Protandim Protects Oligodendrocytes against an Oxidative Insult

Oligodendrocyte damage and loss are key features of multiple sclerosis (MS) pathology. Oligodendrocytes appear to be particularly vulnerable to reactive oxygen species (ROS) and cytokines, such as tumor necrosis factor-α (TNF), which induce cell death and prevent the differentiation of oligodendrocyte progenitor cells (OPCs). Here, we investigated the efficacy of sulforaphane (SFN), monomethyl fumarate (MMF) and Protandim to induce Nrf2-regulated antioxidant enzyme expression, and protect oligodendrocytes against ROS-induced cell death and ROS-and TNF-mediated inhibition of OPC differentiation. OLN-93 cells and primary rat oligodendrocytes were treated with SFN, MMF or Protandim resulting in significant induction of Nrf2-driven (antioxidant) proteins heme oygenase-1, nicotinamide adenine dinucleotide phosphate (NADPH): quinone oxidoreductase-1 and p62/SQSTM1, as analysed by Western blotting. After incubation with the compounds, oligodendrocytes were exposed to hydrogen peroxide. Protandim most potently promoted oligodendrocyte cell survival as measured by live/death viability assay. Moreover, OPCs were treated with Protandim or vehicle control prior to exposing them to TNF or hydrogen peroxide for five days, which inhibited OPC differentiation. Protandim significantly promoted OPC differentiation under influence of ROS, but not TNF. Protandim, a combination of five herbal ingredients, potently induces antioxidants in oligodendrocytes and is able to protect oligodendrocytes against oxidative stress by preventing ROS-induced cell death and promoting OPC differentiation

The Role of the Intestinal Microbiome in Multiple Sclerosis—Lessons to Be Learned from Hippocrates

Based on recent advances in research of chronic inflammatory conditions, there is a growing body of evidence that suggests a close correlation between the microbiota of the gastrointestinal tract and the physiologic activity of the immune system. This raises the idea that disturbances of the GI ecosystem contribute to the unfolding of chronic diseases including neurodegenerative pathologies. Here, we overview our current understanding on the putative interaction between the gut microbiota and the immune system from the aspect of multiple sclerosis, one of the autoimmune conditions accompanied by severe chronic neuroinflammation that affects millions of people worldwide

A new venue of TNF targeting

The first Food and Drug Administration-(FDA)-approved drugs were small, chemically-manufactured and highly active molecules with possible off-target effects, followed by protein-based medicines such as antibodies. Conventional antibodies bind a specific protein and are becoming increasingly important in the therapeutic landscape. A very prominent class of biologicals are the anti-tumor necrosis factor (TNF) drugs that are applied in several inflammatory diseases that are characterized by dysregulated TNF levels. Marketing of TNF inhibitors revolutionized the treatment of diseases such as Crohn's disease. However, these inhibitors also have undesired effects, some of them directly associated with the inherent nature of this drug class, whereas others are linked with their mechanism of action, being pan-TNF inhibition. The effects of TNF can diverge at the level of TNF format or receptor, and we discuss the consequences of this in sepsis, autoimmunity and neurodegeneration. Recently, researchers tried to design drugs with reduced side effects. These include molecules with more specificity targeting one specific TNF format or receptor, or that neutralize TNF in specific cells. Alternatively, TNF-directed biologicals without the typical antibody structure are manufactured. Here, we review the complications related to the use of conventional TNF inhibitors, together with the anti-TNF alternatives and the benefits of selective approaches in different diseases

Grey matter demyelination and neurodegeneration in Multiple Sclerosis: a new animal model for studying disease mechanisms

Multiple sclerosis is the commonest neurological disease affecting young adults. Whilst the initial relapsing-remitting disease phase is associated with inflammatory demyelination and is treatable with immunomodulatory drugs, the secondary progressive phase (SP-MS) is associated with ongoing axonal loss and cortical atrophy and is currently untreatable. Studies of SP-MS have revealed the presence of extensive subpial demyelinated lesions within the cerebral cortex. This pathology is associated with a high level of meningeal inflammation, a gradient of cell loss from the cortical surface and high levels of microglia activation. To test the hypothesis that pro-inflammatory cytokines diffusing from the cerebral meninges could be responsible, we have established an animal model mimicking cortical grey matter pathology. Female DA rats were immunised with 5μg recombinant myelin oligodendrocyte glycoprotein (rmMOG) in incomplete Freunds adjuvant (IFA). This dose was insufficient to initiate encephalomyelitis, but did initiate an anti-MOG humoral immune response in the periphery. Twenty days post-immunisation animals received an injection of tumour necrosis factor (TNF) and interferon gamma (IFNγ) into the subarachnoid space at the sagittal sulcus. Immunohistochemistry revealed areas of subpial demyelination extending through cortical layers I–III. Lesions were maximal after 7 days and had resolved by remyelination at 14 days. A gradient of microglia/macrophage activation was present from the cortical surface. The extent of demyelination correlated with activation of microglia in the cortex and macrophages within the meninges. Activated microglia were observed contacting myelin, oligodendrocytes and neurons. In the demyelinated cortex, expression of the TNF receptors TNFR1A and TNFR1B was upregulated on oligodendrocytes and perivascular macrophages respectively. CD8+ T cells were observed in the meninges, corpus callosum and scattered throughout the grey matter, whereas CD4+ T cells and CD79a+ B cells were restricted to the meninges. Oligodendrocyte numbers were reduced in the upper cortical layers prior to demyelination (days 1 and 3 post-injection), but were still present in demyelinated lesions at day 7. Numbers of neurons and astrocytes were not changed. Control animals immunised with IFA and injected with cytokines had increased presence of inflammatory cells within the meninges but no demyelination. Animals immunised with rmMOG and injected with PBS had no demyelination or immune response within the meninges or cortex. Thus, acute subpial demyelination was dependent on a pre-existing immune response against myelin protein, coupled with generalised pro-inflammatory signalling within the meninges. These findings support our hypothesis of a role for meningeal inflammation in the cortical pathology of MS and describe for the first time an animal model that can be used to study the molecular mechanisms involved. Future research will aim to maintain meningeal inflammation and produce a model of chronic demyelination

Mechanisms and specificity of lentivirus neurotoxicity

Lentiviruses such as Maedi-Visna Virus (MVV) in sheep and Human Immunodeficiency Virus (HIV) often cause a variety of neurological symptoms in later stages of infection. In sheep, MVV infection results in paralysis and ataxia, with post-mortem investigations revealing astrocytosis, demyelination and axonal damage. At present it is unclear how such neurodegeneration is mediated as MVV, like HIV, does not directly infect neurons. There is much debate as to the mechanisms involved in lentivirus related brain damage, centred around three main possibilities. Firstly, there may be a direct detrimental action of a viral fragment on neuronal cell types. Secondly, MVV infected cells may be releasing diffusible mediators such as cytokines which could cause neural damage or act on astrocytes and microglia causing them to produce factors to induce neurodegeneration. Thirdly, the prospect of an immune response occuring within the brain, involving infected macrophages or microglia, is highly probable.In order to investigate the possible mechanisms at work in lentivirus induced neurodegeneration, several sets of experiments were carried out. Firstly, a putative model of MVV encephalopathy was established in severe combined immunodeficient (,scid) mice. Scid mice lack T and B cells and are thus unable to mount any immune response. Blood derived macrophages were obtained from healthy sheep and infected with MVV in vitro. These were intracerebrally injected into scid mice, as were uninfected macrophages, in order to assess the occurrence of any neural cell damage. Cell preparations of peripheral blood mononuclear cells (PBMCs), uninfected, supplemented with virus, or obtained from productively MVV infected sheep, were also administered to scid mice in the same manner, to evaluate the role of immune cells in causing any neurodegeneration. The results show that the administration of MVV infected macrophages produces greater gliosis and astrocytosis in the brains of these mice as compared to those injected with control uninfected macrophages or vehicle. However, the injections of PBMCs did not cause any detectable differences in astrocytes and microglia between the groups. No neuronal loss or demyelination was evident with any of the injections. These results suggest that neural cell disruption in MVV encephalopathy may be due to the presence of MVV infected macrophages in the Central Nervous System (CNS) of infected sheep, acting indirectly through the release of diffusible mediators.To investigate further the underlying causes of these changes, the possible detrimental contribution of cytokines in this model was evaluated through the use of the reverse transcriptase-polymerase chain reaction (RT-PCR) of MVV infected and uninfected blood derived macrophages in vitro. The results show an increase in the levels of the pro-inflammatory cytokine interleukin-1 beta (TL-113) mRNA in MVV infected macrophages. Interpretation of this model therefore suggests that MVV exerts an indirect disruptive effect on neural cells via the release of pro-inflammatory cytokines from infected macrophages, causing microgliosis and astrocytosis which in turn lead to major disturbances of brain function.The possibility of a neurotoxic action of a viral product was also investigated. Peptides derived from the basic region of the transactivating protein Tat from both MVV and HIV have previously been shown to be lethal to neurons in vivo and in vitro, through a mechanism believed to involve Nitric Oxide (NO)-mediated glutamate neurotoxicity and cytokines. The actions of the MVV tat peptide when injected intracerebrally in rats are diminished one week post-operatively by the Nmethyl-D-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,ll-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine maleate (MK801) and the Nitric Oxide Synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Here, its short term effects were examined and its neurotoxic mechanisms probed through the use of in vivo stereotaxic injections and pharmacologic manipulations. The results show that the MVV and HIV tat peptides were very rapidly neurotoxic, causing neuronal cell death within 0.5 hours, and displayed a distinctive and unusual lesion profile. Changes in astrocytes and microglia were also observed. The acute effects of the MVV tat peptide were blocked by MK801, an NMDA receptor antagonist. However, the administration of L-NAME, a NOS inhibitor, alpha melanocyte stimulating hormone (aMSH), a TNFa inhibitor, or 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo- (F)-quinoxaline (NBQX), a non-NMDA glutamate receptor antagonist, did not reduce the volume of the lesion. This suggests that the MVV tat peptide acts at the NMDA receptor to cause cell death either directly or via an excitotoxic mechanism

Inflammatory Cascades in the Pathogenesis of Multiple Sclerosis Lesions

Multiple sclerosis (MS) is a disease of the human central nervous system (CNS) characterised by inflammation and demyelination. Initially the MS lesion has a distinct histopathological picture with myelin-positive microglia in the midst of apparently intact myelin but minimal perivascular inflammation. Inflammatory mediators produced by these activated microglia may precipitate the infiltration of mononuclear cells and the overt myelin loss seen in actively demyelinating MS lesions. Nuclear factor-κB (NF-κB) is a transcriptional regulator of proteolytic enzymes, adhesion molecules and inflammatory cytokines which rapidly translates extracellular signals into protein synthesis. The immunocytochemical detection of the transcriptionally active form of NF-κB, but not the inhibitory protein IκBα, in the nuclei of microglia in normal human CNS white matter indicates the capability of microglia to respond rapidly to pathological stimuli in the CNS. Activation of NF-κB in MS plaques, evident from the nuclear localisation of the NF-κB subunits RelA, c-Rel and p50 in macrophages, may propagate inflammatory demyelination through upregulation of NF-κB-controlled macrophage genes for inflammatory mediators. In demyelinating disease the plasmin-matrix metalloprotease (MMP) enzymatic cascade promotes blood-brain barrier (BBB) damage, generation of encephalitogenic myelin peptides and activation of pro-inflammatory cytokines. Constitutive expression of MMPs 1, 2, 3 and 9 in glial cells in normal control white matter was demonstrated by immunocytochemistry. However, the lack of tissue (t-PA) and urokinase (u-PA) plasminogen activators in glial cells and the absence of caseinolytic activity as shown by in situ zymography emphasises the latent nature of the plasmin-MMP cascade in normal CNS tissue. In contrast, the co-localisation of t-PA and u-PA, rate-limiting serine-proteases, and MMPs in macrophages and astrocytes in active MS lesions forms the basis of a functional enzymatic cascade. Furthermore, increased amounts and activity of u-PA and MMP-9 in homogenates of active MS plaques coupled with the presence of caseinolytic activity in foamy macrophages implicates these cells as the major source of MMPs, which cause proteolytic damage in MS. Insulin-like growth factors (IGFs) play an important role in development and myelination in the CNS but can also stimulate phagocytosis and production of inflammatory mediators by macrophages. In active MS lesions binding of IGF-II to the IGF receptor on foamy macrophages may induce mitogenic responses and invasiveness of macrophages which can be further enhanced by MMP-mediated proteolytic removal of inhibitory IGF-binding proteins. Similarly, the potent mitogens IGF-I and insulin may stimulate astrocytosis and gliosis. In contrast, oligodendrocytes in normal-appearing white matter do not express IGFs or IGF-I receptor which implies that the oligodendrocyte response to these remyelinating growth factors is impaired. Therefore, the prevailing role of IGFs in MS lesions may be in line with pro-inflammatory mediators promoting macrophage and astrocyte responses to tissue damage. In conclusion, NF-κB activation in microglia and macrophages upregulates the production of PAs and inflammatory cytokines which trigger the plasmin-MMP cascade, leading to BBB damage and enhanced inflammatory cell migration and demyelination in white matter. Influx of IGFs through the damaged BBB and their increased local production may promote myelin phagocytosis and reactive astrocytosis. In turn IGF-mediated upregulation of PAs in glial cells could provide a feedback amplification of the MMP cascade. Therefore, the findings from these studies bring together three systems of mediators, NF-κB, MMPS and IGFs, into a hypothetical model for the propagation of demyelination in MS lesions

The role of macrophages in the formation and repair of myelin

Foetal rat brain aggregate cultures resemble the developing brain providing a unique system to investigate myelinogenesis, demyelination and repair. Supplementing aggregate cultures with macrophages accelerated cellular organisation and increased myelin deposition over time without affecting activity of the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Pro-inflammatory cytokines and anti-myelin oligodendrocyte glycoprotein (MOG) antibodies induced demyelination in myelinated aggregate cultures while oligodendrocytes were spared. Demyelination was associated with increased levels of a myelin basic protein (MBP) degradation peptide indicating proteolysis of myelin. MBP continued to accumulate following removal of demyelinating agents while peptide levels declined. Myelinogenesis in the aggregates was associated with patterns of growth factor mRNA expression comparable with those of the developing brain. The mRNA levels of platelet-derived growth factor-A (PDGF-A), a potent mitogen for oligodendrocyte progenitors, rose rapidly while fibroblast growth factor-2 (FGF-2) and ciliary neurotrophic factor (CNTF) mRNA increased gradually as MBP accumulated. The peak of transforming growth factor-β1 (TGF-β1) and neurotrophin-3 (NT-3) mRNA expression coincided with the appearance of MBP mRNA, while that of insulin-like growth factor-I (IGF-I) was more closely associated with the detection of MBP protein. Enhanced myelination in macrophage-enriched cultures was associated with reduced expression of CNTF and increased levels of TGF-β1 and FGF-2 mRNA both of which promote oligodendrocyte development in vitro. Demyelination induced a distinct pattern of expression of many myelination-associated growth factors. A rapid rise in CNTF mRNA in standard cultures closely followed by increases in FGF-2 and IGF-I was in contrast to the delayed induction of PDGF-A mRNA. In macrophage-enriched aggregates the rise in IGF-I mRNA following demyelination preceded that in standard cultures suggesting that macrophage- enrichment instigates a faster IGF-I response during remyelination. Since macrophage-rich demyelinating multiple sclerosis lesions also display signs of remyelination, macrophages, as a source of growth factors, have the potential to promote myelination and repair

The effect of stress on the neuropathogenesis of Theiler's virus-induced demyelination as an animal model of multiple sclerosis

Stressful life events have been associated with the onset and/or exacerbation of multiple sclerosis (MS). To investigate the effects of stress on the pathogenesis of MS, we employed restraint stress (RST) in the TheilerâÂÂs virus-induced demyelination (TVID) model, an animal model for human MS. Intracerebral inoculation of susceptible strain of mice with TheilerâÂÂs murine encephalomyelitis virus (TMEV) results in a biphasic disease â an acute encephalomyelitis and chronic demyelination. The establishment of persistent viral infection is critical in inducing immune-mediated demyelination during the chronic disease. The exposure of mice to RST prior to viral infection produced a stress response as evidenced by elevated circulating corticosterone (CORT). To further study the effect of stress on the immune response to TMEV infection and demyelination, we first examined the cytokine and chemokine response during the acute TMEV infection. We demonstrated that RST down-regulated the virus-induced expression of chemokines, Ltn, IP-10, RANTES, and pro-inflammatory cytokines, TNF, IFN and LT in both the brain and spleen during early infection. Histologically, a decreased pattern of inflammation was observed in the brain of restrained mice as compared to non-restrained mice. The increased viral titer was noted in the CNS of restrained mice and was correlated with the decreased production of pro-inflammatory cytokine, suggesting an impaired immune response by RST. Secondly, the duration of stress on the late demyelination was investigated. Repeated and chronically stressed SJL/J mice developed an early onset of clinical signs and a delayed onset was observed in acutely stressed mice. Both acute and chronic RST suppressed the antibody response to TMEV and stressed displayed a higher incidence of demyelination than non-restrained mice. Axonal loss was also noted in chronic stressed mice. Additionally, RST caused an increased systemic viral infection in extraneural organs during the acute infection and cardiotropic TMEV was isolated from the heart of stressed mice. Taken together, stress resulted in profound immunsuppression during acute infection, which may consequently increase the incidence of demyelination. The present study may be generalized in human MS which is potentially triggered by viral infection