AB5864 Sigma-AldrichAnti-Myelin Basic Protein Antibody

The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia and macrophages, which can be polarized to distinct functional phenotypes: pro-inflammatory (M1) and anti-inflammatory or immunoregulatory (M2). We found that a switch from an M1- to an M2-dominant response occurred in microglia and peripherally derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 cell conditioned media and impaired in vivo following intra-lesional M2 cell depletion. M2 cell densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger mouse and in multiple sclerosis lesions that normally show remyelination. Blocking M2 cell-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Thus, our results indicate that M2 cell polarization is essential for efficient remyelination and identify activin-A as a therapeutic target for CNS regeneration.

Previous studies have indicated that 2,2'-dipyridyl, a lipid-soluble ferrous iron chelator, can reduce brain injury after cerebral ischemia and reduce cerebral vasospasm after subarachnoid hemorrhage. In this study, we examined the efficacy of 2,2'-dipyridyl after intracerebral hemorrhage (ICH) in 12-month-old mice. ICH was modeled by intrastriatal injection of collagenase or autologous whole blood. 2,2'-Dipyridyl or vehicle was administered intraperitoneally 2h before ICH (pretreatment) or 6h after ICH (post-treatment) and then once daily for up to 3days. Mice in the pretreatment group were sacrificed 1 or 3days after ICH and examined for iron deposition, neuronal death, oxidative stress, microglial/astrocyte activation, neutrophil infiltration, and white matter damage. Mice in the post-treatment group were examined for brain lesion volume and edema on day 3 and for neurologic deficits on days 1, 3, and 28 after ICH. Pretreatment with 2,2'-dipyridyl decreased iron accumulation and neuronal death, attenuated production of reactive oxygen species, reduced microglial activation without affecting astrocytes or neutrophil infiltration, and attenuated white matter damage. Post-treatment reduced brain lesion volume and edema and improved neurologic function. These results indicate that the lipid-soluble ferrous iron chelator 2,2'-dipyridyl can reduce brain injury and improve functional outcome after ICH.

The myelin sheath provides electrical insulation of mechanosensory Aβ-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs) damage the myelin sheath. The resulting electrical instability of Aβ-fibers is believed to activate the nociceptive circuitry in Aβ-fibers and initiate pain from innocuous tactile stimulation (mechanical allodynia). The precise molecular mechanisms, responsible for the development of this neuropathic pain state after nerve injury (for example, chronic constriction injury, CCI), are not well understood.Using mass spectrometry of the whole sciatic nerve proteome followed by bioinformatics analyses, we determined that the pathways, which are classified as the Infectious Disease and T-helper cell signaling, are readily activated in the nerves post-CCI. Inhibition of MMP-9/MMP-2 suppressed CCI-induced mechanical allodynia and concomitant TNF-α and IL-17A expression in nerves. MMP-9 proteolysis of myelin basic protein (MBP) generated the MBP84-104 and MBP68-86 digest peptides, which are prominent immunogenic epitopes. In agreement, the endogenous MBP69-86 epitope co-localized with MHCII and MMP-9 in Schwann cells and along the nodes of Ranvier. Administration of either the MBP84-104 or MBP68-86 peptides into the naïve nerve rapidly produced robust mechanical allodynia with a concomitant increase in T cells and MHCII-reactive cell populations at the injection site. As shown by the genome-wide expression profiling, a single intraneural MBP84-104 injection stimulated the inflammatory, immune cell trafficking, and antigen presentation pathways in the injected naïve nerves and the associated spinal cords. Both MBP84-104-induced mechanical allodynia and characteristic pathway activation were remarkably less prominent in the T cell-deficient athymic nude rats.These data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1 day post-injury is critical to the generation of tactile allodynia, neuroinflammation, and the immunodominant MBP digest peptides in nerve. These MBP peptides initiate mechanical allodynia in both a T cell-dependent and -independent manner. In the course of Wallerian degeneration, the repeated exposure of the cryptic MBP epitopes, which are normally sheltered from immunosurveillance, may induce the MBP-specific T cell clones and a self-sustaining immune reaction, which may together contribute to the transition of acute pain into a chronic neuropathic pain state.

Myelin damage can lead to the loss of axonal conduction and paralysis in multiple sclerosis and spinal cord injury. Here, we show that acrolein, a lipid peroxidation product, can cause significant myelin damage in isolated guinea pig spinal cord segments. Acrolein-mediated myelin damage is particularly conspicuous in the paranodal region in both a calcium dependent (nodal lengthening) and a calcium-independent manner (paranodal myelin splitting). In addition, paranodal protein complexes can dissociate with acrolein incubation. Degraded myelin basic protein is also detected at the paranodal region. Acrolein-induced exposure and redistribution of paranodal potassium channels and the resulting axonal conduction failure can be partially reversed by 4-AP, a potassium channel blocker. From this data, it is clear that acrolein is capable of inflicting myelin damage as well as axonal degeneration, and may represent an important factor in the pathogenesis in multiple sclerosis and spinal cord injury.© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

The aim of this study was to characterize myelin loss as one of the features of white matter abnormalities across three common dementing disorders. We evaluated post-mortem brain tissue from frontal and temporal lobes from 20 vascular dementia (VaD), 19 Alzheimer's disease (AD) and 31 dementia with Lewy bodies (DLB) cases and 12 comparable age controls. Images of sections stained with conventional luxol fast blue were analysed to estimate myelin attenuation by optical density. Serial adjacent sections were then immunostained for degraded myelin basic protein (dMBP) and the mean percentage area containing dMBP (%dMBP) was determined as an indicator of myelin degeneration. We further assessed the relationship between dMBP and glutathione S-transferase (a marker of mature oligodendrocytes) immunoreactivities. Pathological diagnosis significantly affected the frontal but not temporal lobe myelin attenuation: myelin density was most reduced in VaD compared to AD and DLB, which still significantly exhibited lower myelin density compared to ageing controls. Consistent with this, the degree of myelin loss was correlated with greater %dMBP, with the highest %dMBP in VaD compared to the other groups. The %dMBP was inversely correlated with the mean size of oligodendrocytes in VaD, whereas it was positively correlated with their density in AD. A two-tier regression model analysis confirmed that the type of disorder (VaD or AD) determines the relationship between %dMBP and the size or density of oligodendrocytes across the cases. Our findings, attested by the use of three markers, suggest that myelin loss may evolve in parallel with shrunken oligodendrocytes in VaD but their increased density in AD, highlighting partially different mechanisms are associated with myelin degeneration, which could originate from hypoxic-ischaemic damage to oligodendrocytes in VaD whereas secondary to axonal degeneration in AD.

Certain genetic defects in LRRK2 and parkin are pathogenic for Parkinson's disease (PD) and both proteins deposit in the characteristic Lewy bodies. LRRK2 is thought to be involved in the early initiation of Lewy bodies. The involvement of LRRK2 and parkin in the similar cellular deposition of fibrillar alpha-synuclein in glial cytoplasmic inclusions (GCI) in multiple system atrophy (MSA) has not yet been assessed. To determine whether LRRK2 and parkin may be similarly associated with the abnormal deposition of alpha-synuclein in MSA GCI, paraffin-embedded sections from the basal ganglia of 12 patients with MSA, 4 with PD and 4 controls were immunostained for LRRK2, parkin, alpha-synuclein and oligodendroglial proteins using triple labelling procedures. The severity of neuronal loss was graded and the proportion of abnormally enlarged oligodendroglia containing different combinations of proteins assessed in 80-100 cells per case. Parkin immunoreactivity was observed in only a small proportion of GCI. In contrast, LRRK2 was found in most of the enlarged oligodendroglia in MSA and colocalised with the majority of alpha-synuclein-immunopositive GCI. Degrading myelin sheaths containing LRRK2-immunoreactivity were also observed, showing an association with one of the earliest oligodendroglial abnormalities observed in MSA. The proportion of LRRK2-immunopositive GCI was negatively associated with an increase in neuronal loss and alpha-synuclein-immunopositive dystrophic axons. Our results indicate that an increase in LRRK2 expression occurs early in association with myelin degradation and GCI formation, and that a reduction in LRRK2 expression in oligodendroglia is associated with increased neuronal loss in MSA.

In multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system (CNS), are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor. Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis.EAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination. TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.Intravenously applied bone marrow-derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE. TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

p25alpha is an oligodendroglial protein that can induce aggregation of alpha-synuclein and accumulates in oligodendroglial cell bodies containing fibrillized alpha-synuclein in the neurodegenerative disease multiple system atrophy (MSA). We demonstrate biochemically that p25alpha is a constituent of myelin and a high-affinity ligand for myelin basic protein (MBP), and in situ immunohistochemistry revealed that MBP and p25alpha colocalize in myelin in normal human brains. Analysis of MSA cases reveals dramatic changes in p25alpha and MBP throughout the course of the disease. In situ immunohistochemistry revealed a cellular redistribution of p25alpha immunoreactivity from the myelin to the oligodendroglial cell soma, with no overall change in p25alpha protein concentration using immunoblotting. Concomitantly, an approximately 80% reduction in the concentration of full-length MBP protein was revealed by immunoblotting along with the presence of immunoreactivity for MBP degradation products in oligodendroglia. The oligodendroglial cell bodies in MSA displayed an enlargement along with the relocalization of p25alpha, and this was enhanced after the deposition of alpha-synuclein in the glial cytoplasmic inclusions. Overall, the data indicate that changes in the cellular interactions between MBP and p25alpha occur early in MSA and contribute to abnormalities in myelin and subsequent alpha-synuclein aggregation and the ensuing neuronal degeneration that characterizes this disease.

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple sclerosis plaques. However, it failed to recognize any structures in normal human, rat, or guinea pig brain. The antiserum recognized the synthetic peptide QDENPVV, which corresponds to human myelin basic protein residues 82 to 88. Immunoabsorption with this peptide eliminated immunohistochemical staining. By contrast, several commercial antibodies recognizing nearby sequences of human myelin basic protein intensely stained all myelinated structures in both normal and multiple sclerosis tissue. The unusual epitope recognized by anti-EP appears to be accessible only in areas of myelin degeneration. If insults occur that repeatedly expose a region of MBP normally sheltered from immunosurveillance, a self-sustaining immune reaction might result.