Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway

Inflammation and oxidative stress are thought to promote tissue damage in multiple sclerosis. Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for multiple sclerosis treatment. BG00012 is an oral formulation of dimethylfumarate. In a phase II multiple sclerosis trial, BG00012 demonstrated beneficial effects on relapse rate and magnetic resonance imaging markers indicative of inflammation as well as axonal destruction. First we have studied effects of dimethylfumarate on the disease course, central nervous system, tissue integrity and the molecular mechanism of action in an animal model of chronic multiple sclerosis: myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis in C57BL/6 mice. In the chronic phase of experimental autoimmune encephalomyelitis, preventive or therapeutic application of dimethylfumarate ameliorated the disease course and improved preservation of myelin, axons and neurons. In vitro, the application of fumarates increased murine neuronal survival and protected human or rodent astrocytes against oxidative stress. Application of dimethylfumarate led to stabilization of the transcription factor nuclear factor (erythroid-derived 2)-related factor 2, activation of nuclear factor (erythroid-derived 2)-related factor 2-dependent transcriptional activity and accumulation of NADP(H) quinoline oxidoreductase-1 as a prototypical target gene. Furthermore, the immediate metabolite of dimethylfumarate, monomethylfumarate, leads to direct modification of the inhibitor of nuclear factor (erythroid-derived 2)-related factor 2, Kelch-like ECH-associated protein 1, at cysteine residue 151. In turn, increased levels of nuclear factor (erythroid-derived 2)-related factor 2 and reduced protein nitrosylation were detected in the central nervous sytem of dimethylfumarate-treated mice. Nuclear factor (erythroid-derived 2)-related factor 2 was also upregulated in the spinal cord of autopsy specimens from untreated patients with multiple sclerosis. In dimethylfumarate-treated mice suffering from experimental autoimmune encephalomyelitis, increased immunoreactivity for nuclear factor (erythroid-derived 2)-related factor 2 was detected by confocal microscopy in neurons of the motor cortex and the brainstem as well as in oligodendrocytes and astrocytes. In mice deficient for nuclear factor (erythroid-derived 2)-related factor 2 on the same genetic background, the dimethylfumarate mediated beneficial effects on clinical course, axon preservation and astrocyte activation were almost completely abolished thus proving the functional relevance of this transcription factor for the neuroprotective mechanism of action. We conclude that the ability of dimethylfumarate to activate nuclear factor (erythroid-derived 2)-related factor 2 may offer a novel cytoprotective modality that further augments the natural antioxidant responses in multiple sclerosis tissue and is not yet targeted by other multiple sclerosis therapies

JC Polyomavirus Abundance and Distribution in Progressive Multifocal Leukoencephalopathy (PML) Brain Tissue Implicates Myelin Sheath in Intracerebral Dissemination of Infection

Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath

VP1 colocalizes with nearby myelin in recently lysed cells.

<p>Representative 5-μm confocal Z-axis stack images of regions of recent virus-induced cell lysis in otherwise intact white matter (A, B, D, E) or gray matter (C) of human PML, or white matter in an SIV-positive rhesus macaque with SV40 PML-like CNS disease (F), stained for VP1 (green in A-F, A″–F″, and A*-F*) and MBP (red in A-C, F, A′–C′, F′, and A*-C*, F*), GFAP (red in D, D′, D*), or IBA1 (red in E, E′, E*). A*–F* show representative colocalization (coloc) images from a single image from each Z-stack, with magenta pixels designating VP1/MBP colocalization (magenta arrows); Image quantitation of these representative Z-stack images revealed 89% and 86% of VP1 colocalized with MBP in white matter (A* and B*, respectively); 34% colocalized in gray matter (C*); and 13% and 2.9% of VP1 colocalized with GFAP or IBA1 (D* and E*, respectively). In SV40 PML-like disease (F), a subset of VP1 was dispersed in a linear pattern and colocalized with partially demyelinated MBP-positive axons (magenta arrow in F*). Occasional cytoplasmic VP1 viral aggregates were seen in cytoplasm of microglia (green arrow in E) or astrocytes, but nuclei of these cell types did not show VP1 positivity that would be indicative of productive infection. Scale bar in panel A = 10 μm.</p

JCV sequence analysis.

<p>(A) Schematic of circular viral DNA showing sites of transcription initiation and directions of early and late transcription (arrows) adjacent to the NCCR. Regions encoding Early T-Ag (T and t) and late agnoprotein (Agno) and VP1, VP2, and VP3 proteins are indicated by colored boxes. (B) Summary of NCCR variants, with the archetype NCCR shown for reference consisting of an origin of replication (ORI) followed by intact sequence blocks A-F. The percent of sequences showing the schematized rearrangements, duplications, and/or mutant/deleted sequence blocks (indicated by lower case letters) are listed. (C) VP1 variants identified. WT (1B) = wild type, strain 1B. (D) Agnoprotein variants identified; del(51-end) = deletion of C-terminal 21 amino acids. (E) VP2 variants identified; del(283-end) = deletion of C-terminal 60 amino acids. P174S and R207G were identified in the VP1 amplicon, which overlaps the C-terminal region of VP2/3, with asterisks designating percent of VP1 region clones that harbored the VP2 mutation. Representative DNA sequences and alignments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155897#pone.0155897.s002" target="_blank">S1 File</a>, and have been deposited at NCBI under accession nos. KX216358-KX216371.</p

Histopathology and virus protein distributions in SV40 PML-like disease.

<p>Representative section of macaque cerebrum was fixed, stained for H&E (A, D, G), TAg (B, E, H), or VP1 (C, F, I). (A-C) Black asterisks mark cortical area with severe demyelination, and red asterisks mark corpus callosum. Gray (G) and white (W) matter regions are designated, with areas in red and black boxes in A-C magnified in D–F and G–I, respectively. Note foci of infection within white matter (blue arrow) and tracking along gray-white junction (blue arrowheads). (D-F) Magnified area of infected gray-white matter junction, showing actively infected area with TAg-positive cells (red arrow) and VP1-positive infected cells with dispersed VP1 (red arrowhead). (G–I) End-stage lesion showing dispersed TAg and VP1 amid sheets of macrophages (black arrowhead). Inset images in lower right corners of D-I show low magnification view of entire tissue section. Scale bar: 5 mm in A–C, 100 μm in D–I.</p

Quantitation of virus components from each tissue block.

<p>Quantitation of virus components from each tissue block.</p

JCV protein distributions during the viral life cycle.

<p>Representative confocal images of JCV-infected white matter oligodendrocytes at various stages of infection, stained for TAg (red), VP1 (green), and DNA (blue), from early to late stages (left to right). Early infection (left two columns) is marked by nuclear foci of TAg (arrow), which increase in number as the nucleus enlarges. Cells transitioning between early and late stages (middle column) show abundant nuclear TAg foci with peripheral nuclear VP1 denoting early viral assembly (arrow). At late stages of infection (right two columns), TAg is absent, and VP1 fills the cytoplasm and proximal extent of some cell processes but does not appear to extend to processes that ensheathe nearby axons (arrow). Cytoplasmic lipofuscin (arrowheads) in some cells was captured with TAg staining with AF594 detection (red channel). Scale bar = 10 μm.</p

Tissue blocking and experimental scheme.

<p>Fixed postmortem sections corresponding to T2 brain MRI images at 6 weeks post-presentation, ~1.5 weeks prior to death. For indicated brain regions, L, PL, and NL tissue blocks, designated L1–3, PL1–3, and NL1–3, were studied as indicated. T2 - T2-weighted; qPCR—quantitative PCR; MRM/MS–multiple reaction monitoring mass spectrometry; IHC–immunohistochemistry; IF–immunofluorescence; ISH—in-situ hybridization.</p

Spectrum of PML histopathology.

<p>H&E (A-C) and IHC (brown, with blue hematoxylin counterstain) for NFP to label neuronal processes (D-F), MBP to label myelin (G-I), GFAP to label astrocytes (black arrows) (J-L), and IBA1 to label microglia/macrophages (blue arrowheads) (M-O) from grossly unaffected, actively infected, and end-stage white matter (blocks NL3, PL3, and L3, respectively). NL3 (left column) had largely normal white matter composed of linear axons/myelin throughout the neuropil, oligodendrocytes with characteristic perinuclear “fried egg” halos (green arrowheads), astrocytes with thin and elongated processes, and microglia with short, thick processes. PL3 (middle column) showed active infection with viral nuclear inclusions (red arrows), hypertrophied astrocytes with thickened processes, swollen myelin, and some enlarged macrophages (this section also had foci of demyelination, not shown here). L3 (right column) had end-stage lesions with rare viral inclusions, reduced number and fragmentation of axons, residual axonal myelin (magenta arrowhead), and variable amounts of MBP within engorged macrophages and massively hypertrophied (“bizarre”) astrocytes. Asterisks in B and C designate thinning of neuropil secondary to axonal loss. Scale bar = 30 μm for all panels.</p

Histopathology of demyelinated foci.

<p>Adjacent sections of NL1 stained with H&E (A), IHC for VP1 using PAB597 at 0.01 μg/mL (B) or 1.0 μg/mL (C), NFP (D), MBP (E), or IBA1 (F). Thumbnail image in right upper corner of each panel shows low magnification image of stained section. Grey (G) / white (W) matter junction is denoted by a dashed line, and asterisks designate area around the focus of demyelination magnified in each panel. VP1 antibody at 0.01 μg/mL detects individually infected cells (red arrow, B), whereas 1 μg/mL additionally reveals broadly distributed VP1 (red asterisk, C), with no VP1 staining in nearby uninfected white matter (black asterisk) or gray matter. In demyelinated foci, there is no decrease in NFP stain (D) in areas with reduced MBP (E), and increased numbers of IBA+ engorged macrophages (arrowhead, F). Scale bar = 50 μm.</p