Curated Optogenetic Publication Database

Abstract: Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells. In this review, we focus on the mechanisms that control cell contraction in space and time in non-muscle cells, and how they can be investigated by light-based methods. The review particularly focusses on signal networks and cytoskeletal components that together control subcellular contraction patterns to perform functions on the level of cells and tissues, such as directional migration and multicellular rearrangements during development. Key features of light-based methods that enable highly local and fast perturbations are highlighted, and how experimental strategies can capitalize on these features to uncover causal relationships in the complex signal networks that control cell contraction.

Abstract: Calcium ions (Ca2+) play pivotal roles in regulating diverse brain functions, including cognition, emotion, locomotion, and learning and memory. These functions are intricately regulated by a variety of Ca2+-dependent cellular processes, encompassing synaptic plasticity, neuro/gliotransmitter release, and gene expression. In our previous work, we developed 'monster OptoSTIM1' (monSTIM1), an improved OptoSTIM1 that selectively activates Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane through blue light, allowing precise control over intracellular Ca2+ signaling and specific brain functions. However, the large size of the coding sequence of monSTIM1 poses a limitation for its widespread use, as it exceeds the packaging capacity of adeno-associated virus (AAV). To address this constraint, we have introduced monSTIM1 variants with reduced coding sequence sizes and established AAV-based systems for expressing them in neurons and glial cells in the mouse brain. Upon expression by AAVs, these monSTIM1 variants significantly increased the expression levels of cFos in neurons and astrocytes in the hippocampal CA1 region following non-invasive light illumination. The use of monSTIM1 variants offers a promising avenue for investigating the spatiotemporal roles of Ca2+-mediated cellular activities in various brain functions. Furthermore, this toolkit holds potential as a therapeutic strategy for addressing brain disorders associated with aberrant Ca2+ signaling.

Abstract: Alzheimer's disease (AD) is the most common form of dementia, resulting in disability and mortality. The global incidence of AD is consistently surging. Although numerous therapeutic agents with promising potential have been developed, none have successfully treated AD to date. Consequently, the pursuit of novel methodologies to address neurodegenerative processes in AD remains a paramount endeavor. A particularly promising avenue in this search is optogenetics, enabling the manipulation of neuronal activity. In recent years, research attention has pivoted from neurons to glial cells. This review aims to consider the potential of the optogenetic correction of astrocyte metabolism as a promising strategy for correcting AD-related disorders. The initial segment of the review centers on the role of astrocytes in the genesis of neurodegeneration. Astrocytes have been implicated in several pathological processes associated with AD, encompassing the clearance of β-amyloid, neuroinflammation, excitotoxicity, oxidative stress, and lipid metabolism (along with a critical role in apolipoprotein E function). The effect of astrocyte-neuronal interactions will also be scrutinized. Furthermore, the review delves into a number of studies indicating that changes in cellular calcium (Ca2+) signaling are one of the causes of neurodegeneration. The review's latter section presents insights into the application of various optogenetic tools to manipulate astrocytic function as a means to counteract neurodegenerative changes.

Abstract: Calcium transients drive cells to discharge prostaglandin E2 (PGE2). We visualized PGE2-induced protein kinase A (PKA) activation and quantitated PGE2 secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE2-producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 or Gq-DREADD, respectively. Second, we prepared reporter cells expressing the Gs-coupled PGE2 reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer (FRET). Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE2 to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE2 diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, we estimated that a single HeLa cell secretes 0.25 fmol PGE2 upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE2 discharge rate is comparable to the diffusion rate. Thus, our method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE2.Key words: prostaglandin E2, imaging, intercellular communication, biosensor, quantification.

Abstract: Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, we developed OptoTrap, a light-inducible protein trapping system that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. We generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed us to instantly disrupt microtubules and inhibit the kinesin-1 motor in specific dendritic branches of Drosophila sensory neurons. Using OptoTrap, we obtained direct evidence that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Abstract: Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.

Abstract: Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

Abstract: CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.

Abstract: Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform – Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially-designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Abstract: Cells communicate with each other to jointly regulate cellular processes during cellular differentiation and tissue morphogenesis. This multiscale coordination arises through spatiotemporal activity of morphogens to pattern cell signaling and transcriptional factor activity. This coded information controls cell mechanics, proliferation, and differentiation to shape the growth and morphogenesis of organs. While many of the molecular components and physical interactions have been identified in key model developmental systems, there are still many unresolved questions related to the dynamics involved due to challenges in precisely perturbing and quantitatively measuring signaling dynamics. Recently, a broad range of synthetic optogenetic tools have been developed and employed to quantitatively define relationships between signal transduction and downstream cellular responses. These optogenetic tools can control intracellular activities at the single cell or whole tissue scale to direct subsequent biological processes. In this brief review, we highlight a selected set of studies that develop and implement optogenetic tools to unravel quantitative biophysical mechanisms for tissue growth and morphogenesis across a broad range of biological systems through the manipulation of morphogens, signal transduction cascades, and cell mechanics. More generally, we discuss how optogenetic tools have emerged as a powerful platform for probing and controlling multicellular development.

Abstract: The cGAS-STING signaling pathway has emerged as a promising target for immunotherapy development. Here, we introduce a light-sensitive optogenetic device for control of the cGAS/STING signaling to conditionally modulate innate immunity, called 'light-inducible SMOC-like repeats' (LiSmore). We demonstrate that photo-activated LiSmore boosts dendritic cell (DC) maturation and antigen presentation with high spatiotemporal precision. This non-invasive approach photo-sensitizes cytotoxic T lymphocytes to engage tumor antigens, leading to a sustained antitumor immune response. When combined with an immune checkpoint blocker (ICB), LiSmore improves antitumor efficacy in an immunosuppressive lung cancer model that is otherwise unresponsive to conventional ICB treatment. Additionally, LiSmore exhibits an abscopal effect by effectively suppressing tumor growth in a distal site in a bilateral mouse model of melanoma. Collectively, our findings establish the potential of targeted optogenetic activation of the STING signaling pathway for remote immunomodulation in mice.

Abstract: The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.

Abstract: Inflammasomes are multiprotein platforms which control caspase-1 activation, leading to the processing of proinflammatory cytokines into mature and active cytokines IL-1β and IL-18, and to pyroptosis through the cleavage of gasdermin-D (GSDMD). Inflammasomes assemble upon activation of specific cytosolic pattern recognition receptors (PRRs) by damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs). They converge to the nucleation of apoptosis-associated speck-like containing a caspase activation and recruitment domain (ASC) to form hetero-oligomers with caspase-1. Studying inflammasome encoding activities remains challenging because PAMPs and DAMPs are sensed by a large diversity of cytosolic and membranous PRRs. To bypass the different signals required to activate the inflammasome, we designed an optogenetic approach to temporally and quantitatively manipulate ASC assembly (i.e. in a PAMP- or DAMP-independent manner). We reveal that controlling light-sensitive oligomerization of ASC is sufficient to recapitulate the classical features of inflammasomes within minutes, and enabled us to decipher the complexity of volume regulation and pore opening during pyroptosis. Overall, this approach offers interesting perspective to decipher PRR signaling pathways in the field of innate immunity.

Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Abstract: Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington’s Disease, and Alzheimer’s Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Abstract: Intra-thymic T cell development is coordinated by the regulatory actions of SATB1 genome organizer. In this report, we show that SATB1 is involved in the regulation of transcription and splicing, both of which displayed deregulation in Satb1 knockout murine thymocytes. More importantly, we characterized a novel SATB1 protein isoform and described its distinct biophysical behavior, implicating potential functional differences compared to the commonly studied isoform. SATB1 utilized its prion-like domains to transition through liquid-like states to aggregated structures. This behavior was dependent on protein concentration as well as phosphorylation and interaction with nuclear RNA. Notably, the long SATB1 isoform was more prone to aggregate following phase separation. Thus, the tight regulation of SATB1 isoforms expression levels alongside with protein post-translational modifications, are imperative for SATB1's mode of action in T cell development. Our data indicate that deregulation of these processes may also be linked to disorders such as cancer.

Abstract: Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ('cell doublet'). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

Abstract: Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro's components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Abstract: High-performance biosensors are crucial for elucidating the spatiotemporal regulatory roles and dynamics of membrane lipids, but there is a lack of improvement strategies for biosensors with low sensitivity and low-content substrates detection. Here we developed universal optogenetic strategies to improve a set of membrane biosensors by trapping them into specific region and further reducing the background signal, or by optically-controlled phase separation for membrane lipids detection and tracking. These improved biosensors were superior to typical tools and light simulation would enhance their detection performance and resolution, which might contribute to the design and optimization of other biosensors.

Abstract: An increasing experimental evidence points to physiological importance of space-time correlations in signalling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of bio-molecules between cells allows the collectives to perform more complex tasks and better tackle environmental challenges. To understand this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signalling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We make a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells induce ERK activity waves with different size, duration, and frequency. The open-source implementations of ARCOS are available as R and Python packages, and as a plugin for napari image viewer to interactively quantify collective phenomena without prior programming experience.

Abstract: Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.

Abstract: Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, the mechanical properties of embryonic epithelial tissues and the factors that control these properties are not well understood. Actomyosin generates contractile tensions and contributes to the mechanical properties of cells and cytoskeletal networks in vitro, but it remains unclear how the levels and patterns of actomyosin activity contribute to embryonic epithelial tissue mechanics in vivo. To dissect the roles of cell-generated tensions in the mechanics of flowing epithelial tissues, we use optogenetic tools to manipulate actomyosin contractility with spatiotemporal precision in the Drosophila germband epithelium, which rapidly flows during body axis elongation. We find that manipulating actomyosin-dependent tensions by either optogenetic activation or deactivation of actomyosin alters the solid-fluid mechanical properties of the germband epithelium, leading to changes in cell rearrangements and tissue-level flows. Optogenetically activating actomyosin leads to increases in the overall level but decreases in the anisotropy of tension in the tissue, whereas optogenetically deactivating actomyosin leads to decreases in both the level and anisotropy of tension compared to in wild-type embryos. We find that optogenetically activating actomyosin results in more solidlike (less fluidlike) tissue properties, which is associated with reduced cell rearrangements and tissue flow compared to in wild-type embryos. Optogenetically deactivating actomyosin also results in more solidlike properties than in wild-type embryos but less solidlike properties compared to optogenetically activating actomyosin. Together, these findings indicate that increasing the overall tension level is associated with more solidlike properties in tissues that are relatively isotropic, whereas high-tension anisotropy fluidizes the tissue. Our results reveal that epithelial tissue flows in developing embryos involve the coordinated actomyosin-dependent regulation of the mechanical properties of tissues and the tensions driving them to flow in order to achieve rapid tissue remodeling.

Abstract: Regulated cell death plays a key role in immunity, development, and homeostasis, but is also associated with a number of pathologies such as autoinflammatory and neurodegenerative diseases and cancer. However, despite the extensive mechanistic research of different cell death modalities, the direct comparison of different forms of cell death and their consequences on the cellular and tissue level remain poorly characterized. Comparative studies are hindered by the mechanistic and kinetic differences between cell death modalities, as well as the inability to selectively induce different cell death programs in an individual cell within cell populations or tissues. In this method, we present a protocol for rapid and specific optogenetic activation of three major types of programmed cell death: apoptosis, necroptosis, and pyroptosis, using light-induced forced oligomerization of their major effector proteins (caspases or kinases).

Abstract: The integrated stress response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered a photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics, we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR, which demonstrated the dependence of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. A record of this paper's transparent peer review process is included in the supplemental information.

Abstract: Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are primarily neurodegenerative diseases with progressive decline in motor function. Aggregates composed of alpha-synuclein, which are known as Lewy bodies, are a neuropathological hallmark of synucleinopathies; their pathogenesis has been attributed to neuronal loss owing to intracellular alpha-synuclein accumulation. However, the mechanism of alpha-synuclein aggregation remains unclear. Here we show that the RNA G-quadruplexes assembly forms scaffolds for alpha-synuclein aggregation, thereby contributing to neurodegeneration. RNA G-quadruplexes undergo phase separation and form scaffolds for co-aggregation with & alpha-synuclein. Upon pathogenic alpha-synuclein seeds-induced cellular stress and an optogenetic assembly of RNA G-quadruplexes, phase-separated RNA G-quadruplexes served as scaffolds for & alpha-synuclein phase transition, and the co-aggregates initiated synaptic dysfunction and Parkinsonism in mice. Treatment with 5-aminolevulinic acid and protoporphyrin IX, which prevents RNA G-quadruplexes phase separation, attenuates alpha-synuclein phase transition, neurodegeneration, and motor deficits in synucleinopathy model mice. Together, the RNA G-quadruplexes assembly accelerates alpha-synuclein phase transition and aggregation owing to intracellular Ca2+ homeostasis, thereby contributing to the pathogenesis of synucleinopathies.