17β‐estradiol protects against hypoxic/ischemic white matter damage in the neonatal rat brain

Developing oligodendrocytes (pre‐OLs) are highly vulnerable to hypoxic‐ischemic injury and associated excitotoxicity and oxidative stress. 17β‐Estradiol plays an important role in the development and function of the CNS and is neuroprotective. The sudden drop in circulating estrogen after birth may enhance the susceptibility of developing OLs to injury. Estrogen receptor (ER)–α and ER‐β are both expressed in OLs. We examined the effect of 17β‐estradiol on oxygen‐glucose deprivation and oxidative stress–induced cell death in rat pre‐OLs in vitro and on hypoxic‐ischemic brain injury in vivo. Pre‐OLs in culture were subjected to oxygen‐glucose deprivation (OGD) or glutathione depletion in the presence or absence of 17β‐estradiol. LDH release, the Alamar blue assay, and phase‐contrast microscopy were used to assess cell viability. Hypoxic‐ischemic injury was generated in 6‐day‐old rats (P6) by unilateral carotid ligation and hypoxia (6% O₂ for 1 hr). Rat pups received one intraperitoneal injection of 300 or 600 μg/kg 17β‐estradiol or vehicle 12 hr prior to the surgical procedure. Injury was assessed by myelin basic protein (MBP) immunocytochemistry at P10. 17β‐Estradiol produced significant protection against OGD‐induced cell death in primary OLs (EC₅₀ = 1.3 ± 0.46 × 10^?9 M) and against oxidative stress. Moreover, 17β‐estradiol attenuated the loss of MBP labeling in P10 pups ipsilateral to the carotid ligation. These results suggest a potential role for estrogens in attenuation of hypoxic‐ischemic and oxidative injury to developing OLs and in the prevention of periventricular leukomalacia. © 2009 Wiley‐Liss, Inc.     

Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitis

There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE-susceptible (LEW and LEW.1AV1) and one EAE-resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real-time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS.     

Developmental changes of glial fibrillary acidic protein and myelin basic protein in perinatal leukomalacia: relationship to a predisposing factor

Focal white matter necrosis is frequently seen in the brains of infants with perinatal cerebral hypoperfusion. Periventricular leukomalacia (PL) occurs in the deep white matter of premature and term neonates and subcortical leukomalacia (SL) in the subcortical white matter of young infants. Using immunoperoxidase methods in normal infants, glia positive for glial fibrillary acidic protein (GFAP) were found first in the deep zones of white matter and with increasing age they became more prominent in the subcortical zone. They increased diffusely in the deep or subcortical zones of the cases of PL or SL, respectively. The number of myelin basic protein-positive glia is much larger than that of GFAP-positive glia in the cases of old PL. These findings suggest that an increased number of positive glia may be a reaction to hypoxic, ischemic, or toxic insults, or this shifting, transient increase of positive glia in cerebral white matter may be one of several predisposing factors leading to perinatal leukomalacia. Furthermore, positive staining of GFAP and MBP for reactive astrocytes in old PL suggests that at a certain stage of gliogenesis both GFAP and myelin basic protein may be present within the same cell.     

CSF antibodies to myelin basic protein and oligodendrocytes in multiple sclerosis and other neurological diseases

Cerebrospinal fluid (CSF) from 18 multiple sclerosis (MS) patients, 13 subacute sclerosing panencephalitis (SSPE) patients, 22 other neurological disease (OND) patients, and 7 neurotic patients as controls were tested in an 125I-labeled anti-human F(ab')2 binding assay for the presence of antibodies to normal human brain cells from tissue culture, human fibroblasts, plasma membranes of MS and normal human brain, myelin basic protein (MBP) and bovine oligodendrocytes. Antibodies to MBP and to oligodendrocytes were found in the CSF of MS, SSPE and OND patients. Absorption of CSF with bovine CNS myelin significantly diminished binding activity to oligodendrocytes. Antibodies in the CSF against MBP and oligodendrocytes, on which some myelin determinants are expressed, seem to be a common feature of diseases in which demyelination is a component.     

Calcium receptor expression and function in oligodendrocyte commitment and lineage progression: potential impact on reduced myelin basic protein in CaR-null mice

Oligodendrocytes develop from oligodendrocyte progenitor cells (OPCs), which in turn arise from a subset of neuroepithelial precursor cells during midneurogenesis. Development of the oligodendrocyte lineage involves a plethora of cell-intrinsic and -extrinsic signals. A cell surface calcium-sensing receptor (CaR) has been shown to be functionally expressed in immature oligodendrocytes. Here, we investigated the expression and function of the CaR during oligodendrocyte development. We show that the order of CaR mRNA expression as assessed by quantitative polymerase chain reaction is mature oligodendrocyte > neuron > astrocyte. We next determined the rank order of CaR expression on inducing specification of neural stem cells to the neuronal, oligodendroglial, or astrocytic lineages and found that the relative levels of CaR mRNA expression are OPC > neuron > astrocytes. CaR mRNA expression in cells at various stages of development along the oligodendrocyte lineage revealed that its expression is robustly up-regulated during the OPC stage and remains high until the premyelinating stage, decreasing thereafter by severalfold in the mature oligodendrocyte. In OPCs, high Ca(2+) acting via the CaR promotes cellular proliferation. We further observed that high Ca(2+) stimulates the mRNA levels of myelin basic protein in preoligodendrocytes, which is also CaR mediated. Finally, myelin basic protein levels were significantly reduced in the cerebellum of CaR-null mice during development. Our results show that CaR expression is up-regulated when neural stem cells are specified to the oligodendrocyte lineage and that activation of the receptor results in OPC expansion and differentiation. We conclude that the CaR may be a novel regulator of oligodendroglial development and function.     

Human T lymphocytes specific for the immunodominant 83–99 epitope of myelin basic protein: Recognition of golli MBP HOG 7

Myelin basic protein (MBP) is a candidate auto-antigen in the disease multiple sclerosis. Although MBP was thought to be sequestered behind the blood-brain barrier, isoforms of MBPs have recently been demonstrated in lymphoid tissues. These isoforms, termed golli MBPs, contain sequences that are shared with “classic” MBP within the CNS. In the present study, we have determined that epitopes within golli MBP isoforms may be recognized by human T lymphocyte clones specific for classic MBP. Ten of 12 T-cell clones recognized golli MBP. Although 11 clones were specific for the immunodominant 83–99 sequence, the clones differed with respect to human leukocyte antigen (HLA) restriction, T-helper phenotype, cytolytic activity, and T-cell receptor usage. Greater responses to classic MBP than to golli MBP suggested a difference in the ability of the two proteins to be processed and to present epitopes therein. These data advance the hypothesis that golli MBP sequences expressed within lymphoid tissues may be recognized by classic MBP-specific T lymphocytes during central or peripheral tolerance. © 1996 Wiley-Liss, Inc.     

Adherence of cells to myelin basic protein I. Adherence of red and white blood cells from patients with multiple sclerosis to myelin basic protein

Red blood cells (RBC) and white blood cells (WBC) of patients with multiple sclerosis (MS) show decreased adherence to myelin basic protein (MBP) immobilized on plastic surfaces compared to the binding of cells from patients with other neurological diseases (OND), or such other autoimmune diseases as psoriasis (PS), and to that of healthy controls (HC). No similar phenomenon occurred to basic and non-basic type proteins other than MBP, for example, to histone (HIS), lysozyme (LYS) and ovalbumin (OVA). Thus, decreased adherence of RBC and WBC in MS patients to MBP appears to be a unique feature of the disease if compared with OND or PS.     

IL‐17A activates ERK1/2 and enhances differentiation of oligodendrocyte progenitor cells

Inflammatory signals present in demyelinated multiple sclerosis lesions affect the reparative remyelination process conducted by oligodendrocyte progenitor cells (OPCs). Interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), and interleukin (IL)?6 have differing effects on the viability and growth of OPCs, however the effects of IL‐17A are largely unknown. Primary murine OPCs were stimulated with IL‐17A and their viability, proliferation, and maturation were assessed in culture. IL‐17A‐stimulated OPCs exited the cell cycle and differentiated with no loss in viability. Expression of the myelin‐specific protein, proteolipid protein, increased in a cerebellar slice culture assay in the presence of IL‐17A. Downstream, IL‐17A activated ERK1/2 within 15 min and induced chemokine expression in 2 days. These results demonstrate that IL‐17A exposure stimulates OPCs to mature and participate in the inflammatory response. GLIA 2015;63:768–779     

Chronic Theiler's virus infection in mice: appearance of myelin basic protein in the cerebrospinal fluid and serum antibody directed against MBP

Myelin basic protein (MBP) appears frequently in the cerebrospinal fluid (CSF) of mice with chronic demyelination following intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV); antibody to MBP can frequently be found in the sera. The peaks of the immune responses to both MBP and TMEV coincide with the time course of the clinical signs of disease. Adsorption of mouse sera with TMEV or MBP indicate the non-identity of the antigens and the specificity of the antisera as measured by ELISA. Immunoblot analysis of sera confirmed the ELISA findings. The mechanism of induction of antibody directed against MBP and its role in TMEV-associated demyelination remain to be determined.     

Purification and spectroscopic characterization of the recombinant BG21 isoform of murine golli myelin basic protein

A recombinant form of the murine Golli-myelin basic protein (MBP) isoform BG21 (rmBG21) has been expressed in E. coli, and isolated to 96% purity via metal chelation chromatography. Characteristic yields were 6-8 mg protein per liter of culture in either minimal M9 or standard Luria-Bertani media. Circular dichroism spectroscopy showed that rmBG21 had a large proportion of random coil in aqueous solution, but gained alpha-helix in the presence of monosialoganglioside G(M1) and PI(4)P, as well as in the membrane-mimetic solvent trifluoroethanol. Bioinformatics analyses of the amino acid sequence of rmBG21 predicted an N-terminal calmodulin (CaM)-binding site. It was determined by fluorescence spectroscopy and dynamic light scattering that rmBG21 and CaM interacted weakly in a 1:1 ratio in a Ca(2+)-dependent manner. Solution NMR spectra of uniformly [(13)C(15)N]-labeled protein in aqueous buffer were consistent with it being an extended protein; spectral quality was independent of temperature. Thus, like "classic" MBP and the Golli-MBP isoform J37, rmBG21 is intrinsically disordered, implying multi functionality, and that its conformation depends on its environment and bound ligands.     

Two neuronal cell lines expressing the myelin basic protein gene display differences in their in vitro survival and in their response to glia

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature-sensitive SV-40 large-T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron-specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34°C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39°C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39°C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39°C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal–glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons. J. Neurosci. Res. 54:309–319, 1998. © 1998 Wiley-Liss, Inc.     

Protection against experimental autoimmune encephalomyelitis requires both CD4 T suppressor cells and myelin basic protein-primed B cells

Suppressor cells that regulate experimental autoimmune encephalomyelitis (EAE) are present in rats that recover from the disease and can protect against the development of active EAE when transferred to normal recipients. Both CD4+ T suppressor cells, known to regulate EAE effector cell lymphokine production, and myelin basic protein (MBP)-primed B cells are required to transfer protection against EAE to normal recipients. Neither CD4+ T suppressor cells nor MBP-primed B cells alone could transfer protection. Moreover, the co-transfer of normal B cells with CD4+ T suppressor cells did not provide protection against EAE. These results suggest that the regulation of EAE and perhaps the recovery from acute clinical disease requires the interaction of two specific subpopulations of regulatory lymphocytes.     

Nonactivated astrocytes downregulate T cell receptor expression and reduce antigen-specific proliferation and cytokine production of myelin basic protein (MBP)-reactive T cells

Astrocytes express variable levels of MHC class II antigens depending on their activation status or exposure to certain cytokines, notably IFN-γ. When they are induced to express higher surface densities of MHC class II molecules, astrocytes are capable of stimulating syngeneic myelin basic protein (MBP)-reactive T cells to proliferate at a modest rate and to secrete proinflammatory cytokines, such as TNF-α, in response to antigen. In the present investigation evidence is presented that uninduced astrocytes, whether fresh or established as clones, on which surface MHC class II molecules are expressed at a very low density, promote an antigen-dependent reduction of TCR on the surface of syngeneic T cells. Accompanying this effect on the TCR is an induction of T cell hyporeactivity and little or no production of proinflammatory cytokines. These observations suggest that the ability of the astrocyte, through varying their surface MHC class II molecules, can control the effect of antigen-induced T cell responses. In their normal state of low MHC II expression astrocytes are expected to induce no or partial, rather than full, activation of autoreactive T cells that enter the CNS, resulting in T cell hyporeactivity. Since astrocytes usually diminish the production of proinflammatory cytokines by T cells that enter the CNS, the status and control of MHC class II expression on astrocytes should be important determinants of the suppression or enhancement of in situ immune responses in the CNS.     

T cell receptor Vβ gene usage in the recognition of myelin basic protein by cerebrospinal fluid- and blood-derived T cells from patients with multiple sclerosis

Because of its proximity to the central nervous system, the cerebrospinal fluid (CSF) represents an important source of T cells that potentially could mediate putative autoimmune diseases such as multiple sclerosis (MS). To overcome the low CSF cellularity, we evaluated culture conditions that could expand CSF T cells, with a focus on the expression of T-cell receptor Vβ genes utilized by T cells specific for the potentially encephalitogenic autoantigen myelin basic protein (BP). Expansion of “activated” CSF cells with IL-2/IL-4 plus accessory cells optimally retained BP-responsive T cells that over-expressed Vβ1, Vβ2, Vβ;5, or Vβ;18, compared to expansion using supernatants from PHA-stimulated blood cells, or anti-CD3 antibody that led to different V gene bias and rare reactivity to BP. Sequential evaluation of paired CSF and blood samples from a relapsing remitting MS patient indicated that BP-reactive T cells were present in CSF during the period of clinical activity, and the pattern of BP recognition in CSF was partially reflected in blood, even after CSF reactivity had dissipated during remission. Over-expressed Vβ genes were not always constant, however, since in three sequential evaluations of a chronic progressive MS patient, Vβ genes over-expressed in the first BP-reactive CSF switched to a different Vβ gene bias that was present in the second and third CSF samples. Blood samples reflected each pattern of CSF Vβ gene bias, but retained the initial bias for at least 4 months after its disappearance from CSF. These data indicate that selective expansion of IL-2/Il-4-responsive CSF cells favors growth of the BP-reactive subpopulation, and, in a limited number of patients studied, reflected clinical disease activity. In comparison, blood T cells provided a partial but longer lasting reflection of the CSF BP reactivity and Vβ gene bias. © 1994 Wiley-Liss, Inc.     

MHC-restriction,cytokine profile,and immunoregulatory effects of human T cells specific for TCR Vβ CDR2 peptides: Comparison with myelin basic protein-specific T cells

HLA-DR2+ patients with multiple sclerosis (MS) that respond to vaccination with TCR Vβ5.2-38-58 peptides have increased frequencies of TCR peptide-specific T cells, reduced frequencies of myelin basic protein (MBP)-specific T cells, and a better clinical course than non-responders. To evaluate possible network regulation of MBP responses by TCR peptide-specific T cells, we compared properties of both cell types. Both MBP- and TCR peptide-specific T cell clones were CD4⁺ and predominantly HLA-DR restricted. HLA-DR2, which is in linkage disequilibrium in MS patients, preferentially restricted TCR peptide-specific clones as well as MBP-specific responses in HLA-DR2 and DR2,3+ donors. Within the DR2 haplotype, however, both DRβ1*1501 and DRβ5*0101 alleles could restrict T cell responses to Vβ CDR2 peptides, whereas responses to MBP were restricted only by DRβ5*0101. TCR peptide-specific clones expressed message for Th2 cytokines, including IL-4, IL-5, IL-6, IL-10, and TGF-β, whereas MBP-specific T cell clones expressed the Th1 cytokines IFN-γ and IL-2. Consistent with the Th2-like cytokine profile, TCR peptide-specific T cell clones expressed higher levels of CD30 than MBP-specific T cells. Culture supernatants from TCR peptide-specific T cell clones, but not from MBP- or Herpes simplex virus-specific T cells, inhibited both proliferation responses and cytokine message production of MBP-specific T cells. These results demonstrate distinct properties of MBP and TCR peptide-specific T cells, and indicate that both target and bystander Th1 cells can be inhibited by Th2 cytokines secreted by activated TCR peptide-specific T cells. These data support the rationale for TCR peptide vaccination to regulate pathogenic responses mediated by oligoclonal T cells in human autoimmune diseases. © 1996 Wiley-Liss, Inc.     

Reprint of “The developing oligodendrocyte: key cellular target in brain injury in the premature infant”

Brain injury in the premature infant, a problem of enormous importance, is associated with a high risk of neurodevelopmental disability. The major type of injury involves cerebral white matter and the principal cellular target is the developing oligodendrocyte. The specific phase of the oligodendroglial lineage affected has been defined from study of both human brain and experimental models. This premyelinating cell (pre-OL) is vulnerable because of a series of maturation-dependent events. The pathogenesis of pre-OL injury relates to operation of two upstream mechanisms, hypoxia-ischemia and systemic infection/inflammation, both of which are common occurrences in premature infants. The focus of this review and of our research over the past 15–20 years has been the cellular and molecular bases for the maturation-dependent vulnerability of the pre-OL to the action of the two upstream mechanisms. Three downstream mechanisms have been identified, i.e., microglial activation, excitotoxicity and free radical attack. The work in both experimental models and human brain has identified a remarkable confluence of maturation-dependent factors that render the pre-OL so exquisitely vulnerable to these downstream mechanisms. Most importantly, elucidation of these factors has led to delineation of a series of potential therapeutic interventions, which in experimental models show marked protective properties. The critical next step, i.e., clinical trials in the living infant, is now on the horizon.