Normal cortical concentration of cholecystokinin-like immunoreactivity with reduced choline acetyltransferase activity in senile dementia of Alzheimer type

Life Sciences, Vol. 29, pp. 405-410 Printed in the U.S.A.

NORMAL CORTICAL IMMUNOREACTIVITY ACTIVITY IN M.N. C.Q.

Pergamon Press

CONCENTRATION OF CHOLECYSTOKININ-LIKE WITH REDUCED CHOLINE ACETYLTRANSFERASE SENILE DEMENTIA OF ALZHEIMER TYPE

Rossor, Mountjoy

J.F. Rehfeld*, 2, M. Roth 2,

P.C. Emson**, L.L. Iversen**.

Departments of Neurological Surgery & Neurology and Psychiatry 2, Addenbrooke's Hospital, Hills Road, Cambridge * I n s t i t u t e of M e d i c a l B i o c h e m i s t r y , U n i v e r s i t y of Aarhus, D K - 8 0 0 0 Arhus C, D e n m a r k **MRC Neurochemical Research Council

Pharmacology Centre, Medical Road, Cambridge

Unit, Medical School, Hills

(Received in final form May 28, 1981)

Summary Choline acetyltransferase (CHAT) activity and cholecystokinin immunoreactivity (CCK-I) were determined in ten brains from patients dying with a diagnosis of senile dementia of Alzheimer type (SDAT) and in ten brains from control cases. The post-mortem stability of CCK-I was hi~, as determined using a mouse brain model. Although ChAT activity was significantly reduced in the cerebral cortex, hippocampus and caudate nucleus in the SDAT cases, there was no difference in CCK-I content between the two groups in any brain area. Thus the population of intrinsic cortical cells which contains CCK-I does not appear to be significantly affected in SDAT. Cholecystokinin (CCK) peptides, are found in s u b s t a n t i a l a m o u n t s in the m a m m a l i a n brain as well as in the gut. T h e localisation of CCK-I to synaptic v e s i c l e s (I), its c a l c i u m d e p e n d e n t r e l e a s e from brain tissue slices (I), and its potent excitatory a c t i o n on c o r t i c a l n e u r o n e s (2) all argue for the c l a s s i f i c a t i o n of C C K as a p u t a t i v e neurotransmitter (for r e v i e w see Emson(3) and Rehfeld(4)). C h e m i c a l a n a l y s i s suggests that the p r i n c i p l e C C K in~nunoreactivity in the brain c o r r e s p o n d s to the C - t e r m i n a l octapeptide, CCK-8 (5,6). R e g i o n a l d i s t r i b u t i o n studies in pig and g u i n e a - p i g brain have d e m o n s t r a t e d large amounts of CCK-I in cerebral cortex, hippocampus, amygdala, h y p o t h a l a m u s and basal g a n g l i a (5,7) and CCK-I has also been demonstrated in human p o s t - m o r t e m c e r e b r a l c o r t e x and basal g a n g l i a (8,9). I m m u n o h i s t o c h e m i c a l studies in the rat i n d i c a t e that C C K - I - c o n t a i n i n g cells in the cerebral c o r t e x correspond to n o n - s p i n y b i p o l a r and n o n - s p i n y s t e l l a t e neurones and are d i s t i n c t from g l u t a m i c acid d e c a r b o x y l a s e p o s i t i v e (GABAergic n e u r o n e s ) and v a s o a c t i v e intestinal p o l y p e p t i d e (VIP)0024-3205/81/040405-06502.-0/0 Copyright (c) 1981 Pergamon Press Ltd.

406

positive

CCK and ChAT in Senile Dementia

neurones

Vol. 29, No. 4, 1981

(I0).

Senile dementia of the Alzheimer type (SDAT) is characterised histologically by the occurence of numerous senile plaques and intraneuronal neurofibrillary tangles. These histological changes are most marked in the hippocampus and cerebral cortex. Neurochemical studies of SDAT post-mortem brain have demonstrated marked reductions in choline acetyltransferase (CHAT) activity and less profound decreases in noradrenaline (for review see Terry and Davies(ll)), both of which are normally found in cortical afferent terminals. Measurements of cortical peptides have shown a significant reduction in somatostatin (SRIF) content (12,13,14) but no change in VIP (15), indicating that the loss of cortical neurones in SDAT may not affect all categories of cells equally. It was, thus, of interest to determine whether CCK, another neuropeptide present in cortical inter-neurones, was reduced in SDAT. The present paper describes the results of a post-mortem study of CCK-I in various regions of the brains from patients dying with a diagnosis of SDAT, together with data on the post-mortem stability of CCK-I using an animal brain model (16). Methods Ten brains were examined from patients dying with a clinical diagnosis of SDAT (3 males, 7 females; mean age ± SD = 80 ± 9years; mean autopsy delay ± SD = 47 ± 22h). All of these patients had been assessed during life on a dementia scale and an informationconcentration-memory test (17) and the diagnosis of SDAT was confirmed histologically. For comparison ten brains were examined from patients dying without a history of intellectual impairment (5 males, 5 females; mean age ± SD = 78 ± 8years; mean autopsy delay ± SD = 60 ± 26h). All control cases were normal on histological examination. All the brains were divided mid-sag±tally and one half fixed in formalin fo~ histological examination and the other half frozen at -70oc for subsequent biochemical studies. The details of the storage and dissection of the frozen half have been described previously (18). Eight brain areas were examined: Brodmann area I0 (prefrontal cortex), Brodmann area 4 (motor cortex), Brodmann area 7 (parietal cortex), Brodmann area 21 (temporal cortex), Brodmann area 17 (occipital cortex), hippocampus, caudate nucleus, lateral segment of globus pallidus and substantia nigra pars compacta. The tissue samples for ChAT assay were homogenized in water and assayed by the method of Fonnum (19). Tissue samples for CCK-I estimation were first extracted in boiling water, centrifuged and the pellet further extracted in IM acetic acid. The supernatants from both the water and acetic acid extracts were pooled and freeze dried. Freeze dried samples were reconstituted in buffer and the CCK-I reactivity determined by the radioimmunoassay procedure of Rehfeld (20). The predominant molecular form (>80%) measured by this assay in post-mortem cerebral cortex corresponds to CCK-8 as determined by gel chromatography (9). Extraction and assay of control and SDAT tissue were performed in parallel. Protein determinations were performed according to the method of Lowry et al.(21).

Vol. 29, No. 4, 1981

CCK and ChAT in Senile Dementia

407

The mouse brain cooling model of Spokes and Koch(16) was used to determine the likely post-mortem stability of CCK-I in brain. Mouse brains were slowly cooled from 37oc to 4oc to simulate the time course of cooling observed in human brain when cadavers are placed in a 4oc mortuary refrigerator. Mouse brains were removed from the cooling incubator at various intervals up to 72h postmortem and extracted for determination of their CCK-I content.

250-

2OO-

i[

150-

o?

50-

o

HOURS

Fig. I Post-Mortem

S t a b i l i t y of M o u s e Brain Immunoreactivity

Cholecystokinin

M o u s e c a d a v e r s were slowly c o o l e d from 37oc to 4°C to s i m u l a t e the c o o l i n g curve o b s e r v e d in human c a d a v e r s (16). M o u s e brains w e r e r e m o v e d at the times indicated and extracted for CCK-I d e t e r mination. F i g u r e s are m e a n s ± S.E.M. for 5-7 brains. None of the v a l u e s d i f f e r e d s i g n i f i c a n t l y from that at zero time. Results T h e CCK-I content of w h o l e m o u s e brains did n o t alter s i g n i f i c a n t l y for up to 72h p o s t - m o r t e m (Fig. I). CCK-I also a p p e a r s to be r e m a r k a b l y stable in human p o s t - m o r t e m tissue, since there was no c o r r e l a t i o n b e t w e e n CCK-I content and a u t o p s y delay. Furthermore, gel c h r o m a t o g r a p h y has d e m o n s t r a t e d the p r e s e n c e of CCK-8, in cerebral c o r t e x samples o b t a i n e d at autopsy in similar a m o u n t s to that found in b i o p s i e d cortex (9). P r e v i o u s human post-mortem and m o u s e brain c o o l i n g studies have d e m o n s t r a t e d that C h A T is very stable after d e a t h (16,18).

408

CCK and ChAT in Senile Dementia

Vol. 29, No. 4, 1981

T h e r e s u l t s of the C h A T and CCK-I d e t e r m i n a t i o n s are s u m m a r i s ed in T a b l e I. C h A T a c t i v i t y was s i g n i f i c a n t l y lower in the SDAT brains in B r o d m a n n areas I0, 4, 7, and 21, and in the h i p p o c a m p u s and c a u d a t e nucleus. T h e fall in C h A T a c t i v i t y was g r e a t e s t in the temporal cortex and h i p p o c a m p u s (54%). In contrast to the reduction in C h A T a c t i v i t y in the SDAT brains, there was no d i f f e r e n c e in CCK-I content in any of the brain areas examined. TABLE Cholecystokinin-I

Content Activity in

Brain Area

I

and Choline Control and

Acetyltransferase SDAT Brains

ChAT

CCK-8 equivalents (pmol/g tissue) Control

(CHAT)

(~mol/h/g protein)

SDAT

SDAT

Control

Cerebral Cortex: Brodmann Area

10

456

±

53

384

± 70

6.76

± 0.45

3.89

± 0.49***

Brodmann Area

4

604

± 163

409

± 80

6.06

± 0.54

3.58

± 0.61"*

Brodmann Area

7

325

±

44

345

± 49

4.68

± 0.53

2.93

i

Brodmann Area

21

603

±

76

495

± 85

8.06

± 0.63

3.70

$ 0.59***

Brodmann Area

17

280

±

29

315

± 52

3.34

± 0.26

2.39

± 0.45

Hippocampus

405

±124(9)

380

± 57

8.44

± 1.79(9)

Caudate

237

±

40

270

± 61

279.1

Globus Pallidus/ lateral segment

46

±

14

51 ± 18

58.1

± 8.1

35.8

± 11.6

Substantia nigra pars compacta

125

±

30(9)

15.88

± 1.89(9)

11.43

± 2.95

Nucleus

161

± 48

18.13

± 1.48(9) ± 30

188.5

0.42*

± 20.8*

Figures are means S.E.M. for I0 control and I0 SDAT brains except where indicated in parenthesis. Brodmann area I0 = prefrontal cortex; Brodmann area 4 = motor cortex; Brodmann area 7 = parietal cortex; Brodmann area 21 = middle temporal gyrus; Brodmann area 17 = occipital cortex. The protein contents of the control and SDAT group did not differ significantly. The lowest concentration of CCK that could be distinguished from zero with 95% confidence was 0.i pmol/g tissue. *p<0.05;

**p<0.01;

***p<0.001,

Students

t-test

(two-tailed).

DiscUssion R e d u c e d C h A T a c t i v i t y in SDAT b r a i n s has b e e n w i d e l y r e p o r t e d (for r e v i e w see T e r r y and D a v i e s (11)). The r e d u c t i o n in C h A T activ i t y r e p o r t e d here, and in e a r l i e r p u b l i c a t i o n s (12,13,15), is less p r o f o u n d than that r e p o r t e d by P e r r y (22) and D a v i e s (23), a l t h o u g h similar to the r e d u c t i o n s found in the temporal cortex by Bowen et a1.(24). T h i s r e d u c t i o n in c o r t i c a l C h A T p r o b a b l y r e f l e c t s a loss of a f f e r e n t c h o l i n e r g i c t e r m i n a l s r a t h e r than ~ intrinsic n e u r o n e s (15,22), and the losses of c o r t i c a l n o r a d r e n a l i n e (25) and of dopamine-B-hydroxylase (26) also indicate d a m a g e to a s c e n d i n g t e r m i n a l s rather than to intrinsic c o r t i c a l neurones.

Vol. 29, No. 4, 1981

CCK and ChAT in Senile Dementia

409

The question of whether substantial numbers of cortical cells are lost in SDAT, when compared with age matched controls is still unanswered (27,28). VIP, which is found in intrinsic cortical cells, is unchanged in SDAT brains (15). On the other hand, we have found that the concentration of somatostatin, which is also a marker of intrinsic cortical cells, is reduced by nearly half in the temporal cortex of SDAT brains (12,13) and a more widespread reduction of somatostatin has also been reported (14). The finding of a normal content of cortical CCK-I another marker of intrinsic cells, is thus of particular interest. Although the possibility remains that tissue shrinkage may have masked an absolute loss of CCK-neurones, the results suggest that these neurones are relatively unaffected. The normal content of cortical CCK-I and VIP (15) suggests that any significant loss of cortical neurones in SDAT must be confined to specific populations of cells. Acknowledgeme.nts We would like to thank the many clinicians and pathologists involved in the collection of tissue and in particular Professor Austin Gresham and members of staff in the Department of Morbid Anatomy at Addenbrooke's Hospital, Cambridge. We also acknowledge the excellent technical assistance of Mr N Garrett, Mrs C RekstenSmith, and Miss Bodil Basse. The study was supported by grants from the Danish MRC and the NOVO foundation and the European Training Programme in Brain and Behaviour Research. References

I. 2.

3. 4. 5. 6. 7.

8. 9. i0. ii. 12.

13~ 14. 15. 16.

P.C. EMSON, C.M. LEE and J.F. REFELD, Life Sciences 26 21572163 (1980) J. DODD and J.S. KELLY, J.Physiol. 295 61-62P (1979) P.C. EMSON, Progr.Neurobiol. 13 61-I-~ (1979) J.F. REHFELD, Trends in Neurosciences 3 65-67 (1980) J.F. REHFELD, J.Biol.Chem. 253 4022-4050 (1978) G.J. DOCKRAY, R.A. GREGORY, J.B. HUTCHISON, I.I. HARRIS and M.J. RUNSWICK, Nature 274 711-713 (1978) L-I. LARSSON and J.F. REHFELD, Brain Res. 165 201-218 (1979) P.C. EMSON, J.F. REHFELD, H. LANGEVIN and M. ROSSOR, Brain Res. 198 497-500 (1980) P.C. ~-SON, M. ROSSOR, S.P. HUNT, V. CLEMENT-JONES, J.FAHRENKRUG and J. REHFELD, Transmitter. B.iochem.istry of the Human Brain, Eds. E. Usdin and P. Riederer, MacMillan 1981 (in press) P.---~EMSON, S.P. HUNT, J.F. REHFELD, N. GOLTERMAN and J. FAHRENKRUG, Neural Peptides and Neu.rona! Con~..unication, Eds. E. Costa and M. Trabucchi, pp 63-74, Raven Press (1980) R.D. TERRY and P. DAVIES, Ann. Rev. Neurosci. 3 77-95 (1980) M.N. ROSSOR, P.C. EMSON, L.L. IVERSEN, C.Q. MOUNTJOY, M. ROTH, J. HAWTHORN, V.T.Y. ANG, J.S. JENKINS and J. FAHRENKRUG, Metabolic Disorders of the Nervous System, Ed. F.C. Rose, Pitman (in press) M.N. ROSSOR, P.C. EMSON, C.Q. MOUNTJOY, M. ROTH and L.L. IVERSEN, Neurosci.Lett. 20 373-377 (1980) P. DAVIES, R. KATZMA~--and R.D. TERRY, Nature 288 279-280 (1980) M. ROSSOR, J. FAHRENKRUG, P. EMSON, C. MOUNTJOY, L. IVERSEN and M. ROTH, Brain Res. 201 249-253 (1980) E.G.S. SPOKES and D.J, KOCH, J.Neurochem. 31 381-383 (1978)

410

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

CCK and ChAT in Senile Dementia

Vol. 29, No. 4, 1981

~A ROTH and B. HOPKINS, J.Ment.Sci. 99 439-450 (1953) E G.S. SPOKES, Brain 102 333-346 (1979) F FONNUM, Biochem. J.--~5 465-472 (1969) J F. REHFELD, J.Biol.Chem. 253 4016-4021 (1978) O H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J Biol.Chem. 193 265-275 (1951) E K. PERRY, Age and Ageing 9 1-8 (1980) P. DAVIES, Brain Res. 171 3T9-327 (1979) D.M. BOWEN, P. WIIITE, J.A. SPILLANE, M.J. GOODHARDT, G. CURZON, P. IWANGOFF, W. ~.~EIER-RUGE and A.N. DAVISON, Lancet 11-14 (1979) R. ADOLFSSON, C.G. GOTTFRIES, B.E. ROOS and B. WINBLAD, Brit. J.Psychiat. 135 216-223 (1979) A.J. CROSS, T.J. CROW, E.K. PERRY, R.H. PERRY, G. BLESSED and B.E. TOMLINSON, Brit.Med. J. 282 93-94 (1981) E.J. COLON, Acta neuropath. (Berl) 23 281-290 (1973) R.D. TERRY, Fed.Proc. 37 2837-2840 ~978)