Hyper-photosensitive opsin-based optogenetic tools for activating gq-coupled signaling and / or activating cells

Abstract

The present invention relates to methods for rapidly, reversibly and accurately activating G q signaling and / or activating isolated photopsin proteins of cells.

Description

Background Technology

[0001] G protein-coupled receptors (GPCRs) regulate many intracellular signaling pathways and represent some of the most thoroughly investigated drug targets (Hauser et al., 2017). Upon ligand binding, GPCRs undergo a conformational change and deliver the G protein to a heterotrimeric G protein, which contains G proteins. α and closely integrated G βγ A multi-subunit complex of subunits. G q Protein, a heterotrimer G α This subfamily of subunits, coupled with a class of GPCRs, mediates cellular responses to neurotransmitters, sensory stimuli, and hormones throughout the body. Their primary downstream signaling targets include phospholipase Cβ (PLC-β), which catalyzes the hydrolysis of phosphatidylinositol diphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 triggers the release of calcium from intracellularly stored... 2+ Released into the cytoplasm, and Ca 2+ It activates protein kinase C (PKC) together with DAG. Several tools, including chemogenetics and photoactivated small molecules, have been developed to study G. q Coupled GPCRs and intracellular Ca 2+ The released signal transduction mechanism and physiological function.

[0002] Optogenetics utilizes light-responsive proteins to perturb cellular activities with genetic specificity and high spatiotemporal precision through optical control. Since the early discovery of optogenetic tools using light-sensitive ion channels and transport proteins, a diverse range of techniques have been developed that now support optical intervention in intracellular second messengers, protein interactions and degradation, and gene transcription. Opt-a1AR, a creatively designed G… q The coupled rhodopsin-GPCR chimera can respond to prolonged light stimulation (60 s) and induce intracellular Ca2+. 2+ Increased (Airan et al., 2009). However, this tool has not been widely used, likely due to its limitations related to photosensitivity and response kinetics (Tichy et al., 2019). Most animals use GPCR-based photoreceptors to detect light, where the photoreceptor contains a protein motif (opsin) and a vitamin A derivative (retinaldehyde) that functions as both a ligand and a chromophore. Thousands of opsins have been identified to date. Two recent studies reported the use of GPCR-based [photoreceptors] from mosquitoes and lampreys. i The use of opsins for presynaptic terminal inhibition in neurons briefly demonstrates that certain naturally occurring photoreceptors are suitable as efficient optogenetic tools. Regarding G... qSignal transduction; opioid 4 (Opn4) in a subset of mammalian retinal ganglion cells is a G... q Opn4 is a conjugated opsin that mediates visual functions that do not form images. However, HEK293 or Neuro-2a cells heterologously expressing Opn4 exhibit a weak light response and require additional retinal in the culture medium. Opn5 (neuropsin) and its orthologs have been reported to be conjugated with G in many vertebrates. i Protein-coupled ultraviolet (UV) sensitive opsins. Summary of the Invention

[0003] This invention relates to an isolated photosensitive opsin for rapid, reversible, and precise activation of G. q Signal transduction and / or cell activation.

[0004] In a first aspect, the present invention relates to an isolated photosensitive opsin for activating G... q Signal transduction and / or cell activation.

[0005] In some embodiments, the light has a wavelength in the range of 360nm-520nm, preferably 450-500nm, and more preferably 460-480nm.

[0006] In some embodiments, the isolated opsin is an isolated opsin from an organism, its homolog, its ortholog, its paralog, its fragment, or a variant, and has the ability to activate G. q Signal transduction and / or activation of cell activity.

[0007] In some embodiments, the isolated opsin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with wild-type opsin, its homologs, its orthologs, its paralogs, its fragments, or variants in the organism, and has the ability to activate G... q Signal transduction and / or activation of cell activity.

[0008] In some implementations, the organism is an animal.

[0009] In some embodiments, the isolated opsin is an isolated opsin 5 (Opn5) from an animal, its homolog, its ortholog, its paralog, its fragment, or a variant, and has the ability to activate G. q Signal transduction and / or activation of cell activity.

[0010] In some embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with wild-type opsin 5 (Opn5), its homologs, its orthologs, its paralogs, its fragments, or variants in the animal, and has the ability to activate G... q Signal transduction and / or activation of cell activity.

[0011] In some embodiments, the animal is a vertebrate.

[0012] In some embodiments, the animal is a bird, reptile, fish, amphibian, or mammal.

[0013] In some embodiments, the animal is a bird, including but not limited to chickens, ducks, geese, ostriches, emus, rheas, sandpipers, cassowaries, turkeys, quails, chickens, falcons, eagles, falcons, pigeons, long-tailed parrots, cockatoos, macaws, parrots, passerines (e.g., songbirds), jays, blackbirds, finches, warblers, and sparrows.

[0014] In some embodiments, the animal is a reptile, including but not limited to lizards, snakes, alligators, sea turtles, crocodiles, and tortoises.

[0015] In some embodiments, the animal is a fish, including but not limited to catfish, eel, shark and swordfish.

[0016] In some embodiments, the animal is an amphibian, including but not limited to toads, frogs, salamanders, and fire salamanders.

[0017] In some embodiments, the isolated opsin 5 (Opn5) is an isolated wild-type opsin 5 (Opn5) from chicken or a fragment or variant thereof, and has the function of activating G. q Signal transduction and / or activation of cell activity.

[0018] In some embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the wild-type opsin 5 (Opn5) from chicken, and has the ability to activate G... q Signal transduction and / or activation of cell activity.

[0019] In some embodiments, the isolated opsin 5 (Opn5) is an isolated wild-type opsin 5 (Opn5) or a fragment or variant thereof from a sea turtle, and has the function of activating G. q Signal transduction and / or activation of cell activity.

[0020] In some embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the wild-type opsin 5 (Opn5) from sea turtles, and has the ability to activate G... q Signal transduction and / or activation of cell activity.

[0021] In some embodiments, the isolated opsin 5 (Opn5) has the amino acid sequence (cOpn5) shown in SEQ ID NO: 1 or a fragment or variant thereof, and has activating G... q Signal transduction and / or activation of cell activity.

[0022] In some embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1, and has the ability to activate G... q Signal transduction and / or activation of cell activity.

[0023] In some embodiments, the isolated opsin 5 (Opn5) has the amino acid sequence (tOpn5) shown in SEQ ID NO: 2 or a fragment or variant thereof, and has activating G... q Signal transduction and / or activation of cell activity.

[0024] In some embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence (tOpn5) shown in SEQ ID NO: 2, and has the ability to activate G. q Signal transduction and / or activation of cell activity.

[0025] The isolated opsin 5 (Opn5) can be used as a convenient optogenetic tool to precisely activate intracellular G... q Signal transduction and / or cell activation.

[0026] Secondly, the present invention relates to an isolated nucleic acid that encodes the isolated opsin described in the first aspect.

[0027] In some embodiments, the isolated nucleic acids encode wild-type opsins, their homologs, their orthologs, their paralogs, their fragments, or variants in organisms, which have the ability to activate G... q Signal transduction and / or activation of cell activity.

[0028] Thirdly, the present invention relates to a chimeric gene comprising the isolated nucleic acid sequence described in the second aspect, wherein the verification sequence is operatively linked to a suitable regulatory sequence.

[0029] Fourthly, the present invention relates to a vector comprising the isolated nucleic acid described in the second aspect or the chimeric gene described in the third aspect.

[0030] The vector is a eukaryotic vector, a prokaryotic expression vector, a viral vector, or a yeast vector.

[0031] In some embodiments, the vector is a herpes simplex virus vector, a vaccinia virus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or an insect vector.

[0032] Preferably, the vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVS, AAVO, or AAV10.

[0033] In some embodiments, the carrier is an expression carrier.

[0034] In some embodiments, the vector is a gene therapy vector.

[0035] Fifthly, the present invention relates to an isolated cell or cell culture comprising the isolated nucleic acid described in the second aspect, the chimeric gene described in the third aspect, or the vector described in the fourth aspect.

[0036] For example, expression of cOpn5 in HEK 293T cells strongly mediates blue light-triggered Gamma rays. q Dependency Ca 2+ Increase from intracellular storage.

[0037] For example, optogenetic activation of astrocytes expressing cOpn5 in the mouse brain induces a large release of ATP.

[0038] Sixthly, the present invention relates to the use of the isolated opsin described in the first aspect, the isolated nucleic acid described in the second aspect, the chimeric gene described in the third aspect, the vector described in the fourth aspect, or the isolated cell or cell culture described in the fifth aspect, for the treatment of G-mediated diseases. q Diseases or conditions mediated by signal transduction and / or activation of cells, or involving the activation of G cells. q Diseases or conditions that affect signal transduction and / or cell activation.

[0039] cOpn5-mediated optogenetics can be used to activate neurons in a circuit-dependent manner and control animal behavior.

[0040] Seventhly, the present invention relates to a treatment for activating G... q Diseases or conditions mediated by signal transduction and / or activation of cells, or involving the activation of G cells. q A method for signal transduction and / or activation of cells in a disease or condition, the method comprising administering an isolated opsin as described in the first aspect, an isolated nucleic acid as described in the second aspect, a chimeric gene as described in the third aspect, a vector as described in the fourth aspect, or an isolated cell or cell culture as described in the fifth aspect.

[0041] In some implementations, the activation of G... q Diseases or conditions mediated by signal transduction and / or activation of cells, or involving the activation of G cells. q Diseases or conditions involving signal transduction and / or activation of cells, including but not limited to those involving activation of G cells. q Diseases or conditions that benefit from signal transduction and / or cell activation, such as those that benefit from activation of astrocytes, strong ATP release, or increased neuronal activity.

[0042] In some implementations, the activation of G... q Signal transduction and / or activation of cells mediated or involved in the activation of G cells q Diseases or conditions that benefit from signal transduction and / or cell activation include, but are not limited to, diseases or conditions that benefit from activated cells such as pancreatic islet cells, immune cells, nerve cells such as central neurons, astrocytes, glial cells, muscle cells, bone cells, endothelial cells, epithelial cells, nervous system cells, skin cells, lung cells, kidney cells and liver cells, cardiomyocytes or vascular endothelial cells.

[0043] In some implementations, the disease or condition includes, but is not limited to, diabetes, immunosuppressive diseases, Alzheimer's disease, depression, anxiety neurosis, cerebral hemorrhage, etc.

[0044] In some embodiments, the method further includes applying blue light with a wavelength range of 360nm-550nm, preferably 450-500nm, and more preferably 460-480nm.

[0045] In some embodiments, the method further includes two-photon activation using long-wavelength (≥920 nm) light.

[0046] The isolated opsin described in this invention is photosensitive to wavelengths in the range of 360-550 nm, preferably 450-500 nm, and more preferably 460-480 nm. Specifically, 470 nm blue light induces the strongest Ca2+ reaction in cells. 2+ Transient, which means that the isolated opsin described in this invention is hypersensitive to light with a wavelength of 470 nm.

[0047] This invention covers all combinations of the specific embodiments described herein. Attached Figure Description

[0048] Figure 1 This demonstrates cOpn5-mediated light-induced G in HEK 293T cells. q Strong activation of signal transduction.

[0049] Figure 2 The cOpn5 and G are shown q Signal conduction coupling but not with G i Signal conduction coupling.

[0050] Figure 3 This demonstrates that cOpn5 sensitively mediates G with high spatiotemporal resolution. q Optical control of signal transmission.

[0051] Figure 4 It was shown that cOpn5 mediates a faster and more sensitive response to light than opto-a1AR, hM3Dq, or opn4.

[0052] Figure 5 This demonstrates that cOpn5 effectively mediates the activation of astrocytes.

[0053] Figure 6 This study demonstrates that cOpn5-mediated activation of astrocytes induces significant ATP flashes and neuronal activation in vivo.

[0054] Figure 7 This demonstrates that cOpn5 mediates sustained and reliable ATP release in astrocytes and activation of surrounding neurons.

[0055] Figure 8 This study demonstrates how cOpn5-mediated optogenetics alters mouse behavior in a neural circuit-dependent manner.

[0056] Figure 9 This demonstrates that cOpn5-mediated optogenetic activation of neurons is reliable.

[0057] Figure 10 The injection location and placement of the optical fiber are shown. Detailed Implementation

[0058] In this invention, the capabilities of opsins from multiple species, particularly Opn5 orthologs, were tested, and it was found that many opsins sensitively and strongly mediate light-induced Gq signaling activation and / or cell activation.

[0059] Preferably, the Opn5 ortholog is a chicken ortholog (abbreviated as cOpn5) or a sea turtle ortholog (abbreviated as tOpn5).

[0060] Detailed characterization of Opn5, especially cOpn5, reveals its ultrasensitivity to blue light (μW / mm). 2 Level, compared to existing G-based q The opsin-conjugating tools opto-a1AR and opn4 have high sensitivity (up to 3 orders of magnitude higher) and high temporal (response to a 10 ms light pulse, up to 3 orders of magnitude faster than opto-a1AR or opn4) and spatial (subcellular level) resolution, and do not require the addition of chromophores. Specifically, endogenous retinaldehyde is sufficient, and the addition of retinaldehyde is unnecessary.

[0061] This invention further demonstrates that cOpn5 optogenetics is a highly efficient method for activating astrocytes in vivo to induce massive ATP release, and for activating neurons to produce robust behavioral changes in freely moving mice. These findings confirm that cOpn5 and potentially other Opn5 orthologs can be used as powerful optogenetic tools to support the observation of G-related changes in both non-excitable and excitable cells. q Experimental studies were conducted on signal transduction and / or activation of cell-related physiological and behavioral functions.

[0062] cOpn5-mediated optogenetic activation and / or activation of cells in Gq signaling

[0063] Specifically, in this invention, Opn5 orthologs from chickens, turtles, humans, and mice (which share 80-90% protein sequence identity with each other) were tested to determine their ability to mediate blue light-induced Gq signaling activation in HEK 293T cells. Blue light and the red intracellular calcium indicator Calbryte were used for stimulation. TM 630AM dye is used to monitor relative Ca 2+ Response. An immediate and intense photoinduced Ca2+ response was found to be mediated by Opn5 orthologs from chickens (cOpn5) and sea turtles (tOpn5). 2+ An increase in signal was observed (~3ΔF / F), while no photoeffect was observed in cells expressing human or mouse Opn5 orthologs. As exemplified by the chicken ortholog, cOpn5 co-localizes with the EGFP-CAAX membrane marker, indicating efficient transport to the plasma membrane. No exogenous retinol was required to be added to the culture medium, suggesting that endogenous retinol is sufficient for cOpn5 to function. 2+ Signals on extracellular Ca 2+ The removal of Ca showed resistance, thus indicating that Ca... 2+ Released from intracellular storage. In two cell lines expressing cOpn5, pre-incubation with a Gq protein inhibitor (e.g., YM-254890, a highly selective Gq protein inhibitor) reversibly eliminated light-induced Ca2+.2+ Transient. A light-induced increase in the level of inositol phosphate (IP1), a rapid degradation product of IP3, was detected in cells expressing cOpn5 but not human OPN5; furthermore, treatment with YM-254890 reduced the extent of this increase. In cOpn5-expressing cells (e.g., HEK 293T cells), blue light also triggered phosphorylation of the MARCKS protein (a recognized PKC target) in a PKC activity-dependent manner. In contrast, in the presence of retinaldehyde, blue light irradiation effectively reduced cAMP levels in cells expressing human and mouse Opn5, but in the absence of retinaldehyde, blue light irradiation did not have this effect in cOpn5-expressing cells. In conclusion, these data support the ability of blue light irradiation to couple cOpn5 to the Gq signaling pathway in HEK 293T cells.

[0064] cOpn5-mediated optogenetics is sensitive and precise.

[0065] Specifically, the photoactivation properties of cOpn5 were characterized in this invention. cOpn5 can be heterologously expressed in cells (e.g., HEK 293T cells). Although Opn5 was previously considered a UV-sensitive photoreceptor, at a fixed light intensity (100 μW / mm²), its photoactivation properties were... 2 The following plot, using a set of wavelengths in the 365–630 nm range, reveals that blue light at 470 nm induces the strongest Ca2+. 2+ The light intensity was transient, while UVA light (365 and 395 nm) was less efficient, and longer wavelengths of visible light (561 nm or above) were completely ineffective. The effects of different light durations on HEK 293T cells expressing cOpn5 were tested, with short light pulses (1, 5, 10, 20, 50 ms; 16 μW / mm²) applied. 2 Stimulation at 470nm showed that Ca2+ was effective when the illumination duration exceeded 10ms. 2+ The response reached saturation mode. At this illumination intensity (16 μW / mm²), 2 At 470 nm, longer illumination durations did not further increase the Ca2+ signal amplitude. Exposure to 470 nm light at different intensities showed that ~4.8 μW / mm and 16 μW / mm blue light produced approximately half-maximal and maximum responses, respectively. These data indicate that the photosensitivity of cOpn5 is 2–3 orders of magnitude higher than that of rhodopsin-2 (ChR2), a commonly reported optogenetic tool. In summary, the results of this invention demonstrate that cOpn5 can function as a single-component optogenetic tool without additional retinal, and that cOpn5 is hypersensitive to blue light, requiring low light intensity (16 μW / mm) for complete activation. 2 ) and short duration (10ms).

[0066] The performance of cOpn5 was compared with that of opto-a1AR, a chimeric GPCR engineered by mixing rhodopsin with a Gq-coupled adrenergic receptor. Following previously reported protocols, it was found that very long exposure to strong radiation (60 s; 7 mW / mm²) was required. 2 Only then can slow and small (~0.5ΔF / F) Ca2+ activation be triggered in HEK 293T cells expressing opto-a1AR. 2+ Signal enhancement was observed, but 15 seconds of irradiation was ineffective. The performance of cOpn5 was compared to that of opn4, a natural opsin reportedly used for Gq signaling activation. It was found that prolonged exposure to strong irradiation (25 seconds; 40 mW / mm²) was required. 2 Only with the addition of retinaldehyde can slow (~1ΔF / F) Ca2+ be triggered in HEK 293T cells expressing opn4. 2+ The signal is enhanced. Therefore, compared with existing opsin-based tools (opto-a1AR and opn4), cOpn5 has much higher photosensitivity (sensitivity is ~3 orders of magnitude higher), requires a much shorter exposure time (10 ms vs. 60 s), and produces a stronger response.

[0067] Furthermore, the performance of cOpn5 was compared with that of the popular Gq-coupled chemogenetic tool hM3Dq, which is activated by the addition of the exogenous small molecule ligand clozapine-N-oxide (CNO). The light-induced activation of cOpn5-expressing HEK 293T cells showed similar Ca²⁺ values ​​to the CNO-induced activation of hM3Dq-expressing HEK 293T cells. 2+ The peak response amplitude of the signal. Furthermore, compared to HEK 293T cells expressing hM3Dq, HEK 293T cells expressing cOpn5 exhibited a faster and more temporally precise response, as well as a faster recovery time. These results indicate that cOpn5-mediated optogenetics is more controllable in terms of temporal accuracy than hM3Dq.

[0068] cOpn5 optogenetics allows for spatially precise control of cellular activity. Restricting a brief light stimulus (63 ms) to a subcellular region of a single cOpn5-expressing HEK 293T cell resulted in immediate activation of that single cell. Interestingly, in regions of high cell aggregation, Ca2+... 2+The signal propagated to surrounding cells, suggesting intercellular communication between HEK 293T cells via an undefined mechanism. cOpn5 was expressed in primary astrocyte cultures derived from neonatal mouse brains using an AAV vector for bicistronic expression of the cOpn5 and EGFP marker proteins. Ca2+ levels were monitored using Calbryte 630AM dye, and blue light irradiation of cOpn5-expressing astrocytes revealed a strong Ca2+ production. 2+ Transient (~8ΔF / F). When the light stimulus (63 ms) was precisely restricted to a subcellular region of a single cOpn5-expressing astrocyte, Ca was observed. 2+ The signal propagates within a single cell. Similar to the test in HEK 293T cells, Ca was observed. 2+ The signal propagates in a wave-like pattern from stimulated astrocytes to more distant, unstimulated astrocytes. Therefore, these experiments demonstrate that cOpn5 optogenetics allows for precise spatial control and suggest that studying the dynamics of astrocyte networks initially discovered using neurochemical and mechanical stimulation may be useful.

[0069] Optogenetic activation of cOpn5 in astrocytes induces significant ATP release and neuronal activation in vivo.

[0070] The in vivo performance of cOpn5-mediated optogenetics was tested. Astrocytes represent an important population of non-excitable cells in the central nervous system, and optogenetic tools have so far achieved limited success in this population. ATP is known to be the messenger for communication between astrocytes; however, intracellular Ca2+... 2+ The real-time effects on ATP release have not yet been visualized. The recently reported GPCR-activated ultrasensitive ATP sensor GRAB was utilized. ATP This was used to monitor changes in extracellular ATP levels. Specifically, after injection of an AAV vector containing the GfaABC1D promoter (which is commonly used for driver gene expression in astrocytes), cOpn5 and GRAB were expressed in the mouse S1 sensory cortex. ATP sensor.

[0071] GRAB from head-fixed, awake, and behaviorally normal mice ATP The signal is used for two-photon imaging. Notably, the 920nm light projected for imaging itself expresses cOpn5 and GRAB... ATPNumerous ATP flashes were triggered in mice, but not in mice expressing ATP sensors but lacking cOps5 expression. Blue light pulses were not required to stimulate ATP signaling. The diameter of a single ATP flash typically ranged from 20 to 100 μm and lasted for approximately 1 minute. After an initial resting period of approximately 1 minute, the flash frequency gradually increased, peaking within approximately 5 minutes in the imaging region (640 × 640 μm). 2 The rate reached approximately 50 flashes per minute. Furthermore, high-frequency ATP flashes were observed in repeated experiments. In GRAB expression alone... ATP In mice, sporadic ATP events were observed (~0.3 flashes / min within the imaging region); 8 hours after intraperitoneal injection of lipopolysaccharide (LPS) for pro-inflammatory treatment, ATP flash events increased to nearly 6 times the baseline level (~2 flashes / min), but exhibited a fairly stable frequency, confirming that inflammation induces ATP release in the brain. Given that the ATP flash frequency observed in cOpn5-expressing mice was ~25 times higher than that in non-Opn5-expressing mice after pro-inflammatory treatment, this confirms that cOpn5-mediated photoactivation of astrocytes induces a continuous and substantial release of ATP in vivo.

[0072] Astrocytes release ATP, and other glial neurotransmitters also act on neuronal receptors to regulate neuronal activity. Using cOpn5-mediated astrocyte activation, neuronal Ca2+ activity was modulated in head-fixed, awake, and behaviorally normal mice. 2+ Two-photon imaging was performed using the signal. Cells expressing cOpn5 (n=406) co-localized with GFAP staining (n=397), but not with neurons expressing GCaMP7b. Original follow-up cases and cohort data showed that cOpn5-mediated astrocyte activation significantly enhanced neuronal activity at 15–20 min compared to 0–5 min. cOpn5 is strictly expressed in astrocytes, as confirmed by the co-localization of cOpn5-expressing cells with GFAP staining signals (which differ from the GCaMP7b signal in neurons). This confirms that cOpn5-mediated photoactivation of astrocytes enhances the activity of peripheral neurons in vivo. Furthermore, this invention demonstrates that long-wavelength (920 nm) light in the pulsed laser used for two-photon imaging can activate cOpn5, suggesting that cOpn5 may possess two-photon optogenetics.

[0073] cOpn5 optogenetics activates neurons and modulates animal behavior.

[0074] The application of cOpn5-mediated optogenetics in neurons was explored. First, it was examined whether cOpn5 could mediate light-induced Ca2+. 2+Signal. Using AAV and the panneuronal SYN promoter, cOpn5 and genetically encoded Ca2+ were expressed in mouse cortical neurons. 2+ Sensor jRGECO1a( Figure 8 a). Blue light pulses (10 s; 100 μW / mm) were applied to brain slice samples. 2 (473nm) reliably induces Ca in neurons 2+ Transient ( Figure 8 b、 Figure 8 c). Therefore, cOpn5 can also be photoinduced to activate neurons.

[0075] The effects of light-induced cOpn5 activation on the electrophysiological properties of neurons in slice samples from the motor cortex, hippocampus, and dorsal striatum were investigated. Figure 8 d). Two types of activation modes were observed. In most recorded neurons, blue light pulses induced small depolarization currents (~20 pA) in a voltage-clamp mode and delayed but intense firing of action potentials in a current-clamp mode. Figure 8 e, left side, n = 12 neurons). Under higher frequency light pulses, cOpn5 drives more spike potentials after the initial light pulse, with a shorter latency (~5s to ~3s), while the inward current is not significantly affected. Figure 9 a). In another neuronal subpopulation, brief light pulses rapidly evoke strong inward currents (100-1000 pA) and drive bursting firing of action potentials. Figure 8 e, right side, n=6 neurons). Neurons stimulated repeatedly at 10Hz, 10ms / pulse, exhibited a non-decaying firing rate pattern in repeated light stimulation trials. Figure 9 (b) It is worth noting that, unlike those generated by ChR2 optogenetics, the action potentials elicited by cOpn5 photostimulation are not time-synchronized with the light pulses.

[0076] Finally, the use of cOpn5-mediated optogenetics in regulating animal behavior was evaluated. The lateral hypothalamus (LH) is a brain center known to function in reward processing and feeding behavior. We expressed cOpn5 in LH GABAergic neurons of VGAT-Cre mice and implanted optical fibers to deliver light pulses to the LH of mice in free-behavioral environments. Figure 8 g). This relates to previously discovered activation of LH GABA neurons driving feeding behavior. 56Consistently, light stimulation (20 Hz; 5 ms / pulse; 473 nm; 0.75 mW, output from the fiber tip) induced a significant increase in food intake in cOpn5-expressing mice, but not in EGFP-expressing control mice. Figure 8 h). The role of cOpn5-mediated optogenetic activation of GABA neurons in the zone of indeterminate origin (ZI) was also tested using a foraging behavior task. Figure 8 i), where the indeterminate band is a region known to drive compulsive feeding. Compared with mice expressing EGFP, mice expressing cOpn5 showed a significant increase in the time spent foraging for high-fat food particles after repeated light stimulation. Figure 8 j). Electrophysiological recordings were performed on cOpn5-expressing neurons in LH and ZI to characterize their response spectra. The injection site and placement of the optical fiber were confirmed using whole-brain slices. Figure 10 (a-10c). Notably, the mice maintained the aforementioned behavior (eating behavior or foraging for high-fat foods) when the lights were on and immediately ceased this behavior when the lights were off. Therefore, cOpn5 effectively, rapidly, accurately, and reversibly modulates the behavioral state of animals.

[0077] Here, the present invention confirms that Opn5 of the present invention serves as a means for activating G. q The use of optogenetics as an extremely efficient tool for signal transduction and / or cell activation. Previous studies have characterized mammalian Opn5 as a UV-sensitive G... i Coupled with opsins; we present a surprising finding that visible blue light can induce rapid Ca2+ uptake in mammalian cells expressing Opn5 (e.g., cOpn5 or tOpn5). 2+ Transient, IP1 accumulation, and PKC activation. The Opn5 (particularly cOpn5) of this invention effectively mediates photoelicited strong ATP release and enhances neuronal activity in mouse astrocytes in vivo. This invention also demonstrates that Opn5, particularly cOpn5, allows for rapid, robust, and reversible optical activation of neurons and its application to the selective regulation of animal behavior. Importantly, the Opn5 (e.g., cOpn5) of this invention is a robust and easy-to-use single-component system that does not require exogenous chromophores. This invention envisions that Opn5-based optogenetics, such as cOpn5-based optogenetics, will be a method for studying the effects of G1 on neuronal activity in both non-excitable and excitable cells. q Techniques for coupled signal transduction and / or activation of important physiological and behavioral functions regulated by cells.

[0078] Table 6 lists the enabling characteristics of cOpn5 by directly comparing its response amplitude, photosensitivity, temporal resolution, and requirement for additional chromophores with other optogenetic tools. For cells expressing cOpn5, only 16 μW / mm... 2 A 10ms blue light pulse of high intensity can awaken Ca. 2+ The signal increases rapidly, with a peak amplitude of 3–8 ΔF / F. In contrast, prior to this invention, activation of opto-a1AR or mammalian Opn4 (two proposed optogenetic tools for Gq signal transduction) has been shown to require ~3 times higher light intensity (7–40 mW / mm²). 2 (and prolonged light exposure (20–60 s), and only weak Ca2+ is produced) 2+ The signal (0.25–0.5 ΔF / F) is observed. Therefore, opto-a1AR or mammalian Opn4 cannot mimic the rapid activation spectrum of endogenous Gq-coupled receptors, which typically trigger strong Gq signaling subseconds after application of their respective ligands. The striking physiological and behavioral effects exhibited in vivo in response to cOpn5-mediated optogenetic activation of astrocytes and neurons confirm the optogenetic capabilities of cOps5. In contrast, recent systematic characterization has shown that opto-a1A and Opn4-mediated optogenetic stimulation does not increase Ca2+. 2+ The amplitude of the signal, and even after prolonged exposure, only slightly adjusts Ca. 2+ The frequency of transient and synaptic events (Gerasimov et al., 2021; Mederos et al., 2019). By overcoming the limitations in photosensitivity, temporal resolution, and response amplitude associated with opto-a1AR and Opn4-mediated optogenetics, the Opn5 of this invention (especially cOpn5 or tOpn5) will be used in the study of G in a large number of cells and tissues. q It has broad applicability in the mechanisms and functions of signal transduction and / or cell activation.

[0079] Optogenetics based on Opn5 (particularly cOpn5 or tOpn5) of this invention also offers advantages in terms of safety and convenience. Although Opn5 from many species has been reported to be UV-responsive (Kojima et al., 2011), cOpn5 is most suitable for activation by 470 nm blue light, which has better penetration than UV and avoids UV-related cytotoxicity. Its hypersensitivity to light also minimizes potential heating artifacts. It can be activated by two-photon using long-wavelength (≥920 nm) light, suggesting its suitability for activation of deeper tissues using pulsed lasers. cOpn5 or tOpn5 is strongly and reproducibly activated by light without the need for exogenous retinaldehyde, likely because cOpn5 or tOpn5 is a bistable opsin covalently bound to endogenous retinaldehyde, thus exhibiting resistance to photobleaching (Koyanagi and Terakita, 2014; Tsukamoto and Terakita, 2010). In contrast, Opn4 requires additional retinaldehyde for mammalian experiments and has a long response time and low photosensitivity. The Opn5 of this invention (particularly cOpn5 or tOpn5) is particularly useful as a single-component system for in vivo studies because it avoids the burden of delivering the compound to tissues during experiments.

[0080] Compared to chemogenetics and uncaging tools, the optogenetics of Opn5 of this invention (especially cOpn5 or tOpn5 optogenetics) offers several important advantages. It is far more precise in time and provides spatial resolution for single cells and even subcellular structures. Although CNO / hM3Dq-mediated chemogenetics has been used to study the physiological and behavioral functions of non-excitable cell types, such as astrocytes in the brain (Agulhon et al., 2013; Shen et al., 2021), its in vivo use typically requires several minutes for CNO to reach its target cells and tissues. The diffuse nature of the compound suggests that it provides spatial resolution for G at both cellular and subcellular levels. q Signal transduction via chemogenetic stimulation is virtually impossible. The Opn5 of this invention (particularly cOpn5 or tOpn5) also differs from cage-locking compound-based "cage-breaking" tools (e.g., cage-locking calcium and cage-locking IP3) because these tools require pre-loading of compounds and only partially mimic G. q Signal transduction and / or activation of cell-related calcium 2+Related pathways. Other "unlocking" tools exist, such as cage-locking glutamate and cage-locking ATP targeting endogenous GPCRs (Ellis-Davies, 2007; Lezmy et al., 2021). However, these cage-locking compounds require introduction into extracellular media or intracellular cytoplasm, which limits their application in behavioral animals (Adams and Tsien, 1993b). The advantages of the Opn5 of this invention (especially cOpn5 or tOpn5) over "unlocking" tools have been clearly demonstrated experimentally. The Opn5 of this invention (especially cOps5 or tOpn5) is similar to existing ChR2-based tools and can be used just as conveniently to regulate animal behavior.

[0081] The optogenetics of Opn5 (especially cOps5 or tOpn5) of this invention are particularly suitable for the precise activation of intracellular G cells. q Signal transduction and / or cell activation, which subsequently triggers the release of intracellular calcium. 2+ Release and activate PKC. The Opn5 of this invention (particularly cOpn5 or tOpn5) differs from current channel-based optogenetic tools, such as ChR2 or its variants that transfer cations across the plasma membrane. ChR2 and its variants have made significant contributions to dissecting neural circuits by controlling cell membrane potentials, thereby controlling action potential firing; however, their success is more limited in studying non-excitable cells lacking active ion channels that generate action potentials (Gourine et al., 2010). In addition to its application to non-excitable cells, the optogenetics of the Opn5 of this invention (particularly cOpn5 or tOpn5) can also stimulate G in neurons. q Signal transduction and / or activation of neurons, controlling animal behavior in a circuit-dependent manner. Notably, Gq-coupled GPCRs may indiscriminately recruit G proteins and generate variable downstream signal transduction in receptor- and cell-specific ways. In fact, we have observed that the same illumination parameters produce different activation patterns in different neurons. The Opn5-mediated optogenetic activation of this invention (particularly cOpn5 or tOpn5) does not produce precisely time-locked action potential bursts in neurons like ChR2. This can be useful because it avoids artificially synchronizing neuronal activation. However, if precise temporal control of action potential bursts is required, we recommend using ion channel-based optogenetic tools.

[0082] Beyond technological advancements, our findings also reveal functional implications for signal transduction and / or activation in astrocytes in vivo. Although ATP is considered a crucial glial neurotransmitter, previous studies have revealed multiple release mechanisms that depend on processing methods, including extracellular Ca2+. 2+The presence of G in cells and tissues, and the exact form of sample preparation (Figueiredo et al., 2014; Hamilton and Attwell, 2010). Furthermore, the presence of ATP release is usually monitored indirectly. It is unclear whether activation of astrocytes triggers ATP release, and if so, how this release manifests in vivo. Here, we demonstrate for the first time that stimulation of G in astrocytes... q Signal transduction pathways trigger massive ATP release in the form of ATP flashes. The optogenetics of Opn5 (particularly cOpn5 or tOpn5) of this invention provides an ideal technique for studying the molecular and cellular mechanisms of ATP release. In addition to ATP, astrocyte activation leads to the release of other glial neurotransmitters such as D-serine, glutamate, and GABA. ATP can also be converted into other metabolites, such as adenosine. Glial neurotransmitters and their metabolites can exert complex regulatory effects on neuronal excitability and synaptic strength. For example, while ATP can activate neurons through various P2X and P2Y receptors, adenosine strongly inhibits neurons through A1 receptors (Lezmy et al., 2021; Zhang et al., 2003). It is unclear how these different effects integrate to regulate neuronal activity in vivo. Here, we reveal that optogenetic activation of astrocytes expressing cOpn5 significantly stimulates neurons in the mouse S1 cortex. Therefore, the optogenetics of Opn5 (especially cOpn5 or tOpn5) of this invention lays the foundation for dissecting the molecular, cellular, and circuit mechanisms of the complex interactions between astrocytes and neurons. In vivo experiments have also demonstrated that the Opn5 (especially cOpn5 or tOpn5) of this invention, in conjunction with optogenetic probes and imaging sensors (e.g., genetically encoded Ca2+), 2+ Indicators and GPCR-based neurotransmitter sensors are compatible. The Opn5 of this invention (especially cOpn5 or tOpn5), together with these sensors, potentially allows for the instantaneous activation of GPCRs using an all-optical approach. q Signal transduction and / or cell activation, while simultaneously monitoring related effects.

[0083] In summary, this invention demonstrates that the Opn5 of this invention (particularly cOpn5 or tOpn5), as a blue photosensitive opsin, can rapidly, reversibly, and precisely activate Gq signaling and / or activate cells. This invention also establishes the Opn5 of this invention (particularly cOpn5 or tOpn5) as a powerful and easy-to-use optogenetic tool for activating non-excitable cells and neurons. Given Gq... q The importance of conjugated GPCRs, with cOpn5 expected to play a crucial role in analyzing G in all major cell types and tissues. q It has a wide range of applications in terms of signal transmission mechanisms and functions.

[0084] Descriptions of specific implementation methods and embodiments are provided for illustrative purposes and not for limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

[0085] Example

[0086] Materials and methods:

[0087] Table 1: Primers used for cloning

[0088]

[0089]

[0090] Table 2: Recombinant DNA

[0091] EGFP-CAAX Gift from Li Yulong pLJM1-EGFP Addgene plasmid #19319 pAAV-GfaABC1D-hM3D(Gq)-mCherry Addgene plasmid #50478 pAAV-EF1a-DIO-eGFP-WPRE-pA N / A pAAV-hSyn-GOI N / A pLJM1-cmv-cOpn5 N / A pLJM1-cmv-tOpn5 N / A pLJM1-cmv-hOPN5 N / A pLJM1-cmv-mOpn5 N / A pLJM1-cmv-V5-Opn5 N / A pLJM1-cmv-cOpn5-T2A-eGFP N / A PAAV-hSyn-cOpn5-T2A-eGFP-WPR-pA N / A PAAV-GfaABC1D-cOpn5-T2A-eGFP-WPR-pA N / A pAAV-EF1a-DIO-cOpn5-T2A-eGFP-WPRE-pA N / A PAAV-GfaABC1D-cOpn5-T2A-mCherry-WPR-pA N / A

[0092] Table 3: Virus strains

[0093]

[0094] Table 4: Photoexcitation Sources

[0095]

[0096] Table 5: Microscope Equipment

[0097]

[0098] Table 6: Statistical Analysis

[0099]

[0100]

[0101] Example 1. cOpn5-mediated G q Optogenetic activation of signal transduction

[0102] The study tested whether heterologous expression of Opn5 orthologs from chickens, turtles, humans, and mice (which share 80-90% protein sequence identity) could mediate blue light-induced Gamma globulin production in HEK 293T cells. q The ability to activate signal transduction ( Figure 1 (a and Table 7). Stimulation was performed using blue light, with the red intracellular calcium indicator Calbryte used. TM 630AM dye to monitor relative Ca 2+ response( Figure 1 b). Opn5 orthologs from chickens (cOpn5) and turtles (tOpn5) mediate Ca2+.2+ The signal showed an immediate and strong light-induced increase (~3ΔF / F), while no light effect was observed in cells expressing human or mouse Opn5 orthologs. Figure 1 d and Figure 2 a, Figure 2 b). As exemplified by chicken orthologs, cOpn5 colocalizes with the EGFP-CAAX membrane marker, indicating its efficient transport to the plasma membrane. Figure 1 c). No exogenous retinol was added to the culture medium, indicating that endogenous retinol was sufficient for cOpn5 to function. 2+ Signals on extracellular Ca 2+ The removal of Ca exhibits tolerance, thus indicating that Ca... 2+ Release from intracellular storage ( Figure 2 c). YM-254890 (High Selectivity G) q protein inhibitors 33 Pre-incubation reversibly abolished light-induced Ca2+ in two cOpn5-expressing cells. 2+ Transient ( Figure 1 e). In cells expressing cOpn5 (but not human OPN5), a light-induced increase in the level of inositol phosphate (IP1), a rapid degradation product of IP3, was detected; furthermore, treatment with YM-254890 reduced the extent of this increase. Figure 1 f and Figure 2 d). In HEK 293T cells expressing cOpn5, blue light also triggers the activation of the MARCKS protein (a recognized target of PKC) in a PKC-dependent manner. 34 phosphorylation of ) Figure 1 g and Figure 2 e). In contrast, blue light irradiation effectively reduced cAMP levels in retinaldehyde-expressing human and mouse Opn5 cells, but this effect was not observed in retinaldehyde-expressing cOpn5 cells. Figure 2 f). In summary, these data support the conclusion that blue light irradiation can enhance cOpn5 and G in HEK 293T cells. q Signal conduction path coupling.

[0103] Table 7: Opsins and Species

[0104] Chicken Opn5 cOpn5 Red junglefowl (Gallus gallus) GenBank NM_001130743.1 Sea turtle Opn5 tOpn5 Green sea turtle (Chelonia mydas) GenBank XM_007068312.4 Opn5 hOPN5 Homo sapiens GenBank AY377391.1 Mouse Opn5 mOpn5 Mouse (Mus musculus) GenBank NM_181753.4

[0105] Figure 1 This demonstrates the role of cOpn5 in mediating light-induced G in HEK 293T cells. q Strong activation of signal transduction.

[0106] a. A hypothetical schematic diagram of intracellular signaling in response to light-induced cOpn5 activation. PLC: Phospholipase C; PIP2: Phosphatidylinositol-4,5-bisphosphate; IP3: Inositol-1,4,5-triphosphate; IP1: Inositol monophosphate; DAG: Diacylglycerol; PKC: Protein kinase C; YM-254890: A selective G... q Protein inhibitors.

[0107] b. In HEK 293T cells expressing Opn5 from three species (Gallus gallus, Homo sapiens, and Musmusculus), under blue light stimulation (10 s; 100 μW / mm²), 2 Before and after Ca (488nm) 2+ A pseudo-color image of the signal. Scale bar, 10 μm.

[0108] c. In HEK 293T cells, Cy3-counted V5-cOpn5 fusion protein (red) co-localizes with the membrane tag EGFP-CAAX (green). DAPI counterstaining (blue) indicates the cell nucleus. Scale bar, 10 μm.

[0109] Figures d and c show the light-evoked Ca2+ in cells. 2+ The time-course variation of the signal.

[0110] e, G q The protein inhibitor YM-254890 (10 nM) reversibly blocks cOpn5-mediated light-induced Ca2+. 2+ Signal.

[0111] f, In HEK 293T cells expressing cOpn5, YM inhibition was achieved by continuous light stimulation (3 min; 100 μW / mm). 2 IP1 accumulation evoked at 470nm (left). ***P<0.0001, *P=0.0128; Tukey's multiple comparison test.

[0112] g. Phosphorylation of MARCKS in cOpn5-expressing HEK 293T cells in the control group (no light stimulation), the light-stimulated group, and the light + staphylococcalin (ST, a PKC inhibitor) group. The amount of p-MARCKS in each fraction was normalized relative to the amount of α-tubulin. **P = 0.0096, ***P = 0.0004; Tukey's multiple comparison test.

[0113] Figure 2 This shows cOpn5 coupled to G q Signal conduction, but not coupled to G i Signal Transduction

[0114] a, In HEK 293T cells expressing Opn5 from a sea turtle species (Chelonia mydas), under blue light stimulation (10 s; 100 μW / mm²), 2 Before and after Ca (488nm) 2+ A pseudo-color image of the signal. Scale bar, 10 μm (left); Ca2+ in response to light-evoked signals in cells. 2+ The time history variation of the signal (right).

[0115] b. The Gq protein inhibitor YM-254890 (10 nM) reversibly blocks cOpn5 and turtle Opn5-mediated light-induced Ca2+. 2+ Group data of the signal. ****P<0.0001, unidirectional ANOVA. Error bars represent SEM.

[0116] c, in the absence of extracellular Ca 2+ Under these conditions, Ca generated using light stimulation (10 ms; 16 μW / mm²; 470 nm) 2+ The time-course variation of the signal.

[0117] d, IP1 accumulation in HEK 293T cells expressing human Opn5 with or without light stimulation (right). ns, no significant difference; unpaired t-test.

[0118] e. A representative result of MARCKS phosphorylation in cOpn5-expressing HEK 293T cells in the control group (no light stimulation), light stimulation group, and light + staphylococcalin group. The amount of p-MARCKS in the same fraction was normalized relative to the amount of α-tubulin.

[0119] f. In the absence of retinal supplementation in the culture medium, light had no effect on cAMP levels in HEK 293T cells expressing cOpn5 (pre-incubation with 10 μM retinal) (left panel). The right panel shows the effect of light stimulation on cAMP concentrations in HEK 293T cells expressing four different species of Opn5 after pre-incubation with 10 μM retinal.

[0120] The error bars in d and f represent SEM.

[0121] Example 2. cOpn5-mediated optogenetics is sensitive and precise.

[0122] The photoactivation properties of cOpn5 heterologously expressed in HEK 293T cells were characterized. Although Opn5 was previously thought to be a UV-sensitive photoreceptor... 27 However, at a fixed light intensity (100 μW / mm²), 2The following plot, using a set of wavelengths in the 365–630 nm range, reveals that blue light at 470 nm induces the strongest Ca2+. 2+ Transient, while UVA light (365 and 395 nm) is less efficient, and longer wavelengths of visible light (561 nm or above) are completely ineffective. Figure 3 a). The effects of different light durations on HEK 293T cells expressing cOpn5 were tested. Short light pulses (1, 5, 10, 20, 50 ms; 16 μW / mm) were used. 2 ;470nm) stimulation, showing that Ca 2+ The response reaches saturation mode ( Figure 3 b). At this light intensity (16 μW / mm) 2 At 470 nm, longer illumination duration did not further improve Ca 2+ Signal amplitude ( Figure 4 a) Display using 470nm light at different intensities, ~4.8μW / mm 2 and 16μW / mm 2 Blue light produces approximately half and maximum responses, respectively. Figure 3 c and Figure 4 b). Therefore, the photosensitivity of cOpn5 is 3–4 orders of magnitude higher than that of reported photosensitivity Gq-coupled GPCRs, and even 2–3 orders of magnitude higher than that of the commonly used optogenetic tool channel rhodopsin-2 (ChR2) (Lin, 2011; Zhang et al., 2006) (Table 8). In summary, these results indicate that cOpn5 can function as a single-component optogenetic tool without additional retinal, and that cOpn5 is hypersensitive to blue light because its complete activation requires low light intensity (16 μW / mm). 2 ) and short duration (10ms).

[0123] Table 8: Comparison of cOpn5 with other optogenetic tools

[0124]

[0125]

[0126]

[0127] The performance of cOpn5 was compared with that of opto-a1AR, where opto-a1AR is a product of the interaction between rhodopsin and G... q Chimeric GPCRs engineered with conjugated adrenergic receptors. Following previously reported protocols. 14 It was found that a very long exposure to strong radiation (60s; 7mW / mm) was required.2 Only then can slow and small (~0.5ΔF / F) Ca2+ activation be triggered in HEK 293T cells expressing opto-a1AR. 2+ Signal increase, 15s of irradiation is ineffective. Figure 4 c. Figure 4 d). The performance of cOpn5 was compared with that of opn4, a natural opsin reportedly used as a tool for Gq signaling activation. It was found that prolonged exposure to strong irradiation (25 s; 40 mW / mm²) was required. 2 Only with additional retinaldehyde can slow (~1ΔF / F) Ca2+ activation be triggered in HEK 293T cells expressing opn4. 2+ Signal increase ( Figure 4 e Figure 4 f). Therefore, compared with existing opsin-based tools (opto-a1AR and opn4), cOpn5 has much higher photosensitivity (by ~3 orders of magnitude), requires much shorter exposure time (10 ms vs. 60 s), and produces a stronger response.

[0128] Furthermore, the performance of cOpn5 is compared with that of popular G... q The performance of the coupled chemogenetic tool hM3Dq was compared, which is activated by adding the exogenous small molecule ligand clozapine-N-oxide (CNO). 37-39 The light-induced activation of HEK 293T cells expressing cOpn5 and the CNO-induced activation of HEK 293T cells expressing hM3Dq have similar Ca2+ values. 2+ The peak response amplitude of the signal. Meanwhile, compared to HEK 293T cells expressing hM3Dq, HEK 293T cells expressing cOpn5 exhibited a faster and more time-precise response, as well as a faster recovery time. Figure 4 g~ Figure 4 i). These results indicate that cOpn5-mediated optogenetics is more controllable in terms of temporal accuracy than hM3Dq.

[0129] cOpn5 optogenetics allows for precise spatial control of cellular activity. Restricting a brief light stimulus (63 ms) to a subcellular region of a single cOpn5-expressing HEK 293T cell resulted in immediate activation of that single cell. Interestingly, in regions of high cell aggregation, Ca2+... 2+ The signal propagates to surrounding cells, suggesting intercellular communication between HEK 293T cells via an unidentified mechanism. Figure 3 d、 Figure 3e). These findings were extended to primary cell cultures. In primary astrocyte cultures prepared from neonatal mouse brains, cOpn5 was expressed using an AAV vector for bicistronic expression of cOpn5 and the EGFP marker protein. Figure 5 a) Monitoring Ca using Calbryte 630AM dye. 2+ At the level, it was found that irradiation of astrocytes expressing cOpn5 with blue light produced a strong Ca2+. 2+ Transient (~8ΔF / F)( Figure 5 b、 Figure 5 c). When the light stimulation (63 ms) was precisely restricted to a subcellular region of a single cOpn5-expressing astrocyte, Ca was observed. 2+ Signals propagate within a single cell ( Figure 3 f). Similar to the test in HEK 293T cells, Ca was observed. 2+ The signal propagates in a wave-like pattern from the stimulated astrocytes to more distant, unstimulated astrocytes. Figure 3 g、 Figure 3 Therefore, these experiments confirm that cOpn5 optogenetics allows for precise spatial control and demonstrate the dynamics of astrocyte networks initially discovered using neurochemical and mechanostimulation. 40,41 It might be useful.

[0130] Figure 3 The display shows that cOpn5 mediates G sensitively with high temporal and spatial resolution. q Optical control of signal transmission.

[0131] a. Selected wavelengths (365, 395, 470, 515, 561, 590, and 630 nm; left figure) and the response of HEK 293T cells expressing cOpn5 to light stimulation at different wavelengths. 2+ Signal amplitude (2s; 100μW / mm) 2 (See right figure) Schematic diagram. Error bars represent SEM.

[0132] b, at different light stimulation durations (1, 5, 10, 20 or 50 ms; 16 μW / mm) 2 The response amplitude at 470 nm. The error bars represent the SEM.

[0133] c, Ca mediated by cOpn5 under different light intensities 2+ The time-history variation of the signal (0, 4.8, 8, 16 or 32 μW / mm) 2 ; 10ms; 470nm; 16μW / mm for 10ms 2For stimulation, 10% peak activation = 1.36 ± 0.55 s; 90% peak activation = 2.37 ± 0.87 s; decay time τ = 18.66 ± 4.98 s, mean ± SEM; n = 10 cells).

[0134] d, Photoinduced CaOpn5 levels in HEK 293T cells expressing cOpn5 (63 ms; 17 μW; arrow points to stimulation area) 2+ Image of signal propagation. Scale bar, 10 μm.

[0135] e, showing the Ca of d 2+ A pseudo-color image of the signal propagation process across time (frames N / (N-1)>1). The frame interval is 500ms, and the count is performed once per frame.

[0136] f, light-induced Ca2+ in single cOpn5-expressing primary astrocytes stimulated in subcellular regions. 2+ Image of signal propagation (stimulus size 4×4μm) 2 (Frame interval is 300ms). Scale bar: 10μm.

[0137] g, in primary astrocytes expressing cOpn5, light-induced Ca 2+ Image of signal propagation. Scale bar, 10 μm.

[0138] h, showing the Ca of g 2+ A pseudo-color image of the signal propagation process across time (frames N / (N-1)>1). The frame interval is 500ms, and the count is performed once per frame.

[0139] Figure 4 It was shown that cOpn5 mediates a faster and more sensitive response to light than opto-a1AR, hM3Dq, or opn4.

[0140] a, using optical pulses (16μW / mm) 2 ; 470nm; 1, 5, 10, 20 or 50ms) Ca 2+ The time-course variation of the signal.

[0141] b, at different light intensities (0, 4.8, 8, 16 or 32 μW / mm) at 10 ms and 470 nm. 2 The response amplitude under ).

[0142] c, In HEK 293T cells expressing opto-a1AR, baseline and peak Ca 2+ Pseudo-color image of the signal (ΔF / F0). The culture medium buffer contains 10 μM all-trans retinaldehyde. Scale bar, 30 μm.

[0143] d, In HEK 293T cells expressing opto-α1AR, 60s light stimulation had an effect on Ca2+. 2+ Effects of 15s light stimulation on Ca (n=15 cells; top figure) 2+ The signal is unaffected (see image below).

[0144] e, in HEK 293T cells expressing human OPN4, baseline and peak Ca 2+ Pseudo-color image of the signal (ΔF / F0). The culture medium buffer contains 10 μM all-trans retinaldehyde. Scale bar, 30 μm.

[0145] f, In HEK 293T cells expressing OPN4, 25s of light stimulation with 10 μM ATR resulted in the reduction of Ca2+. 2+ The effect (n=12 cells; red line) on Ca in the absence of ATR 2+ The signal is unaffected (black image).

[0146] g, In HEK 293T cells expressing cOpn5, light stimulation had an effect on Ca 2+ The effect of the signal. The top figure shows pseudo-color images of the baseline and peak responses. The bottom figure shows the Ca2+ mediated optogenetic stimulation elicited in cOpn5-expressing HEK 293T cells across five consecutive trials. 2+ Heatmap of the signal. Scale bar, 20 μm.

[0147] h, in HEK 293T cells expressing hM3Dq, chemogenetic stimulation affects Ca 2+ The influence of the signal.

[0148] i, Ca2+ induced by cOpn5-mediated optogenetic stimulation (10s) and hM3Dq-mediated chemogenetic stimulation with a small amount of CNO (100 nM; 10s), respectively. 2+ The time-course variation of the signal.

[0149] Figure 5 This demonstrates that cOpn5 effectively mediates the activation of astrocytes.

[0150] a. cOpn5 was expressed in cultured primary astrocytes using AAV-cOpn5-T2A-EGFP (green). Astrocytes were identified by GFAP immunostaining (red). Scale bar, 20 μm.

[0151] b, Baseline and peak Ca2+ levels in astrocytes expressing cOpn5 after light stimulation. 2+ A pseudo-color image of the signal. Scale bar, 20 μm.

[0152] c, Ca2+ The signal graph and the Ca throughout the experiment 2+ Heatmap of the signal (n=25 cells).

[0153] Example 3: Optogenetic activation of cOpn5 in astrocytes induces massive ATP release and neuronal activation in vivo.

[0154] The performance of cOpn5-mediated optogenetics in vivo was tested. Astrocytes represent an important population of non-excitable cells in the central nervous system, and optogenetic tools have so far achieved limited success in this population. 42 ATP is known to be the messenger for communication between astrocytes; however, intracellular calcium... 2+ The real-time effects on ATP release have not yet been visualized. The GRAB, an ultrasensitive ATP sensor based on GPCR activation, was utilized. ATP This was used to monitor changes in extracellular ATP levels. Specifically, after injection of an AAV vector containing the GfaABC1D promoter (which is commonly used for driver gene expression in astrocytes), cOpn5 and GRAB were expressed in the mouse S1 sensory cortex. ATP sensor( Figure 6 a).

[0155] GRAB from head-fixed, awake, and behaviorally normal mice ATP Two-photon imaging of signals ( Figure 6 a) Initially, it was anticipated that, in addition to the 920 nm pulsed laser light used for two-photon imaging, blue light pulses would also be needed to stimulate ATP signaling. Notably, the 920 nm light projected for imaging itself expresses cOpn5 and GRAB. ATP Numerous ATP flashes were triggered in mice with ATP sensor expression but lacking cOps5 expression, but no ATP flashes were triggered in mice with ATP sensor expression but lacking cOps5 expression. The diameter of a single ATP flash typically ranged from 20 to 100 μm and lasted for approximately 1 minute. After an initial resting period of approximately 1 minute, the flash frequency gradually increased, reaching a peak within approximately 5 minutes in the imaging region (640 × 640 μm). 2 The speed reaches approximately 50 flashes per minute. Figure 6 b、 Figure 6 c and Figure 7 a). Furthermore, high-frequency ATP flashes were also observed in repeated experiments ( Figure 7 b). In the separate expression of GRAB ATPIn mice, sporadic ATP events were observed (~0.3 flashes / min within the imaging area); 8 hours after intraperitoneal injection of lipopolysaccharide (LPS) for pro-inflammatory treatment, ATP flash events increased to nearly 6 times the baseline level (~2 flashes / min), but exhibited a fairly stable frequency, confirming that inflammation induces ATP release in the brain. Given that the ATP flash frequency observed in cOpn5-expressing mice was ~25 times higher than that in non-Opn5-expressing mice after pro-inflammatory treatment... Figure 6 d、 Figure 7 c) confirmed that cOpn5-mediated photoactivation of astrocytes induces a continuous release of large amounts of ATP in vivo.

[0156] Astrocytes release ATP, and other glial neurotransmitters also act on neuronal receptors to regulate neuronal activity. Using cOpn5-mediated astrocyte activation, neuronal Ca2+ in head-fixed, awake, and behaviorally normal mice was analyzed. 2+ Two-photon imaging of signals ( Figure 6 e). Cells expressing cOpn5 (n=406) colocalized with GFAP staining (n=397), but did not colocalize with neurons expressing GCaMP7b. Figure 6 f). Original tracking instance and cohort data showed that cOpn5-mediated astrocyte activation significantly improved neuronal activity at 15–20 min compared to 0–5 min. Figure 6 g、 Figure 6 h and Figure 7 d) cOpn5 is strictly expressed in astrocytes, as confirmed by co-localization of cOpn5-expressing cells with GFAP staining signals (which differ from the GCaMP7b signal in neurons). We demonstrated that cOpn5-mediated photoactivation of astrocytes enhances the activity of peripheral neurons in vivo. Furthermore, our data show that long-wavelength (920 nm) light in pulsed lasers used for two-photon imaging can activate cOpn5, suggesting that cOpn5 may possess two-photon optogenetics.

[0157] Figure 6 The study showed that cOpn5-mediated activation of astrocytes induced significant ATP flashes and neuronal activation in vivo.

[0158] a. Schematic diagram of the experimental setup for in vivo two-photon imaging (920 nm) of ATP release following cOpn5-mediated astrocyte activation. The image shows cOpn5 (red) expression in astrocytes and GRAB in the mouse S1 cortex. ATP Expression of the sensor (green) in astrocytes. Scale bar, 100 μm.

[0159] b. Changes in the number of ATP flash events in astrocytes over time (0–10 min) in control mice (without cOpn5 expression), LPS-treated mice (without cOpn5 expression), and mice expressing cOpn5 (right side).

[0160] c. Total ATP flash events in control mice (without cOpn5 expression), LPS-treated mice (without cOpn5 expression), and mice expressing cOpn5. The left column shows the original GRAB at basal levels (before photodelivery). ATP The image, with the middle column showing GRAB at 5 minutes. ATP The right column shows the pseudo-color encoded ATP flash events accumulated during the 0-20 min period.

[0161] d, corresponding to the data shown in c, is a raster plot of ATP flash events in astrocytes over time.

[0162] e. Schematic diagram of the experimental setup used for in vivo two-photon imaging (920 nm) of neuronal calcium imaging following cOpn5-mediated astrocyte activation. The image shows the expression of cOpn5 (red) and GCaMP7b (green) in astrocytes within the mouse S1 cortex. Scale bar, 100 μm.

[0163] f, Cells expressing cOpn5 (red) co-localize with GFAP cells counterstained with 647nm dye (purple), and cells expressing GCaMP7b (green) are neurons. 406 red cells and 397 purple cells, scale bar, 100μm.

[0164] g, time traces of 10 neurons expressing GCaMP7b in the 0–5 min and 15–20 min time periods, coupled with cOpn5-mediated astrocyte activation.

[0165] Analysis of calcium events in neurons expressing GCaMP7b during 0–5 min and 15–20 min, coupled with cOpn5-mediated astrocyte activation. N = 193 neurons, ****P < 0.0001, unpaired t-test.

[0166] Figure 7 This study demonstrated that cOpn5 mediates sustained and reliable ATP release in astrocytes and activation of surrounding neurons.

[0167] a, An example of astrocyte ATP flashing events detected in mice expressing cOpn5, with different colors indicating individual flashes.

[0168] b. ATP flash events (0–20 min) in mice expressing cOpn5 during a repeat test 1 hour later.

[0169] c. Quantification of ATP flash events in the control, LPS, and cOpn5 groups. Control-LPS comparison: **P = 0.0066; LPS-cOpn5 comparison: **P = 0.0031; Control-cOpn5 comparison: ***P = 0.0002, unpaired t-test.

[0170] d. Analysis of decoding spike firing rate in neurons expressing GCaMP7b during 0–5 min and 15–20 min, coupled with cOpn5-mediated astrocyte activation. N = 193 neurons, ***P < 0.0001, unpaired t-test.

[0171] Example 4: Optogenetic activation of neurons and regulation of animal behavior

[0172] The application of cOpn5-mediated optogenetics in neurons was explored. First, it was examined whether cOpn5 could mediate light-induced Ca2+. 2+ Signal. Using AAV and the panneuronal SYN promoter, cOpn5 and genetically encoded Ca2+ were expressed in mouse cortical neurons. 2+ Sensor jRGECO1a( Figure 8 a). Blue light pulses (10 s; 100 μW / mm) were applied to brain slice samples. 2 (473nm) reliably induces Ca in neurons 2+ Transient ( Figure 8 b、 Figure 8 c). Therefore, cOpn5 can also be photoinduced to activate neurons.

[0173] Next, the effects of light-induced cOpn5 activation on the electrophysiological properties of neurons in slice samples from the motor cortex, hippocampus, and dorsal striatum were investigated. Figure 8 d). Two types of activation modes were observed. In most recorded neurons, blue light pulses induced small depolarization currents (~20 pA) in a voltage-clamp mode and delayed but intense firing of action potentials in a current-clamp mode. Figure 8 e, left side, n = 12 neurons). Under higher frequency light pulses, cOpn5 drives more spike potentials after the initial light pulse, with a shorter latency (~5s to ~3s), while the inward current is not significantly affected. Figure 9 a). In another neuronal subpopulation, brief light pulses rapidly evoke strong inward currents (100-1000 pA) and drive bursts of action potentials. Figure 8e, right side, n=6 neurons). Neurons stimulated repeatedly at 10 Hz and 10 ms / pulse exhibited a non-decaying firing rate pattern in repeated light stimulation trials. Figure 9 b). It is worth noting that, with ChR2 optogenetics 53 The difference is that the action potential evoked by cOpn5 light stimulation is not synchronized with the light pulse in time.

[0174] Finally, the utility of cOpn5-mediated optogenetics in regulating animal behavior was evaluated. The lateral hypothalamus (LH) is a brain center known to function in reward processing and feeding behavior. 54,55 cOpn5 was expressed in the LH GABAergic neurons of VGAT-Cre mice, and optical fibers were implanted to deliver light pulses to the LH neurons of freely behaving mice. Figure 8 g). This relates to previously discovered activation of LH GABA neurons driving feeding behavior. 56 Consistently, light stimulation (20 Hz; 5 ms / pulse; 473 nm; 0.75 mW, output from the fiber tip) induced a significant increase in food intake in cOpn5-expressing mice, but not in EGFP-expressing control mice. Figure 8 h). The role of cOpn5-mediated optogenetic activation of GABA neurons in the zone of indeterminate origin (ZI) was also tested using a foraging behavior task. Figure 8 i), where the indeterminate zone is a region known to drive compulsive feeding. 57 Compared to mice expressing EGFP, mice expressing cOpn5 showed a significantly increased time spent foraging for high-fat food particles after repeated light stimulation. Figure 8 j). Electrophysiological recordings were performed on cOpn5-expressing neurons in LH and ZI to characterize their response spectra. The injection site and placement of the optical fiber were confirmed using whole-brain slices. Figure 10 a~ Figure 10 c). Notably, the mice maintained the aforementioned behaviors (eating or foraging for high-fat foods) when the lights were on and immediately ceased these behaviors when the lights were off. Therefore, cOpn5 can effectively, rapidly, accurately, and reversibly modulate the behavioral state of animals.

[0175] Figure 8 This study demonstrated that cOpn5-mediated optogenetics alters mouse behavior in a neural circuit-dependent manner.

[0176] a, showing the use of optogenetic stimulation and Ca 2+ A schematic diagram of the imaging experimental setup.

[0177] b, showing the effect of light stimulation (10 s; 100 μW / mm²). 2Ca before and after 473nm 2+ A pseudo-color image of the signal. Scale bar, 10 μm.

[0178] c shows the Ca of the 6 neurons in b. 2+ Signal trace diagram.

[0179] d, a schematic diagram depicting optogenetic stimulation and whole-cell patch-clamp recording of neurons expressing cOpn5 in the cortex, striatum, and hippocampus.

[0180] e. Representative diagrams of two types of neurons, one of which responds to light pulses (1Hz, 5s, 10ms / pulse) and exhibits strong, delayed bursts of action potentials, along with small, sustained inward currents; the other exhibits rapid membrane potential depolarization and large inward currents.

[0181] f, After applying a pulse signal of 473 nm light stimulation (1 Hz, 5 s), the firing rate of neurons increased (***P = 0.0005, n = 18, unpaired t test).

[0182] g. Schematic diagram of the experimental setup used for optogenetics and food intake assays. cOpn5-EGFP is expressed in GABAergic neurons in the lateral hypothalamus (LH) of VGAT-Cre mice. EGFP expression serves as a control.

[0183] Summary of cOpn5-mediated activation of feeding behavior induced by light (20 Hz; 5 ms / pulse; 473 nm; 0.75 mW, output from fiber tip). ***P = 0.0003; ns, not significant; n = 6 mice; unpaired t-test. Error bars represent SEM.

[0184] i. Schematic diagram of the experimental setup used for foraging behavior. High-fat food particles were used. cOpn5-EGFP was expressed in GABAergic neurons within the zone of indeterminate in mice (ZI). EGFP expression served as a control.

[0185] Summary of j,cOpn5-mediated foraging behavior. The percentage of foraging time after light stimulation until the mice found the hidden food was calculated. ****P<0.0001; n=6 mice; unpaired t-test. Error bars represent SEM.

[0186] Figure 9 This demonstrates that cOpn5-mediated optogenetic activation of neurons is reliable.

[0187] a, a representative neuron that responds to light pulses of 1, 10 and 20 Hz (5 s; 10 ms / pulse).

[0188] b shows the original traces of reliable and reproducible neuronal photoactivation mediated by cOpn5.

[0189] c. Summary of firing rates in repeated light stimulation experiments.

[0190] Figure 10 The injection location and placement of the optical fiber are shown.

[0191] Image a shows the expression of EGFP control and bicistronic cOpn5 in LH (white dashed line). The lesion site and blue dashed line indicate the placement of the optical fiber. Scale bar, 500 μm.

[0192] b, Injection location and fiber placement in ZI. Scale bar, 500 μm.

[0193] Electrophysiological recordings of cOpn5-expressing neurons in c, LH, and ZI were used to characterize cOpn5-mediated photoactivation.

[0194] Example 5

[0195] Experimental Description: Table 9 below is a partial list of cOpn5 orthologs from the subphylum Vertebrata tested in this invention. The complete genomes of all reported opsin 5 orthologs from the subphylum Vertebrata (including Rotundia, cartilaginous fish, bony fish, amphibians, reptiles, birds, and mammals) were synthesized and expressed in HEK 293T cells. Calcium imaging was performed with or without 470 nm blue light stimulation to test the light sensitivity of the opsin 5 orthologs. Light-induced temporal changes in calcium signaling revealed the activation level of the Gq signaling pathway and the sensitivity of these orthologs.

[0196] Table 9:

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206] References

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Claims

1. The use of isolated photosensitive opsins in drug preparation. in, The drug is used to treat conditions caused by G12 activation in astrocytes. q Diseases or conditions that benefit from signal transduction-induced ATP release and neuronal activation, wherein the disease or condition is depression. The photosensitive opsin is an isolated opsin 5 from animals, and it has the ability to activate G... q Signal transduction and / or activation of cell activity, The animal in question is either a chicken or a sea turtle. The GenBank accession number for the chicken's opsin 5 is NM_001130743.1, and the GenBank accession number for the sea turtle's opsin 5 is XM_007068312.

4.

2. Use of the isolated nucleic acid molecule in the preparation of a drug, said nucleic acid molecule encoding opsin 5 derived from chicken or turtle. The drug is used to treat G12 cells activated in astrocytes. q Diseases or conditions that benefit from signal transduction-induced ATP release and neuronal activation, wherein the disease or condition is depression. in, The GenBank accession number for the chicken's opsin 5 is NM_001130743.1, and the GenBank accession number for the sea turtle's opsin 5 is XM_007068312.

4.

3. Use of a chimeric gene in the preparation of a drug, said chimeric gene comprising a nucleic acid sequence encoding opsin 5 from chickens or turtles, said nucleic acid sequence being operatively linked to a suitable regulatory sequence. The drug is used to treat G12 disorders caused by activation of G12 cells in central neurons or astrocytes. q Diseases or conditions that benefit from signal transduction-induced ATP release and neuronal activation, wherein the disease or condition is depression. in, The GenBank accession number for the chicken's opsin 5 is NM_001130743.1, and the GenBank accession number for the sea turtle's opsin 5 is XM_007068312.

4.

4. Use of a vector in the preparation of a drug, said vector comprising: an isolated nucleic acid molecule encoding opsin 5 from chickens or turtles; or, a chimeric gene comprising a nucleic acid sequence encoding opsin 5 from chickens or turtles. The drug is used to treat G12 disorders caused by activation of G12 cells in central neurons or astrocytes. q Diseases or conditions that benefit from signal transduction-induced ATP release and neuronal activation, wherein the disease or condition is depression. in, The GenBank accession number for the chicken's opsin 5 is NM_001130743.1, and the GenBank accession number for the sea turtle's opsin 5 is XM_007068312.

4.

5. The use according to claim 4, wherein the vector is a eukaryotic vector, a prokaryotic expression vector, or a viral vector.

6. The use according to claim 4, wherein the carrier is a yeast carrier.

7. The use according to claim 4, wherein the vector is a herpes simplex virus vector, a vaccinia virus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or an insect vector.

8. The use according to claim 7, wherein the carrier is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVS, AAVO, or AAV10.

9. The use according to claim 7, wherein the vector is an expression vector or a gene therapy vector.

10. Use of isolated cells or cell cultures in the preparation of a medicament, said cells or cell cultures comprising: an isolated nucleic acid molecule encoding opsin 5 from chickens or turtles; a chimeric gene comprising a nucleic acid sequence encoding opsin 5 from chickens or turtles; or, a vector comprising said nucleic acid molecule or chimeric gene. in, The drug is used to treat conditions caused by G12 activation in astrocytes. q Diseases or conditions that benefit from signal transduction-induced ATP release and neuronal activation, wherein the disease or condition is depression. The GenBank accession number for the chicken's opsin 5 is NM_001130743.1, and the GenBank accession number for the sea turtle's opsin 5 is XM_007068312.

4.

11. The use according to claim 1, 2, 3, 4 or 10, wherein the treatment further comprises applying light in the wavelength range of 360 nm to 550 nm.

12. The use according to claim 1, 2, 3, 4 or 10, wherein the treatment further comprises applying light in the wavelength range of 450-500 nm.

13. The use according to claim 1, 2, 3, 4 or 10, wherein the treatment further comprises applying blue light in the wavelength range of 460-480 nm.

14. The use according to claim 1, 2, 3, 4 or 10, wherein the treatment further comprises applying blue light in the wavelength range of 470 nm.

15. The use according to claim 1, 2, 3, 4 or 10, wherein the treatment further comprises two-photon activation using light with a wavelength ≥920 nm.

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