Intercellular communication atlas reveals Oprm1 as a neuroprotective factor for retinal ganglion cells.

Previous studies of neuronal survival have primarily focused on identifying intrinsic mechanisms controlling the process. This study explored how intercellular communication contributes to retinal ganglion cell (RGC) survival following optic nerve crush based on single-cell RNA-seq analysis. We observed transcriptomic changes in retinal cells in response to the injury, with astrocytes and Müller glia having the most interactions with RGCs. By comparing RGC subclasses characterized by distinct resilience to cell death, we found that the high-survival RGCs tend to have more ligand-receptor interactions with neighboring cells. We identified 47 interactions stronger in high-survival RGCs, likely mediating neuroprotective effects. We validated one identified target, the µ-opioid receptor (Oprm1), to be neuroprotective in three retinal injury models. Although the endogenous Oprm1 is preferentially expressed in intrinsically photosensitive RGCs, its neuroprotective effect can be transferred to other subclasses by pan-RGC overexpression of Oprm1. Lastly, manipulating the Oprm1 activity improved visual functions in mice.

Molecular regulation of retinal regeneration is context specific.

Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, akin to disease conditions, are undefined. Combining a novel retinal ganglion cell (RGC) ablation-based glaucoma model, single cell omics, and rapid CRISPR/Cas9-based knockout methods to screen 100 genes, we identified 18 effectors of RGC regeneration kinetics. Surprisingly, 32 of 33 previously known/implicated regulators of retinal tissue regeneration were not required for RGC replacement; 7 knockouts accelerated regeneration, including sox2, olig2, and ascl1a . Mechanistic analyses revealed loss of ascl1a increased "fate bias", the propensity of progenitors to produce RGCs. These data demonstrate plasticity and context-specificity in how genes function to control regeneration, insights that could help to advance disease-tailored therapeutics for replacing lost retinal cells. One sentence summary: We discovered eighteen genes that regulate the regeneration of retinal ganglion cells in zebrafish.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes through Müller glia (MG) reprogramming and asymmetric cell division that produces a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, do MG reprogram to a developmental retinal progenitor cell (RPC) state? Second, to what extent does regeneration recapitulate retinal development? And finally, does loss of different retinal cell subtypes induce unique MG regeneration responses? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. Here we show that injury induces MG to reprogram to a state similar to late-stage RPCs. However, there are major transcriptional differences between MGPCs and RPCs, as well as major transcriptional differences between activated MG and MGPCs when different retinal cell subtypes are damaged. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes.

Transition to Chronic Fibrosis in an Animal Model of Retinal Detachment With Features of Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of surgically repaired rhegmatogenous retinal detachment (RRD). Chemically induced and cell injection PVR models do not fully simulate the clinical characteristics of PVR in the post-RRD context. There is an unmet need for translational models in which to study mechanisms and treatments specific to RRD-PVR. Methods: RRD was induced in adult Dutch Belted rabbits. Posterior segments were fixed or processed for RNA sequencing at 6 hours and 2, 7, 14, and 35 days after induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein, alpha smooth muscle actin, vascular endothelial growth factor receptor 2, CD68, and RPE 65 kDa protein were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. Results: Acute histopathological changes included intravitreal and intraretinal hemorrhage, leukocytic vitritis, chorioretinitis, and retinal rarefaction. Chronic lesions showed retinal atrophy, gliosis, fibrotic subretinal membranes, and epiretinal fibrovascular proliferation. Fibrillar collagen was present in the fibrocellular and fibrovascular membranes in chronic lesions. Moderate to strong labeling of glia and vasculature was detected in chronic lesions. At day 14, most cells profiled by single cell sequencing were identified as Mϋller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes was upregulated in chronic lesions. Conclusions: Histological and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intraretinal and periretinal fibrosis. This animal model of RRD with features of PVR will enable further research on targeted treatment interventions.

Robust reprogramming of glia into neurons by inhibition of Notch signaling and NFI factors in adult mammalian retina.

Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for neurodegenerative diseases. Despite successful applications in vitro , in vivo implementation has been hampered by low efficiency. In this study, we present a highly efficient strategy for reprogramming retinal glial cells into neurons by simultaneously inhibiting key negative regulators. By suppressing Notch signaling through the removal of its central mediator Rbpj, we induced mature Müller glial cells to reprogram into bipolar and amacrine neurons in uninjured adult mouse retinas, and observed that this effect was further enhanced by retinal injury. We found that specific loss of function of Notch1 and Notch2 receptors in Müller glia mimicked the effect of Rbpj deletion on Müller glia-derived neurogenesis. Integrated analysis of multiome (scRNA- and scATAC-seq) and CUT&Tag data revealed that Rbpj directly activates Notch effector genes and genes specific to mature Müller glia while also indirectly represses the expression of neurogenic bHLH factors. Furthermore, we found that combined loss of function of Rbpj and Nfia/b/x resulted in a robust conversion of nearly all Müller glia to neurons. Finally, we demonstrated that inducing Müller glial proliferation by AAV (adeno-associated virus)-mediated overexpression of dominant- active Yap supports efficient levels of Müller glia-derived neurogenesis in both Rbpj - and Nfia/b/x/Rbpj - deficient Müller glia. These findings demonstrate that, much like in zebrafish, Notch signaling actively represses neurogenic competence in mammalian Müller glia, and suggest that inhibition of Notch signaling and Nfia/b/x in combination with overexpression of activated Yap could serve as an effective component of regenerative therapies for degenerative retinal diseases.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

A clock-dependent brake for rhythmic arousal in the dorsomedial hypothalamus.

Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. Here, we show, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca2+ imaging reveals elevated DMHmWAKE activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMHmWAKE neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMHmWAKE neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMHmWAKE neurons promote arousal. Surprisingly, our data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMHmWAKE neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Intercellular communication atlas reveals Oprm1 as a neuroprotective factor for retinal ganglion cells.

The progressive death of mature neurons often results in neurodegenerative diseases. While the previous studies have mostly focused on identifying intrinsic mechanisms controlling neuronal survival, the extracellular environment also plays a critical role in regulating cell viability. Here we explore how intercellular communication contributes to the survival of retinal ganglion cells (RGCs) following the optic nerve crush (ONC). Although the direct effect of the ONC is restricted to the RGCs, we observed transcriptomic responses in other retinal cells to the injury based on the single-cell RNA-seq, with astrocytes and Müller glia having the most interactions with RGCs. By comparing the RGC subclasses showing distinct resilience to ONC-induced cell death, we found that the high-survival RGCs tend to have more ligand-receptor interactions with other retinal cells, suggesting that these RGCs are intrinsically programmed to foster more communication with their surroundings. Furthermore, we identified top 47 interactions that are stronger in the high-survival RGCs, likely representing neuroprotective interactions. We performed functional assays on one of the receptors, µ opioid receptor (Oprm1), a receptor known to play roles in regulating pain, reward, and addictive behavior. Although Oprm1 is preferentially expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), its neuroprotective effect could be transferred to multiple RGC subclasses by specific overexpressing Oprm1 in pan-RGCs in ONC, excitotoxicity, and glaucoma models. Lastly, manipulating Oprm1 activity improved visual functions and altered pupillary light response in mice. Our study provides an atlas of cell-cell interactions in both intact and post-ONC retina and an effective strategy to predict molecular mechanisms in neuroprotection, underlying the principal role played by extracellular environment in supporting neuron survival.

Intercellular communication atlas reveals Oprm1 as a neuroprotective factor for retinal ganglion cells.

The progressive death of mature neurons often results in neurodegenerative diseases. While the previous studies have mostly focused on identifying intrinsic mechanisms controlling neuronal survival, the extracellular environment also plays a critical role in regulating cell viability. Here we explore how intercellular communication contributes to the survival of retinal ganglion cells (RGCs) following the optic nerve crush (ONC). Although the direct effect of the ONC is restricted to the RGCs, we observed transcriptomic responses in other retinal cells to the injury based on the single-cell RNA-seq, with astrocytes and Müller glia having the most interactions with RGCs. By comparing the RGC subclasses with distinct resilience to ONC-induced cell death, we found that the high-survival RGCs tend to have more ligand-receptor interactions with other retinal cells, suggesting that these RGCs are intrinsically programmed to foster more communication with their surroundings. Furthermore, we identified the top 47 interactions that are stronger in the high-survival RGCs, likely representing neuroprotective interactions. We performed functional assays on one of the receptors, µ-opioid receptor (Oprm1), a receptor known to play roles in regulating pain, reward, and addictive behavior. Although Oprm1 is preferentially expressed in intrinsically photosensitive retinal ganglion cells (ipRGC), its neuroprotective effect could be transferred to multiple RGC subclasses by selectively overexpressing Oprm1 in pan-RGCs in ONC, excitotoxicity, and glaucoma models. Lastly, manipulating Oprm1 activity improved visual functions or altered pupillary light response in mice. Our study provides an atlas of cell-cell interactions in intact and post-ONC retina, and a strategy to predict molecular mechanisms controlling neuroprotection, underlying the principal role played by extracellular environment in supporting neuron survival.

Molecular mechanisms controlling vertebrate retinal patterning, neurogenesis, and cell fate specification.

This review covers recent advances in understanding the molecular mechanisms controlling neurogenesis and specification of the developing retina, with a focus on insights obtained from comparative single cell multiomic analysis. We discuss recent advances in understanding the mechanisms by which extrinsic factors trigger transcriptional changes that spatially pattern the optic cup (OC) and control the initiation and progression of retinal neurogenesis. We also discuss progress in unraveling the core evolutionarily conserved gene regulatory networks (GRNs) that specify early- and late-state retinal progenitor cells (RPCs) and neurogenic progenitors and that control the final steps in determining cell identity. Finally, we discuss findings that provide insight into regulation of species-specific aspects of retinal patterning and neurogenesis, including consideration of key outstanding questions in the field.

Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery, and the molecular changes leading to this aberrant wound healing process are currently unknown. Our ultimate goal is to study PVR pathogenesis by employing single-cell transcriptomics to dissect cellular heterogeneity. Design: Here we aimed to compare single-cell RNA sequencing (scRNA-seq)  and single-nucleus RNA-sequencing (snRNA-seq) of retinal PVR samples in the rabbit model. Participants: Unilateral induction of PVR lesions in rabbit eyes with contralateral eyes serving as controls. Methods: Proliferative vitreoretinopathy was induced unilaterally in Dutch Belted rabbits. At different timepoints after PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for scRNA-seq or snRNA-seq. Main Outcome Measures: Single cell and nuclei transcriptomic profiles of retinas after PVR induction. Results: Single-cell RNA sequencing and snRNA-seq were conducted on retinas at 4 hours and 14 days after disease induction. Although the capture rate of unique molecular identifiers and genes were greater in scRNA-seq samples, overall gene expression profiles of individual cell types were highly correlated between scRNA-seq and snRNA-seq. A major disparity between the 2 sequencing modalities was the cell type capture rate, however, with glial cell types overrepresented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Müller glia were overrepresented in snRNA-seq samples, whereas reactive Müller glia were overrepresented in scRNA-seq samples. Trajectory analyses were similar between the 2 methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq data sets. Conclusions: These findings highlight limitations of both scRNA-seq and snRNA-seq analysis and imply that use of both techniques together can more accurately identify transcriptional networks critical for aberrant fibrogenesis in PVR than using either in isolation. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

A super Sonic circadian synchronizer.

Sonic Hedgehog signaling and primary cilia control the core mammalian circadian clock.

Single-cell transcriptome analysis of xenotransplanted human retinal organoids defines two migratory cell populations of nonretinal origin.

Human retinal organoid transplantation could potentially be a treatment for degenerative retinal diseases. How the recipient retina regulates the survival, maturation, and proliferation of transplanted organoid cells is unknown. We transplanted human retinal organoid-derived cells into photoreceptor-deficient mice and conducted histology and single-cell RNA sequencing alongside time-matched cultured retinal organoids. Unexpectedly, we observed human cells that migrated into all recipient retinal layers and traveled long distances. Using an unbiased approach, we identified these cells as astrocytes and brain/spinal cord-like neural precursors that were absent or rare in stage-matched cultured organoids. In contrast, retinal progenitor-derived rods and cones remained in the subretinal space, maturing more rapidly than those in the cultured controls. These data suggest that recipient microenvironment promotes the maturation of transplanted photoreceptors while inducing or facilitating the survival of migratory cell populations that are not normally derived from retinal progenitors. These findings have important implications for potential cell-based treatments of retinal diseases.

Elevated nuclear TDP-43 induces constitutive exon skipping.

Cytoplasmic inclusions and loss of nuclear TDP-43 are key pathological features found in several neurodegenerative disorders, suggesting both gain- and loss-of-function mechanisms of disease. To study gain-of-function, TDP-43 overexpression has been used to generate in vitro and in vivo model systems. Our study shows that excessive levels of nuclear TDP-43 protein lead to constitutive exon skipping that is largely species-specific. Furthermore, while aberrant exon skipping is detected in some human brains, it is not correlated with disease, unlike the incorporation of cryptic exons that occurs after loss of TDP-43. Our findings emphasize the need for caution in interpreting TDP-43 overexpression data, and stress the importance of controlling for exon skipping when generating models of TDP-43 proteinopathy. Understanding the subtle aspects of TDP-43 toxicity within different subcellular locations is essential for the development of therapies targeting neurodegenerative disease.

A neuroepithelial wave of BMP signalling drives anteroposterior specification of the tuberal hypothalamus.

The tuberal hypothalamus controls life-supporting homeostatic processes, but despite its fundamental role, the cells and signalling pathways that specify this unique region of the central nervous system in embryogenesis are poorly characterised. Here, we combine experimental and bioinformatic approaches in the embryonic chick to show that the tuberal hypothalamus is progressively generated from hypothalamic floor plate-like cells. Fate-mapping studies show that a stream of tuberal progenitors develops in the anterior-ventral neural tube as a wave of neuroepithelial-derived BMP signalling sweeps from anterior to posterior through the hypothalamic floor plate. As later-specified posterior tuberal progenitors are generated, early specified anterior tuberal progenitors become progressively more distant from these BMP signals and differentiate into tuberal neurogenic cells. Gain- and loss-of-function experiments in vivo and ex vivo show that BMP signalling initiates tuberal progenitor specification, but must be eliminated for these to progress to anterior neurogenic progenitors. scRNA-Seq profiling shows that tuberal progenitors that are specified after the major period of anterior tuberal specification begin to upregulate genes that characterise radial glial cells. This study provides an integrated account of the development of the tuberal hypothalamus.

Amyloid-beta and tau pathologies act synergistically to induce novel disease stage-specific microglia subtypes.

BACKGROUND: Amongst risk alleles associated with late-onset Alzheimer's disease (AD), those that converged on the regulation of microglia activity have emerged as central to disease progression. Yet, how canonical amyloid-ß (Aß) and tau pathologies regulate microglia subtypes during the progression of AD remains poorly understood. METHODS: We use single-cell RNA-sequencing to profile microglia subtypes from mice exhibiting both Aß and tau pathologies across disease progression. We identify novel microglia subtypes that are induced in response to both Aß and tau pathologies in a disease-stage-specific manner. To validate the observation in AD mouse models, we also generated a snRNA-Seq dataset from the human superior frontal gyrus (SFG) and entorhinal cortex (ERC) at different Braak stages. RESULTS: We show that during early-stage disease, interferon signaling induces a subtype of microglia termed Early-stage AD-Associated Microglia (EADAM) in response to both Aß and tau pathologies. During late-stage disease, a second microglia subtype termed Late-stage AD-Associated Microglia (LADAM) is detected. While similar microglia subtypes are observed in other models of neurodegenerative disease, the magnitude and composition of gene signatures found in EADAM and LADAM are distinct, suggesting the necessity of both Aß and tau pathologies to elicit their emergence. Importantly, the pattern of EADAM- and LADAM-associated gene expression is observed in microglia from AD brains, during the early (Braak II)- or late (Braak VI/V)- stage of the disease, respectively. Furthermore, we show that several Siglec genes are selectively expressed in either EADAM or LADAM. Siglecg is expressed in white-matter-associated LADAM, and expression of Siglec-10, the human orthologue of Siglecg, is progressively elevated in an AD-stage-dependent manner but not shown in non-AD tauopathy. CONCLUSIONS: Using scRNA-Seq in mouse models bearing amyloid-ß and/or tau pathologies, we identify novel microglia subtypes induced by the combination of Aß and tau pathologies in a disease stage-specific manner. Our findings suggest that both Aß and tau pathologies are required for the disease stage-specific induction of EADAM and LADAM. In addition, we revealed Siglecs as biomarkers of AD progression and potential therapeutic targets.

Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis.

An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation.

IReNA: Integrated regulatory network analysis of single-cell transcriptomes and chromatin accessibility profiles.

Recently, single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) have been developed to separately measure transcriptomes and chromatin accessibility profiles at the single-cell resolution. However, few methods can reliably integrate these data to perform regulatory network analysis. Here, we developed integrated regulatory network analysis (IReNA) for network inference through the integrated analysis of scRNA-seq and scATAC-seq data, network modularization, transcription factor enrichment, and construction of simplified intermodular regulatory networks. Using public datasets, we showed that integrated network analysis of scRNA-seq data with scATAC-seq data is more precise to identify known regulators than scRNA-seq data analysis alone. Moreover, IReNA outperformed currently available methods in identifying known regulators. IReNA facilitates the systems-level understanding of biological regulatory mechanisms and is available at https://github.com/jiang-junyao/IReNA.