Transgenic expression of the HERV-W envelope protein leads to polarized glial cell populations and a neurodegenerative environment.

The human endogenous retrovirus type W (HERV-W) has been identified and repeatedly confirmed as human-specific pathogenic entity affecting many cell types in multiple sclerosis (MS). Our recent contributions revealed the encoded envelope (ENV) protein to disturb myelin repair by interfering with oligodendroglial precursor differentiation and by polarizing microglial cells toward an axon-damage phenotype. Indirect proof of ENV's antiregenerative and degenerative activities has been gathered recently in clinical trials using a neutralizing anti-ENV therapeutic antibody. Yet direct proof of its mode of action can only be presented here based on transgenic ENV expression in mice. Upon demyelination, we observed myelin repair deficits, neurotoxic microglia and astroglia, and increased axon degeneration. Experimental autoimmune encephalomyelitis activity progressed faster in mutant mice equally accompanied by activated glial cells. This study therefore provides direct evidence on HERV-W ENV's contribution to the overall negative impact of this activated viral entity in MS.

A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage.

Stroke is a major reason for persistent disability due to insufficient treatment strategies beyond reperfusion, leading to oligodendrocyte death and axon demyelination, persistent inflammation and astrogliosis in peri-infarct areas. After injury, oligodendroglial precursor cells (OPCs) have been shown to compensate for myelin loss and prevent axonal loss through the replacement of lost oligodendrocytes, an inefficient process leaving axons chronically demyelinated. Phenotypic screening approaches in demyelinating paradigms revealed substances that promote myelin repair. We established an ex vivo adult organotypic coronal slice culture (OCSC) system to study repair after stroke in a resource-efficient way. Post-photothrombotic OCSCs can be manipulated for 8 d by exposure to pharmacologically active substances testing remyelination activity. OCSCs were isolated from a NG2-CreERT2-td-Tomato knock-in transgenic mouse line to analyze oligodendroglial fate/differentiation and kinetics. Parbendazole boosted differentiation of NG2+ cells and stabilized oligodendroglial fate reflected by altered expression of associated markers PDGFR-α, CC1, BCAS1 and Sox10 and GFAP. In vitro scratch assay and chemical ischemia confirmed the observed effects upon parbendazole treatment. Adult OCSCs represent a fast, reproducible, and quantifiable model to study OPC differentiation competence after stroke. Pharmacological stimulation by means of parbendazole promoted OPC differentiation.

Teriflunomide as a therapeutic means for myelin repair.

BACKGROUND: Promotion of myelin repair in the context of demyelinating diseases such as multiple sclerosis (MS) still represents a clinical unmet need, given that this disease is not only characterized by autoimmune activities but also by impaired regeneration processes. Hence, this relates to replacement of lost oligodendrocytes and myelin sheaths-the primary targets of autoimmune attacks. Endogenous remyelination is mainly mediated via activation and differentiation of resident oligodendroglial precursor cells (OPCs), whereas its efficiency remains limited and declines with disease progression and aging. Teriflunomide has been approved as a first-line treatment for relapsing remitting MS. Beyond its role in acting via inhibition of de novo pyrimidine synthesis leading to a cytostatic effect on proliferating lymphocyte subsets, this study aims to uncover its potential to foster myelin repair. METHODS: Within the cuprizone mediated de-/remyelination model teriflunomide dependent effects on oligodendroglial homeostasis and maturation, related to cellular processes important for myelin repair were analyzed in vivo. Teriflunomide administration was performed either as pulse or continuously and markers specific for oligodendroglial maturation and mitochondrial integrity were examined by means of gene expression and immunohistochemical analyses. In addition, axon myelination was determined using electron microscopy. RESULTS: Both pulse and constant teriflunomide treatment efficiently boosted myelin repair activities in this model, leading to accelerated generation of oligodendrocytes and restoration of myelin sheaths. Moreover, teriflunomide restored mitochondrial integrity within oligodendroglial cells. CONCLUSIONS: The link between de novo pyrimidine synthesis inhibition, oligodendroglial rescue, and maintenance of mitochondrial homeostasis appears as a key for successful myelin repair and hence for protection of axons from degeneration.

Interplay between activation of endogenous retroviruses and inflammation as common pathogenic mechanism in neurological and psychiatric disorders.

Human endogenous retroviruses (ERVs) are ancestorial retroviral elements that were integrated into our genome through germline infections and insertions during evolution. They have repeatedly been implicated in the aetiology and pathophysiology of numerous human disorders, particularly in those that affect the central nervous system. In addition to the known association of ERVs with multiple sclerosis and amyotrophic lateral sclerosis, a growing number of studies links the induction and expression of these retroviral elements with the onset and severity of neurodevelopmental and psychiatric disorders. Although these disorders differ in terms of overall disease pathology and causalities, a certain degree of (subclinical) chronic inflammation can be identified in all of them. Based on these commonalities, we discuss the bidirectional relationship between ERV expression and inflammation and highlight that numerous entry points to this reciprocal sequence of events exist, including initial infections with ERV-activating pathogens, exposure to non-infectious inflammatory stimuli, and conditions in which epigenetic silencing of ERV elements is disrupted.

Susceptibility and resilience to maternal immune activation are associated with differential expression of endogenous retroviral elements.

Endogenous retroviruses (ERVs) are ancestorial retroviral elements that were integrated into the mammalian genome through germline infections and insertions during evolution. While increased ERV expression has been repeatedly implicated in psychiatric and neurodevelopmental disorders, recent evidence suggests that aberrant endogenous retroviral activity may contribute to biologically defined subgroups of psychotic disorders with persisting immunological dysfunctions. Here, we explored whether ERV expression is altered in a mouse model of maternal immune activation (MIA), a transdiagnostic environmental risk factor of psychiatric and neurodevelopmental disorders. MIA was induced by maternal administration of poly(I:C) on gestation day 12 in C57BL/6N mice. Murine ERV transcripts were quantified in the placentae and fetal brains shortly after poly(I:C)-induced MIA, as well as in adult offspring that were stratified according to their behavioral profiles. We found that MIA increased and reduced levels of class II ERVs and syncytins, respectively, in placentae and fetal brain tissue. We also revealed abnormal ERV expression in MIA-exposed offspring depending on whether they displayed overt behavioral anomalies or not. Taken together, our findings provide a proof of concept that an inflammatory stimulus, even when initiated in prenatal life, has the potential of altering ERV expression across fetal to adult stages of development. Moreover, our data highlight that susceptibility and resilience to MIA are associated with differential ERV expression, suggesting that early-life exposure to inflammatory factors may play a role in determining disease susceptibility by inducing persistent alterations in the expression of endogenous retroviral elements.

Siponimod Modulates the Reaction of Microglial Cells to Pro-Inflammatory Stimulation.

Siponimod (Mayzent®), a sphingosine 1-phosphate receptor (S1PR) modulator which prevents lymphocyte egress from lymphoid tissues, is approved for the treatment of relapsing-remitting and active secondary progressive multiple sclerosis. It can cross the blood-brain barrier (BBB) and selectively binds to S1PR1 and S1PR5 expressed by several cell populations of the central nervous system (CNS) including microglia. In multiple sclerosis, microglia are a key CNS cell population moving back and forth in a continuum of beneficial and deleterious states. On the one hand, they can contribute to neurorepair by clearing myelin debris, which is a prerequisite for remyelination and neuroprotection. On the other hand, they also participate in autoimmune inflammation and axonal degeneration by producing pro-inflammatory cytokines and molecules. In this study, we demonstrate that siponimod can modulate the microglial reaction to lipopolysaccharide-induced pro-inflammatory activation.

Cladribine treatment improves cortical network functionality in a mouse model of autoimmune encephalomyelitis.

BACKGROUND: Cladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice. METHODS: In the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo. RESULTS: Oral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient. CONCLUSIONS: Our results indicate that cladribine exerts a neuroprotective effect after crossing the blood-brain barrier independently of its peripheral immunosuppressant action.

Deficiency of the Two-Pore Potassium Channel KCNK9 Impairs Intestinal Epithelial Cell Survival and Aggravates Dextran Sodium Sulfate-Induced Colitis.

BACKGROUND & AIMS: The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells. METHODS: Kcnk9-/- mice were challenged with 3% dextran sulfate sodium (DSS) to induce experimental acute colitis. Primary cultures of intestinal epithelial cells were generated, and expression of potassium channels as well as cytosolic calcium levels and susceptibility to apoptosis were evaluated. Furthermore, we evaluated whether KCNK9 deficiency was compensated by the closely related 2-pore potassium channel KCNK3 in vivo or in vitro. RESULTS: Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9-dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk9-/- mice but did not prevent apoptosis after DSS exposure. Conversely, increased levels of KCNK9 in Kcnk3-/- mice were associated with an ameliorated course of DSS-induced colitis. CONCLUSIONS: KCNK9 enhances mitochondrial stability, reduces apoptosis, und thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3-/- resulted in less mitochondrial damage and apoptosis and was associated with beneficial outcomes in DSS-induced colitis.

Myelin repair is fostered by the corticosteroid medrysone specifically acting on astroglial subpopulations.

BACKGROUND: Multiple sclerosis is characterised by inflammation, oligodendrocyte loss and axonal demyelination and shows an additional impact on astrocytes, and their polarization. Although a certain degree of spontaneous myelin repair can be observed, disease progression, and aging impair regeneration efforts highlighting the need to better understand glial cell dynamics to establish specific regenerative treatments. METHODS: Applying a chronic demyelination model, we here analysed demyelination and remyelination related effects on astrocytes and stem cell niches and studied the consequences of medrysone application on myelin repair, and astrocyte polarization. FINDINGS: Medrysone induced recovery of mature oligodendrocytes, myelin expression and node formation. In addition, C3d/S100a10 co-expression in astrocytes was enhanced. Moreover, Timp1 expression in C3d positive astrocytes revealed another astrocytic phenotype with a myelination promoting character. INTERPRETATION: Based on these findings, specific astrocyte subpopulations are suggested to act in a myelin regenerative way and manner the regulation of which can be positively modulated by this corticosteroid. FUNDING: This work was supported by the Jürgen Manchot Stiftung, the Research Commission of the medical faculty of the Heinrich-Heine-University of Düsseldorf, the Christiane and Claudia Hempel Foundation for clinical stem cell research and the James and Elisabeth Cloppenburg, Peek and Cloppenburg Düsseldorf Stiftung.

Increased Remyelination and Proregenerative Microglia Under Siponimod Therapy in Mechanistic Models.

BACKGROUND AND OBJECTIVES: Siponimod is an oral, selective sphingosine-1-phosphate receptor-1/5 modulator approved for treatment of multiple sclerosis. METHODS: Mouse MRI was used to investigate remyelination in the cuprizone model. We then used a conditional demyelination Xenopus laevis model to assess the dose-response of siponimod on remyelination. In experimental autoimmune encephalomyelitis-optic neuritis (EAEON) in C57Bl/6J mice, we monitored the retinal thickness and the visual acuity using optical coherence tomography and optomotor response. Optic nerve inflammatory infiltrates, demyelination, and microglial and oligodendroglial differentiation were assessed by immunohistochemistry, quantitative real-time PCR, and bulk RNA sequencing. RESULTS: An increased remyelination was observed in the cuprizone model. Siponimod treatment of demyelinated tadpoles improved remyelination in comparison to control in a bell-shaped dose-response curve. Siponimod in the EAEON model attenuated the clinical score, reduced the retinal degeneration, and improved the visual function after prophylactic and therapeutic treatment, also in a bell-shaped manner. Inflammatory infiltrates and demyelination of the optic nerve were reduced, the latter even after therapeutic treatment, which also shifted microglial differentiation to a promyelinating phenotype. DISCUSSION: These results confirm the immunomodulatory effects of siponimod and suggest additional regenerative and promyelinating effects, which follow the dynamics of a bell-shaped curve with high being less efficient than low concentrations.

Translational value of choroid plexus imaging for tracking neuroinflammation in mice and humans.

Neuroinflammation is a pathophysiological hallmark of multiple sclerosis and has a close mechanistic link to neurodegeneration. Although this link is potentially targetable, robust translatable models to reliably quantify and track neuroinflammation in both mice and humans are lacking. The choroid plexus (ChP) plays a pivotal role in regulating the trafficking of immune cells from the brain parenchyma into the cerebrospinal fluid (CSF) and has recently attracted attention as a key structure in the initiation of inflammatory brain responses. In a translational framework, we here address the integrity and multidimensional characteristics of the ChP under inflammatory conditions and question whether ChP volumes could act as an interspecies marker of neuroinflammation that closely interrelates with functional impairment. Therefore, we explore ChP characteristics in neuroinflammation in patients with multiple sclerosis and in two experimental mouse models, cuprizone diet-related demyelination and experimental autoimmune encephalomyelitis. We demonstrate that ChP enlargement-reconstructed from MRI-is highly associated with acute disease activity, both in the studied mouse models and in humans. A close dependency of ChP integrity and molecular signatures of neuroinflammation is shown in the performed transcriptomic analyses. Moreover, pharmacological modulation of the blood-CSF barrier with natalizumab prevents an increase of the ChP volume. ChP enlargement is strongly linked to emerging functional impairment as depicted in the mouse models and in multiple sclerosis patients. Our findings identify ChP characteristics as robust and translatable hallmarks of acute and ongoing neuroinflammatory activity in mice and humans that could serve as a promising interspecies marker for translational and reverse-translational approaches.

Identification of novel myelin repair drugs by modulation of oligodendroglial differentiation competence.

BACKGROUND: In multiple sclerosis loss of myelin and oligodendrocytes impairs saltatory signal transduction and leads to neuronal loss and functional deficits. Limited capacity of oligodendroglial precursor cells to differentiate into mature cells is the main reason for inefficient myelin repair in the central nervous system. Drug repurposing constitutes a powerful approach for identification of pharmacological compounds promoting this process. METHODS: A phenotypic compound screening using the subcellular distribution of a potent inhibitor of oligodendroglial cell differentiation, namely p57kip2, as differentiation competence marker was conducted. Hit compounds were validated in terms of their impact on developmental cell differentiation and myelination using both rat and human primary cell cultures and organotypic cerebellar slice cultures, respectively. Their effect on spontaneous remyelination was then investigated following cuprizone-mediated demyelination of the corpus callosum. FINDINGS: A number of novel small molecules able to promote oligodendroglial cell differentiation were identified and a subset was found to foster human oligodendrogenesis as well as myelination ex vivo. Among them the steroid danazol and the anthelminthic parbendazole were found to increase myelin repair. INTERPRETATION: We provide evidence that early cellular processes involved in differentiation decisions are applicable for the identification of regeneration promoting drugs and we suggest danazol and parbendazole as potent therapeutic candidates for demyelinating diseases. FUNDING: This work was supported by the Jürgen Manchot Foundation, Düsseldorf; Research Commission of the Medical Faculty of Heinrich-Heine-University Düsseldorf; Christiane and Claudia Hempel Foundation; Stifterverband/Novartisstiftung; James and Elisabeth Cloppenburg, Peek and Cloppenburg Düsseldorf Stiftung and International Progressive MS Alliance (BRAVEinMS).

Meeting report: "Human endogenous retroviruses: HERVs or transposable elements in autoimmune, chronic inflammatory and degenerative diseases or cancer", Lyon, France, november 5th and 6th 2019 - an MS scientist's digest.

The Third International Workshop on Human Endogenous Retroviruses and disease (www.hervanddisease.com), addressing HERVs or transposable elements in autoimmune, chronic inflammatory and degenerative diseases or cancer, in Lyon, France on November 5-6th 2019, once again gathered an international group of basic and clinical scientists investigating the involvement of human endogenous retroviruses (HERVs) in human diseases.

The Molecular Basis for Remyelination Failure in Multiple Sclerosis.

Myelin sheaths in the central nervous system (CNS) insulate axons and thereby allow saltatory nerve conduction, which is a prerequisite for complex brain function. Multiple sclerosis (MS), the most common inflammatory autoimmune disease of the CNS, leads to the destruction of myelin sheaths and the myelin-producing oligodendrocytes, thus leaving behind demyelinated axons prone to injury and degeneration. Clinically, this process manifests itself in significant neurological symptoms and disability. Resident oligodendroglial precursor cells (OPCs) and neural stem cells (NSCs) are present in the adult brain, and can differentiate into mature oligodendrocytes which then remyelinate the demyelinated axons. However, for multiple reasons, in MS the regenerative capacity of these cell populations diminishes significantly over time, ultimately leading to neurodegeneration, which currently remains untreatable. In addition, microglial cells, the resident innate immune cells of the CNS, can contribute further to inflammatory and degenerative axonal damage. Here, we review the molecular factors contributing to remyelination failure in MS by inhibiting OPC and NSC differentiation or modulating microglial behavior.

Neural Cell Responses Upon Exposure to Human Endogenous Retroviruses.

Human endogenous retroviruses (HERVs) are ancient retroviral elements, which invaded the human germ line several million years ago. Subsequent retrotransposition events amplified these sequences, resulting in approximately 8% of the human genome being composed of HERV sequences today. These genetic elements, normally dormant within human genomes, can be (re)-activated by environmental factors such as infections with other viruses, leading to the expression of viral proteins and, in some instances, even to viral particle production. Several studies have shown that the expression of these retroviral elements correlates with the onset and progression of neurological diseases such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Further studies provided evidence on additional roles for HERVs in schizophrenia (SCZ). Since these diseases are still not well understood, HERVs might constitute a new category of pathogenic components that could significantly change our understanding of these pathologies. Moreover, knowledge about their mode of action might also help to develop novel and more powerful approaches for the treatment of these complex diseases. Therefore, the main scope of this review is a description of the current knowledge on the involvement of HERV-W and HERV-K in neurological disease specifically focusing on the effects they exert on neural cells of the central nervous system.

pHERV-W envelope protein fuels microglial cell-dependent damage of myelinated axons in multiple sclerosis.

Axonal degeneration is central to clinical disability and disease progression in multiple sclerosis (MS). Myeloid cells such as brain-resident microglia and blood-borne monocytes are thought to be critically involved in this degenerative process. However, the exact underlying mechanisms have still not been clarified. We have previously demonstrated that human endogenous retrovirus type W (HERV-W) negatively affects oligodendroglial precursor cell (OPC) differentiation and remyelination via its envelope protein pathogenic HERV-W (pHERV-W) ENV (formerly MS-associated retrovirus [MSRV]-ENV). In this current study, we investigated whether pHERV-W ENV also plays a role in axonal injury in MS. We found that in MS lesions, pHERV-W ENV is present in myeloid cells associated with axons. Focusing on progressive disease stages, we could then demonstrate that pHERV-W ENV induces a degenerative phenotype in microglial cells, driving them toward a close spatial association with myelinated axons. Moreover, in pHERV-W ENV-stimulated myelinated cocultures, microglia were found to structurally damage myelinated axons. Taken together, our data suggest that pHERV-W ENV-mediated microglial polarization contributes to neurodegeneration in MS. Thus, this analysis provides a neurobiological rationale for a recently completed clinical study in MS patients showing that antibody-mediated neutralization of pHERV-W ENV exerts neuroprotective effects.

Rescuing the negative impact of human endogenous retrovirus envelope protein on oligodendroglial differentiation and myelination.

Remyelination in the adult CNS depends on activation, differentiation, and functional integration of resident oligodendroglial precursor cells (OPCs) and constitutes the only spontaneous neuroregenerative process able to compensate for functional deficits upon loss of oligodendrocytes and myelin sheaths as it is observed in multiple sclerosis. The proteins encoded by p57kip2- and by human endogenous retrovirus type W (pHERV-W) envelope genes were previously identified as negative regulators of OPC maturation. We here focused on the activity of the ENV protein and investigated how it can be neutralized for an improved myelin repair. We could demonstrate that myelination in vitro is severely affected by this protein but that application of an anti-ENV neutralizing antibody, currently investigated in clinical trials, can rescue the generation of internodes. We then compared p57kip2 and ENV dependent inhibitory mechanisms and found that a dominant negative version of the p57kip2 protein can equally save OPCs from myelination failure in response to ENV-mediated TLR4 activation. Additional experiments addressing p57kip2's underlying mode of action revealed a direct interaction with ATP6v1d, a central component of a vascular ATPase. Its pharmacological blocking was then shown to exert an analogous myelination rescue effect in presence of the ENV protein. Therefore, our study provides mechanistic insights into oligodendroglial inhibition processes and presents three different means to counteract the anti-myelination effect of the ENV protein. These observations are therefore of interest in light of understanding the complexity of the numerous oligodendroglial inhibitors and might promote the establishment of novel regenerative therapies.