A new procedure for determining ganglioside GD3 a potential glial cell activation marker in cerebrospinal fluid

NEUROCHEMISTRY International Neurochem[ Int[ 22 "0887# 092Ð097

A new procedure for determining ganglioside GD2 a potential glial cell activation marker in cerebrospinal ~uid Annika Lekman\ Pam Fredman Institute of Clinical Neuroscience\ Department of Psychiatry and Neurochemistry\ Gotebor` University\ Sweden Received 08 November 0886^ accepted 12 April 0887

Abstract Increased amounts of ganglioside GD2 ðII2 "NeuAc#1!LacCerŁ\ associated with reactive gliosis\ have been documented in a variety of neurodegenerative disorders[ GD2 expression has also been reported in microglial cells\ not only during development but also in reactive states\ where the glial activation is considered to be part of the repair process[ It is important to _nd markers in cerebrospinal ~uid that will enable us to identify damage and register changes in pathological processes within the brain[ A sensitive and practically applicable method for determination of GD2 ganglioside in cerebrospinal ~uid has been developed[ The procedure\ which includes extraction\ puri_cation on silica gel and thin!layer enzyme!linked immunostaining\ also allows determination of sulphatide\ a marker of demyelinating processes\ in the same portion of CSF[ The method has been applied to CSF samples from 090 normal individuals aged 1Ð72 years[ The GD2 concentration was found to be signi_cantly correlated to age and re~ecting the concentrations within the brain[ GD2 ganglioside analysis by means of this method might be useful for studying glial changes during brain maturation as well as in brain disorders[ Þ 0887 Elsevier Science Ltd[ All rights reserved[ Key words] GD2^ glycolipids^ sulphatide^ microglia^ neurodegeneration^ demyelination^ gangliosides

0[ Introduction Brain damage is often associated with astrocytic acti! vation as part of the natural repair process and astrocytes form the main macroglial component of this reactive response "Laming\ 0878#[ Essentially all genetic disorders of the central nervous system "CNS# that involve tissue destruction exhibit reactive gliosis "Norton et al[\ 0881#[ This knowledge is based on pathological post mortem investigations[ It is today di.cult to register ongoing processes in the brain including gliosis[ Imaging tech! niques have improved and are valuable tools for rec! ording pathological processes[ However\ these techniques are still limited in resolution and most of them cannot discriminate between ongoing and past processes[ Extended knowledge of these processes in the patient will help us to get a better picture of the development of the disease and thereby also to _nd more e}ective treatments[ From a clinical point of view\ it is also important to be able to register possible e}ects of treatment\ e[g[ dece! lerating gliosis[ Consequently\ it is of great importance Corresponding author[ Institute of Clinical Neuroscience\ Depart! ment of Psychiatry and Neurochemistry\ Sahlgrenska University Hospi! tal\ 320 79 Molndal\ Sweden[ Tel] ¦35!20!751!398^ Fax] ¦35!20!751! 315^ E!mail] annika[lekmanÝms[se 9086Ð9075:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 7 # 9 9 9 1 6 Ð 7

to identify biochemical markers of gliosis that can be assayed in cerebrospinal ~uid "CFS#[ The most com! monly used markers today are glial _brillary acidic pro! tein "GFAP# which is a structural protein of the astroglial _lament "Bignami and Dahl\ 0865^ Reuger et al[\ 0868# and S!099 protein "Moor\ 0854#[ Another possible marker is the ganglioside GD2[ Gan! gliosides are glycolipids localised mainly to the outer surface of the plasma membrane and are continuously released from the neuronal surface to the intercellular space\ which is in direct contact with the CSF "Doljanski and Kapeller\ 0865#[ Ganglioside GD2 is one of the major glycolipid components in the immature CNS but its con! tent decreases signi_cantly during early development "Svennerholm et al[\ 0880#[ It is still present in mature brain tissue but then comprises a minor part of the glyco! lipids[ The observation that GD2 is expressed by imma! ture\ undi}erentiated CNS cells has led to the suggestion that this molecule might be a marker for multipotential precursor cells "Goldman et al[\ 0875#[ Phenotypic alter! ations which take place in association with reactive glial changes includes upregulation of ganglioside GD2 expression on the astrocyte surface "LeVine et al[\ 0875#[ Recent studies have reported GD2 expression also in microglial cells\ not only during development but also in reactive states "Reynolds and Wilkin\ 0882^ Wolswijk\

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0883^ Amat et al[\ 0885^ Goldman and Reynolds\ 0885#[ Increased amounts of ganglioside GD2 have been found to be associated with reactive gliosis in CNS tissue from patients with various neurodegenerative diseases] in mul! tiple sclerosis "Yu et al[\ 0863#\ subacute sclerosing leuko! encephalitis "Ledeen et al[\ 0857#\ Creutzfeld!Jakob dis! ease "Tamai et al[\ 0867#\ metachromatic leukodystrophy "Suzuki\ 0856#\ Krabbe|s disease "Svennerholm and Vanier\ 0861# and in adrenoleukodystrophy "Igarashi et al[\ 0865#[ Our knowledge regarding ganglioside GD2 in CSF is\ however\ limited[ Previously reported data on GD2 in individual samples have included only a few controls and the analytical procedure required 4 ml of CSF "Davidsson et al[\ 0889^ Trobojevic!Cepe et al[\ 0880#[ For routine clinical determinations it is\ in general\ di.cult to use as much as 4 ml of CSF just for one speci_c analysis and especially from children\ from whom at most 2 ml is usually drawn[ The aim of the study was to elaborate a sensitive and practically applicable method that allowed determination of GD2 ganglioside in at most 0 ml of CSF and with the requirement that sulphatide could be measured in the same portion of CSF[ Demyelination is a common feature in neurodegenerative disorders and this emphasis the importance of measuring sulphatide\ a marker of demy! elination in association with GD2 to follow the activity of an ongoing process[

1[ Experimental procedures

by isoelectric focusing "Fredman\ 0881#[ Thus\ there were no signs of intrathecal IgG or IgM production[ 1[1[ Chemicals Silica gel\ 129Ð399 mesh was obtained from Merck AG\ Darmstadt\ FRG and polygram Sil Gþ precoated plastic sheets "09×19 cm# from Marchery!Nagel\ Duren\ FRG[ The substrate 4!bromo!3!chloro!2!indolyl!phosphate "BCIP#\ potassium salt\ was purchased from Sigma\ St[ Louis\ MO\ USA[ Spectraporþ dialysis tubing "12 mm in diam\ pore size 9[9901# was obtained from Spectrum Medical industries\ Los Angeles\ CA[ Ganglioside GD2\ used as standard\ was isolated from bovine brain and structurally characterised in our laboratory[ Tritiated ganglioside GM1\ used for recovery determinations\ was labelled in the sphingosine base with the sodium borohy! dride procedure described by Schwartzmann "0867# and diluted with unlabelled ganglioside to a _nal speci_c activity of 439999 dpm:nmol[ ð2HŁsulphatide was pre! pared and labelled as described elsewhere "Fredman et al[\ 0877# and diluted to a speci_c activity of 7×092 dpm:nmol[ 1[2[ Antibodies The monoclonal anti!GD2 antibody used was DMAb!7\ speci_c for NeuAca1!7NeuAca1!2Galb0!3 Glc!epitope found in GD2 "He et al[\ 0878#[ A.nity puri_ed alkaline phosphatase conjugated goat anti!mouse IgG¦IgM "H¦L# was obtained from Jackson Immunoresearch Laboratories\ West Grove\ PA\ USA[

1[0[ Cerebrospinal ~uid 1[3[ Method CSF samples\ 01 ml from adults and 2 ml from children "³04 years#\ were drawn by lumbar puncture and sub! jected to protein analysis for exclusion of underlying infection[ Out of the 80 adult control subjects\ 64 were healthy volunteers[ The remaining 05 adults and the 09 control children shown minor symptoms like headache but no sign of neurological disease and CSF samples had been taken and sent to our laboratory for protein determinations to exclude CNS infections[ The CSF included in the study were those where the parameters described below were found to be normal[ All the CSF samples contained less than 2×095 mononuclear and no polynuclear cells per litre[ The inclusion criteria for the adults were an albumin content ³299 mg:l\ IgG ³31 mg:l and IgM ³9[35 mg:l[ Corresponding inclusion cri! teria for the control children|s "0[4Ð04 years# CSF were albumin content ³049 mg:l\ IgG ³29 mg:l and IgM ³9[1 mg:l[ The albumin quotient was not allowed to exceed 4[9 in children 0[4Ð04 years of age\ 5[7 in adults 04Ð34 years of age and 09 in adults over 34 years of age[ The controls all had normal IgG and IgM indices and there were no detectable oligoclonal IgG bands as veri_ed

The procedure is a modi_cation of a previously described method "Davidsson et al[\ 0880#[ Lipids from 0 ml of CSF in a Kimax tube with a te~on!lined screw cap were extracted by addition of chloroform"C#:methanol"M# to a _nal proportion of C:M:CSF of 3]7]2 by volume[ The tube was centrifuged at 0999×` for 09 min at room temperature\ after which the supernatant was transferred to a new kimax tube\ evaporated and redissolved in 9[4 ml C:M:water"W# "59]29]3[4 by volume#[ The sample was applied to a column with 9[3 g silica gel packed in C:M:W "59]29]3[4 by volume#[ Sulphatide was eluted with 0[4 ml C:M:W "59]29]3[4 by volume#\ after which all the gangliosides were eluted with 5[9 ml C:M:W "29]59]19 by volume#[ The ganglioside fraction was evaporated to dryness under a stream of air in a water!bath at 39>C\ dissolved in 9[4 ml water\ transferred to a dialysis tube and desalted by dialysis against running tap water for three days[ After dialysis\ the ganglioside fraction was evaporated to dryness and redissolved in 9[0 ml C:M:W "59]29]3[4 by volume#[ ð2HŁ!GM1 was used as an internal standard in the serial determinations of GD2 ganglioside

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094

in CSF and 099 pmol "10499 cpm# was added to each sample after the silica gel chromatography[ Triplicate samples of CSF ganglioside fraction cor! responding to 49 and 099 ml of CSF and known amounts of ganglioside GD2 ranging from 9[7 to 5[2 pmol were spotted as 4 mm lanes on plastic backed TLC!plates and chromatographed in C:M:9[14) aqueous KCl "49]39]09 by volume#[ The plate was dipped twice "for 4 and 2 min# in 9[4) polyisobutylmethacrylate in hexane\ dried and unspeci_c binding blocked by preincubation for 29 min at room temperature in Tris!BSA "49 mM TrisÐHCL\ pH 7[9\ 9[14 M NaCl and 0) bovine serum albumin\ BSA#[ Thereafter the plate was incubated overnight at room temperature\ with 19 ml monoclonal anti!GD2 antibody "9[14 mg:ml# in TrisÐBSA[ After rinsing for 3×0 min with phosphate bu}ered saline "PBS] 9[04 M NaCl and 9[90 M sodium phosphate\ pH 6[3#\ the plate was incubated with 19 ml alkaline phosphatase!conjugated goat anti! mouse antibody "9[5 mg:ml\ IgG¦IgM# in Jackson bu}er "9[92 M TrisÐHCl\ pH 7[9\ 9[14 M NaCl and 0[4) BSA# for 2h at room temperature[ Thereafter the plate was rinsed again with PBS and the bound antibody was visu! alised by addition of a substrate solution "9[2 nM BCIP in 9[0 mol:l glycine bu}er\ pH 09[3\ containing 0 mM ZnCl1 and 0 mM MgCl1# and incubated for 09Ð19 min in an oven at 26>C[ The ganglioside antigens were quanti_ed by densitometric scanning of the plates at 519 nm on a CAMAG TLC Scanner II[ The recovery of GD2 in the extraction and puri_cation steps was assayed by addition of known amounts of GD2 to CSF aliquots\ which thereafter were extracted\ applied to silica gel columns and analysed according to the described method[ The procedure was repeated without standards in order to obtain a CSF blank value which was subtracted from the recovery samples before calculation[ The described method was developed to allow deter! mination of sulphatide\ a potential marker for demyelina! tion "Davidsson et al[\ 0880^ Fredman et al[\ 0881^ Gisslen et al[\ 0885#\ in the same CSF sample[ The procedure normally used for determination of the so!called ganglio! tetraose series gangliosides in CSF "Lekman et al[\ 0884# includes extraction and dialysis but no column!chromato! graphy and allows determination of all gangliosides including GD2[ The method does not allow deter! mination of sulphatide in the same portion of CSF\ how! ever[ For comparison between the two methods\ 09 samples of pooled CSF were analysed[ The albumin quotient\ CSF!albumin "mg:l# divided by serum!albumin "g:l#\ was used as the measure of the bloodÐbrain barrier function[

1[5[ Statistical analysis

1[4[ Ethics

Fig[ 0[ Recovery of GD2 in cerebrospinal ~uid[ Known amount of GD2 was added to CSF!samples[ The absorbance of the CSF was substracted from the obtained value and the net absorbances of GD2 after the experiment was plotted against a standard curve[ P!value "Spearman rank correlation test# for the correlation is shown[

The study was approved by the Ethical Committee of Gothenburg University[

Comparisons between di}erent age!groups of patients were performed using Wilcoxon|s rank sum tests[ Values of p³9[94 were considered signi_cant[ Correlations were tested using Spearman rank correlations[

2[ Results and discussion The present method allows determination of GD2 gan! glioside in 0 ml of CSF and the determination is sensitive enough to allow measurements in CSF from children[ The procedure also allows determination of gangliotetraose series gangliosides and sulphatide in the same CSF sample "Davidsson et al[\ 0880^ Lekman et al[\ 0884#[ Ganglioside GD2 might be measured with the pro! cedure used for determination of the gangliotetraose series gangliosides "GM0\ GD0a\ GD0b and GT0b# "Lekman et al[\ 0884#[ This method has the advantage that it does not include any separation steps and thus exclude selective loss of individual gangliosides[ However\ this method does not allow determination of sulphatide in the same portion of CSF[ The comparison estimated on 09 pooled CSF samples\ resulted in a con! centration of 38[626 nM "mean2S[D[# with the pro! cedure presented in this study and 33[825 nM with the previously described "Lekman et al[\ 0884#[ Thus no di}erence was found with regard to GD2 recovery between the two methods\ which demonstrates that all GD2 was eluted in our ganglioside fraction[ Recovery of GD2 was also tested by adding increasing amounts of GD2 to CSF!samples "Fig[ 0# and the results showed a linear recovery of GD2[ In the recovery studies\ the absorbance value of the CSF was subtracted from the

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value obtained on the samples to which GD2 had been added[ The net absorbances of ganglioside GD2 were compared with the standard curve "Fig[ 0#[ A linear func! tion describing the relationship between known amount of added GD2 and estimated GD2 after the experiment was calculated^ linear regression analysis yielded a cor! relation coe.cient of r9[8833[ In addition\ the same series of GD2 standards without any CSF were submitted to the same procedure and the recovery was 82Ð092)[ The most critical step regarding the recovery of GD2 was found to be the transfer of the sample to and from the dialysis tube[ In order to compensate for varying losses of gangliosides during the dialysis step\ ð2HŁ!GM1 was added as an internal ganglioside standard[ ð2HŁ!GM1 is the ganglioside normally used in our routine deter! minations of gangliotetraose series gangliosides "Lekman et al[\ 0884# and as ð2HŁ!GM1 does not interfere with either the gangliotetraose series ganglioside or the gan! glioside GD2 determinations\ it was preferred as an internal standard[ The optimal procedure would have been to apply ð2HŁ!GM1 to the CSF!sample before extraction but it had to be added after the silica gel chromatography\ as this ganglioside was partially eluted in the C:M:W "59]29]3[4 by volume# fraction used for the quantitative elution of sulphatide[ The recovery result for GM1 in each individual sample was used to extrapolate the CSF ganglioside values to 099)[ A linear relationship between the absorbance and the concentration of GD2 was found in the range 9[7Ð5[2 pmol and the correlation coe.cient varied between 9[87 and 0[99[ The CSF samples with a GD2!concentration below 5 nM were found to give a falsely low value[ In these cases\ a volume of ganglioside fraction cor! responding to 199 ml of CSF had to be applied to the HPTLC!plate[ The imprecisions of this method\ calculated on 7 sam! ples of pooled CSF for the within!run and 09 samples for the between!run determinations\ were 09 and 01)\ respectively[ It is important to use one CSF sample to measure a variety of glycolipids each re~ecting di}erent cellular involvement in a pathological process in the brain[ Sul! phatide re~ects demyelinating processes regardless of eti! ology and will give valuable information\ especially in combination with ganglioside determinations\ which are allowed by the present method[ The recovery of sul! phatide was assayed using liquid scintillation counting of ð03CŁ sulphatide and calculated on 09 samples was found to be 74Ð095) "7825)#[ 2[0[ GD2 concentrations in adults and children In the controls\ the GD2 concentrations in CSF cor! related signi_cantly with age "p³9[990^ Fig[ 1#[ The con! centration of GD2 ganglioside in children aged 1Ð19 years was found to be 1328 nM "mean2S[D[#[ When

Fig[ 1[ Developmental changes of GD2 in CSF from normal individuals[ Signi_cant correlation "p³9[990# was found between GD2 con! centration in CSF and age[ The data obtained in each age!group are expressed as the mean2S[D[

dividing the adults into smaller groups\ we found the group aged 10Ð39 years to have a signi_cantly "p9[9927# lower concentration level "2527 nM#\ than the older groups "49200 nM#[ "There was no di}erence between the group 30Ð59 years and ×59 years#[ No sex di}erence was found except in the oldest group "×59 years of age#\ where a signi_cantly "p9[9924# higher level was found for men\ with a concentration of 53[5202 nM\ while the level for women was 34[8209 nM[ There was no signi_cant di}erence in GD2 concentration between the di}erent control groups "p9[0879#[ Pre! viously reported "Davidsson et al[\ 0889^ Trobojevic!Cepe et al[\ 0880# values of GD2 ganglioside content in CSF di}er from our results\ probably due to di}erent ana! lytical procedures[ Furthermore\ the results in one of these studies were based on pooled CSF samples and in none of the studies the controls were subjected to rigorous inclusion criteria and could therefore not be considered as normal[ Increased passage of blood components to CSF is a well known problem in the presence of bloodÐCSF bar! rier dysfunction[ Interference by blood components of the CSF!samples might also depend on bleeding during lumbar puncture or as a result of neurological damage[ GD2 represents 29) of the glycolipid concentration in serum "Kundu et al[\ 0874# and reduced function of the bloodÐbrain barrier due to damage or disease might lead to leakage of plasma gangliosides into CSF[ This is a confounding factor when the analysed CSF marker also occurs in serum[ Accordingly\ the increase of GD2 in elderly individuals could be due to a less e.cient bloodÐ CSF barrier function[ Determination of the con! centration of albumin in CSF and serum\ that is the CSF:S albumin ratio\ is the most commonly used method for assessing bloodÐCSF barrier dysfunction "Blennow et al[\ 0882#[ However\ we found no correlation between

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the concentration of GD2 and the albumin quotient with the inclusion criteria used in this study "Fig[ 2#[ We there! fore propose that the increased amount of GD2 found in CSF most likely originates from cells within the brain and re~ects an increased number and activation of astro! cytes or microglia[ In the brain GD2 constitutes 7Ð09) of total gan! glioside sialic acid at the end of the _rst trimester\ dimin! ishes to 2) at the end of the second trimester and then starts to increase again\ accounting for 3Ð4) of total ganglioside sialic acid from 1 years of age "Svennerholm et al[\ 0878#[ The concentration of GD2 in cerebral cortex increased by 39) from 19 years to old age\ which was suggested to re~ect an increased glial activity with aging "Svennerholm et al[\ 0883#[ Our results regarding age! related changes of GD2 in CSF are in accordance with those in brain tissue[ A larger variation of GD2 was found in the oldest group "×59 years#\ as can be expected among individuals with di}erent degrees of age!depen! dent changes during physiological aging[ GFAP\ today the most common marker of astrocytes\ is known to be highly susceptible to degradation by cal! cium!dependent proteinases and the CSF samples must by frozen immediately[ Gangliosides\ however\ are extremely stable\ which facilitates the handling of CSF samples and adds to clinical usefulness[ We propose that an increase of GD2 in CSF probably re~ects activation or changed metabolism of astrocytes\ but might even re~ect proliferation of astrocytes as well as microglia[ The present method also has the advantage of allowing determination of various glycolipid markers in the same portion of CSF re~ecting di}erent degenerative pro! cesses[ In summary analysis of ganglioside GD2\ by means of this method\ might be useful for studying glial changes during brain maturation as well as registering and moni! toring di}erent pathological processes[

Fig[ 2[ GD2 concentration in CSF was plotted against the albumin quotient[ The linear regression curve is shown[

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Acknowledgements This study was supported by Landstingsgemensamt FoU!rad "Bohuslandstinget\ Sweden# and the Swedish Medical Research Council "Project]K86!01X!98898! 95A#[

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