Ganglioside alterations in guinea pig brains at end stages of experimental Creutzfeldt-Jakob disease

Journal o f the Neurological Sciences, 1978, 35 : 15-.23 © Elsevier/North-Holland Biomedical Press

15

G A N G L I O S I D E A L T E R A T I O N S IN G U I N E A PIG BRAINS AT END STAGES OF E X P E R I M E N T A L C R E U T Z F E L D T - J A K O B DISEASE

ROBERT K. YU* and ELIAS E. MANUELIDIS Departments of Neurology and Pathology, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.)

(Received 2 June, 1977)

SUMMARY Gangliosides were isolated from guinea pig brains at the end stages of experimental Creutzfeldt-Jakob disease. Quantitative analyses revealed marked decreases of ganglioside levels in pathologically devastated tissues such as cerebral cortex (--21 ~), basal ganglia and thalamus (--18 ~), and brain stem ( - - 2 3 ~ ) . The cerebellum revealed only minor pathological abnormalities and its ganglioside level remained unchanged. Thin-layer chromatography of the Creutzfeldt-Jakob brain gangliosides showed aberrant ganglioside patterns in all regions studied, including the cerebellum. With some exceptions, a trend in ganglioside pattern changes was detected which consisted of proliferation of GM3,GD3,GD2 and loss of GM1, GDla, GDlband GTlb**.

INTRODUCTION In the group of subacute spongiform virus encephalopathies, 4 naturally occurring diseases in man and animals are included; kuru and Creutzfeldt-Jakob (C-J) disease of man, scrapie of the sheep and transmissible mink encephalopathy (Gibbs and Gajdusek 1972). In these diseases, microscopic changes are found only in the central nervous system and consist of neuronal destruction, status spongiosus and astrocytosis. It was of fundamental importance that kuru, an exotic subacute neurological disorder occurring in New Guinea, was transmitted from man to the chimpanzee by Gajdusek, Gibbs and Alpers (1966). Subsequently, C-J disease, a subacute degenerative disease of the central nervous system characterized by rapidly progressive presenile dementia with pyramidal, extrapyramidal and occasionally cerebellar signs has also * To whom reprint requests should be sent. ** The ganglioside nomenclature system of Svennerholm (1963)is used. The structures of some of the gangliosides have been compiled recently (Ledeen and Yu 1976).

16 been successfully transmitted from man to the chimpanzee by Gibbs, Gajdusek, Asher, Alpers, Beck, Daniel and Matthews (1968). Both kuru and C-J disease have been transmitted to domestic cats (Gibbs and Gajdusek 1973). However, Gibbs and Gajdusek (1973) were unable to transmit these diseases to small hosts such as mice, rats, hamsters and guinea pigs. Successful transmission of C-J disease to guinea pigs in serial passages has been reported from this laboratory (Manuelidis 1975; Manuelidis, Kim, Angelo and Manuelidis 1976); more recently this disease has been also transferred to hamsters (Manuetidis, Angelo, Gorgarz and Manuelidis 1977) and mice (Manuelidis et al., unpublished). The availability of the guinea pig as an animal model provides a valuable adjunct for biochemical studies of C-J disease. Biochemical analyses of the brains of humans suffering from C-J disease revealed, among other abnormalities, general decreases in total lipids, total phosphatides, and RNA content; and increases in proteolipid-protein and lipid-to-protein ratio (Suzuki and Chen 1966; Bass, Hess and Pope 1974). Brain ganglioside content was also severely reduced, particularly in morphologically devastated cortical areas, in C-J disease (Suzuki and Chen 1966; Bass et al. 1974). Such alteration in ganglioside content as well as minor changes in ganglioside pattern were also observed in chimpanzee brains infected with the C-J and the kuru agents (Yu, Ledeen, Gajdusek and Gibbs 1974). Since gangliosides are constituents of plasma membranes and are particularly abundant in neuronal membranes (Derry and Wolfe t 967; Lapetina, Soto and De Robertis 1967; Morgan, Wolfe, Mandel and Gombos 1971), they may serve as excellent markers for monitoring changes in neuronal membrane structure as the result of slow virus infection. In addition, the study of altered ganglioside composition could provide some clue to the metabolic basis in the pathogenesis of these diseases. It is therefore of special interest to delineate the brain ganglioside alterations in guinea pigs terminally affected with C-J disease. MATERIALS AND METHODS The initial transmission of the C-J agent was made from a human patient to guinea pigs. The guinea pigs (Hartley strain, 9-10 months old) used in this study received the C-J agent about 8 months earlier through serial propagation (Manuelidis 1975; Manuelidis et al. 1976). All the animals showed typical clinical signs of the disease at the terminal stage (223-246 days post-inoculation) and became totally prostrated. The animals were then anesthetized with ether and sacrificed by cervical dislocation. Pathological examination of the C-J guinea pig brains revealed features characteristic of subacute spongiform virus encephalopathies. These included marked enlargement of the ventricular system with concomitant hydrocephalus. There was extensive status spongiosus of the neuropil and the neuronal perikarya, and neuronal loss and astrocytic proliferation in the cerebral cortex and subcortical gray structures. Light microscopic examination of the cerebellum revealed only minor abnormalities. Details of these findings have been described elsewhere (Manuelidis 1975; Manuelidis et al. 1976). Four brains were used for lipid analysis. Each brain was dissected into 4 regions: cerebral cortex, basal ganglia (including thalamus), brain stem (including pons and

17 medulla) and cerebellum. Each portion was weighed immediately and then lyophilized in order to obtain the dry weight. As controls, the brains from 4 normal guinea pigs of the same strain (Hartley) and of the same age were used. The dried tissues were softened by the addition of 1-2 ml of water. The tissues were homogenized with 10 vol of chloroform-methanol (1:1). The insoluble residue was removed by filtration using a medium porosity sintered glass funnel. The gangliosides were then isolated and purified using the method of Ledeen, Yu and Eng (1973) which employed a combination of DEAE-Sephadex (acetate form, A-25) and Unisil (Clarkson Chemical Co., Williamsport, Pa.) column chromatographic techniques. The ganglioside content of each fraction was determined by analyzing the lipidbound sialic acid by a gas-liquid chromatographic method (Yu and Ledeen 1970). The ganglioside patterns were obtained by thin-layer chromatography (TLC) performed with Merck precoated (Silica Gel 60) plates (EM Laboratories, Darmstadt, G.F.R.). The plates were activated before sample application by heating at 110 °C for 30-40 rain. Developing solvents included chloroform-methanol-water (60:40:9) and chloroform-methanol-2.5 N ammonium hydroxide (60:40:9). Gangliosides were revealed by the resorcinol-hydrochloric acid (Svennerholm 1957) spray followed by heating the covered plate at 90-95 °C for 30-40 min. The distribution of ganglioside sialic acid was obtained by direct densitometric scanning of the thin-layer plate using a Transidyne RFT scanning densitometer (Transidyne, Ann Arbor, Mich.) and peak areas were determined by a Hewlett-Packard Model 3380A Electronic Integrator. Details of the densitometric method will be published elsewhere (Yu et al., in preparation).

RESULTS AND DISCUSSION

The most conspicuous and consistent gross pathological finding in intracerebrally inoculated guinea pig brains was the moderate to marked enlargement of the ventricular system with cortical atrophy (Manuelidis et al. 1976). Analysis of the morphologically devastated cortical and subcortical (basal ganglia and thalamus) areas revealed reduction in wet and dry weights (Table 1). A slight decrease in tissue weight was also found in the brain stem. No changes in tissue weights in the grossly normal-appearing cerebellum were observed. The water content in cerebral cortical gray matter also showed a slight but statistically significant increase. Quantitative analysis of ganglioside concentrations in C-J guinea pig brains revealed reductions of 18-23 ~ in affected brain tissues (Table 1). The decrease in gangli0side concentrations can probably be attributed to the profound neuronal atrophy characteristic of the affected brain regions, as was pointed out previously in human cases of C-J disease (Suzuki and Chen 1966; Bass et al. 1974) and in C-J and kuru chimpanzee gray matter (Yu et al. 1974). It has been noted that the more severe the neuronal loss, the greater the decrease in gangliosides (Bass et al. 1974; Yu et al. 1974), Thus, the decrease in gangliosides in our C-J guinea pig brains lends support to the notion that the primary damage was to the neurons. The C-J guinea pig cerebellum

2.06 ± 0.09

0.57 ± 0.04

0.55 ± 0.14

0.60 + 0.04

Cerebral cortex

Basal ganglia and thalamus

Brain stem

Cerebellum

Average ± S.D.

Ftg SA

138.1 ± 10.5

1 4 6 . 7 + 40.8

137.7 ~ 13.3

446.3 ± 12.5

77.0 ± 0.2

73.4 ± 0.6

75.7 ± 0.9

78.4 ± 0.2

278 ± 10

503 ± 17

788 ± 42

853 -k 34

g wet wt.

0.59 ± 0,07

0.41 ± 0.11

0.30 i 0.07 (--47.4 %, P < 0.01)

1.72 ± 0.14 (--16.5%, P < 0.05)

127.4 ± 5.7

115.2 ± 28.6

67.6 ± 12.2 (--50.9 %, P < 0.01)

346.4 ± 25.1 (--22.4 %, P < 0.01)

Dry wt. (mg)

Wet wt. (g)

Water (~o)

Wet wt. (g)

Dry wt. (mg)

Creutzfeldt-Jakob (4)

Normal (4)

GANGLIOSIDE CONCENTRATIONS IN G U I N E A PIG BRAIN

TABLE 1

78.3 --4-1.4

71.9 ± 0.8

76.9 ± 1.5

79.9 ± 0.7 (+1.9%, P < 0.01)

Water (%)

268 ~ 32

389 ± 43 (---.23 o/£, P < 0.01)

647 ± 35 (--18% , P < 0.01)

674 ± 48 (--21%, P < 0.01)

/zg SA g wet wt.

O¢

19

GM4 GM3 GM2 GM~ GD3 Gola GD2 Golb GT~

I

2

3

4

5

Fig. 1. Thin-layer chromatogram of guinea pig brain gangliosides. Lane 1 : normal human white matter ganglioside mixture; 2: normal cerebral cortex; 3: C-J cerebral cortex; 4: normal basal ganglia plus thalamus; 5: C-J basal ganglia plus thalamus. Each lane contained 20 pg of lipid-bound sialic acid. The plate was developed once with the solvent system of chloroform-methanol-water (60:40:9). See text for experimental details.

Fig. 2. Thin-layer chromatogram of guinea pig brain gangliosides. Lane 1 : normal human white matter ganglioside mixture; 2: normal brain stem; 3 : C-J brain stem; 4: normal cerebellum; 5 : C-J cerebellum. Each lane contained 20/tg of lipid-bound sialic acid. The plate was developed once with the solvent system of chloroform-methanol-water (60:40:9).

20 again did not show any change in ganglioside concentration. As already stated, the cerebellum in infected guinea pigs showed only mild changes morphologically. TLC of the C-J guinea pig brain ganglioside showed altered ganglioside patterns as compared to normal in all regions studied (Figs. 1-2). The changes were more prominent in pathologically devastated tissues such as cerebral cortex, basal ganglia plus thalamus, and brain stem. In these tissues, there was a general increase in the relative proportions of minor gangliosides such as GMa, GD3 and GD2 (except in the brain stem which showed a slight but insignificant decrease in GD2) and decreases in GDla, GDlb and GTlb (Table 2). G m also showed a decline in cerebral cortical and subcortical tissues. Several non-specific changes including increases in GM4 and GM2 were observed in the cerebral cortex and brain stem, and an increase in G~I in basal ganglia plus the thalamus and brain stem. The cerebellum, on the other hand, also showed slight but significant elevations in GD3 and GD2 and a reduction in Gma. The overall pattern changes were strikingly similar to those found in the human C-J brains (Suzuki and Chen 1966) and to a lesser degree to the pattern changes found in C-J and kuru chimpanzee brains (Yu et al. 1974). The qualitative changes were also strongly reminiscent of the drastically altered patterns in another chronic virus disease, subacute sclerosing panencephalitis (SSPE) studied by Norton, Poduslo and Suzuki (1966) and Ledeen, Salsman and Cabrera (1968). The proliferating ganglioside species in SSPE brains were the usually minor brain gangliosides GM3, G:~2, GD3, GD2, whereas other major brain gangliosides, GM1, GDI~, GDlb and GTlb, suffered relative and absolute depletion. The severe neuronal depletion and accompanying astrocytosis are the major contributing factors to the quantitative and qualitative ganglioside changes found in the infected guinea pig brains. However, it may be an over-simplification to explain the aberrant ganglioside patterns solely on this basis. Recent studies indicated that neurons, astrocytes and oligodendrocytes isolated from brains appeared to have similar ganglioside patterns which were similar to those of whole brain (Hamberger and Svennerholm 1971; Norton, Abe, Poduslo and De Vries 1975). Our own study also showed that, with the exception of GM4, there was striking similarity between the ganglioside patterns of human neurons and oligodendrocytes (Yu and Iqbal 1977). Thus the loss of neurons may not be the only reason for the altered ganglioside pattern. Alternative explanations may therefore seem necessary. The only possibility we favor, which has also been suggested earlier (Yu et al. 1974), is the perturbation of ganglioside metabolism in the nerve cells as a result of viral infection. In recent years there have been large volumes of data documenting altered ganglioside metabolism in cultured cells transformed by conventional, oncogenic viruses (Hakamori 1973; Brady and Fishman 1974; Richardson, Baker, Morre and Keenan 1975; Fishman and Brady 1976). Similar metabolic disturbances could conceivably occur in slow virus diseases such as the C-J disease studied here. In the present context, it is worth noting that several glycosyltransferases and glycosidases related to glyeoprotein metabolism have been found to be altered in scrapie (Hunter 1972; Hunter and Millson 1973; Millson and Bountiff 1973; Suckling and Hunter 1974) and in Semliki Forest virus-infected mouse brains (Suckling, Webb, Chew-Lira and Oaten 1976). As a working hypothesis, we would like to propose

4- 0.8 4- 0.3 4- 0.9 4- 0.6 4- 0.1

32.1 15.4 17.6 4.5 1.1

27.8 13.4 16.4 4.5 0.8

444d: 4-

44444-

2.7 4.0 5.8 19.3 6.6 17.3 5.5 17.0 15.3 5.3 1.0

4- 0.2 4- 0.2* 4- 1.5 4- 1.0 4- 0.3* 4- 0.7* 4- 0.2* d: 0.6* 4- 0.2* 4- 0.4* 4- 0.2

44444444444-

0.3 0.1 0.2 0.6 0.2 0.5 0.2 0.6 0.4 0.4 0.3

± 4444444+ 44-

0.7* 0.7* 0.3** 0.8 0.5* 0.1" 0.1 0.8* 0.3*** 0.1" 0.2

3.4 2.5 3.2 13.7 7.5 7.0 9.3 19.0 20.7 10.6 1.5

4- 0.2 4- 0.4 4- 0.6 4- 0.5 4- 0.4 4- 0.4 4- 0.3 4- 0.3 4- 0.4 4- 1.0 4- 0.1

4.9 3.1 4.0 21.2 5.7 7.8 6.0 21.5 16.6 8.1 1.1

3.3 5.8 4.4 17.3 6.0

0.8*** 3.3 4- 1.2 0.9* 1.8 + 0.4 0.4* 5.9 4- 1.5 0.5* 19.7 4- 1.0 0.2* 4.0 4- 0.3 21.9 4- 0.3 1.0" 3.4 4- 0.1 0.4* 18.8 4- 0.4 0.4** 16.8 4- 0.7 0.2 3.9 + 0.4 0.1 0.6 4- 0.1

3.5 1.0 3.5 20.7 3.9 12.6 6.3 23.3 17.5 6.7 0.8

0.1 0.3 0.6 0.2 0.1

44444-

1.5 0.9 2.2 20.0 4.6

Values expressed as mean ± S.D. * P < 0.01. ** P < 0.05. *** P < 0.02.

GM4 G~ta G~ Gm GD3 GDla GD~ GDlb GTlb Gqs Origin

Normal (4)

C-J (4)

Normal (4)

C-J (4)

Normal (4)

Normal (3)

C-J (4)

Cerebellum

Brain stem

Basal ganglia + thalamus

Cerebral cortex

DISTRIBUTION OF L I N D - B O U N D SIALIC ACID IN G U I N E A PIG BRAIN

TABLE 2

2.8 3.2 3.0 13.7 10.1 5.0 10.0 19.1 20.3 12.3 0.4

+ 0.7 + 0.5 -4- 1.0 4- 0.9 4- 0.2* ± 0.2* 4- 0.2*** 4- 0.7 -4- 1.0 4- 0.6 4-0.1

C-J (4)

to

22 that the ganglioside alterations in brains of subacute spongiform virus encephalopathies may be due primarily to changes of cell glycosyltransferase or glycosidase activities as a result of viral action. As the disease progresses, increasingly severe altera t i o n of m e m b r a n e ganglioside c o m p o s i t i o n would occur. The cells would be converted to a n o n - f u n c t i o n a l or physiologically unstable state, which eventually leads to their complete destruction. We are currently studying several enzymes involved in ganglioside m e t a b o l i s m in order to test this hypothesis. ACKNOWLEDGEMENT This work was supported by U S P H S G r a n t s NS-11853 a n d NS-12674, a n d in part by grants from The Kroc F o u n d a t i o n a n d The Klingenstein F u n d .

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