MRS of Canavan’s Disease,an Exercise in QuantitationResolution of Discrepancies

Bryan Ross and Stefan BlumlHuntington Medical Research Institute, Pasadena, CA

Canavan’s disease, a spongiform brain disorder thatresults from deficiency of N-aspartyl acylase (EC 3.5.1.15),leads to accumulation of the enzyme’s substrate N-acety-laspartic acid (NAA). NAAis excreted in the urine and accu-mulates in the brain. The ease with which NAA is detectedby in vivo MRS has led to many demonstrations of anincreased cerebral NAA/Cr ratio in patients with Canavandisease (see Barker et al., 1991), but the one quantitativeMRS study (see ref. 2) singularly failed to confirm Matalon’soriginal in vitro tissue analysis. This is “bad news” for MRSand for quantitation generally.

We have now studied a single patient with the diseaseand suggest that the results (1) confirm all previous stud-ies and (2) resolve the apparent discrepancies between them.

NAA/Cr is increased in short-echo time spectra as wellas in long-echo time spectra. Altered T2 is not a factor; sim-ilarly T1 is unimportance, since acquisitions at TR = 1.5 and5.0 s appear similar. In contrast to the findings of Barkerand coworkers (see ref. 2), NAA is increased 25–50% in thetwo hemispheres of our patient. Contributing to this increaseis the alteration of brain water (increased from 82 to 97fi)and a considerable degree of ventricular dilatation, with csfcontributing 32% to the volume. If these corrections hadbeen applied to the data of Barker and coworkers (ratherthan the assumption of the brain water—80%, and neglectof csf contributions) NAA would, indeed, have beenreported 30–40% higher in the Canavan patients than incontrols.

This should remind us that quantitation is important inin vivo MRS. However, without rigorous technique and adegree of standardization between investigators, major dif-ferences will appear and clutter the literature with mis-conceptions.

Reference

Barker P. J., Kumar A. J., and Naidu S. (1991) 1H NMR spec-troscopy of Canavan’s disease. 10th Proc. Soc. Magn.Reson. Med. 1, 381.

Remyelination in the Central Nervous SystemHow Might This Be Achieved?

Ian D. DuncanUniversity of Wisconsin, Madison, WI

The central nervous system (CNS) has a limited capac-ity to remyelinate itself in both the genetic leukodystro-phies and in multiple sclerosis (MS). It used to be consideredthat remyelination did not occur in MS, but more recentstudies have conclusively shown that remyelination is pre-sent early in the course of the disease, but in chronic MS,demyelination persists (Prineas et al., 1990). In the inher-ited myelin disorders, such as Pelizaeus Merzbacher dis-ease, there is a failure of myelination, so most axons in theCNS remain persistently nonmyelinated or dysmyelinated.In contrast, in such disorders as metachromatic leukodys-trophy or adrenoleukodystrophy (ALD), myelination pro-ceeds normally, but myelin subsequently is broken down.This demyelination presumably persists as oligodendro-cytes are genetically “disabled” and are unable to re-ensheath denuded axons. It is not clear, however, whetherabortive attempts at remyelination do, in fact, occur in thesedisorders.

Amajor challenge in these diseases, therefore, is to devisestrategies aimed at repair or remyelination of the CNS. Atpresent, two approaches to remyelination can be consid-ered (Duncan and Milward, 1995). In the first, oligoden-drocytes might be replaced by transplantation, either indiseases where they had been lost or where they are genet-ically incapable of myelinating or maintaining a myelinsheath. In the second approach, endogenous remyelinationwould be enhanced by the delivery of substances known

Journal of Molecular Neuroscience© Copyright 1999 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN0895-8696/99/12:179–183/$11.25

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Studies on Myelin and Potential for RemyelinationJames Powers, Chairperson

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to protect or promote host oligodendrocytes or their prog-enitors (Duncan et al., 1997). Clearly the second approachwould only be useful in acquired myelin disorders, such asMS, and not in the genetic diseases.

Transplantation of glia has been extensively performedin investigation of certain aspects of glial cell biology.However, it has been noted in different circumstances thatextensive myelination by transplanted cells of the oligo-dendrocyte lineage can occur, and it has therefore been sug-gested that this approach could have therapeutic value.These experiments have utilized two approaches, first usingthe myelin mutants as hosts of donor cells (Duncan andMilward, 1995), and second, transplanting myelinating cellsinto focal demyelinating lesions in the spinal cord (Blake-more and Franklin, 1991). In the former approach, severalmyelin mutants have been used, but studies in the shiverer(shi) mouse (Vignais et al., 1993) and in the myelin-deficient(md) rat (Duncan and Milward, 1995) have been most numer-ous. We have used the md rat in a wide variety of experi-mental approaches. In summary, we have shown that:

1. Primary oligodendrocytes derived from neonatal oradult donors can myelinate md axons.

2. Mature md rats can be myelinated by donor cells.3. Cells sorted by FACS or immunopanning can myeli-

nate md axons.4. Allografts and xenografts (in immunosuppressed recip-

ients) both make myelin in the md rat.5. Oligodendrocyte cell lines can be used as a source of

myelinating cells.6. md axons can be myelinated by cryopreserved oligo-

dendrocytes.7. Focal myelination of md axons by transplantation

restores conduction velocity.

In order to extend these data in terms of increase in myelinproduction by the transplanted cells, it is essential to findthe best cell for repair. The oligodendrocyte undergoes anintricate series of developmental “steps” as it matures fromthe earliest stem cell through the oligodendrocyte progen-itor, then to an immature and then mature oligodendrocyte(Richardson et al., 1990). These stages have been charac-terized in vitro, and it appears likely that cells at the earli-est stages of the lineage will be the best candidates. At thesestages, the cells are highly mitotic and migratory, two keyfeatures for extensive repair.

We have recently been investigating the potential ofoligospheres, aggregates of free-floating cells isolated fromthe striatum, grown in the presence of B 104 conditionedmedium, to myelinate the md rat CNS. In culture, these cellsgive rise predominately to oligodendrocytes. We have nowshown that when transplanted into the md rat, they migrate

extensively along the spinal cord and myelinate large areas(Zhang and Duncan, unpublished). Although these dataare encouraging, it is still not clear that human cells can bemanipulated in a similar fashion. At present, it appears asif the mitogens known to promote division of rodent gliaare not as effective in human cells, so identifying new mito-gens remains a major challenge (Duncan et al., 1997).

In many of the myelin disorders, not only is there myelinloss and oligodendrocyte depletion, but there is concomi-tant inflammation in the neuropil. Since it is likely that theseinvading cells are producing significant cytokines, a numberof which are proinflammatory (Merrill and Benveniste, 1996)and some thought to be injurious to oligodendrocyte func-tion, it is essential to take this into account prior to trans-planting normal cells. Thus, in both MS and ALD (Powerset al., 1992), inflammation may be a barrier to successfultransplant-induced remyelination. However, the cytokinecascade seen in vivo may be crucial for oligodendrocytefunction as several lines or evidence indicate. Thus, it maybe that inflammation has a positive and not a negativeinfluence on remyelination. This issue urgently requiresresolution.

References

Blakemore W. F. and Franklin R. J. M. (1991) Transplanta-tion of glial cells into the CNS. Trends Neurosci. 14,323–327.

Duncan I. D. and Milward E. A. (1995) Glial cell transplants:Experimental therapies of myelin diseases. Brain Pathol.5, 301–310.

Duncan I. D., Grever W. E., and Zhang S. C. (1997) Repairof myelin disease: Strategies and progress in animalmodels. Mol. Med. Today 3, 554–561.

Merrill J. E. and Benveniste E. N. (1996) Cytokines in inflam-matory brain lesions: Helpful and harmful. Trends Neu-rosci. 19(8), 331–338.

Powers J. M., Liu Y., Moser A. B., and Moser H. W. (1992)The inflammatory myelinopathy of adreno-leukodys-trophy: Cells, effector molecules, and pathogenic impli-cations. J. Neuropathol. Exp. Neurol. 51, 630–643.

Prineas J. W., McDonald W. I., Graham D. I., and Lantos P.L. (eds.) (1997) Greenfield’s Neuropathology, 6th ed. vol.13, Demyelinating Diseases, pp. 813–881.

Richardson W. D., Raff M., and Noble M. (1990) The oligo-dendrocyte-type-2-astrocyte lineage. The Neurosciences2, 445–454.

Vignais L., Oumesmar B. N, Mellouk F., Gout O., Labour-dette G., Baron-Van Evercooren A., et al. (1993) Trans-plantation of oligodendrocyte precursors in the adultdemyelinated spinal cord: Migration and remyelination.Int. J. Dev. Neurosci. 11, 603–612.

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Examining the Role of IL-6in Globoid Cell LeukodystrophySteven M. LeVine and Tanya PedchenkoUniversity of Kansas Medical Center, Kansas City, KS

Twitcher mice are an authentic animal model of globoidcell leukodystrophy (Krabbe’s disease). Cytokines havebeen postulated to play a pathogenic role in this and otherdemyelinating diseases. Initially, we set out to determinewhich cytokines are expressed in twitcher mice tumor necro-sis factor (TNF)-αwas found to be expressed in macrophagesand globoid cells. These cells were present at active sites ofdemyelination. This result indicates that TNF-α is posi-tioned to participate in demyelination events in twitchermice. Interleukin-6 (IL-6) was expressed in reactive astro-cytes and microglia in white matter and several gray matterregions in twitcher mice. Although IL-6 was also expressedin normal mice, the number of cells and expression levelswere dramatically increased in twitcher mice. The spatialdistribution of IL-6 was extended beyond that for TNF-α,but everywhere TNF-α was found, IL-6 was also present.In order to address the function of IL-6 in twitcher mice, weproduced double-mutant mice that carry the twitcher muta-tion and a mutation in IL-6. If these double-mutant micedisplayed an improved clinical and pathological coursecompared to twitcher mice, then this would indicate a path-ogenic role for IL-6. In our initial evaluation of these mice,we observed the following clinical and pathological features:

1. The day of onset for twitching was 23.8 d for the double-mutant mice compared to 29.6 d for twitcher mice.

2. The moribund day was 35.2 d for double-mutant micecompared to 34.3 d for twitcher mice.

3. Astrocyte gliosis was more pronounced in double-mutant mice compared to twitcher mice.

4. The number of globoid cells and macrophages was sim-ilar in these mice.

These data suggest that IL-6 does not amplify diseaseprocesses, but instead, it may help to modulate pathogenicresponses by astrocytes. This modulation may influence theclinical course of this disease.

CoQ10-Responsive MitochondrialEncephalomyopathy Owing to an InbornError of Ubiquinone SynthesisA. Rötig,1 E. L. Appelkvist,2 V. Geromel,1D. Chretien,1 B. Parfait,1 N. Kadhom,1 P. Edery,1M. Lebideau,1 G. Dallner,2 L. Ernster,2A. Munnich,1 and P. Rustin1

1INSERM U393 and Department of Genetics,Hopital Necker, 75743 Cedex 15, Paris, France; and2Department of Biochemistry, Arrhenius Laboratory,Stockholm, Sweden

Ubiquinone transfers electrons from complex I and com-plex II to complex III in the respiratory chain. We reportmultiple respiratory enzyme deficiencies with severeencephalomyopathy and renal failure in two siblings bornto nonconsanguinous parents. Investigation of the mito-chondrial respiratory chain in different tissues revealedreduced complex I + III and complex II + III activities andabnormal activity ratios. By contrast, complex I, II, and IIIactivities were in the normal range. In vitro stimulation ofthe enzyme activities by decylubiquinone as well as thevery low quinone content in lymphoblastoid cell lines sug-gested a CoQ10 deficiency. Growing skin fibroblasts inmedium-supplemented idebenone (quinone analog)resulted in a decrease of decylubiquinone stimulation andan increase of respiratory chain activities. CoQ10 synthesisbranches on cholesterol synthesis pathway, as farnesylpyrophosphate is transformed into decaprenyl pyrophos-phate by iterative prenyl transfers prior to condensationwith 4-hydroxybenzoate and quinone synthesis. Accumu-lation of farnesyl diphosphate and geranyl diphosphate incultured fibroblasts suggested a deficiency of decaprenyl-transferase in our patients. After 6 mo of quinone admin-istration, the physical and intellectual performances of thetwo patients have markedly improved, and respiratorychain activities in circulating lymphocytes have recovered.A widespread expression of the CoQ10 deficiency has notbeen hitherto reported. The extensive investigation of thesepatients and the identification of the disease-causing genewill shed light on an hitherto unknown group of inheritedmetabolic disorders in humans.

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Monoclonal Antibodies and Their Effectson Experimental RemyelinationClaudia F. Lucchinetti, K. Asakura,D. J. Miller, and Moses RodriguezMayo Clinic, Rochester, MN

The traditional concept that remyelination in the centralnervous system (CNS) occurs rarely has been discardedbased on both clinical and experimental observations ofCNS remyelination in both multiple sclerosis (MS) andexperimental models of CNS demyelination. Although thetypical chronic MS lesion shows minimal remyelinationgenerally limited to the periphery of the lesion, the obser-vation of substantial remyelination in some acute MS lesionssuggests that the potential for myelin repair is present inthe CNS. These observations emphasize that CNS demyeli-nating diseases like MS are not simply a static and irre-versible process of myelin destruction as originally thought,but rather represent a dynamic interaction between patho-genic and reparative factors, which results in a delicate equi-librium between demyelination and remyelination. Theidentification of factors that could shift this equilibriumtoward remyelination may have major therapeutic impli-cations not only for MS, but for other myelin diseases aswell. Several experimental models of CNS demyelinationprovide the opportunity to investigate these factors andattempt to develop therapeutic strategies designed to pro-mote remyelination.

We have used a model induced by Theiler’s murineencephalomyelitis virus (TMEV) to study the mechanismsof demyelination and remyelination in the CNS. Followingintracerebral injection of the Daniel’s strain of TMEV intoSJL mice, there is extensive demyelination with relativeabsence of remyelination in the spinal cord. After about 1–3mo, mice develop spasticity, weakness of the lower extrem-ities, and bladder incontinence. Inflammatory infiltrates areclosely associated with areas of demyelination. We consid-ered the possibility that differences in remyelination in theTMEV model may be determined by the immune response.Lang et al. (1984) demonstrated that when SJL mice chron-ically infected with TMEV are immunized with SCH or MBPplus galatocerebroside, they showed substantial remyeli-nation compared to control animals. To test the possibilitythat the humoral response directed against CNS antigenswas responsible for promoting this remyelination, normalsyngeneic mice were hyperimmunized with a homogenateof spinal cord. Serum from these mice was transferred tomice chronically infected with TMEV. This resulted in afour- to sixfold increase in the area of CNS remyelination.In subsequent hyperimmune serum transfer experiments,we confirmed that IgG contained within the anti-SCH serumwas responsible for this effect. These initial experimentssuggested that antibodies directed against self-antigens(normal SCH and possibly other self-antigens) may play abeneficial role by promoting CNS remyelination.

Our recent studies specifically addressed the potentialfor autoantibody-mediated CNS remyelination duringchronic TMEV infection. We identified two monoclonalautoantibodies prepared from splenocytes of mice injectedwith SCH. This involved initially immunizing the mice withSCH and then making hybridomas by fusing splenocyteswith myeloma cells. We then cloned these hybridomas andscreened them for their ability to react with SCH by usingtechniques of cultured glial cell immunostaining, SCH pro-tein dot blot, and SCH ELIZA. Of 100 clones, 5 reacted withSCH, and we expanded these clones via ascites production.We then determined whether any of these clones promotedremyelination in the CNS of infected animals. We intrac-erebrally inoculated the mice and treated them with Mon-oclonal Antibodies (MAbs) for 4.6 wk. Following perfusionand preparation of araldite and frozen sections, we ana-lyzed the spinal cord for evidence of remyelination usingquantitative measurements. We identified two clones (MAB94.03 and 79.08) that promoted remyelination.

MAb 94.03 promoted repair in the CNS of TMEV-infectedmice compared to phosphate-buffered saline (PBS) or IgMantibody-treated control animals. Immunostaining exper-iments demonstrated 94.03 stains oligodendrocytes both onthe surface and in the cytoplasm. Immunofluorescencedouble-labeling experiments for 94.03 and MBP demon-strated that 94.03 stains a relatively late oligodendrocyte(OL) differentiation stage. Localization experiments withradiolabeled 94.03 confirmed that the antibody is accessi-ble to the CNS.

To define the nature of the antigens recognized by SCH94.03, Western blotting was performed with the lysate fromsecondary isolated well-differentiated OLs. Multiple bandswere identified by SCH 94.03. The polyreactivity towardprotein antigen observed by Westem blotting made it impos-sible to determine which band corresponded to the cell-sur-face antigen. In vivo immunoperoxidase and multiple-colorimmunofluorescence staining techniques indicated thatSCH 94.03 is a multiorgan reactive autoantibody. In orderto identify the potential antigens recognized by MAb SCH94.03 a rat brain λ gt 11 cDNA expression library wasscreened with this antibody. Nine independent clones wereidentified. Five clones were identical or highly similar toknown DNAs or protein (rat kinesin light chain, mousethrombospondin 1, mouse oncofetal antigen, RNA poly-meraseβ-subunit and nuclear phosphoprotein). Four cloneswere not homologous to any known genes or proteins. Noneof nine control IgM MAbs showed reactivity with any ofthe nine cDNAclones. By using a solid-phase assay system,we showed reactivity of SCH 94.03 toward several proteinantigens and chemical haptens with prominent reactivitytoward spectrin. Sequence analysis was performed andshowed that SCH 94.03 is encoded by germline genes withno definite somatic mutations.

In summary, SCH 94.03 reacts with several protein anti-gens and chemical haptens, has prominent reactivity towardspectrin, and is encoded by germline immunoglobin genes

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with no definite somatic mutations. These characteristicssuggest that SCH 94.03 has biochemical and molecular char-acteristics of natural or physiologic autoantibodies.

The existence of natural autoantibodies has been demon-strated by numerous laboratories in the past decade. Theyexist naturally and react with autoantigens, are usuallyIgMs, are typically polyreactive, and are encoded directlyby the germline genes with few mutations. The discoverythat certain natural autoantibodies can be beneficial in amodel of CNS demyelination suggests that the presumedpathogenic role of antibodies in demyelinating diseaseneeds to be re-evaluated.

We propose two general mechanisms of autoantibody-mediated remyelination. The first mechanism is based ona direct effect where autoantibodies actively stimulate OLsor cells that secrete factors necessary for OL survival anddifferentiation in vivo as has been demonstrated in vitro.In support of the direct hypothesis, MBASC. 94.03 has beenshown to react with a surface antigen on Los. We are cur-rently testing whether MAb SCH 94.03 has any effect on OLproliferation or differentiation in vitro.

The second general mechanism of autoantibody-medi-ated remyelination is based on an effect where autoanti-bodies inhibit a pathogenetic component of the diseaseprocess, thereby allowing a normal physiologic repairresponse to predominate. We have previously shown thebeneficial effect of immunosuppression in chronic TMEVinfection, thereby indicating that a T-cell-mediated immuneresponse may inhibit remyelination. There are several poten-tial mechanisms whereby beneficial autoantibodiesproduced after myelin destruction could promoteremyelination via modulation of a pathogenetic immuneresponse, including depletion of pathogenic lymphocytes,inhibition of lymphocyte or macrophage activation, or directneutralization of cytokines preventing remyelination.

There is a strong need for a new therapeutic approachto demyelinating and dysmyelinating diseases. Resultsusing immunosuppression have thus far been disappoint-ing. The development of experimental strategies to promoteCNS remyelination in animal models of CNS demyelina-tion is the first step toward promoting myelin repair inhumans.

References

Asakura K., Miller D. J., Muffay K., Bansal R., Pfeiffer S. E.,and Rodriguez M. (1996a) Monoclonal autoantibodySCH 94.03 which promotes central nervous systemremyelination recognizes an antigen on the surface ofoligodendrocytes. J. Neurosci. Res. 43, 273–281.

Asakura K., Pogulis R., Pease L. R., and Rodriguez M. (1996b)Amonoclonal autoantibody which promotes central ner-vous system remyelination is highly polyreactive to mul-tiple known and novel antigens. J. Neuroimmunol. 65,11–19.

Miller D. J. and Rodriguez M. (1995) A monoclonal autoan-tibody that promotes central nervous system remyeli-nation in a model of multiple sclerosis is a naturalautoantibody encoded by germline immunoglobulingenes. J. Immunol. 154, 2460–2469.

Miller D. J., Sanborn K. S., Katzmann J. A., and RodriguezM. (1994) Monoclonal autoantibodies promote centralnervous system repair in an animal model of multiplesclerosis. J. Neurosci. 14, 6230–6238.

Rodriguez M. and Lennon V. A. (1990) Immunoglobulinspromote remyelination in the central nervous system.Ann. Neurol. 27, 12–17.

Rodriguez M., Lennon V. A., Benveniste E. N., and MerrillJ. E. (1997) Remyelination by oligodendrocytes stimu-lated by antiserum to spinal cord. J. Neuropathol. Exp.Neurol. 46, 84–95.