Laurie Zoecklein

Gamma Interferon Is Critical for Neuronal Viral Clearance and Protection in a Susceptible Mouse Strain following Early Intracranial Theiler's Murine Encephalomyelitis Virus Infection

ABSTRACT We evaluated the role of gamma interferon (IFN-γ) in protecting neurons from virus-induced injury following central nervous system infection. IFN-γ−/− and IFN-γ+/+ mice of the resistant major histocompatibility complex (MHC) H-2b haplotype and intracerebrally infected with Theilers murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-γ+/+H-2b mice also cleared virus from the spinal cord white matter, whereas IFN-γ−/−H-2b mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-γ−/− mice of the susceptible H-2q haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-γ−/−H-2q mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-γ−/−H-2q mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-γ−/−H-2q mice. IFN-γ deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-γ−/−H-2q mice was lower than those in IFN-γ+/+H-2q mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-γ exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-γ plays a critical role in protecting spinal cord neurons from persistent infection and death.

Quantitative assessment of neurologic deficits in a chronic progressive murine model of CNS demyelination

The precise factors involved in the development of a progressive motor dysfunction, a hallmark of immune-mediated demyelinating diseases such as multiple sclerosis, are not well defined. The ability to identify neurologic deficits that result in impaired motor performance early in disease may allow for the identification of therapeutic interventions that slow or eliminate the progression toward a permanent dysfunction. Here we describe the use of three objective, quantitative functional assays (spontaneous activity box, rotarod, and footprint analysis) to detect early neurologic deficits following the initiation of a demyelinating disease with Theilers murine encephalomyelitis virus (TMEV). The results show that the assays are capable of revealing neurologic deficits at the early stages of the demyelinating disease process. These findings are the first to objectively characterize neurologic function in an animal model of progressive CNS demyelination.

Demyelinated Axons and Motor Function Are Protected by Genetic Deletion of Perforin in a Mouse Model of Multiple Sclerosis

Axon injury is a major determinant of the loss of neurological function in patients with multiple sclerosis. It is unclear, however, whether damage to axons is an obligatory consequence of demyelination or whether it is an independent process that occurs in the permissive environment of demyelinated lesions. Previous investigations into the role of CD8+ T cells and perforin in the Theiler murine encephalomyelitis virus model of multiple sclerosis have used mouse strains resistant to Theiler murine encephalomyelitis virus infection. To test the role of CD8+ T cells in axon injury, we established a perforin-deficient mouse model on the H-2q major histocompatibility complex background thereby removing confounding factors related to viral biology in this Theiler murine encephalomyelitis virus-susceptible strain. This permitted direct comparison of clinical and pathological parameters between perforin-competent and perforin-deficient mice. The extent of demyelination was indistinguishable between perforin-competent and perforin-deficient H-2q mice, but chronically infected perforin-deficient mice exhibited preservation of motor function and spinal axons despite the presence of spinal cord demyelination. Thus, demyelination is necessary but insufficient for axon injury in this model; the absence of perforin protects axons without impacting demyelination. These results suggest that perforin is a key mediatorof axon injury and lend additional support to the hypothesisthat CD8+ T cells are primarily responsible for axon damage in multiple sclerosis.

Direct Comparison of Demyelinating Disease Induced by the Daniel's Strain and BeAn Strain of Theiler's Murine Encephalomyelitis Virus

We compared CNS disease following intracere‐bral injection of SJL mice with Daniels (DA) and BeAn 8386 (BeAn) strains of Theilers murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.

Tumor Necrosis Factor α is Reparative via TNFR1 in the Hippocampus and via TNFR2 in the Striatum after Virus-Induced Encephalitis

Differentiating between injurious and reparative factors facilitates appropriate therapeutic intervention. We evaluated the role of tumor necrosis factor α (TNFα) in parenchymal brain pathology resolution following virus‐induced encephalitis from a picornavirus, Theilers murine encephalomyelitis virus (TMEV). We infected the following animals with TMEV for 7 to 270 days: B6/129 TNF−/− mice (without TNFα expression), B6/129 TNFR1−/− mice (without TNFα receptor 1 expression), and B6/129 TNFR2−/− mice (without TNFα receptor 2 expression). Normal TNFα‐expressing controls were TMEV‐infected B6, 129/J, B6/129F1 and B6/129F2 mice. Whereas all strains developed inflammation and neuronal injury in the hippocampus and striatum 7 to 21 days postinfection (dpi), the control mice resolved the pathology by 45 to 90 dpi. However, parenchymal hippocampal and striatal injury persisted in B6/129 TNF−/− mice following infection. Treating virus‐infected mice with active recombinant mouse TNFα resulted in less hippocampal and striatal pathology, whereas TNFα‐neutralizing treatment worsened pathology. T1 “black holes” appeared on MRI during early infection in the hippocampus and striatum in all mice but persisted only in TNF−/− mice. TNFR1 mediated hippocampal pathology resolution whereas TNFR2 mediated striatal healing. These findings indicate the role of TNFα in resolution of sublethal hippocampal and striatal injury.

Assessment of hindlimb gait as a powerful indicator of axonal loss in a murine model of progressive CNS demyelination

Identifying the role of axonal injury in the development of permanent, irreversible neurologic disability is important to the study of central nervous system (CNS) demyelinating diseases. Our understanding of neurologic dysfunction in demyelinating diseases and the ability to assess therapeutic interventions depends on the development of objective functional assays that can non-invasively measure axonal loss. In this study, we demonstrate in a murine model of progressive CNS demyelination that assessment of the hindlimb width of stride provides a powerful indicator of axonal loss and can dissociate between deficits induced by demyelination versus axonal loss.

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis

Theilers murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L‐dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV‐induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H‐2b mice. CD40L‐mediated interactions were also necessary for development of protective H‐2b‐restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody‐ and cell‐mediated protective immune responses in distinct phases of TMEV‐mediated pathology.

A natural human IgM that binds to gangliosides is therapeutic in murine models of amyotrophic lateral sclerosis

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a devastating, fatal neurological disease that primarily affects spinal cord anterior horn cells and their axons for which there is no treatment. Here we report the use of a recombinant natural human IgM that binds to the surface of neurons and supports neurite extension, rHIgM12, as a therapeutic strategy in murine models of human ALS. A single 200 µg intraperitoneal dose of rHIgM12 increases survival in two independent genetic-based mutant SOD1 mouse strains (SOD1G86R and SOD1G93A) by 8 and 10 days, delays the onset of neurological deficits by 16 days, delays the onset of weight loss by 5 days, and preserves spinal cord axons and anterior horn neurons. Immuno-overlay of thin layer chromatography and surface plasmon resonance show that rHIgM12 binds with high affinity to the complex gangliosides GD1a and GT1b. Addition of rHIgM12 to neurons in culture increases α-tubulin tyrosination levels, suggesting an alteration of microtubule dynamics. We previously reported that a single peripheral dose of rHIgM12 preserved neurological function in a murine model of demyelination with axon loss. Because rHIgM12 improves three different models of neurological disease, we propose that the IgM might act late in the cascade of neuronal stress and/or death by a broad mechanism. Summary: A single peripheral dose of a recombinant natural human IgM increases lifespan and delays neurological deficits in mouse models of human ALS.

Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.

Naturally Occurring Monoclonal Antibodies and Their Therapeutic Potential for Neurologic Diseases

IMPORTANCE Modulating the immune system does not reverse long-term disability in neurologic disorders. Better neuroregenerative and neuroprotective treatment strategies are needed for neuroinflammatory and neurodegenerative diseases. OBJECTIVE To review the role of monoclonal, naturally occurring antibodies (NAbs) as novel therapeutic molecules for treatment of neurologic disorders. EVIDENCE REVIEW Peer-reviewed articles, including case reports, case series, retrospective reviews, prospective randomized clinical trials, and basic science reports, were identified in a PubMed search for articles about NAbs and neurologic disorders that were published from January 1, 1964, through June 30, 2015. We concentrated our review on multiple sclerosis, Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis. FINDINGS Many insults, including trauma, ischemia, infection, inflammation, and neurodegeneration, result in irreversible damage to the central nervous system. Central nervous system injury often results in a pervasive inhibitory microenvironment that hinders regeneration. A common targeted drug development strategy is to identify molecules with high potency in animal models. Many approaches often fail in the clinical setting owing to a lack of efficacy in human diseases (eg, less than the response demonstrated in animal models) or a high incidence of toxic effects. An alternative approach is to identify NAbs in humans because these therapeutic molecules have potential physiologic function without toxic effects. NAbs of the IgG, IgA, or IgM isotype contain germline or close to germline sequences and are reactive to self-components, altered self-components, or foreign antigens. Our investigative group developed recombinant, autoreactive, natural human IgM antibodies directed against oligodendrocytes or neurons with therapeutic potential for central nervous system repair. One such molecule, recombinant HIgM22, directed against myelin and oligodendrocytes completed a successful phase 1 clinical trial without toxic effects with the goal of promoting remyelination in multiple sclerosis. CONCLUSIONS AND RELEVANCE Animal studies demonstrate that certain monoclonal NAbs are beneficial as therapeutic agents for neurologic diseases. This class of antibodies represents a unique source from which to develop a new class of disease-modifying therapies.