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Retinitis pigmentosa (RP) is a collection of retinal disorders characterized by the progressive degeneration of photoreceptor cells, leading to significant visual impairment and, in severe cases, blindness. RP affects individuals worldwide and can be inherited through various genetic patterns, making it a genetically diverse condition. Despite considerable advancements in diagnostic methods and supportive therapies, there is currently no cure for RP. The focus of existing management strategies is on slowing the progression of the disease and improving the quality of life for those affected. This comprehensive review explores the latest therapeutic approaches in the management of RP, highlighting advancements in genetic therapies, such as gene augmentation and editing, as well as cell-based treatments including stem cell transplantation and induced pluripotent stem cell (iPSC) technologies. Emerging methods like optogenetics and pharmacological interventions designed to preserve retinal function are also discussed. Additionally, the review examines technological innovations, including retinal prosthetics and the use of artificial intelligence, which hold the potential to revolutionize RP treatment. The challenges and limitations associated with these novel therapies, such as safety concerns, accessibility issues, and regulatory hurdles, are critically evaluated. By providing an overview of current research and future directions, this review aims to inform clinicians and researchers about the state of the art in RP treatment and the prospects for achieving significant therapeutic advancements.

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Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

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Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors.

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Optical methods for studying the brain offer powerful approaches for understanding how neural activity underlies complex behavior. These methods typically rely on genetically encoded sensors and actuators to monitor and control neural activity. For microendoscopic calcium imaging, injection of a virus followed by implantation of a lens probe is required to express a calcium sensor and enable optical access to the target brain region. This two-step process poses several challenges, chief among them being the risks associated with mistargeting and/or misalignment between virus expression zone, lens probe and target brain region. Here, an adeno-associated virus (AAV)-eluting polymer coating is engineered for gradient refractive index (GRIN) lenses enabling the expression of a genetically encoded calcium indicator (GCaMP) directly within the brain region of interest upon implantation of the lens. This approach requires only one surgical step and guarantees alignment between GCaMP expression and lens in the brain. Additionally, the slow virus release from these coatings increases the working time for surgical implantation, expanding the brain regions and species amenable to this approach. These enhanced capabilities should accelerate neuroscience research utilizing optical methods and advance the understanding of the neural circuit mechanisms underlying brain function and behavior in health and disease.

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The integration of functional magnetic resonance imaging (fMRI) with advanced neuroscience technologies in experimental small animal models offers a unique path to interrogate the causal relationships between regional brain activity and brain-wide network measures-a goal challenging to accomplish in human subjects. This review traces the historical development of the neuromodulation techniques commonly used in rodents, such as electrical deep brain stimulation, optogenetics, and chemogenetics, and focuses on their application with fMRI. We discuss their advantageousness roles in uncovering the signaling architecture within the brain and the methodological considerations necessary when conducting these experiments. By presenting several rodent-based case studies, we aim to demonstrate the potential of the multimodal neuromodulation approach in shedding light on neurovascular coupling, the neural basis of brain network functions, and their connections to behaviors. Key findings highlight the cell-type and circuit-specific modulation of brain-wide activity patterns and their behavioral correlates. We also discuss several future directions and feature the use of mediation and moderation analytical models beyond the intuitive evoked response mapping, to better leverage the rich information available in fMRI data with neuromodulation. Using fMRI alongside neuromodulation techniques provide insights into the mesoscopic (relating to the intermediate scale between single neurons and large-scale brain networks) and macroscopic fMRI measures that correlate with specific neuronal events. This integration bridges the gap between different scales of neuroscience research, facilitating the exploration and testing of novel therapeutic strategies aimed at altering network-mediated behaviors. In conclusion, the combination of fMRI with neuromodulation techniques provides crucial insights into mesoscopic and macroscopic brain dynamics, advancing our understanding of brain function in health and disease. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.

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Rationale: Optogenetically engineered facultative anaerobic bacteria exhibit a favorable tendency to colonize at solid tumor sites and spatiotemporally-programmable therapeutics release abilities, attracting extensive attention in precision tumor therapy. However, their therapeutic efficacy is moderate. Conventional photothermal agents with high tumor ablation capabilities exhibit low tumor targeting efficiency, resulting in significant off-target side effects. The combination of optogenetics and photothermal therapy may offer both tumor-targeting and excellent tumor-elimination capabilities, which unfortunately has rarely been investigated. Herein, we construct a bacteria-based cascade near-infrared optogentical-photothermal system (EcNαHL-UCNPs) for enhanced tumor therapy. Methods: EcNαHL-UCNPs consists of an optogenetically engineered Escherichia coli Nissle 1917 (EcN) conjugated with lanthanide-doped upconversion nanoparticles (UCNPs), which are capable of locally secreting α-hemolysin (αHL), a pore-forming protein, in responsive to NIR irradiation. Anti-tumor effects of EcNαHL-UCNPs were determined in both H22 and 4T1 tumors. Results: The αHL not only eliminates tumor cells, but more importantly disrupts endothelium to form thrombosis as an in situ photothermal agent in tumors. The in situ formed thrombosis significantly potentiates the photothermic ablation of H22 tumors upon subsequent NIR light irradiation. Besides, αHL secreted by EcNαHL-UCNPs under NIR light irradiation not only inhibits 4T1 tumor growth, but also suppresses metastasis of 4T1 tumor via inducing the immune response. Conclusion: Our studies highlight bacteria-based cascade optogenetical-photothermal system for precise and effective tumor therapy.

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Alteration of synaptic function in the dorsal horn (DH) has been implicated as a cellular substrate for the development of neuropathic pain, but certain details remain unclear. In particular, the lack of information on the types of synapses that undergo functional changes hinders the understanding of disease pathogenesis from a synaptic plasticity perspective. Here, we addressed this issue by using optogenetic and retrograde tracing ex vivo to selectively stimulate first-order nociceptors expressing Nav1.8 (NRsNav1.8) and record the responses of spinothalamic tract neurons in spinal lamina I (L1-STTNs). We found that spared nerve injury (SNI) increased excitatory postsynaptic currents (EPSCs) in L1-STTNs evoked by photostimulation of NRsNav1.8 (referred to as Nav1.8-STTN EPSCs). This effect was accompanied by a significant change in the failure rate and paired-pulse ratio of synaptic transmission from NRsNav1.8 to L1-STTN and in the frequency (not amplitude) of spontaneous EPSCs recorded in L1-STTNs. However, no change was observed in the ratio of AMPA to NMDA receptor-mediated components of Nav1.8-STTN EPSCs or in the amplitude of unitary EPSCs constituting Nav1.8-STTN EPSCs recorded with extracellular Ca2+ replaced by Sr2+ In addition, there was a small increase (approximately 10%) in the number of L1-STTNs showing immunoreactivity for phosphorylated extracellular signal-regulated kinases in mice after SNI compared with sham. Similarly, only a small percentage of L1-STTNs showed a lower action potential threshold after SNI. In conclusion, our results show that SNI induces presynaptic modulation at NRNav1.8 (consisting of both peptidergic and nonpeptidergic nociceptors) synapses on L1-STTNs forming the lateral spinothalamic tract.

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Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

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Teaching aspects of neuroscience to large undergraduate classes can be difficult in terms of the cost of equipment involved such as microscopes and electrophysiology equipment, the time taken to master techniques such as dissection or intracellular recording, and ethical concerns when using vertebrates. Here, I describe a practical that uses behavioral readouts and optogenetics on Drosophila that can be implemented with minimal cost as well as reduced ethical concerns and uses mostly observational techniques. The practical can be used to teach aspects of genetics and the tools for manipulating neuronal activity for ascribing neuronal function. The practical can be customized to fit different undergraduate levels and learning objectives.

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BACKGROUND: Functional activation of the focal ischemic brain has been reported to improve outcomes by augmenting collateral blood flow. However, functional activation also increases metabolic demand and might thereby worsen outcomes. Indeed, preclinical and clinical reports have been conflicting. Here, we tested the effect of functional activation during acute ischemic stroke using distal middle cerebral artery occlusion in anesthetized mice. METHODS: Using transgenic mice expressing channelrhodopsin-2 in neurons, we delivered functional activation using physiological levels of transcranial optogenetic stimulation of the moderately ischemic cortex (ie, penumbra), identified using real-time full-field laser speckle perfusion imaging during a 1-hour distal microvascular clip of the middle cerebral artery. Neuronal activation was confirmed using evoked field potentials, and infarct volumes were measured in tissue slices 48 hours later. RESULTS: Optogenetic stimulation of the penumbra was associated with more than 2-fold larger infarcts than stimulation of the contralateral homotopic region and the sham stimulation group (n=10, 7, and 9; 11.0±5.6 versus 5.1±4.3 versus 4.1±3.7 mm3; P=0.008, 1-way ANOVA). Identical stimulation in wild-type mice that do not express channelrhodopsin-2 did not have an effect. Optogenetic stimulation was associated with a small increase in penumbral perfusion that did not explain enlarged infarcts. CONCLUSIONS: Our data suggest that increased neuronal activity during acute focal arterial occlusions can be detrimental, presumably due to increased metabolic demand, and may have implications for the clinical management of hyperacute stroke patients.

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Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i.e., "turned off") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a "turn-on mode" insertion.

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Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5'-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics.

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Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

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Plant phytochromes perceive red and far-red light to elicit adaptations to the changing environment. Downstream physiological responses revolve around red-light-induced interactions with phytochrome-interacting factors (PIF). Phytochromes double as thermoreceptors, owing to the pronounced temperature dependence of thermal reversion from the light-adapted Pfr to the dark-adapted Pr state. Here, we assess whether thermoreception may extend to the phytochrome:PIF interactions. While the association between Arabidopsis (Arabidopsis thaliana) PHYTOCHROME B (PhyB) and several PHYTOCHROME-INTERACTING FACTOR (PIF) variants moderately accelerates with temperature, the dissociation does more so, thus causing net destabilization of the phytochrome:PIF complex. Markedly different temperature profiles of PIF3 and PIF6 might underlie stratified temperature responses in plants. Accidentally, we identify a photoreception mechanism under strong continuous light, where the extent of phytochrome:PIF complexation decreases with red-light intensity rather than increases. Mathematical modeling rationalizes this attenuation mechanism and ties it to rapid red-light-driven Pr⇄Pfr interconversion and complex dissociation out of Pr. Varying phytochrome abundance, e.g., during diurnal and developmental cycles, and interaction dynamics, e.g., across different PIFs, modify the nature and extent of attenuation, thus permitting light-response profiles more malleable than possible for the phytochrome Pr⇄Pfr interconversion alone. Our data and analyses reveal a photoreception mechanism with implications for plant physiology, optogenetics, and biotechnological applications.

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To understand the function of cells such as neurons within an organism, it can be instrumental to inhibit cellular function, or to remove the cell (type) from the organism, and thus to observe the consequences on organismic and/or circuit function and animal behavior. A range of approaches and tools were developed and used over the past few decades that act either constitutively or acutely and reversibly, in systemic or local fashion. These approaches make use of either drugs or genetically encoded tools. Also, there are acutely acting inhibitory tools that require an exogenous trigger like light. Here, we give an overview of such methods developed and used in the nematode Caenorhabditis elegans.

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Spiral ganglia neurons (SGNs) impairment can cause deafness. One important therapeutic approach involves utilizing stem cells to restore impaired auditory circuitry. Nevertheless, the inadequate implementation of research methodologies poses a challenge in accurately assessing the functionality of derived cells within the circuit. Here, we describe a novel method for converting human embryonic stem cells (hESCs) into otic neurons (ONs) and assess their functional connectivity using an optogenetic approach with cells or an organotypic slice of rat cochlear nucleus (CN) in coculture. Embryonic stem cell-derived otic neurons (eONs) exhibited SGN marker expression and generated functional synaptic connection when cocultured with cochlear nucleus neurons (CNNs). Synapsin 1 and VGLUT expression are found in the cochlear nucleus of brain slices, where eONs projected processes during the coculture of eONs and CN brain slices. Action potential spikes and INa+/IK+ of CNNs increased in tandem with light stimulations to eONs. These findings provide further evidence that eONs may be a candidate source to treat SGN-deafness.

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Lysosomes are essential degradative organelles and signaling hubs within cells, playing a crucial role in the regulation of macroautophagy/autophagy. Dysfunction of lysosomes and impaired autophagy are closely associated with the development of various neurodegenerative diseases. Enhancing lysosomal activity and boosting autophagy levels holds great promise as effective strategies for treating these diseases. However, there remains a lack of methods to dynamically regulate lysosomal activity and autophagy levels in living cells or animals. In our recent work, we applied optogenetics to manipulate lysosomal physiology and function, developing three lysosome-targeted optogenetic tools: lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2. These new actuators enable light-dependent regulation of key aspects such as lysosomal membrane potential, lumenal pH, hydrolase activity, degradation processes, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy via the MTOR pathway while it promotes Aß clearance through autophagy induction in cellular models of Alzheimer disease. Furthermore, lyso-ChR2 activation reduces Aß deposition and alleviates Aß-induced paralysis in Caenorhabditis elegans models of Alzheimer disease. Our lysosomal optogenetic actuators offer a novel method for dynamically regulating lysosomal physiology and autophagic activity in living cells and animals.

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Macrophages measure the "eat-me" signal immunoglobulin G (IgG) to identify targets for phagocytosis. We tested whether prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc receptor. To temporally control Fc receptor activation, we engineered an Fc receptor that is activated by the light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that subthreshold Fc receptor activation primes mouse bone-marrow-derived macrophages to be more sensitive to IgG in future encounters. Macrophages that have previously experienced subthreshold Fc receptor activation eat more IgG-bound human cancer cells. Increased phagocytosis occurs by two discrete mechanisms-a short- and long-term priming. Long-term priming requires new protein synthesis and Erk activity. Short-term priming does not require new protein synthesis and correlates with an increase in Fc receptor mobility. Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.

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Recently developed optogenetic technology, which allows high-fidelity control of neuronal activity, has been applied to investigate the neural circuits underlying sensory processing and behavior. The nasal cavity is innervated by the olfactory nerve and trigeminal nerve, which are closely related to common symptoms of rhinitis, such as impairment of smell, itching, and sneezing. The olfactory system has an amazing ability to distinguish thousands of odorant molecules at trace levels. However, there are many issues in olfactory sensing mechanisms that need to be addressed. Optogenetics offers a novel technical approach to solve this dilemma. Therefore, we review the recent advances in olfactory optogenetics to clarify the mechanisms of chemical sensing, which may help identify the mechanism of dysfunction and suggest possible treatments for impaired smell. Additionally, in rhinitis patients, alterations in the other nerve (trigeminal nerve) that innervates the nasal cavity can lead to hyperresponsiveness to various nociceptive stimuli and central sensitization, causing frequent and persistent itching and sneezing. In the last several years, the application of optogenetics in regulating nociceptive receptors, which are distributed in sensory nerve endings, and amino acid receptors, which are distributed in vital brain regions, to alleviate overreaction to nociceptive stimuli, has gained significant attention. Therefore, we focus on the progress in optogenetics and its application in neuromodulation of nociceptive stimuli and discuss the potential clinical translation for treating rhinitis in the future.

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Singularity phenomena are rare events that occur only with a probability of one in tens of thousands and yet play an important role in the fate of the entire system. Recently, an ultra-wide-field microscopy imaging systems, AMATERAS, have been developed to reliably capture singularity phenomena. However, to determine whether a rare phenomenon captured by microscopy is a true singularity phenomenon-one with a significant impact on the entire system-, causal analysis is required. In this section, we introduce the CALI method, which uses light to inactivate molecules as one of the techniques enabling causal analysis. In addition, we discuss the technical innovations of the CALI method that are required to contribute to the future development of singularity biology.