Journal of Neurochemistry Raven Press, Ltd.. New York 0 1989 International Society for Neurochemistry

Down’s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

*?Stephen Kish, ?Harry Karlinsky, $$Lawrence Becker, “Joseph Gilbert, *Michelle Rebbetoy, *Li-Jan Chang, *Linda DiStefano, and *J.’Oleh Hornykiewicz

*Human Brain Laboratory, Clarke Institute of Psychiatry, Toronto, Departments of ?Psychiatry and SNeuropathology, University of Toronto, Q The Hospital for Sick Children, Toronto, I‘ Victoria Hospital, London, Ontario, Canada; and ’The

Institute of Biochemical Pharmacology and University of Vienna, Vienna, Austria

Abstract: We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and ace- tylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down’s syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer’s disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic en- zyme activity was normal in all brain regions of the individ- uals with infant DS with the exception of above-normal ac-

tivity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic in- tervention to prevent the development of brain cholinergic changes in patients with DS. Key Words: Down’s syndrome- Cholinergic-Neuron-Choline acetyltransferase-Acetyl- cholinesterase-Alzheimer’s disease. Kish S . et al. Down’s syndrome individuals begin life with normal levels of brain cholinergic markers. J. Neurochem. 52, 1183-1 187 (1989).

Down’s syndrome (DS, trisomy 2 1) has several im- portant features in common with Alzheimer’s disease (AD) (for extensive review see Karlinsky, 1986). In this regard, virtually all DS individuals surviving beyond the age of 40 develop the histopathological brain changes typical of AD, i.e., overabundance of senile plaques, neurofibrillary tangles, and granulovacuolar degeneration (Wisniewski et al., 1985). Similarly, a brain cholinergic deficiency, as demonstrated neuro- chemically by reduced levels of choline acetyltransfer- ase (ChAT) and acetylcholinesterase (AChE) (Yates et al., 1980, 1985; Godridge et al., 1987), and histologi- cally by loss of cholinergic neurons in the nucleus bas- alis (cf. Mann et al., 1984; Casanova et al., 1985; Cum- mings and Benson, 1987), has now been demonstrated both in AD and in elderly DS individuals. Circum- stantial evidence suggests that the cognitive impairment in AD and, by analogy, in elderly DS individuals may be due in part to reduced cholinergic innervation of cerebral cortical and limbic brain regions (for reviews see Cummings and Benson, 1987; Perry, 1987).

Although a brain cholinergic deficiency in elderly

DS individuals now appears to be conclusively dem- onstrated, it is at present unknown whether DS indi- viduals begin life with a compromised brain cholinergic system. The relative paucity of such neurochemical data is mostly due to the difficulties in obtaining both infant DS and control brain material as well as the technical problems of accurate and reproducible dis- section of newborn and infant brain for neurochemical study.

We now present neurochemical results of our ex- amination of the cholinergic marker enzymes ChAT and AChE in brains of seven infants aged 1 year and less with DS as compared with an age-matched control group.

PATIENTS AND METHODS

Autopsied brain was obtained from seven infants with DS (age range 14-52 weeks) and seven infants [four sudden infant death syndrome (SIDS), three non-SIDS] who died without any clinical or histopathological evidence of neurological disease (age range: 10-37 weeks). The intervals between death

Received May 20, 1988; final revised manuscript received S e p tember 21, 1988; accepted September 25, 1988.

Address correspondence and reprint requests to Dr. S. Kish at Human Brain Laboratory, Clarke Institute of Psychiatry, 250 College Street, Toronto, Ontario, M5T 1R8 Canada.

Abbreviations used: AChE, acetylcholinesterase; AD, Alzheimer’s disease; ChAT, choline acetyltransferase; DS, Down’s syndrome; SIDS, sudden infant death syndrome.

1183

1184 S. KISH ET AL.

and freezing of the brain at -80°C ranged from 2 to 24 h for the DS and from 12 to 30 h for the controls. Activities of ChAT (Perry and Perry, 1980) and AChE (Atack et al., 1986) have been shown to be stable postmortem for at least 24 h after death. A karyotype of standard trisomy ;!1 was obtained in five of the seven DS patients. In the two DS individuals for whom a karyotype had not been obtained (DS patients 3 and 4), the diagnosis of DS had been based on characteristic clinical and neuropathological features of this disorder.

Following identification of the central and temporall sulci, the cerebral cortical areas on the undissected half brain were removed according to the Brodmann classification. The hemisphere was then cut into approximately 2 mm-thick coronal slices. Subcortical brain areas were dissected from the coronal slices and identified according to the atlas of Riley (1960). Both in control and DS cases areas from identical slices were analyzed. For the caudate head the intermediate subdivision of slice 4 was analyzed whereas the intermediate subdivision of slice 7 was taken for the putamen (for details see Methods section of Kish et al., 19883).

ChAT was estimated by the radiochemical procedure of Fonnum (1975). AChE was determined using the spectro- photometric procedure of Ellman et al. (1961) as modified by Bonham et al. (1981).

RESULTS

Selected clinical and neuropathological details for DS and control cases are presented in Tables 1 and 2.

All of the DS brains showed the characteristic neuro- pathological features of DS with narrowing of the su- perior temporal gym.

The mean protein concentration (expressed per mil- ligram of tissue wet weight) was not significantly dif- ferent in DS and control brain ( p =- 0.05), with the exception of a slight but statistically significant reduc- tion in the cerebellar cortex (- 14%, p < 0.05; data not shown).

Table 3 compares the mean enzyme values for AChE and ChAT in the DS cases versus the seven control patients. Since the mean enzyme values of the four SIDS cases were not significantly different from the values of the three non-SIDS cases, the biochemical data for the SIDS and non-SIDS cases were combined for the purposes of comparison with the DS group. Mean activities of AChE and ChAT were normal in the DS group with the exception of a statistically sig- nificant elevation in occipital cortex (+49% AChE ac- tivity, p < 0.05) and putamen (+44% ChAT activity, p < 0.05). A statistically significant correlation between ChAT and AChE activity was observed only in control occipital cortex (v = 0.78, p < 0.05) and hippocampal gyrus (Y = 0.87, p < 0.05).

Figures 1 and 2 show the age dependence of the in- dividual enzyme values in frontal cortex of the control and DS cases. Although a trend was observed for in- creasing ChAT activity and declining AChE activity

TABLE 1. Selected clinical and neurupathulugical findings in infants with DS

Patient Age no. (weeks) Sex Clinical history

Brain weight" Postmortem Medications (2 weeks prior to death) Cause of death (9) delay (h)

1 14

2 16

3 20

4 24

5 30

6 34

7 52

F Laryngotracheomalacia with recurrent aspiration

M Congenital heart disease: died I day postopera- tively following cardiac surgery

F Congenital heart disease: cardiac surgery 3 weeks prior to death (remained on ventilator postopera- tively until death)

F Congenital heart disease with increasing respira- tory failure (ventilated for final 16 days of life)

M Recurrent bronchiolitis

F Congenital heart disease

M Congenital heart disease, di- abetes mellitus

Furosemide, spironolactone-hydrochlorothiazide, chloral h,ydrate, tlucloxacillin

Preoperatively: digoxin, spironolactone-hydro- chlorothi,azide. dl-hyoscyamine; intra- and postoperatively: dopamine, glyceryl trinitrate, epinephrine HCI, morphine sulfate, furose- mide, pancuronium bromide, cefazolin so- dium, magnesium

Dopamine, furosemide, digoxin, potassium, so- dium bicarbonate, morphine sulfate, spirono- lactone, nifedipine, cloxacillin, gentamycin, diazepam, phenoxybenzamine, chloral hydrate

Dopamine, glyceryl trinitrate, potassium, mor- phine sulfate, pancuronium bromide, furose- mide, sodium bicarbonate, digoxin, tobramy- cin, ticarcillin, piperacillin, trimethopnm

Salbutamol, orciprenaline sulfate

Digoxin, spironolactone

Insulin

Cor pulmonale

Cardiac arrest

476 (516) 24

581 (540) 8

Cardiac failure

Interstitial pneu- monitis

Bronchopneumonia

Dehydration (sec- ondary to fulmi- nant diarrhea)

Apneic episode

544 (575) 2

516 (644) 18

825 (691) 12

720 (714) 5

772 (925) 12

All brains showed characteristic appearance for DS including narrow superior temporal gyms. No other gross neuropathological abnormalities were observed with

a Normal brain weights were taken from Coppoletta and Wolbach (1933). the exception of softening of the calcarine cortex (case 4) and softening and discoloration of the basal ganglia and thalamus (case 7).

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BRAIN CHOLINERGIC MARKERS IN DOWN’S SYNDROME

TABLE 2. Selected clinical and neuropathological jindings in young controls

1185

Medications Control Age (2 weeks prior to Brain Postmortem

no. (weeks) Sex Clinical history death) Cause of death weight” (9) delay (h)

1 10 M Well None SIDS 615 (516) 24

2 16 F Well None SIDS 751 (540) 23

3 16 M Well None SIDS 721 (540) 18

4 17 F Mild urinary tract infection 7 days prior to None SIDS 737 (540) 12 death

5 30 M Born 2% months premature, otherwise well Erythromycin Cardiogenic 812 (691) 30

6 33 M Well None M yocarditis 1,025 (714) 15

shock

7 37 F Well None Dehydration, 696 (750) 18 secondary to viral gastroenteritis

~~

No gross neuropathological abnormalities were observed with the exception of congestion of white matter in cases 2 and 5. Normal brain weights taken from Coppoletta and Wolbach (1933).

with advancing age, these changes did not attain sta- tistical significance ( p > 0.05).

DISCUSSION

In brains of elderly DS individuals having Alzhei- mer’s-type neuropathology, i.e., increased number of senile plaques and neurofibrillary tangles, decreased activities of the cholinergic marker enzymes ChAT and AChE have been previously described by Yates et al. (1 980, 1985), Godridge et al. (1987), and Smith et al. (1988). With respect to the status of the brain cholin- ergic system in infant individuals with DS only anec- dotal single case information is available (see below). Thus, it has yet to be clearly established whether the neurochemical abnormality in adult DS reflects a su-

perimposed degenerative process or a preexisting de- velopmental failure-an interpretational dilemma first highlighted by Davidoff ( 1928), in relation to his find- ings of reduced neuronal number in adult DS cerebral cortex.

The results of our study in a group of seven DS cases aged 1 year and less demonstrate normal or somewhat above-normal activities of the specific (ChAT) and less specific (AChE) cholinergic markers in cortical and subcortical brain areas. Although the possibility exists that different results could have been obtained had we been able to compare our DS cases with a pure non- SIDS control group, this seems unlikely since our brain cholinergic data for SIDS and non-SIDS were similar. These data are consistent with the earlier findings in two single case studies in infant DS. In this regard,

TABLE 3. AChE and ChAT activity in infant brain: control versus DS

ChAT (nmol/mg protein/ AChE (mmol/g protein/h) 10 min)

Brain region Controls DS Controls DS

Frontal cortex (Area 10) Temporal cortex (Area 21) Parietal cortex (Area 7b) Occipital cortex (Area 17) Cerebellar cortex Ammon’s horn Dentate gyms Hippocampal gyms Am ygdala Caudate head Putamen

0.58 f 0.05 0.61 f 0.06 0.63 t 0.1 1 0.37 f 0.04 2.99 t 0.28 1.61 t 0.28 1.60 k 0.14 1.39 f 0.17 3.68 t 0.52 27.3 f 1.3 27.3 t 1.8

0.64 f 0.08 0.67 f 0.08 0.63 f 0.08 0.55 f 0.07“ 3.43 f 0.23 1.38 f 0.20 1.93 f 0.43 1.73 f 0.36 3.51 f 0.71 27.8 f 1.6 31.3 f 1.1

1.24 k 0.13 1.33 f 0.16 1.21 ? 0.18 0.87 2 0.1 1 3.15 f 0.96 2.83 + 0.58 3.05 + 0.54 4.11 f 0.87 4.65 f 1.01 28.6 t 4.9 33.4 f 5.0

1.45 f 0.20 1.26 + 0.20 1.17 f 0.10 0.95 f 0.22 5.67 k 1.14 3.19 + 0.57 4.83 + 1.40 4.27 f 1.48 6.94 f 2.27 38.0 ? 5.2 48.2 f 4.9”

Values represent means k SEM of seven controls and five to seven DS individuals. “ p < 0.05, Student’s two-tailed t test.

J. Neurochem., Vol. 52, No. 4, 1989

1186 S . KISH ET AL.

-2 .- : 2‘5 I 2.0 -

1.5 -

1.0 -

0.5 -

0

_,”-k’ _--- __--- __--- +--a-------o 0 --- _--- 0 0

” 10 20 30 40 50 60

Age (weeks)

FIG. 1. ChAT activity in frontal cortex of controls and patients with DS. Linear regression analysis was used to provide the best fitting straight line for the controls (solid) and DS (dashed).

Brooksbank and co-workers (1978) observed normal cortical ChAT and AChE levels in an infant (38 weeks gestational age) with probable DS who died at lbirth. Similarly, “normal” ChAT activity has been observed in cerebral cortex, caudate, and amygdala of n 10- month-old DS infant as compared with adult (mean age 68 years) control values (Yates et al., 1985). In contrast, McGeer et al. (1 985) reported relatively low values of ChAT in cerebral cortex of a 5.5-month-old DS infant as compared with adult controls andl two infants with SIDS. These investigators also reported extraordinarily high levels of AChE in most brain re- gions (cortical and subcortical) in both the case with infant DS and the two cases with SIDS (values 20-30 times adult control levels). This is in contrast to our own observations and those of Brooksbank e:t al. (1 978), who reported AChE activity in cerebral cortex of infants (newborn to 1 year) to be at most only twice adult levels (compare AChE values in Table 3 with adult levels shown in the figure in Kish et al., 1988~).

A comparison of the brain ChAT values obtained for the infants in the present study versus adult control values also obtained in our laboratory (mean age 39 years; Kish et al., 1988~) revealed that cerebral cortical and hippocampal ChAT levels in infant and adult brain are similar, indicating that a marked age-related deicline in the activity of this specific cholinergic marker en- zyme does not occur in these brain areas between the infant and middle age period. Interestingly, the mean activity of ChAT in cerebellar cortex of infant brain is about five times adult levels. Although the physiological function of acetylcholine in cerebellum is not under- stood, some immunohistological evidence indicates that, in the human cerebellum, ChAT may be localized preferentially in mossy fibers (Kan et al., 1980). Thus, our biochemical data suggest the possibility of an age- dependent loss of mossy fiber innervation in huiman cerebellum. Activity of the less specific cholinergic marker, AChE, was similar in cerebellum and occilpital cortex of infants and adults whereas in several brain

areas (frontal cortex, temporal cortex, hippocampus) enzyme levels were moderately higher (f40-70%) in the infants.

Our demonstration in a representative number of cases of normal cholinergic marker enzyme levels in cerebral and cerebellar cortex, limbic brain (hippo- campus, amygdala), and basal ganglia (caudate, puta- men, substantia nigra) suggests that the DS individual begins life with a normal complement of cholinergic neurons innervating these brain areas. However, since partial neuronal loss is known to activate neurotrans- mitter metabolism in the remaining neurons, the “normal” ChAT values in the DS brain could be the result of such compensation for moderate cholinergic neuron loss. This question requires verification (now in progress) through histological examination of cho- linergic neuron density in the nucleus basalis of Mey- nert, the medial septum, and the diagonal band of Broca; these regions provide the major cholinergic in- nervation of cerebral cortex and limbic forebrain areas (Mesulam et al., 1983). To date, the pertinent published neuropathological data are still insufficient and con- tradictory, consisting of anecdotal single case infor- mation suggesting normal (Kirkpatrick and Hicks, 1984) or possibly low (McGeer et al., 1985) nucleus basalis cell density in infant DS.

Although the ultimate cause of DS can be regarded as being inherent in the extra chromosome 21, the neurobiological basis of the mental retardation in this condition is unknown. The results of our neurochem- ical study suggest that any cognitive deficits already present in the infant DS individual are unlikely to be a consequence of reduced brain cholinergic function. More likely, such impairment in young DS may be explained by other neurobiological abnormalities pres- ent in infant DS brain, such as reduced dendritic spine number or altered synaptic morphology (Scott et al., 1983).

Recently, the mouse trisomic for chromosome 16 has been proposed as an animal model of human tri-

1.2 i- I

w o.2 t 0 ’ ’ I ” ’ I ’ I * I ’

10 20 30 40 50 60

Age (weskr)

FIG. 2. AChE activity in frontal cortex of controls and patients with DS. Linear regression analysis was used to provide the best fitting straight line for the controls (solid) and DS (dashed).

J . Neurochern., Vol. 52, No. 4, 1989

BRAIN CHOLINERGIC MARKERS IN DOWN’S SYNDROME 1187

somy 21 (DS) (Miyabara et al., 1982). Although the trisomy 16 mouse does not survive the perinatal period, the reduced levels of cholinergic, serotonergic, and noradrenergic markers in the fetal brain have been taken to represent an analogy to the brain changes in DS adults with the typical neurochemical and mor- phological Alzheimer brain changes (Ozand et al., 1984; Singer et al., 1984). However, the mouse 16 tri- somy model does not seem to reflect the biochemical brain changes found in human infant DS patients who, as we have demonstrated, begin life with normal levels of brain ChAT. Therefore, the reduced ChAT levels in the brains of adult DS patients seem to be the result of a decline from normal levels at birth rather than a congenital brain cholinergic deficiency.

With respect to clinical implications, our biochem- ical results allow, in principle, for the possibility of early therapeutic prevention, in DS, of the development of the brain cholinergic deficiency and possibly associated cognitive decline. Such possibilities may also exist for other hereditary neurodegenerative disorders such as familial AD and some cases of dominantly inherited olivopontocerebellar atrophy (Kish et al., 19884b) in which the brain cholinergic reduction present in adult- hood may develop later in life.

Acknowledgmenk This study was supported by the Clarke Institute of Psychiatry. S. Kish is a Career Scientist of the Ontario Ministry of Health.

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