Ependymal cells undergo astrocyte-like reactivity in response to neuroinflammation.

Ependymal cells form a specialized brain-cerebrospinal fluid (CSF) interface and regulate local CSF microcirculation. It is becoming increasingly recognized that ependymal cells assume a reactive state in response to aging and disease, including conditions involving hypoxia, hydrocephalus, neurodegeneration, and neuroinflammation. Yet what transcriptional signatures govern these reactive states and whether this reactivity shares any similarities with classical descriptions of glial reactivity (i.e., in astrocytes) remain largely unexplored. Using single-cell transcriptomics, we interrogated this phenomenon by directly comparing the reactive ependymal cell transcriptome to the reactive astrocyte transcriptome using a well-established model of autoimmune-mediated neuroinflammation (MOG35-55 EAE). In doing so, we unveiled core glial reactivity-associated genes that defined the reactive ependymal cell and astrocyte response to MOG35-55 EAE. Interestingly, known reactive astrocyte genes from other CNS injury/disease contexts were also up-regulated by MOG35-55 EAE ependymal cells, suggesting that this state may be conserved in response to a variety of pathologies. We were also able to recapitulate features of the reactive ependymal cell state acutely using a classic neuroinflammatory cocktail (IFNγ/LPS) both in vitro and in vivo. Taken together, by comparing reactive ependymal cells and astrocytes, we identified a conserved signature underlying glial reactivity that was present in several neuroinflammatory contexts. Future work will explore the mechanisms driving ependymal reactivity and assess downstream functional consequences.

Cellular rejuvenation protects neurons from inflammation mediated cell death.

In multiple sclerosis (MS), the invasion of the central nervous system by peripheral immune cells is followed by the activation of resident microglia and astrocytes. This cascade of events results in demyelination, which triggers neuronal damage and death. The molecular signals in neurons responsible for this damage are not yet fully characterized. In MS, retinal ganglion cell neurons (RGCs) of the central nervous system (CNS) undergo axonal injury and cell death. This phenomenon is mirrored in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. To understand the molecular landscape, we isolated RGCs from mice subjected to the EAE protocol. RNA-sequencing and ATAC-sequencing analyses were performed. Pathway analysis of the RNA-sequencing data revealed that RGCs displayed a molecular signature, similar to aged neurons, showcasing features of senescence. Single-nucleus RNA-sequencing analysis of neurons from human MS patients revealed a comparable senescence-like phenotype., which was supported by immunostaining RGCs in EAE mice. These changes include alterations to the nuclear envelope, modifications in chromatin marks, and accumulation of DNA damage. Transduction of RGCs with an Oct4 - Sox2 - Klf4 transgene to convert neurons in the EAE model to a more youthful epigenetic and transcriptomic state enhanced the survival of RGCs. Collectively, this research uncovers a previously unidentified senescent-like phenotype in neurons under pathological inflammation and neurons from MS patients. The rejuvenation of this aged transcriptome improved visual acuity and neuronal survival in the EAE model supporting the idea that age rejuvenation therapies and senotherapeutic agents could offer a direct means of neuroprotection in autoimmune disorders.

Comparing RNA-sequencing datasets from astrocytes, oligodendrocytes, and microglia in multiple sclerosis identifies novel dysregulated genes relevant to inflammation and myelination.

Central nervous system (CNS) inflammation is a key factor in multiple sclerosis (MS). Invasion of peripheral immune cells into the CNS resulting from an unknown signal or combination of signals results in activation of resident immune cells and the hallmark feature of the disease: demyelinating lesions. These lesion sites are an amalgam of reactive peripheral and central immune cells, astrocytes, damaged and dying oligodendrocytes, and injured neurons and axons. Sustained inflammation affects cells directly located within the lesion site and further abnormalities are apparent diffusely throughout normal-appearing white matter and grey matter. It is only relatively recently, using animal models, new tissue sampling techniques, and next-generation sequencing, that molecular changes occurring in CNS resident cells have been broadly captured. Advances in cell isolation through Fluorescence Activated Cell Sorting (FACS) and laser-capture microdissection together with the emergence of single-cell sequencing have enabled researchers to investigate changes in gene expression in astrocytes, microglia, and oligodendrocytes derived from animal models of MS as well as from primary patient tissue. The contribution of some dysregulated pathways has been followed up in individual studies; however, corroborating results often go unreported between sequencing studies. To this end, we have consolidated results from numerous RNA-sequencing studies to identify and review novel patterns of differentially regulated genes and pathways occurring within CNS glial cells in MS. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology.

3-Dimensional Immunostaining and Automated Deep-Learning Based Analysis of Nerve Degeneration.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models.

Self-delivering RNAi compounds as therapeutic agents in the central nervous system to enhance axonal regeneration after injury.

The therapeutic use of RNAi has grown but often faces several hurdles related to delivery systems, compound stability, immune activation, and on-target/off-tissue effects. Self-delivering RNAi (sdRNA) molecules do not require delivery agents or excipients. Here we demonstrate the ability of sdRNA to reduce the expression of PTEN (phosphatase and tensin homolog) to stimulate regenerative axon regrowth in the injured adult CNS. PTEN-targeting sdRNA compounds were tested for efficacy in vivo by intravitreal injection after adult rat optic nerve injury. We describe critical steps in lead compound generation through the optimization of nucleotide modifications, enhancements for stability in biological matrices, and screening for off-target immunostimulatory activity. The data show that PTEN expression in vivo can be reduced using sdRNA and this enhances regeneration in adult CNS neurons after injury, raising the possibility that this method could be utilized for other clinically relevant nervous system indications.

To grow and to stay, both controlled by RhoA: Opposing cellular effects on axon regeneration.

In this issue of Neuron, Stern et al. (2021) demonstrate that cell-type-specific ablation of RhoA differentially affects axon regeneration outcomes in spinal cord injury models. Their findings highlight the importance of considering cell-type-specific strategies to promote axon regeneration.

Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth.

Targeting protein-protein interactions (PPIs) is a promising approach in the development of drugs for many indications. 14-3-3 proteins are a family of phosphoprotein-binding molecules with critical functions in dozens of cell signaling networks. 14-3-3s are abundant in the central nervous system, and the small molecule fusicoccin-A (FC-A), a tool compound that can be used to manipulate 14-3-3 PPIs, enhances neurite outgrowth in cultured neurons. New semisynthetic FC-A derivatives with improved binding affinity for 14-3-3 complexes have recently been developed. Here, we use a series of screens that identify these compounds as potent inducers of neurite outgrowth through a polypharmacological mechanism. Using proteomics and X-ray crystallography, we discover that these compounds extensively regulate the 14-3-3 interactome by stabilizing specific PPIs, while disrupting others. These results provide new insights into the development of drugs to target 14-3-3 PPIs, a potential therapeutic strategy for CNS diseases.

Collapsin Response Mediator Protein 4 (CRMP4) Facilitates Wallerian Degeneration and Axon Regeneration following Sciatic Nerve Injury.

In contrast to neurons in the CNS, damaged neurons from the peripheral nervous system (PNS) regenerate, but this process can be slow and imperfect. Successful regeneration is orchestrated by cytoskeletal reorganization at the tip of the proximal axon segment and cytoskeletal disassembly of the distal segment. Collapsin response mediator protein 4 (CRMP4) is a cytosolic phospho-protein that regulates the actin and microtubule cytoskeleton. During development, CRMP4 promotes growth cone formation and dendrite development. Paradoxically, in the adult CNS, CRMP4 impedes axon regeneration. Here, we investigated the involvement of CRMP4 in peripheral nerve injury in male and female Crmp4-/- mice following sciatic nerve injury. We find that sensory axon regeneration and Wallerian degeneration are impaired in Crmp4-/- mice following sciatic nerve injury. In vitro analysis of dissociated dorsal root ganglion (DRG) neurons from Crmp4-/- mice revealed that CRMP4 functions in the proximal axon segment to promote the regrowth of severed DRG neurons and in the distal axon segment where it facilitates Wallerian degeneration through calpain-dependent formation of harmful CRMP4 fragments. These findings reveal an interesting dual role for CRMP4 in proximal and distal axon segments of injured sensory neurons that coordinately facilitate PNS axon regeneration.

Profiling the microRNA signature of the peripheral sensory ganglia in experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: Multiple sclerosis is an autoimmune disease with a distinct female bias, as well as a high prevalence of neuropathic pain in both sexes. The dorsal root ganglia (DRG) contain the primary sensory neurons that give rise to pain, and damage to these neurons may lead to neuropathic pain. Here, we investigate the sex differences of the DRG transcriptome in a mouse model of MS. METHODS: Next-generation sequencing was used to establish RNA and microRNA profiles from the DRG of mice with MOG35-55-induced EAE, a model of CNS inflammation that mimics aspects of MS. Differential expression and multiple meta-analytic approaches were used to compare expression profiles in immunized female and male mice. Differential expression of relevant genes and microRNAs were confirmed by qPCR. RESULTS: Three thousand five hundred twenty genes and 29 microRNAs were differentially expressed in the DRG of female mice with MOG35-55-EAE, while only 189 genes and 3 microRNAs were differentially expressed in males with MOG35-55-EAE. Genes related to the immune system were uniquely regulated in immunized female mice. Direct comparison of sex within disease indicates significant differences in interferon and phagosomal pathways between the sexes. miR-21a-5p is the primary dysregulated microRNA in both sexes, with females having additional dysregulated microRNAs, including miR-122-5p. CONCLUSIONS: This study provides evidence that females are uniquely affected by MOG35-55-EAE and that this difference may result from additional signaling not present in the male. The altered transcriptome of females correlates with other studies finding hyperactivity of pain-sensing neurons and suggests underlying sex-specific pathways for neuropathic pain.

MicroRNA-223 protects neurons from degeneration in experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling.

microRNA dysregulation in neurodegenerative diseases: A systematic review.

While the root causes for individual neurodegenerative diseases are distinct, many shared pathological features and mechanisms contribute to neurodegeneration across diseases. Altered levels of microRNAs, small non-coding RNAs involved in post transcriptional regulation of gene expression, are reported for numerous neurodegenerative diseases. Yet, comparison between diseases to uncover commonly dysregulated microRNAs during neurodegeneration in general is lagging. We performed a systematic review of peer-reviewed publications describing differential microRNA expression in neurodegenerative diseases and related animal models. We compiled the results from studies covering the prevalent neurodegenerative diseases in the literature: Alzheimer's disease, amyotrophic lateral sclerosis, age-related macular degeneration, ataxia, dementia, myotonic dystrophy, epilepsy, glaucoma, Huntington's disease, multiple sclerosis, Parkinson's disease, and prion disorders. MicroRNAs which were dysregulated most often in these diseases and their models included miR-9-5p, miR-21-5p, the miR-29 family, miR-132-3p, miR-124-3p, miR-146a-5p, miR-155-5p, and miR-223-3p. Common pathways targeted by these predominant miRNAs were identified and revealed great functional overlap across diseases. We also identified a strong role for each microRNA in both the neural and immune components of diseases. microRNAs regulate broad networks of genes and identifying microRNAs commonly dysregulated across neurodegenerative diseases could cultivate novel hypotheses related to common molecular mechanisms underlying neurodegeneration.

Neuronal microRNA regulation in Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease but the molecular mechanisms underlying neurodegenerative aspects of the disease are poorly understood. microRNAs (miRNAs) are powerful regulators of gene expression that regulate numerous mRNAs simultaneously and can thus regulate programs of gene expression. Here, we describe miRNA expression in neurons captured from mice subjected to experimental autoimmune encephalomyelitis (EAE), a model of central nervous system (CNS) inflammation. Lumbar motor neurons and retinal neurons were laser captured from EAE mice and miRNA expression was assessed by next-generation sequencing and validated by qPCR. We describe 14 miRNAs that are differentially regulated in both neuronal subtypes and determine putative mRNA targets though in silico analysis. Several upregulated neuronal miRNAs are predicted to target pathways that could mediate repair and regeneration during EAE. This work identifies miRNAs that are affected by inflammation and suggests novel candidates that may be targeted to improve neuroprotection in the context of pathological inflammation.

The Molecular Interplay between Axon Degeneration and Regeneration.

Neurons face a series of morphological and molecular changes following trauma and in the progression of neurodegenerative disease. In neurons capable of mounting a spontaneous regenerative response, including invertebrate neurons and mammalian neurons of the peripheral nervous system (PNS), axons regenerate from the proximal side of the injury and degenerate on the distal side. Studies of Wallerian degeneration slow (WldS /Ola) mice have revealed that a level of coordination between the processes of axon regeneration and degeneration occurs during successful repair. Here, we explore how shared cellular and molecular pathways that regulate both axon regeneration and degeneration coordinate the two distinct outcomes in the proximal and distal axon segments. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018.

MT3-MMP Promotes Excitatory Synapse Formation by Promoting Nogo-66 Receptor Ectodomain Shedding.

Cell-surface molecules are dynamically regulated at the synapse to assemble and disassemble adhesive contacts that are important for synaptogenesis and for tuning synaptic transmission. Metalloproteinases dynamically regulate cellular behaviors through the processing of cell surface molecules. In the present study, we evaluated the role of membrane-type metalloproteinases (MT-MMPs) in excitatory synaptogenesis. We find that MT3-MMP and MT5-MMP are broadly expressed in the mouse cerebral cortex and that MT3-MMP loss-of-function interferes with excitatory synapse development in dissociated cortical neurons and in vivo We identify Nogo-66 receptor (NgR1) as an MT3-MMP substrate that is required for MT3-MMP-dependent synapse formation. Introduction of the shed ectodomain of NgR1 is sufficient to accelerate excitatory synapse formation in dissociated cortical neurons and in vivo Together, our findings support a role for MT3-MMP-dependent shedding of NgR1 in regulating excitatory synapse development.SIGNIFICANCE STATEMENT In this study, we identify MT3-MMP, a membrane-bound zinc protease, to be necessary for the development of excitatory synapses in cortical neurons. We identify Nogo-66 receptors (NgR1) as a downstream target of MT3-MMP proteolytic activity. Furthermore, processing of surface NgR1 by MT3-MMP generates a soluble ectodomain fragment that accelerates the formation of excitatory synapses. We propose that MT3-MMP activity and NgR1 shedding could stimulate circuitry remodeling in the adult brain and enhance functional connectivity after brain injury.

Maximizing functional axon repair in the injured central nervous system: Lessons from neuronal development.

The failure of damaged axons to regrow underlies disability in central nervous system injury and disease. Therapies that stimulate axon repair will be critical to restore function. Extensive axon regeneration can be induced by manipulation of oncogenes and tumor suppressors; however, it has been difficult to translate this into functional recovery in models of spinal cord injury. The current challenge is to maximize the functional integration of regenerating axons to recover motor and sensory behaviors. Insights into axonal growth and wiring during nervous system development are helping guide new approaches to boost regeneration and functional connectivity after injury in the mature nervous system. Here we discuss our current understanding of axonal behavior after injury and prospects for the development of drugs to optimize axon regeneration and functional recovery after CNS injury. Developmental Dynamics 247:18-23, 2018. © 2017 Wiley Periodicals, Inc.

14-3-3 adaptor protein-protein interactions as therapeutic targets for CNS diseases.

14-3-3s are a family of ubiquitously expressed adaptor proteins that regulate hundreds of functionally diverse 'client proteins.' In humans, there are seven isoforms with conserved structure and function. 14-3-3s typically bind to client proteins at phosphorylated serine/threonine motifs via a linear binding groove. Binding can have a variety of effects on the stability, activity and/or localization of the client protein. 14-3-3s are generating significant interest as potential drug targets for their involvement in cellular homeostasis and disease. They are especially abundant in the central nervous system (CNS) and are implicated in numerous CNS diseases, often through specific interactions with disease-relevant client proteins. Several tool compounds that can modulate 14-3-3 interactions with client proteins to elicit therapeutic effects have recently been described. Here we offer a perspective on the functions of 14-3-3s in neurons and the potential development of drugs to therapeutically target 14-3-3 PPIs for CNS diseases.

Ectodomain shedding of Limbic System-Associated Membrane Protein (LSAMP) by ADAM Metallopeptidases promotes neurite outgrowth in DRG neurons.

IgLONs are members of the immunoglobulin superfamily of cell adhesion proteins implicated in the process of neuronal outgrowth, cell adhesion and subdomain target recognition. IgLONs form homophilic and heterophilic complexes on the cell surface that repress or promote growth depending on the neuronal population, the developmental stage and surface repertoire of IgLON family members. In the present study, we identified a metalloproteinase-dependent mechanism necessary to promote growth in embryonic dorsal root ganglion cells (DRGs). Treatment of embryonic DRG neurons with pan-metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-3, or an inhibitor of ADAM Metallopeptidase Domain 10 (ADAM10) reduces outgrowth from DRG neurons indicating that metalloproteinase activity is important for outgrowth. The IgLON family members Neurotrimin (NTM) and Limbic System-Associated Membrane Protein (LSAMP) were identified as ADAM10 substrates that are shed from the cell surface of DRG neurons. Overexpression of LSAMP and NTM suppresses outgrowth from DRG neurons. Furthermore, LSAMP loss of function decreases the outgrowth sensitivity to an ADAM10 inhibitor. Together our findings support a role for ADAM-dependent shedding of cell surface LSAMP in promoting outgrowth from DRG neurons.

Targeting 14-3-3 adaptor protein-protein interactions to stimulate central nervous system repair.

The goal of developing treatments for central nervous system (CNS) injuries is becoming more attainable with the recent identification of various drugs that can repair damaged axons. These discoveries have stemmed from screening efforts, large expression datasets and an improved understanding of the cellular and molecular biology underlying axon growth. It will be important to continue searching for new compounds that can induce axon repair. Here we describe how a family of adaptor proteins called 14-3-3s can be targeted using small molecule drugs to enhance axon outgrowth and regeneration. 14-3-3s bind to many functionally diverse client proteins to regulate their functions. We highlight the recent discovery of the axon-growth promoting activity of fusicoccin-A, a fungus-derived small molecule that stabilizes 14-3-3 interactions with their client proteins. Here we discuss how fusicoccin-A could serve as a starting point for the development of drugs to induce CNS repair.

Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

Extracellular functions of 14-3-3 adaptor proteins.

14-3-3s are a family of adaptor proteins with a wide range of roles in cell signaling. Although they are primarily localized within the cytosol, 14-3-3s are also known to be present in the extracellular environment. Externalization of 14-3-3 can occur as a result of cell death or physiologically via release in exosomes. Interesting biological activities with relevance for tissue homeostasis and disease are now being described for extracellular 14-3-3s. Moreover, aminopeptidase N (APN) has been identified as a cell surface receptor for 14-3-3s. Here we review the array of bioactivities that have been ascribed to extracellular 14-3-3s and discuss applications as biomarkers and as targets for drug development.