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.

Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways.

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.

Inflammatory Factor IL1α Induces Aberrant Astrocyte Proliferation in Spinal Cord Injury Through the Grin2c/Ca2+/CaMK2b Pathway.

Spinal cord injury (SCI) is one of the most devastating traumas, and the aberrant proliferation of astrocytes usually causes neurological deficits. However, the mechanism underlying astrocyte over-proliferation after SCI is unclear. Grin2c (glutamate ionotropic receptor type 2c) plays an essential role in cell proliferation. Our bioinformatic analysis indicated that Grin2c and Ca2+ transport functions were inhibited in astrocytes after SCI. Suppression of Grin2c stimulated astrocyte proliferation by inhibiting the Ca2+/calmodulin-dependent protein kinase 2b (CaMK2b) pathway in vitro. By screening different inflammatory factors, interleukin 1α (IL1α) was further found to inhibit Grin2c/Ca2+/CaMK2b and enhance astrocyte proliferation in an oxidative damage model. Blockade of IL1α using neutralizing antibody resulted in increased Grin2c expression and the inhibition of astrocyte proliferation post-SCI. Overall, this study suggests that IL1α promotes astrocyte proliferation by suppressing the Grin2c/Ca2+/CaMK2b pathway after SCI, revealing a novel pathological mechanism of astrocyte proliferation, and may provide potential targets for SCI repair.

Glycogen synthase kinase 3 signaling in neural regeneration in vivo.

Glycogen synthase kinase 3 (GSK3) signaling plays important and broad roles in regulating neural development in vitro and in vivo. Here, we reviewed recent findings of GSK3-regulated axon regeneration in vivo in both the peripheral and central nervous systems and discussed a few controversial findings in the field. Overall, current evidence indicates that GSK3ß signaling serves as an important downstream mediator of the PI3K-AKT pathway to regulate axon regeneration in parallel with the mTORC1 pathway. Specifically, the mTORC1 pathway supports axon regeneration mainly through its role in regulating cap-dependent protein translation, whereas GSK3ß signaling might be involved in regulating N6-methyladenosine (m6A) mRNA methylation-mediated cap-independent protein translation. In addition, GSK3 signaling also plays key roles in reshaping the neuronal transcriptomic landscape during neural regeneration. Finally, we proposed some research directions to further elucidate the molecular mechanisms underlying the regulatory function of GSK3 signaling and discover novel GSK3 signaling-related therapeutic targets. Together, we hope to provide an updated and insightful overview of how GSK3 signaling regulates neural regeneration in vivo.

X chromosome encoded histone demethylase UTX regulates mammalian axon regeneration via microRNA-124.

Neurons in the mammalian central nervous system (CNS) gradually lose their intrinsic regeneration capacity during maturation mainly because of altered transcription profile. Recent studies have made great progress by identifying genes that can be manipulated to enhance CNS regeneration. However, as a complex process involving many genes and signaling networks, it is of great importance to deciphering the underlying neuronal chromatin and transcriptomic landscape coordinating CNS regeneration. Here we identify UTX, an X-chromosome associated gene encoding a histone demethylase, as a novel regulator of mammalian neural regeneration. We demonstrate that UTX acts as a repressor of spontaneous axon regeneration in the peripheral nerve system (PNS). In the CNS, either knocking out or pharmacological inhibiting UTX in retinal ganglion cells (RGCs) leads to significantly enhanced neuronal survival and optic nerve regeneration. RNA-seq profiling revealed that deleting UTX switches the RGC transcriptomics into a developmental-like state. Moreover, microRNA-124, one of the most abundant microRNAs in mature neurons, is identified as a downstream target of UTX and blocking endogenous microRNA124-5p results in robust optic nerve regeneration. These findings revealed a novel histone modification-microRNA epigenetic signaling network orchestrating transcriptomic landscape supporting CNS neural regeneration.

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.

UCHL1 facilitates protein aggregates clearance to enhance neural stem cell activation in spinal cord injury.

Activation of endogenous neural stem cells (NSCs) is greatly significant for the adult neurogenesis; however, it is extremely limited in the spinal cord after injury. Recent evidence suggests that accumulation of protein aggregates impairs the ability of quiescent NSCs to activate. Ubiquitin c-terminal hydrolase l-1 (UCHL1), an important deubiquitinating enzyme, plays critical roles in protein aggregations clearance, but its effects on NSC activation remains unknown. Here, we show that UCHL1 promotes NSC activation by clearing protein aggregates through ubiquitin-proteasome approach. Upregulation of UCHL1 facilitated the proliferation of spinal cord NSCs after spinal cord injury (SCI). Based on protein microarray analysis of SCI cerebrospinal fluid, it is further revealed that C3+ neurotoxic reactive astrocytes negatively regulated UCHL1 and proteasome activity via C3/C3aR signaling, led to increased abundances of protein aggregations and decreased NSC proliferation. Furthermore, blockade of reactive astrocytes or C3/C3aR pathway enhanced NSC activation post-SCI by reserving UCHL1 and proteasome functions. Together, this study elucidated a mechanism regulating NSC activation in the adult spinal cord involving the UCHL1-proteasome approach, which may provide potential molecular targets and new insights for NSC fate regulation.

Neuronal Histone Methyltransferase EZH2 Regulates Neuronal Morphogenesis, Synaptic Plasticity, and Cognitive Behavior in Mice.

The histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)-mediated trimethylation of histone H3 lysine 27 (H3K27me3) regulates neural stem cell proliferation and fate specificity through silencing different gene sets in the central nervous system. Here, we explored the function of EZH2 in early post-mitotic neurons by generating a neuron-specific Ezh2 conditional knockout mouse line. The results showed that a lack of neuronal EZH2 led to delayed neuronal migration, more complex dendritic arborization, and increased dendritic spine density. Transcriptome analysis revealed that neuronal EZH2-regulated genes are related to neuronal morphogenesis. In particular, the gene encoding p21-activated kinase 3 (Pak3) was identified as a target gene suppressed by EZH2 and H3K27me3, and expression of the dominant negative Pak3 reversed Ezh2 knockout-induced higher dendritic spine density. Finally, the lack of neuronal EZH2 resulted in impaired memory behaviors in adult mice. Our results demonstrated that neuronal EZH2 acts to control multiple steps of neuronal morphogenesis during development, and has long-lasting effects on cognitive function in adult mice.

scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of PirtEGFPf mice in neuropathic pain condition.

Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using PirtEGFPf mice that selectively express an enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using Fgf3 as a proof-of-principle, RNAscope study provided further evidence of increased Fgf3 in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of Fgf3 in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.

Reprogramming neurons for regeneration: The fountain of youth.

Neurons in the central nervous system (CNS) are terminally differentiated cells that gradually lose their ability to support regeneration during maturation due to changes in transcriptomic and chromatin landscape. Similar transcriptomic changes also occur during development when stem cells differentiate into different types of somatic cells. Importantly, differentiated cells can be reprogrammed back to induced pluripotent stems cells (iPSCs) via global epigenetic remodeling by combined overexpression of pluripotent reprogramming factors, including Oct4, Sox2, Klf4, c-Myc, Nanog, and/or Lin28. Moreover, recent findings showed that many proneural transcription factors were able to convert non-neural somatic cells into neurons bypassing the pluripotent stage via direct reprogramming. Interestingly, many of these factors have recently been identified as key regulators of CNS neural regeneration. Recent studies indicated that these factors could rejuvenate mature CNS neurons back to a younger state through cellular state reprogramming, thus favoring regeneration. Here we will review some recent findings regarding the roles of genetic cellular state reprogramming in regulation of neural regeneration and explore the potential underlying molecular mechanisms. Moreover, by using newly emerging techniques, such as multiomics sequencing with big data analysis and Crispr-based gene editing, we will discuss future research directions focusing on better revealing cellular state reprogramming-induced remodeling of chromatin landscape and potential translational application.

Synchronized cluster firing, a distinct form of sensory neuron activation, drives spontaneous pain.

Spontaneous pain refers to pain occurring without external stimuli. It is a primary complaint in chronic pain conditions and remains difficult to treat. Moreover, the mechanisms underlying spontaneous pain remain poorly understood. Here we employed in vivo imaging of dorsal root ganglion (DRG) neurons and discovered a distinct form of abnormal spontaneous activity following peripheral nerve injury: clusters of adjacent DRG neurons firing synchronously and sporadically. The level of cluster firing correlated directly with nerve injury-induced spontaneous pain behaviors. Furthermore, we demonstrated that cluster firing is triggered by activity of sympathetic nerves, which sprout into DRGs after injury, and identified norepinephrine as a key neurotransmitter mediating this unique firing. Chemogenetic and pharmacological manipulations of sympathetic activity and norepinephrine receptors suggest that they are necessary and sufficient for DRG cluster firing and spontaneous pain behavior. Therefore, blocking sympathetically mediated cluster firing may be a new paradigm for treating spontaneous pain.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

In vivo glial trans-differentiation for neuronal replacement and functional recovery in central nervous system.

The adult mammalian central nervous system (CNS) is deficient in intrinsic machineries to replace neurons lost in injuries or progressive degeneration. Various types of these neurons constitute neural circuitries wired to support vital sensory, motor, and cognitive functions. Based on the pioneer studies in cell lineage conversion, one promising strategy is to convert in vivo glial cells into neural progenitors or directly into neurons that can be eventually rewired for functional recovery. We first briefly summarize the well-studied regeneration-capable CNS in the zebrafish, focusing on their postinjury spontaneous reprogramming of the retinal Müller glia (MG). We then compare the signaling transductions, and transcriptional and epigenetic regulations in the zebrafish MGs with their mammalian counterparts, which perpetuate certain barriers against proliferation and neurogenesis and thus fail in MG-to-progenitor conversion. Next, we discuss emerging evidence from mouse studies, in which the in vivo glia-to-neuron conversion could be achieved with sequential or one-step genetic manipulations, such as the conversions from retinal MGs to interneurons, photoreceptors, or retinal ganglion cells (RGCs), as well as the conversions from midbrain astrocytes to dopaminergic or GABAergic neurons. Some of these in vivo studies showed considerable coverage of subtypes in the newly induced neurons and partial reestablishment in neural circuits and functions. Importantly, we would like to point out some crucial technical concerns that need to be addressed to convincingly show successful glia-to-neuron conversion. Finally, we present challenges and future directions in the field for better neural function recovery.

Strategies to Promote Long-Distance Optic Nerve Regeneration.

Mammalian retinal ganglion cells (RGCs) in the central nervous system (CNS) often die after optic nerve injury and surviving RGCs fail to regenerate their axons, eventually resulting in irreversible vision loss. Manipulation of a diverse group of genes can significantly boost optic nerve regeneration of mature RGCs by reactivating developmental-like growth programs or suppressing growth inhibitory pathways. By injury of the vision pathway near their brain targets, a few studies have shown that regenerated RGC axons could form functional synapses with targeted neurons but exhibited poor neural conduction or partial functional recovery. Therefore, the functional restoration of eye-to-brain pathways remains a greatly challenging issue. Here, we review recent advances in long-distance optic nerve regeneration and the subsequent reconnecting to central targets. By summarizing our current strategies for promoting functional recovery, we hope to provide potential insights into future exploration in vision reformation after neural injuries.

Updates and challenges of axon regeneration in the mammalian central nervous system.

Axon regeneration in the mammalian central nervous system (CNS) has been a long-standing and highly challenging issue. Successful CNS axon regeneration will benefit many human diseases involving axonal damage, such as spinal cord injury, traumatic brain injury, glaucoma, and neurodegenerative diseases. The current consensus is that the diminished intrinsic regenerative ability in mature CNS neurons and the presence of extrinsic inhibitors blocking axon regrowth are two major barriers for axon regeneration. During the past decade, studies targeting the intrinsic axon growth ability via regulation of gene transcription have produced very promising results in optic nerve and/or spinal cord regeneration. Manipulations of various signaling pathways or the nuclear transcription factors directly have been shown to sufficiently drive CNS axon regrowth. Converging evidence reveals that some pro-regenerative transcriptomic states, which are commonly accomplished by more comprehensive epigenetic regulations, exist to orchestrate the complex tasks of injury sensing and axon regeneration. Moreover, genetic reprogramming achieved via transcriptome and epigenome modifications provides novel mechanisms for enhancing axon regeneration. Recent studies also highlighted the important roles of remodeling neuronal cytoskeleton in overcoming the extrinsic inhibitory cues. However, our knowledge about the cellular and molecular mechanisms by which neurons regulate their intrinsic axon regeneration ability and response to extrinsic inhibitory cues is still fragmented. Here, we provide an update about recent research progress in axon regeneration and discuss major remaining challenges for long-distance axon regeneration and the subsequent functional recovery.

Upregulating Lin28a Promotes Axon Regeneration in Adult Mice with Optic Nerve and Spinal Cord Injury.

Severed CNS axons fail to regenerate in adult mammals and there are no effective regenerative strategies to treat patients with CNS injuries. Several genes, including phosphatase and tensin homolog (PTEN) and Krüppel-like factors, regulate intrinsic growth capacity of mature neurons. The Lin28 gene is essential for cell development and pluripotency in worms and mammals. In this study, we evaluated the role of Lin28a in regulating regenerative capacity of diverse populations of CNS neurons in adult mammals. Using a neuron-specific Thy1 promoter, we generated transgenic mice that overexpress Lin28a protein in multiple populations of projection neurons, including corticospinal tracts and retinal ganglion cells. We demonstrate that upregulation of Lin28a in transgenic mice induces significant long distance regeneration of both corticospinal axons and the optic nerve in adult mice. Importantly, overexpression of Lin28a by post-injury treatment with adeno-associated virus type 2 (AAV2) vector stimulates dramatic regeneration of descending spinal tracts and optic nerve axons after lesions. Upregulation of Lin28a also enhances activity of the Akt signaling pathway in mature CNS neurons. Therefore, Lin28a is critical for regulating growth capacity of multiple CNS neurons and may become an important molecular target for treating CNS injuries.

Knocking Out Non-muscle Myosin II in Retinal Ganglion Cells Promotes Long-Distance Optic Nerve Regeneration.

In addition to altered gene expression, pathological cytoskeletal dynamics in the axon are another key intrinsic barrier for axon regeneration in the central nervous system (CNS). Here, we show that knocking out myosin IIA and IIB (myosin IIA/B) in retinal ganglion cells alone, either before or after optic nerve crush, induces significant optic nerve regeneration. Combined Lin28a overexpression and myosin IIA/B knockout lead to an additive promoting effect and long-distance axon regeneration. Immunostaining, RNA sequencing, and western blot analyses reveal that myosin II deletion does not affect known axon regeneration signaling pathways or the expression of regeneration-associated genes. Instead, it abolishes the retraction bulb formation and significantly enhances the axon extension efficiency. The study provides clear evidence that directly targeting neuronal cytoskeleton is sufficient to induce significant CNS axon regeneration and that combining altered gene expression in the soma and modified cytoskeletal dynamics in the axon is a promising approach for long-distance CNS axon regeneration.

The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.