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Circadian system rhythm disorders in aging and Alzheimer's disease. Role of changes inmelatonin, suprachiasmatic nucleus and corticosteroids

Liu, R-Y.

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Citation for published version (APA):Liu, R-Y. (2001). Circadian system rhythm disorders in aging and Alzheimer's disease. Role of changes inmelatonin, suprachiasmatic nucleus and corticosteroids.

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Download date: 24 May 2020

CHAPTERR 6

GlucocorticoidsGlucocorticoids Suppress Vasopressin Gene Expression in the HumanHuman Biological Clock

Rong-Yuu Liu1'2, Unga A. Unmehopa', Jiang-Ning Zhou1'2 & Dick F. Swaab1

'Netherlandss Institute for Brain Research, Netherlands 2Geriatricc Department, First Affiliated Hospital of Anhui Medical University, China

Sleepp impairment is one of the major side effects of glucocorticoid therapy. The mecha-

nismm responsible for this circadian disorder is not understood, but alterations in the su-

prachiasmaticc nucleus (SCN) are presumed to play a major role. In the present study, the

amountt of vasopressin mRNA (AVP mRNA) expression in the SCN, one of its major

neuropeptides,, was investigated in 22 human subjects. The total amount of AVP mRNA

expressedd as masked silver grains in the SCN was two times lower in glucocorticoid-

exposedd patients (n=10; 5,115+1,3 Hum2) than in age- and clock-time-of-death-matched

controlss (n=10; 11,021 1,408pm2) (P=0.006). There was also a 53% decrease in the to-

tall number of profiles in the SCN that expressed AVP mRNA in glucocorticoid-exposed

patientss ) compared with those in controls (31,490+3,816) (P=0.01).No

significantt correlation was found between the amount of AVP mRNA expression and

thee postmortem delay or brain weight in the controls or in the glucocorticoid-exposed

group.. In conclusion, glucocorticoids have an inhibitory effect on AVP mRNA expres-

sionn in the human SCN, which maybe the biological basis of the circadian rhythm dis-

turbancess during glucocorticoid therapy. This effect is rapid and reversible. Since vaso-

pressinn effects from the SCN inhibits the HPA-axis and Cortisol inhibits vasopressin pro-

ductionn in the SCN, the biological clock seems to be included in the physiology of the

feedbackk system of the HPA-axis.

97 7

CHAPTERR 6

Introductio n n

Glucocorticoidd therapy potentially has numerous central side effects, such as insomnia andd depression as well as impairment of memory and cognition, psychosis and convul-sionss (1 -4). When subjects receive glucocorticoid therapy, insomnia occurs early and is usuallyy unavoidable and sleep efficiency is reduced (1,2). The mechanism responsible forr sleep disturbances in glucocorticoid-exposed patients is not known, but alterations inn the suprachiasmatic nucleus (SCN) are presumed to be a key factor. The SCN is the majorr circadian pacemaker of the mammalian brain and coordinates hormonal and behaviorall circadian rhythms (5). One of the endocrine rhythms regulated by the SCN iss that of adrenal activity. The vasopressinergic neurons of the SCN are crucial in the regulationn of this system. In the first place, the SCN-derived vasopressinergic projec-tionn to the dorsomedial and paraventricular nucleus of the hypothalamus (6) is respon-siblee for the diurnal trough of adrenal activity. SCN lesions and microperfusion of vaso-pressinn in rats have shown that this vasopressinergic projection inhibits the hypothalamo-pituitary(HPA)) axis (7). Degeneration of vasopressin (AVP) neurons of the SCN during thee course of aging and in Alzheimer s disease (8,9) is, therefore, thought to be an impor-tantt causal factor for the elevated Cortisol levels and for the decreased amplitude of cir-cadiann rhythms in this disorder (10,11). In the second place, several animal experimen-tall studies have demonstrated that adrenalectomy and dexamethasone affect the levels off AVP or AVPmRNA in the SCN (12,13). The latter observations in rat suggest that the disorderr of circadian rhythms in patients treated with glucocorticoids may be due to theirr action on the SCN. The present study was therefore performed on postmortem SCNN of patients who had been exposed to glucocorticoids during the last premortem period.. It shows for the first time that AVP mRNA expression in the human SCN is in-deedd suppressed by glucocorticoids.

Material ss and Methods

Subjects Subjects

Tenn subjects, who were free of glucocorticoid therapy during the premortem period and

diedd from different diseases, served as controls. Of the ten patients who formed the glu-

cocorticoid-exposedd group, one had been exposed to high levels of endogenous

glucocorticoidss as the result of an adrenal tumor, and nine had been exposed to exog-

enouss glucocorticoid tablet administration at different doses and duration during the

premortemm period until their death. For clinical pathological details see Table I. Two

subjects,, who had received glucocorticoid treatment until 2 months before their death

andd one subject who had taken a low dose of beclomethason inhalation until his death,

weree evaluated separately. The patients in the glucocorticoid-exposed group had no pi-

98 8

CHAPTERR 6

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CHAPTERR 6

tuitaryy disorder as an indication for corticosteroid therapy. After their death, brain au-topsyy was performed on the patients and controls as part of the program of the Nether-landss Brain Bank. Written informed consent for brain autopsy and the use of the tissue andd medical records for research purposes were obtained before subjects entered the study.. The brains of the corticosteroid-treated patients were matched with the 10 con-trolss for age, sex and clock time of death (Table I).

MeasurementMeasurement of AVP mRNA in SCN

Inn situ hybridization was performed on every fiftieth (6-ujn) section of the SCN as de-scribedd extensively before (9). Briefly, the AVP probe (hvp3) consisted of an oligomer of 488 nucleotides complementary to bases 411-458 of the preprovasopressin precursor (14). Thee specificity of the probes has been described previously (9,15) The probe was 3'-end labeledd using terminal deoxynucleotidyl transferase (Boehringer Mannheim) and [a-33S]] dATP as described earlier (16). Each section was incubated with 68 uJ hybridization solutionn containing an approximate 1 x 106 cpm labeled probe. After overnight incuba-tionn at 42 °C, the sections were rinsed in 1 x SSC for 30 min at 50°C, 2 x 30 min 0.1 x SSC att 50°C, and 2 x 30 min 0.1 x SSC at room temperature. Sections were dehydrated at roomm temperature in 300-mM ammonium acetate (pH 5.5)/ethanol 100% at volume ratioss 1:1,3:7,1:9 and 0:1. In order to check the autoradiographic signal, sections were exposedd to p-max hyperfilm (Amersham, UK) for 2 days. Subsequently, slides were dippedd in photographic emulsion (NTB2 Kodak USA) and exposed for 17 days. Slides weree developed for 2 min in Detol Developer (Sigma) at 15°C, briefly rinsed in aquadest att 15°C and fixed in Kodak fixer (Sigma) for 14 min. Sections were washed to remove the fixativee and counter-stained with thionin.

QuantitativeQuantitative analysis of AVP mRNA

Forr quantitative analysis of the in situ signal of the AVP mRNA in the SCN, an IBAS-

KATT image analysis system was connected to a SONY XL-77CE videocamera mounted

onn a Zeiss microscope. The microscope was equipped with plan-neofluar objects and a

scanningg stage. The main principle and procedure of the IBAS measurement have been

extensivelyy described before (9,16) Briefly, the area of the SCN was manually outlined at

loww magnification (2.5x objective) and a grid of fields corresponding to a 40 x objective

wass superimposed. From this grid 50% of the fields indicated in red rectangles were

randomlyy selected and the 40x objective images were stored. Then, at high magnifica-

tionn (40x objective), each field was retrieved at high resolution on the image analysis

monitor.. A mask was superimposed on the silver grains in their images. The total mask

coveringg the silver grains in these profiles was calculated under program control. The

silverr grains of the background were subtracted. Every fiftieth section through the entire

SCNN was measured and stored in the IBAS. Finally, the total number of profiles express-

100 0

CHAPTERR 6

ingg AVP mRNA in the SCN and the total mask of the silver grains over the profiles were

calculatedd as an estimate of total amount of AVP mRNA expression in the SCN.

StatisticalStatistical Analysis

Differencess in amount of AVP mRNA in the SCN or total number of AVP mRNA ex-

pressingg cells between the groups were tested using the Wilcoxon paired matched pairs

test.. Correlation of brain weight, postmortem interval and fixation time versus amount

off AVP mRNA was analyzed by Spearman correlation coefficients in the glucocorticoid-

exposedd group and controls. A significance level of 5% (two-tailed) was used in all sta-

tisticall tests. Throughout this paper, values are expressed as mean standard error of

thee mean (SEM).

Results s

DecreasedDecreased total amount of AVP mRNA in glucocorticoid exposed patients

Thee in situ hybridization procedure on formalin-fixed paraffin-embedded material al-

lowedd us to show and quantify AVP mRNA expressing cell profiles in the human SCN of

alll patients. A clearly decreased amount of AVP mRNA expression was found in the SCN

off the majority of the patients in the glucocorticoid-exposed group (Figure 1 A, IB). The

totall mask of silver grains in cells (an estimate of total amount of AVP mRNA in the

SCN)) was reduced by 46% in glucocorticoid-exposed patients (5,115 1,314um2) as com-

paredd with controls 2) (P=0.006) (Fig 2). There was also a 53% de-

creasee in the total number of profiles in the SCN that expressed AVP mRNA in glucocor-

ticoid-exposedd patients ) compared with those in controls (31,49013,816)

(P=0.01).. Since the SCN is the clock of the brain, the time of death should also be con-

sideredd a possible confounding factor. We excluded this possibility by matching gluco-

corticoidd exposure patients as much as possible with control subjects who had died

aroundd the same time (Table I). Moreover, the differences between controls and gluco-

corticoid-- exposed patients were found at every time point over the entire period of the

dayy and night (Fig. 3).

TheThe doses and duration of glucocorticoids in relation to the AVP mRNA expression in the

SCN:SCN: case studies

Duringg the highest cumulative doses of glucocorticoid administration in our study, which

wass 12 mg of dexamethasone per day for the last 5 months (patient #99096), the total

amountt of AVP mRNA expression and the total number of profiles were around the

meann value of the cortisol-exposed group (6,522|im2 and 11,771, respectively)

Thee total amount of AVP mRNA expression in the SCN for two subjects (patient # 95092

101 1

CHAPTERR 6

Fig.. 1 Thionin-counterstained emulsion autoradiograms in frontal sections (6|0m) of the hu-mann suprachiasmatic nucleus (SCN) at high magnification. Note the black silver deposits that showw the presence of vasopressin (AVP) mRNA in SCN neurons. There are also thionin-stained AVPP mRNA negative cells present in the SCN. A: Control subject. B: Corticosteroid exposed pa-tient.. Note there were fewer AVP mRNA expressing neurons in corticosteroid exposed patients. Scalee bar=20um.

102 2

CHAPTERR 6

250000 -

200000 -

150000 -

AA 10000 -

50000 -

oo -

Totall amount of AVP mRNA in the SCN

• •

t t ! ! t t

• •

• •

T T • • 1 1

1 1

• •

Control l CST T

Fig.. 2 Estimated total amount of AVP mRNA in the SCN (expressed as masked area of silver grains)) of the controls and the corticosteroid-exposed subjects (CST). The bars and error lines representt the mean and standard error of the mean (SEM).

E E

25000 0

20000 0

15000 0

10000 0

5000 0

Totall amount of AVP mRNA in the SCN

o o o o

oo •

- 1 — I — I — I — I — I — I — I — I — I — I — I — II ' I ' I

44 8 12 16 20

Clockk time 24 4

oo Control • CST

Fig.. 3 Day-night fluctuation in the total amount of AVP mRNA of the SCN in controls and in the glucocorticoid-exposedd subjects (CST). Note that at any moment of the day the values for CST aree lower than those of controls.

103 3

CHAPTERR 6

andd 95054), who stopped glucocorticoid treatment 2 months before death was 13,394um2

andd 8,694|lm2, respectively, whereas the total number of profiles that expressed AVP mRNAA in the SCN was 19,893 and 21,606, respectively, all data in the range of the con-trols. .

Thee suppressing effect of prednisone on AVP mRNA expression in the SCN became alreadyy obvious on the second day of administration, since the total amount of AVP mRNAA and the total number of profiles in the SCN of patient # 84026 were only 97jim2

andd 709 respectively. The patient who received a low dose of beclomethason inhalation forr the last 7 days (patient #95051) did, however, not show any difference as compared withh controls, either in the total amount of AVP mRNA expression 2) or in the totall number of cell profiles (14,703).

AVPAVP mRNA expression in the SCN in relation to age, postmortem delay and fixation time

Theree were no statistical differences between the glucocorticoid-exposed group and the

controll group in postmortem delay (P=0.28), brain weight (P=0.95), fixation time

(P=0.79)) or age (P=0.96). No significant correlation was found between the amount of

AVPP mRNA and postmortem delay in controls or the glucocorticoid-exposed group

(r=0.39,, P-0.25; r=-0.13, P=0.70; respectively). Neither in controls, nor in corticoster-

oidd treated patients was a correlation found with brain weight (r=0.20 P=0.61; r=0.19,

P=0.60,, respectively). In a pilot study we found an effect of long fixation times on the

amountt of AVP mRNA expression in the SCN. To further substantiate this observation,

twoo corticosteroid-exposed patients with long fixation times (patients # 93095; 618 days

andd patient #93016; 269 days) were compared with controls who had long fixation times

(controll #96426; 728 days and control #96268 798 days). The results showed that long

fixationn times indeed sharply reduced the signal of AVP mRNA expression in the SCN.

Corticosteroid-exposedd patients with long fixation times (more than 130 days) were

thereforee excluded from the present study.

Discussion n

Duringg glucocorticoid (GC) therapy patients frequently suffer from sleep-wake rhythm

disturbancess (1,4), but the underlying mechanisms of this side effect are not known. In

thee present study, a strongly decreased amount of AVP mRNA expression was found in

thee SCN of the glucocorticoid-exposed group. The total number of cell profiles that ex-

pressedd AVP mRNA in the SCN was also strongly decreased in glucocorticoid-exposed

patientss compared with those in controls, indicating that not only the total AVP produc-

tionn but also the AVP production per cell was diminished. The SCN is considered to be

thee circadian pacemaker of the mammalian brain and to coordinate hormonal and

behaviorall circadian rhythms (5). AVP is one of the major neuropeptides in the SCN

104 4

CHAPTERR 6

andd is involved in the amplification of the amplitude of circadian rhythms (17) and in thee synchronization of the circadian rhythm of a light/dark cycle to the light entrainable oscillatorr (18). Isobe et al. reported that in rat the AVP content in the SCN and plasma corticosteronee level were inversely correlated, although the causality of this relationship didd not become clear from that study (12). The increased release of AVP from SCN ter-minalss during the light period in rat and monkey coincides with the low levels of circu-latingg corticosterone at this time of the day, and the AVP neurons of the SCN are consid-eredd to be a major inhibiting factor for the CRH neurons in the PVN (19,20). An oppo-sitee relationship between a high activity of CRH neurons of the PVN and a decreased activityy of the AVP neurons in the SCN was observed in aging humans and Alzheimer patientss where the increased CRH mRNA level in the PVN (21) and Cortisol in the CSF (10)) go together with a pronounced decline of AVP activity in the SCN (8,9,22). Sleep disruption,, nightly restlessness and other circadian rhythm disturbances are indeed fre-quentlyy seen in aging people and even more so in Alzheimer patients (11,23-25). Clini-call research indicates detrimental effects of corticosteroids on sleep architecture. Turner andd Elson report insomnia and nightmares in cancer patients receiving 8-16mg dexam-ethasonee daily (26). The study by Young et al (27) demonstrated statistically significant reductionss in the percentage of REM sleep and number of REM periods following 20mg off hydrocortisone orally, twice daily.

Thee neurobiological basis of the disturbed sleep architecture in aging, Alzheimer's dis-easeease and during corticosteroid exposure was hypothesized to be a decreased functional activityy of the SCN. Only a few animal studies have so far demonstrated that adrenal steroidss may affect the AVP content of the SCN. Adrenalectomy as well as treatment of thee rats with dexamethasone has shown that the expression of AVP mRNA in the SCN is susceptiblee to alterations in circulating levels of glucocorticoids, at least during a narrow timee window in the diurnal cycle (13). Boyer et al (1979) found that there is a persist-entlyy abnormal Cortisol circadian rhythm in patients with Cushing's disease (28). These observationss and our present data support the hypothesis that glucocorticoid exposure duringg corticosteroid treatment or in Alzheimer's disease could cause circadian rhythm disturbancess because they affect the function of the SCN. Whether or not this is a receptor-mediatedd effect, and whether the SCN is directly or indirectly influenced by corticosteroids,, should be further investigated.

Wee did not find an age-related decline in AVP mRNA in the SCN in the present study. Thiss is probably due to the relatively young average age of the subjects in our study, which wass 5 years. This is much younger than the 80 years of age in which previous studies foundd a change in the number of neurons expressing AVP peptide and the amount of AVPP mRNA in the SCN (8,9). Corticosteroid seems to have a suppressive effect on vari-ouss peptidergic neurons in the hypothalamus. A recent report showed a negative corre-lationn between circulating Cortisol levels and metabolic activity of the hypothalamus

105 5

CHAPTERR 6

(29).. Corticosteroids seem to have a global inhibiting effect on brain metabolism as well

(30).. Earlier studies by our group have shown that corticosteroid decreased the amount

off CRH and AVP in the paraventricular nucleus and AVP in the supraoptic nucleus of

thee human hypothalamus. On the other hand, oxytocin immunocytochemistry in the

PVNN was not affected by corticosteroids, indicating specificity in the effect of

corticosteroidss on hypothalamic neurons (31).

Thee suppressive effect of prednisone on AVP mRNA expression in the SCN seems to

actt rapidly since its effect was already obvious on the second day of administration (pa-

tientt # 84026). On the other hand, the patient who had received low dose beclomethason

inhalationn for the last 7 days (patient #95051) did not show any difference as compared

withh controls, either in the amount of AVP mRNA expression or in the total number of

celll profiles containing AVP mRNA in the SCN, while the same patient showed a de-

creasee in CRH and AVP content in the PVN and SON (31). The SON and PVN thus

seemm to be more sensitive to the effect of beclomethason than the SCN. In a study of low

dosee (0.5mg) oral dexamethasone in depression, there was no change in sleep structure

(32).. Whether these results can be explained by the fact that AVP expressing cells in the

PVNN are more sensitive to glucocorticoids than AVP-expressing cells in the SCN should

bee further investigated. On the other hand, during the highest cumulative doses of glu-

cocorticoidd administration in our study, which was 12mg/day dexamethasone for the

lastt 5 months (patient # 99096), AVP mRNA expression was still detectable around the

meann value of the cortisol-exposed group, suggesting some mechanism of partial adap-

tationn to a long-term, high dosage of corticosteroids. The amount of AVP mRNA was in

thee range of control values 2 months after stopping prednisone therapy, suggesting re-

coveryy from suppression (patient # 95054 and 95092). This agrees with an animal ex-

perimentall study that showed that one dose of dexamethasone did not have an effect on

thee AVP content of the SCN on the next day (12). The inhibiting effect of glucocorticoids

onn the AVP mRNA in the SCN is of special interest in depression. In this disorder high

Cortisoll levels and sleep disorders are often found. These may well be explained by the

inhibitoryy effect of glucocorticoids on the SCN. Indeed, we recently found a decreased

activityy of AVP neurons in the SCN in depression (Zhou et al., 2000 submitted). In con-

clusion,, the glucocorticoids have an inhibitory effect on the AVP mRNA in the SCN.

Thiss effect is rapid and reversible. Since vasopressin efferents from the SCN inhibit the

HPA-axiss (7,33,34) and corticosteroids inhibit the vasopressin production of SCN neu-

rons,, the vasopressin neurons of the SCN seem to be incorporated in the feedback sys-

temm of the HPA-axis.

Acknowledgments s

Wee are grateful to J.J. van Heerikhuize and B. Fisser for their technical assistance, to W.T.P.

Verweijj for secretarial help, to Michel Hofman for his kind help with statistical evalua-

106 6

CHAPTERR 6

tionn and to C. Pool for his IBAS image analysis programs. Brain material was obtained

fromm the Netherlands Brain Bank (coordinator R. Ravid). This study is partly supported

byy the Royal Netherlands Academy of Arts and Sciences (98CDP004; R-Y Liu and J-N

Zhou),, by the Van den Houten foundation (Zhou), by PAD 9607 (D.F. Swaab),and by the

Nationall Key Project for Basic Research of China (G1999054007).

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