Non-cell autonomous impairment of oligodendrocyte differentiation precedes CNS degeneration in the Zitter rat: Implications of macrophage/microglial activation in the pathogenesis
© Sakakibara et al; licensee BioMed Central Ltd. 2008
Received: 20 November 2007
Accepted: 05 April 2008
Published: 05 April 2008
The zitter (zi/zi) rat, a loss-of-function mutant of the glycosylated transmembrane protein attractin (atrn), exhibits widespread age-dependent spongiform degeneration, hypomyelination, and abnormal metabolism of reactive oxygen species (ROS) in the brain. To date, the mechanisms underlying these phenotypes have remained unclear.
Here, we show differentiation defects in zi/zi oligodendrocytes, accompanied by aberrant extension of cell-processes and hypomyelination. Axonal bundles were relatively preserved during postnatal development. With increasing in age, the injured oligodendrocytes in zi/zi rats become pathological, as evidenced by the accumulation of iron in their cell bodies. Immunohistochemical analysis revealed that atrn expression was absent from an oligodendrocyte lineage, including A2B5-positive progenitors and CNPase-positive differentiated cells. The number and distribution of Olig2-positive oligodendrocyte progenitors was unchanged in the zi/zi brain. Furthermore, an in vitro differentiation assay of cultured oligodendrocyte progenitors prepared from zi/zi brains revealed their normal competence for proliferation and differentiation into mature oligodendrocytes. Interestingly, we demonstrated the accelerated recruitment of ED1-positive macrophages/microglia to the developing zi/zi brain parenchyma prior to the onset of hypomyelination. Semiquantitative RT-PCR analysis revealed a significant up-regulation of CD26 and IL1-β in the zi/zi brain during this early postnatal stage.
We demonstrated that the onset of the impairment of oligodendrocyte differentiation occurs in a non-cell autonomous manner in zi/zi rats. Hypomyelination of oligodendrocytes was not due to a failure of the intrinsic program of oligodendrocytes, but rather, was caused by extrinsic factors that interrupt oligodendrocyte development. It is likely that macrophage/microglial activation in the zi/zi CNS leads to disturbances in oligodendrocyte differentiation via deleterious extrinsic factors, such as the cytokine IL1-β or ROS. Atrn might be involved in the activation of brain macrophages/microglia by suppressing excessive migration of monocytes into the CNS, or by accelerating the transformation of brain monocytes into resting microglia. Understanding the pathogenesis of the zi/zi rat may provide novel insights into the developmental interaction betweens macrophages/microglia and cells of an oligodendrocyte lineage.
Emerging evidence suggests that glial cells play an important role in the onset of several neurodegenerative disorders, including multiple system atrophy (MSA), Alzheimer dementia (AD), Parkinson's dementia (PD) and multiple sclerosis (MS). These diseases are characterized by iron deposition in oligodendrocytes, reactive oxygen species (ROS)-mediated oxidative stress, and neuroinflammation accompanied by macrophage/microglial activation in the CNS [1–3].
The zitter (zi/zi) rat is an autosomal recessive spontaneous mutant, the phenotype of which includes curled body hair, bent whiskers, fine tremor that develops at 3 weeks of age, and flaccid paresis of the hind limb at around 6 months of age . It has been reported that the levels of ROS production and oxidative stress are abnormally elevated within the zi/zi brain [5, 6]. Our previous studies suggested that there was a prominent degeneration of dopaminergic neurons in the zi/zi rat substantia nigra owing to increased oxidative stress with age [7–9], similar to the degeneration observed in human PD. Pathologically, the zi/zi rat brain exhibits severe spongiform degeneration and hypomyelination, which is frequently associated with abnormal membranous structures in oligodendrocytes . A previous study demonstrated that the commencement of myelination was not delayed in zi/zi rats compared with control rats, and that the fundamental structures of myelin sheaths are normal in the zi/zi rat during the postnatal period . In addition, the biochemical components of myelin, such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin-associated glycoprotein (MAG), are normally expressed . Ultrastructural studies have indicated that the hypomyelination of zi/zi rats is characterized by a decrease in the density of myelinated fibers as well as aberrant split myelin lamellae in the ventral column of the cervical spinal cord and in the optic nerve [10, 11]. Gross pathological changes in these regions, including vacuole formation and degeneration of myelin sheaths into condensed spheroids, appeared from 3 weeks of age in zi/zi rats [10, 12]. However, curiously, the incidence of these membranous abnormalities was shown to be quite low; they were seen in only about 5% of the oligodendrocytes in random electron microscopic observations . Consistently, our previous electron microscopic study failed to detect any degenerative changes in the oligodendrocytes in the substantia nigra of zi/zi rats until postnatal 4 weeks .
A positional cloning study revealed that the zi/zi mutant phenotype is due to a severe decrease in the level of attractin (atrn) mRNA expression as a consequence of an 8-bp deletion at the splice donor site of the gene . Attractin/mahogany (atrn) mRNA encodes a transmembrane protein containing a C-type lectin domain and four EGF-like motifs, suggesting a function in intercellular interactions through its downstream signaling . Atrn also possesses a CUB (C1r/C1s/Urinary epidermal growth factor, Bone morphogenetic protein) domain, which is found almost exclusively in extracellular and plasma membrane-associated proteins. Many CUB domain-containing proteins are proteases, although the roles of the CUB domain have been largely unexplored . In the CNS, although previous in situ hybridization studies have indicated a ubiquitous distribution of the atrn mRNA in the adult rodent brain , it remains obscure what cell-types express atrn protein within the brain, and what its physiological function is in the nervous system. Several pathological studies have suggested the possibility that atrn is involved in axon-oligodendrocyte interactions or in both the assembly of myelin sheaths and the maintenance of neurons, but the developmental profile of brain oligodendrocytes of the zi/zi rat has not been characterized extensively to date. Moreover, these previous studies have raised the question of whether zi/zi hypomyelination is caused primarily by a developmental failure of oligodendrocytes themselves, or alternatively, by secondary consequences of an impairment of neuronal differentiation; they also raise the question of why the incidence of abnormalities in oligodendrocyes is so low (see above).
It has been suggested that atrn also has multiple functions outside the nervous system, including roles in monocyte responses, hair pigmentation, energy control and obesity [17–19]. In the immune system, membrane-type atrn was recently shown to possess dipeptidyl peptidase IV (DP IV)/CD26-like ectoenzyme activity, and to be expressed on the surfaces of human peripheral blood monocytes , while the in vitro study provided compelling evidence that secreted form of atrn in human plasma has no DP IV activity . CD26 is expressed on the surfaces of hemopoietic stem/progenitor cells, and plays a crucial role in their homing/mobilization ability to/from the bone marrow, through the proteolytic cleavage of a local pool of chemokine SDF-1α . Although atrn exhibits no structural similarity to CD26, it is now thought that atrn is a member of a unique DP IV-family designated the DASH (DP IV activity and/or structure homologues) family based on the comparable substrate specificity of atrn to DP IV . Thus, it is intriguing to find a linkage between the oligodendrocyte degeneration in the zi/zi CNS and the tightly regulated expression of atrn in the immune system.
In the present study, we describe the developmental profile of oligodendrocytes and the brain pathogenesis in the zi/zi rat in detail. Our observations suggest the possibility that the loss of function in atrn might cause the abnormal infiltration of macrophages/microglia into the brain, mediating the oligodendrocyte morbidity in zi/zi rats.
Impaired development of oligodendrocytes in zi/zimutants
Thus, the spatial distribution of Rip immunoreactivity strongly supported the idea that the loss of function in atrn primarily causes defects in oligodendrocyte differentiation within wide areas of the brain, but does not affect neuronal development per se, the process of axonal sprouting or axonal projection. Given that the initial phase of myelination is not severely perturbed in the caudal regions of zi/zi CNS during the early postnatal period, it is likely that the hypomyelination and aberrant myelin sheath formation in zi/zi mutants result from a failure in the terminal differentiation of incipient myelinating oligodendrocytes; that is, defects in the subsequent extension or maintenance of oligodendrocytic processes. Alternatively, it is also possible that the hypomyelination in zi/zi brain might be caused by a toxic effect on oligodendrocytes and myelin after its normal formation.
Iron accumulation in zi/zioligodendrocytes in an age-dependent manner
Absence of attractin expression from cells of an oligodendrocyte lineage
Double-labeling experiments demonstrated that atrn was not expressed by PDGFR-α- or A2B5-positive cells (Fig. 6A–D), confirming the absence of atrn protein in OPCs at the early postnatal stage. During the process of cellular differentiation, atrn protein is likely to be missing in cells of an oligodendrocyte lineage. Consequently, atrn was found to be absent from almost all differentiated oligodendrocytes in the mature brain (Fig. 6F–H). Double immunofluorescence staining revealed non-overlapping expression of atrn and CNPase. Virtually all CNPase-positive oligodendrocytes in the neocortex (Fig. 6G), striatum (data not shown) and the granule cell layer of cerebellum (Fig. 6H) were devoid of staining for atrn. However, it should be noted that there was a small population of oligodendrocytes that were positive for atrn in the adult brain. For example, atrn was expressed in approximately only 2–5% of the oligodendrocytes within the corpus callosum. The vast majority of these cells, which formed clusters in rows, have small and oval cell bodies and resembled typical myelinating oligodendrocytes, were immunonegative for anti-atrn (Fig. 6F). The specificity of the anti-atrn antibody we used in this report was validated by immunohistochemical analysis using wild-type and zi/zi mutant brains. Since we could not find any anti-atrn immunoreactivity in zi/zi mutant brain sections (Fig. 6I,J and ref. ), the possibility that the cellular heterogeneity of oligodendrocytes (positive or negative for atrn) might result from a staining artifact could be excluded. These observations suggest the possibility that atrn does not exert an intrinsic function in most oligodendrocytes residing in the postnatal brain. The detailed characterization of the anti-atrn antibody, together with an analysis of the spatio-temporal expression pattern and subcellular distribution of atrn, has been described in our resent report .
Normal development of zi/zi oligodendrocytes in vitro
Considering the lack of atrn in cells of an oligodendrocyte lineage, and the normal distribution of Olig2-positive OPCs in the zi/zi SVZ, we upheld a hypothesis that the impaired developmental failure of zi/zi oligodendrocytes might be caused by a non-cell autonomous mechanism; that is, that some external factor(s) that was increased or decreased in concentration outside the oligodendrocytes in zi/zi rats detrimentally influences oligodendrocyte development. To assess this possibility, we next examined the developmental potential and the ability for terminal differentiation of zi/zi oligodendrocytes in vitro, using a primary culture of purified OPCs. Purified OPCs were cultured from neonatal zi/zi brains in serum-free defined medium containing the mitogens PDGF and bFGF; thereafter, these progenitors were induced to differentiate into mature oligodendrocytes. Cultures were used for study only if astrocytes comprised <1% and microglia <0.5% of the total cell number, thus eliminating the effect of other glial cell types. Cultures were characterized immunocytochemically using antibodies against the surface antigens A2B5 and galactocerebroside GalC, sequential expression of which defines early progenitors (OPC) and terminally differentiated oligodendrocytes, respectively, as described previously . As shown in Fig. 7, A2B5-positive OPCs prepared from zi/zi rats displayed a typical bipolar or poorly branched morphology (Fig. 7C,E), and could proliferate normally in the presence of mitogens, similar to OPCs from wild-type SD rats (Fig. 7D) (percentage of cells that were A2B5 positive: zi/zi rats, 81 ± 6.1% vs SD rats, 73 ± 8.6%) (Fig. 7I). In a differentiated culture, there was no difference in the frequency of OPCs differentiating into GalC-positive cells between zi/zi and wild-type rats (percentage of cells that were GalC positive: zi/zi rats, 26 ± 3.4% vs SD rats, 32 ± 5.2%) (Fig. 7I). GalC-positive oligodendrocytes developed from zi/zi OPCs exhibited a morphologically mature cell shape and a profuse network of processes and membranes, similar to oligodendrocytes from wild-type rats (Fig. 7F–H). The number of astrocytes or microglia did not increase with differentiation of the cultures (data not shown). These results indicated that cells of an oligodendrocyte lineage from zi/zi rats possess normal potential for proliferation and differentiation, at least in vitro, and supported the idea that the abnormal development of oligodendrocytes in zi/zi rats is not caused by a failure of the intrinsic program in oligodendrocytes themselves, but rather, by changes in extrinsic factors that can influence oligodendrocyte development.
Changes in the expression of proinflammatory cytokines/chemokines in zi/zibrains
One group of candidate extrinsic factors that affect oligodendrocyte development is the proinflammatory cytokines and chemokines. Some proinflammatory cytokines or chemokines, oversupplied from other CNS or non-CNS regions, may exert their toxic effects directly on oligodendrocytes in zi/zi rats from an early developmental stage. To determine the levels of various cytokines in zi/zi brains, semi-quantitative RT-PCR was performed for interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (INF-γ), tumor growth factor β (TGF-β) and tumor necrosis factor-α (TNF-α) at postnatal day 5 (P5) and day30 (P30). We also determined the levels of several CC chemokine mRNAs, including those for MCP-1 (CCL2), MIP-1α (CCL3) and RANTES (CCL5), as well as the levels of the CXC chemokine mRNAs for GRO-1 (CXCL1), MIP-2 (CXCL2) and SDF-1α (CXCL12a), and the two chemokine receptors, CXCR3 and CXCR4. As shown in Figure 7J and 7K, IL-1β mRNA expression was significantly higher (2–4 fold) in zi/zi brains than in control brains, even at P5 (zi/+ vs. zi/zi; 0.392 ± 0.217 vs. 1.575 ± 0.321* at P5, and 0.709 ± 0.212 vs. 1.447 ± 0.315* at P30, values are represented as means ± SEM of the relative expression ratio to the level of GAPDH, P* < 0.01 unpaired t-test). Such elevated expression of IL-1β mRNA was observed at least up to 5 months of age (data not shown). The levels of TGF-β, TNF-α and INF-γ (data not shown) mRNAs were unchanged between zi/zi and control brains. Expression of IL-6 mRNA was undetectable in both groups. Interestingly, gene expression profiling studies of the cerebral cortex and striatum from zi/zi rats, using the Affymetrix Microarray system, indicated a significant up-regulation of IL-1β converting enzyme (ICE) mRNA (data not shown). Among the chemokines we examined, MIP-1α (macrophage inflammatory peptide-1α) mRNA expression was detected at higher levels in zi/zi rats at P5 (×1.71 ± 0.128, relative fold change compared with control zi/+ rats). We could not detect significant changes in the mRNAs for any other chemokines in zi/zi rats.
On the other hand, we unexpectedly discovered a marked upregulation of the mRNA for dipeptidyl peptidase IV (DP IV/CD26) in zi/zi rats. The CD26 mRNA level was upregulated approximately 2–3 fold in zi/zi postnatal brains (P5 and P30), compared with zi/+ brains (Fig. 7K). CD26, a 110 kDa cell surface glycoprotein with a large extracellular domain, a transmembrane segment and a cytoplasmic tail, plays an important role in T cell co-stimulation .
Prolonged activation and accelerated recruitment of macrophages/microglia in the zi/ziCNS
We next attempted to determine the phenotype of EDl-positive cells found in the zi/zi parenchyma. Sections across the pons and cerebellum at P21 were processed for immunofluorescence detection with multiple cell type-specific markers, counterstained with the nuclear dye TOPRO3, and analyzed by confocal microscopy. An antibody to von Willebrand Factor (vWF) was used to label the endothelial cells of blood vessels , and Iba1 (ionized calcium-binding adapter molecule 1), a calcium binding protein, was used to label resident microglia, including both resting and activated forms [41, 42]. As shown in Fig. 8F, a significant number of EDl-positive cells were intimately associated with the vasculature, including capillaries and vessels of larger caliber, throughout the brain. These cells exhibited an amoeboid or elongated morphology, and contained an extensive perinuclear cytoplasm, with few extending projections (Fig. 8L,M). Combined immunostaining with vWF confirmed that these EDl-positive cells resided on the parenchymal side of the endothelium, but never within the lumen (Fig. 8L). Anatomical distribution of these cells suggested that they belong to a subset of macrophages termed perivascular cells, which are not integral components of the vascular wall and are derived from the bone marrow, migrating into the brain parenchyma during postnatal and adult life . Of interest is the observation that almost all ED1-positive perivascular cells were also immunoreactive for the microglial marker Iba1 (Fig. 8M). On the other hand, there were a large number of ED1-positive stellate cells with thick ramified processes in discrete regions of zi/zi brain, as shown in Fig. 8F. They were often deeply implanted in the parenchyma, exhibiting no apparent association with vascular elements. Double immunostaining of sections through the zi/zi cerebellar gray matter at P21 revealed that these cells were a subpopulation of Iba1-positive microglia (Fig. 8N arrows), presumably corresponding to the activated microglial cells characterized, in part, by the ED1-expressing phagocytic lysosomes.
Taken together, recruitment of monocytes/macrophages into the brain parenchyma and the subsequent activation of microglia appeared to be accelerated in the zi/zi CNS during the postnatal stage, prior to the appearance of their abnormal clinical signs for hypomyelination.
Non-cell autonomous impairment of oligodendrocyte differentiation in the zi/ziCNS
The present study provides a comprehensive phenotypic profile of the zi/zi brain during the early postnatal period. We observed the normal projection of neuronal axons and the developmental failure of oligodendrocyte processes in zi/zi rats. Onset of the impairment of oligodendrocyte differentiation might occur in a non-cell autonomous manner in zi/zi rats. Aberrant extension of cellular processes and hypomyelination of oligodendrocytes were not due to the failure of the intrinsic program of oligodendrocytes, but rather, were caused by extrinsic factors that interrupt oligodendrocyte development, based on the facts that zi/zi OPCs exhibit normal competence for proliferation and differentiation into mature oligodendrocytes in an in vitro culture system. The immunohistochemical evidence showing the absence of atrn expression from the early stage of an oligodendrocyte lineage reinforced the idea of non-cell autonomous failure of oligodendrocyte development in zi/zi rats. Such a non-cell autonomous defect of oligodendrocyte development may explain why the penetrance of abnormalities in oligodendrocyes is low (see Background section). If zi/zi oligodendrocytes developed under the control of an impaired intrinsic program, most of these cells should exhibit a common and synchronous abnormality. Nevertheless it should be noted that there was a small population of oligodendrocytes that were positive for atrn in the adult brain [this study, and ref. ]. Thus, it is also possible that atrn bears a critical function in some oligodendrocytes. Loss-of-function of atrn in this small population might be sufficient to initiate vacuolation in the zi/zi rat. Alternatively, atrn may be transiently expressed in most oligodendrocytes at the time when its function is required. We might have failed to detect atrn expression in oligodendrocytes due to the rapid down-regulation of atrn in this cell lineage. A recent study demonstrated that there is a decline in the amount of plasma membrane lipid rafts in the liver and spleen cells prepared from atrn mutant mice, suggesting a cell-autonomous defect in the plasma membrane maintenance of atrn-deficient cells . Therefore, we cannot exclude the possibility that atrn transiently plays an intrinsic role in the maintenance/integrity of the plasma membrane or the myelin sheaths of oligodendrocytes. Another explanation for the low penetrance of myelin defects observed in zi/zi rat is the redundancy of the biochemical pathways in oligodendrocyes in which atrn functions. Atrn and atrnl1 (attractin like-1) gene products were shown to share significant sequence similarity . A genetic study using transgenic and knock-out mice actually demonstrated that over-expression of atrnl1 compensates for the loss of atrn . Given the similar patterns of expression of the two genes in the adult rodent CNS , endogenous atrnl1 might partially compensate to prevent development of a severe phenotype in the zi/zi CNS.
We should further note the possibility of inappropriate expression of the secreted form of atrn in the zi/zi brain. In the rat brain, the atrn gene is transcribed into two different mRNAs with sizes of 9.0 kb and 4.5 kb, by alternative splicing. The 9.0-kb transcript was deduced to encode membrane-type atrn, while the 4.5-kb transcript was deduced to encode the secreted type of atrn corresponding to the secreted form of the human ATRN locus product [13, 46]. Our current and recent studies have shown the absence of membrane-type atrn protein in zi/zi brain using an antibody specific for the cytoplasmic tail of membrane-type atrn [this study and ref. ]; however, it remains to be determined whether an abnormal secreted form of atrn protein is generated in zi/zi brain. However, we could exclude the possibility that the defect in zi/zi oligodendrocytes is due to the inappropriate expression of the secreted form of atrn in the zi/zi rat brain for the following three reasons. (i) The deletion in the atrn zi allele has been identified at the splice donor site of the intron of the atrn gene, which is expected to result in unstable transcripts. Certainly, previous Northern analysis showed a marked decrease in both secreted-type and membrane-type atrn mRNAs in the zi/zi brain . Kuramoto et. al. also mentioned the existence of faint and long multiple atrn-related transcripts in zi/zi brain, although it remains unclear whether or not these transcripts encode a functional protein . (ii) Transgenic rescue experiments showed that membrane-type atrn is responsible for the neuropathological phenotype in zi/zi rats, but that the secreted-type atrn cannot complement this mutant phenotype . This result indicated that the zi/zi brain phenotype is attributable solely to the loss of membrane-type atrn, even though secreted-type atrn might be up-regulated in zi/zi brain. (iii) The mv/mv (myelin vacuolation) rat, a spontaneous mutant harboring a genomic deletion including exon 1 of the atrn gene (atrn mv ), showed no detectable expression of both secreted- and membrane-type atrn mRNA in the brain . The mv/mv rat brain exhibits neurological and neuropathological phenotypes including hypomyelination and vacuolation, which are quite similar to those found in zi/zi brain. This finding strongly suggests that the hypomyelination phenotype observed in atrn mutant rats is not directly linked to the expression of the secreted form of atrn. Nevertheless, it has been largely unknown whether the secreted form of atrn might exert a specific function in the normal rat and human CNS. Moreover, it is of interest to note that the morphological defects found in the zi/zi oligodendrocyte processes appears remarkably similar to the effects of secreted-type human atrn upon dendrite development in differentiating neurons in vitro with "wavy processes that branched irregularly" . In order to explore the function of the secreted form in the CNS, an immunostaining study using an anti-atrn antibody recognizing the secreted form is in ongoing in our laboratory.
Our observations raised the possibility that macrophage/microglia activation in the developing zi/zi brain leads to the disturbance of oligodendrocyte differentiation in discrete regions of the brain. Primitive macrophages/monocytes have been proposed to be the cells of origin of microglia. These cells are thought to enter the developing brain parenchyma from the bloodstream, the ventricular spaces or the meninges, during both the prenatal and postnatal periods [1, 48]. Although the relationship between circulating macrophages and microglial lineage cells is largely undefined, it is possible that the increase in the number of Iba1-positive perivascular cells in the zi/zi parenchyma is due to an increase in the population of monocytes/macrophages recruited from the vessels and destined to become microglia. Since both brain macrophages and CNS resident microglia retain common properties, including phagocytic ability, and are possible sources of ROS, proinflammatory cytokines and chemokines, the increased number of monocytes/macrophages/microglia could initially dominate the development of oligodendrocytes within discrete regions of the axon tract through the production of toxic molecules, such as ROS, IL-1β and MIP-1α, leading to the commencement of hypomyelination in the zi/zi CNS.
Peroxides, including hydrogen peroxide (H2O2), are one of the main ROS leading to oxidative stress . A previous biochemical study  has shown lowered catalase activity and increased activity of d-amino acid oxidase (d-AAO) in zi/zi medulla, pons and cerebellum at P10, before the appearance of morphological vacuolation in the brains of suckling zi/zi rats. D-AAO is known to produce ROS such as superoxide anion (O2•-) and H2O2, while catalase scavenges H2O2. These findings suggest the abnormal metabolism of H2O2 and a subsequent increase in the amount of ROS in zi/zi brain. Consistently, our previous studies demonstrated an accumulation of ROS in the nigrostriatal dopaminergic system leading to axonal degeneration or neuronal cell death being frequently observed in the substantia nigra of aged zi/zi rats [6, 8]. Chronic administration of vitamin E (D, L-α-tocophenol), an effective free radical scavenger in the brain, resulted in a significant increase in the number of surviving dopamine neurons in zi/zi brain , supporting elevated oxidative stress in zi/zi rat brain. Importantly, cells of an oligodendrocyte lineage, especially OPCs, have been shown to be exquisitely vulnerable to oxidative stress [50–52]. Therefore, we assume that free radical injury to developing oligodendrocytes underlies, in part, the pathogenesis of zi/zi rats. Presumably, the elevated level of ROS, which are released from accumulated macrophages/microglia, may impede oligodendrocyte differentiation from OPCs during the early postnatal stage, leading to hypomyelination as well as neuronal injury at a later stage.
Proinflammatory cytokines, the mediator molecules of inflammation, such as IFN-γ, TNF-α and IL-1β, have been shown to be derived from macrophages/microglia and have been implicated in the pathogenesis of demyelinating diseases [53, 54]. In vitro culture studies indicated that the developing oligodendrocytes exhibit higher susceptibility to cytokine-mediated cytotoxicity compared with mature oligodendrocytes [52, 55]. IL-1β is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases. OPCs and differentiated oligodendrocytes are known to express IL-1 receptors, and IL-1β has been shown to block the proliferation of OPCs in vitro . As shown here, expression of IL-1β mRNA is elevated from an early stage. This excess delivery of IL-1β may promote the premature differentiation of OPCs in the zi/zi brain.
Possible functions of Atrn in CNS
It is thought that atrn is involved in the initial axon-oligodendrocyte interaction and the assembly process of the myelin sheath, because of the existence of extracellular functional domains, which are usually required for receptor-ligand interactions [14, 18]. In this context, however, we cannot explain the possible cellular mechanism by which atrn mediates the activation or invasion of macrophages/microglia in the CNS. It was recently shown that membrane-type atrn possesses an activity of dipeptidyl peptidase IV (DP IV)/CD26-like ectoenzyme, and is expressed on the surface of human peripheral blood monocytes , while the in vitro study by Friedrich et al. provided compelling evidence that atrn protein purified from human plasma has no DP IV activity, suggesting that atrn acts as a receptor or adhesion protein rather than a protease . CD26 is an ectoenzyme DP IV (EC 188.8.131.52) that releases N-terminal dipeptides from peptides with proline in the penultimate position, and is known to be expressed as both a secreted form and a membrane-bound form localized on the surfaces of T cells, B cells and natural killer cells . Several chemokines, including RANTES and SDF-1α, have been identified to be hydrolyzed by CD26 . A recent study suggested that CD26 expressed on the surface of hemopoietic stem/progenitor cells plays a crucial role in their homing/mobilization ability to/from the bone marrow, though the proteolytic cleavage of a local pool of SDF-1α . Although atrn exhibits no structural similarity to CD26, it is now thought that atrn is a member of a unique DP IV-family based on the comparable substrate specificity of atrn to DP IV [20, 23]. Our present study indicated that the expression of SDF-1α mRNA in the brain was unaltered in zi/zi rats. However, even though atrn cleaves SDF-1α as a target chemokine with the same specificity as CD26, we could not detect such a truncated form of chemokines, as no available technique can discriminate these from the full-length forms. If CD26 serves similar or redundant functions to the atrn gene in the CNS, upregulation of its mRNA may reflect a functional compensation for the loss of atrn expression in zi/zi rats. In this context, the DP IV-activity of atrn may be crucial for the onset or progression of CNS degeneration in zi/zi rats. Interestingly, it was shown that atrn potentially influences monocyte function through its DP IV activity; inhibition of DP IV activity in stimulated cultured monocytes caused significantly enhanced release of cytokines . Moreover, inhibition of its activity decreased the adhesion of cultured monocytes to the extracellular matrix . It should also be noted that the secreted form of atrn circulating in human plasma functions in the immune response, mediating the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes [17, 58]. Taken together, these findings suggest the possibility that the monocyte/macrophage carrying a mutation of the atrn gene may alter the homing/mobilization ability, directing cells towards the chemokines in the brain, or may disregulate their own adhesion properties through the endothelium, leading to extravasation of macrophages into the zi/zi brain parenchyma. Alternatively, atrn might principally function in promoting the transformation of monocytes into resting microglia in the brain. The accumulation of ED-1-positive monocytes in the zi/zi CNS observed in this report might reflect the fact that ED-1-positive cells fail to become resting microglia and remain active in zi/zi brain. Thus, there are more ED-1-positive monocytes/macrophages in zi/zi rats that express cytokines and chemokines. Considering the expression of atrn in almost all OX42-positive microglia , the loss of atrn may alter the characteristics of microglia themselves, leading to the continuous and increased production of ROS or cytokines/chemokines in zi/zi microglia, and toxicity to oligodendrocytes/myelin as a consequence. Appropriate and persistent expression of atrn in monocytes/macrophages may be required for their proper transform into resting microglia in the brain.
On the other hand, it is well known that the majority of microglia are derived from bone marrow cells during embryogenesis and the early postnatal period. Recent bone-marrow transplantation studies have indicated that there exists slow supplementation of microglia by marrow cells under physiological conditions, even in adult life . Atrn may be involved in brain morphogenesis by suppressing the excessive migration of monocytes into the CNS, or by accelerating the transformation of brain monocytes into resident microglia, but its molecular mechanisms remain to be elucidated. Transplantation studies of EGFP-tagged zi/zi macrophages/microglia into the wild-type brain, and co-culture experiments of activated zi/zi macrophages/microglia with oligodendrocytes, which are currently in progress in our laboratory, will unequivocally reveal the function of this protein.
Relevance to neurodegenerative diseases
Iron-mediated oxidative damage to axons may be an early event that is common to several neurodegenerative diseases, such as AD, PD, MSA and MS [3, 28]. In the zi/zi brain, iron-accumulation became evident in oligodendrocytes with increasing age. Although it is still a matter of debate whether iron accumulation is a primary cause of neurodegeneration or a secondary consequence, there seems to be no doubt that iron-induced oxidative stress contributes to the pathogenesis of neurodegenerative diseases, including animal models . In addition, several lines of evidence also suggest an involvement of macrophages/microglia in these neurodegenerative diseases. Pronounced recruitment and activation of macrophages/microglia into the brain parenchyma have also been implicated in mediating oligodendrocyte and myelin damage, leading to demyelination in MS and its murine model EAE (experimental autoimmune encephalomyelitis) . Recent studies suggest an involvement of CD26/DP IV-activity in the pathogenesis of MS, including T-cell activation, cytokine production and lymphocyte invasion into CNS tissues, through the proteolytic processing of target chemokines [22, 33, 61]. In vivo experiments using a potent synthetic inhibitor of DP IV demonstrated that the clinical signs of EAE could be diminished by DP IV inhibition, both in a preventive and therapeutic fashion, suggesting a crucial role of DP IV activity in the pathogenesis of MS . In view of the compatible DP IV-activity between atrn and CD26, the zi/zi rat might be a useful model for studying the functions of DP IV in pathogenesis linking the immune system with nervous system. To date, we have found no hereditary neurological disorders that map onto the human atrn gene locus. It will be interesting to determine whether the atrn gene is implicated in neurological disorders as a novel modifier.
Our studies provide evidence that onset of the impairment of oligodendrocyte differentiation occur in a non-cell autonomous manner in zi/zi rats. Based on the lack of atrn expression from the early stage of an oligodendrocyte lineage, along with the normal competence of zi/zi OPCs for proliferation and differentiation in vitro, it is highly likely that the hypomyelination of zi/zi oligodendrocytes is not due to the failure of the intrinsic program of oligodendrocytes, but rather, is caused by extrinsic factors that interrupt oligodendrocyte development. An enhanced recruitment and prolonged activation of ED-1-positive monocytes/macrophages into the zi/zi brain parenchyma during the early postnatal stage, prior to the onset of hypomyelination, suggests the possibility that these activated macrophage/microglial lineage cells might induce disturbances in oligodendrocyte differentiation via the secretion of deleterious factors including IL-1β and MIP-1α. Atrn might be involved in the activation of brain macrophages/microglia by suppressing excessive migration of monocytes into the CNS, or by accelerating the transformation of brain monocytes into resting microglia.
Homozygous zitter mutant (zi/zi) rats were maintained as a congenic strain with the genetic background of Sprague-Dawley rats (SD). As control animals, we used age-matched SD rats purchased from Charles River Japan Inc. (Japan), or zitter heterozygote (zi/+) littermates, which were obtained by intercrossing each strain. Rats were handled according to the Guidelines for the Care and Use of Laboratory Animals, Dokkyo Medical University School of Medicine.
The day of birth was designated as P0. Animals were lethally anaesthetized with sodium pentobarbital administered i. p. (10 mg/kg) and perfused transcardially with phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffer, pH 7.4 for immunohistochemistry or by 1% glutalaldehyde, 4% PFA in PB for Perls'/DAB iron staining . Brains and other tissues were dissected, postfixed overnight at 4°C, and cryoprotected in 30% sucrose in PBS at 4°C. Tissue sections for indirect double immunostaining were cut coronally at a thickness of 12 μm using a cryostat, and affixed to 3-aminopropyltriethoxysilane-coated glass slides (Matsunami, Osaka, Japan). For immunostaining with a single primary antibody or Perls'/DAB iron staining, serial free-floating sections were cut at a 30-μm thickness, rinsed with 0.1 M PBS, and stored at 4°C.
Immunohistochemistry and immunofluorescence
Immunolabeling with a single primary antibody was performed using the avidin-biotin-peroxidase technique (Vectastain ABC kit, Vector Laboratories, Burlingame, CA), according to the manufacturer's instructions. Briefly, free-floating sections were quenched for endogenous peroxidase activity by treating them with 0.3% H2O2 in PBS for 30 min at room temperature, blocked for 1 h in 10% normal goat serum, and 0.3% Triton X-100 in PBS, and then incubated with a primary antibody diluted in the same blocking solution overnight at 4°C. Sections were placed in an appropriate biotinylated secondary antibody diluted in blocking solution for 2 h followed by incubation with avidin-conjugated horseradish peroxidase (HRP). Immunoreactivities were visualized using 0.25 mg/ml diaminobenzidine (DAB) and 0.03% H2O2. Each step was followed by four washes in PBS containing 0.3% of Triton X-100 (PBST). For immunostaining with Rip antibody, the sections were quenched with 0.3% H2O2 after incubation with secondary antibody. Double indirect immunostaining was performed on frozen sections with the appropriate combination of primary antibodies. After four washes with PBST, bound antibodies were visualized by incubation with Alexa Fluor 488- or 568-conjugated secondary antibodies (used at a dilution of 1:1000; Molecular Probes, Inc. Eugene, OR). Cells were counter-stained with 10 μM Hoechst 33342 dye (Sigma-Aldrich, St. Louis, MO) or TOPRO-3 (Molecular Probes) to identify nuclei. After being rinsed with PBST, the specimens were examined under a fluorescence microscope equipped with the appropriate epifluorescent filters. Optical sections were viewed using the FV500 scanning laser confocal imaging system (Olympus, Tokyo, Japan).
The following antibodies were used in this study: anti-attractin (affinity purified rabbit polyclonal ); anti- 2', 3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) (mouse monoclonal IgG1, Sigma-Aldrich) diluted 1:200; A2B5 (mouse monoclonal IgM, Chemicon, Temecula, CA), diluted 1:500; O4 (mouse monoclonal IgM, Roche Diagnostics), diluted 1:20; GalC (mouse monoclonal IgG, Sigma), diluted 1:200; anti-PDGFR alpha (rat monoclonal IgG ), diluted 1:500; anti-Ferritin H/L (rabbit antiserum, Sigma), diluted 1:1,000; anti-oligodendrocyte-specific protein, Rip (mouse monoclonal IgG1, Chemicon), diluted 1:10,000; anti-Neurofilament 70/200 kDa (mouse monoclonal IgG1, MP Biomedicals, Costa Mesa, CA), diluted 1:200; ED1 (mouse monoclonal IgG1, Chemicon), diluted 1:1,000; anti-Iba1 (rabbit polyclonal antibody, Wako Pure Chemical Industries, Japan), diluted 1:2,000; MRC OX-6 (mouse monoclonal IgG1, Serotec, UK), diluted 1:300; anti-vWF (von Willebrand factor; rabbit polyclonal antibody, Dako Japan, Japan), diluted 1:300; and anti-human Olig2 (rabbit IgG, Immuno-Biological Laboratories, Japan), diluted 1:250.
Primary rat oligodendrocytes were prepared from the cerebral cortices of zi/zi or SD rats at postnatal day 1 using a shaking method . Briefly, forebrains free of meninges were chopped into 1 mm3 blocks and placed into HBSS containing 0.25% trypsin and 10 μg/ml DNase. After digestion for 15 min at 37°C, the tissue was collected by centrifugation and triturated in DMEM medium containing 10% fetal bovine serum, 40 IU/ml penicillin and 40 μg/ml streptomycin, and passed through a 70 μm sieve. Cells were plated onto poly-D-lysine-coated 25 cm2 flasks at a density of 1 pup brain per flask. Cultures were fed with fresh DMEM medium every other day for 10–11 d at 37°C in a humid atmosphere of 5% CO2. To isolate oligodendrocytes and oligodendrocyte precursor cells (OPCs), mixed glial cultures were shaken for 1 hr in an orbital shaker at 150 rpm at 37°C to remove adherent microglia/macrophages, and the cultures were washed with the same medium and subjected to continuous shaking at 220 rpm for 16 hr to separate OPCs from the astrocyte layer. The suspension was plated onto uncoated Petri dishes and incubated for 1 hr at 37°C to further remove residual microglia and astrocytes adhering to the dishes. The OPCs were then collected by passing through a nylon mesh, followed by centrifugation. Isolated OPCs were plated onto poly-D-lysine-coated (100 μg/ml) glass coverslips in 24-well plates at a cell density of 2.0 × 104 cells per well for morphological and immunocytochemical studies. Purified OPCs were cultured for 7–8 d in a serum-free glial defined medium (GDM: DMEM, 0.1% bovine serum albumin (Roche Diagnostics), 50 μg/ml apo-transferrin (Sigma), 50 μg/ml insulin (Invitrogen), 30 nM sodium selenite (Sigma), 10 nM putrescin (Sigma), 10 nM D-biotin (Sigma), 20 nM progesterone (Sigma), 10 nM hydrocortisone (Sigma)), supplemented with 5 ng/ml platelet-derived growth factor (PDGFAA) (R&D systems, Minneapolis, MN) and 10 ng/ml basic fibroblast growth factor (bFGF) (R&D systems). At 7–8 d, the cultures were composed primarily of progenitors and pre-oligodendrocytes (A2B5+, O4-, GalC-). After 7 d, the culture medium was changed to serum-free GDM medium containing 5 ng/ml ciliary neurotrophic factor (CNTF) and 15 nM 3, 3, 5-triiodo-L-thyronine (T3) for 7 additional days until cells were differentiated into mature oligodendrocytes. Proliferating OPCs or mature oligodendrocytes were fixed with 4% PFA and immunostained with A2B5 or GalC (O1) antibodies.
RNA extraction and semi-quantitative RT-PCR assay of cytokine and chemokine mRNAs
At each time point, zi/zi or zi/+ control rats (n = 3 respectively) were killed and whole brains were rapidly dissected and frozen in liquid nitrogen. Total RNA was extracted using TRIzol reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions and quantified spectrophotometrically. Three micrograms of total RNA was reverse-transcribed according to the manufacturer's protocol (Superscript II, Invitrogen) using random primers, and the cDNA prepared from 30 ng of RNA was subjected to PCR. The primers for rat cytokines were synthesized based on rat cDNA seqeunces for TNF-α (GenBank accession number: 82524821), IL-1β (204905), IL-6 (2170752), TGF-β (11024651), IFN-γ (24475821), CCL5 (RANTES)(124286795), MIP-1α (CCL3) (40254793), MCP-1 (CCL2) (13928713), MIP-2 (CXCL2) (16758459), CXCR4 (82617587), CXCL12 (76496502), GRO-1 (CXCL1) (13540651), CD26 (DPP4) (6978772), CXCR3 (16758151) and GAPDH (9798637) as an internal control. All primer pairs spanned at least one intron in the corresponding genomic DNA. The sequences of oligonucleotide primers and predicted cDNA sizes were as follows: IL-1β, sense 5'-CAAGCACCTTCTTTTCCTTCATC-3' and antisense 5'-GTCGTTGCTTGTCTCTCCTTGTA-3' (241 bp); TNF-α, sense 5'-CCCAGACCCTCACACTCAGAT-3' and antisense 5'-TTGTCCCTTGAAGAGAACCTG-3'(215 bp); INF-γ, sense 5'-GCCAAGGCACACTCATTGAA-3' and antisense 5'-GCTGGTGAATCACTCTGATG-3' (360 bp); IL-6, sense 5'-CAAGAGACTTCCAGCCAGTTGC-3' and antisense 5'-TTGCCGAGTAGACCTCATAGTGACC-3' (101 bp); TGF-β, sense 5'-GAGAGCCCTGGATACCAACTACTG-3'and antisense 5'-GTGTGTCCAGGCTCCAAATGTAG-3' (173 bp); GAPDH, sense 5'-ACCACCATGGAGAAGGCTGG-3' and antisense 5'-CTCAGTGTAGCCCAGGATGC-3' (528 bp); CCL5 (RANTES), sense 5'-CTCCAACCTTGCAGTCGTCT-3' and antisense 5'-GCCTGTGAAGAGCACACCTC-3' (296 bp); MIP-1α (CCL3), sense 5'-TGCTGTTCTTCTCTGCACCA-3' and antisense 5'-GGCTACTTGGCAGCAAACAG-3' (380 bp); MCP-1 (CCL2), sense 5'-TGTAGCATCCACGTGCTGTC-3' and antisense 5'-GCTTGAGGTGGTTGTGGAAA-3' (362 bp); MIP-2 (CXCL2), sense 5'-TCAATGCCTGACGACCCTAC-3' and antisense 5'-ACTCAGACAGCGAGGCACAT-3' (308 bp); CXCR4, sense 5'-GTTTGGTGCTCCGGTAGCTA-3' and antisense 5'-CCAGAAGGGGAGTGTGATGA-3' (317 bp); CXCL12 (SDF-1α), sense 5'-GCTCTGCATCAGTGACGGTA-3' and antisense 5'-CTTTGTGCTGGCAAATCTCAG-3' (382 bp); GRO-1 (CXCL1), sense 5'-AGACAGTGGCAGGGATTCAC-3' and antisense 5'-GAACGACCATCGATGAAACG-3' (375 bp); CD26 (DPP4), sense 5'-GCCTGGGTTTCAGAAGACAGA-3' and antisense 5'-CTGGAACTGGCAGATGTGTTTG-3 (254 bp); and CXCR3, sense 5'-ACAAGTGCCAAAGGCAGAGAAG-3' and antisense 5'-GAGCAGGAAGGTGTCTGTGCT-3' (350 bp). For semiquantitative PCR, target sequences were amplified by 20–35 cycles of PCR (denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 30 sec) using a GeneAmp 9700 thermal cycler (Applied Biosystems Japan, Tokyo Japan). The number of amplification cycles was optimized to ensure that PCR products were quantified during the exponential phase of the amplification. A 10 μl aliquot of each PCR product was size-separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide. The gels were photographed, and the bands were quantified using Image J 1.36 software for Macintosh computers and normalized to the values for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Enhanced Perls' staining for ferric iron
Iron distribution in zi/zi or control brains was examined using the enhanced Perls' method with a minor modification . Free-floating brain sections were incubated in Perl's solution (a 1:1 mixture of 2% potassium ferrocyanide and 2% HCl) for 30 minutes, washed in distilled water, and immersed for 15 min in 0.05% DAB in 0.1 M phosphate buffer (pH 7.4). One ml of 1% hydrogen peroxide was then added for every 200 ml of DAB solution, and sections were incubated in the solution for 15 min. After being rinsed with distilled water for 30 min, sections were mounted on glass slides, lightly counterstained with 0.1% methyl-green or Nissl (0.1% cresyl violet). Some sections were further processed for electron microscopy.
Iron-stained sections were subdissected into smaller rectangular portions that included fields of the substantia nigra within the midbrain. The sections were osmicated with 1% osmium tetroxide solution buffered with 0.05 M phosphate buffer for 2 h and embedded in Epon 812 after dehydration with a graded ethanol series. The specimens were cut using an ultramicrotome and observed using a JEM 2000EX electron microscope (JOEL. Ltd., Japan) after staining with uranyl acetate and lead hydroxide.
SS would like to acknowledge Ms. Y. Yamada and F. Terauchi for technical assistance. SS is also grateful to Dr. Yuki Nakamura for critical reading of the manuscript.
This work was supported by grants, KAKENHI (17590173) (SS) from JSPS, and in part by the "Academic Frontier" Project from MEXT (SU), Japan.
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