Analysis of humoral immune responses to LM1 ganglioside in guinea pigs.

Guillain-Barré syndrome (GBS) is an autoimmune-mediated disease triggered by a preceding infection. A substantial body of evidence implicates antibodies to various gangliosides in subtypes of GBS. A significant proportion of patients with acute demyelinating subset of GBS have IgG antibodies against peripheral nervous system myelin specific neolactogangliosides such as LM1 and Hex-LM1. Although anti-neolactoganglioside antibodies in GBS were described more than two decades ago, their pathogenic role in neuropathy remains unknown due to the lack of suitable experimental models. In this study, we immunized ten guinea pigs with purified LM1 ganglioside mixed with keyhole limpet hemocyanin (KLH) and emulsified in complete Freund's adjuvant (CFA). Control guinea pigs were injected with KLH emulsified in CFA only. The animals were bled every four week intervals. The animals were boosted 3 times every four weeks. Experiments were terminated four months after initial immunization. Nine of 10 guinea pigs immunized with LM1 exhibited antibody responses to LM1. Anti-LM1 IgG titers in nine guinea pigs ranged from 1:400 to 1:12,800 at 16-weeks after initial immunization. Anti-LM1 antibodies were predominantly of IgG2 subclass. One guinea pig with the highest levels of IgG antibodies exhibited mild signs of neuropathy. There was no evidence of demyelination or inflammation in the sciatic nerves of LM1-immunized guinea pigs. Anti-LM1 antibodies bound to rat sciatic nerve myelin and to isolated rat Schwann cells. In summary, our findings suggest that relatively high levels of anti-LM1 IgG antibodies can be induced in guinea pigs and that LM1 is localized in peripheral nerve myelin and in Schwann cells. Further studies are needed to determine the pathogenic potential of anti-neolactoganglioside antibodies in neuropathy.

Developmental regulation of Neuregulin1 isoforms and erbB receptor expression in intact rat dorsal root ganglia.

Neuregulins (NRGs) are a family of growth factors which bind to the erbB family of tyrosine kinase receptors. The exact nature and interaction of specific NRG isoforms and erbB receptors that occur during the development of the nervous system have not been reported. In order to better understand the role that different NRG isoforms and erbB receptors play in the differentiation, proliferation, and survival of neurons and glial cells, we isolated protein and mRNA from dorsal root ganglia of rat pups between embryonic day (E) 13 and postnatal day (P) 15. The relative expression levels of the NRGs and erbB receptors for the different time points were compared using both Western and RT-PCR analyses. NRG1-type1alpha protein levels were highest at E-13 and then decreased by approximately 40% and remained constant through P-15. In contrast, mRNA levels for NRG1-type1alpha remained constant from E-15 to P-15. The protein levels for NRG1-type 1beta were similar to NRG1-type1alpha at E-13 with an approximate 40% increase in the levels at E-15 and E-17 followed by a decrease to E-13 levels for the remainder of the developmental time periods. The mRNA levels for NRG1-type1beta remained constant from E-15 to P-15. The protein and mRNA expression patterns for each erbB receptor were distinctive. The protein levels for erbB-2 were highest at E-19 while erbB-3 levels were highest at E-17 and E-18. ErbB-4 protein levels were highest at E-13 and decreased through P-15. The developmental pattern for erbB-2 and erbB-4 mRNA levels had no relation to that of the corresponding protein levels while the mRNA levels for erbB-3 were highest at E-17 and E-18 similar to the pattern observed for the erbB-3 protein levels. We concluded that both NRG and erbB expression in dorsal root ganglia are mostly translationally controlled and that NRG1 isoforms and their erbB receptors are not coordinately regulated.

Localization of neuregulin isoforms and erbB receptors in myelinating glial cells.

Neuregulins (NRGs) are growth factors present in neurons and glial cells of the central and peripheral nervous systems and play a role in the survival, proliferation, and differentiation of these cells. We now report the localization of the two major isoforms of NRG (alpha and beta) and their receptors (erbB) in cultured Schwann cells and oligodendrocytes isolated from neonatal rat pups. Immunocytochemistry and Western blots for NRG and erbB receptors in defined subcellular fractions were utilized to assess cellular localization. Less differentiated oligodendrocytes contain both NRG isoforms in the cell bodies but not the processes, while only NRG-1beta was found in the nucleus. In contrast, more differentiated oligodendrocytes contained neither isoform in the nucleus while both isoforms were colocalized in the cytoplasm and cell processes. In Schwann cells, both NRG-1beta and NRG-1alpha were colocalized in the cytoplasm and processes. The Schwann cell nucleus had weak immunoreactivity for both NRG-1 isoforms, although NRG-1beta was predominant. ErbB2 and erbB3 receptors, which transduce the NRG-1 signal in Schwann cells, were found throughout the cytoplasm and in the processes and were also localized in the cell nucleus. The nuclear localization of NRG-1 isoforms and/or erbB receptors in both cell types was confirmed by Western blotting of nuclear and cytoplasmic extracts. Stimulation of Schwann cells with mitotic agents increased NRG-1beta expression in the nucleus and dramatically suppressed NRG-1alpha expression throughout the cell. The functional implications of this differential localization in myelinating cells are discussed.

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins.

BACKGROUND: Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. RESULTS: Using confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. CONCLUSION: We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

Phosphorylated neurofilaments and SNAP-25 in cultured SH-SY5Y neuroblastoma cells.

Components of the extracellular matrix (ECM) of mammals have profound effects on the behavior and differentiation of many different cell types. Here, we report the results of biochemical and immunocytochemical investigations of the expression of SNAP-25 and phosphorylated neurofilament proteins (NFs) by cells grown on coverslips, cells cultured in EHS-ECM gels, and cells in situ in rat brain. SNAP-25 and phosphorylated NFs were detected by immunofluorescence in all these environments but were not detectable by Western analysis in extracts of cells grown on coverslips. The results support the interpretation that EHS-ECM induces differentiation of SH-SY5Y cells in culture and suggest this system as a model system for study of nerve tissue formation and repair.