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      Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner

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          Abstract

          Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.

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          Oxidative damage in multiple sclerosis lesions

          Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are currently poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Since mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, we analysed by immunocytochemistry the presence and cellular location of oxidized lipids and oxidized DNA in lesions and in normal-appearing white matter of 30 patients with multiple sclerosis and 24 control patients without neurological disease or brain lesions. As reported before in biochemical studies, oxidized lipids and DNA were highly enriched in active multiple sclerosis plaques, predominantly in areas that are defined as initial or ‘prephagocytic’ lesions. Oxidized DNA was mainly seen in oligodendrocyte nuclei, which in part showed signs of apoptosis. In addition, a small number of reactive astrocytes revealed nuclear expression of 8-hydroxy-d-guanosine. Similarly, lipid peroxidation-derived structures (malondialdehyde and oxidized phospholipid epitopes) were seen in the cytoplasm of oligodendrocytes and some astrocytes. In addition, oxidized phospholipids were massively accumulated in a fraction of axonal spheroids with disturbed fast axonal transport as well as in neurons within grey matter lesions. Neurons stained for oxidized phospholipids frequently revealed signs of degeneration with fragmentation of their dendritic processes. The extent of lipid and DNA oxidation correlated significantly with inflammation, determined by the number of CD3 positive T cells and human leucocyte antigen-D expressing macrophages and microglia in the lesions. Our data suggest profound oxidative injury of oligodendrocytes and neurons to be associated with active demyelination and axonal or neuronal injury in multiple sclerosis.
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            Virtual hypoxia and chronic necrosis of demyelinated axons in multiple sclerosis.

            Multiple sclerosis (MS), an inflammatory demyelinating disease, is a major cause of neurological disability in young adults in the developed world. Although the progressive neurological disability that most patients with MS eventually experience results from axonal degeneration, little is known about the mechanisms of axonal injury in MS. Accumulating evidence suggests that the increased energy demand of impulse conduction along excitable demyelinated axons and reduced axonal ATP production induce a chronic state of virtual hypoxia in chronically demyelinated axons. In response to such a state, key alterations that contribute to chronic necrosis of axons might include mitochondrial dysfunction (due to defective oxidative phosphorylation or nitric oxide production), Na+ influx through voltage-gated Na+ channels and axonal AMPA receptors, release of toxic Ca2+ from the axoplasmic reticulum, overactivation of ionotropic and metabotropic axonal glutamate receptors, and activation of voltage-gated Ca2+ channels, ultimately leading to excessive stimulation of Ca2+-dependent degradative pathways. The development of neuroprotective therapies that target these mechanisms might constitute effective adjuncts to currently used immune-modifying agents.
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              Mitochondrial changes within axons in multiple sclerosis.

              Multiple sclerosis is the most common cause of non-traumatic neurological impairment in young adults. An energy deficient state has been implicated in the degeneration of axons, the pathological correlate of disease progression, in multiple sclerosis. Mitochondria are the most efficient producers of energy and play an important role in calcium homeostasis. We analysed the density and function of mitochondria using immunohistochemistry and histochemistry, respectively, in chronic active and inactive lesions in progressive multiple sclerosis. As shown before in acute pattern III and Balo's lesions, the mitochondrial respiratory chain complex IV activity is reduced despite the presence of mitochondria in demyelinated axons with amyloid precursor protein accumulation, which are predominantly located at the active edge of chronic active lesions. Furthermore, the strong non-phosphorylated neurofilament (SMI32) reactivity was associated with a significant reduction in complex IV activity and mitochondria within demyelinated axons. The complex IV defect associated with axonal injury may be mediated by soluble products of innate immunity, as suggested by an inverse correlation between complex IV activity and macrophage/microglial density in chronic lesions. However, in inactive areas of chronic multiple sclerosis lesions the mitochondrial respiratory chain complex IV activity and mitochondrial mass, judged by porin immunoreactivity, are increased within approximately half of large (>2.5 microm diameter) chronically demyelinated axons compared with large myelinated axons in the brain and spinal cord. The axon-specific mitochondrial docking protein (syntaphilin) and phosphorylated neurofilament-H were increased in chronic lesions. The lack of complex IV activity in a proportion of Na(+)/K(+) ATPase alpha-1 positive demyelinated axons supports axonal dysfunction as a contributor to neurological impairment and disease progression. Furthermore, in vitro studies show that inhibition of complex IV augments glutamate-mediated axonal injury (amyloid precursor protein and SMI32 reactivity). Our findings have important implications for both axonal degeneration and dysfunction during the progressive stage of multiple sclerosis.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: Project administrationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: Project administration
                Role: Formal analysisRole: Investigation
                Role: Project administration
                Role: Formal analysisRole: Investigation
                Role: Project administration
                Role: Project administration
                Role: Project administration
                Role: Formal analysis
                Role: Formal analysis
                Role: Formal analysisRole: Investigation
                Role: Formal analysisRole: Investigation
                Role: Formal analysis
                Role: Project administration
                Role: Project administration
                Role: Project administration
                Role: Project administration
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: Project administration
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 November 2017
                2017
                : 12
                : 11
                : e0187215
                Affiliations
                [1 ] Department of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
                [2 ] Department of Neurology, Osaka General Medical Center, Osaka, Osaka, Japan
                [3 ] Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
                [4 ] Pharmaceutical Development Research Laboratories, Teijin Pharma Ltd., Hino, Tokyo, Japan
                [5 ] Department of Neurology, National Hospital Organization Toneyama National Hospital, Toyonaka, Osaka, Japan
                Nagoya Daigaku, JAPAN
                Author notes

                Competing Interests: Takashi Shirakura and Mizuho Tamura were employees of Teijin Pharma Ltd. The drug febuxostat used in the study was kindly provided by Teijin Pharma Ltd. This does not alter our adherence to PLoS One policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0001-6173-3759
                Article
                PONE-D-17-26460
                10.1371/journal.pone.0187215
                5673182
                29107957
                974048b9-4678-4b85-a7c4-92a92f269c29
                © 2017 Honorat et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 July 2017
                : 16 October 2017
                Page count
                Figures: 4, Tables: 0, Pages: 12
                Funding
                This study is supported in part by the Practical Research Project for Rare/Intractable Diseases of the Japan Agency for Medical Research and Development (AMED), JSPS KAKENHI (grant numbers JP26461293 and JP15K09339), and a Health and Labor Sciences Research Grant for Rare and Intractable Diseases from the Ministry of Health, Labor and Welfare of Japan. Teijin Pharma Ltd. provided support in the form of salaries for authors T.S. and M.T., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.
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