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Published as doi: 10.1096/fj.05-4446fje.
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(The FASEB Journal. 2006;20:1874-1876.)
© 2006 FASEB

Pineal clock gene oscillation is disturbed in Alzheimer’s disease, due to functional disconnection from the "master clock"

Ying-Hui Wu*, David F. Fischer*,1, Andries Kalsbeek*, Marie-Laure Garidou-Boof*, Jan van der Vliet*, Caroline van Heijningen*, Rong-Yu Liu{dagger}, Jiang-Ning Zhou{ddagger} and Dick F. Swaab*,2

* Netherlands Institute for Neuroscience, Amsterdam, The Netherlands;

{dagger} Anhui Geriatrics Institute, The first Affiliated Hospital of Anhui Medical University, Hefei, P. R. China; and

{ddagger} Department of Neurobiology, School of Life Science, University of Science and Technology of China, Hefei, P.R. China

2Correspondence: Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands. E-mail: d.f.swaab{at}nin.knaw.nl

ABSTRACT

The suprachiasmatic nucleus (SCN) is the "master clock" of the mammalian brain. It coordinates the peripheral clocks in the body, including the pineal clock that receives SCN input via a multisynaptic noradrenergic pathway. Rhythmic pineal melatonin production is disrupted in Alzheimer’s disease (AD). Here we show that the clock genes hBmal1, hCry1, and hPer1 were rhythmically expressed in the pineal of controls (Braak 0). Moreover, hPer1 and hß1-adrenergic receptor (hß1-ADR) mRNA were positively correlated and showed a similar daily pattern. In contrast, in both preclinical (Braak I-II) and clinical AD patients (Braak V-VI), the rhythmic expression of clock genes was lost as well as the correlation between hPer1 and hß1-ADR mRNA. Intriguingly, hCry1 mRNA was increased in clinical AD. These changes are probably due to a disruption of the SCN control, as they were mirrored in the rat pineal deprived of SCN control. Indeed, a functional disruption of the SCN was observed from the earliest AD stages onward, as shown by decreased vasopressin mRNA, a clock-controlled major output of the SCN. Thus, a functional disconnection between the SCN and the pineal from the earliest AD stage onward could account for the pineal clock gene changes and underlie the circadian rhythm disturbances in AD.—Wu, Y-H., Fischer, D. F., Kalsbeek, A., Garidou-Boof, M-L., van der Vliet, J., van Heijningen, C., Liu, R-Y., Zhou, J-N., Swaab, D. F. Pineal clock gene oscillation is disturbed in Alzheimer’s disease, due to functional disconnection from the "master clock."


Key Words: circadian system • clock gene • vasopressin mRNA • superchiasmatic nucleus

IN MAMMALS, THE MASTER circadian clock is located within the suprachiasmatic nucleus (SCN) of the hypothalamus, which receives environmental light-dark information and orchestrates circadian rhythms at the organismal level (1 , 2) . Molecular components of the circadian oscillator in mammals are a set of clock genes that involve intracellular transcriptional/translational feedback loops with negative (Per1–3, Cry1–2) and positive limbs (Bmal1 and clock) (1 , 3) . Recent mammalian clock gene studies have revealed molecular clocks in many other brain regions and peripheral tissues, such as the pineal gland and liver, that are probably synchronized by the master clock in the SCN (4 5 6) . Human clock genes are also expressed widely in the brain (7) , although an analysis of rhythmic expression has so far been reported only in peripheral tissues such as oral mucosa, skin, and peripheral blood mononuclear cells (8 9 10) .

A major output of the SCN in mammals, including humans, is the circadian rhythm of melatonin synthesis in the pineal gland, which is involved in the regulation of the circadian system (11 12 13) . Sympathetic innervation of the mammalian pineal is activated at night via a multisynaptic pathway from the SCN to release noradrenalin, which acts on the ß1-adrenergic receptor (ß1-ADR) of the pinealocyte to trigger the cAMP signaling pathway (14) and thus leads to the activation of melatonin biosynthesis (15 , 16) . Clock gene Per1 is rhythmically expressed in the rodent pineal under the same noradrenergic control from the SCN as the one that regulates melatonin synthesis (17 18 19 20) . Thus, the molecular clock of the rodent pineal seems to be synchronized by the central clock in the SCN. Although the role of the pineal molecular clock has not been fully elucidated, its involvement in the gated expression of N-acetyltransferase, the rhythm-generating enzyme of melatonin biosynthesis, has been proposed in rodents (21) .

Alzheimer’s disease (AD) patients often have disturbed sleep-awake behavior, which is one of their major clinical problems (22) . A deterioration of neuronal function in the SCN has been reported in clinical AD patients (23 24 25) . Moreover, melatonin levels are dramatically decreased and the circadian rhythm in melatonin is lost in AD patients (26 27 28) . These changes are considered to be responsible for the circadian disorders in AD. Recently we reported that the circadian noradrenergic regulation of the pineal by the SCN is affected in both preclinical AD subjects (cognitively intact subjects with the earliest AD neuropathological changes, i.e., Braak stages I-II) and clinical AD patients (Braak stages VI), and results in a loss of the circadian rhythm of melatonin synthesis (28) .

In the present study, we hypothesized that 1) day-night synchronization of the clock gene oscillation in the human pineal gland is affected early on in the AD process; 2) the SCN is affected from the earliest AD stages onward, which is responsible for the changes in pineal clock genes. To test the first hypothesis, we studied the diurnal rhythm of clock gene expression in the pineal gland of unaffected controls (Braak stage 0), preclinical AD subjects (Braak stages I-II), and clinical AD patients (Braak stages V-VI) by quantitative polymerase chain reaction (QPCR). To test the second hypothesis, we determined the expression of vasopressin mRNA in the SCN, a clock-controlled major output of the SCN in controls, preclinical and clinical AD subjects by means of quantitative in situ hybridization. To find experimental support for the second hypothesis, the effect of a loss of SCN control on the pineal clock gene oscillation was investigated by measuring pineal clock gene diurnal expression in the rat before and after disruption of the SCN-pineal functional connection, either by superior cervical ganglionectomy (SCG-X) or by lesioning the SCN (SCN-X).

MATERIALS AND METHODS

Subjects
Human postmortem brain material was obtained from the Netherlands Brain Bank (NBB), which supplies postmortem specimens from clinically well-documented and neuropathologically confirmed cases. Autopsies were performed on NBB donors from whom written informed consent had been obtained for a brain autopsy and for the use of the material and of the clinical data for research purposes. Pineal glands from 68 subjects were studied: 24 controls (without any primary neurological or psychiatric disease and devoid of AD neuropathological changes in the brain, i.e., Braak stage 0), 22 preclinical AD subjects (cognitively intact with minor AD neuropathological changes, i.e., Braak stages I-II), and 22 late clinical AD patients [clinically met the NINCDS-ADRDA criteria (29 , 30) and with extensive AD neuropathological changes, i.e., Braak stages V-VI]. To determine and compare diurnal variations of pineal clock genes expression in Braak stages 0, I-II and V-VI, subjects were grouped into four time bins: 1000–1600; 1600–2200; 2200–0400; 0400–1000 (i.e., day period, day/night transition period, night period, and night/day transition period, respectively) based on their clock time of death, and matched according to their distribution in the time bins (Table 1 ). For the human SCN study, 9 controls in Braak stage 0, 9 preclinical AD subjects in Braak stages I-II, and 9 clinical AD patients with Braak stages V-VI were collected and matched for age, gender, and day/night distribution (day: 10 AM-10 PM) (25) according to their clocked time of death (Table 2 ). The following variables were included in the statistical analysis: age, sex, clock time of death, postmortem delay, brain weight, pineal weight, pH of CSF [a measure of agonal state (31) ], fixation time, and the cause of death (Tables 1 , 2) . There was no difference in pH of CSF, pineal weight, and fixation time in control, preclinical, or clinical AD groups (all P>0.05). The Spearman correlation test showed that the differences of brain weight and postmortem delay among groups (P=0.03, P=0.04, respectively) did not seem to affect the results we obtained (all P>0.05).


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  		Table 1. Clinical and pathological data for the controls, preclinical and clinical Alzheimer’s disease patients whose pineal was studied (mean±SE)a


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  		Table 2. Clinical and pathological data for the controls, preclinical and clinical Alzheimer’s disease patients whose SCN was studied (mean±SE)a

Animals
Experiments were conducted on adult male Wistar rats (200–300 g) (12 h light/12 h dark schedule, food and water ad libitum). All experiments were performed in accordance with guidelines on the care of experimental animals of the Animal Experimentation Committee of the Netherlands Institute for Neuroscience (NIN) and approved by the European Communities Council Directive. Rats were divided into five groups: intact controls (n=48), sham SCN lesions (n=6), sham SCG lesions (n=6), SCN lesions (SCN-X) (n=36), and SCG lesions (SCG-X) (n=12). Control rats (6 per time point) were sacrificed at different time points of a day: ZT2, 6, 10, 14, 18, and 22 (ZT12 is the time when the light was switched off). Using quantitative PCR, we revealed that rat Cry1 and Per1 were diurnally expressed in the pineal, with a peak at ZT18 and a trough at ZT8 (see Results). Bmal1 mRNA also showed a significant diurnal pattern, with a peak at ZT2. Bmal1 mRNA was significantly higher at ZT18 than at ZT8 (see Results). We therefore chose to sacrifice the SCN-X and SCG-X rats at ZT18 and ZT8. Sham lesioned rats did not differ from intact control rats as far as clock gene expression was concerned and so were added to the control group.

Suprachiasmatic nucleus lesion (SCN-X)
SCN-X was performed in 36 rats as described previously (32 , 33) . To make a preselection of the effective SCN lesions we measured water intake during a 3 wk period after a 2 wk recovery period. The rats that drank >33% of their 24 h water intake during 8 h of the 12 h light period (from ZT2 to ZT10) were considered arrhythmic (34) . This resulted in a group of 14 animals, which were decapitated and processed for immunohistochemistry. The histological control of vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) staining in the SCN area or SCN target areas revealed nine animals with complete SCN-X (32 , 33) .

Superior cervical ganglia ectomy (SCG-X)
Animals (n=12) were deprived of the sympathetic innervation of the pineal gland by bilateral removal of the SCG (35) 10 days before they were sacrificed. The success of the surgery was checked by observing the ptosis of both eyelids, resulting in a group of 10 rats.

Pineal RNA isolation, reverse transcription, and quantitative PCR (QPCR)
Each frozen human or rat pineal gland was homogenized and total RNA was isolated with TRIzol Reagent (Invitrogen, Breda, the Netherlands). cDNA was synthesized using superscript II reverse transcriptase (Invitrogen) in 5 min at 30°C, 5 min at 37°C, and 90 min at 42°C. We used elongation factor 1 alpha (EF-1-alpha) and E2 ubiquitin conjugating enzyme (Ube2d2) to normalize the target gene expression data. The primers were designed with Primer Express software (Applied Biosystems, Foster City, CA, USA). The efficiency of each primer pair was calculated using cDNA dilution curves and linear regression. Details of the primers, GenBank accession numbers, and the efficiency of each primer pair are given in Table 3 . QPCR was performed using the SYBR Green PCR kit (Applied Biosystems) and an Applied Biosystems Model ABI 5700 Prism Sequence Detection System. An RT-polymerase chain reaction (RT-PCR) volume of 20 µl was used. All samples were run in duplicate using an annealing temperature of 60°C. The amount of every target gene is calculated by raising the primer efficiency of the gene to the power of minus cycle threshold (–CT), normalizing this, and dividing it by the average of the two normalized housekeeping gene expression levels (28) .


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  		Table 3. GenBank accession code, sequence of PCR primer pairs for the HUMAN and RAT target genes and reference genes, and amplification efficiency of each primer pair

In situ hybridization and quantitative analysis of AVP mRNA in the SCN
In situ hybridization was performed on every fiftieth (6 µm) section of the human postmortem SCN as extensively described before (25) . We used an IBAS-KAT image analysis system for quantitative analysis of the in situ signal of the AVP mRNA in the SCN as described (25) .

Statistical analysis
Differences among the groups were statistically evaluated by the Kruskal-Wallis multiple comparison test. Differences between groups were tested by the Mann-Whitney test. Diurnal rhythmicity of clock gene expression was determined first by the Kruskal-Wallis test over the four time bins. If a significant difference was identified among the four time bins, each combination of two time bins was then compared by the Mann-Whitney test. Correlations were analyzed by the Spearman correlation test. Statistical significance was considered at the P < 0.05 level (2-tailed). Data are expressed as mean ±SE.

RESULTS

Diurnal rhythmic expression of hBmal1, hCry1, hPer1 is lost in the pineal gland of both preclinical (Braak stages I-II) and clinical AD subjects (Braak stages V-VI).

We found a significant diurnal rhythmic profile of clock genes hBmal1, hCry1, and hPer1 in the pineal gland of control subjects, as well as hß1-adrenergic receptor (hß1-ADR) mRNA (P=0.01; P=0.04; P=0.03; P=0.006, respectively) (Fig. 1 a–c, e). There were no significant daily variations in hClock gene expression (Fig. 1d , P=0.76). Gene expression of hBmal1 showed a trough at the 1000–1600 time bin (0.86±0.07) compared with the other three time bins (all P<0.02). hCry1 had a nocturnal peak at 2200–0400 (1.31±0.08), which represents a 75% increase compared with the 1000–1600 time bin (0.75±0.12) (P=0.01). Two significant daily peaks in hPer1 expression were detected at 0400–1000 (1.24±0.16) and at 1600–2200 (1.22±0.19) (i.e., during the light-dark transition periods), which represents an increase of ~85% compared with the 1000–1600 time bin (0.67±0.11, both P=0.01) (Fig. 1c ). Diurnal variations of hß1-ADR expression were similar to the hPer1 diurnal expression pattern, also with two peaks in the light-dark transition periods 0400–1000 (1.14±0.13) and 1600–2200 (1.09±0.20) compared with the 1000–1600 (0.70±0.04) and the 2200–0400 time bin (0.44±0.23) (all P<0.05, respectively) (Fig. 1e ). Moreover, hß1-ADR and hPer1 mRNA were positively correlated (r=0.50, P=0.02, n=24).


Figure 1
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  		Figure 1. Daily gene expression pattern of hBmal1, hCry1, hPer1, hClock, hß1-adrenergic receptor (hß1-ADR) in the human pineal gland of aged controls ({blacksquare}: Braak stage 0), preclinical AD ({circ}: Braak stages I-II) and clinical AD ({Delta}: Braak stages V-VI). The significant daily variation of hBmal1, hCry1, hPer1, hß1-ADR mRNA only appears in Braak stage 0 (aged controls). *Among Braak stage 0, values significantly higher than 1000–1600 (a–c) or significantly higher than 1000–1600 and 2200–0400 (e). #hCry1 mRNA levels are higher in Braak stages V-VI than Braak stage 0 and I-II (b). ##hCry1 mRNA levels are higher in Braak stages V-VI and Braak stages I-II than Braak stage 0 (b).

Intriguingly, diurnal rhythmic expression of hBmal1, hCry1, hPer1, and hß1-ADR mRNA was lost in both preclinical AD (Braak stages I-II) (P=0.20, P=0.45, P=0.82, P=0.35; respectively) and clinical AD (Braak stages V-VI) (P=0.30, P=0.20, P=0.11, P=0.25; respectively) (Fig. 1) . No significant correlation was found between hß1-ADR and hPer1 gene expression either in Braak stages I-II (r=0.09, P=0.68, n=22) or Braak stagesV-VI (r=0.25, P=0.26, n=22).

Remarkably, we found that the daytime (0400–1000 and 1000–1600) levels of hCry1 mRNA were significantly increased in Braak stages V-VI (1.53±0.17) compared with those in Braak stage 0 (0.94±0.09) (P=0.004) and in Braak stages I-II (0.99±0.14) (P=0.014), while no such change was observed between Braak stages 0 and I-II (P=0.9) (Fig. 1b ). There was no significant difference of hß1-ADR, hPer1, hBmal1, hClock mRNA levels among Braak stages 0, I-II and V-VI either in the average level of 24 h (P=0.18; P=0.36; P=0.49, P=0.26, respectively) or in the average level of any time bins (all P>0.05).

Rat pineal gland deprived of SCN control showed similar clock gene alterations as the human pineal gland in AD
As the pineal gland is synchronized to the environment through the SCN, we investigated the effect of the loss of SCN control on the pineal clock gene oscillation. We compared pineal clock gene diurnal expression in the rat before and after disruption of the SCN-pineal functional connection either by superior cervical ganglionectomy (SCG-X) or by lesioning the SCN (SCN-X).

In control rat pineal gland, Cry1, Per1, Bmal1, and ß1-ADR mRNA showed clear diurnal rhythms (P<0.001; P<0.001; P=0.01; P=0.02) (Fig. 2 ), but not clock (P=0.55). Moreover, Per1 and ß1-ADR mRNA were highly correlated during 24 h (r=0.61, P<0.001, n=58) or at their trough and peak times (ZT 8 and ZT18) (r=0.76, P<0.001, n=22).


Figure 2
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  		Figure 2. Daily expression patterns of clock genes in control rat pineal glands (a, c, e, g), and effects of suprachiasmatic nucleus lesion (SCN-X) and superior cervical ganglionectomy (SCG-X) on the daily fluctuations of pineal clock genes (b, d, f, h). a) Cry1; *P < 0.05 vs. ZT 6, 8, 10. c) Per1; *P < 0.05 vs. ZT 2, 6, 8, 10. e) ß1-ADR; *P < 0.05 vs. ZT 8, 10. g) Bmal1; *P < 0.05 vs. ZT 6, 8, 10. SCN-X, SCG-X and their controls (CON) were sampled at ZT 8 (day) and ZT 18 (night), the nadir and peak of the Cry1, Per1, and ß1-ADR diurnal profile, respectively. b) Cry1; *P < 0.05 vs. control ZT 8. d) Per1; *P < 0.05 vs. control ZT 8. f) ß1-ADR; *P < 0.05 vs. control ZT 8 and SCG-X ZT18. h) Bmal1; *P < 0.05 vs. control ZT 8. The numbers in the bars indicate the number of animals in each group.

After experimental denervation, the day-night (ZT 8 and ZT 18) differences in Per1, Cry1, ß1-ADR, and Bmal1 gene expression completely disappeared in both SCN-X and SCG-X rats (Fig. 2b, d, f, h ). Moreover, the correlation between Per1 and ß1-ADR mRNA on ZT8 and ZT18 was lost in both SCN-X (r=0.58, P=0.10; n=9) and SCG-X rats (r=0.46, P=0.17, n=10). Strikingly, daytime (ZT8) Cry1 mRNA levels were higher in SCN-X (0.83±0.06) (P=0.036) and SCG-X rats (1.00±0.04) (P=0.003) compared with control rats (0.66±0.04) (Fig. 2b ).

Remarkably, these changes in denervated rat pineal mirrored the clock gene alterations in the AD pineal, suggesting that the synchronization of the pineal through output from the SCN is lost in both preclinical and clinical AD.

Decreased AVP mRNA levels in the SCN of both preclinical and clinical AD subjects
AVP is a major rhythmic neuropeptide output of the SCN clockwork. It regulates the rhythm of activity within the SCN and induces the rhythmicity in other brain regions (36 37 38 39) . A significant decrease of AVP mRNA in the SCN was observed from the earliest preclinical AD stages (Braak stages I-II) onward. The total masked area of silver grains as an estimate of total amount of AVP mRNA in the SCN, was decreased by 45% in Braak stages I-II (4334±1099 µm2) (n=9) (P=0.038) and strongly reduced, by 70%, in Braak stages V-VI (2381±871 µm2) (n=9) (P=0.004) compared with Braak stage 0 (7861±1302 µm2) (n=9) (Fig. 3 a). There was no significant difference in the AVP mRNA amount between Braak stages I-II and Braak stages V-VI (P=0.085) (Fig. 3a ). The total number of AVP mRNA-expressed cell profiles in the SCN markedly decreased in Braak stages V-VI (10270±2140), i.e., 20% of that in Braak stage 0 (53968±14192) (P=0.001) and 30% of that in Braak stages I-II (34228±6393) (P=0.003) (Fig. 3b ). No difference was found between Braak stages 0 and I-II as far as the number of AVP mRNA-expressed cell profiles was concerned (P=0.40) (Fig. 3b ). These data indicate that the activity of the SCN, but not the cell number, is reduced very early on in the AD pathogenesis.


Figure 3
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  		Figure 3. Total amount of AVP mRNA (estimated by total mask area of silver grains, µm2) and total number of AVP mRNA expressing cell profiles in the SCN in Braak stage 0 (aged controls), Braak stages I-II (preclinical AD) and Braak stages V-VI (clinical AD stage). a) Total amount of AVP mRNA in the SCN. b) Total number of AVP mRNA expressing cell profiles in the SCN. Note that in Braak stages I-II the total AVP mRNA levels are already decreased while the number of AVP mRNA expressing cell profiles is not significantly different. *P < 0.05; **P < 0.01.

DISCUSSION

This is the first study to show a rhythmic expression of a number of clock genes (hBmal1, hCry1, and hPer1) in the human pineal gland of controls, i.e., subjects without neurological disease or AD neuropathological changes. Furthermore, we found that the rhythmic expression of pineal clock genes was lost in both preclinical and clinical AD patient groups, which may be due to a decreased output of the master clock, the SCN.

We observed in the rat pineal a 24 h rhythm in the expression of the clock genes Per1, Cry1, Bmal1, but not in the expression of clock, in agreement with previous studies in the rat pineal (17 , 20 , 40) . Moreover, the temporal phasing of Per1 and Cry1 mRNA daily rhythm in rat pineal is in good accordance with previous rat studies with the same 12/12 light/dark conditions (17 , 20) . Bmal1 mRNA showed a peak at ZT2 in our study rather than at ZT5, as reported in a previous study, which may be attributed to the difference between the two studies in light/dark conditions (LD 12/12 instead of LD 8/16, respectively) (40) . Our observation of a diurnal rhythmic expression of hPer1, hCry1, hBmal1 mRNA in the human pineal gland agrees with these animal studies. However, the amplitude of clock gene daily rhythm is generally lower in the human pineal gland than in the rat pineal gland. Similarly, sheep pineal gland also showed lower amplitude in the diurnal rhythm of Per1 mRNA compared with rodents (41) . These data indicate that there are species differences in diurnal expression of pineal clock genes that deserve further investigation.

In the rodent, the pineal clock gene Per1 is under the noradrenergic control of the SCN through a ß-adrenergic cAMP signaling pathway (17 , 19 , 20 , 42) . ß1-ADR acts as a key factor in this cAMP signaling pathway and is directly linked to the multisynaptic noradrenergic pathway derived from the SCN (14 , 43) . The highly positive correlation between Per1 and ß1-ADR mRNA observed in the rat pineal, which actually disappeared in the SCN or SCG lesioned rat pineal (both lacking the noradrenergic SCN control of the pineal gland), may thus reflect the SCN regulation of Per1. Intriguingly, in the pineal gland of human control subjects, hPer1 and hß1-ADR mRNA were also positively correlated and showed a similar daily expression pattern. Moreover, functionally active cAMP-responsive elements (CREs) have been found in the promoter region of the hPer1 gene of human (44) . It therefore seems likely that the clock gene hPer1 in the human pineal gland is also controlled by the SCN via the ß-adrenergic cAMP-signaling pathway. Thus, by activating hPer1, the sympathetic input could potentially trigger the clockwork oscillation in the human pineal gland.

The diurnal rhythmic expression of hPer1, hCry1, and hBmal1 was lost in both preclinical and clinical AD, which suggests that pineal clock gene oscillation is disrupted very early on in the AD process. Moreover, the positive correlation between hPer1 and hß1-ADR mRNA, which probably indicates SCN control of hPer1, is disturbed in both preclinical and clinical AD. The pineal gland itself does not suffer from AD neuropathology (45) , nor does it show alterations in calcium deposition or total protein content in AD compared with aged controls (28 , 46) . Therefore, we propose that changes in clock gene expression in the pineal gland in the AD process are due to a disrupted SCN control. This possibility is strongly supported by our animal experimental data. The rat pineal that was deprived of SCN control showed alterations of clock gene expression remarkably similar to those we observed in the AD pineal. This holds for the loss of rhythmic clock gene expression, the loss of correlation between Per1 and ß1-ADR mRNA, and in particular for the increased Cry1 mRNA. Although the mechanism underlying the increased hCry1 in AD pineal is not yet clear, our studies suggest it may be due to a complete lack of sympathetic input. It has been shown that CRY1 protein is able to regulate Per1 transcription by binding both to activators (CLOCK and BMAL1) and inhibitors (PER1, PER2) in the circadian feedback loop (47) . We hypothesize that if the sympathetic regulation of Per1 expression from the SCN is lacking, CRY1 may be needed to act as the main regulator for the transcription-translation loop that leads to the increased Cry1 gene expression, as observed in late AD and denervated rat pineal. Moreover, the recent identification of the orphan nuclear receptor REV-ERB-alpha as a negative regulator of Cry1 (48 , 49) suggests that this signaling pathway might also be involved.

The pineal clock genes have been suggested to gate melatonin synthesis through the cAMP signaling cascade in the pineal gland and retina of rodents (21 , 50) . cAMP signaling can influence both transcription and post-transcriptional regulation (e.g., proteasomal degradation and inhibition/activation switch) of AA-NAT (the rate-limiting enzyme for melatonin synthesis), and thus melatonin production (51 , 52) . Notably, the gated regulation of melatonin synthesis has also been observed in humans. In healthy human subjects, evening administration of ß-ADR agonist or antagonist strongly affected plasma melatonin concentrations (53) whereas daytime administration had no such effect (54 , 55) . In rodents, melatonin synthesis is controlled on both AA-NAT transcriptional and post-transcriptional levels, whereas in primates it is probably mainly controlled on the post-transcriptional level of AA-NAT regulation (28 , 56) . Thus, we hypothesize that the pineal clock genes gate the regulation of melatonin synthesis through a cAMP-dependent post-transcriptional pathway in humans. Further studies are needed to elucidate this mechanism.

We and others have reported typical cytoskeletal alterations and degenerative changes in the SCN, both on AVP protein and mRNA levels (23 24 25 , 57) . Remarkably, in the present study we found that AVP mRNA levels in the SCN decreased from the earliest AD neuropathological stages onward. AVP is a major rhythmic neuropeptide output of the SCN clockwork, and regulates the rhythm of activity within the SCN and induces the rhythmicity in other brain regions (36 37 38 39) . The AVP neuropeptide pattern in the SCN is driven by AVP gene expression (58) , which is strictly controlled by the molecular clock in the SCN, as its rhythm had disappeared and its mRNA levels were dramatically decreased in the SCN of clock mutated mice (59) . Therefore, our data suggest that the SCN has a diminished output and a disrupted clock function from the earliest AD stages onward. Moreover, it supports the possibility that the SCN control of the pineal gland is disturbed very early in the AD process. The mechanisms underlying such early functional changes of the SCN in AD certainly deserve further study.

The finding that human pineal clock gene changes in the AD process are mimicked in the rat pineal by SCN lesion or SCG ectomy strongly suggests that the functional connection between the SCN and the pineal gland is affected in both preclinical and clinical AD stages. Other functional disturbances of brain network connections could underlie the cognitive deficits in Alzheimer’s disease. Indeed, a loss of functional connectivity between prefrontal cortex and hippocampus has been reported recently in living Alzheimer patients (60) , and a loss of synapses has been proposed to be one of the earliest changes in the disease process (61) . Synchronization of the pineal clock gene oscillation to environmental cues has provided us with a rare opportunity to study functional connectivity in the postmortem brain.

In addition to the pineal gland, many peripheral tissues in mammals express oscillating clock genes (4 , 62) that may be synchronized by the master clock in the SCN and contribute to many aspects of circadian physiology. Alterations of the master clock in AD that disrupt clock gene oscillation in the pineal gland could also affect clock gene oscillation in other peripheral tissues, and in this way contribute to the circadian disorders in AD.

Considerable variability was found between subjects within the groups. Ante- and postmortem confounding factors such as age, sex, agonal state, and clock time of death, which may contribute to such variations (63 , 64) , were excluded as much as possible in the present study by matching. Information on the exact influence of each of these factors on clock gene mRNA levels, however, is still very limited.

In conclusion, the decreased activity of the SCN already present at the moment of the occurrence of the very first tangles in the transentorhinal cortex (Braak stage I) most likely affects pineal clock gene synchronization. The loss of functional connectivity between the master clock (the SCN) and peripheral clocks (e.g., the pineal gland) may underlie the circadian disturbances in the course of AD. Apparently, the circadian system is extraordinarily vulnerable to AD pathogenesis. We propose that circulating melatonin levels and their daily rhythmicity may thus provide information about the very first AD stages that cannot be monitored in any other way at this time.

ACKNOWLEDGMENTS

This study was supported by the China committee of the Royal Netherlands Academy of Arts and Sciences (01CDP019, 02CDP014 and 04CDP026), and by the Hersenstichting Nederland. We are grateful to W.T.P. Verweij for her secretarial help and to H. Stoffels for the illustrations. We thank C. Cailotto and L. Harthoorn for their technical help.

FOOTNOTES

1 Present address: BioFocus, Galapagos Genomics, Leiden, The Netherlands.

Received for publication January 12, 2006. Accepted for publication April 17, 2006.

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