Altered cerebellar ganglioside pattern in rett syndrome

Neurochem. Int. Vol. 19, No. 4, pp. 505-509, 1991

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ALTERED CEREBELLAR GANGLIOSIDE PATTERN IN RETT S Y N D R O M E ANNIKA LEKMAN,I BENGT HAGBERG2 and LARS SVENNERHOLM1. ~Department of Psychiatry and Neurochemistry and :Department of Pediatrics, University of G6teborg, Sweden (Received 26 February 1991 ; accepted 1 May 1991)

Abstract--The membrane lipids were examined in the cerebellum from five patients who died with Rett syndrome (RS). The major lipids ofcerebellar folia and white matter did not show any difference compared with age-matched controls. There were slightly low values for cerebrosides, a biochemical marker for myelin, in cerebellar folia but high values in white matter of corpus medullare. The ganglioside concentration was reduced in one case which had shown marked astrocytosis at histological examination. Astrocyte associated gangliogides were significantly increased in this case, but their proportion was also increased in the four other patients. Lacto series acidic glycosphingolipids, 3'-LM1 and LKI, closely associated with Purkinje cells were reduced in the Rett cases which fits well with neuropathological examination demonstrating the loss of Purkinje cells. The most prominent finding was a decreased proportion of gangliosides GDla and GT Ib in cerebellar folia and white matter. The decreased proportion of GD 1a might reflect an abnormality of synaptogenesis in RS and would be compatible with the clinical profile of this disease.

The Rett syndrome (RS) is a neurodegenerative disorder affecting girls in late infancy. Current data suggest that RS is a genetic disorder, sporadic in most instances, and best explained by an X-linked dominant mutation. In the absence of a specific biochemical marker, the condition is defined by clinical criteria for inclusion and exclusion (Hagberg et al., 1985; Hagberg, 1989). The girls develop normally until 6 12 months of age, and subsequently develop a progressive loss of motor and cognitive skills. In later years slowly progressive neuromotor deterioration occurs while contact and communicative abilities partly return (Hagberg, 1989). The most striking clinical feature is the peculiar movement of the hands which is stereotyped and described as hand washing, hand wringing or clapping. There is an early stagnation of head growth and the hands and feet tend to remain remarkably small. A successively appearing

quite characteristic abnormal E E G pattern is a comm o n finding (Hagne et al., 1989). Neuropathological examinations have revealed microencephaly with a decrease in brain weight and diffuse cortical atrophy, both related to the duration of the disorder (Jellinger et al., 1986). Isolated abnormal neurites and reactive or degenerative axonal swellings were seen in frontal cortex and caudate nucleus (Jellinger et al., 1988). Degeneration and loss of spinal ganglion cells in addition to gliosis were found in spinal cord (Oldfors et al., 1988). Mitochondrial changes were observed in peripheral nerves (Wakai et al., 1990) as well as mild distal axonopathy without demyelination (Jellinger, 1990). Oldfors et al. (1990) examined the cerebellar pathology in RS. All five cerebella studied had markedly reduced brain weights with proportionally small cerebella. Cerebellar atrophy increased with age and occurred focally along the folia. Microscopic exam*Author to whom all correspondence should be addressed ination showed loss of Purkinje cells, atrophy and at: Department of Psychiatry and Neurochemistry, St astrocytic gliosis of the molecular and granular cell Jrrgen Hospital, 42203 Hisings Backa, Sweden. layer, and gliosis and loss of myelin in the white Abbreviations: GM4, I3NeuAc~3alCer; GM3, lI3NeuA~ matter. LacCer ; GM2, II3NeuAc GgOse3Cer ; GM 1, II3NeuA~In a previous study of the frontal and temporal GgOse4Cer ; GD3, II3(NeuAc)2-LacCer ; GD2, II3(NeuAc)2 GgOse3Cer; GDla, IV3NeuAc.ll3NeuAc cerebral lobes (Lekman et al., 1991) a selective loss of GgOse4Cer; GDlb, II3(NeuAc)z~GgOse4Cer; GTIb, myelin-associated lipids, and a remarkable enrichIV3NeuAc, ll3(NeuAc)2~GgOse4Cer; GQIb, IV3(NeuAc)2 ment of gangliosides in temporal white matter was II3(NeuAc)2~_igOse4Cer; 3'-LM1, IV3NeuAc-nLcOse4 found. The ganglioside pattern showed a significant Cer; 3'-isoLM 1, IV3NeuAc LcOseaCer ; LK1, sulfate-3Gluc nLcOseaCer. increase of astroglial cell-associated gangliosides and 505

506

ANNIKA LEKMAN el aZ

low proportions o f two major gangliosides, G D 1a and G T l b . The aim o f this study was to investigate the m e m b r a n e lipids in cerebellum, with special emphasis on determination o f Purkinje cell-associated gangliosides. Brain autopsy specimens from three o f the five Rett females studied by Oldfors et al. (1990) and two additional Swedish Rett females have been available for biochemical examinations.

EXPERIMENTAL PROCEDURES

ChemicaL~' Analytical reagent, solvents and high-performance TLC (HPTLC) plates were obtained from Merck AG (Darmstadt, Fed. Rep. Germany). 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) was obtained from Sigma (St Louis, M,O. U.S.A.). Two monoclonal antibodies (Mabs) used for the determination of gangliosides were produced in our laboratory: the Mab LS-I (binding epitope, NeuAca2-3Galfll4GIcNacfll-) for 3'LMI and Mab SL-50 (binding epitope, NeuAc~2-3Galfll-3GlcNacfll-) for 3'-isoLM 1. The HNK-1 murinc hybridoma (Abo and Balch, 1981 ; Abo et aL, 1982) was obtained from American Type Culture Collection (Rockville, MD, U.S.A.) for determination of the glucuronylglycosphingolipid LK 1. Ganglioside standards were isolated and characterized by FAB-MS in our laboratory. Alkaline phosphatase conjugated goat anti-mouse IgM and lgG (H + L) were purchased from Jackson lmmunoresearch Lab. (West Grove, PA, U.S.A.). Tissue Brain tissue from five patients with classical RS was available for analysis of cerebellar folia and from four of these patients for analysis of cerebellar white matter. The patients* ages were 12, 18, 20, 28 and 30 years. The clinical records of cases S-35 (12 yr), S-44 (18 yr), S-12 (20 yr), S-92 (28 yr) and S-21 (30 yr) have been described previously (Lekman et al., 1989, 1991 ; Oldfors et al., 1988). All had typical RS in the fourth stage of the disease (Hagberg and Witt-Engerstr6m, 1986). Control tissue was available from 10 individuals, 4 women and 6 men, 15 34 years of age. Each subject had died suddenly in an accident or from acute cardiac disease. None of the subjects has complained about neurological or psychiatric symptoms. All had normal brain weights for their age. There were no differences in measured parameters between men and women in the control subjects. The cadavers were placed in a cold room after death and autopsies were performed within 48 h post mortem. Cer ebellar folia and white matter from corpus medullare were dissected and the samples frozen and stored at -- 20:C until analyzed. Lipid extraction The lipid extraction has been described in detail previously (Lekman et al., 1991), as well as the methods used for determination of major membrane lipids. Detection (?['gan,qlioside antigen by T L G EL1SA The TLC ELISA procedure has been described elsewhere (Da,,idsson et al., 1989). Portions of the ganglioside extracts

and known amounts of the ganglioside antigens were applied to high performance TLC-plates and chromatographed in chloroform methanol4).25% aqueous KC1 or 2.5 M ammonia (50:40: 10, by vol.). The plate was dipped in 0.5% polyisobutylmethacrylate and unspecific binding blocked by preincubation in Tris-BSA (50 mM Tris HC1, pH 8.0, 0.15 M NaCI and I% bovine serum albumin, BSA). lmmunostaining was then performed by incubation, m subsequent order, with a specific monoclonal antibody in Tris BSA, alkaline phosphatase conjugated anti-mouse antibody in Tris BSA and finally a substrate solution, 0.0 [ % (w:v) BCI P in 0.1 M glycine, pH 10.4, containing [ mM ZnCI2 and I mM MgCI> Quantitation of the visualized ganglioside antigens was performed by densitometric scanning of the HPTLC-plate at 620 nm. The detection limit for ganglioside antigens with lnonoclonal antibodies in the TLC ELISA procedure was ca 1.0 pmol for 3"-LMI, 3"-isoLM1 and LKI. RESUI,TS Cerebellar Jblia

The major lipid composition o f cerebellar lblia (Table 1) showed no difference c o m p a r e d with the controls. One o f the brains (S-12) had lost some water during storage (more than three years) as evident from an increase o f dry weight, and its concentration o f major lipids was therefore higher. One other, S-44, had a marked decrease o f neuronal gangliosides, which was also reflected in the ganglioside pattern with a significantly increased p r o p o r t i o n o f GD3 and a reduced p r o p o r t i o n o f G D 1a. The astrocyte-associated ganglioside, GM3, was proportionally increased in all Rett cases. The lacto series gangliosides, Y - L M 1, as well as the sulfoglucuronylsphingolipid, LK1, was reduced in the Rett patients (Figs 1 and 2).

•

14

Rett syndrome

& ControLs

g <'~

g

&

=L

I

....~

g

':

""

"

L 12

16

20

24

28

32

A g e (yrsl

f=ig. 1. Ganglioside 3'-LM I in cerebellar folia from subjects with RS and controls. Values are expressed as nmol ganglioside'g tissue.

Cerebellar gangliosides in Rett syndrome

==

34 years of age for the determination of the lacto series gangliosides. Y-LM1 was not detected in Rett cases and controls. Y-isoLMl was only discovered in two Rett cases, 0.3 nmol/g in S-35 and 1.7 nmol/g in S-44. The LKI value in the controls was 1.3___0.2 nmol/g tissue compared with 0.04 nmol/g in the most severely affected girl, case S-44, and 0.18 in case S-12. Case S-35 showed a normal value of 1.0 nmol/g. No material was available for the two oldest cases.

• Rett syndrome z~ Controts

t.4

~' 1.o z~

~

z~ A

0.6

507

0.2 12

16

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DISCUSSION

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Fig. 2. Sulfoglucuronylsphingolipid LKI in cerebellar folia from subjects with RS and controls. Values are expressed as nmol glycolipid/g tissue.

Cerehellar white m a t t e r

Phosphotipids, cholesterol and particularly cerebrosides were increased in the white matter in three Rett cases S-44, S-12 and S-21 (Table 2). They are in increased order myelin associated lipids, and as the dry weight is not significantlyincreased with exception for S-12, the results suggest an increased proportion of myelin. This suggestion is further confirmed by the increased proportion of the myelin-associated ganglioside GM4. White matter was not available from the three controis 15-19 years of age, but only for six controls 20-

It was not possible to dissect the gray matter from the white matter in the cerebellar folia of the Rett samples, and therefore the whole folia from cerebellar vermis were examined in controls and affected cases. The neuropathological examination of cerebellar folia (Oldfors et al., 1990) revealed loss of Purkinje cells, astrocytic gliosis of the molecular and granular cell layer, and gliosis and loss of myelin, which was most pronounced in the tips of the folia. Despite these histological changes, the biochemical determination showed that the reduction of membrane lipids was remarkably little, if any. The concentration of cerebroside was in fact higher in cerebellar folia of S-21 and only slightly lower in S-35, S-44 and S-92 than in the controls. In the central white matter the cerebroside concentration was also higher which contradicts any significant myelin loss. The gangliosides, which are biochemical markers for neuronal membranes (Svennerholm, 1957), did

Table 1. Lipid composition ofcerebellarfolia in Rett and control brains

Protein (%) Dry weight (%) Cholesterol (,umol/g) Phospholipids (#tool/g) Cerebrosides (,umol/g) Gangliosides (#mol NeuAc/g) Protein-bound sialic acid (/tmol NeuAc/g)

$35 12 yr

$44 18 yr

S12 29 yr

$92 28 yr

$21 30 yr

Controls (n = 10) 15-34 yr

9.6 18.3 34.2 51.9 6.7 2.38 0.74

8.8 18.3 39.5 50.7 7.2 1.66 1.01

10.6 22.2 46.0 64.4 9.9 2.98 1.19

8.6 17.8 35.3 48.9 5.8 1.92 0.75

9.9 19.8 44.3 57.1 11.2 2.08 0.98

9.2_+ 1.3 18.9_+ 1.3 39.6 _+6.0 53.7 _+4.1 9.1 _+ 1.7 2.36 + 0.22 0.84 _+0.05

2.8 2.9 1.1 6.2 9.8 10.5 5.1 23.7 31.2 6.7

1.2 2.5 1.4 6.9 20.6 7.9 3.4 24.8 24.2 6.9

1.6 2.4 1.3 6.7 9.0 9.3 3.7 26.0 31.9 7.8

1.4 1.6 1.0 7.5 10.2 13.7 5.8 24.1 28.9 5.8

1.4 3.2 1.1 9.0 12.2 16.0 3.3 21.3 26.6 5.9

1.5_+0.4 1.3_+0.3 1.0_+0.4 8.3+ 1.3 8.4+ 1.2 14.1 + 1.7 4.3 + 0.7 21.5+ 1.4 31.8 ± 3.5 6.7_+0.8

Percentage composition of ganglioside sialic acid GM4 GM3 GM2 GMI GD3 GDIa GD2 GDIb G T 1b GQIb

508

ANNIKA LEKMAN et al. Table 2. Lipid composition of cerebellar white matter in Rett and control brains

$35 12 yr Protein (%) Dry weight (%) Cholesterol (p.mol/g) Phospholipids (/~mol/g) Cerebrosides (,umol/g) Gangliosides (,umol NeuAc/g) Protein-bound sialic acid (pmol NeuAc, g)

9.4 28.2 123.8 108.9 56.2

$44 18 yr

SI2 20 yr

8.4 31.6 149.4 125.6 65.8

$21 30 yr

8.8 34.3 167.0 138.6 69.6

9.4 32.5 L64.5 133.0 76.II

1.28

1.28

1.38

I, 14

/).50

11.71

0.36

11.42

Controls (n = 10) 15 34 yr 10.1 ~0.9 30.5 + 0.8 125.7 + 6.3 113.0±~6.3 52.6 ± 8.0 1.38 ±. 0.12 I).61 + O.12

Percenta~w composition o/ gan.qlioside sialic acid GM4 GM3 GM2 GM1 GD3 GDIa GD2 GDIb GTIb GQIb

13.4 3.3 2.5 20.8 6.0 5.2 3.5 20.3 18.8 6.2

not show any marked change except S-44, in which the concentration of gangliosides of cerebellar folia was reduced. The ganglioside pattern in S-44 also showed a reduced proportion of G D l a and GTlb. A slightly decreased proportion of G D l a and GT1 b was also found in other Rett cases in cerebellar folia but also in cerebral cortices of frontal and temporal lobes (Lekman et al., 1991). A common denominator for G D I a and G T l b is that the same sialyltransferase catalyses their formation from their immediate precursors GMI and G D l b (Schwarzmann and Sandhoff, 1990), which is shown in the biosynthetic scheme outlined below :

13.3 1.2 2.8 23.9 6.9 6.2 4.8 237 13.0 4.2

14.7 3.6 3.2 23.8 4.7 6.3 2.8 21.2 14.7 5.11

[6.1 1.6 4.4 28.7 2.8 5.6 3.8 20.0 12.9 4. I

10.5:+2.8 2.6+ 1.2 2.4+0.6 22.1 +2.1 7.0± 1.4 7.0+ 1.6 3.5+0.8 2t).9 :~ 1.8 18.4± 1.3 5.5+ 1.0

enriched in the synaptic membranes. The accretion of G T l b will proceed slower and its maximum level is reached first at 20 years of age. It is tempting to speculate that an inhibition of sialytransferase IV which catalysis the formation of ganglioside G D l a occurs in RS, and the slight reduction of this ganglioside reflects an abnormality of synaptogenesis which might lead to the early infantile symptoms of stagnation, then followed by communicative regression in late infantile development (Hagberg, 1989). The other changes in the ganglioside pattern, an increase of GM3 and GD3, are a common finding in

CMP NeuAc Sialyltransferase IV Gal GalNAc-GaI-GIc Cer I , Gal GalNAc Gal Glc Ccr

I

NeuAc GMI

I

NeuAc GDla

I

NeuAc

CM~NeuAc Sialyltransferase 1V Gal GalNAc-GaI-GIc Cert , GaI-GalNAc-Gal Glc-Cer NeuAc

NeuAc

I

I

NeuAc GDIb The accretion o f G D l a ganglioside in human brain will increase to its maximum level at two years of age when the outgrowth of nerve endings and synapses is most intense (Svennerholm et al., 1989). It has therefore been assumed that this ganglioside is, particularly

NeuAc NeuAc

GTlb neurodegenerative disorders with increased astrocytosis (Ledeen et al., 1968 : Svennerholm and Vanier, 1972). We have shown that a new ganglioside, Y-isoLM1, is associated with reactive astrocytes (Svennerholm et al., 1987) it is pertinent that the

Cerebellar gangliosides in Rett syndrome highest level of this ganglioside was demonstrated in S-44, who showed the most marked biochemical signs of astrocytosis (Oldfors et al., 1990). The sulfoglucuronylglycosphingolipid, LK1, which has the same terminal epitope as the myelin-associated glycoprotein of peripheral nerve ( M A G ) , has been shown to be associated with the cerebellar Purkinje cells (Chou et al., 1988). Figure 1 demonstrates that LKI is reduced in the four oldest Rett cases and that the most seriously affected woman, S-44, had the lowest level. The neuropathological examination has already demonstrated the loss of Purkinje cells in Rett cases (Oldfors et al., 1990), but the biochemical result is of value for the quantitative evaluation o f this loss. Acknowledgements--This investigation was supported by

Swedish Medical Research Council (03X-627). We are grateful to Professor Alan K. Percy, Baylor College of Medicine, Houston, TX, for valuable suggestions during the study.

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