The mRNA of transferrin is expressed in Schwann cells during their maturation and after nerve injury

Transferrin, the iron carrier protein, has been shown to be involved in oligodendroglial cell differentiation in the central nervous system but little is known about its role in the peripheral nervous system. In the present work, we have studied the presence of transferrin and of its mRNA in rat sciatic nerves and in Schwann cells isolated at embryonic and adult ages as well as during the regeneration process that follows nerve crush. We have also studied the correlation between the expression of the mRNAs of transferrin and the expression of mature myelin markers in the PNS. We show that transferrin is present in whole sciatic nerves at late stages of embryonic life as well as at postnatal day 4 and in adult rats. We demonstrate for the first time, that in normal conditions, the transferrin mRNA is expressed in Schwann cells isolated from sciatic nerves between embryonic days 14 and 18, being absent at later stages of development and in adult animals. In adult rats, 3 days after sciatic nerve crushing, the mRNA of transferrin is expressed in the injured nerve, but 7 days after injury its expression disappears. Transferrin protein in the sciatic nerve closely follows the expression of its mRNA indicating that under these circumstances, it appears to be locally synthesized. Transferrin in the PNS could have a dual role. During late embryonic ages it could be locally synthesized by differentiating Schwann cells, acting as a pro-differentiating factor. A similar situation would occur during the regeneration that follows Wallerian degeneration. In the adult animals on the other hand, Schwann cells could pick up transferrin from the circulation or/and from the axons, sub serving possible trophic actions closely related to myelin maintenance.     

Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions

Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.     

The complex etiology of multiple sclerosis

Multiple sclerosis is a demyelinating disease which is presumed to be a consequence of infiltrating lymphocytes autoreactive to myelin proteins. This is substantiated by several lines of clinical evidence and supported by correlative studies in preclinical models. The development of new therapeutics for MS has been guided by this perspective; however, the pathogenesis of MS has proven to be quite complex as observations exist which question the role of autoreactive lymphocytes in the etiology of MS. In addition the current immunomodulatory therapeutics do not prevent most patients from progressing into more serious forms of the disease. The development of truly transformational therapeutics for MS will likely require a broad assault that expands beyond the concept of MS being an autoimmune disease.     

Identification and functional characterization of mouse TPO1 as a myelin membrane protein

TPO1 is a member of the AIGP family, a unique group of proteins that contains 11 putative transmembrane domains. Expression of the rat TPO1 gene is upregulated in cultured oligodendrocytes (OLs) during development from pro-oligodendroblasts to postmitotic OLs. However, the distribution of native TPO1 protein in cultured OLs and in the brain has not been elucidated. We investigated the distribution and cellular function of TPO1 in myelinating cells of the nervous system. In mice, TPO1 gene expression was detected in the central (CNS) and peripheral (PNS) nervous systems and was markedly upregulated at postnatal days 10-20, an early phase of myelination in the mouse brain. To investigate TPO1 localization, we generated affinity-purified antibodies to synthetic peptides derived from mouse TPO1. Immunohistochemical analysis showed that TPO1 was expressed in OLs and Schwann cells but not in neurons and astrocytes. Schwann cells from trembler mice, which lack PNS myelin, had significantly decreased TPO1 expression and an altered localization pattern, suggesting that TPO1 is a functional myelin membrane protein. In OL lineage cell cultures, TPO1 was detected in A2B5+ bipolar early progenitors, A2B5+ multipolar Pro-OLs, GalC+ immature OLs and MBP+ mature OLs. The subcellular localization of TPO1 in OL lineage cells was mapped to the GM130+ Golgi in cell bodies and Fyn+ cell processes and myelin-like sheets. Furthermore, TPO1 selectively colocalized with non-phosphorylated Fyn and promoted Fyn autophosphorylation in COS7 cells, suggesting that TPO1 may play a role in myelin formation via Fyn kinase activation in the PNS and CNS.     

A double mutation of MBP(83-99) peptide induces IL-4 responses and antagonizes IFN-gamma responses

A number of treatment options are available to multiple sclerosis patients, however this needs to be improved. Herein, we designed and synthesized a number of peptides by mutating principal TCR contact residues based on MBP(83-99) peptide epitope. Immunization of SJL/J mice with MBP(83-99) and mutant [A(91)]MBP(83-99), [E(91)]MBP(83-99), [F(91)]MBP(83-99), [Y(91)]MBP(83-99), and [R(91), A(96)]MBP(83-99) peptides, induced IFN-gamma, and only [R(91), A(96)]MBP(83-99) mutant peptide was able to induce IL-4 secretion by T cells. T cells against the native MBP(83-99) peptide cross-reacted with all peptides except [Y(91)]MBP(83-99) and [R(91),A(96)]MBP(83-99). The double mutant [R(91), A(96)]MBP(83-99) was able to antagonize IFN-gamma production in vitro by T cells against the native MBP(83-99) peptide. Antibodies generated to [R(91), A(96)]MBP(83-99) did not cross-react with whole MBP protein. Molecular modeling between peptide analogs and H2 I-A(s) demonstrated novel interactions. The [R(91), A(96)]MBP(83-99) double mutant peptide analog is the most promising for further therapeutic studies.     

急性CO中毒患者血清S100B、NSE、MBP的动态改变及其意义

目的探讨急性一氧化碳中毒患者血清S100B、神经元特异性烯醇化酶（NSE）、髓鞘碱性蛋白（MBP）的动态改变及其与患者的病情、预后的关系。方法检测30名健康体检者和185例一氧化碳中毒患者入院时SIOOB、NSE、MBP水平,并对其中60例中重型住院病人第2、3天这些指标进行检测,分析其与患者预后的关系。结果入院时,中重型一氧化碳中毒患者血清S100B、NSE水平明显高于对照组和轻型一氧化碳中毒患者;中重型患者血清MBP水平高于轻型,轻型高于对照组。对中重型一氧化碳中毒患者,死亡患者第2天SIOOB、NSE水平明显高于人院时水平,第3天与第2天相比无统计学差异;而MBP水平在3天中无明显改变（P〉0．05）;存活患者中,SIOOB、NSE、MBP3天无明显改变。结论一氧化碳中毒患者入院时MBP水平与患者病情密切相关,而动态检测NSE、SIOOB水平能有效地判断对患者预后。     

Insulin-like growth factor type 1 receptor signaling in the cells of oligodendrocyte lineage is required for normal in vivo oligodendrocyte development and myelination

Insulin-like growth factor-I (IGF-I) has been shown to be a potent agent in promoting the growth and differentiation of oligodendrocyte precursors, and in stimulating myelination during development and following injury. To definitively determine whether IGF-I acts directly on the cells of oligodendrocyte lineage, we generated lines of mice in which the type 1 IGF receptor gene (igf1r) was conditionally ablated either in Olig1 or proteolipid protein expressing cells (termed IGF1R(pre-oligo-ko) and IGF1R(oligo-ko) mice, respectively). Compared with wild type mice, IGF1R(pre-oligo-ko) mice had a decreased volume (by 35-55%) and cell number (by 54-70%) in the corpus callosum (CC) and anterior commissure at 2 and 6 weeks of age, respectively. IGF1R(oligo-ko) mice by 25 weeks of age also showed reductions, albeit less marked, in CC volume and cell number. Unlike astrocytes, the percentage of NG2(+) oligodendrocyte precursors was decreased by approximately 13% in 2-week-old IGF1R(pre-oligo-ko) mice, while the percentage of CC1(+) mature oligodendrocytes was decreased by approximately 24% in 6-week-old IGF1R(pre-oligo-ko) mice and approximately 25% in 25-week-old IGF1R(oligo-ko) mice. The reduction in these cells is apparently a result of decreased proliferation and increased apoptosis. These results indicate that IGF-I directly affects oligodendrocytes and myelination in vivo via IGF1R, and that IGF1R signaling in the cells of oligodendrocyte lineage is required for normal oligodendrocyte development and myelination. These data also provide a fundamental basis for developing strategies with the potential to target IGF-IGF1R signaling pathways in oligodendrocyte lineage cells for the treatment of demyelinating disorders.     

The in vivo and in vitro induction of anterior chamber associated immune deviation to myelin antigens in C57BL/6 mice

Introduction of antigens into the anterior chamber (AC) of the eye generates a specific systemic form of tolerance that is termed AC-associated immune deviation (ACAID). Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human CNS demyelinating diseases, including multiple sclerosis (MS) and acute disseminated encephalomyelitis. We investigated whether the encephalitogenic antigens myelin oligodendrocyte glycoprotein (MOG_35–55) or myelin basic protein (MBP) induce ACAID in the EAE-prone C57BL/6 mice. We hypothesized that injection of MOG_35–55/MBP induces antigen-specific tolerance whether via the AC route, the adoptive transfer of in vitro-generated MOG_35–55-specific/MBP-specific ACAID antigen presenting cells (APCs), or the adoptive transfer of MOG_35–55-specific/MBP-specific ACAID T regulatory cells (Tregs). ACAID is characterized by the specific impairment of delayed-type hypersensitivity (DTH) responses. Thus, DTH assays were used to test for ACAID following the AC injection of MOG_35–55/MBP, or the intravenous injection of MOG_35–55-specific/MBP-specific ACAID APCs. The functional local adoptive transfer (LAT) assays were used to examine the putative regulatory functions of in vitro generated MOG_35–55-specific/MBP-specific Tregs. This report is the first to demonstrate the in vivo and in vitro induction of MOG_35–55-specific/MBP-specific ACAID-mediated tolerance in C57BL/6 mice. These findings highlight the need for novel immunotherapeutic strategies for MS and optic neuritis.     

Myelin basic protein induces inflammatory mediators from primary human endothelial cells and blood–brain barrier disruption: implications for the pathogenesis of multiple sclerosis

T. G. D'Aversa, E. A. Eugenin, L. Lopez and J. W. Berman (2013) Neuropathology and Applied Neurobiology 39, 270–283 Myelin basic protein induces inflammatory mediators from primary human endothelial cells and blood–brain barrier disruption: implications for the pathogenesis of multiple sclerosis Aim: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by demyelination of white matter, loss of myelin forming oligodendrocytes, changes in the blood–brain barrier (BBB) and leucocyte infiltration. Myelin basic protein (MBP) is a component of the myelin sheath. Degradation of myelin is believed to be an important step that leads to MS pathology. Transmigration of leucocytes across the vasculature, and a compromised BBB participate in the neuroinflammation of MS. We examined the expression and regulation of the chemokine (C–C motif) ligand 2 (CCL2) and the cytokine interleukin‐6 (IL‐6) in human endothelial cells (EC), a component of the BBB, after treatment with MBP. Methods: EC were treated with full‐length MBP. CCL2 and IL‐6 protein were determined by ELISA. Western blot analysis was used to determine signalling pathways. A BBB model was treated with MBP and permeability was assayed using albumin conjugated to Evan's blue dye. The levels of the tight junction proteins occludin and claudin‐1, and matrix metalloprotease (MMP)‐2 were assayed by Western blot. Results: MBP significantly induced CCL2 and IL‐6 protein from EC. This induction was partially mediated by the p38 MAPK pathway as there was phosphorylation after MBP treatment. MBP treatment of a BBB model caused an increase in permeability that correlated with a decrease in occludin and claudin‐1, and an induction of MMP2. Conclusion: These data demonstrate that MBP induces chemotactic and inflammatory mediators. MBP also alters BBB permeability and tight junction expression, indicating additional factors that may contribute to the BBB breakdown characteristic of MS.