- Wiley Online Library

J. Pineal Res. 2005; 38:10–16
Copyright Ó Blackwell Munksgaard, 2004
Journal of Pineal Research
Doi:10.1111/j.1600-079X.2004.00169.x
Reduced hippocampal MT2 melatonin receptor expression in
Alzheimer’s disease
Abstract: The aim of the present study was to identify the distribution of the
second melatonin receptor (MT2) in the human hippocampus of elderly
controls and Alzheimer’s disease (AD) patients. This is the first report of
immunohistochemical MT2 localization in the human hippocampus both in
control and AD cases. The specificity of the MT2 antibody was ascertained
by fluorescence microscopy using the anti-MT2 antibody in HEK 293 cells
expressing recombinant MT2, in immunoblot experiments on membranes
from MT2 expressing cells, and, finally, by immunoprecipitation experiments
of the native MT2. MT2 immunoreactivity was studied in the hippocampus of
16 elderly control and 16 AD cases. In controls, MT2 was localized in
pyramidal neurons of the hippocampal subfields CA1-4 and in some granular
neurons of the stratum granulosum. The overall intensity of the MT2 staining
was distinctly decreased in AD cases. The results indicate that MT2 may be
involved in mediating the effects of melatonin in the human hippocampus,
and this mechanism may be heavily impaired in AD.
Egemen Savaskan1, Mohammed
A. Ayoub2, Rivka Ravid3, Debora
Angeloni4, Franco Fraschini5,
Fides Meier1, Anne Eckert1, Franz
Müller-Spahn1 and Ralf Jockers2
1
Psychiatric University Clinic, Basel,
Switzerland; 2Institut Cochin, Paris, France;
3
Netherlands Brain Bank, Amsterdam, the
Netherlands; 4Scuola Superiore S.Anna, Pisa;
5
Department of Pharmacology, University of
Milan, Milan, Italy
Key words: Alzheimer’s disease,
hippocampus, melatonin receptor, MT2
Address reprint requests to E. Savaskan,
Psychiatric Clinic, University of Basel, Wilhelm
Klein-Str.27, CH-4025 Basel, Switzerland.
E-mail: [email protected]
Received May 10, 2004;
accepted July 13, 2004.
Introduction
The pineal secretory product melatonin provides a circadian and seasonal signal in vertebrates regulating responses
to changes in day length [1–3]. The biological clock in the
suprachiasmatic nucleus (SCN), entrained by the light-dark
cycle, controls the rhythm of melatonin synthesis in the
pineal gland with the highest levels of melatonin production
occurring during the night [1–5]. Melatonin provides a
hormonal signal of darkness. The physiological effects of
melatonin include retinal [6], antioxidative [7], neuroprotective [8, 9], vasoactive [10–12], immunological [13] and
oncostatic [14] properties.
In mammals melatonin acts through two specific highaffinity membrane receptors, MT1 (previously termed
Mel1a) and MT2 (previously termed Mel1b), which have
been cloned and characterized [15–18]. They share a
common seven-transmembrane structure and transduce
signals via G protein-coupling. Whereas MT1 is coupled
to different G proteins that mediate adenylyl cyclase
inhibition and phospholipase Cb activation, MT2 is also
coupled to inhibition of adenylyl cyclase and additionally
inhibits cyclic GMP levels via the soluble guanylyl cyclase
pathway [5, 19]. A third melatonin receptor, Mel1c, was
cloned from Xenopus [20] and birds [21] but is not found in
mammals, so that of the family of three melatonin receptor
subtypes in vertebrates, only two are present in mammals
[5].
MT1 mRNA distribution has been studied in different
mammalian brains including rodents [22] and humans [23,
24]. In human central nervous system (CNS) MT1 mRNA
10
is expressed in SCN [23], cerebellum, occipital, parietal,
frontal and temporal cortex, thalamus and hippocampus
[24]. There is also immunohistochemical evidence for the
presence of MT1 in human hippocampus [24], cerebrovascular tissue [11] and retina [25]. The presence of MT2
mRNA in CNS, on the other hand, has been shown in
rodent SCN using in situ hybridization [26] and quantitative receptor autoradiography [27]. Thus far, in the human
CNS, MT2 mRNA has been found in the cerebellum [28],
and RT-PCR revealed MT2 expression in the hippocampus
[15]. However, the exact cellular distribution of MT2 in
human CNS is still not known.
As specific antibodies against MT2 have been developed
recently [29], we studied MT2 distribution in human
hippocampus using immunohistochemistry. The results
were compared with findings in the hippocampus of
Alzheimer’s disease (AD) patients, because melatonin
alterations may contribute to AD symptomatology and
pathology, and hippocampus is a brain region highly
implicated in AD-related neurodegeneration [3, 8, 9]. This
study provides the first immunohistochemical description
of MT2 distribution in human hippocampus and provides
evidence for altered expression of MT2 in patients affected
with AD.
Materials and methods
Melatonin receptor-specific antibodies
The polyclonal anti-MT2-specific antibody is directed
against a peptide (GVQHQADAL) corresponding to the
Hippocampal MT2 in Alzheimer’s disease
sequence found at the C terminus of the human MT2 [29].
The polyclonal anti-MT1-specific antibody is directed
against a peptide (KWKPSPLMTNNNVVKVDSV) corresponding to the sequence found at the C terminus of the
human MT1 [19].
Cell culture and transfection
HEK 293 cells were grown in complete medium [DMEM
supplemented with 10% (v/v) FBS, 4.5 g/L glucose, 100 U/
mL penicillin, 0.1 mg/mL streptomycin, 1 mm glutamine;
all from Life Technologies, Gaithersburg, MD, USA).
Stable clones expressing 25 fmol/mg of total protein of the
human MT2 receptor tagged with a six copies of the Myc
epitope (EQKLISEEDL) at its N-terminus and 150 fmol/
mg of total protein of the human MT1 tagged with a Flag
epitope (DYKDDDDK) at its N-terminus were used [30].
Transfection was performed using the transfection reagent
FuGene 6 (Hoffman LaRoche AG, Basel, Switzerland)
according to supplier instructions.
Immunofluorescence microscopy
HEK 293 cells transiently or stably transfected with the
N-terminally Myc-tagged MT2 or stably expressing
the N-terminally Flag-tagged MT1 [30] were grown on
polylysine-coated cover glasses and fixed in PBS 4%
formaldehyde. Cells were permeabilized in PBS, 0.1%
Triton X-100 for 10 min at room temperature, saturated in
BSA 3% for 15 min at 4°C and incubated with the antiMT2 receptor-specific serum (1/1000) antibody in PBS,
BSA 0.3% (buffer A) for 1 hr at 4°C. Cells were washed
three times with buffer A and then incubated in buffer A
supplemented with FITC-labeled anti-rabbit IgG antibody
(0.75 lg/mL) (Jackson ImmunoResearch Laboratories,
West Grove, PA, USA) for 45 min at 4°C. Cells were
washed three times, cover glasses mounted and observed by
confocal fluorescence microscopy using FITC filter settings.
SDS-PAGE/immunoblotting
Membranes were denatured in 62.5 mm Tris/HCl (pH 6.8),
5% SDS, 10% glycerol, 0.05% bromophenol blue at room
temperature overnight. Proteins were separated by 10%
SDS-PAGE and transferred to nitrocellulose. Immunoblot
analysis was carried out with a polyclonal A-14 anti-Mycspecific antibody (0.2 lg/mL) (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), or the anti-MT2-specific antibody
(1/500). Immunoreactivity was revealed using appropriate
secondary antibodies coupled to horseradish peroxidase
and the ECL chemi-luminescent reagent (Amersham,
Aylesbury, UK).
Crude membrane preparation, radioligand binding,
solubilization and immunoprecipitation
Crude membranes were prepared from cells stably expressing MT1 or MT2 receptors and receptors were labeled with
2-[125I]iodo-melatonin (400 pm) (NEN, Boston, MA, USA)
as described [19]. Labeled receptors were solubilized with
1% digitonin, a detergent known to maintain melatonin
receptors in a native conformation, and cleared lysates
incubated with the polyclonal anti-MT1-specific 536 antibody (1/40) [19] or the anti-MT2-specific antibody (1/500)
overnight at 4°C. Protein A-agarose was added for 2 hr at
4°C to precipitate antibody-receptor complexes. Precipitates were washed two times with ice-cold buffer (75 mm
Tris pH 7.4, 12 mm MgCl2, 2 mm EDTA, 0.2% digitonin)
and then counted using a c-counter.
Human brain tissue
Paraffin-embedded human hippocampus samples were
kindly provided by the Netherlands Brain Bank. Consecutive, coronal, 10 lm-thick serial sections were made and
stained for MT2. Sixteen control (mean age 78 ± 10.3 yr;
mean postmortem delay 6 hr 27 min ± 2 hr 10 min) and
16 AD (mean age 79.1 ± 10.3; mean postmortem delay
4 hr 52 min ± 1 hr 13 min) cases were included in the
study (Table 1). The demented patients are clinically
assessed and the diagnosis of probable AD is based on
exclusion of other possible causes of dementia by history,
physical examination and laboratory tests. The clinical
diagnosis is performed according the NINCDS-ADRDA
criteria [31]. The postmortem diagnosis based on the
presence and distribution of the classical hallmarks for
the disease investigated [32]. The Netherlands Brain Bank
uses a scoring system in which the density of senile plaques,
neurofibrillary tangles, disrupted interneuronal-network,
neuropil threads, congophylic plaques and vessels are
estimated in Bodian and Congo stains in four neocortical
areas; frontal, temporal, parietal and occipital. For the
stageing of the various pathological hallmarks, a combination of a quantitative grading system and the neuropathological Braak stageing, and, additionally, apolipoprotein E
(ApoE) allele frequency were determined for each case
(Table 1). Braak stageing differentiates six neuropathological stages in AD according to the distribution pattern of
the neurofibrillary tangles [33]. The E4 allele of ApoE, on
the other hand, is a major risk factor for sporadic AD,
promoting amyloid-b (Ab) precipitation into insoluble
plaques and inhibiting neurite growth and dendritic plasticity [34].
Immunohistochemistry
The observed antigen, MT2, was visualized by peroxidase
staining using the peroxidase substrate 3-amino-9-ethylcarbazole. The staining method has been previously reported in detail [11, 25, 35]. 1:500 was the optimum
concentration experimentally determined for the primary
antibody. Adjacent sections to MT2-stained hippocampus
samples were stained simultaneously to serve as control
samples, using the same procedure with the exception that
primary antibodies were omitted.
Results
To study the specificity of the anti-MT2 antibody, HEK 293
cells expressing recombinant human MT1 tagged with a
Flag epitope at their N-terminus and cells expressing
recombinant human MT2 tagged with a Myc epitope at
11
Savaskan et al.
Table 1. Data of control and Alzheimer’s disease cases including
postmortem delays in minutes (pmd), Braak staging (BS), apolipoprotein E allele differentiation (ApoE) and semiquantitative data
tabulating the intensity of MT2 immunoreactivity. Netherlands
Brain Bank autopsy numbers (NBB)
Gender
pmd (min)
BS
ApoE
MT2
52
72
76
78
83
88
92
62
78
89
86
85
75
83
72
78
F
F
F
M
M
F
F
M
M
F
F
M
M
M
M
F
410
405
290
415
409
340
425
395
335
260
810
295
425
265
270
450
0
1
1
1
1
2
1
0
1
2
2
3
3
1
0
2
33
43
32
33
33
33
32
43
43
33
43
43
33
33
33
43
+
+
+++
+++
++
+++
+
+
+
+++
++
+
+
+
+++
++
F
F
F
M
F
F
M
F
F
M
F
M
F
M
M
F
395
405
305
305
355
470
205
200
225
225
195
205
295
315
315
260
5
5
5
5
5
5
4
5
5
5
6
6
6
4
5
5
33
33
44
43
33
43
43
33
43
44
43
43
33
33
43
33
+
)
+
+
+
+
)
)
)
)
+
)
)
)
)
)
Alzheimer’s disease
98-186
58
01-042
71
00-091
76
01-092
79
97-009
89
01-071
91
90-105
90
91-085
86
91-098
93
91-111
75
91-093
82
91-096
87
92-103
63
91-095
83
93-019
72
94-117
71
M: male, F: female; MT2 staining intensity: ()) no reaction, (+)
slight, (++) moderate and (+++) high immunoreactivity.
their N-terminus were used. MT2 were readily detected
using the anti-MT2 antibody by fluorescence microscopy in
permeabilized cells expressing Myc-MT2 but not in cells
expressing Flag-MT1 (Fig. 1). In immunoblot experiments
on membranes from Myc-MT2-expressing cells, a major
immunoreactive band with an apparent molecular weight of
60 kDa was revealed (Fig. 2). Minor immunoreactive forms
of lower apparent molecular weights (40–50 kDa), most
likely represent immature, nonglycosylated receptor forms,
were also observed. Diffuse high molecular weight forms
(>100 kDa) were also detectable and may represent
oligomeric forms of the receptor as described previously
[30]. The absence of these immunoreactive bands in
membrane samples prepared from Flag-MT1-expressing
cells confirmed the specificity of detected bands for the
MT2. An identical pattern of specific immunoreactive
bands were observed when Myc-MT2 was visualized using
anti-Myc antibodies. Taken together, anti-MT2 antibodies
recognize the recombinant MT2 as a 60 kDa protein.
We then tested whether anti-MT2 antibodies are able to
immunoprecipitate native MT2. Receptors were labeled
12
Fig. 1. Detection of MT2 expression by immunofluorescence analysis. Myc-MT2 expression was monitored in stably (A) or transiently (B) transfected HEK 293 cells using confocal fluorescence
microscopy. HEK 293 cells stably expressing the Flag-MT1 construct were used as a negative control (C, D).
1
2
3
1
2
66-
3
55-
-66
-55
45-
-45
Anti-Myc
kDa
Controls
00-050
00-017
00-127
00-049
01-045
00-106
00-137
95-011
00-015
93-035
95-016
90-080
95-072
92-026
93-005
96-084
Age
kDa
Case (NBB)
Anti-MT2
Fig. 2. Immunoblot analysis of MT2. Membranes prepared from
cells stably expressing Flag-MT1 (lane 1) or Myc-MT2 stably (lane
2) or transiently (lane 3) were submitted to SDS-PAGE and
revealed by immunoblot analysis using anti-MT2 antibodies.
with 2-[125I]iodo-melatonin and solubilized with digitonin,
a detergent known to solubilize the receptor in its native
form. Lysates were incubated with anti-MT2 antibodies and
after Protein A-agarose addition, approximately 80% of
labeled MT2 was precipitated (Fig. 3). No cross-reactivity
could be observed when MT1-expressing cells were used for
the experiment confirming the specificity of the precipitation. The presence of functional MT1 in these lysates was
confirmed by the specific precipitation of labeled MT1 using
the anti-MT1-specific 536 antibody. Taken together, antiMT2 antibodies specifically recognize the recombinant MT2
in its native form (immunoprecipitation) and in its SDSdenatured form as a protein with an apparent molecular
mass of 60 kDa in immunoblots. Furthermore, MT2 is
easily detectable in cells by fluorescence microscopy.
The description of the cytoarchitectural classification of
the hippocampal subfields follows detailed previous reports
[36]. The data summarizing the intensity of the MT2
immunoreactivity has been tabulated in semiquantitative
form both for control and AD cases (Table 1).
Hippocampal MT2 in Alzheimer’s disease
Immunoprecipitated 125I-MLT
(% of total)
100
75
50
25
0
Antibody
Anti-MT1
Anti-MT2
Receptor
MT1
MT1
MT2
MT2
Fig. 3. Specific recognition of the native MT2. Membranes prepared from HEK 293 cells stably expressing Flag-MT1 or MycMT2 receptors were labeled with 2-[125I]iodo-melatonin. Labeled
receptors were solubilized with digitonin and cleared lysates were
subjected to immunoprecipitation using the indicated antibodies.
Data are mean ± S.E.M. of two independent experiments each
performed in duplicate. Data were statistically significant compared with controls as determined by a paired Student’s t-test.
MT2 immunoreactivity was localized to pyramidal neurons in the CA4 (dentate gyrus), CA3, CA2 and CA1
subfields of the hippocampus (Fig. 4A,B). In addition,
some granular neurons in the stratum granulosum surrounding the CA4 subfield were immunoreactive for MT2
(Fig. 4D). These MT2-containing cells of the stratum
granulosum will be hereinafter referred to as granular
neurons. In the pyramidal neurons the MT2 immunoreactivity was apparent on cell somata and apical dendrites
(Fig. 4A,B). In general, there were more MT2 immunoreactive neurons in the subfields CA4 and CA3 than in CA2
and CA1. The pyramidal neurons in different subfields were
morphologically distinguishable: whereas MT2 immunoreactive neurons in the CA4 were more ovoid in form,
pyramidal neurons in the other subfields were triangular
(Fig. 4A,B). In some sections small, round but densely
packed granular neurons in the stratum granulosum
surrounding the CA4 subfield revealed a distinct MT2
immunoreactivity (Fig. 4D). In the stratum granulosum the
MT2 immunoreactivity was not found in all granular
neurons, but as patches marking a group of cells (Fig. 4D).
There was no MT2 immunoreactivity either in intrahippocampal or superficial vessels. Individual differences in
overall staining intensity (tabulated in Table 1) did not
correlated with postmortem delay, Braak stageing or ApoE
allele frequency.
The overall intensity of MT2 immunoreactivity in single
neurons was clearly decreased in AD cases, even absent in
some AD hippocampi (Fig. 4E,F; Table 1). In addition, in
AD cases, the number of MT2 immunoreactive neurons was
reduced when compared with control cases (Fig. 4E,F).
Distorted neurons probably indicating neurodegenerative
changes common in this region were often found in AD
hippocampi. In those AD cases with slight MT2 immunoreactivity, the regional distribution of MT2 immunoreactive
cells in different hippocampal subfields was similar to the
control cases, i.e. most immunoreactive pyramidal neurons
were found in the CA4 and CA3 subfields. The pale
perikaryal immunoreaction was localized to pyramidal
neurons (Fig. 4E,F). Granular neurons and intrahippocampal vessels did not reveal MT2 immunoreactivity in any
AD case.
In AD cases, the decrease in individual MT2 staining did
not correlate with postmortem delay, Braak stageing or
ApoE allele frequency. The statistical analysis of the subject
data in Table 1 revealed following differences between
control and AD cases: age, Mann–Whitney U-test,
Z ¼0.226, not significant; gender, chi-square ¼ 0.508,
Fisher’s exact P-value ¼ 0.7224, not significant; postmortem delay, Mann–Whitney U-test, corrected for ties,
Z ¼ 2.452, P ¼0.0142; MT2 staining intensity, Mann–
Whitney U-test, corrected for ties, Z ¼ 4.174, P < 0.0001.
Discussion
The production and characterization of the polyclonal MT2
antibody has been previously described [29]. In those
experiments using the anti-MT2 antibody, NIH3T3 cells
stably transfected with MT2 cDNA gave intense reaction
and neither the preimmune serum nor cross-tested antisera
showed any reactivity [29]. Our results ascertain the
specifity of the MT2 antibody confirming these findings.
First, anti-MT2 antibody was able to detect the receptor on
HEK 293 cells expressing recombinant human MT2 as
shown by fluorescence microscopy. Secondly, in immunoblot experiments, the antibody revealed a 60 kDa immunoreactive band corresponding to MT2 on membranes of
MT2-expressing cells, but not on cells expressing MT1.
Finally, the anti-MT2 antibody was able to immunoprecipitate the native MT2 without any cross-reactivity for MT1.
These findings indicate that the present anti-MT2 antibody
provides a suitable and specific tool for studying the cellular
localization of MT2.
The statistical analysis of the subject data included in the
present study revealed that both control and AD cohorts
were balanced in term of age and gender. MT2 staining
intensity was significantly higher in controls when compared with AD cases, even, taking in consideration that the
controls had significantly longer postmortem delays. MT2
has been immunohistochemically localized to pyramidal
and granular neurons of the hippocampus in control cases
in the present study. Although the presence of MT2
expression has been demonstrated in the human hippocampus using RT-PCR [15], the present study is the first
showing the exact cellular distribution of MT2 in the
hippocampus. In rodents, the hippocampus belonged to the
brain regions expressing MT2, which was localized to
pyramidal neurons [37–39]. This is in accordance with our
results. These previous studies did not mentioned granular
neurons as expression sites, but as synaptosomal preparations [37] and hippocampal slices [38] were investigated,
methodical or interspecies differences may account for
differences in the results. In accordance with our results,
13
Savaskan et al.
Fig. 4. MT2 immunoreactive (red deposits) structures in the human hippocampus.
Pyramidal neurons are MT2 immunoreactive in the (A) CA4 and (B) CA3 subfields of a control case (NBB-Nr.: 00-106).
(C) Control section revealing no MT2
immunoreactivity. CA4 subfield stained
simultaneously following the same procedure, with the exception that the primary
antibody was omitted (control case, NBBNr: 00-106). (D) Granular cells surrounding the CA4 subfield reveal MT2
immunoreactivity (NBB-Nr.: 00-106). (E)
MT2 immunoreactivity is distinctly
decreased in CA4 and (F) CA3 pyramidal
neurons of an AD case (NBB-Nr.:
01-092). Scale bar ¼ 50 lm for all figures.
previous RT-PCR data localized MT2 transcripts in all
hippocampal subfields [39].
The presence of MT1 in the human hippocampus is well
established [23, 24, 35]. Our data additionally provides
evidence for the hippocampal localization of the second
melatonin receptor. Similar to MT1 [35], MT2 has been
shown in pyramidal neurons of all hippocampal subfields.
Whereas MT1 was predominantly present in the CA1
subfield [35], more MT2 immunoreactive pyramidal neurons were detectable in the CA4 and CA3 subfields in the
present study, indicating regional differences in the distribution of both receptors. Axons of the CA1 have been
considered the main output of the hippocampus, whereas
CA4 and CA3 pyramidal neurons are the main targets of
the axons of granular neurons which receive glutamatergic
excitatory input from the entorhinal cortex via the perforant path [36]. As MT2 was localized in granular neurons,
but MT1 was not [35], it may be predominantly MT2 which
is responsible for transmitting melatonin’s effects on the
afferent hippocampal connections, consisting of granular
and pyramidal neurons in the CA4 subfield.
Melatonin has been reported to have both enhancing [39]
and inhibitory [40] effects on excitability of hippocampal
neurons, most likely through MT2 [40]. As MT2 is localized
presynaptically in the retina [41] and can affect glutamate
uptake and release at this site [42], a similar neuronal
regulatory mechanism has been postulated for the hippocampus [40] where glutamate is also the major excitatory
neurotransmitter [36]. The MT2-mediated effect of melatonin in the hippocampus attenuates hippocampal evoked
14
potentials in a concentration-dependent manner [40].
Therefore, the activity of the hippocampal neurons has
been assumed to be influenced by the circadian rhythm of
melatonin secretion, and may be of importance in regulating memory processes [40]. Thus, a diurnal expression
pattern has been postulated also for melatonin receptors in
the hippocampus, which may be responsible for the
melatonin-sensitive enhancement of excitability of hippocampal neurons during the night [39].
Melatonin is known to be a vasoactive substance [10–
12], and MT1 may be responsible for transducing
melatonin’s vascular effects in the hippocampus [11]. In
contrast, MT2 was not found in intrahippocampal or
superficial vessels in the present study. On the other
hand, MT2 has been localized in peripheral arteries
including aorta, left ventricle and coronary arteries, and
MT2 expression has been found to be altered in coronary
heart disease [12].
Melatonin is a highly neuroprotective substance [8, 9].
Besides having antioxidative properties scavenging free
radicals [7], melatonin has been shown to be able to
protect neurons against Ab-induced neuropathology in
AD [8, 9]. Ab, generated during the course of neurodegenerative disorders, particularly in the hippocampus and
cerebral cortex, induces oxidative damage in neurons and
melatonin can attenuate this effect [8, 9]. Thus, Ab
generation is reduced by melatonin. Nocturnal amplitude
of melatonin secretion declines in the elderly when
compared with younger humans and seem to be more
depressed in AD patients than in normal elderly [43, 44].
Hippocampal MT2 in Alzheimer’s disease
Therefore, alterations in melatonin receptor expression
may additionally affect melatonin’s beneficial effects in
AD. We previously reported that MT1 was increased in
the hippocampus of AD patients [35]; the present data
shows that MT2 is decreased in the AD hippocampus.
Both melatonin receptors may be adversely impaired in
AD. This may be a common effect in AD, as the MT2
decrease was not correlated with Braak stageing or ApoE
allele frequency. Not only the intensity of the MT2
immunoreactivity in single cells was reduced in AD cases,
but also the number of MT2 immunoreactive neurons
was decreased in AD hippocampi, which may reflect ADrelated neurodegeneration in this highly affected brain
region. Besides the cellular loss of MT2, the overall
neuronal degeneration in the hippocampus may contribute to MT2 decrease. Interestingly, in contrast to
controls, MT2 immunoreactivity was missing in the
granular neurons of all AD hippocampi. Whether this
finding is indicative of selective impairment of melatonin
receptors in the hippocampal afferents remains to be
elucidated.
Acknowledgments
We specially thank to Prof. A. Wirz-Justice, Centre for
Chronobiology, Psychiatric University Clinic, Basel, Switzerland, for her invaluable advice and editing of the
manuscript, and to Dr M. Masson-Pevet, Laboratoire de
Neurobiologie des Rhythmes, UMR-CNRS 7518, Université Louis Pasteur, Strasbourg, France, for help in obtaining the anti-MT2 antibody. R. Jockers was supported by
grants from the INSERM, CNRS, the Université Paris V,
the Association pour la Recherche sur le Cancer (ARC No.
5513 and 7537).
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