Activation of early components of complement targets myelin and oligodendrocytes in the aged rhesus monkey brain
Introduction
The white matter of the brain is the site of a number of the major changes that occur in the central nervous system with age. Imaging studies have shown age-related decreases in white matter volume [2], [19] while structural and biochemical studies have demonstrated an age dependent disruption of the myelin membrane with an overall loss of myelin [10], [27], [45], [57]. Myelin loss has been found to be associated with an increase in degraded myelin proteins as revealed by the fractionation of myelin membranes isolated from brain white matter [57]. Calpain-1, a calcium dependent cysteine protease, whose known substrates include myelin proteins [3], [57], was detected in an active form in degraded myelin and white matter in young and old animals [57]. Increases in the active enzyme accompanied age-related increases in degraded myelin, suggesting the participation of calpain in the observed catabolic breakdown and loss of myelin. These changes in myelin and calpain could contribute to the reported decline in cognitive function in aged subjects by impairing axonal conduction [20], [25].
In vitro studies suggest that the complement system may play a role in the activation of calpain and the degradation of myelin. These studies have shown that the terminal complement complex, C5b-9, generated by complement activation induced by myelin, is capable of activating calcium dependent cysteine proteases in the myelin membrane with resulting proteolysis of myelin proteins [62], [63].
In addition to C5b-9, the activation of early components of the complement system may have an affect on myelin and the myelin producing oligodendrocytes. These products include the anaphylatoxins, C3a, C4a and C5a and the opsonins, C3b, C3bi and C4b. They have been reported to induce the cellular release of mediators which could alter myelin metabolism [14], [15], [50]. These mediators include cytokines, reactive oxygen species and eicosanoids [7], [16], [24], [28], [34], [37], [39], [46], [54]. The opsonins also have been shown to enhance myelin phagocytosis and to be essential for the phagocytic removal of damaged myelin from injured myelinated nerves [6], [9], [47], [61], [65].
The activation of early and terminal components of the complement pathways is controlled by regulatory proteins, which can promote or inhibit the complement cascade. The action of these proteins have been described in a comprehensive review [32]. The activation of early complement components are derived from the degradation of C3 and C4 with C3d and C4d being the final catabolic products of these respective parent proteins. The terminal complement complex, C5b-9, results from the assembly of its protein components on the target membranes. This process is controlled by both fluid phase and membrane protein regulators.
The three major pathways of complement activation are the classical, alternate and lectin pathways. The classical cascade is initiated by the activation of the C1q subcomponent of C1 while the alternate system is induced by cleavage of C3. The lectin pathway is initiated by the cleavage of C2 and C4 by serine proteases associated with mannose binding lectin and ficolins [22], [33]. Both lectin and classical complement activation lead to the formation of the classical form of C3 and C5 convertases. In addition to immune complexes and injured or apoptotic cells, myelin has been shown to be an important activator of the classical system [8], [62].
The aim of the present study was to determine whether complement activation might play a role in age-related changes in brain myelin. The findings demonstrate a widespread distribution of complement containing oligodendrocytes (CAOs) containing C3d and C4d activation products bound to oligodendrocytes and myelinated nerve fibers, occurring in brain white and gray matter of both young and aged rhesus monkeys. Activation of terminal complement products was not demonstrable in these CAOs, which were present in larger numbers in aged monkeys than in young animals. Previous studies by other workers have described similar findings of CAOs but only within diseased brains; including Parkinson's disease, amyotrophic lateral sclerosis and Alzheimer's disease [66]. Recent studies have reported C4d containing plaques in multiple sclerosis and have implicated these CAOs in demyelination [51]. There are no published reports on the comparison of CAOs in normal young and aged humans. The monkey CAOs appear morphologically indistinguishable from the human CAOs. The findings in the present study suggest a role of myelin in initiating the activation of the complement system and the generation of C3 and C4 activation products. They, furthermore, suggest that activation of early components of complement is a normal biological function of the brain which involves the metabolism of myelin and oligodendrocytes and which up-regulates with age.
Section snippets
Non-human primates
Sixteen rhesus monkeys (Macaca mulatta) acquired from the colony of the Yerkes Regional Primate Research Center were behaviorally tested and their brains processed to evaluate age-related changes. All monkeys spent several years free ranging in social groups maintained at the Yerkes Regional Primate Research Center before being housed individually for 1–3 years during behavioral testing at either the Yerkes Regional Primate Research Center or the Laboratory Animal Science Center at Boston
C3d and C4d localize to oligodendrocytes and myelinated fibers
The immunohistochemical distribution of complement proteins in white and gray matter was investigated to assess if complement activation occurred and whether it changed with age. Cryostat whole hemisphere brain sections of both aged and young monkeys were immunohistochemically stained using monoclonal antibodies to the neo-epitopes of C3d (C3d-neo) and C4d (C4d-neo) (Quidel). The distribution of C3d and C4d appeared to be the same with both fragments localizing in the same areas of the white
Discussion
The present study demonstrated the wide spread distribution of complement containing CAOs in the brains of both young and aged monkeys, with the number of CAOs being significantly increased with age. Immunohistochemical staining of the CAOs revealed that the C3d and C4d complement fragments closely associated to oligodendrocytes and myelinated fibers and distinctly separated from microglia of the plaque, although localization to astrocytes was not excluded. These findings were supported by
Acknowledgements
The authors would like to thank M.B. Moss and D.L. Rosene for assistance in behavioral assessment of animals and tissue processing, J.D. Hinman for the myelin purification, and H.J. Cabral for invaluable assistance with statistical analysis. This work was supported by NIH-NIA AG00001.
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