Journal of the Neurological Sciences, 1985, 67:345-350 345 Elsevier

JNS 02459

Brain Glutamate Decarboxylase and Cholinergic Enzyme Activities in Scrapie Khalid Iqbal, Robert A. Somerville*, Christopher H. Thompson and Henryk M. Wisniewski

New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314 (U. S.A )

(Received 4 April, 1984) (Revised, received 10 October, 1984) (Accepted 18 October, 1984)

SUMMARY

C57BL/6J mice, age 6-8 weeks were inoculated intracerebrally with brain homo- genate from mice previously infected with the 139A strain of scrapie; control mice were identically treated with brain homogenate from non-infected normal mice. The activities of choline acetyltransferase (CAT), acetyl cholinesterase (ACHE), and glutamic acid decarboxylase (GAD) were determined in the forebrain and hindbrain of these animals after 67, 126 and 151 days post-inoculation.

There were no significant differences in the activities of CAT and GAD between scrapie and control mice at early, middle or late stages of the disease in the scrapie- infected animals; there was an about 20% decline in AChE activity in the scrapie brain.

Key words: Acetyl cholinesterase - Alzheimer disease~senile dementia - Choline acetyl- transferase - Cholinergic ac t iv i t y - Glutamic acid decarboxylase - Scrapie

INTRODUCTION

Scrapie is a transmissible degenerative disease of the central nervous system of sheep and goats. Several strains of scrapie have been transmitted to mice and hamsters. Some of these strains produce histopathological changes in the brain, specifically neuritic (senile) plaques (Fraser and Bruce 1973; Wisniewski et al. 1975) and neuronal

These studies were supported in part by NIH Grant NS-17487. * Dr. Somerville's present address is: A. R. C. & M. R. C. Neuropathogenesis Unit, West Mains Road,

Edinburgh EH 3 JF, U.K. Please send all correspondence to: Dr. Khalid Iqbal, N.Y.S. Institute for Basic Research in

Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NYI0314, U.S.A., Phone: (212) 494-5259.

0022-510X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

346

degeneration (Fraser 1979a,b) but not Alzheimer neurofibrillary tangles, which bear similarities to those found in Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT) (Dickinson et al. 1983). It should, however, be pointed out that firstly the plaques are found in only a few scrapie agent and host strain combinations and secondly the number of plaques in scrapie is sparse whereas in AD/SDAT it is one of the most prominent lesions. Thus though scrapie is being increasingly referred in the literature as an animal model for AD/SDAT (Dickinson et al. 1979) it is by no means adequate.

In AD/SDAT a marked decrease in the activities of the cholinergic enzyme, choline acetyltransferase (CAT) and acetyl cholinesterase (ACHE) have been reported in cerebrum (Bowen et al. 1976; Davies and Maloney 1976; Perry et al. 1977; Dziedzic et al. 1980). McDermott et al. (1978) reported a reduction in CAT activity in clinically ill C57BL mice infected with several strains of scrapie including two which form neuritic (senile) plaques. These authors observed the most marked changes in CAT activity i.e. a decline of up to 70~o activity in mice infected with the scrapie agent 79A which does not form plaques. We investigated the activities of CAT, AChE and in addition the GABAergic enzyme, glutamic acid decarboxylase (GAD) in the brains of C57BL mice infected with scrapie agent 139A at early, middle and late stages of the disease; the agent 139A is very similar to the agent 79A in incubation period, clinical and histopathological changes (Dickinson and Outram 1983). Our results show that in the scrapie model examined there is no significant change in the activities of either CAT or GAD; the activity of AChE is about 20~ reduced in scrapie.

MATERIALS AND METHODS

Female C57BL/6J mice, 6-8 weeks old were inoculated intracerebrally with 25 #1 of 1~o brain homogenate from mice previously infected with 139A strain of scrapie. Control animals were identically injected with normal mouse brain homogenate. The two groups of 6 animals each were housed under identical conditions but in separate cages and were killed by cervical dislocation on 67, 126 and 151 days post-inoculation. The rationale for choosing these 3 time points was to determine changes in enzyme activities at preclinical, early clinical, and late clinical stages of the disease. Brain was removed and divided by cutting vertically between the cerebrum and the cerebellum into a forebrain and a hindbrain region. The forebrain contained both cerebral hemispheres and most of the thalamus. The hindbrain consisted of the cerebellum, pons, and medulla. The tissue was weighed and a 10~ (w/v) homogenate of the tissue was prepared in 10 mM sodium potassium phosphate buffer, pH 7.2, using a glass Teflon homogenizer.

CAT and AChE assays were performed by the modified radiochemical method of Fonnum (1975). For CAT assays the tissue homogenate was diluted to 0.2~/o and 0.4~ for forebrain and hindbrain, respectively, with 1 mM EDTA in 0.1~o Triton X-100, pH 7.0. The enzyme activity was linear with up to 45 min at 37 °C. Incubations at 37 °C for 20 min were carried out for all subsequent assays.

The AChE assays were carded out on the homogenate diluted to 0.04~o and 0.08 ~ for forebrain and hindbrain, respectively, with the EDTA/Triton X-100 solution

347

(see above). The enzyme reaction was carried out in the presence of 0.5 mM etho- propazine which inhibits butyrylesterase activity. The enzyme activity was linear up to 20 min of incubation at 37 °C. Incubations for 15 min at 37 °C were carried out for the routine assays.

GAD assays were performed by measuring the 14COz formed from L-[ 1-14C]gl u- tamic acid (Roberts and Simonsen 1963). The tissue homogenate for the GAD assay was diluted to 5 ~ with 250 mM potassium phosphate buffer, pH 7.35. The enzyme assays were carried out in a final volume of 1 ml of 250 #moles potassium phosphate buffer, pH 7.35, containing 10 #moles glutathione (reduced), 0.2 #moles pyridoxal phosphate, 1~ Triton X-100 and 2 #moles of L-glutamic acid (with 0.2 #Ci of L-[ 1-14C]glutamic acid). The enzyme activity was linear with up to 75 rain of incubation at 37 °C. For the routine assays incubation at 37 °C for 60 min was used.

The statistical analysis on differences in each enzyme activity between scrapie and control mice was carried out by the Student t-test. The changes in enzyme activities with age from 67 days post-inoculation to 151 days post-inoculation within each group were analyzed by the dependent t-test.

RESULTS

Scrapie-inoculated mice had early signs of disease i.e. a reduction in motor activity and competency at the time of their killing on day 126 and severe clinical disease (weight loss and extreme difficulty in moving) by day 151; by 151 days these animals also showed histopathological changes including a diffuse vacuolation of gray and white matter but no neuritic plaques. The scrapie-infected mice killed on day 67 and all the control animals showed no clinical symptoms of scrapie. Although the brain weight of the scrapie-infected animals was normal both at 67 and 126 days post-inocu- lation, a 10~ decrease in hindbraln (0.07 + 0.003 g vs 0.08 + 0.003 g) and in forebrain (0.30 + 0.005 g vs 0.33 + 0.006 g) wet weight was noted at 151 days post-inoculation.

The activities of the cholinergic enzymes, CAT and AChE in the hindbrain were about 40 ~o of that in the forebrain (Table 1). Interestingly the activity of the GABAergic enzyme, GAD was about the same in both the forebrain and the hindbrain in these animals by 67 days post-inoculation but was about 25-30 ~o lower in the hindbrain than the forebrain by 126 days and 151 days post-inoculation.

There were no significant differences in the activities of both CAT and GAD between scrapie and control mice at early, middle or late stages of the disease in the infected animals (Table 1); a slightly low GAD activity in the forebrain of scrapie mice at 67 days post-inoculation was an exception probably unrelated to the disease. However, there was significantly lower AChE activity in scrapie-infected mice than in the control animals at all 3 stages of the disease; these differences in AChE activity were more pronounced in hindbrain than in the forebrain.

DISCUSSION

This study has revealed that there is about 20~ lower activity of AChE in scrapie than in the control C57BL/6J mice and that this difference is observed on day 67

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post-inoculation when the scrapie-infected animals have no clinical signs i.e. loss of weight and motor activity and minimal histopathological changes; the difference in the AChE activity between scrapie, and control animals is not enhanced with the pro- gression of the disease in the scrapie-infected animals from 67 days to 151 days post-inoculation.

Since AChE is not a specific marker of the cholinergic neurons, reduction in this enzyme activity may be associated with widespread histological changes found in this model of scrapie.

Interestingly no significant changes in the activities of CAT and GAD were found between scrapie and controls in the combination of scrapie strain and mouse strain examined. Accordingly these results do not confn'm the previous findings of up to 70 decrease in CAT activity in C57BL mice infected with the 79A strain of scrapie although more modest losses were found with some but not all scrapie strains (McDermott et al. 1978). In subsequent studies a smaller reduction in CAT activity comparable to that found in other strains was found in 79A-infected mice and only in terminally ill animals (Dickinson, personal communication). The differences between the present results and those of McDermott et al. (1978) are surprising since the strain of scrapie used, 139A, is closely related to 79A and indeed it is now thought that 139A is a complex mixture of agents a major component of which is 79A (Dickinson and Outram 1983).

Previously we showed normal CAT activity in spinal cord of rabbits with aluminum-induced neurofibriUary changes (Hetnarski et al. 1980) although others (Yates et al. 1980) have found some reduction. Like the aluminum-induced ence- phalomyelopathy, an acute model of neurofibrillary changes, scrapie, a chronic model of CNS degenerative changes does not show the central cholinergic deficit seen in AD/SDAT. According to Price and his colleagues (Whitehouse et al. 1981, 1982; Price et al. 1982; Coyle et al. 1983) the cholinergic deficit in AD/SDAT brain is due to a massive (up to 90~o) loss of cholinergic neurons in the nucleus basalis of Meynert of the basal forebrain which innervates the neocortex. In Parkinson disease a loss of neurons in the nucleus basalis even more marked than in AD/SDAT is not accompanied by any significant loss ofcholinergic activity in the neocortex (Lloyd et al. 1975; Reisine et al. 1977; Candy et al. 1983). Thus the cause of the loss of neurons in this specific nucleus in AD/SDAT and its relationship to the loss of the activities of the cholinergic enzymes and to the histopathology seen in the neocortex remains to be understood.

ACKNOWLEDGEMENTS

The scrapie agent 139A was obtained from Dr. R. H. Kimberlin, A. R. C. & M. R. C. Neuropathogenesis Unit, Edinburgh, U.K. The manuscript was typed by Patricia Calimano.

REFERENCES

Bowen, D.M., C.B. Smith, P. White and A.N. Davison (1976) Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99: 459-496.

Candy, J.M., R.H. Perry, E.K. Perry, D. Irving, G. Blessed, A.F. Fairbairn and B.E. Tomlinson (1983)

350

Pathological changes in the nucleus of Meynert in Alzheimer's and Parkinson's diseases, J. Neurol. Sci., 59: 277-289.

Coyle, J.T., D. L. Price and M.R. DeLong (1983) Alzheimer's disease - - A disorder of cortical cholinergic innervation, Science, 219:1184-1190.

Davies P. and A.J.F. Maloney (1976) Selective loss of cholinergic neurons in Alzheimer disease, Lancet, ii: 1403.

Dickinson, A.G. and G.W. Outram (1983) In: L. Court (Ed.), Procs. of Symp. on Virus Non Conventionnels et Affections du Syst~me Nerveux Central, Masson, Paris, pp. 3-16.

Dickinson, A.G, H. Fraser and M. Bruce (1979) In: A.I.M. Glen and L.J. Whalley (Eds.), Alzheimer's Disease - - Early Recognition of Potentially Reversible Deficits, Churchill Livingstone, Edinburgh, London, New York, pp. 42-45.

Dickinson, A.G., M. E. Bruce and J. R. Scott, (1983) In: R. Katzman (Ed.), Banbury Report 15 : Biological Aspects of Alzheimer's Disease, Cold Spring Harbor Laboratory, pp. 387-398.

Dziedzic, J.D., K. Iqbal and H.M. Wisniewski (1980) Central cholinergic activity in Alzheimer dementia, J. Neuropath. Exp. Neurol., 39: 1351.

Fonnum, F. (1975) Radiochemical assays for choline acetyltransferase and acetylcholinesterase. In: N. Marks and R. Rodnight (Eds.), Research Methods in Neurochemistry, Vol. 3, Plenum Press, New York, pp. 253-275.

Fraser, H. (1979a) Neuropathology of scrapie - - The precision of the lesions and their diversity. In: S.B. Prusiner and W. J. Hadlow (Eds.), Slow Transmissible Diseases of the Nervous System, Academic Press, New York, pp. 387-406.

Fraser, H. (1979b) The pathogenesis and pathology of scrapie. In: D. A. J. Tyrrell (Ed.), Aspects of Slow and Persistent Virus Infections, Martinus Nijhoff, The Hague, pp. 30-57.

Fraser, H. and M. Bruce (1973) Argyrophilic plaques in mice inoculated with scrapie from particular sources, Lancet, i: 617-618.

Hetnarski, B., H. M. Wisniewski, K. Iqbal, J.D. Dziedzic and A. Lajtha (1980) Central cholinergic activity in aluminum-induced neurofibrillary degeneration, Ann. Neurol., 7: 489-490.

Lloyd, K. G., H. Mohler, P. H. Heitz and G. Bartholini (1975) Distribution of choline acetyltransferase and glutamate de-carboxylase with the substantia nigra and other brain regions from control and Parkin- sonian patients, J. Neurochem., 25: 789-795.

McDermott, J.R., H. Fraser and A.G. Dickinson (1978) Reduced choline-acetyltransferase activity in scrapie mouse brain, Lancet, ii: 318.

Perry, E.K., R.H. Perry, G. Blessed and B.E. Tomlinson (1977) Necropsy evidence of central cholinergic deficits in senile dementia, Lancet, i: 189.

Price, D.L., P.J. Whitehouse, R.G. Struble, A.W. Clark, J.T. Coyle, M.R. DeLong and J.C. Hedreen (1982) Basal forebrain cholinergic systems in Alzheimer's disease and related dementias, Neurosci. Comment., 1: 84-92.

Reisine, T.D., J.Z. Fields, I. Yamamura, E.D. Bird, E. Spokes, P.S. Schreiner and S.I. Enna (1977) Neurotransmitter receptor alterations in Parkinson's disease, Life Sci., 21 : 335-344.

Roberts, E. and D.G. Simonsen (1963) Some properties of L-glutamic decarboxylase in mouse brain, Biochem. Pharmacol., 12:113-134.

Whitehouse, P.J., D.L. Price, A.W. Clark, J.T. Coyle and M.R. DeLong (1981) Alzheimer disease - - Evidence for selective loss of cholinergic neurons in the nucleus basalis, Ann. Neurol., 10: 122-126.

Whitehouse, P.J., D. L. Price, R.G. Struble, A. W. Clark, J.T. Coyle and M. R. DeLong (1982) Alzheimer's disease and senile dementia - - Loss of neurons in the basal forebrain, Science, 215: 1237-1239.

Wisniewski, H. M., M. E. Bruce and H. Fraser (1975) Infectious etiology of neuritic (senile) plaques in mice, Science, 190:1108-1110.

Yates, C.M., J. Simpson, D. Russell and A. Gordon (1980) Cholinergic enzymes in neurofibrillary degeneration produced by aluminum, Brain Res., 197: 269-274.