respond to them by inhibiting seizures, either directly inhibiting principal neurons using halorhodopsin or

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stimulating inhibitory GABAergic neurons using channelrhodopsin-2 (Fig 3.). This study shows how optogenetics can alter medical interventions in brain diseases.

Nevertheless, presented methods are not ideal in terms of side-effects. For example, in the case of TLE, chloride concentrations inside the neuron usually alter [12]. That affects the effectiveness of GABAergic chloride channels, even making them excitatory in some cases.

Halorhodopsin therapy would exacerbate this alteration. Taking it into account, one can declare the importance of further search for optogenetic tools with different ion selectivity.

2.2.2 New discoveries in brain research

At the beginning of 21 century, several studies showed that neural plasticity in different populations of basolateral amygdala complex (BLA) neurons might mediate both positive and negative memories. However, the properties of these populations remained unknown.

In 2015 group of scientists from Cambridge optogenetically investigated two populations of neurons in basolateral amygdala complex (BLA) [13]. One population consist of neurons projecting to the nucleus accumbens (NAc), another one consist of neurons projecting to the centromedial amygdala (CeM). In the end, they found out that these two types of neurons from the same region are responsible for opposite synaptic changes following reward and fear conditioning, respectively.

They used retrogradely infectious viruses to label first NAc projectors, and then to label CeM projectors with channelrhodopsin-2 (Fig. 4). This procedure allowed them to activate separately two populations of neurons and define whether activation of specific cells impairs or enhances fear and reward conditioning. Also, they obtained the same procedure with chloride-pump halorhodopsin, which allowed them to inhibit the activity of these cells. These

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optogenetic interventions proved the hypothesis that NAc and CeM projectors are responsible for opposite behavioral reactions.

Figure 4. Optogenetic stimulation of BLA neurons projecting to NAc and CeM. After retrograde virus injection into NAc or CeM, animals were tested under positive (intracranial self-stimulation) and negative (real-time place avoidance) reinforcement conditions. [13]

Although optogenetics played an indispensable role in this survey, scientists significantly strengthened the work using retrobeads (retrogradely traveling fluorescent beads) to label BLA neurons of the two populations. It made it possible to sort cells (they used manual sorting) and investigate their transcriptional profiles. Thus they obtained levels of expression of different proteins in two populations of neurons that are located in the same place in the brain.

This remarkable study brought new knowledge about the structure of the brain. Importantly, it could not have been done using the electrical stimulation technique because it does not allow an investigator to stimulate only one type of cells in a specific region.

2.2.3 Optogenetic memory retrieval

Alzheimer’s disease is the most common variant of dementia of aged people. It has a wide range of symptoms: at the beginning difficulty in remembering recent events, as the disease advances, it affects language, mood, motivation and behavior, spatial orientation - in the later stages this leads to self-care disability. This disease has a dramatic social and significant economic effects in developed countries.

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Currently, there is no competent medical cure for Alzheimer’s disease for now. However, a prolonged survey of the scientific group from MIT led in 2016 to the first optogenetic approach for extracting memories in AD mice.

Scientists from the Tonegawa laboratory in 2012 managed to extract optogenetically fear memories in healthy mice [14]. They labeled engram cells in mice (which were active during training in fear context, where mice are electrically shocked) with channelrhodopsin-2 (Fig.

5a,b). Mice with labeled engrams froze in neutral conditions when were optically stimulated.

This experiment showed the possibility to extract memories optogenetically. More importantly, this experiment showed that activation of engram cells is sufficient to retrieve the memory, which had a tremendous impact on neuroscience.

In 2013 the same group of scientists used a similar methodology for making false fear memories in living animals [15]. They labeled mice engrams activated during neutral context A with channelrhodopsin-2. Afterward, they stimulated these engrams in distinct fear context B. That made mice brain encode fear into engrams corresponding to neutral context A as well as into engrams of context B. After this procedure mice froze in context A without any optical stimulation (in control neutral context C mice were still active). Therefore, this methodology allowed the authors to create a false memory.

In 2014 another group of scientists showed that activation of engram neurons is also necessary for memory retrieval [16]. They conducted a similar experiment except for expressing silencing archaerhodopsin-3 in engrams. The silencing of these neurons significantly reduced the recall of the fear memory. Therefore, the theorem: “Neurons of dentate gyrus activated during contextual fear conditioning are both necessary and sufficient for fear memory retrieval” was proven. Q.E.D.

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Figure 5. Optogenetic memory retrieval in AD mice. (a) Two-virus technology for activity-dependent neuron labeling. (b) DG engram labeled with CrChR2 (green). (c) Memory retrieval by optogenetic activation of DG engrams (d) EC engram labeled with ChIEF (red) and DG engram labeled with eYFP (yellow). (e) Dendrite spines in healthy mice DG. (f) Dendrite spines in AD mice DG. (g) Recovered dendrite spines in AD mice DG after optogenetic activation of long-term potentiation (h) Memory rescue in AD mice. Behavioral schedule (left) and freezing restoration Dashed line represents control mice freezing. [14], [17]

Using accumulated knowledge Tonegawa’s group reached the goal [17]. They proved the possibility to retrieve memory in healthy and AD mice optogenetically by activation of fear- engrams (Fig. 5c). Importantly, their study showed no difference in optogenetic memory recall between healthy and AD mice. That means AD impairs recall of memory rather than the encoding of memory. Moreover, researchers could fix the dendritic spines deficit in early AD mice by long-term potentiation induced by light-activation of ChIEF-labeled engrams in the entorhinal cortex (EC) (Fig. 5d,e,f,g). The spines restoration, in its turn, rescued term memory retrieval: AD mice froze in fear conditions without additional light-stimulation (Fig. 5h).

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Investigations described above are examples of significant impacts on neuroscience via optogenetic interventions. Moreover, they showed possible medical alternatives in curing neurodegenerative diseases.

2.2.4 Optogenetic sight and hearing restoration

Optogenetics can be applicable in curing hereditary and acquired diseases depriving a person of sight of hearing. According to World Health Organization (WHO), over 5% of the world’s population are people with hearing loss (~ 466 mln. ~34 mln of them are children) and 39 mln completely blind people (~246 mln people have low vision). There are plenty of diseases causing such disorders. Optogenetics may help to treat at least some of them. Moreover, it can be more effective than already existing techniques.

2.2.4.1 Hearing restoration

Nowadays, electrical cochlear implants are widely used for hearing restoration. Although they can effectively restore hearing for speech comprehension under noiseless conditions, it is not enough for listening to music and talk in a noisy environment. Spiral ganglion neurons (SGNs) in different parts of a cochlear are responsible for different sound wave frequencies. Electrical implants stimulate these spiral ganglion neurons.

A broad spread of electrical activation allows using only 10 to 20 stimulation channels along the cochlear [18] (Fig. 6a). Moreover, the loudness growth function typical for electrical stimulation has several times more narrow dynamic range than for the natural stimulation [19].

These facts lead to low frequency and intensity resolution. Optogenetic stimulation of SGNs can solve these problems (Fig. 6b). Optical stimulation can be effectively focused; thus, the sound frequency can be precisely transformed into the activation of corresponding SGNs.

Spatial precision has also impact on intensity resolution. During natural listening, loudness

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increase leads to activation of neighboring SNGs in addition to frequency-defined ones.

Besides that, intensity increase leads to activation of SNGs with high spontaneous firing rates, which can also be mimicked by different channelrhodopsin expression levels in different SGNs.

Figure 6. Schematic overview of сochlear operating principles. (a) A broad spread of activation by

electrical cochlear implants. (b) Optogenetic activation of SGNs promises a significant increase in frequency resolution.[18]

The optogenetic approach can also be used in patients with the injured auditory nerves. CrChR2 was reported to be used for auditory brainstem implants as well as for cochlear implants [20].

However, CrChR2 is not an optimal optogenetic tool for hearing restoration. SGNs are among the fastest neurons in mammals, which can fire with up to 1000 Hz rate. Therefore, rhodopsins with fast closing kinetics are needed. Chronos is a fast-closing blue-light absorbing naturally existing channelrhodopsin [21]. It was reported to elicit auditory brainstem responses even at 1000 Hz of stimulation frequency [22]. Besides, a red-shifted ultrafast channelrhodopsin variant was obtained by rational optimization and shown to be capable of restoring hearing in deaf mice [23].

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Microbial opsins can also be used for vision restoration. It is especially convenient due to the presence of human rhodopsin in the eye. There are several possible optogenetic strategies of sight restoration, depending on the severity of the disease.

In brief, photoreception in the eye is arranged as follows. There are several different types of cells present in the retina: cones, rods, on and off bipolar neurons, ganglion neurons, amacrine, and horizontal cells (Fig. 7a). All cells communicate with each other through neurotransmitters, which are released in response to changes in membrane potential. Thus, the illumination of the retina in a healthy state results in depolarization and hyperpolarization of specific types of cells.

In particular, rods and cones, as well as off-bipolar cells and off-ganglion neurons, are hyperpolarized in response to light. On the contrary, on-bipolar neurons and on-ganglion cells are hyperpolarized under the illumination of the retina. However, only cones and rods are light- sensitive: they absorb light and transmit the signal to bipolar cells, which in turn transmit the signal to ganglion neurons. Axons of ganglion cells assemble into the optic nerve that transmits visual information to the brain. Horizontal cells and amacrine cells modulate signal transmission from photosensitive cells to bipolar cells and from bipolar cells to ganglion neurons, respectively.

In 2004 the Zhuo-Hua Pan laboratory submitted a manuscript, one of the first works, concerning optogenetic eye treatment in mice. Work was published in 2 years [24].

Optogenetic tools can be expressed in the retina in different ways corresponding to the state of the disease [25]. For instance, if only cones outer photosensitive segments degenerated, then optogenetic inhibitors can be expressed in cones for sight restoration [26], [27] (Fig. 7b). If cones fully degenerated, then optogenetic activators can be expressed in on-bipolar cells, and the inhibitors in off-bipolar cells or optogenetic activators can be expressed in amacrine cells

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(Fig. 7c). If the layer of the bipolar cells also degenerates, then optogenetic activation/suppression of on/off ganglion cells can help [28] (Fig. 7d).

Figure 7. Stages of retinal degradation and optogenetic therapy for blindness. Blue/red colors show expression of CrChR2/NpHR in certain types of cells, respectively, as a therapy. (a) A healthy retina consists of cones and rods, on/off bipolar cells, and ganglion neurons. (b) Rods degenerated, cones outer segments lost: restoration of cones. (c) Cones degenerated: optogenetic intervention into bipolar cells of amacrine cells. (d) Bipolar cells degenerated: optogenetic intervention into ganglion cells. [29]

However, an efficient application of these approaches requires future improvements [29].

Optogenetic tools respond to a much narrower intensity range of light than healthy cones and rodes. Although it can be solved by using special goggles that adjust light intensity, it is preferable to have more sensitive optogenetic tools. Besides that, all approaches described above can not restore color vision: one has to express several rhodopsins with different spectra at a time. Finally, retina cells membrane potentials are finely modulated in a healthy state,

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which can hardly be controlled using only one optogenetic inhibitor/actuator at a time. At least some of the described problems can be solved by the rational design of optogenetic tools.

2.2.5 Deep brain stimulation therapy using microbial rhodopsins

Deep brain stimulation (DBS) is a neurosurgical procedure. An electrical neurostimulator is implanted into the patient brain. It sends electrical impulses to defined brain regions and thereby intervene in neural work. Nowadays, DBS is one of the primary methods used to treat neuronal disorders such as Parkinson’s disease (PD), dystonia, obsessive-compulsive disorder, epilepsy.

Despite the great importance and widespreadness, the exact mechanism underlying DBS is still not known. For instance, DBS can treat many symptoms of PD, including tremor, rigidity, and bradykinesia - but not others, such as speech impairment, depression, and dementia. It is evident that conventional DBS methodology lacks cell-type specificity, spatial resolution, and is invasive. In 2009 researchers made progress in the understanding of DBS treatment of PD using optogenetics [30]. They stimulated and inhibited distinct neural circuits in parkinsonian rodents. Thus the authors showed that neither inhibition with eNpHR nor excitation with CrChR2 of subthalamic nucleus (STN, the brain region linked to movement impairments in PD patients) neurons did not affect rodents’ behavior. Therefore the scientists tested if effects of DBS arise from activation of axonal projections that enter the STN. Such activation led to the complete restoration of the normal healthy behavior of the parkinsonian rodents. Thus, optogenetics has contributed to the understanding of DBS and PD symptoms.

In 2018 group of scientists invented a non-invasive near-infrared optogenetic solution for DBS [31]. They expressed channelrhodopsin-2 in mice neurons of interest and stimulated them using 980 nm laser. This highly red-shifted light penetrated through the brain well enough without neurosurgical implanting of fiber-optics. However, the wavelength was inappropriate for the

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activation of channelrhodopsin-2. To deal with the problem, scientists used upconversion nanoparticles (UCNPs). These particles can absorb near-infrared light (980 nm) and emit blue light instead (450 and 475 nm). This approach allowed scientists to introduce DBS without neurosurgical interventions.

Nevertheless, this methodology still needs to deliver not natural compounds UCNPs inside the brain. Engineering of rhodopsin channels with a highly red-shifted spectrum and sufficient quantum efficiency would allow making DBS without any additional particles. Rational mutagenesis of rhodopsins and bioinformatic approaches have already optimized optogenetic working spectral range. However, further investigations are necessary to provide optogenetic tools absorbing in far-infrared.

2.3 Optogenetic toolbox: microbial rhodopsins and synergetic technologies

Various optogenetic applications require different tools. Moreover, the widespread use of optogenetics resulted in the rapid development of synergetic technologies. Such technologies are necessary to perform specific experiments, especially in living animals. Therefore, the development of these technologies is of great importance.

Nevertheless, namely, microbial rhodopsins play a central role in optogenetic research.

Therefore, the absence of the optimal microbial rhodopsin often limits optogenetic application.

Since 2005 scientists expanded the diversity of optogenetic tools dramatically. However, both science and medicine require further development of optogenetic tools.

2.3.1 Microbial rhodopsins and alternative optogenetic approaches

The first reported microbial rhodopsin was the famous bacteriorhodopsin (HsBR) – light- driven retinylidene light-driven proton pump [32]. In 1971 the protein was isolated from the purple membranes [33]. Since then, bacteriorhodopsin was a subject of a considerable number

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In 1994 german scientists reported successful expression of HsBR in the inner mitochondrial membrane of the eukaryotic cell Schizosaccharomyces pombe [35], a naturally non-photoactive organism. Hoffmann et al. transformed it into a light-sensitive organism that uses light energy instead of glucose. This experiment can be considered the first optogenetic intervention into a eukaryotic organism with microbial rhodopsin. Although modern optogenetics mainly focuses on manipulations of neurons.

Despite the great interest of the scientific community, only a few microbial rhodopsins genes were identified by 1999, and all of them were archaeal proteins. Thanks to genomic analysis, the breakthrough happened when the rhodopsin gene was found in bacteria. The protein named proteorhodopsin turned out to be a new bacterial light-driven proton pump [36].

In the early 2000s, several groups independently submitted sequences of two microbial rhodopsins from Chlamydomonas reinhardtii after large scale expressed sequence tag (EST) sequencing of the organism [37]–[39]. These two rhodopsins are targetly expressed in eye-spot being responsible for phototaxis of the algae. Later the rhodopsins were named channelrhodopsin-1 and channelrhodopsin-2, highlighting their particular function. It was reported that both rhodopsins function as light-gated cation channels: CrChR1 - proton- selective channel [40] and CrChR2 - non-selective cation channel [1].

In a short time era of optogenetics began with the first usage of CrChR2 as a light-sensitive actuator for the neurons [2], [24]. These reports significantly increased the interest of the scientific community in microbial rhodopsins. However, CrChR2 was not an ideal tool for neuronal manipulations: it had blue-light shifted absorption spectrum and slow closing kinetics for some neuroscientific applications. Therefore, searching for new rhodopsins and optimization of the known ones was and still is a scientific task of prominent importance.

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There are several main parameters, which have to be optimized in order to obtain better and complementary optogenetic tool: absorption spectrum, on and off kinetics, the strength of the effect, quantum efficiency, target expression, and selectivity. The growing interest in optogenetics, together with the development of genome sequencing technologies, led to the discovery of multiple microbial rhodopsin genes. Researchers found them across all domains of life, and they cover broad spectra of desirable properties. Besides that, researchers assembled a lot of mutants and chimera constructs, optimizing rhodopsins for optogenetics.

2.3.1.1 Optogenetic actuators

In the next years, researchers made multiple CrChR2 variants and obtained actuators with different properties. In 2005 german scientists investigated the possibility to evoke muscle contractions in Caenorhabditis elegans [41]. They performed mutation H134R, which enhanced the magnitude of the light-driven effect of CrChR2, albeit this mutation leads to deceleration of channels closing kinetics as well. That was the first report of optogenetics working in vivo. Later T159C mutation was shown to increase CrChR2 photocurrents in a factor of ten [42].

Nevertheless, neuroscience needs faster control of activation. Researchers from the University of California designed several chimeric channelrhodopsins comprising domains from CrChR1 and CrChR2 [43]. They combined it with site-directed mutagenesis and obtained several perspective channelrhodopsin variants. The variants were different in terms of current magnitude, desensitization level, and closing kinetics. The best variant called ChIEF (Ch for the channel, I for I170V mutation, EF for chimera crossing site in EF-loop) had almost six times higher stationary currents and 1.5 times faster closing kinetics than CrChR2. ChIEF could reliably evoke action potentials in neurons with frequency up to 25 Hz.

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Red-shifted spectrum was always preferable variant for optogenetics since red-light can penetrate deeper in living tissue. The first channelrhodopsin variant with a red-shifted absorption spectrum (maximum at 545 nm) was extracted from different algae Volvox carteri [44]. Later chimeric rhodopsins comprising domains of channelrhodopsins from Volvox carteri were reported. C1V1(T/T) channelrhodopsin was able to fire neurons with frequency up to 40 Hz utilizing 560 nm light [45]. Presented later ReaChR (ChIEF/VChR1/VChR2 chimera with L171I mutation) had much better plasma membrane localization, and consequently, larger photocurrents [46]. Besides that, its spectral maximum was ~600 nm. Later, ReaChR was modified for enhanced expression in the long-range projections of neurons as well as accelerated channel kinetics, resulting in bReaChES [47].

Meanwhile, a bunch of papers was published describing CrChR2 optimized by mutagenesis.

Researchers showed DC-gate (C128 and D156 positions in CrChR2) to have a significant impact on channel closing kinetics [45], [48]. CrChR2-C128T/A/S and CrChR2-C128S- D156A variants called step-function-opsin (SFO) and stabilized-step-function-opsin (SSFO) can be used for prolonged activation of neurons (closing time constant up to ~30 min, Fig. 8g).

They can be triggered on and off by short light pulses (470 nm - on, 590 nm - off). Later SSFO- mutations were combined with T159C making SFO opsin variant with higher light-sensitivity, called SOUL [49]. In opposite, another mutation in close proximity of retinal Schiff base E123T significantly accelerates closing kinetics of the channel [50]. The protein called ChETA had a closing time constant ~4.4 ms allowing ultrafast neuronal manipulations up to 200 Hz (Fig. 8a,b).

Besides closing kinetics, mutagenesis allowed to alter the selectivity properties of the channels.

For instance, single mutation L132C in CrChR2 (CatCh) increased relative permeability for calcium by a factor of 6 and also increase light sensitivity by a factor of 70 [51]. Amazingly,

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single mutation E90K/R could convert CrChR2 into a light-sensitive chloride-selective channel [52].

Genome mining had expanded optogenetic toolkit significantly as well. In 2014 scientists from MIT characterized opsins from over 100 species of alga [21] physiologically. Among investigated proteins, there were two most promising ones: Chrimson (from Chlamydomonas noctigama) and Chronos (from Stigeoclonium helveticum). The Chronos had extremely fast closing kinetics (~3.2 ms, Fig. 8d). As was mentioned above, later, this advantage has been used in spiral ganglion neurons for hearing restoration [22]. It turned out that the Chronos closing time constant at 36°C is less than 1 ms, which helped to activate ultrafast SNGs at 1000 Hz.

In contrast, Chrimson was channelrhodopsin with a red-shifted absorption spectrum (600 nm, Fig. 8c). Together with Chronos, which absorption maximum is 500 nm, the two channelrhodopsins had been proved to allow independent stimulation of different neuronal populations with the light of different wavelengths. Chrimson’s slow closing kinetics (~21 ms) was sped up by K176R mutation (~15.8 ms) in the original work and later was accelerated more by additional mutations: fChrimson (Chrimson Y261F/S267M, ~5.7 ms, Fig. 8e,f) and vfChrimson (Chrimson K176R/Y261F/S267M, ~2.7 ms, Fig. 8e) [23].

In 2018 international group of researchers performed topological inversion of some channelrhodopsins in the membrane by fusing with additional N-terminal domain of Neurexin 1B-delta [53]. Such inversion affected the selectivity and function of rhodopsin. Inverted CrChR2 functioned as a cation pump; thus, it could be used for neuronal silencing. Inverted Chrimson had a significant effect on selectivity: relative permeability for potassium increased 7 times.

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In 2016 another clade of rhodopsin was shown to function as light-sensitive cation channels [54], [55]. Unlike known channelrhodopsins, they share high functional residues similarity to those of archaeal proton pumps. Later, member of this clade ChRmine was shown to induce high depolarizing photocurrent (~4 nA, Fig. 8h) in neurons and fire them at frequencies up to 40 Hz [56].

Besides channelrhodopsins, Russian and German scientists reported of an alternative approach to activate neurons [57]. Proteins of newly found rhodopsin clade [58] were shown to function as light-sensitive inward proton pumps (Fig. 8i) [57], [59]. NsXeR was capable of driving spikes in hippocampal neurons with a frequency of at least 40 Hz (Fig. 8j). Recently, metagenomic searches resulted in the discovery of Schizorhodopsins [60]. These rhodopsins function as inward proton pumps as well [61]. Thus, they also can expand a variety of optogenetic actuators.

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Figure 8. Different optogenetic tools. (a) CrChR2 fires action potential in neurons at 20 Hz and misses spikes. (b) Fast mutant called ChETA reliably fires interneurons at 200 Hz. (c) Spectra channelrhodopsins obtained by genome mining. Chrimson has a red-shifted spectrum. (d) Closing-kinetics of channelrhodopsins obtained by genome mining. Chronos has fast off-kinetics. (e) Optimization of Chrimson. Mutations accelerated red-shifted Chrimson: fChrimson (Chrimson Y261F/S267M, ~5.7 ms) and vfChrimson (Chrimson K176R/Y261F/S267M, ~2.7 ms). (f) fChrimson drives interneurons to their frequency limit of 300 Hz. (g) CrChR2 stable step-function mutant has closing kinetics of 30 min. (h) ChRmine is a bacteriorhodopsin-like channelrhodopsin capable of inducing photocurrents over 1 nA utilizing far-red 650 nm light. (i) Inward-proton pump NsXeR current-voltage relation without reversal. (j) NsXeR is capable of driving rat hippocampal neurons at 40 Hz. (k) eNpHR3.0 is a version of inward Chloride-pump halorhodopsin in neurons with optimized plasma membrane localization. (l) eNpHR3.0 can reliably silence neurons. (m) Engineered ACR iC1C2 is capable of neuron silencing. (n) Step-function ACR SwiChR turns neuronal firing on and off. [50], [21], [23], [56], [45], [57], [62], [63]

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34 2.3.1.2 Optogenetic inhibitors

The other mandatory need for neuroscience was to hyperpolarize the cell membrane. The idea of using rhodopsins to hyperpolarize neurons evolved after the big success of channelrhodopsin. In 2005 there were already known several microbial rhodopsins that perform a desirable function: proton pumps and chloride pumps. Since then, multiple attempts have been made to build reliable optogenetic inhibitors.

Natronomonas pharaonis halorhodopsin (NpHR) is an inward chloride pump [64]. It was one of the first proteins to be successfully used as light-sensitive neuronal silencer [65], [66]. NpHR was complementary to the CrChR2 silencing tool since it had a red-shifted spectral maximum.

However, photocurrents in neurons were not sufficient (~50 pA) to use it reliably in a variety of possible applications. Therefore, the NpHR gene has undergone several significant optimizations [67], [62]. In brief, the target expression of NpHR in the plasma membrane of neurons was optimized by fusion of its gene with several signal peptides: N-terminal signal peptide ensured membrane insertion, ER export, and Golgi export signal peptides, and membrane trafficking signal to ensure surface expression. The final best variant called eNpHR3.0, which induced photocurrents of ~700 pA on average and was capable of hyperpolarizing neurons for -100 mV when illuminated with 593 nm light (Fig. 8k,l). Besides, eNpHR3.0 could sufficiently depolarize neurons under illumination with light of far red-shifted wavelength up to 680 nm.

Meanwhile, proton pumps were also shown to have optogenetic potential. Famous bacteriorhodopsin was optimized in the same way as NpHR [62]. Although optimization made HsBR capable of hyperpolarizing neurons up to -10 mV, it still was a much less effective tool than eNpHR3.0. Several proton pumps from different organisms were compared to NpHR [68].

Three of them had larger photocurrents than chloride pumps: Arch (Halorubrum sodomense),

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Mac (Leptosphaeria maculans), and cruxrhodopsin-1 (Haloarcula), thanks to proper plasma membrane localization. Moreover, Mac could be used simultaneously with NpHR utilizing light of different wavelengths (for instance, Mac and NpHR can absorb light of 470 nm and 630 nm, respectively). Later, genome mining helped to find an even better archaerhodopsin variant called ArchT. It was 3-times more sensitive to light [69], which allowed silencing of a twice larger brain tissue volume.

In 2013 Japanese scientists reported about the first light-driven non-proton cation pump KR2 [70]. It pumped sodium ions, which was unexpected as the long-standing paradigm said that rhodopsin could not pump any positive ion other than the proton. Therefore, this discovery had great importance for science and optogenetics, as a pump with new selectivity, and for future engineering of new rhodopsins. KR2 structure was reported by two groups independently, and both groups showed the possibility to engineer a light-driven potassium pump [71], [72]. This possibility is of uttermost importance for optogenetics. Voltage-gated potassium channels are usually in charge of repolarization of neuronal membranes, therefore silencing with potassium might minimize possible side-effects. The KR2 was also shown to silence neurons with sodium ions, however, with limited efficacy.

Optimizations of KR2 lead to engineering eKR2 with much better plasma membrane localization[73]. Besides that, it was reported that one mutation R109Q makes KR2 passively permeable for potassium, although with residual sodium pumping activity [74]. Passive-K⁺ to active-Na⁺ transports ratio further increased by additional S70A mutation [74].

However, there was a fundamental question if cation channelrhodopsins could be converted into anion conducting channels. The main physiological anion is chloride, which has its Nernst equilibrium potential at around -80 mV. Anion-conducting channelrhodopsins with a high level of plasma membrane expression might have been used for stable neuronal silencing without

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huge hyperpolarization of neurons to non-physiological potentials. As mentioned above, structure-guided rational mutagenesis revealed that single residue replacement E90 with positively charged residues converts CrChR2 into chloride-conducting channel [52].

Meanwhile, another group of scientists conducted extensive mutagenesis of chimera C1C2 and came up with other variants of engineered ACRs called iC1C2 and SwiChR [63]. The iC1C2 was a ninefold mutant of C1C2 (Fig. 8m) chimera, while SwiChR had additional “step- function” mutation (Fig. 8n). Both proteins had current-voltage relation reversed at around -65 mV, which was enough to keep neurons from firing. Besides that, SwiChR had slower closing kinetics (~7.3 s compared to 24 ms for iC1C2) and could be activated and deactivated with short light pulses similar to SFO channels. Later, researchers performed additional mutagenesis and came up with iC++ and SwiChR++, which had 15-fold increased photocurrents compared to iC1C2 and SwiChR [75]. However, engineered ACRs had small residual cation conductance, which increased reversal potential and thus lowered its silencing efficacy.

A new success came later, after full genome sequencing of the cryptophyta Guillardia theta, two genes coding anion conducting channelrhodopsins (ACR) were discovered [76]. The photocurrent of the natural anion channelrhodopsin GtACR2 in cultured pyramidal neurons reversed exactly at Nernst potential for chloride. GtACR2 photocurrents exceeded the maximal photocurrent of Arch at thousandth lower light intensities. In comparison with engineered chloride channels, GtACR2 had much higher light sensitivity while maintaining fast kinetics.

Interestingly, researchers could invert channel gating of GtACR1 by introducing single E68R mutation: the mutant channel was open under dark conditions and closed when illuminated.

Later, structural based rational mutagenesis helped to engineer GtACR1 double mutant R83Q/N239Q called FLASH, which was capable of effective silencing of neurons with fast

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closing kinetics [77]. Further research for natural ACRs revealed MerMAIDs: intensely desensitized ACRs [78].

2.3.1.3 Other types of optogenetic cell manipulation

Besides modulation of electrically active cells, microbial rhodopsins are prospective candidates for other types of genetic interventions.

Light-driven protons pump Arch was targetly expressed and used for acidification of lysosomes and synaptic vesicles [79]. The generation of electrochemical potential on the synaptic vesicle membrane is crucial for the accumulation of neurotransmitters inside them. Therefore, this approach could indirectly influence signal transmission by chemical synapses. On the other hand, acidification of lysosomes could have effects on the fundamental processes of any cells in the organisms (not only electrically active). Moreover, such tools can speed up research concerning physiological aspects of pH maintenance in different cell compartments.

Another notable example is the target-mitochondrial expression of rhodopsin for control of mitochondrial membrane potential [80]. By controlling the mitochondrial membrane potential, it became possible to control its calcium uptake indirectly. Moreover, it was shown that one could optogenetically control beatings of rat cardiomyocytes via this technique [80].

However, microbial rhodopsins allow not only electrogenic control of biological processes.

The first microbial rhodopsin capable of triggering cellular cascades with light was reported in 2012. Such rhodopsins called enzymerhodopsins. They consist of two main parts: N-terminal light-sensitive rhodopsin domain and C-terminal effector domain. At the moment, several enzymerhodopsins are known. Based on their function, they are divided into three classes:

histidine kinase rhodopsins (HKR) [81], rhodopsin guanylyl cyclases (RhGC) [82] and

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rhodopsin phosphodiesterases (RhoPDE) [83]. Despite the great and obvious potential of enzymerhodopsins, they are still to be integrated into optogenetic research.

Search for new rhodopsin genes is productive, as never before. Each year thousands of new genes are discovered. Investigations of their functions take a considerable amount of time.

Currently, there is no reliable approach to predict their functions and properties. However, the history of optogenetics showed that some of them might find its application in it.

2.3.2 Development of techniques synergetic to rhodopsins

The progress of optogenetics relies on the development of specific technical aspects as well as search for rhodopsins with new properties.

First of all, neural research needs precise delivery of optogenetic tools. Modern working spatial precision is not just cellular but subcellular. In order to obtain reliable expression of certain rhodopsin in neuronal dendrites and axons or cellular organelles, researchers have to develop new methods of targeting. As already mentioned above, the fusion of rhodopsin with signal peptides plays an essential role in target expression [62], [73].

Besides that, modern viral technologies allow researchers to increase the spatial precision of stimulation [84]. First, scientists achieve opsin expression in the desired tissue of wild-type animals by placing genes under the cell-type specific promoters (Fig. 9a). Moreover, one can exclusively stimulate neurons, which project their axons in a specific brain region, placing stimulation fiber above the axonal projection (Fig. 9c). By combining, one can stimulate only neurons specified by a particular promoter and projecting their axons to a particular region.

Second, Cre-Lox recombination is widely used in order to specify cell types and regions in the brain. In this technology Cre-recombinase dependent opsin gene is delivered to a desirable region (Fig. 9b). The opsin expression occurs either in the Cre-positive or Cre-negative

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neurons. Double-floxed Inverted Orientation (DIO or Cre-on) or Double-floxed in Orientation (DO or Cre-off) genes are used for that, respectively. Double-floxed means opsin gene is placed between two different pairs of Lox-sequences, which allows expressing the gene in an inverted orientation in the presence of Cre (Cre-on), and in forward orientation in the absence of Cre (Cre-off). A cell-specific promoter regulates the expression of the Cre-recombinase gene. The gene can either already exist in transgenic animals or be delivered by the second virus (Fig. 9d left).

Figure 9. Cell-specific viral technologies for opsin-gene delivery. (a,b) The high-titer virus is delivered to the brain region of interest. Cell-specificity is achieved by using either cell-specific promoter in wild- type animal or recombinase-dependent virus in transgenic animal or by the second injection of virus, carrying recombinase-gene. (c) AAVs carrying opsin-gene under control of the cell-specific promoter are injected into the region of interest. The stimulating light can either be delivered over cell bodies (down- left) or be delivered over neuronal axons projecting to the desired brain region (down-right). (d) Projecting- region dependent opsin expression is achieved by injection of DIO opsin gene in combination with retrograde CAV virus, carrying Cre-recombinase (left). Another approach is to inject the retrograde rabies virus, carrying the opsin gene. [84]

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Third, retrograde viruses can be used in order to label neurons with projections to a specific region (Fig. 9d right). Insertion of the Cre-recombinase gene into the genome of the retrograde virus allows researchers to label neurons of defined type and brain region.

To sum up, advances in genome editing, virus gene delivery, and light-targeting have a significant impact on optogenetic research.

Brain discoveries also benefit a lot from the development of complementary to optogenetics technologies. For instance, a combination of in vivo electrophysiological recording with optogenetics allowed closed-loop optogenetic interventions when optical stimulation is driven by ongoing activity. Indeed, researchers detected initiation of epileptic seizures in the thalamus and, at the moment, silenced thalamocortical neurons with halorhodopsin in order to terminate the epileptic activity [85].

As already mentioned above, GECIs play an essential role in modern optogenetics. They allow researchers to stimuli and read-out the activity of neurons in an optical way. However, GECIs have too slow kinetics to resolve action potentials in vivo. It may be possible to use genetically encoded voltage indicators (GEVI) instead. Nevertheless, optimization of spectral parameters (to separate them from opsins) and kinetics is still to be done for GEVI as well [86].

Besides all of that, there is also a methodology of activity-guided optogenetic stimulation. As described above, it is possible to recall fear memory in AD mice by activating their engrams [17]. The engrams were labeled with CrChR2 by placing its gene under immediate early gene (IEG) c-Fos promoter, which is activated when corresponding neurons fire action potentials.

Thus, activity-guided channelrhodopsin expression is a powerful approach, and its development would undoubtedly have a considerable impact on optogenetics and, in particular, neuroscience.

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41 2.4 Microbial rhodopsins diversity

Massive genome sequencing reveals thousands of rhodopsins each year. Nowadays, the superfamily of rhodopsins is considered as the most diverse and widespread family of light- harvesting proteins among organisms on Earth [87].

Discoveries of proteorhodopsins [36] and heliorhodopsins [4] significantly expanded the phylogenetic tree of rhodopsins (Fig. 10). The current conventional opinion is that rhodopsins have been evolving for a long time. Now they are capable of performing an enormously diverse set of biological functions. Furthermore, rhodopsins are abundant among all domains of life and also in viruses [5].

In some cases, like the case of HsBR [32] or channelrhodopsins, function and biological role are already explored. Outward proton pumps provide cells with energy when the channelrhodopsins provide unicellular organisms with a kind of “eye” for phototaxis [38].

In other cases, like for xenorhodopsins, researchers have already only revealed molecular function [57], [59]. Although their function as inward proton pumping is already used for optogenetics, the biological role is still to be explored. Another rhodopsins clade Schizorhodopsins were proven to be inward proton pumps as well [61], which shows an example of divergent evolution of this property. Thus, inward proton pumps play an important biological role, which is still to be revealed.

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Figure 10. Phylogenetic tree of microbial rhodopsins. An unrooted maximum-likelihood tree of microbial rhodopsins of type-1 (including channelrhodopsins and viral rhodopsins), heliorhodopsins, and schizorhodopsins. Protein sequences were adapted from Bulzu et al. [60]

Viral rhodopsins comprise another group of rhodopsin without explored biological roles [5].

Giant viruses, carrying genes of rhodopsins, are widely distributed in the World Ocean [88].

They infect different organisms, both initially sensitive to light and not. At the moment, x-ray structures of VirRDTS (viral rhodopsins group 1)and OLPVR2 presented (viral rhodopsins group 2) [88], [89]. However, the biological role of these rhodopsins is not yet clear. Both proteins were shown to have proton pumping activity, but there were no patch-clamp data to verify channeling activity. Distinctive structural features of these proteins suggest that they

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possess a new function. Thus, viral rhodopsins are unique proteins, which might have a significant impact on ecology.

Some cases are not clear from both points of view: neither function nor biological role is disclosed. Heliorhodopsins constitute one of these classes. Nevertheless, the wide distribution and diversity of heliorhodopsins lead to the belief in their potential for application, including optogenetics.

Taking together, genomic discoveries of new rhodopsins have an impact not only in a field of optogenetics. The biodiversity of rhodopsins raises the question of their effect on global ecology. That is indeed is a question of uttermost importance, which has to be answered in the future.

2.5 Structural insights into channelrhodopsins function

2.5.1 X-Ray structures of cation and anion conducting channelrhodopsins

An in-depth understanding of structural features responsible for the function of certain rhodopsin can help in engineering new optogenetic tools with desirable properties. As already mentioned, many optogenetic tools were optimized using structure-based mutagenesis. A deep investigation of only one molecule C1C2 resulted in the engineering of new tools for both activation and inhibition, with increased sensitivity and optimized kinetics. X-ray structures helped to perform such optimizations more rationally.

After the discovery of optogenetics, researchers around the globe started to optimize CrChR2, relying on general knowledge of rhodopsins organization and HsBR structure [90]. There were considerable efforts to obtain the X-ray structure of channelrhodopsin. The first successful attempt was obtained with the chimera of CrChR1 and CrChR2 and reported 2012 [91]. In this study, structural insights were confirmed by extensive mutagenesis and electrophysiological

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experiments. At the moment, three X-Ray structures of cation conducting channelrhodopsins and two X-Ray structures of anion conducting channelrhodopsins are available (Fig. 11) [91]–

[93], [77], [94].

In brief, channelrhodopsins, as rhodopsins in general, comprise of 7-transmembrane helices and co-factor retinal covalently bonded to specific lysine in G-helix. Presumably, channelrhodopsins have a pore protruding through the complete protein in the open state, passing nearby of the retinal Schiff base. However, at the moment, there is no structure of the open state of any channelrhodopsin. Meanwhile, in the ground state, one can either see several cavities separated by constriction sites (case of CrChR2 and Chrimson, Fig. 11B, C) or complete pore going from extracellular space up to the central gate region (case of C1C2 and GtACR1, Fig. 11A, D). The surfaces of the pores are shown to be electronegative and electropositive for cation and anion conducting rhodopsins, respectively. Thus, the pores serve as selectivity filters for the channelrhodopsins.

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Figure 11. Overall X-ray crystal structures of channelrhodopsins. Blue arrows indicate a putative cation-conducting pathway. Conservative glutamates, lining cation pathway, are indicated in each structure as “E” in red (namely, E82, E83, E90, E97, E101, E123, E235, according to CrChR2 numeration). R120 is shown as “R” in blue. Red arrows indicate a putative anion conducting pathway in the GtACR1. Gray lines represent two surfaces of the lipid membranes. (A) The structure of the chimera C1C2. (B) The structure of the CrChR2. (C) The structure of the red-shifted channelrhodopsin Chrimson. (D) The structure of the anion channelrhodopsin GtACR1; alterations of glutamates are indicated in black.

Rational design of optogenetic tools: from bioinformatic genomic data analysis to electrophysiological validation