Retrograde AAV-mediated gene modulation reveals chloride intracellular channel proteins as potent regulators of retinal ganglion cell death.

Most projection neurons, including retinal ganglion cells (RGCs), undergo cell death after axotomy proximal to the cell body. Specific RGC subtypes, such as ON-OFF direction selective RGCs (ooDSGCs) are particularly vulnerable, whereas intrinsically photosensitive RGCs (ipRGCs) exhibit resilience to axonal injury. Through the application of RNA sequencing and fluorescent in situ hybridization, we show that the expression of chloride intracellular channel protein 1 and 4 (Clic1 and Clic4) are highly increased in the ooDSGCs after axonal injury. Toward determining a gene's role in RGCs, we optimized the utility and efficacy of adenovirus associated virus (AAV)-retro expressing short hairpin RNA (shRNA). Injection of AAV2-retro into the superior colliculus results in efficient shRNA expression in RGCs. Incorporating histone H2B gene fused with mGreenLantern results in bright nuclear reporter expression, thereby enhancing single RGC identification and cell quantitation in live retinas. Lastly, we demonstrate that AAV2-retro mediated knockdown of both Clic1 and Clic4 promotes RGC survival after injury. Our findings establish an integrated use of AAV2-retro-shRNA and real-time fundus imaging and reveal CLICs' contribution to RGC death.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Single-nucleus RNA sequencing of developing superior colliculus identifies neuronal diversity and candidate mediators of circuit assembly.

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment of neural connectivity. Here we perform single-nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Using these data, we find that Pax7 expression is restricted to a subset of GABAergic neurons. Our data provide a valuable resource for interrogating the mechanisms of circuit development and identifying markers for manipulating specific SC neuronal populations and circuits.

Optic nerve diseases and regeneration: How far are we from the promised land?

The retinal ganglion cells (RGCs) are the sole output neurons that connect information from the retina to the brain. Optic neuropathies such as glaucoma, trauma, inflammation, ischemia and hereditary optic neuropathy can cause RGC loss and axon damage, and lead to partial or total loss of vision, which is an irreversible process in mammals. The accurate diagnoses of optic neuropathies are crucial for timely treatments to prevent irrevocable RGCs loss. After severe ON damage in optic neuropathies, promoting RGC axon regeneration is vital for restoring vision. Clearance of neuronal debris, decreased intrinsic growth capacity, and the presence of inhibitory factors have been shown to contribute to the failure of post-traumatic CNS regeneration. Here, we review the current understanding of manifestations and treatments of various common optic neuropathies. We also summarise the current known mechanisms of RGC survival and axon regeneration in mammals, including specific intrinsic signalling pathways, key transcription factors, reprogramming genes, inflammation-related regeneration factors, stem cell therapy, and combination therapies. Significant differences in RGC subtypes in survival and regenerative capacity after injury have also been found. Finally, we highlight the developmental states and non-mammalian species that are capable of regenerating RGC axons after injury, and cellular state reprogramming for neural repair.

Single-Nucleus RNA Sequencing of Developing and Mature Superior Colliculus Identifies Neuronal Diversity and Candidate Mediators of Circuit Assembly.

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment and refinement of neural connectivity. Here we performed single nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We used these data to identify Pax7 as a marker for an anatomically homogeneous population of GABAergic neurons. Lastly, we report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Our data provide a valuable resource for interrogating the mechanisms of circuit development, and identifying markers for manipulating specific SC neuronal populations and circuits.

GSDMD deficiency ameliorates hyperoxia-induced BPD and ROP in neonatal mice.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are among the most common morbidities affecting extremely premature infants who receive oxygen therapy. Many clinical studies indicate that BPD is associated with advanced ROP. However, the mechanistic link between hyperoxia, BPD, and ROP remains to be explored. Gasdermin D (GSDMD) is a key executor of inflammasome-induced pyroptosis and inflammation. Inhibition of GSDMD has been shown to attenuate hyperoxia-induced BPD and brain injury in neonatal mice. The objective of this study was to further define the mechanistic roles of GSDMD in the pathogenesis of hyperoxia-induced BPD and ROP in mouse models. Here we show that global GSDMD knockout (GSDMD-KO) protects against hyperoxia-induced BPD by reducing macrophage infiltration, improving alveolarization and vascular development, and decreasing cell death. In addition, GSDMD deficiency prevented hyperoxia-induced ROP by reducing vasoobliteration and neovascularization, improving thinning of multiple retinal tissue layers, and decreasing microglial activation. RNA sequencing analyses of lungs and retinas showed that similar genes, including those from inflammatory, cell death, tissue remodeling, and tissue and vascular developmental signaling pathways, were induced by hyperoxia and impacted by GSDMD-KO in both models. These data highlight the importance of GSDMD in the pathogenesis of BPD and ROP and suggest that targeting GSDMD may be beneficial in preventing and treating BPD and ROP in premature infants.

Awakening dormant neurons long after spinal cord injury.

Neurons lack the ability to regenerate after injury. A new Preregistered Article in PLOS Biology finds that pharmacologically boosting regenerative capacity long after injury in mice, together with an enriched animal environment, promotes axonal and synaptic plasticity.

Subtype-specific survival and regeneration of retinal ganglion cells in response to injury.

Retinal ganglion cells (RGCs) are a heterogeneous population of neurons that function synchronously to convey visual information through the optic nerve to retinorecipient target areas in the brain. Injury or disease to the optic nerve results in RGC degeneration and loss of visual function, as few RGCs survive, and even fewer can be provoked to regenerate their axons. Despite causative insults being broadly shared, regeneration studies demonstrate that RGC types exhibit differential resilience to injury and undergo selective survival and regeneration of their axons. While most early studies have identified these RGC types based their morphological and physiological characteristics, recent advances in transgenic and gene sequencing technologies have further enabled type identification based on unique molecular features. In this review, we provide an overview of the well characterized RGC types and identify those shown to preferentially survive and regenerate in various regeneration models. Furthermore, we discuss cellular characteristics of both the resilient and susceptible RGC types including the combinatorial expression of different molecular markers that identify these specific populations. Lastly, we discuss potential molecular mechanisms and genes found to be selectively expressed by specific types that may contribute to their reparative capacity. Together, we describe the studies that lay the important groundwork for identifying factors that promote neural regeneration and help advance the development of targeted therapy for the treatment of RGC degeneration as well as neurodegenerative diseases in general.

Retinal ganglion cell expression of cytokine enhances occupancy of NG2 cell-derived astrocytes at the nerve injury site: Implication for axon regeneration.

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

Identification of long noncoding RNAs in injury-resilient and injury-susceptible mouse retinal ganglion cells.

BACKGROUND: Emerging evidence indicates that long noncoding RNAs (lncRNAs) are important regulators of various biological processes, and their expression can be altered following certain pathological conditions, including central nervous system injury. Retinal ganglion cells (RGCs), whose axons form the optic nerve, are a heterogeneous population of neurons with more than 40 molecularly distinct subtypes in mouse. While most RGCs, including the ON-OFF direction-selective RGCs (ooDSGCs), are vulnerable to axonal injury, a small population of RGCs, including the intrinsically photosensitive RGCs (ipRGCs), are more resilient. RESULTS: By performing systematic analyses on RNA-sequencing data, here we identify lncRNAs that are expressed in ooDSGCs and ipRGCs with and without axonal injury. Our results reveal a repertoire of different classes of lncRNAs, including long intergenic noncoding RNAs and antisense ncRNAs that are differentially expressed between these RGC types. Strikingly, we also found dozens of lncRNAs whose expressions are altered markedly in response to axonal injury, some of which are expressed exclusively in either one of the types. Moreover, analyses into these lncRNAs unraveled their neighboring coding genes, many of which encode transcription factors and signaling molecules, suggesting that these lncRNAs may act in cis to regulate important biological processes in these neurons. Lastly, guilt-by-association analysis showed that lncRNAs are correlated with apoptosis associated genes, suggesting potential roles for these lncRNAs in RGC survival. CONCLUSIONS: Overall, the results of this study reveal RGC type-specific expression of lncRNAs and provide a foundation for future investigation of the function of lncRNAs in regulating neuronal type specification and survival.

Proteomics and systems biology in optic nerve regeneration.

We present an overview of current state of proteomic approaches as applied to optic nerve regeneration in the historical context of nerve regeneration particularly central nervous system neuronal regeneration. We present outlook pertaining to the optic nerve regeneration proteomics that the latter can extrapolate information from multi-systems level investigations. We present an account of the current need of systems level standardization for comparison of proteome from various models and across different pharmacological or biophysical treatments that promote adult neuron regeneration. We briefly overview the need for deriving knowledge from proteomics and integrating with other omics to obtain greater biological insight into process of adult neuron regeneration in the optic nerve and its potential applicability to other central nervous system neuron regeneration.

Neural Cadherin Plays Distinct Roles for Neuronal Survival and Axon Growth under Different Regenerative Conditions.

Growing axons in the CNS often migrate along specific pathways to reach their targets. During embryonic development, this migration is guided by different types of cell adhesion molecules (CAMs) present on the surface of glial cells or other neurons, including the neural cadherin (NCAD). Axons in the adult CNS can be stimulated to regenerate, and travel long distances. Crucially, however, while a few axons are guided effectively through the injured nerve under certain conditions, most axons never migrate properly. The molecular underpinnings of the variable growth, and the glial CAMs that are responsible for CNS axon regeneration remain unclear. Here we used optic nerve crush to demonstrate that NCAD plays multifaceted functions in facilitating CNS axon regeneration. Astrocyte-specific deletion of NCAD dramatically decreases regeneration induced by phosphatase and tensin homolog (PTEN) ablation in retinal ganglion cells (RGCs). Consistent with NCAD's tendency to act as homodimers, deletion of NCAD in RGCs also reduces regeneration. Deletion of NCAD in astrocytes neither alters RGCs' mammalian target of rapamycin complex 1 (mTORC1) activity nor lesion size, two factors known to affect regeneration. Unexpectedly, however, we find that NCAD deletion in RGCs reduces PTEN-deletion-induced RGC survival. We further show that NCAD deletion, in either astrocytes or RGCs, has negligible effects on the regeneration induced by ciliary neurotrophic factor (CNTF), suggesting that other CAMs are critical under this regenerative condition. Consistent with this notion, CNTF induces expression various integrins known to mediate cell adhesion. Together, our study reveals multilayered functions of NCAD and a molecular basis of variability in guided axon growth.

Multi-Omic Analyses of Growth Cones at Different Developmental Stages Provides Insight into Pathways in Adult Neuroregeneration.

Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.

Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells.

Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.

3D Visualization of Individual Regenerating Retinal Ganglion Cell Axons Reveals Surprisingly Complex Growth Paths.

Retinal ganglion cells (RGCs), the sole output cells of the retina, are a heterogeneous population of neurons that project axons to visual targets in the brain. Like most CNS neurons, RGCs are considered incapable of mounting long distance axon regeneration. Using immunolabeling-enabled 3D imaging of solvent-cleared organs (iDISCO) in transgenic mice, we tracked the entire paths of individual RGC axons and show that adult RGCs are highly capable of spontaneous long-distance regeneration, even without any treatment. Our results show that the Thy1-H-YFP mouse sparsely labels RGCs, consisting predominantly of regeneration-competent α-type RGCs (αRGCs). Following optic nerve crush, many of the YFP-labeled RGC axons extend considerable distances proximal to the injury site with only a few penetrating through the lesion. This tortuous axon growth proximal to the lesion site is even more striking with intravitreal ciliary neurotrophic factor (CNTF) treatment. We further demonstrate that despite traveling more than 5 mm (i.e., a distance equal to the length of mouse optic nerve), many of these circuitous axons are confined to the injury area and fail to reach the brain. Our results re-evaluate the view that RGCs are naturally incapable of re-extending long axons, and shift the focus from promoting axon elongation, to understanding factors that prevent direct growth of axons through the lesion and the injured nerve.

Optic nerve regeneration in mammals: Regenerated or spared axons?

Intraorbital optic nerve crush in rodents is widely used as a model to study axon regeneration in the adult mammalian central nervous system. Recent studies using appropriate genetic manipulations have revealed remarkable abilities of mature retinal ganglion cell (RGC) axons to regenerate after optic nerve injury, with some studies demonstrating that axons can then go on to re-innervate a number of central visual targets with partial functional restoration. However, one confounding factor inherent to optic nerve crush injury is the potential incompleteness of the initial lesion, leaving spared axons that later on could erroneously be interpreted as regenerating distal to the injury site. Careful examination of axonal projection pattern and morphology may facilitate separating spared from regenerating RGC axons. Here we discuss morphological criteria and strategies that may be used to differentiate spared versus regenerated axons in the injured mammalian optic nerve.

Regenerative Responses and Axon Pathfinding of Retinal Ganglion Cells in Chronically Injured Mice.

Purpose: Enhanced regeneration of retinal ganglion cell (RGC) axons can be achieved by modification of numerous neuronal-intrinsic factors. However, axon growth initiation and the pathfinding behavior of these axons after traumatic injury remain poorly understood outside of acute injury paradigms, despite the clinical relevance of more chronic settings. We therefore examined RGC axon regeneration following therapeutic delivery that is postponed until 2 months after optic nerve crush injury. Methods: Optic nerve regeneration was induced by virally mediated (adeno-associated virus) ciliary neurotrophic factor (AAV-CNTF) administered either immediately or 56 days after optic nerve crush in wild-type or Bax knockout (KO) mice. Retinal ganglion nerve axon regeneration was assessed 21 and 56 days after viral injection. Immunohistochemical analysis of RGC injury signals and extrinsic factors in the optic nerve were also examined at 5 and 56 days post crush. Results: In addition to sustained expression of injury response proteins in surviving RGCs, we observe axon regrowth in wild-type and apoptosis-deficient Bax KO mice following AAV-CNTF treatment. Fewer instances of aberrant axon growth are seen, at least in the area near the lesion site, in animals given treatment 56 days after crush injury compared to the animals given treatment immediately after injury. We also find evidence of long distance growth into a visual target in Bax KO mice despite postponed initiation of this regenerative program. Conclusions: These studies provide evidence against an intrinsic critical period for RGC axon regeneration or degradation of injury signals. Regeneration results from Bax KO mice imply highly sustained regenerative capacity in RGCs, highlighting the importance of long-lasting neuroprotective strategies as well as of RGC axon guidance research in chronically injured animals.

Retinal ganglion cell survival and axon regeneration after optic nerve injury in naked mole-rats.

In the adult mammalian central nervous system (CNS), axonal damage often triggers neuronal cell death and glial activation, with very limited spontaneous axon regeneration. In this study, we performed optic nerve injury in adult naked mole-rats, the longest living rodent, with a maximum life span exceeding 30 years, and found that injury responses in this species are quite distinct from those in other mammalian species. In contrast to what is seen in other mammals, the majority of injured retinal ganglion cells (RGCs) survive with relatively high spontaneous axon regeneration. Furthermore, injured RGCs display activated signal transducer and activator of transcription-3 (STAT3), whereas astrocytes in the optic nerve robustly occupy and fill the lesion area days after injury. These neuron-intrinsic and -extrinsic injury responses are reminiscent of those in "cold-blooded" animals, such as fish and amphibians, suggesting that the naked mole-rat is a powerful model for exploring the mechanisms of neuronal injury responses and axon regeneration in mammals. J. Comp. Neurol. 525:380-388, 2017. © 2016 Wiley Periodicals, Inc.

Wnt signaling promotes axonal regeneration following optic nerve injury in the mouse.

Adult mammalian CNS axons generally do not regenerate, creating an obstacle to effective repair and recovery after neuronal injury. The canonical Wnt/ß-catenin signaling pathway is an essential signal transduction cascade that regulates axon growth and neurite extension in the developing mammalian embryo. In this study, we investigated whether a Wnt/ß-catenin signaling activator could be repurposed to induce regeneration in the adult CNS after axonal injury. We used a retinal ganglion cell (RGC) axon crush injury model in a transgenic Wnt reporter mouse, and intravitreal injections were used to deliver Wnt3a or saline to the RGC cell bodies within the retina. Our findings demonstrated that Wnt3a induced Wnt signaling in RGCs and resulted in significant axonal regrowth past the lesion site when measured at two and four weeks post-injury. Furthermore, Wnt3a-injected eyes showed increased survival of RGCs and significantly higher pattern electroretinography (PERG) amplitudes compared to the control. Additionally, Wnt3a-induced axonal regeneration and RGC survival were associated with elevated activation of the transcription factor Stat3, and reducing expression of Stat3 using a conditional Stat3 knock-out mouse line led to diminished Wnt3a-dependent axonal regeneration and RGC survival. Therefore, these findings reveal a novel role for retinal Wnt signaling in axonal regrowth and RGC survival following axonal injury, which may lead to the development of novel therapies for axonal regeneration.