/~eurochem Int Vol 20, No 3, pp 421M.31, 1992 0197-0186/9255 00+0 00 Printed m Great Britain All rights reserved Copyright ,c 1992 Pergamon Press plc

GANGLIOSIDES IN THE HUMAN BRAIN DEVELOPMENT AND AGING

IVICA KRACUN, t']" HARALD ROSNER, ~ VALERIJA DRNOVSEK,~ ZELJKA VUKELIC, CEDOMIR COSOVIC,~ MILICA TRBOJEVIC-CEPE ~ and MILOVAN KUBAT 2

~Department of Chemistry and Bmchemistry. Neurochemical Laboratory and 2Department of Forensic Medmme, School of Me&clne, University of Zagreb, Salata 3, 41000 Zagreb, Croatia

3Institute of Zoology, University of Hohenhelm, Stuttgart, Fed Rep Germany

( Recen,ed 10 July 1991, accepted 20 July 1991 )

Abstract--In this study, brain ganghosldes in prenatal and postnatal human life were analyzed Immuno- hlstochemlcally, the presence of"c"-pathway of ganghosldes (GQIc) m embryonic brain was only recorded at 5 weeks of gestation Biochemical results indicated a twofold increase in human cortex ganglioslde concentration between 16 and 22 weeks of gestation The increasing ganghoslde concentration was based on an increasing GDIa ganghostde fraction m all regions analyzed except cerebellar cortex, which was characterized by increasing GTIb In this developmental period, GD3 was found to be localized in the ventncular zone of the cortical wall After birth, GDlb ganghoside in neuropd of granular cell layer corresponding to growing mossy fibers was expressed in eerebellar cortex Between birth and 20/30 years of age, a cerebral neocortical &fference of ganghoside composition was observed, characterized by lowest GDIa m wsual cortex Analyzing the composmon of gangliomdes m cortical regmns during aging, they were observed to follow region-specific alterauons. In frontal cortex, there was a greater decrease m GDla and GM1 than in GTlb and GDIb, but in occipital (visual) cortex there was no change in individual ganghomdes In hlppocampus, GDla moderately decreased, whereas other fractmns were stable In cer- ebellar cortex, GD I b and GT I b fractmns decreased with aging

*Parts of the results of this refereed paper were presented at the ESN Satellite Meeting Chmcal and Behavtoural A wet t~ o[ GanghosMe Research, held in Magdeburg, 28 31 July 1990 The Satellite Meeting was organized by Dr H Schenk, The Me&cal Academy of Magdeburg, Fed Rep Germany, and Dr G. Tettamanti, The Uni- versity of Milan, Milan, Italy

+Author to whom all correspondence should be addressed ~bbreviations a/b ratio, G D l a ( G T l a ) + G M 1 / G Q l b +

G T l b + G D l b , CP, cortical plate, ELISA, enzyme- linked immunosorbent assay, HPTLC, high-perform- ance thin-layer chromatography, LBSA, lipid-bound siahc acid, MAb, monoclonal antibody/ies, PBS, phos- phate buffered saline, SL, subplate layer, wm, white matter, vz, ventncular zone, M, molecular zone, Ad, adult, B, birth, gcl, granular cell layer, fw, fresh weight

Ganghoside abbreviations follow the nomenclature system of Svennerholm (1963) and designations according to IUPAC IUB Recommendations (1977) [GM3] ll ~ NeuAc-Lac~Cer, [GMI] II~ NeuAc-GgOse4Ser, [GDla] IV 3 NeuAc , II 3 NeuAc4GgOse~?er, [GDIb] II ' NeuAc,43gOse4~Cer, [GTlb] IV ~ NeuAc , II ~ NeuAc2~gOse4-Cer, [GQIb] IV ~ NeuAc, , I1 ~ NeuAc,~-JgOse4-Cer

Gangho-senes gangllosldes of &fferent pathways of syn- thesis are discussed as "'a"-pathway (GMI,GDIa, GTIa), "b"-pathway (GDIb,GTIb,GQIb) and "c ' - pathway ganghosldes (GTIc,GQlc,GPIc) (Pohlentz et al, 1988)

Gangltosldes of the so-called gangho-serles are pri- mary neurona l m e m b r a n e glycosphmgohplds impor- tant for dtfjerentzatton (Rolsen et a l , 1984), synap- togenests (Dreyfus et a l , 1980), neurotransmzsston (Svennerholm, 1980), bioelectrogenesis (F l shman and Brady, 1976), and regeneration of neurons (Cuello et al,, 1989)

Concern ing cellular & s t n b u t l o n of ganghosldes in the nervous system, all the three major groups of cells (neurons, ol igodendrocytes, astrocytes) conta in ganghosldes (Wlegandt , 1967, Ledeen, 1978) However, a difference has been observed m gan- ghostde synthesis between mammal i an neurons and ghal cells (Byrne et a l , 1988; vanEchten and Sandhoff, 1989)

Astrogha and ohgodendrogha mamly possess simple ganghosldes GM3, GD3 and aslalohp~ds, respectively (Byrne et al., 1988), and only traces of the gangllo- series ganghosldes. DescrtpUve studies on ganghomde dis t r ibut ion mdt f fe ren t b ram regions of rats (Rosner and R a h m a n m 1987), mtce (Irwin and Irwin, 1979) and h u m a n s (SuzukL 1965 ; Kracun et a l , 1984) sup- port the concept of different ganghomde metabol ism m different bra in regtons. Findings in G M 2 gan- ghosldosis have cont r ibu ted addmona l evidence on

421

422 I KRACUN et al

dlfferentml ganghoslde metabohsm in human brain Namely. both accumulation of ganghoslde GM2 and residual fl-hexosammldase actlwty (Bolhws et al , 1987) as well as neuronal degeneration (Escola, 1961) markedly vary between different bram areas.

Concerning the brain ganghosldes pattern during human development, aging and Alzhelmer's &sease, the question which challenged us was whether all areas of the human brain suffered equally from increasing age and diseases or certain regions underwent more changes than others Data on Alzhelmer 's disease brain are reported in the accompanying paper (Kra- cun et a l , 1991)

EXPERIMENTAL PROCEDURES

Material for btochemwal analy~ts o[ development, agm9 and A lzhetmer'~ disease

Bram aamphny For biochemical analysis, human fetal brains collected from legal abortions (O Novosel Hospital, Zagreb) were 16, 22 and 30 weeks old (on the basis of crown rump length) Brains of newborns (4 months) were from Institute of Pathology (School of Medicine, Zagreb) Adult human brains were collected from the Department of For- enslc Medicine from subjects who had &ed m accidents For aging study, human brains (n = 15) were &wded into three age groups (I) 20-30 years (21, 24, 24, 27, 30), (II) 40 50 years (43, 46, 49, 50, 51) and (III) 80-90 years (79, 80, 80, 81, 90)

For lmmunohlstochemlcal analysis of ganghosldes in the developing human brain, the brains at 5, 17, 22 and 32 weeks of gestation were used Postnatal human brain maturation was studied on one brain of a child (1 5 years of age) and an adult brain (34 years) Embryonic, fetal and infant brains were collected at the Department of Pathology, Banja Luka Clinical Hospital Immunohlstochemlstry was performed on frontal, occipital and cerebellar cortex, and hlppocampus

Neuroanatomz~al pro~edure~ of samples isolation The brains were freed from blood vessels and meninges,

and carefully washed with running water The samples of cortical layers of the human fetal brains

were isolated by dissection of cortical plate (CP, including marginal zone, M) and subplate layer (SL) as well as cortical anlage including all the three layers (Fig 1) Fetal cerebella and hlppocampl were isolated in toto, the latter by dissection of inferior ventrlcular horn

Immediately after receiving the adult brain material, sam- ples of frontal cortex (Brodmann's 10,11), occipital cortex (Brodmann's 17, area strlata), cerebellar cortex and hlp- pocampus were isolated for biochemical analysis Cortex was carefully dissected from underlying white matter

All brain samples were processed within 8-27 h after death and frozen at - 2 0 'C until biochemical analysis

Blo~ heroic al procedures The samples were homogenized in ice-cold water The

total proteins (Lowry et al, 1951) were determined from

Fig 1 Specific lamination of the human fetal cortex at 28 weeks of gestation (A, Nlssl-stammg with Cresyl Violet) and acetylchohnesterase distribution (B, ACHE, EC 3 1 1 7 ) demonstrated by Lewls's modification of Koelle's method (Krnjevic and Silver, 1966) The figure illustrates the presence of AChE in most differentiating cortical layer (subplate layer) and growing fibres (thalamocortlcal) but not in cort- ical plate at 28 weeks of gestation Arrow--front of growing

fibres (gf)

homogenates in duplicate From the homogenate remainder, the ganghosldes were ~solated according to the method of Svennerholm and Fredman (1980) Two allquots of lipid extracts were used for quantitative determination of lipid- bound slahc acids (LBSA, ganghosldes), according to the method of Svennerholm (1957) as modified by Mlettlnen and Takkl-Luukalnen (1959) The ganghoslde content was expressed as/~g LBSA per mg proteins

The composition of ganghosldes was determined by high- performance thin-layer chromatography (HPTLC, Slhcagel 60 plates, Merck, Darmstadt) by use of a double solvent system according to Rosner (1980) (I) C M 12 mM MgCI2 NH4OH, 60 36 62 04, by volume, (II) C M 12 mM MgC12, 60 40 I0, by volume The ganglloslde spots were visualized by spraying the plates with resorcinol/HC1 reagent (Svennerholm, 1957), as suggested by Yu and Ando (1980) The percentage of individual ganghosides was deter- mined by laser densItometry (PMQ 3, Zelss) and the absolute amount of IndIwdual ganghosldes calculated from the total content The so-called a/b ratio of ganghosldes was cal- culated from absolute values (/Jg LBSA/mg proteins) of GTIa + GDIa + G M I / G Q I b + G T I b + G D l b

Immunoht~tochemlcal pro~edure~ and monoelonal antibodies (MAb)

The brain slabs ol different regions were fixed in 1% for- mahn buffered wlth PBS (phosphate buffered saline) for 24 h and stored in hquld nitrogen until immunohlstochemlstry Thin and semi-thin sections were made on a Re~chert-Jung

Ganghosides in the human brain development and aging 423

cryostat, mounted on the chromalaun-gelatme coated glass plates and processed according to standard procedure (Rosner et al., 1988)

The following monoclonal antibodies (MAb) were tested for lmmunoreacUvity m the developing human brain. (1) anti-GD2, (2) anti-GD3, (3) anti-GD2+GDlb; (4) antl- GQlc (Q211); (5) antI-GM3 (MACG1) (Schnever et al, 1989), and (6) Gmix MAb produced in our laboratory

Monoclonal antibody Gmlx was prepared by lmmu- mzauon of Balb/c mice with microsomal fraction of the adult human brain cortex Hybrids were screened according to reactivity against a mixture of gangliostdes containing GM l, GDla, GDlb and GTlb (obtained from Fldia Res. Labs., Abano Terme, Italy) in ELISA test The antibody Is of IgM class and was proven to be equally reacUve against the afore- mentioned ganghoslde fraction in ELISA test However, the lmmunoreactwity was not confirmed in chromatogram-hind- mg assay 0mmunoblot) In spite of this feature, we tested Gmlx MAb was tested by ~mmunohistochemlcal analysis of the fetal and adult brains. Monoclonal antibodies (1)-(3) were purchased from Biotechmk (Munich, Germany) whereas MAb (4) was from Rosner's laboratory. Mon- oclonal antibody against GM3 recognizing one subspecies of GM3 (up to now unidentified) was a generous gift of Dr F Schnever

R E S U L T S

Biochemical results on human brain ganghostdes durmg development

In prenatal human brains two major cortical layers (cortical plate and subplate layer) of the frontal and occipital cortex [Fig. 1 (A)-(B)] as well as hlppo- campus and cerebeilar cortex, were examined. The biochemical values of ganghoside concentrations (#g

LBSA/mg prot.) in these brain parts are shown in Figs 2 and 3

Between 16 and 22 weeks of gestation, ganghoside concentration increased twofold in frontal cortex (Figs 2 and 3), whereas in hippocampus (Fig. 4) this increase was about 30% Cerebellar cortex of a 16- week old fetus was not analyzed. In general, at 16 weeks of gestation human fetal neocortex (frontal, occipital) and archicortex (hlppocampus) possessed high ganglioside concentrations (6-8 #g LBSA/mg prot.) (Fig. 4) In cortical regions analyzed, a high ganglios]de concentration persisted up to 30 weeks of gestation, except in cerebellar cortex where an increas- ing concentration of gangliosides was observed to per- sist into the postnatal period (4th months) (Figs 2 and 4). In cortical plates and subplate layers of neopalhum between 22 and 30 weeks, a high concentration of gangliosides remained in the subplate layer (16 #g LBSA/mg prot.) of frontal and occipital cortex, whereas in the cortical plate it dropped to lower values (8-9/tg LBSA/mg prot.) (Fig. 2).

On the basis of ganghoside composition between 16 and 22 weeks of gestation, in all cortical regions the increasing ganghoside concentration was based on G D l a fraction (up to 4-fold) (Fig. 3). After 22 weeks of gestation, in all cortical areas a moderate decrease of GDla occurred. The pattern of gangliosides in fron- tal cortex from 16 to 30 weeks of gestation in com- parison to postnatal and adult brain was also dem- onstrated by thin-layer chromatography (Fig. 5), showing the presence of ganghosIde fractions mlgrat-

2 0

16

• , 1:)

cn _J

3

Fron ta l c o r t e x

~/ ~ / co.ic., .,=.

I

x ~

I I I I I - - _ J 16 2: ) 3 0 w B 4 m Ad

Occipital corteK

l ° y e r T

ortlcol p la te

f: " x

x a x

I I I I I - - - - J 16 2Z 3 0 w B 4 m Ad

Fig. 2 Variation of total ganghosldes in frontal and occipital cortical layers (see Fig 1), subplate layer (SL) and cortical plate (CP), durmg prenatal development and after birth (B) (4-month old child and adult bram, Ad) ND, not determined, a/b ratlo---a/b ratio of ganghostdes was calculated from ;tg LBSA of

GTIa+GDla+GM1/GQlb+GTIb+GDIb m SL (®) and CP ( x ),

424 l KRACUN et a/

.J

8

6

4

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C P Frontol cortex ° i ' matter

o

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SL Frontol cortex

White matter

1

'I- -~'- - y - -- i _ _

16 :>2 3 0 w B 4 rn Ad

C P OcclpltoI cortex (vzStml)

Grey

rnatie r

o

- r _ _ J

SL Occzp~tal cortex (vzsuol)

16 2 2 30w B 4 m Ad

Fig 3 The composmon of gangllosldes of the cort]cal layers of the frontal and Occipital cortex during human prenatal development m comparison to postnatal and adult brain (see also Fig 2) It is ~mportant to note that fetal cortical subplate layer postnatally mainly corresponds to underlying white ~natter (Kostowc and Goldman-Raklc, 1983) ND, not determined GM1 (A), GDla (©), GDlb (0) , GTlb

(&), GQlb ( I ) , GD3 ( × I

mg as polysmloganghosldes (pentasmlo-'9 at 16 weeks of gestation In frontal cortical plate, between 30 weeks and fourth postnatal month there was a mod- erate increase m G D I a again, accompamed by a GM1 increase In the subplate layer (postnatally wh]te mat- ter), there was a moderate decrease m G D l a as well as m other frachons m parallel w~th an increase m G M I (frontal subplate) and G D I b (occipital sub- plate) In hlppocampus, a decreasing ganglios]de con- centrat~on between 30 weeks of gestation and fourth postnatal month was primarily due to a decrease in G D I a , whereas other ganghoslde speczes remained stable (Fig 4). Cerebellar cortex in the perInatal period (30 weeks~4 months) was characterized by the most prominent rise in GTI b concentration

Alter birth, changes of ganghosldes proceeded in a regionally specific pattern (Fig 6) Postnatally, until 20/30 years of age, a regional neocortlcal difference m ganghoslde composmon occurred in parallel to a decrease in total ganghosldes, particularly G D I a In this period, a rapid decrease of G D l a in occxpltal cortex is a major difference from frontal neocortex.

in which the concentrauon of G D I a exceeded other ganghosldes (Fig 4)

In hlppocampus, the concentraUon of gangllos~des decreased m parallel with a decreasing proporhon of G D I a until 20/30 years of age Cerebellar cortex also showed a decreasing ganghos]de concentration until 20/30 years of age (Fig. 4), however, m parallel with a decreasing G T l b (and moderately GM1 and G D I b ) A specific feature of cerebellar cortex in all developmental, maturation and aging periods was a significant proportion of ganghoslde GD3 m com- parison to other cortical regions (Fig 4)

Immunohz~tochemwal re~ult~ on antt,qanghosMe rnono- chmal anttbodte,s reactn,t O' m the developm 9 and adult human brain

The results on lmmunohlstochem~ca] reactivity of different MAb against ganghosldes are presented in Table I Although the analysis of ganghoslde lmmu- noreactwity was merely quahtatlve, &ffercntml tem- poro-spatml expression of the antigens was dem- onstrated

6

4

2

Ganghosldes in the human brain development and aging

/~, ~, Frontal cortex 16

12

8

4

425

D 2 Q.

E

.,_1

>

Occipital cortex [vlsuol)

~ ' l - - f / / I 1 ~ 11

t 6 I I I

12 L

Q,

<

II1 _1

H,ppocompu$ -- 12

4 /" - - ~ +

6 I 4

2

I 16w

Cerebellar cortex

. + _ ~ ~ __-- " + . . . . . 4 ~ . .

22w 30w B 4m 20 /30y 40 /50y 80190

12

8

4

Fig 4 Variation of total and mdwldual ganghosldes (expressed in #g lipid-bound slahc acids, LBSA) in human brain cortex d u n n g prenatal development and aging For abbreviations of ganghoslde species see Fxg 3 In brains of 16, 22 and 30 weeks of gestation and 4th postnatal mon th values of total ganghosldes are expressed as s tandard error of mean, whereas m brains of 20/30 (n = 5), 40/50 (n = 5) and 80/90 (n = 5) years of age values are expressed as mean standard deviation Left ordinate, #g LBSA of individual

ganghosldes per mg proteins GM1 (A) , G D l a (O) , G D I b ( 0 ) , G T l b (A) , G Q l b (11), GD3 ( × )

In the embryonic human brain at 5 weeks of gestation, the presence of GD3 (Fig 5), GDlb , and GQ l c ganghosides was demonstrated.

MAb against GD2 ganghosIde was reactive only in 17 weeks old fetal frontal cortex sections.

G D I b ganghoslde was consistently positive in all material analyzed, however, different cellular dis- tributions were observed Ependymal layer hnmg the roof of the fourth ventricle expressed G D l b gan- ghoslde at 17 weeks of gestation. In cerebral cortex,

426 I KRA('UN t ' l t l /

F

Q

' I ' ' l l l

Q

O

3M3 M2

D3

Dtb Tlb 01b

St. St,

22,w 30,w 30,w Ad

I-'lg 5 Thin-layer chromatographic separahon of ganghosJdes of the human frontal cortex (~hole cortlc,d anlage, including molecular zone, corhcal plate and subplate layer) during prenatal development ]n comparison to adult human cortex (Ad) St. standard ganglzoslde mixture Ganghoszde fractions were

designated by numbers probabl~ corresponding to GTIa (1), GTIc (2), GPlc (3) and GH (4)

including frontal, visual and h lppocampus , the dis- t r ibut ion of G D I b was even wi thout laminar appear- ance However, in cerebellar cortex of bo th child and adult, GD1 b was expressed in distinct cortical (fiber) lamina (within granular layer) [Fig 6 (D) and (E)]

The presence of GD 3 ganghoslde was demonst ra ted m h u m a n fetal neocortlcal anlage at 17 weeks of ges- tation. Cellular expression of G D 3 m h u m a n fetal h lppocampus at 17 weeks of gestat ion was restricted to the ~entncular zone [Fig. 6 (B) and (C)] This fraction remained strongly and permanent ly present in the white mat ter of fetal and postnata l cerebellum

Finally, our M A b Gmlx showed cellular lmlnuno- reactlwty to be more p ronounced m fetal than m adult h u m a n brains (Fig 7).

Bto~ hemt~ al re~sults on human hram ganqhomdes' durmq a qtny

After 20/30 years of age. changes m the con- centra t lon of individual ganghosides in brain cortical regions were observed to be region-specific (Fig 4) A decrease in ganghoside concent ra t ion was observed between the third and n in th decade (ca 10 20% in h lppocampus and cerebellar cortex, 40% in frontal cortex, and no changes in occipital cortex) Com- parison of frontal cortex at 20/30 years of age dis-

~losed a much more p ronounced decrease In G D l a and G M I than In G T l b and G D I b In contrast , occipital cortex in the same aging interval showed a stable ganghoslde concent ra t ion and composi t ion, with a pe rmanen t predominance o f " b " - p a t h w a y gan- ghosldes ( G D l b , G T l b ) In hlppocampus, we observed a decreased concent ra t ion of G D l a and GM1 accompanied by an increase in G D I b , as com- pared to brains at 20/30 and 80/90 ~ears of age Never- theless, at 80/90 years of age, h lppocampus still pos- sessed G D l a as a dominan t ganghoslde species In contras t to all o ther human cortical regions, cerebellar cortex sho~ ed a decrease in gangltosldes G T 1 b, G D I b and G D l a between 20/30 and 80/90 years of age. whereas other ganghostdes underwent little change

In conclusion, dur ing the prenatal h u m a n devel- opment , ~ariatxons in brain ganghosldes in four human cortical regions showed changes (comparing "'a"- and - b " - p a t h w a y ganghosldes) that were dlffel- ent in neocortcx (frontal and occipital), archlcortex (h lppocampus) and cerebellar cortex After birth, until 20/30 years of age, region-specific ganghoslde pat tern between polar cortex developed, cont r ibut ing to a low concent ra t ion of G D l a in occipital (visual area, sensory cortex) in compar ison to |¥ontal (motor) cortex Finally, dur ing aging, the change in partlcula~

Ganghosldes m the human brain development and aging 427

Fig 6 (A) Pnmordmm of the brain m the filth week of gestation reactive toward MAb against GD3 ganghoslde The arrows ln&cate mitotically actwe cells (B) (C) lmmunoh~stochemlcal reactwltly of MAb against GD3 ganghoslde (B) in human fetal hlppocampus at 17 weeks of gestation (vz, ventncular zone) (C) &ckdlummatlon picture of brain section on (B) (D) lmmunofluorescence pattern of MAb against GDIb (+GD2) m cerebellar cortex of 1 5 year old chdd and adult brain (E) The arrows point to the

lmmunoreactwe band corresponding to the granular cell layer

ganghoslde species was observed which was not com- mon to all cortical areas analyzed

DISCUSSION

Our results on h u m a n bra in ganghosldes are in agreement with previous findings of the studies deal- mg with deve lopment (Vamer et a l , 1971, Yusuf et a l , 1977, Mar tmez and Bal labnga , 1978, Kracun et

a l , 1986 ; Svennerholm et al., 1991), adul t d is t r ibut ion (Suzuki, 1965, Kracun et a l , 1984) and aging (Segler- Stahl et a l , 1983; Svennerholm et al., 1989) An evidence c o m m o n to all reports ~s tha t m the prenatal deve lopment of h u m a n brain, the most increasing concent ra t ion of ganghoslde G D l a occurs m parallel with mos t mtenswe cortical synaptogenes~s (16-30 weeks of gestat ion) (S~dman and Rak~c, 1973, Kostovlc and Krmpot l c -Nemamc, 1976) Recently,

4 2 8 I K R A ( U N el a[

T a b l e I l m m u n o h l s t o c h e m l c a l react l~mV o f a n t l g a n g h o s l d e s m o n - oc lona l a n n b o d m s m the d e v e l o p i n g a n d adu l t h u m a n b r a i n

M A b 17w 22w 32v, I 5y 34y

I t tHIIH] ( Ot [ e x

G D 2 + - G D 2 + G D I b + + + + + G D 3 - - - G M 3 ( M A C G 1 ) N D N D N D ( h m \ + ~ + W e a k GQI~. (Q21 [)

I I~ll~l[ ¢ OI [U \ ¢ ()~ ( Ipll¢l[ J

( , I ) 2 ( _ , D 2 + G D I b ~- + + + + (J[):~ +

(]M:~ ( M A ( ' G I ) ( . l i ] ] l x ' {. - L __

( , Q l t (Q211) ( t't t'l~t'/ltlt ( Ot It' \

O D 2 - + -- G D 2 + G D I b + + + ÷ +

( JD~ t + + ( ; M ~ ( M A ( ( ; I I W e a k + N D ( J lmx + ÷ N D W e a k W e a k ( , Q I c (Q211) . . . .

Illpprn tIIIIDlt~

(~D2 N D ( J D 2 + G D I b + + + + N D G D 3 + + + + N D ( , M ~ ( M A C G I } N D - N D Omlx + + + + N D (7,Q I c {Q211) W e a k - - N D

, po~l t txc, n e g a n v e , N D , no t d e t e r m i n e d

Svennerholm's group (Percy et al., 1991) dem- onstrated a marked increase of GM l-synthase actlvlty and a decline of GD3-synthase activny in a similar developmental interval (10-22 weeks of gestation), whmh might explain a rapid synthesis of"a"-pathway of ganghosldes (GDIa) in this period of most inten- sive cortmal synaptogenesls in the human fetal brain We observed that fetal cortical layer, the so-called subplate layer [SL, first described by Kostovlc and Molhver (1974)]. was characterized by a higher con- centrauon of G D l a at 30 weeks of gestation than cortical plate (CP) in frontal and occipital cortex This reformation on higher G D l a In SL than in CP of human fetal neopalhum (Kracun et a l , 1983, this study) correlated well with neuroanatomlcal evidence on the subplatc layer to contain the most differ- entlatcd neurons of cerebral cortex in this devel- opmental interval (McConnell et a l . 1989) A high proportion of G D l a remains m human h,ppocampus throughout the hfc (Kracun et a l , 1987) Cerebellar cortex could be distinguished prenatally by a high concentranon of "'b"-pathway ganghosldes (GTIb) and a low proportion of GDIa m comparison to archmortex and neocortex

In addition, we found that the pattern dlft'erence of neocortical ganghosldes developed after birth, by the

age of 20/30, and was charactenzed by much lowcl G D l a m visual cortex than in frontal cortex. It might be that this difference, developed during the postnatal funcnonal cortical maturation, corresponds to the cytoarchltectonic and/or functional &fferences between these cortical areas, since vmual cortex IS d typical heterotyplc granular cortex (containing small pyramidal neurons), whereas frontal cortex (motor) ~s a typical heterotyplc agranular or homotypm cortex (containing large pyramidal neurons) (Economo and Koskmas, 1925) We speculate that the &fference in GDIa content between frontal and v,sual cortices might be due to different ganghoslde synthesis in small and large pyramidal neurons

Immunohlstochemlcally, we found the presence of "'c"-pathway gangllosldes recognized by MAb Q211 only at a very early stage of human brain development (5 week old embryo) and weak lmmunoreactlvlty m 17 week old human fetal hlppocampus The presence of"c"-pathway ganghosldes m fish brain is well estab- hshed (Hllblg and Rahmann, 1987). They were also lmmunohlstochemmally localized in distinct corneal lamina containing migrating cells and growing fibres of the developing chicken and rat brain (Rosner et a l .

1988, Grins and Rosner, 1990) The lmmunohistochemlcal localization of GDIb in

the cerebellar cortex of a 1.5 year old child clearb corresponded to the granular cell layer. The pattern of fluorescence is not cellular. The premse localization of this fraction remains unsettled but it might be that it is contained in growing mossy fibres (Seyfried and Yu, 1990).

GD3 ganglioside has been documented to charac- terize prohferatmg neurons in ventncular zone of the developmg rat and chmken cortmes (Rosner et a l .

1988) Here, we also confirmed its expression in pro- hferatlng cells of human fetal pallium 0 e ventrlcular zone)

Monoclonal antibody MACGI against a sub- species of GM3 was found to be reactive only in fetal cerebella at 17 and 22 weeks of gestation Since a true antigen (Schnever et al , 1989) has not been identified. we cannot speculate on the meaning of this particular pattern of MACGI mamunostalnlng in human fetal brain Gmlx monoclonal antibody was demonstrated to recognize ganghoserles ganghosldes m ELISA test. cellular lmmunoreactlwty m human brain, however. failing to bind ganghosldes in lmmunoblot on thin- layer plates The reason for this failure might be that the actual epltope is generated in concert with a mem- brane protein, similarly to ~-GDla MAb showing the same immunochemlcal characteristics (Rosner's observations)

Gangllosldes In the human brain development and aging 429

Fig 7 Cellular lmmunoreactlvlty (arrowheads) of MAb Gmlx on the human fetal hlppocampal shces at 18 weeks (A) and 32 weeks (B) of gestation

Characteristic regional changes in the ganghoslde composition of human cortex are observed to occur between the third and ninth decade. Namely, we found GDla and GM1 to suffer a more pronounced decrease in senescent frontal cortex than "b"-pathway ganghosldes Our results are in accordance with recent studies on the frontal pole (Svennerholm et a l , 1989). However, in occipital and cerebellar cortex GD 1 a and GM1 were stable between 20/30 and 80/90 years of age That means that the increase in the relative con- centratlon of "b"-pathway gangllosldes obtained by analysis of whole human brain is not a general phenomenon of the human brain aging

There are at least two explanations for the change in the ratio between "a"- and "b"-pathway of gan- ghosldes during human aging, i.e. metabolic and/or cytoarchltectonlc

We believe that changes in cortical ganglioslde pat- tern during human aging follow a regional (-specific) cytoarchltectonlc changes (neuron gha ratio, number

of dendrites and synapses, etc ) (McNedl, 1983) rather than a shift in biosynthesis of ganghosldes from "a"- to "b"-pathway suggested by Segler-Stahl et al. (1983) and recently by Svennerholm et al. (1989) based upon recent 'competitive' model of ganghoslde biosynthesis (Pohlentz et al., 1988).

During human brain aging, a significant increase in ghal population with a decrease in neuron-to-gha ratio was observed (Uemura and Hartmann, 1978) In precentral frontal gyrus of human newborn brain, dominant cells in external and internal granular layers are granular cells, whereas in senescent brain small pyramidal neurons prevail (Brody; 1955) It might be that pyramidal neurons are characterized by "b"- pathway of ganghosldes, whereas granular cells are characterized by "a"-pathway of gangllosides So, it is possible that neurons containing "a"-pathway gan- ghosldes ("a"-neurons) are more vulnerable by aging processes than "b"-neurons expressing "b"-pathway ganghosldes

430 l KRA(t'N e/ a/

4¢hnow&dqement~--Supported by Mlmstry of Sciences, Technolog) and Informatmns of Croatm (1-08-136, I K ) The authors express their thanks to Anatonlja Redovmkowc, B A , for her skdful ed~torml work

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