Ingo Gerhauser

MMP-12, MMP-3, and TIMP-1 Are Markedly Upregulated in Chronic Demyelinating Theiler Murine Encephalomyelitis

Abstract Theiler murine encephalomyelitis (TME) represents a highly relevant viral model for multiple sclerosis. Matrix metalloproteinases (MMPs) degrade extracellular matrix molecules and are involved in demyelination processes. To elucidate their impact on demyelination in TME, spinal cords of TME virus (TMEV)-infected SJL/J mice were taken at 9 different time points postinfection (pi) ranging from 1 hour to 196 days pi and investigated for the expression of TMEV, MMP-2, -3, -7, -9, -10, -11, -12, -13, -14, -15, -24, and TIMP-1 to -4 by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). High TMEV RNA levels were detectable throughout the observation period using RT-qPCR. In addition, TMEV RNA was visualized within demyelinated lesions by in situ hybridization. MMP-3 mRNA was significantly upregulated at 1 day pi and again in the late phase of infection. TIMP-1 mRNA was significantly elevated throughout the observation period. MMP-12 mRNA was most prominently upregulated in the late phase of infection and MMP-12 protein was localized in intralesional microglia/macrophages and astrocytes by immunohistochemistry. In summary, in early TMEV infection, MMP-3 and TIMP-1 mRNA upregulation might be directly virus-induced, whereas persistent TMEV infection directly or indirectly stimulated MMP-12 production in microglia/macrophages and astrocytes and might account for ongoing demyelination in TME.

Matrix metalloproteinases and their inhibitors in the developing mouse brain and spinal cord: a reverse transcription quantitative polymerase chain reaction study.

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are essential for coordinated extracellular matrix turnover during central nervous system development. Reverse transcription quantitative polymerase chain reaction was employed to evaluate the mRNA expression of MMP-2, -3, -7, -9, -10, -11, -12, -13, -14, -15, and -24, and TIMP-1, -2, -3, and -4 in the prosencephalon, rhombencephalon, and spinal cord of 1- to 40-week-old mice. The molecular data were interpreted in the context of morphological observations. Significantly higher expression levels of MMP-2, -11, -13, -14, -15, and -24, and TIMP-1 and -3 were found in the brain and spinal cord 1 week after birth compared to later time points, while MMP-9 and TIMP-2 upregulation was restricted to the brain. This upregulation coincided with the maximal extension of the transient cerebellar external granular layer, a marker of neuronal progenitor proliferation and migration. MMP-12 was significantly upregulated at later time points and found to be positively correlated with myelination in the rhombencephalon and spinal cord. MMP-3, -7, and -10 mRNA expressions remained unchanged or were negligible. In summary, while most of the MMPs and TIMPs studied seem to be involved in cell proliferation and migration, MMP-12 might be decisive for myelination.

Brain inflammation, neurodegeneration and seizure development following picornavirus infection markedly differ among virus and mouse strains and substrains

Infections, particularly those caused by viruses, are among the main causes of acquired epilepsy, but the mechanisms causing epileptogenesis are only poorly understood. As a consequence, no treatment exists for preventing epilepsy in patients at risk. Animal models are useful to study epileptogenesis after virus-induced encephalitis and how to interfere with this process, but most viruses that cause encephalitis in rodents are associated with high mortality, so that the processes leading to epilepsy cannot be investigated. Recently, intracerebral infection with Theilers murine encephalomyelitis virus (TMEV) in C57BL/6 (B6) mice was reported to induce early seizures and epilepsy and it was proposed that the TMEV mouse model represents the first virus infection-driven animal model of epilepsy. In the present study, we characterized this model in two B6 substrains and seizure-resistant SJL/J mice by using three TMEV (sub)strains (BeAn-1, BeAn-2, DA). The idea behind this approach was to study what is and what is not necessary for development of acute and late seizures after brain infection in mice. Receiver operating characteristic (ROC) curve analysis was used to determine which virus-induced brain alterations are associated with seizure development. In B6 mice infected with different TMEV virus (sub)strains, the severity of hippocampal neurodegeneration, amount of MAC3-positive microglia/macrophages, and expression of the interferon-inducible antiviral effector ISG15 were almost perfect at discriminating seizing from non-seizing B6 mice, whereas T-lymphocyte brain infiltration was not found to be a crucial factor. However, intense microglia/macrophage activation and some hippocampal damage were also observed in SJL/J mice. Overall, the TMEV model provides a unique platform to study virus and host factors in ictogenesis and epileptogenesis.

Ets-1 represents a pivotal transcription factor for viral clearance, inflammation, and demyelination in a mouse model of multiple sclerosis

Demyelination of Theilers murine encephalomyelitis (TME) depends on viral persistence and on the mouse genotype. Ets-1 expression, a transcription factor involved in T cell activation and cytokine expression, was investigated in the spinal cord during TME using RT-qPCR and immunohistochemistry. Resistant C57BL/6 mice lacking virus persistence and demyelination demonstrated a stronger upregulation of Ets-1 mRNA transcripts in the early phase of TME compared to susceptible SJL/J mice probably linked to viral clearance. Though strong Ets-1 expression in resident glial cells such as astrocytes might inhibit lesion development, delayed Ets-1 activation in inflammatory cells seemed to promote demyelination in the late phase of TME in SJL/J mice.

Induction of Activator Protein-1 and Nuclear Factor-κB as a Prerequisite for Disease Development in Susceptible SJL/J Mice After Theiler Murine Encephalomyelitis

Theiler murine encephalomyelitis (TME) represents an important mouse model of multiple sclerosis. Activator protein and nuclear factor-&kgr;B proteins are interacting transcription factors controlling the expression of cytokines involved in the demyelination process. However, specific expression patterns of these transcription factors in susceptible and resistant mouse strains and their relationship to demyelination remains to be determined. The expression of activator protein-1 (c-fos and c-jun) and nuclear factor-&kgr;B (p50 and p65) genes, TME virus, tumor necrosis factor-&agr;, and interferon-&ggr; was investigated in the spinal cord of TME virus (BeAn strain)-infected SJL/J and C57BL/6 mice until 196 days postinfection (dpi) using reverse transcription-quantitative polymerase chain reaction. Additionally, c-fos, c-jun, and p50 expression was examined by applying immunohistochemistry. In susceptible SJL/J mice, in contrast to resistant C57BL/6 mice, all investigated mRNA transcripts were upregulated in the early (0-7 days dpi) and late phases (28-196 days dpi) of TME. In addition, white matter lesions of SL/J mice were characterized by c-jun-positive astrocytes and p50-positive mononuclear immune cells. Upregulation of activator protein-1 and nuclear factor-&kgr;B in resident glial cells in the early phase followed by strong downstream tumor necrosis factor-&agr; production might account for disease development in susceptible SJL/J mice. In the late phase, the formation of JUN/JUN homodimers in intralesional astrocytes might contribute to the sustained release of proinflammatory cytokines, thereby promoting disease progression.

Spatio-temporal expression of immediate early genes in the central nervous system of SJL/J mice

Gene products of immediate early genes (IEGs) interact with specific binding sites in promoter regions of inducible and constitutively expressed genes. Thereby, they control transcription of down‐stream targets, like pro‐ and anti‐apoptotic genes and matrix‐metalloproteinases (MMPs), known to play an important role in development, plasticity, response to injury and repair of the central nervous system (CNS). A real‐time quantitative RT‐PCR and immunohistochemical investigation was performed to study mRNA expression levels and protein distribution patterns of IEGs in cerebrum, cerebellum, and spinal cord of SJL/J mice between postnatal weeks 1 and 40. A down‐regulation of c‐jun, NF‐κB1, Max, Ets‐1, and p53 mRNA, and an up‐regulation of c‐fos mRNA was noticed. Down‐regulations of Ets‐1 and p53 were most prominent between week 1 and 3. The prominent role in CNS development for c‐jun, Ets‐1 and Max was supported by immunohistochemistry. One‐week‐old mice were strongly positive for all three proteins in cerebral cortex, medulla oblongata, and gray matter of the spinal cord. A high staining intensity was detected in the developing granule cell layer of the cerebellum for c‐jun and Ets‐1, and in the Purkinje cell layer of the cerebellum for Max. In addition to the general down‐regulation of most mRNAs, minor up‐regulations of all IEG proteins could be detected in restricted parts of the CNS indicating regional variations and differential expression and translation during development. Apoptosis was demonstrated using immunohistochemistry for active caspase‐3. The expression patterns of IEGs might represent the key to understand the balance of proteolytic activities by MMPs, myelination, and the induction of apoptosis during the development of the CNS.

Periventricular Demyelination and Axonal Pathology Is Associated with Subependymal Virus Spread in a Murine Model for Multiple Sclerosis

Objectives: Theiler’s murine encephalomyelitis virus (TMEV) infection of mice is a widely used animal model for demyelinating disorders, such as multiple sclerosis (MS). The aim of the present study was to identify topographical differences of TMEV spread and demyelination in the brain of experimentally infected susceptible SJL/J mice and resistant C57BL/6 mice. Methods: Demyelination was confirmed by Luxol fast blue and cresyl violet staining and axonal damage by neurofilament-specific and β-amyloid precursor protein-specific immunohistochemistry. Viral dissemination within the central nervous system (CNS) was quantified by immunohistochemistry and in situ hybridization. Further, the phenotype of infected cells was determined by confocal laser scanning microscopy. Results: An early transient infection of periventricular cells followed by demyelination and axonopathies around the fourth ventricle in SJL/J mice was noticed. Periventricular and brain stem demyelination was associated with a predominant infection of microglia/macrophages and oligodendrocytes. Conclusions: Summarized, the demonstration of ependymal infection and subjacent spread into the brain parenchyma as well as regional virus clearance despite ongoing demyelination and axonal damage in other CNS compartments allows new insights into TME pathogenesis. This novel aspect of TMEV CNS interaction will enhance the understanding of region-specific susceptibilities to injury and regenerative capacities of the brain in this MS model.

Morbillivirus Control of the Interferon Response: Relevance of STAT2 and mda5 but Not STAT1 for Canine Distemper Virus Virulence in Ferrets

ABSTRACT The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. IMPORTANCE The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors.

Macrophage depletion by liposome-encapsulated clodronate suppresses seizures but not hippocampal damage after acute viral encephalitis

Viral encephalitis is a major risk factor for the development of seizures and epilepsy, but the underlying mechanisms are only poorly understood. Mouse models such as viral encephalitis induced by intracerebral infection with Theilers virus in C57BL/6 (B6) mice allow advancing our understanding of the immunological and virological aspects of infection-induced seizures and their treatment. Previous studies using the Theilers virus model in B6 mice have indicated that brain-infiltrating inflammatory macrophages and the cytokines released by these cells are key to the development of acute seizures and hippocampal damage in this model. However, approaches used to prevent or reduce macrophage infiltration were not specific, so contribution of other mechanisms could not be excluded. In the present study, we used a more selective and widely used approach for macrophage depletion, i.e., systemic administration of clodronate liposomes, to study the contribution of macrophage infiltration to development of seizures and hippocampal damage. By this approach, almost complete depletion of monocytic cells was achieved in spleen and blood of Theilers virus infected B6 mice, which was associated with a 70% decrease in the number of brain infiltrating macrophages as assessed by flow cytometry. Significantly less clodronate liposome-treated mice exhibited seizures than liposome controls (P<0.01), but the development of hippocampal damage was not prevented or reduced. Clodronate liposome treatment did not reduce the increased Iba1 and Mac3 labeling in the hippocampus of infected mice, indicating that activated microglia may contribute to hippocampal damage. The unexpected mismatch between occurrence of seizures and hippocampal damage is thought-provoking and suggests that the mechanisms involved in degeneration of specific populations of hippocampal neurons in encephalitis-induced epilepsy are more complex than previously thought.

Vacuolation and mineralisation as dominant age-related findings in hamster brains

Syrian golden hamsters (Mesocricetus auratus) are laboratory animals increasingly used for research and toxicological studies. Despite the need for an adequate knowledge of spontaneously occurring lesions, studies investigating the background pathology of different organ systems in hamsters are lacking. The aim of this study was to investigate the occurrence of spontaneous, age-dependent lesions in the central nervous system of this species. Multiple brain and spinal cord transverse sections of 520 hamsters of 1, 3, 6, 12, and 24 months of age were investigated using histology and immunohistochemistry. Vacuolation of grey matter neuropil and mineralisation especially in the brain stem were the most prominent findings. They gradually increased in severity and frequency with age. Vacuolation and mineralisation affected approximately 100% and 50% of 24-month-old hamsters, respectively. In addition, pigment deposition and mast cell infiltration were commonly detected. Whether vacuolation and mineralisation represent an incidental finding or are related to a cognitive dysfunction syndrome remains to be determined.