No abstract

Activation of the classical complement system is known to play a central role in autoimmune demyelination. We have analyzed the role of complement component C5 in experimental autoimmune encephalomyelitis (EAE) using C5-deficient (C5-d) and C5-sufficient (C5-s) mice. Both groups of mice displayed early onset EAE, a short recovery phase, and similar stable chronic courses. However, in contrast to the clinical similarities, marked differences were apparent by histopathology. During acute EAE in C5-d, a delay in inflammatory cell infiltration and tissue damage was observed along with restricted lesion areas, which in C5-s mice were more extensive and diffuse. More striking were the differences in chronic lesions. In C5-d mice, inflammatory demyelination and Wallerian degeneration were followed by axonal depletion and severe gliosis, while in C5-s, the same initial signs were followed by axonal sparing and extensive remyelination. In C5-d, immunohistochemistry and Western blotting showed an increase in glial fibrillary acidic protein and a decrease in neurofilament protein, proteolipid protein, and several pro-inflammatory markers. These results in the EAE model indicate that absence of C5 resulted in fiber loss and extensive scarring, whereas presence of C5-favored axonal survival and more efficient remyelination.

Glial cell Ca2+ signals play a key role in glial-neuronal and glial-glial network communication. Numerous studies have thus far utilized cell-permeant and injected Ca2+ indicator dyes to investigate glial Ca2+ signals in vitro and in situ. Genetically encoded fluorescent Ca2+ indicators have emerged as novel probes for investigating cellular Ca2+ signals. We have expressed one such indicator protein, the YC 3.60 cameleon, under the control of the S100β promoter and directed its expression predominantly in astrocytes and Schwann cells. Expression of YC 3.60 extended into the entire cellular cytoplasmic compartment and the fine terminal processes of protoplasmic astrocytes and Schwann cell Cajal bands. In the brain, all the cells known to express S100β in the adult or during development, expressed YC 3.60. While expression was most extensive in astrocytes, other glial cell types that express S100β, such as NG2 and CNP-positive oligodendrocyte progenitor cells (OP cells), microglia, and some of the large motor neurons in the brain stem, also contained YC 3.60 fluorescence. Using a variety of known in situ and in vivo assays, we found that stimuli known to elicit Ca2+ signals in astrocytes caused substantial and rapid Ca2+ signals in the YC 3.60-expressing astrocytes. In addition, forepaw stimulation while imaging astrocytes through a cranial window in the somatosensory cortex in live mice, revealed robust evoked and spontaneous Ca2+ signals. These results, for the first time, show that genetically encoded reporter is capable of recording activity-dependent Ca2+ signals in the astrocyte processes, and networks.

Complement activation is involved in the initiation of Ab-mediated inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE). At a sublytic dose, the C5b-9 membrane attack complex protects oligodendrocytes (OLG) from apoptosis. Using C5-deficient (C5-d) mice, we previously showed a dual role for C5: enhancement of inflammatory demyelination in acute EAE, and promotion of remyelination during recovery. In this study, we investigated the role of C5 in apoptosis in myelin-induced EAE. In acute EAE, C5-d and C5-sufficient (C5-s) mice had similar numbers of total apoptotic cells, whereas C5-s had significantly fewer than C5-d during recovery. In addition, although both groups of mice displayed TUNEL+ OLG, there were significantly fewer in C5-s than in C5-d during both acute EAE and recovery. Gene array and immunostaining of apoptosis-related genes showed that Fas ligand expression was higher in C5-s. In C5-s mice, Fas+ cells were also higher than in C5-d mice in acute EAE; however, these cells were significantly reduced during recovery. Together, these findings are consistent with the role of C5, possibly by forming the membrane attack complex, in limiting OLG apoptosis in EAE, thus promoting remyelination during recovery.

Complement activation plays a central role in autoimmune demyelination. To explore the possible effects of C5 on post-inflammatory tissue repair, we investigated the transcriptional profile induced by C5 in chronic experimental allergic encephalomyelitis (EAE) using oligonucleotide arrays. We used C5-deficient (C5-d) and C5-sufficient (C5-s) mice to compare the gene expression profile and we found that 390 genes were differentially regulated in C5-s mice as compared to C5-d mice during chronic EAE. Among them, a group of genes belonging to the family of insulin-like growth factor binding proteins (IGFBP) and transforming growth factor (TGF)-β3 were found most significantly differentially regulated by C5. The dysregulation of these genes suggests that these proteins might be responsible for the gliosis and lack of remyelination seen in C5-d mice with chronic EAE.