Host genetic diversity drives variable central nervous system lesion distribution in chronic phase of Theiler’s Murine Encephalomyelitis Virus (TMEV) infection

Host genetic background is a significant driver of the variability in neurological responses to viral infection. Here, we leverage the genetically diverse Collaborative Cross (CC) mouse resource to better understand how chronic infection by Theiler’s Murine Encephalomyelitis Virus (TMEV) elicits diverse clinical and morphologic changes in the central nervous system (CNS). We characterized the TMEV-induced clinical phenotype responses, and associated lesion distributions in the CNS, in six CC mouse strains over a 90 day infection period. We observed varying degrees of motor impairment in these strains, as measured by delayed righting reflex, paresis, paralysis, seizures, limb clasping, ruffling, and encephalitis phenotypes. All strains developed neuroparenchymal necrosis and mineralization in the brain, primarily localized to the hippocampal regions. Two of the six strains presented with axonal degeneration with myelin loss of the nerve roots in the lumbar spinal cord. Moreover, we statistically correlated lesion distribution with overall frequencies of clinical phenotypes and phenotype progression to better understand how and where TMEV targets the CNS, based on genetic background. Specifically, we assessed lesion distribution in relation to the clinical progression of these phenotypes from early to late TMEV disease, finding significant relationships between progression and lesion distribution. Finally, we identified quantitative trait loci associated with frequency of lesions in a particular brain region, revealing several loci of interest for future study: lysosomal trafficking regulator (Lyst) and nidogen 1 (Nid1). Together, these results indicate that the genetic background influences the type and severity of clinical phenotypes, phenotypic resilience to TMEV, and the lesion distribution across strains.

Efficacy of vafidemstat in experimental autoimmune encephalomyelitis highlights the KDM1A/RCOR1/HDAC epigenetic axis in multiple sclerosis

Background Vafidemstat (ORY-2001) is a clinical stage inhibitor of the Lysine Specific Demethylase KDM1A in development for treatment of neurodegenerative and psychiatric diseases. KDM1A demethylates H3K4me1/2 and together with the histone deacetylases HDAC1/2, it forms part of co-repressor complexes recruited by zinc finger factors to control transcription. The exact role of KDM1A in neuroinflammation remained to be explored. Methods Compounds were administered p.o. gavage to mice with MOG35-55 induced experimental autoimmune encephalomyelitis or mice infected with Theiler’s murine encephalomyelitis virus. Immune cell infiltration was analyzed by immunohistochemistry. Cytokine and chemokine levels were analyzed by ELISA. Genome wide gene expression in spinal cord and brain were analyzed by two-color microarray analysis and qRT-PCR. Results ORY-2001 improved the clinical score in mouse experimental autoimmune encephalomyelitis and in mice infected with the Theiler’s murine encephalomyelitis virus. The compound reduced lymphocyte egress and infiltration of immune cells in the spinal cord and prevented demyelination. ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the induction of the inflammatory gene expression signature in the central nervous system more potently. Gene expression changes and axonal protection in animals, and protection against glutamate excitoxicity in spinal cord explants support that ORY-2001 has neuroprotective qualities. Conclusions ORY-2001 exerts therapeutic activity in two mouse models of multiple sclerosis. The anti-inflammatory properties of ORY-2001 are being tested in a Phase IIa clinical trial in patients with relapse remitting and secondary progressive multiple sclerosis, and in severely ill COVID-19 patients at risk for acute respiratory distress syndrome.

Efficacy of vafidemstat in experimental autoimmune encephalomyelitis highlights the KDM1A/RCOR1/HDAC epigenetic axis in multiple sclerosis

Background Vafidemstat (ORY-2001) is a clinical stage inhibitor of the Lysine Specific Demethylase KDM1A in development for treatment of neurodegenerative and psychiatric diseases. KDM1A demethylates H3K4me1/2 and together with the histone deacetylases HDAC1/2, it forms part of co-repressor complexes recruited by zinc finger factors to control transcription. The exact role of KDM1A in neuroinflammation remained to be explored. Methods Compounds were administered p.o. gavage to mice with MOG35-55 induced experimental autoimmune encephalomyelitis or mice infected with Theiler’s murine encephalomyelitis virus. Immune cell infiltration was analyzed by immunohistochemistry. Cytokine and chemokine levels were analyzed by ELISA. Genome wide gene expression in spinal cord and brain were analyzed by two-color microarray analysis and qRT-PCR. Results ORY-2001 improved the clinical score in mouse experimental autoimmune encephalomyelitis and in mice infected with the Theiler’s murine encephalomyelitis virus. The compound reduced lymphocyte egress and infiltration of immune cells in the spinal cord and prevented demyelination. ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the induction of the inflammatory gene expression signature in the central nervous system more potently. Gene expression changes and axonal protection in animals, and protection against glutamate excitoxicity in spinal cord explants support that ORY-2001 has neuroprotective qualities. Conclusions ORY-2001 exerts therapeutic activity in two mouse models of multiple sclerosis. The anti-inflammatory properties of ORY-2001 are being tested in a Phase IIa clinical trial in patients with relapse remitting and secondary progressive multiple sclerosis, and in severely ill COVID-19 patients at risk for acute respiratory distress syndrome.

Efficacy of vafidemstat in experimental autoimmune encephalomyelitis highlights the KDM1A/RCOR1/HDAC epigenetic axis in multiple sclerosis

Background Vafidemstat (ORY-2001) is a clinical stage inhibitor of the Lysine Specific Demethylase KDM1A in development for treatment of neurodegenerative and psychiatric diseases. KDM1A demethylates H3K4me1/2 and together with the histone deacetylases HDAC1/2, it forms part of co-repressor complexes recruited by zinc finger factors to control transcription. The exact role of KDM1A in neuroinflammation remained to be explored. Methods Compounds were administered p.o. gavage to mice with MOG35-55 induced experimental autoimmune encephalomyelitis or mice infected with Theiler’s murine encephalomyelitis virus. Immune cell infiltration was analyzed by immunohistochemistry. Cytokine and chemokine levels were analyzed by ELISA. Genome wide gene expression in spinal cord and brain were analyzed by two-color microarray analysis and qRT-PCR. Results ORY-2001 improved the clinical score in mouse experimental autoimmune encephalomyelitis and in mice infected with the Theiler’s murine encephalomyelitis virus. The compound reduced lymphocyte egress and infiltration of immune cells in the spinal cord and prevented demyelination. ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the induction of the inflammatory gene expression signature in the central nervous system more potently. Gene expression changes and axonal protection in animals, and protection against glutamate excitoxicity in spinal cord explants support that ORY-2001 has neuroprotective qualities. Conclusions ORY-2001 exerts therapeutic activity in two mouse models of multiple sclerosis. The anti-inflammatory properties of ORY-2001 are being tested in a Phase IIa clinical trial in patients with relapse remitting and secondary progressive multiple sclerosis, and in severely ill COVID-19 patients at risk for acute respiratory distress syndrome.

Efficacy of vafidemstat in experimental autoimmune encephalomyelitis highlights the KDM1A/RCOR1/HDAC epigenetic axis in multiple sclerosis

Background Vafidemstat (ORY-2001) is a clinical stage inhibitor of the Lysine Specific Demethylase KDM1A in development for treatment of neurodegenerative and psychiatric diseases. KDM1A demethylates H3K4me1/2 and together with the histone deacetylases HDAC1/2, it forms part of co-repressor complexes recruited by zinc finger factors to control transcription. The exact role of KDM1A in neuroinflammation remained to be explored. Methods Compounds were administered p.o. gavage to mice with MOG35-55 induced experimental autoimmune encephalomyelitis or mice infected with Theiler’s murine encephalomyelitis virus. Immune cell infiltration was analyzed by immunohistochemistry. Cytokine and chemokine levels were analyzed by ELISA. Genome wide gene expression in spinal cord and brain were analyzed by two-color microarray analysis and qRT-PCR.Results ORY-2001 improved the clinical score in mouse experimental autoimmune encephalomyelitis and in mice infected with the Theiler’s murine encephalomyelitis virus. The compound reduced lymphocyte egress and infiltration of immune cells in the spinal cord and prevented demyelination. ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the induction of the inflammatory gene expression signature in the central nervous system more potently. Gene expression changes and axonal protection in animals, and protection against glutamate excitoxicity in spinal cord explants support that ORY-2001 has neuroprotective qualities.Conclusions ORY-2001 exerts therapeutic activity in two mouse models of multiple sclerosis. The anti-inflammatory properties of ORY-2001 are being tested in a Phase IIa clinical trial in patients with relapse remitting and secondary progressive multiple sclerosis, and in severely ill COVID-19 patients at risk for acute respiratory distress syndrome.

Reactivity and increased proliferation of NG2 cells following central nervous system infection with Theiler’s murine encephalomyelitis virus

Background Neuron-glial antigen 2 (NG2) cells are a glial cell type tiled throughout the gray and white matter of the central nervous system (CNS). NG2 cells are known for their ability to differentiate into oligodendrocytes and are commonly referred to as oligodendrocyte precursor cells. However, recent investigations have begun to identify additional functions of NG2 cells in CNS health and pathology. NG2 cells form physical and functional connections with neurons and other glial cell types throughout the CNS, allowing them to monitor and respond to the neural environment. Growing evidence indicates that NG2 cells become reactive under pathological conditions, though their specific roles are only beginning to be elucidated. While reactive microglia and astrocytes are well-established contributors to neuroinflammation and the development of epilepsy following CNS infection, the dynamics of NG2 cells remain unclear. Therefore, we investigated NG2 cell reactivity in a viral-induced mouse model of temporal lobe epilepsy. Methods C57BL6/J mice were injected intracortically with Theiler’s murine encephalomyelitis virus (TMEV) or PBS. Mice were graded twice daily for seizures between 3 and 7 days post-injection (dpi). At 4 and 14 dpi, brains were fixed and stained for NG2, the microglia/macrophage marker IBA1, and the proliferation marker Ki-67. Confocal z stacks were acquired in both the hippocampus and the overlying cortex. Total field areas stained by each cell marker and total field area of colocalized pixels between NG2 and Ki67 were compared between groups. Results Both NG2 cells and microglia/macrophages displayed increased immunoreactivity and reactive morphologies in the hippocampus of TMEV-injected mice. While increased immunoreactivity for IBA1 was also present in the cortex, there was no significant change in NG2 immunoreactivity in the cortex following TMEV infection. Colocalization analysis for NG2 and Ki-67 revealed a significant increase in overlap between NG2 and Ki-67 in the hippocampus of TMEV-injected mice at both time points, but no significant differences in cortex. Conclusions NG2 cells acquire a reactive phenotype and proliferate in response to TMEV infection. These results suggest that NG2 cells alter their function in response to viral encephalopathy, making them potential targets to prevent the development of epilepsy following viral infection.

Efficacy of vafidemstat in experimental autoimmune encephalomyelitis highlights the KDM1A/RCOR1/HDAC epigenetic axis in multiple sclerosis

Background Vafidemstat (ORY-2001) is a clinical stage inhibitor of the Lysine Specific Demethylase KDM1A in development for treatment of neurodegenerative and psychiatric diseases. KDM1A demethylates H3K4me1/2 and together with the histone deacetylases HDAC1/2, it forms part of co-repressor complexes recruited by zinc finger factors to control transcription. The exact role of KDM1A in neuroinflammation remained to be explored. Methods Compounds were administered p.o. gavage to mice with MOG35-55 induced experimental autoimmune encephalomyelitis or mice infected with Theiler’s murine encephalomyelitis virus. Immune cell infiltration was analyzed by immunohistochemistry. Cytokine and chemokine levels were analyzed by ELISA. Genome wide gene expression in spinal cord and brain were analyzed by two-color microarray analysis and qRT-PCR.Results ORY-2001 improved the clinical score in mouse experimental autoimmune encephalomyelitis and in mice infected with the Theiler’s murine encephalomyelitis virus. The compound reduced lymphocyte egress and infiltration of immune cells in the spinal cord and prevented demyelination. ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the induction of the inflammatory gene expression signature in the central nervous system more potently. Gene expression changes and axonal protection in animals, and protection against glutamate excitoxicity in spinal cord explants support that ORY-2001 has neuroprotective qualities.Conclusions ORY-2001 exerts therapeutic activity in two mouse models of multiple sclerosis. The anti-inflammatory properties of ORY-2001 are being tested in a Phase IIa clinical trial in patients with relapse remitting and secondary progressive multiple sclerosis, and in severely ill COVID-19 patients at risk for acute respiratory distress syndrome.

Structural and molecular basis for protein-stimulated ribosomal frameshifting in Theiler’s murine encephalomyelitis virus

The 2A protein of Theiler’s murine encephalomyelitis virus (TMEV) is required for stimulating programmed −1 ribosomal frameshifting (PRF) during infection. However, the amino acid sequence of TMEV 2A shares only 27% identity with the 2A orthologue from the related cardiovirus encephalomyocarditis virus (EMCV) for which a structure has been recently determined. Here we present the X-ray crystal structure of TMEV 2A, revealing that, despite the low sequence identity, the overall beta-shell architecture is retained, and the location of previously described mutations on this structure suggests a common RNA binding mode. We determine the minimal stimulatory element in the viral RNA required for 2A binding and show that 2A binds to this element with 1:1 stoichiometry and nanomolar affinity. We also demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence that an alternative pseudoknot-like conformation recently demonstrated for EMCV is a conserved feature of cardiovirus stimulatory elements. We go on to examine frameshifting in infected cells by ribosome profiling and metabolic labelling. We observe PRF efficiencies of up to 85%, highlighting this as the most efficient example of −1 PRF in any natural system thus far characterised. Furthermore, we document a series of ribosomal pauses in and around the site of PRF with potential implications for our understanding of translational control in cardioviruses.

Infection and Activation of B Cells by Theiler’s Murine Encephalomyelitis Virus (TMEV) Leads to Autoantibody Production in an Infectious Model of Multiple Sclerosis

Theiler’s murine encephalomyelitis virus (TMEV) induces immune-mediated inflammatory demyelinating disease in susceptible mice that is similar to human multiple sclerosis (MS). In light of anti-CD20 therapies for MS, the susceptibility of B cells to TMEV infection is particularly important. In our study, direct viral exposure to macrophages and lymphocytes resulted in viral replication and cellular stimulation in the order of DCs, macrophages, B cells, and T cells. Notably, B cells produced viral proteins and expressed elevated levels of CD69, an activation marker. Similarly, the expression of major histocompatibility complex class II and costimulatory molecules in B cells was upregulated. Moreover, TMEV-infected B cells showed elevated levels of antigen-presenting function and antibody production. TMEV infection appeared to polyclonally activate B cells to produce autoantibodies and further T cell stimulation. Thus, the viral infection might potentially affect the outcome of autoimmune diseases, and/or the development of other chronic infections, including the protection and/or pathogenesis of TMEV-induced demyelinating disease.