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Keywords:

  • ceramide galactosyltransferase;
  • cerebroside;
  • cholesterol;
  • GeneChip;
  • myelin basic protein;
  • myelin

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down-regulated during demyelination and up-regulated during remyelination. Genes related to microglia/macrophages appeared in a time-course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.

Abbreviations used
CGT

ceramide galactosyltransferase

HMG

hydroxymethylglutaryl

MBP

myelin basic protein

MOBP

myelin-associated oligodendrocyte basic protein

SDS

sodium dodecyl sulfate.

Inclusion of the copper chelator, cuprizone (bis-cyclohexanone oxalydihydrazone), in the diet of young adult mice produces a massive demyelination of certain brain regions (Suzuki and Kikkawa 1969; Blakemore 1973a; Ludwin 1978; more recently Hiremath et al. 1998; Morell et al. 1998). In the late stages of such exposure, animals exhibit neurological symptoms. Recovery, including remyelination, occurs if the metabolic insult is removed (Blakemore 1973b; Ludwin 1994). We have utilized such a model, adjusting the dosing schedule to 6 weeks of exposure to cuprizone in the diet, sufficient to achieve considerable demyelination and produce some neurological symptoms. After a period of recovery (6 weeks on a normal diet), animals appear normal neurologically and some myelin is regained (for review, see Matsushima and Morell 2001). The corpus callosum is one area that is preferentially affected by exposure to cuprizone, and morphological studies of this structure have been conducted over the time-course of demyelination and remyelination. Useful as morphology-based studies are, it is also important to carry out biochemical and molecular biological investigations to more fully understand the underlying metabolic processes involved. Some biochemical studies with cuprizone-dosed mice have been done (e.g. Carey and Freeman 1983). More recently, our laboratory has presented results concerning metabolism of myelin lipids, correlating this metabolism with gene expression at the level of mRNA expression (Jurevics et al. 2001). Qualitatively, the biochemical results for whole brain tracked the morphological observations with respect to loss of myelin-specific components during demyelination, and synthesis and accumulation of myelin components during remyelination. However, there were quantitative and temporal discrepancies that can be attributed to the fact that the biochemistry was done on whole brain, and thus averaged the results from brain regions severely affected and those only somewhat affected. In contrast, morphological evaluation was conducted on defined areas of the severely affected corpus callosum.

We have now expanded this model to allow for brain regional analysis of lipids and lipid metabolism. We determined these myelination-specific biochemical parameters for the corpus callosum region dissected over the time-course of cuprizone-induced demyelination and subsequent remyelination. Perturbations for this brain region were considerably greater than those for cerebellum and brainstem. We further extended the utility of this model by applying Affymetrix GeneChip technology to mRNA expression in corpus callosum. This allowed us to determine how expression of many genes is altered with time during the sequence of demyelination and remyelination. In this more discretely defined model, it is likely that during demyelination, many of the metabolic and gene-expression changes observed are directly related to the pathophysiology of cuprizone intoxication. Importantly, it also allows tracking of the sequence of events involved in recovery from demyelination.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Under protocols approved by the University of North Carolina Institutional Animal Care and Use Committee and described previously (Jurevics et al. 2001), C57BL/6J mice 8 weeks of age and weighing about 24 g were placed on milled Purina Laboratory chow containing 0.2% cuprizone (Sigma Chemical Co., St Louis, MO, USA) for 2–12 weeks. Some animals were removed from cuprizone exposure after 6 weeks and returned to a normal diet for up to 6 additional weeks. Animals used as controls were maintained on Purina Laboratory chow. Mice whose brain regions were used for analysis of protein or mRNA were killed at the stated ages. Mice utilized for studies of brain lipid concentration and metabolism were injected intraperitoneally with 25 mCi 3H2O and maintained for 4 h prior to being killed by exposure to CO2. Each brain was removed and cerebellum was separated from brainstem by severing the peduncles and medullary vela. Wet weights of cerebellum for all control animals (8–20 weeks of age) was 51.9 ± 4.3 mg and for those exposed to cuprizone 50.7 ± 6.0 mg. Brainstem was separated from the cerebral hemispheres rostral to the superior colliculi dorsally and the cerebral peduncles ventrally. Wet weights of this region were 97.2 ± 6.9 mg for controls and 95.2 ± 10.7 for cuprizone-exposed animals. Portions of corpus callosum, dissected from both cerebral hemispheres following mid-sagittal sectioning of the forebrain and containing mostly the genu and trunk directly over the lateral ventricles, weighed approximately 4–6 mg.

Lipid analysis

Preparation and analysis of samples is detailed in a recent publication (Jurevics et al. 2001). Briefly, lipids were extracted from brain samples with organic solvents. An aliquot of the total lipid was benzoylated and desulfated, and cerebrosides and sulfatides were separated into hydroxy and non-hydroxy-containing fatty acid classes by HPLC. Galactolipids were detected by absorption at 230 nm and each chromatography peak was quantitated by comparison with authentic standards. Another aliquot of the lipid extract was subjected to reverse-phase HPLC and material present in the peaks for cholesterol and desmosterol was collected and quantitated by comparing absorption at 210 nm with that of authentic standards. Radioactivity in material under the peaks was also determined.

Data for lipid mass obtained from ultraviolet absorption data was used to calculate lipid concentration and changes in lipid concentration with time during the course of demyelination/remyelination. As detailed previously (Jurevics and Morell 1994; Muse et al. 2001), the amount of radioactivity present in the isolated lipid allowed calculation of the rate of synthesis of cerebroside and sterol. The underlying principle is that for each molecule of cholesterol synthesized, there is incorporation of 22 hydrogens that are equilibrated with the hydrogens of body water (Andersen and Dietschy 1979; Dietschy and Spady 1984; Belknap and Dietschy 1988); for cerebroside this number is 33 (Muse et al. 2001). The absolute amount of cholesterol or cerebroside synthesized can be calculated from the radioactivity in each lipid and the specific activity of the whole-body water precursor pool (determined from a serum sample). Corrections are made for the approximately 1% of brain sterol present as the cholesterol precursor, desmosterol, and for the fact that over the 4 h course of incorporation of label from radioactive water, almost half the incorporated radioactivity is still present in this precursor sterol.

Isolation and analysis of RNA

Tissue samples were homogenized in 350 µL lysis buffer using a Potter-Elvehjem tissue grinder and RNA was then isolated with ‘RNeasy’ mini-prep spun-columns (Qiagen, Valencia, CA, USA), equipped with ‘Qiashredder’ column attachments. RNA species were separated according to molecular weight on denaturing 0.8% agarose gels containing formaldehyde and then transferred to Zeta-Probe nylon blotting membranes (Bio-Rad Laboratories, Richmond, CA, USA). Filters were hybridized with 32P-labeled cDNA probes synthesized as described by Toews et al. (1990), and radioactivity distribution quantified using a Molecular Dynamics Typhoon Variable Mode Imager (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Values obtained were normalized to the amount of ribosomal RNA in each lane, as assayed with 32P-end-labeled oligonucleotides specific to defined sequences in the 28S subunit.

Affymetrix GeneChip analysis of mRNA expression

For each sample analyzed, 7 µg of total RNA was used to synthesize cDNA, using a custom cDNA kit (Invitrogen Life Technologies, Carlsbad, CA, USA) with a T7-(dT)24 primer. Biotinylated cRNA was then generated from the cDNA using the BioArray High Yield RNA Transcript Kit. The cRNA was fragmented in fragmentation buffer (5X fragmentation buffer: 200 mm Tris-acetate, pH 8.1, 500 mm potassium acetate, 150 mm magnesium acetate) at 94°C for 35 min prior to chip hybridization. 15 µg of fragmented cRNA was then added to a hybridization cocktail (0.05 µg/µL fragmented cRNA, 50 pm control oligonucleotide B2, BioB, BioC, BioD, and cre hybridization controls, 0.1 mg/mL herring sperm DNA, 0.5 mg/mL acetylated BSA, 100 mm MES, 1 m NaCl, 20 mm EDTA, 0.01% Tween 20). Affymetrix (Santa Clara, CA, USA) murine genome U74Av2 GeneChip probe arrays were hybridized for 16 h in the GeneChip Fluidics Station 400, washed, and scanned with a dedicated Hewlett-Packard GeneArray Scanner. During washing, the cRNA probe was labeled with R-phycoerythrin streptavidin. Affymetrix GeneChip Microarray Suite 4.0 software was used for washing, scanning, and basic analyses. Sample quality was assessed by examination of 3′−5′ intensity ratios of certain genes. GeneChip data were analyzed using GeneSpring microarray data analysis software (Silicon Genetics, Redwood City, CA, USA). Intensity values for each gene are generated by Affymetrix software; the total intensity of the 16 negative mismatch sequences is subtracted from the total intensity of the 16 positive sequences and then divided by 16 to give the average difference (now called signal in the newest Affymetrix software) for a given gene. This average difference value was used in all analyses by GeneSpring.

Western blot analysis of myelin basic protein (MTP) levels

Proteins in the dissected tissue samples were solubilized by homogenization in 1% sodium dodecyl sulfate (SDS) and boiling for 15 min. The insoluble residue obtained following centrifugation was discarded and 2–3 µg protein in the SDS-soluble fraction was subjected to electrophoretic separation and western blotting (Muse et al. 2001).

Statistical analysis

Single-factor analysis of variance (mnova) was performed on data generated in experiments using control and cuprizone-fed mice. Differences were considered significant at p < 0.05. Differences between groups at certain time points were analyzed by Student's t-test and considered significantly different at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Loss of cerebroside is most profound in corpus callosum and varies in other brain regions

In the corpus callosum, loss of the myelin-specific lipid, cerebroside, was 65% after 6 weeks of cuprizone exposure; after 12 weeks of exposure, the deficit was 78%(Fig. 1a, p < 0.05, mnova) This deficit was almost double that reported for whole brain using the same exposure model (Jurevics et al. 2001). The cerebroside deficit present in corpus callosum by 6 weeks of cuprizone exposure was only partially compensated following removal of the metabolic insult and allowing for 6 weeks of recovery. This also contrasts to whole brain data, where the lesser cerebroside deficit on a whole brain basis was largely eliminated by 6 weeks into the recovery phase. This discrepancy is probably due to the greater precision of analysis when only the severely affected region is analyzed. The differential response to cuprizone exposure for different brain regions is clear when results for cerebellum (maximum of 33% demyelination, Fig. 1d) and brainstem (maximum of 13% demyelination, Fig. 1g) are examined. As for corpus callosum, if a recovery phase is included, there is a hint of partial restoration of cerebroside levels.

image

Figure 1. Effects of cuprizone exposure on cerebroside concentration, cerebroside synthesis rates, and ceramide galactosyltransferase (CGT) steady-state mRNA levels in corpus callosum, cerebellum, and brainstem of mice. Animals at 8 weeks of age were exposed to cuprizone in their diet for up to 12 weeks. In some cases, cuprizone was removed after 6 weeks of exposure and animals allowed to recover for up to 6 additional weeks. Data for cerebroside concentration and synthesis rates are means ± SEM (n = 3); data for mRNA levels are single determinations and are plotted relative to the mean of the three controls.

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Changes in rate of cerebroside synthesis precede changes in accumulation

For corpus callosum, the rate of synthesis of cerebroside is qualitatively as expected (Fig. 1b). The alteration in rate of synthesis of cerebroside is not, however, directly proportional to subsequent changes in cerebroside level. Even in the control situation, although the concentration of cerebroside is constant from 8 to 20 weeks postnatal (Fig. 1a), there is a decrease in rate of synthesis of cerebroside, presumably reflecting a developmental slowing of cerebroside turnover. If animals are exposed to cuprizone, there is an immediate and significant down-regulation of cerebroside synthesis (Fig. 1b, p < 0.03, mnova); as expected, this precedes the physical loss of cerebroside. Yet the down-regulation may not be as marked as expected in consideration of the profound loss of cerebroside noted in Fig. 1(a). Consider that over the 12-week period, the control animals synthesize a total of 10.9 nmoles cerebroside/mg wet weight of corpus callosum (proportional to the area under the closed circle data line of Fig. 1b– after correction to weekly values). The equivalent calculation after cuprizone treatment (area under the closed diamond line) is 7.7 nmoles cerebroside/mg wet weight of corpus callosum. Thus, the decrease in cerebroside synthesized over 12 weeks is 30%; in contrast, the actual decrement in cerebroside level is 78% (noted earlier with respect to Fig. 1a). This suggests there is not just a lack of renewal of cerebroside undergoing normal metabolic turnover, but also pathophysiological events that result in increased catabolism or removal of cerebroside. As expected, during recovery, the total amount of cerebroside synthesized (5.1 nmol/mg wet weight) was more than enough to account for the amount of cerebroside accumulation (3.8 nmol/mg wet weight).

The data relating to cerebellum is less striking; a 20% decrease in total synthesis of cerebroside and a 32% decrease in concentration of cerebroside. Interestingly, the mild 12% cerebroside deficit in brainstem is less than the 30% deficit in cerebroside newly synthesized over the 12-week period (see Discussion).

Changes in levels of mRNA coding for CGT also reflect demyelination/remyelination

There is a profound down-regulation of mRNA for CGT (ceramide galactosyltransferase, the rate-limiting enzyme for cerebroside biosynthesis) in corpus callosum (Fig. 1c). Even in the presence of continued challenge with cuprizone, there is complete return to normal levels of mRNA. If cuprizone exposure is continued, there is another fall in mRNA levels followed by partial recovery. This pattern is as expected (Matsushima and Morell 2001); the available evidence suggests that upon cuprizone challenge, oligodendroglia go through apoptosis, myelin is removed, and precursor cells infiltrate and differentiate into myelinating glia – a cycle which can be repeated (Mason et al. 2001). Again, the pattern in corpus callosum is an exaggerated version of that noted previously in whole brain (Jurevics et al. 2001). Data for CGT mRNA from the other brain regions (Figs 1f and i) are less dramatic, but compatible with the data for corpus callosum.

Alterations in cholesterol levels are as predicted for loss of myelin

During cuprizone-induced demyelination, cholesterol levels in corpus callosum were also significantly reduced (Fig. 2a, p < 0.02, mnova). The reductions in cholesterol levels, if calculated in terms of nanomole deficit, are roughly as expected to match the cerebroside deficits. For example, after 6 weeks of exposure to cuprizone in the diet, the deficiency in cerebroside relative to initiation of exposure is 12.5 nmol/mg wet weight, and the deficit in cholesterol is 22 nmol/mg wet weight. The molecular ratio of cholesterol to galactolipid lost is 1.8, only slightly off the expected value of 2.0 characteristic of myelin (Norton 1977). However, in contrast with the myelin and oligodendroglial-specific distribution of cerebroside, cholesterol is distributed in surface membranes of all cells. Thus the cholesterol deficit in terms of percentage decrement of the total is less dramatic than for cerebroside. Similarly, cuprizone exposure induced decrements in the rate of synthesis of cholesterol (Fig. 2b) and of steady state levels of the mRNA coding for the rate-limiting enzyme in cholesterol synthesis (HMG-CoA reductase, Fig. 2c) that are proportionately less than analogous measures for cerebroside. As expected, for cerebellum and brainstem, the decrements in levels of cholesterol (Figs 2d and g), rates of cholesterol synthesis (Figs 2e and h), and HMG-CoA reductase mRNA (Figs 2f and i) are milder than those for corpus callosum.

image

Figure 2. Effects of cuprizone exposure on cholesterol concentration, cholesterol synthesis rates, and HMG-CoA reductase (HMG-CoA Red) steady-state mRNA levels in corpus callosum, cerebellum, and brainstem of mice. See Fig. 1 legend for additional details. Data for cholesterol concentration and synthesis rates are means ± SEM (n = 3); data for mRNA levels are single determinations, and are plotted relative to the mean of the three controls.

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Myelin basic protein levels – another marker for myelin loss

Although cerebroside is generally accepted to be a good marker for oligodendroglial cells and myelin (Kagawa et al. 1996), we also assayed corpus callosum for levels of myelin basic protein (MBP). The deficit resulting from cuprizone exposure and the subsequent recovery are very similar to alterations in cerebroside levels during demyelination/remyelination (compare Fig. 3a with Fig. 1a). This is of interest since presumably MBP is susceptible to many extracellular and intracellular proteases and does not survive the destruction of the myelin sheath. The correlation of the removal of MBP and cerebroside suggests that cerebroside is also rapidly degraded as myelin becomes disrupted. Levels of MBP mRNA also were altered in a manner very similar to those for CGT (compare Fig. 3b with Fig. 1c).

image

Figure 3. Effects of cuprizone exposure on myelin basic protein (MBP) steady-state mRNA and protein levels. See Fig. 1 for additional details.

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GeneChip analysis of RNA from corpus callosum

The corpus callosum, as dissected from brains of cuprizone-exposed animals, represents a preparation in which the sequence and time-course of events is quite reproducible. As we have shown above, due to the severity of the demyelination, specific biochemical alterations related to demyelination and remyelination are easy to discern above the general metabolic background. This should be true also for gene expression. Thus, we used Affymetrix GeneChip technology to analyze mRNA expression in corpus callosum at various stages of demyelination and remyelination (all GeneChip data are for 6 weeks of cuprizone exposure followed by 6 weeks of recovery). We did not consider as significantly altered any gene whose peak expression was less than one-third of that of the ‘average’ gene on the chip, although we realize this arbitrary discriminator may exclude genes whose up-regulation is of physiological significance.

Preexisting information regarding the cuprizone exposure model system allows for testing for specific expectations. GeneChip data for CGT (GenBank accession # U48896) is similar to that obtained by northern analysis (compare Fig. 4, closed circles, to that in Fig. 1c). Other data in Fig. 4 indicate the expected lesser alterations for HMG-CoA reductase mRNA (M62766). Two other profiles for mRNA species involved in more general aspects of lipid metabolism are included; those for fatty acid synthase (X13135) and stearoyl desaturase-1 (M21285) also lack the cuprizone-induced profound down/up-regulation of the more myelin-specific CGT mRNA.

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Figure 4. GeneChip analysis of effects of cuprizone exposure on steady-state mRNA levels of selected enzymes involved in lipid synthesis in corpus callosum of mice. Data are from animals exposed to cuprizone for up to 6 weeks and then (for later time points) allowed to recover. Relative mRNA levels were determined from Affymetrix GeneChip data using GeneSpring analysis software. Average difference values for each time point were normalized to the overall average value for all genes on the chip; these values were then plotted relative to the corresponding control value for that gene obtained from three control samples.

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The patterns of some genes coding for membrane components present in myelin or in oligodendroglial cells are shown in Fig. 5(a). These include the well characterized proteolipid protein (PLP, AI85038) and myelin-associated glycoprotein (MAG; M31811; for references, see Morell and Quarles 1999). Also included are results for myelin-oligodendrocyte glycoprotein (MOG, L20942; for review see Johns and Bernard 1999), myelin-associated oligodendrocyte basic protein (MOBP, U81317, Yamamoto et al. 1994), myelin and lymphocyte protein (MAL, Y07812, Schaeren-Wiemers et al. 1995), and oligodendrocyte transmembrane protein (OTP, U19582, Szuchet et al. 2001). We do not report results for a gene listed by Affymetrix as myelin basic protein (M11533), since they were not readily interpretable. We do not have access to the sequences on the GeneChip and suspect this may be some representation of isoforms of MBP or of sequences from the Golli gene (this larger and earlier-expressed gene includes the MBP sequence; for references, see Campagnoni and Skoff 2001).

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Figure 5. GeneChip analysis of effects of cuprizone (exposure for 6 weeks, followed by recovery) on steady-state mRNA levels of myelin or myelin-related proteins (a) and of other genes showing temporal patterns of mRNA expression similar to MOG (b). Data were analyzed and plotted as described in Fig. 4.

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We used GeneSpring data analysis software to search for genes that are expressed in the same pattern as MOG, which showed the most dramatic down-/up-regulation among the myelin/oligodendroglial-specific genes. Some of these genes are plotted Fig. 5(b): synectin (AF104358), presumed to be involved in syndecan-4-dependent interactions, with a possible role in assembly of the syndecan-4 signaling complex (Gao et al. 2000); BCL7C (Y11905), related to a gene deleted in Williams' syndrome, possibly resulting from some impaired developmental process (Jadayel et al. 1998); G-protein coupled receptor 26 (GPC R-26; U13370), distantly related to some known receptors and found in brain (Lee et al. 2000); heterogeneous nuclear ribonucleoprotein U (hnRNP U; AF073992), interacts with class III POU factors (Malik et al. 1997) that are well expressed in the oligodendrocyte lineage (Schreiber et al. 1997); and NK6 transcription factor related (NK6 TFR; L08074, also called Gtx), this gene is expressed during myelination and re-expressed during remyelination after experimentally induced demyelination (Sim et al. 2000); kinesin family 5C, kinesin heavy chain (Kinesin HC; X61435); several expressed sequence tags (ESTs) also showed similar temporal patterns of expression − one of these (AA797709) is shown in the figure.

The temporal expression pattern of genes associated with myelin is consistent with demyelination followed by remyelination; other genes with similar temporal expression patterns may be linked to these processes as well. We caution that these microarray data are preliminary – comparison of one chip at each age point to the mean of three control chips. The validity of the interpretations rests upon the normalization procedures built into the analysis of data from each chip and, importantly, the demonstration of expected temporal specificity as related to chemical analysis and histology. With respect to these results and those presented below, we argue only for the utility of this model system and that the observations are worth follow-up with more quantitative techniques such as northern blots or real-time PCR.

Another reference point for temporal analysis of microarray data is the time-course of accumulation of microglia/macrophages in corpus callosum. Lysozyme is a good marker for activated macrophages (Cross et al. 1988; Venezie et al. 1995). Up-regulation of lysozyme mRNA in cuprizone-exposed mice correlates with immunohistochemical evidence for recruitment of microglia/macrophages and consequent demyelination (Morell et al. 1998). Expression of lysozyme M (M21050) and other genes with similar temporal patterns are shown in Fig. 6. Note that these genes go from a normally very low level to a significant level of expression. Thus a plot of mRNA levels ‘relative to control values’ might give misleading results since the control level of base expression is very low but variable. Therefore, we have included in Fig. 6 the magnitude of the peak response relative to the average expression level of all the genes on the chip. Among the representative genes plotted, some are directly involved as macrophage components or are factors involved in their differentiation: macrophage metalloelastase (MME; M82831); histocompatibilty 2 antigen 1-E (HC-2A; X00958); leukocyte common antigen (L-CA, M23158); hematopoietic lineage switch 2 protein (HLS2, AF009513); involved in the erythroid to myeloid lineage switch.

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Figure 6. GeneChip analysis of effects of cuprizone (exposure for 6 weeks, followed by recovery) on steady-state mRNA levels of the macrophage marker, lysozyme M, and of other genes showing temporal patterns of mRNA expression similar to lysozyme M. Data were analyzed and plotted as described in Fig. 4.

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Also induced were markers less specific but clearly with functions relevant to recruitment and activation of phagocytic cells. Examples include phospholipase D1 (PLase D1; U87868), a widely distributed activity involved in membrane trafficking (see Jones et al. 1999 for review). Other molecules involved in signaling possibly related to such recruitment and activity include TrkB (M3385), known to play a role in neural development (e.g. Frost 2001) and possibly in glial cell biology (Althaus and Richter-Landsberg 2000), and R-ras (M21019), a monomeric GTPase known to activate αMβ2 integrin and thus promote phagocytosis (Self et al. 2001). The above are representative of genes whose increased expression during microglial/macrophage recruitment might be expected. Many genes were identified with similar patterns of expression in response to cuprizone exposure, and these might be relevant for future targeted study. Again, these include a number of ESTs; AI019327 is plotted in Fig. 6 as an example.

Another group of genes of interest (Fig. 7) includes those with a temporal pattern similar to insulin-like growth factor 1 (IGF-1, X04480). Appearance of IGF-1 in this model is prominent in the corpus callosum only after apoptosis of oligodendroglial cells (Mason et al. 2000). A literature suggests IGF-1 acts to promote early differentiation of oligodendroglial precursors (e.g. Ye et al. 1995; see Mason et al. 2000, 2001 for other references). Other potential signaling molecules that may promote oligodendroglial precursor recruitment and differentiation could include those with similar temporal patterns of expression (Fig. 7). Some of these genes could also be involved in recruitment or stimulation of microglia/macrophage. Expression of mRNA species with the temporal pattern characteristic of IGF-1 include: JE protein (M19681); monocyte chemoattractant protein-1 (MCP-1), a CC chemokine involved in recruitment of monocytes; MIP-1 gamma (U49513), mouse macrophage inflammatory protein-1 gamma, a CC chemokine family member; LIX precursor (U2727), a lipopolysaccharide response gene possibly involved in recruitment of inflammatory cells; SOCS-3 (U88328), suppressor of cytokine signaling-3 protein; MIP (M58004), a macrophage inflammatory protein related to MIP-1; CCR5 (AF022990), CC chemokine receptor-5, a coreceptor for HIV-1; T-cell receptor (L2G25B), similar to macrophage inflammatory protein-1 alpha precursor; and angiogenin (U22516), a blood vessel-inducing protein.

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Figure 7. GeneChip analysis of effects of cuprizone (exposure for 6 weeks, followed by recovery) on steady-state mRNA levels of insulin-like growth factor-1 (IGF-1) and on other genes showing temporal patterns of mRNA expression similar to IGF-1. Data were analyzed and plotted as described in Fig. 4.

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Some of the examples above have been examined for function, such as MIP-1α, or are currently being examined (McMahon et al. 2001).

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

A goal of the present study was to develop a model system in which perturbations in chemical composition and metabolism reflect primarily events involved in demyelination and subsequent remyelination. Many models of demyelination exist and some of them can be coupled with studies of remyelination. Examples are experimental allergic encephalomyelitis (for reviews, see Raine 1984; Martin et al. 1992) and virally induced demyelination (Knobler et al. 1983; Miller et al. 1996; Stohlman and Hinton 2001). Local application of lysolecithin and ethidium bromide (Woodruff and Franklin 1999) offers a model in which remyelination is not confounded by continuing immunological or viral challenge. Transgenic manipulation approaches also exist (Mathis et al. 2000). Morphological studies with these and other systems have given considerable insight into the pathophysiology involved in demyelination and remyelination. Still, the total loss of myelin is slight, and the lesions sufficiently diffuse, so that it is difficult to dissect and analyze reproducibly compositional alterations in affected brain regions. We have now demonstrated the feasibility of conducting metabolic and biochemical studies of the corpus callosum of mouse brain during cuprizone-induced demyelination and remyelination. The marked changes observed reflect events related to demyelination and remyelination, and the temporal patterns of metabolic parameters allow for a close correlation with previously published morphological studies of these processes (for review, see Matsushima and Morell 2001).

That the decrement in myelin is profound and preferential to the corpus callosum is evident simply from analysis of the myelin and oligodendroglial cell-specific component, cerebroside; the maximal deficit of 77% is twice that of whole brain (compare present results to those previously reported for whole brain; Jurevics et al. 2001). The interpretation of cerebroside decrement reflecting closely the loss of myelin is supported by the demonstration that the changes in levels of MBP mimic those for cerebroside. That the deficit of membranous material is relatively specific for myelin is evident from the data regarding cholesterol levels. Cholesterol is present in membranes of all cell types, including the axolemma and other membranes present in axons, as well as in membranes of any other cellular elements present. Assuming a molecular ratio of 2 : 1 for cholesterol to cerebroside, in control mice some 70% of the cholesterol in corpus callosum is associated with myelin. The loss of cholesterol is almost entirely accounted for by loss of myelin.

Correlation of analytical data with metabolic data supports the suggestion (Jurevics et al. 2001) that, normally, most cerebroside synthesis in the adult mouse brain is to compensate for metabolic turnover of myelin. Study of dissected brain regions increases the level of resolution of the analysis. An early response to the metabolic insult of cuprizone exposure is down-regulation of cerebroside synthesis, presumably reflecting a decrease in steady-state levels of CGT mRNA. The lag in reduction of cerebroside levels may reflect the lag time for myelin to fall apart and be phagocytosed. It might also represent some initial compensation to insult by decreasing the normal rate of metabolic turnover of myelin components. For example, in brainstem the decrement in cerebroside is less than the down-regulation of cerebroside synthesis, suggesting that metabolic turnover of at least cerebroside can be reduced without proportional destruction of myelin.

Our data indicate that, subsequent to cuprizone exposure, major changes in biochemical and metabolic parameters in the corpus callosum reflect primarily events related to demyelination and remyelination. This made it feasible to examine correlated changes in gene expression while minimizing the background of changes in gene expression of cell types not directly involved in demyelination/remyelination. In this context, we acknowledge the utility of combining local lesioning techniques and in situ methods to analyze gene expression during remyelination, as exploited by others (e.g. Sim et al. 2000). This approach allows good correlation of gene expression with cell type, resolution not attainable by even careful dissection. We suggest RNA isolation and analysis as complementary and useful for screening and gene exploration.

As indicated in Fig. 5(a), many of the genes presumed to code for markers relatively specific for myelin or myelinating cells are expressed in the expected pattern. There are other genes of interest expressed in the same temporal pattern (Fig. 5b), and therefore possibly also playing an important role in remyelination. It is not our intent to review functions of known ‘myelin genes’ or to present and discuss exploratory data in detail. We present only a few of the genes selected by the GeneSpring program under stringent criteria – that the peak value for each gene be at least one-third of the whole-chip average. Commonly used less-stringent criteria would provide hundreds of candidate genes to discuss. Restricting ourselves to a few interesting examples, we note the presence of genes involved in signaling, such as synectin and G-protein coupled receptor 26. There are genes that may be involved in early differentiation such as hnRNP U and BCL7C. The involvement of kinesin family 5C-kinesin heavy chain is interesting in view of a recent report of the presence in ‘myelin sheath assembly sites’ of mRNA coding for some proteins originally implicated in axonal transport (Gould et al. 2000). Although not shown in the figures, a number of expressed sequence tags (not identified and therefore of unknown function) also met criteria of being expressed like myelin genes. We suggest this model system may be useful in study of the repertoire of genes expressed by oligodendroglia during myelination.

We also present some data on a gene presumed to be of microglial/macrophage origin (lysozyme M) and of genes with similar temporal expression patterns. Again, many genes are identified in such a screen and we have listed a few whose involvement in recruitment and late differentiation of phagocytic cells can be easily rationalized. Absence of one of these, MIP-1α, has been shown to decrease slightly the recruitment of microglia/macrophages and delay onset of cuprizone induced severe demyelination (McMahon et al. 2001). Analogous to the situation with ‘myelin’ genes, even with the stringent selection criteria noted, there are many genes and expressed sequence tags identified whose function in the context of microglia is not obvious. As a starting point for speculation, we note that our previous work indicates that cuprizone induces apoptosis of oligodendroglial cells; this is followed by recruitment of microglia/macrophages which phagocytose myelin (Mason et al. 2000). We speculated that phagocytic cells might release factors involved in recruitment and differentiation of oligodendroglial precursors required for remyelination. Candidate genes for such a function might show up in the screen for genes temporally expressed with lysozyme M.

There is prominent early up-regulation of mRNA levels for a number of growth factors and cytokines (Fig. 7). Genes in this classification of temporal appearance might well be involved in recruitment of progenitor oligodendroglia or, earlier on, in recruitment of microglia/macrophages. Indeed, IL-1β and TNF-α have been recently shown to be involved in promoting repair of demyelination lesions (Arnett et al. 2001; Mason et al. 2001). Of further interest is SOCS-3, a gene presumably involved in the control of cytokine signaling (Starr et al. 1997). Sampling of more time points should give the greater temporal resolution required to differentiate more subtle differences in timing of gene expression (e.g. Hinks and Franklin 1999). Negative data (genes which did not change significantly with respect to mRNA expression) might also be useful in testing hypotheses concerning the pathophysiology of cuprizone-induced demyelination and subsequent remyelination. Noted also is the possibility of comparing gene expression in corpus callosum to that in the much less severely affected brainstem; this might give insight into why some brain regions are preferentially resistent to this metabolic insult. Finally, we remind readers that the data for any specific gene identified in our GeneChip study be considered with caution, since results have not been verified by more specific and quantitative methods. Nevertheless, we believe the weight of the evidence supports our claim of results from microarray analysis as a potentially important tool in studies of the pathophysiology of demyelination and of the mechanisms of remyelination.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Drs Arthur Roach and Leroy Hood for kindly providing the MBP clone, Drs Norbert Stahl and Brian Popko for the CGT clone, and Drs Kevin Strait and Jack Oppenheimer for the HMG-CoA reductase clone. We thank Dr Brian Popko, Michael Vernon and Krystle Strand for advice and assistance with GeneChip analysis. This study was supported by USPHS grants NS-11615, NS-37815, and NS-35372, and grants from the National Multiple Sclerosis Society RG-3052-A, RG-2754-A, and RG-3291-A.

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