U.S. patent application number 15/159222 was filed with the patent office on 2016-09-08 for compositions for classifying a human pediatric subject having adolescent idiopathic scoliosis (ais).
The applicant listed for this patent is CHU SAINTE-JUSTINE. Invention is credited to ALAIN MOREAU.
Application Number | 20160258947 15/159222 |
Document ID | / |
Family ID | 27762090 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160258947 |
Kind Code |
A1 |
MOREAU; ALAIN |
September 8, 2016 |
COMPOSITIONS FOR CLASSIFYING A HUMAN PEDIATRIC SUBJECT HAVING
ADOLESCENT IDIOPATHIC SCOLIOSIS (AIS)
Abstract
A composition for classifying a human pediatric subject having
adolescent idiopathic scoliosis (AIS), comprising (a) a cell sample
of the subject; and (b) a known melatonin-signaling pathway
agonist, wherein said melatonin signaling pathway in said cell
sample has been artificially induced by said melatonin-signaling
pathway agonist, wherein the cell sample is selected from the group
consisting of blood cell sample, osteoblast cell sample, osteoclast
cell sample and myoblast cell sample, and whereby the composition
enables the detection of the presence or absence of at least one
impairment in the melatonin-signaling pathway in cells from said
cell sample and thereby enables the classification of subject.
Inventors: |
MOREAU; ALAIN; (MONTREAL,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHU SAINTE-JUSTINE |
MONTREAL |
|
CA |
|
|
Family ID: |
27762090 |
Appl. No.: |
15/159222 |
Filed: |
May 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14143811 |
Dec 30, 2013 |
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15159222 |
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13726416 |
Dec 24, 2012 |
8652791 |
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14143811 |
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13165226 |
Jun 21, 2011 |
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13726416 |
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12232141 |
Sep 11, 2008 |
7989175 |
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13165226 |
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10505951 |
Aug 27, 2004 |
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PCT/CA2003/000286 |
Feb 28, 2003 |
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12232141 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/56966 20130101;
G01N 33/6893 20130101; G01N 33/74 20130101; C12Q 1/02 20130101;
G01N 33/566 20130101; G01N 2800/10 20130101; G01N 2333/726
20130101; C12Q 1/527 20130101 |
International
Class: |
G01N 33/566 20060101
G01N033/566; G01N 33/74 20060101 G01N033/74 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2002 |
CA |
2373854 |
Claims
1. A composition for classifying a human pediatric subject having
adolescent idiopathic scoliosis (AIS), comprising (a) a cell sample
of the subject; and (b) a known melatonin-signaling pathway
agonist, wherein said melatonin signaling pathway in said cell
sample has been artificially induced by said melatonin-signaling
pathway agonist, wherein the cell sample is selected from the group
consisting of blood cell sample, osteoblast cell sample, osteoclast
cell sample and myoblast cell sample, and whereby the composition
enables the detection of the presence or absence of at least one
impairment in the melatonin-signaling pathway in cells from said
cell sample and thereby enables the classification of subject.
2. The composition of claim 1, wherein said cell sample is a blood
cell sample selected from the group consisting of lymphocyte cell
sample and monocyte cell sample.
3. The composition of claim 1, wherein said cell sample is a
lymphocyte cell sample.
4. The composition of claim 1, wherein said cell sample is a
monocyte cell sample.
5. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is a melatonin analog.
6. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is melatonin.
7. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is a GTP analog.
8. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is GTP.
9. The composition of claim 1, further comprising a known activator
of adenylyl cyclase.
10. The composition of claim 9, wherein said known activator of
adenylyl cyclase is forskolin.
11. The composition of claim 1, further comprising artificial bone
or dentin matrix, wherein said sample is an osteoclast cell
sample.
12. The composition of claim 1, further comprising
[.sup.3H]-thymidine, wherein said cell sample is an osteoblast cell
sample.
13. A composition for classifying a human pediatric subject having
idiopathic scoliosis (AIS), comprising (a) a lysed cell sample of
the subject; and (b) reagents for detecting cAMP accumulation,
wherein, prior to being lysed, said cell sample has been contacted
with a known activator of adenylyl cyclase to artificially induce
Cyclic Adenosine Monophosphate (cAMP) accumulation and subsequently
with a known melatonin-signaling pathway agonist, wherein the cell
sample is selected from the group consisting of blood cell sample,
osteoblast cell sample, osteoclast cell sample and myoblast cell
sample, and whereby the composition enables the detection of the
presence or absence of at least one impairment in the
melatonin-signaling pathway in cells from said cell sample and
thereby enables the classification of the subject.
14. The composition of claim 13, wherein said known activator of
adenylyl cyclase is forskolin.
15. The composition of claim 13, wherein said known
melatonin-signaling pathway agonist is a melatonin analog.
16. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is melatonin.
17. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is a GTP analog.
18. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is GTP.
19. The composition of claim 1, wherein said reagents for detecting
cAMP comprises an antibody which binds to cAMP.
20. The composition of claim 1, wherein said known
melatonin-signaling pathway agonist is at a pharmacological
concentration
21. The composition of claim 21, wherein said cell sample has been
contacted with a pharmacological concentration of said know
melatonin-signaling pathway agonist.
22. The composition of claim 1, further comprising an antibiotic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/143,811 now pending, which is a divisional
application of U.S. patent application Ser. No. 13/726,416 filed
Dec. 24, 2012, now issued under U.S. Pat. No. 8,652,791, which is a
continuation application of U.S. patent application Ser. No.
13/165,226 filed Jun. 21, 2011 now abandoned, which is a
continuation application of U.S. patent application Ser. No.
12/232,141 filed Sep. 11, 2008 now issued under U.S. Pat. No.
7,989,175, which is a continuation application of U.S. patent
application Ser. No. 10/505,951 filed Aug. 27, 2004 now abandoned,
which is a National Entry Application of PCT application no.
PCT/CA2003/00286, filed on Feb. 28, 2003 and published in English
under PCT Article 21(2), which itself claims priority on Canadian
Patent Application 2,373,854, filed on Feb. 28, 2002. All documents
above are incorporated herein in their entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions for
classifying a human pediatric subject having adolescent idiopathic
scoliosis (AIS). More specifically, the present invention is
concerned with neuroendocrinal compositions for classifying
AIS.
BACKGROUND OF THE INVENTION
[0003] The etiology of adolescent idiopathic scoliosis (AIS), a
disease affecting 0.2 to 6% of the population, is unclear. AIS
affects mainly girls in number and severity but in spite of several
studies suggesting a genetic predisposition, the form of
inheritance remains uncertain (3; 4; 4-6). Several divergent
perspectives have been postulated to better define this etiology
(reviewed in (2; 21-23)). Genetics, growth hormone secretion,
connective tissue structure, muscle structure, vestibular
dysfunction, melatonin secretion, and platelet microstructure are
major areas of focus. The current opinion is that there is a defect
of central control or processing by the central nervous system
(CNS) that affects a growing spine and that the spine's
susceptibility to deformation varies from one individual to
another.
CNS Hypothesis: B. Muscle Spindle Ontology and AIS
[0004] In 1999, Dubousset suggested that AIS is probably caused by
a proprioception control problem, a neuromuscular disorder in
relation with the neurotransmitter involved with the bipedal
condition.
[0005] Muscles spindles are skeletal muscle sensory organs that
provide axial and limb position information (proprioception) to the
CNS. It has been proposed that muscle spindles act as muscle
receptors involved in the detection of movement, both passive and
active (5). Spindles consist of encapsulated muscles fibers
(intrafusal fibers) that are innervated by specialized motor and
sensory axons. Indeed, histologic and histochemical analysis of the
distribution of muscle spindles in paraspinal musculature of
patients suffering from AIS show few muscle spindles in the
scoliotic muscle (6). Although the mechanism involved in spindle
ontogeny are poorly understood, the innervation of a subset of
developing myotube (type I) by peripheral sensory afferents (group
Ia) is a critical event for inducing intrafusal fiber
differentiation and subsequent spindle formation. The inactivation
of Egr3, a zinc-finger transcription factor, by gene targeting
generates mice exhibiting gait ataxia, increased frequency of
perinatal mortality, scoliosis, resting tremors and ptosis.
Egr3-deficient mice lacked muscles spindles, a finding that is
consistent with their profound gait ataxia. Egr3 is highly
expressed in developing muscle spindles, but not in Ia afferent
neurons or their terminals during developmental periods that
coincide with the induction of spindle morphogenesis by sensory
afferent axons. This indicates that type I myotubes are dependent
upon Egr3-mediated transcription for proper spindle
development.(7-9) In addition, Rodgers et al., reported the
detection of Pax7 expression, a member of the Pax family of
transcription factor, in the capsules surrounding adult mouse
muscle spindles where it may be implicated in the formation and
maintenance of neuromuscular contacts within the muscle spindles
throughout life.(10) The recent report of Ichikawa et al.,(11)
showing in muscle spindles the presence of OPN-immunoreactive
spiral axon terminals suggest that OPN could behave as a molecular
mechanoreceptor within the spindles. This aspect is further
supported from the fact that OPN-null mice, which are normal and
viable, are not responding to biomechanical stimuli.
Neuroendocrine Hypothesis
[0006] Recent experiments involving pinealectomy in chicken and
more recently in rats maintained in a bipedal mode led to an
alternate hypothesis. These surgeries produced a scoliosis (7; 8;
8-10) resembling in many aspects the human disease and pointed to a
neuroendocrine hypothesis involving a melatonin deficiency as the
source for AIS. Treatment after pinealectomy in both animal models
with melatonin, the major hormone of the pineal gland, prevented
the formation of scoliosis (11).
[0007] The biological relevance of melatonin in AIS remains
controversial however since no significant decrease in circulating
melatonin level has been observed in a majority of studies (12; 13;
13; 14).
[0008] There is therefore a need for a useful method for diagnosing
AIS and other diseases involving spinal deformities and for
identifying compounds for treating these diseases.
SUMMARY OF THE INVENTION
[0009] The present invention demonstrates for the first time that
AIS patients exhibit a melatonin-signaling pathway impairment and
that this impairment can be observed through various
manifestations.
[0010] In particular, the present invention demonstrates for the
first time a dysfunction of melatonin-signaling in bone-forming,
muscle-forming cells and blood cells of AIS patients. In addition,
the present invention demonstrates for the first time that
non-functional Gi proteins normally coupled to melatonin receptors
MT1 and MT2 is related to such dysfunction.
[0011] More specifically, in accordance with the present invention,
there is provided a method for diagnosing an increased risk for a
disease characterized by a dysfunctional melatonin-signaling
pathway in an animal, comprising detecting the presence or absence
of at least one impairment in melatonin-signaling pathway in at
least one of the animal's cells, wherein the presence of at least
one impairment in melatonin-signaling pathway indicates that the
animal possesses an increased risk of developing said adolescent
idiopathic scoliosis or other disease.
[0012] The method of the present invention may also be
advantageously used to diagnose a particular type of disease
characterized by a dysfunctional melatonin-signaling pathway by
determining whether the results of the assay correspond to those of
a previously tested animal affected by this particular type of
disease. For instance, it would be possible to determine with the
method for diagnosing of the present invention, whether an animal
is affected by AIS of group 1, 2 or 3 (as described in Table 2
below) by determining its osteoblast responsiveness to melatonin
treatment. This is particularly interesting if the most effective
drug for treating or preventing AIS varies between the groups (1, 2
or 3). The method for diagnosing of the present invention therefore
permits a better selection of the drug to be used for a particular
patient.
[0013] According to another embodiment of the present invention,
there is also provided a method of screening for a compound useful
in the treatment of a disease characterized by a dysfunctional
melatonin-signaling pathway, said method comprising the steps of
contacting a candidate compound with at least one cell expressing
at least one melatonin-signaling pathway impairment in the presence
of a known melatonin-signaling pathway agonist, wherein the
candidate compound is selected if said melatonin-signaling pathway
impairment is reduced in the presence of the candidate compound as
compared to that in the absence thereof. This method can be used
for screening for compounds able to modulate melatonin-signaling
impairment generally. It can however also be used to determine
which compound is the most effective for modulating and in
particular reducing or counteracting the melatonin-pathway
impairment in cells from a specific group of patient or for a
specific patient. Indeed, the most effective compound for these
purposes may vary from one patient to the next. The method of
screening of the present invention may therefore be used to
identify which compound is the most effective in counteracting the
melatonin-signaling pathway impairment in a specific group of
patients or in one patient in particular.
[0014] In a specific embodiment, said disease characterized by a
dysfunctional melatonin-signaling pathway is adolescent idiopathic
scoliosis or an other disease involving spinal deformities. In
another specific embodiment, said disease characterized by a
dysfunctional melatonin-signaling pathway is adolescent idiopathic
scoliosis. In another specific embodiment, said impairment is
detected by an accumulation of cyclic adenosine 5'-monophosphate
(cAMP) in at least one of said cells. In another specific
embodiment, said accumulation of cyclic adenosine 5'-monophosphate
(cAMP) is induced by a known activator of adenylyl cyclase, and
wherein the inhibition of said accumulation by a known
melatonin-signaling pathway agonist is detectably reduced in at
least one said cells as compared to that obtained in a control
cell. In another specific embodiment, said known
melatonin-signaling pathway agonist is melatonin or an analog
thereof. In another specific embodiment, said known
melatonin-signaling pathway agonist is GTP or an analog thereof. In
another specific embodiment, said known activator of adenylyl
cyclase is forskolin or an analog thereof. In another specific
embodiment, said impairment is detected by an absence of
proliferation of in at least one of said cells in presence of a
known melatonin-signaling pathway agonist. In another specific
embodiment, said impairment is detected by a reduction of
inhibition of osteoclasts resorption activity by the known
melatonin-signaling pathway agonist, and wherein the candidate
compound is selected if said reduction of inhibition of osteoclasts
resorption activity is inhibited in the presence of the candidate
compound as compared to that in the absence thereof. In another
specific embodiment, said cells are selected from the group
consisting of osteoblasts, osteoclasts, lymphocytes, monocytes and
myoblasts. In another specific embodiment, said cells are blood
cells. In another specific embodiment, said cells are lymphocytes.
In another specific embodiment, said impairment is detected by an
accumulation of cyclic adenosine 5'-monophosphate (cAMP) in said
cell as compared to that in a control cell. In another specific
embodiment, the method further comprises the step of artificially
inducing said accumulation of cyclic adenosine 5'-monophosphate
(cAMP) by a known activator of adenylyl cyclase.
[0015] According to a further embodiment of the present invention,
there is also provided a method of formulating a drug useful in the
treatment of a disease characterized by a dysfunctional
melatonin-signaling pathway, said method comprising the steps of
contacting a candidate compound with at least one cell expressing
at least one melatonin-signaling pathway impairment, wherein the
candidate compound is selected if said melatonin-signaling pathway
impairment is reduced in the presence of the candidate compound as
compared to that in the absence thereof, and formulating said drug
with said selected candidate compound.
[0016] The present invention discloses such compounds able to
modulate melatonin-signaling pathway impairment including
melatonin, forskolin and estradiol.
[0017] According to specific embodiments of the present invention,
the disease characterized by a dysfunctional melatonin-signaling
pathway is adolescent idiopathic scoliosis or another disease
involving spinal deformities. More specifically, the impairment may
be detected by an accumulation of cyclic adenosine 5'-monophosphate
(cAMP) in a cell of the animal, an absence of said cells
proliferation in presence of the known melatonin-signaling pathway
agonist, and a reduction of inhibition of osteoclasts resorption
activity induced by the known melatonin-signaling pathway agonist,
wherein the candidate compound is selected if said reduction of
inhibition of osteoclasts resorption activity is inhibited in the
presence of the candidate compound as compared to that in the
absence thereof. Note that any cell from tissues targeted by
melatonin or expressing melatonin signalisation and wherein other
pathway members do not mask melatonin-signaling impairments may be
used in accordance with the methods of the present invention. The
cells used herein were selected in part for their accessibility.
Hence, cells such as osteoblasts, osteoclasts, lymphocytes,
monocytes and myoblasts are advantageously accessible and may
conveniently be used in the methods of the present invention. Blood
cells in particular are particularly accessible and provide for a
more rapid testing. In specific embodiment, said known
melatonin-signaling pathway agonist is melatonin, GTP or analogs
thereof. Any other known melatonin-signaling pathway agonist may be
used in accordance with the present invention. In a specific
embodiment, the known activator of adenylyl cyclase is
forskolin.
Melatonin-Signaling Pathways
[0018] Melatonin-signaling pathways have been better characterized
in the brain, the pituitary gland and few peripheral tissues than
bone or other musculoskeletal tissues. Melatonin exerts its effects
through specific, high-affinity receptors (12-14). These melatonin
receptors are coupled to guanine nucleotide-binding proteins (G
proteins), and their activation leads to the inhibition of adenylyl
cyclases, which are responsible for the accumulation of cyclic
adenosine 5'-monophosphate (cAMP) (15). Through molecular cloning,
three G protein-coupled melatonin receptor subtypes have been
identified in vertebrates. The ligand-binding properties and
signaling mechanisms of these receptors are remarkably similar.
Each receptor subtypes is coupled to inhibition of cAMP
accumulation. The MT1 (MelR1a) and MT2 (MelR1b) receptor genes are
present in mammals and several lines of evidence demonstrated that
MT1 is the receptor that mediates the reproductive and circadian
responses to melatonin. The third receptor, MelR1c (two isoforms
.alpha. and .beta.) has been only detected in the chicken and in
Xenopus. A second type of melatonin receptor called MT3 has been
first discovered based on its pharmacological properties that are
quite distinct from the MT1 and MT2 receptor subtypes. Recently,
the human and mouse MT3 receptors have been cloned and correspond
to protein encoded by the quinone reductase 2 gene (QR2) (16). The
precise role of this gene in melatonin signal transduction remains
to be determined. Besides the membranous receptors, the orphan
nuclear receptors RZR.alpha. and .beta., have been proposed to
interact with melatonin but such interaction remains elusive.
Interestingly, estrogens markedly inhibit the expression and
synthesis of G protein .alpha.-subunits (Gi1-3 and Gs) in
osteoblast cultures suggesting that melatonin-signaling may be
modulated by estrogens (17). Furthermore, estrogens are able to
increase calmodulin expression, independently of both estrogens
receptors (ER.alpha. and .beta.) (18). This is particularly
interesting because calmodulin and melatonin exert a mutual
antagonism,(19; 20) and membrane-bound calmodulin is able to
interact with melatonin as demonstrated in Xenopus.(21) Moreover,
melatonin has the property to destabilize the ERs DNA-binding on
their cognate sequence.(22)
Fundamental Aspects of Melatonin Signal Transduction
[0019] Expression analysis revealed that melatonin up-regulates key
osteoblasts terminal differentiation markers like osteocalcin (OC),
osteopontin (OPN) and bone sialoprotein (BSP). This activation was
already detectable after only 10 min of stimulation suggesting that
melatonin stimulates osteoblast differentiation in vitro through
specific interactions with one of its membranous receptors. It was
then determined whether this transcriptional activation was
mediated by MT1 or MT2 receptor subtype, and demonstrated that both
receptor subtypes are expressed although time-course expression
analysis revealed that MT2 receptor expression was predominantly
detected. At the protein level, IHC experiments demonstrated the
presence of both melatonin receptors at the cell surface. In
parallel, co-immunoprecipitation assays with osteoblast purified
membranes demonstrated for the first time a preferential
pre-coupling of Gi proteins, Gi3>Gi2, to MT2 receptors in
absence of ligand while in presence of melatonin both proteins
binding were increased. Similar analysis with MT1 receptor revealed
that only Gi3 was pre-coupled to this receptor. No interaction was
detected between Gi1 proteins and both melatonin receptors.
Bone Mineral Density in Pinealectomized Chicken
[0020] Scoliotic and non-scoliotic pinealectomized chicken showed a
similar and significant decrease in bone mineral density suggesting
that bone tissue is indeed a target of melatonin action. EMG
analysis performed with the same set of chicken showed a 75%
increase in muscular tone of paraspinal muscles on both sides while
a 60% asymmetrical increase of the muscular activity was measured
on the left side, which correlated with the side of the scoliosis
curve (99% left sided).
[0021] As used herein, the expression "melatonin-signaling pathway
impairment" or "dysfunction" is meant to refer to any impairment in
this pathway that characterizes cells from patients with AIS and
related syndromes causing spinal deformities and includes but is
not limited to: absence of inhibition of osteoclasts resorption
activity, accumulation of cAMP in an animal cell, an
hypofunctionality of Gi proteins, a phosphorylation state of Gi
proteins distinct from that of normal cells, an absence of
proliferation of certain cells in response to melatonin, a mutation
in a gene encoding a member of the melatonin signaling pathway.
[0022] As used herein, the term "control cells" is used to refer to
any cell not expressing the melatonin-signaling pathway of the cell
under scrutiny. It includes cells from non-scoliotic animals and
cells from animals displaying other types of scoliosis.
[0023] As used herein, the expression "analog thereof" is meant to
include any compound displaying the same activity as that for which
the compound of reference is used. For instance, Gpp(NH)p is an
analog of GTP.
[0024] In a specific embodiment, the cAMP accumulation may have
been artificially induced by a known adenylyl cyclase activator
such as forskolin and inhibited by a melatonin-signaling pathway
agonist such as melatonin itself or any agonist known for
inhibiting cAMP accumulation such as GTP or Gpp(NH)p. An absence of
cAMP accumulation by these known agonists is interpreted as a
melatonin-signaling impairment of the subject cell and of the
animal from which the cell was isolated.
[0025] As used herein, the methods for diagnosing AIS and related
syndromes causing spinal deformities in an animal comprises
detecting any melatonin-signaling pathway impairment in at least
one of the animal cells such as but not limited to lymphocytes,
monocytes, osteoclastes, osteoblasts, myoblasts from the animal,
and according to specific embodiments the animal is a human.
Assays to Identify Peptides of the Present Invention
[0026] Preferred methods for testing the ability of candidate
compounds to modulate (antagonize or agonize) the
melatonin-signaling pathway are presented herein. It will be
understood that the invention is not so limited. Indeed, often
assays well known in the art can be used in order to identify such
compounds.
[0027] It should be understood that candidate compounds to be
tested according to the method of the present invention include
non-peptides drug candidates (small molecules) as well as peptides
targeting defective proteins involved in the melatonin-signaling
pathway impairment, or oligonucleotides such as antisens molecules
targeting a defective gene involved in the melatonin-signaling
pathway impairment.
[0028] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments thereof, given
by way of example only with reference to the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In the appended drawings:
[0030] FIG. 1 graphically shows the inhibitory effect of melatonin
on adenylyl cyclase activity in human normal osteoblasts and in AIS
osteoblasts. Distribution of single data points obtained for each
AIS patients and control subjects (congenital scoliosis, cong; and
other scoliotic type, other/s) tested at physiological dose of
melatonin (10.sup.-9M) on forskolin-stimulated osteoblasts. The
black bars represent the mean of each group;
[0031] FIG. 2 graphically shows the inhibitory effect of melatonin
on adenylyl cyclase activity in human normal osteoblasts and in AIS
osteoblasts. Representative experiments showing the effect of
increasing concentrations of melatonin (10.sup.-11 to 10.sup.-5M)
on forskolin-stimulated adenylyl cyclase activity in osteoblasts
from control subject and patients with AIS (group 1, 2 and 3). Data
are expressed as mean.+-.SEM;
[0032] FIG. 3 illustrates through photographs the detection of MT1
and MT2 melatonin receptors in human osteoblasts from patients with
AIS and from control subjects. Each panel illustrates
representative IHC experiments performed with MT1 receptor
antibodies (upper panels) and MT2 receptor antibodies (lower
panels) on primary human osteoblast cultures prepared from patients
with AIS (AIS1-2) and compared with a control subjects;
[0033] FIG. 4 graphically shows the Gpp(NH)p inhibitory effect on
adenylyl cyclase activity on human osteoblasts from patients with
AIS and on control subjects. Distribution of single data points
obtained for each AIS patients and control subjects in presence of
Gpp(NH)p (10.sup.-7M) on forskolin-stimulated osteoblasts. The
black bars represent the mean of each group;
[0034] FIG. 5 illustrates through photographs the detection of Gi3
proteins coupled to MT2 receptors. Immunoblots were revealed with
specific antibodies reacting with individual Gi with the exception
of anti-Gi3 antibodies, which cross-react also with human Gi1
proteins. Note the presence of 60 kDa bands corresponding to
phosphorylated Gi3 proteins. Lane 1 and 2 are from control subject
(Marfan) not treated and treated with melatonin (10.sup.-7 M)
respectively. Lanes 3-4 and 5-6 come from two different AIS
patients. Lanes 7-8-9 are positive control peptides for Gi1, Gi2
and Gi3 respectively;
[0035] FIG. 6 graphically shows the proliferation of human normal
osteoblasts and that of AIS osteoblasts through time-courses of
[3H]thymidine uptake;
[0036] FIG. 7 shows the expression analysis of melatonin receptors
in human osteoblasts;
[0037] FIG. 8 shows the effect of Gpp(NH)p (10.sup.-7M) on
forskolin-stimulated adenylyl cyclase activity in osteoblasts from
control subjects and in osteoblasts from patients with AIS.
Distribution of single data points obtained from each patient with
AIS and control subjects tested;
[0038] FIG. 9 shows Gi proteins coupled to MT2 melatonin receptor.
Black and white arrows correspond to unphosphorylated and
phosphorylated forms of Gi proteins respectively;
[0039] FIG. 10 shows the detection of phosphoserine residues in
phosphorylated Gi proteins. The immunoblot in FIG. 9 was stripped
and reprobed with antibodies recognizing antiphosphoserine
residues. Numbering corresponds to cell cultures conditions: 1)
untreated; 2) with melatonin; 3) with Na3VO4, a tyrosine
phosphatase inhibitor; and 4) with genistein, a tyrosine kinase
inhibitor;
[0040] FIG. 11 graphically shows the bone mineral density in
scoliotic and control chicken;
[0041] FIG. 12 graphically shows the bone mineral density in
scoliotic and control chicken in four different plans;
[0042] FIG. 13 graphically shows EMG activity in paraspinal
musculature of pinealectomized chicken;
[0043] FIG. 14 graphically shows EMG activity in paraspinal
musculature of pinealectomized chicken in movement;
[0044] FIG. 15 graphically shows the inhibitory effect of melatonin
on adenylyl cyclase activity in human normal myoblasts and in AIS
myoblasts;
[0045] FIG. 16 illustrates through photographs the detection of MT1
and MT2 melatonin receptors in human osteoclasts from normal human
subjects. Panels labeled MT1 and MT2 represent corresponding
receptor subtype detected by IHC with specific primary antibodies
and distinct secondary antibodies conjugated to different
fluorochromes (red, phycoerythrin; green, FITC). The panel labeled
h-OC corresponds to a human surface antigen specific for mature
osteoclasts. Negative control has been generated by omission of the
primary antibodies;
[0046] FIG. 17 illustrates through photographs the detection of MT1
and MT2 melatonin receptors in human osteoclasts from AIS human
patients. Panels labeled MT1 and MT2 represent corresponding
receptor subtype detected by IHC with specific primary antibodies
and distinct secondary antibodies conjugated to different
fluorochromes (red, phycoerythrin; green, FITC). The panel labeled
h-OC corresponds to a human surface antigen specific for mature
osteoclasts. Negative control has been generated by omission of the
primary antibodies;
[0047] FIG. 18 graphically shows the measurement of osteoclasts
activity (pit resorption assay) on bone matrix. The inhibitory
effect of melatonin on osteoclasts activity was performed using
normal human osteoclasts derived from peripheral blood. Mel,
melatonin; luz, luzindole, a specific MT2 antagonist;
[0048] FIG. 19 graphically shows the effect of estrogen on the
melatonin-signaling pathway impairment of patients with AIS;
[0049] FIG. 20 shows Gi proteins coupled to MT2 melatonin receptor.
The cells used were prepared from human MG-63 osteoblast culture
(panel A) and osteoblast cultures from AIS patient (case 22 of
Table 1, panel B); and
[0050] FIG. 21 shows Gi proteins coupled to MT2 melatonin receptor.
The cells used were prepared from osteoblast cultures from AIS
patient. Panel A, case 37 of Table 1; panel B, case 29 of Table
1.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0051] In vitro assays were performed with bone-forming and
muscle-forming cells isolated from 41 patients with adolescent
idiopathic scoliosis (AIS) patients and 17 control subjects
demonstrating that patients with this disease exhibit a dysfunction
of the melatonin-signaling pathway in tissues targeted by this
hormone.
[0052] Osteoblast and myoblast cultures prepared from specimens
obtained intraoperatively during spine surgeries were used to test
the ability of melatonin and Gpp(NH)p, a GTP analogue, to block
cAMP accumulation induced by forskolin. In parallel, melatonin
receptors and Gi proteins functions were evaluated by
immunohistochemistry, binding assays with [.sup.125I]-iodomelatonin
and by co-immunoprecipitation experiments. The cAMP assays
demonstrated that melatonin-signaling was severely impaired in
osteoblasts and myoblasts isolated from AIS patients allowing their
classification in 3 distinct groups based upon their responsiveness
to melatonin or Gpp(NH)p. Melatonin-signaling is clearly impaired
in osteoblasts and myoblasts of all AIS patients and DD patients
tested. Classification of AIS patients in 3 groups suggests the
presence of distinct mutations interfering with the melatonin
signal transduction. Post-translational modifications affecting Gi
protein function should be considered as one possible
mechanism.
[0053] Experimental data showed a melatonin-signaling dysfunction
in osteoblasts and myoblasts isolated from 100% AIS patients
tested.
[0054] Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be
modified, without departing from the spirit and nature of the
subject invention as defined in the appended claims.
Example 1
Clinical Characteristics of AIS Patients and Control Subjects from
which Osteoblasts were Isolated
[0055] The clinical characteristics of the examined AIS and control
subjects are shown in Table 1.
TABLE-US-00001 TABLE 1 Clinical data on patients with adolescent
idiopathic scoliosis and control subjects Case Diagnostic Curve
Pattern Gender Age at surgery Cobb Angle Heredity Basal cAMP
Induced cAMP Melatonin Group 1 AIS L lumbar M 16.33 45 Yes 0.01
12.06 3 2 AIS L thoracolumbar F 16.17 37 Yes 2.01 21.77 2 3 AIS L
thoracolumbar F 17.08 47 No 0.10 15.84 2 4 AIS L thoracolumbar F
17.92 50 na 0.16 3.81 1 5 AIS R thoracic M 16.00 49 No 0.10 24.52 1
6 AIS R thoracic M 15.42 48 No 0.01 3.50 2 7 AIS R thoracic M 13.25
75 No 0.01 11.90 2 8 AIS R thoracic F 14.92 54 Yes 0.10 16.36 1 9
AIS R thoracic F 14.92 30 Yes 0.10 19.85 1 10 AIS R thoracic F
16.67 57 Yes 0.40 12.20 2 11 AIS R thoracic F 12.67 67 Yes 0.88
48.95 3 12 AIS R thoracic F 17.25 53 Yes 0.26 31.71 3 13 AIS R
thoracic F 15.25 53 No 0.70 20.37 2 14 AIS R thoracic F 18.42 34 No
0.46 61.08 2 15 AIS R thoracic F 13.75 61 No 0.01 14.70 2 16 AIS R
thoracic F 13.00 48 No 0.10 15.84 2 17 AIS R thoracic F 16.25 60 No
0.01 13.35 2 18 AIS R thoracic F 14.75 67 No 0.03 3.41 2 19 AIS R
thoracic F 15.08 30 No 0.40 16.78 3 20 AIS R thoracic F 14.67 32 No
0.10 21.20 3 21 AIS R thoracic F 15.83 43 No 0.10 20.81 3 22 AIS R
thoracolumbar M 18.67 61 Yes 0.10 22.74 1 23 AIS R thoracolumbar F
14.08 50 Yes 0.90 18.70 3 24 AIS R/L double scoliosis M 17.25 46-30
Yes 0.10 4.45 1 25 AIS R/L double scoliosis M 17.17 70-50 No 1.90
67.78 3 26 AIS R/L double scoliosis F 14.17 70-48 Yes 0.03 51.64 2
27 AIS R/L double scoliosis F 12.75 53-55 Yes 0.19 7.25 2 28 AIS
R/L double scoliosis F 14.75 41-50 Yes 0.10 9.18 3 29 AIS R/L
double scoliosis F 16.25 51-30 No 0.10 69.91 1 30 AIS R/L double
scoliosis F 18.92 29-35 No 0.28 5.39 2 31 AIS R/L double scoliosis
F 14.33 57-65 No 1.20 63.40 2 32 AIS R/L double scoliosis F 19.17
45-60 No 0.10 11.49 2 33 AIS R/L double scoliosis F 11.75 74-56 No
0.10 11.47 2 34 AIS R/L double scoliosis F 11.42 57-38 No 0.10
14.64 2 35 AIS R/L double scoliosis F 18.33 23-35 No 0.41 2.30 2 36
AIS R/L double scoliosis F 14.33 90-66 No 0.36 4.84 2 37 AIS R/L
double scoliosis F 12.58 61-46 No 0.23 27.28 3 38 AIS R/L double
scoliosis F 15.08 90-90 No 0.20 26.53 3 39 AIS R/L double scoliosis
F 14.33 56-53 No 0.01 17.04 3 40 AIS R/L double scoliosis F 13.50
48-42 No 0.48 4.94 3 41 AIS R/L double scoliosis F 12.83 59-57 No
0.10 24.99 3 42 Congenital L lumbar F 18.42 53 No 0.01 1.00 2* 43
Congenital R thoracic M 14.17 45 No 0.08 12.26 2* 44 Congenital R
thoracic M 13.08 70 No 0.95 55.36 2* 45 Congenital R thoracic F
7.42 75 No 0.10 6.74 2* 46 Cancer/spine none F 10.00 0 No 0.10
45.34 Control 47 Cancer/spine L thoracic F 16.33 19 No 0.12 19.55
Control 48 Chiari L thoracic M 19.92 51 Yes 0.32 15.19 Control 49
DMD none M 14.00 0 No 0.16 8.80 Control 50 encephalopath R/L double
scoliosis M 17.67 60-30 No 0.10 18.24 Control 51 Marfan L
thoracolumbar F 19.42 38 No 0.10 8.00 Control 52 Marfan/spondylo
R/L double scoliosis F 12.92 0 Yes 0.09 15.51 Control 53
NF/scoliosis R thoracolumbar F 15.75 115 No 0.10 21.48 Control 54
Noonan R thoracic F 18.75 49 No 0.01 13.97 Control 55 spondylo L
lumbar F 19.00 0 No 0.10 15.34 Control 56 spondylo R lumbar F 16.42
0 No 0.10 36.20 Control 57 spondylo R thoracolumbar M 14.50 0 No
0.10 5.95 Control 58 Traumatic R thoracic F 17.75 40 No 0.01 9.29
Control cyphosis
AIS: adolescent idiopathic scoliosis; R, right; L, left; na, not
available; NF, neurofibromatosis Basal and induced cAMP values are
given as pmoles/1.times.10.sup.5 cells
TABLE-US-00002 TABLE 2 Clinical data associated with individual AIS
groups Cobb's Basal Induced Patients Age angle Heredity cAMP cAMP
Groupe 1 F 57% 15.5 48.degree.-30.degree. 4/7 (57%) 0.11 23.09 M
43% 17 Groupe 2 F 79% 14.2 54.degree.-47.degree. 1/14 (7%) 0.20
15.15 M 21% 16 Groupe 3 F 75% 14.3 55.degree.-52.degree. 3/8 (38%)
0.42 28.51 M 25% 16.5 Controls mean values basal cAMP: 0.12;
induced cAMP: 7.58
Example 2
Study Design for Assays Performed with Osteoblasts
[0056] The melatonin signal transduction pathway functionality was
investigated in osteoblasts from patients with clinically
well-defined AIS (n=41) and compared with age- and gender-matched
subjects presenting or not a scoliosis (n=17) (Table 1).
Example 3
Isolation of Human Osteoblasts
[0057] Osteoblasts were obtained intraoperatively from bone
fragments reduced in smaller pieces mechanically with a bone cutter
in sterile conditions and incubated at 37.degree. C. in 5% CO.sup.2
in a 100 mm culture dish in presence of DMEM medium containing 10%
FBS (certified FBS, Invitrogen, Burlington, ON, Canada) and 1%
penicillin/streptomycin (Invitrogen). After a 30-day period,
osteoblasts emerged from the bone pieces were separated at
confluence from the remaining bone fragments by trypsinization.
Example 4
Assay for Detecting Melatonin-Signaling Pathway in AIS
Osteoblasts-cAMP Accumulation Using Melatonin as Known Agonist of
the Melatonin-Signaling Pathway
[0058] Osteoblasts from patients with AIS and control subjects were
seeded in quadruplet on 24-wells plate (1.times.10.sup.5
cells/well) and incubated either with the vehicle alone, dimethyl
sulphoxide (DMSO, Sigma.TM., Oakville, ON, Canada) or the diterpene
forskolin (10.sup.-5M, Sigma.TM.) to stimulate the cAMP formation.
Inhibition curves of cAMP production were generated by adding
melatonin to the forskolin-containing samples in concentrations
ranging from 10.sup.-11M to 10.sup.-5M in a final volume of 1 ml of
DMEM media with 0.5% bovine serum albumin (BSA, Sigma.TM.). After a
30-minute incubation at 37.degree. C., the cells were lysed and the
sample centrifuged at 4.degree. C. The cAMP content was determined
in 200 .mu.l aliquot of the supernatant using an enzyme immunoassay
kit (Amersham-Pharmacia Biosciences, Mississauga, ON, Canada). All
assays were performed in duplicate.
[0059] In osteoblasts from control subjects, melatonin produced a
dose-dependent inhibition of forskolin-stimulated adenylyl cyclase
activity detectable by a reduction of cAMP levels of about 60-70%
(FIGS. 1-2). In contrast, osteoblasts from patients with AIS showed
a lack of inhibition of forskolin-stimulated adenylyl cyclase
activity by melatonin (FIGS. 1-2). The distributions of single data
points obtained with patients with AIS, in comparison with control
subjects are reported in FIG. 1. Further analysis allowed
classifying patients with AIS into 3 distinct groups according to
their osteoblast responsiveness to melatonin treatment (FIG. 2). In
group 1, melatonin increased cAMP accumulation in treated
osteoblasts, which contrasted with the normal inhibitory values
obtained with control subjects (FIG. 2). In group 2, osteoblasts
did not response to melatonin since no significant inhibition of
cAMP accumulation was observed even at pharmacological dose
(10.sup.-7M) or higher (10.sup.-5M) as illustrated by the cAMP
curve inhibition (FIG. 2). Finally, the third group showed only a
weak response toward melatonin treatment, although at physiological
dose (10.sup.-9M) no significant inhibition was measured (FIG. 2).
Under standard assay conditions, basal and induced cAMP levels
increased from group 1 to group 3 when compared to control subjects
(data not shown).
[0060] In addition, 57% of the patients in the first group (1)
showed the strongest heredity link when compared with the two other
groups of patients. The second group of AIS patients did not
respond to melatonin treatment even at pharmacological doses
(10.sup.-7M) and showed basal cAMP levels slightly elevated as
compared with the first group and the control subjects. The third
group remained resistant to melatonin, although at higher doses of
melatonin, it was possible to measure some significant inhibitory
effects on adenylyl cyclase activity. Both basal and
forskolin-stimulated cAMP levels were increased in that particular
group when compared with the other groups and the control subjects
(Tables 1 and 2). Interestingly, the severity of the disease seems
to be correlated by the augmentation of both basal and induced cAMP
levels since the third AIS group is composed of the youngest mean
age of female patients at the time of the surgery exhibiting also
the highest Cobb's angle degrees pre-op in double scoliosis (Table
2). In spite of the heterogeneity of both groups, AIS patients
displayed a more significant dysfunction of melatonin-signaling
over the other types of scoliotic patients. Comparison with control
subjects exhibiting also a scoliosis suggested that spinal
deformities observed in distinct diseases and syndromes could share
a common pathogenic mechanism interfering with the melatonin signal
transduction.
Example 5
Assay for Detecting Melatonin-Signaling Pathway Impairment in AIS
Osteoblasts--cAMP Accumulation Using GTP or Gpp(NH)p as Known
Agonists of the Melatonin-Signaling Pathway
[0061] The functionality of Gi proteins was assessed by
investigating their ability to inhibit adenylyl cyclase activity in
osteoblasts. To obtain inhibition curve of cAMP production the
non-hydrolysable analogue of GTP, Gpp(NH)p (guanilyl
5'-imidophosphate, Sigma.TM.) was added to the forskolin-containing
samples in concentrations ranging from 10 nM to 100 .mu.M. The cAMP
content was determined as described above in similar assays with
melatonin.
[0062] In vitro assays with Gpp(NH)p reduced cAMP levels in
osteoblasts from control subjects in contrast to patients with AIS
tested, which showed no inhibitory effect for a majority of AIS
patients. The distribution of single data points obtained from each
patient is reported in FIG. 8. The values reported in FIG. 8 were
detected after the administration of 10.sup.-9M Gpp(NH)p, a GTP
non-hydrolysable analogue.
[0063] The ability of the non-hydrolysable GTP analogue to inhibit
adenylyl cyclase activity was detected. This enzymatic activity was
previously amplified by forskolin. The impaired capability to
inhibit forskolin-stimulated adenylyl cyclase activity was also
observed with the non-hydrolysable GTP analogue, Gpp(NH)p (FIG. 8).
Considering the multiplicity of GTP-binding proteins present in
osteoblasts (24), it may seem difficult to detect a defect in a
particular G-protein subtype with the use of a GTP analogue.
However, not only a preferential affinity of Gpp(NH)p towards Gi
proteins in the range of concentrations used has been evidenced
(25; 26), but also a widely different expression of each type of G
protein is documented, indicating that Gi are commonly 10 times
more abundant than Gs(24).
[0064] Analysis of the data obtained with the Gpp(NH)p assays
revealed again the presence of three distinct groups although
different groups of AIS patients (based upon the melatonin assays)
are retrieved. This may suggest again that at least 3 distinct
genes or type of mutations contribute to decrease or modify Gi
proteins function in AIS, which matches well with the clinical
variables associated with each AIS patients.
Example 6
Statistical Analysis
[0065] Results from the cAMP accumulation assays are given as the
mean.+-.SEM. Data were analyzed with StatView software.
Example 7
Level of Melatonin Receptors in Osteoblasts
[0066] Patients with AIS and control subjects were also
investigated to exclude the possibility that melatonin signaling
dysfunction observed in that assay could be secondary to a reduced
level of melatonin receptors. RT-PCR were performed with specific
primers corresponding to melatonin receptor subtypes, MT1 (panel A,
FIG. 7), MT2 (panel B, FIG. 7) and OPN (panel C, FIG. 7) using 2
.mu.g of RNA isolated every 2 h during a 24 h cycle from MC3T3-E1
cells treated (+) or not (-) with melatonin (10.sup.-7M). Panel D,
FIG. 7 represents a similar analysis with osteoblast cultures
obtained from one scoliotic patient (AIS female, 15 year old) and a
non-scoliotic subject (NS female, 15 year old) without (c) and with
melatonin (m, 10-7M). PCR products were separated on a 1.5% agarose
gel and visualized by ethidium bromide staining. Note that both
melatonin receptor subtypes are expressed in MC3T3-E1 cells and in
human osteoblasts but in MC3T3-E1 cells, only MT1 subtype is down
regulated in presence of melatonin while MT2 subtype is the
predominant form. No significant difference was observed between
AIS and NS subjects. Expression analysis by RT-PCR and IHC
experiments indicated no significant variation in melatonin
receptor levels although it cannot be ruled-out that their function
could be altered in AIS.
Example 8
Assay for Determining Mel Receptors Function and Distribution in
Osteoblasts: Radioligand Binding and IHC Assays
[0067] Radioligand-binding assays with 2-.sup.125I-iodomelatonin
and IHC assays with MT1 and MT2 specific antibodies were performed
to assess whether the dysfunction of melatonin-signaling observed
could be secondary to either a reduced level of melatonin receptors
or to mutations affecting their function.
[0068] To determine whether or not melatonin receptor function is
affected in osteoblasts from patients with AIS, total binding
assays were conducted using the radioligand 2-[.sup.125I]
iodomelatonin (500 pM, Amersham-Pharmacia Biosciences) in the
absence (total binding) or presence (non-specific binding) of
melatonin (1 .mu.M, Sigma.TM.). All reactions were run in
duplicate. The data were expressed as femtomoles of receptor per
milligram of protein. Protein determination was made by the method
of Bradford using BioRad.TM. protein assay reagents (BioRad,
Mississauga, ON, Canada). Receptor subtype localization and
distribution were determined in osteoblastic cells by
immunohistochemistry (IHC) assays with anti-human MT1 and
anti-human MT2 antibodies (kind gifts from Prof. F. Fraschini and
Dr D. Angeloni, University of Milan, Italy).
[0069] Results obtained with the radioligand binding assays showed
no significant variation in the function of melatonin receptors
(data not shown). This correlated well with IHC analysis, which
revealed no significant variation in the synthesis and distribution
of both melatonin receptor subtypes in both patients with AIS and
control subjects (FIG. 3).
Example 9
Assay for Determining Gi Protein Coupling to Individual Melatonin
Receptors and Phosphorylation State in Osteoblasts
[0070] Co-immunoprecipitation assays were performed with anti-MT1
and anti-MT2 specific antibodies (kind gifts of Dr Debora Angeloni
and Prof Franco Fraschini, University of Milan, Italy) to identify
Gi proteins coupled to individual melatonin receptor in human MG-63
osteoblasts (FIG. 9).
[0071] The specific antibodies were incubated with membrane
fractions purified from osteoblasts untreated and treated with
melatonin (10.sup.-9M), genistein or herbimycin (1 .mu.M, tyrosine
kinase inhibitors, Sigma.TM.) or sodium orthovanadate,
Na.sub.3VO.sub.4, (1 mM, tyrosine phosphatase inhibitor, Sigma.TM.)
for at least 16 h. Presence of coupled Gi proteins in respective
immune-complexes were determined by SDS-PAGE and Western blot with
specific Gi antibodies (Santa Cruz Biotechnology Inc., Santa Cruz,
Calif., USA) and phosphorylation status of these coupled Gi
proteins were determined using anti-phosphoserine, phosphothreonine
and phosphotyrosine antibodies (Sigma.TM.) using the same membrane
after stripping. Purified recombinant Gi proteins were used as
control for antibody specificity.
[0072] These assays showed a predominant coupling of Gi3 proteins
with MT2 receptor in purified osteoblast membrane fractions treated
or not with melatonin (FIG. 9) Interestingly, Western blot analysis
with Gi3 antibodies revealed the presence of an additional higher
molecular weight band corresponding to a phosphorylated form of Gi3
proteins (the presence of 60 kDa bands corresponding to
phosphorylated Gi3 proteins in FIG. 5).
[0073] Immunodetection assay with specific antibodies reacting with
phosphoproteins confirmed the presence of at least one
phosphoserine residue in those higher molecular weight Gi3 proteins
(FIGS. 9-10). Furthermore, similar assays with osteoblasts isolated
from two AIS patients revealed a distinct phosphorylation pattern
with and without melatonin addition. Western blot analysis
performed with respective membrane fractions using antibodies
reacting against individual Gi proteins did not reveal any
significant variation in the level of the three Gi proteins present
in human osteoblast (FIG. 5).
[0074] FIG. 20 also illustrate how the phosphorylation pattern of
cells isolated from patients with a melatonin-signaling impairment
differs from those of control cells. Identification of Gi proteins
coupled to MT2 melatonin receptor. Co-immunoprecipitation assays
were performed with specific anti-MT2 antibodies using purified
membrane fractions prepared from human MG-63 osteoblast culture
(panel A) and osteoblast cultures from AIS patient (case 22, panel
B) treated overnight in different conditions: 1-5-9) untreated;
2-6-10) with melatonin; 3-7-11) with Na.sub.3VO.sub.4, a tyrosine
phosphatase inhibitor; and 4-8-12) with genistein, a tyrosine
kinase inhibitor. Immunoblots were revealed with specific
antibodies reacting with individual Gi with the exception of
anti-Gi3 antibodies, which cross-react also with human Gi1
proteins. Lanes 1-4 with anti-Gi1; lanes 5-8 with anti-Gi2 and
lanes 9-12 with anti-Gi3. Lanes 13-15 correspond to purified
recombinant Gi1-3 proteins respectively and were used as control
for antibody specificity. The 60 kDa and 43 kDa bands correspond to
the phosphorylated (inactive) and unphosphorylated (active) forms
of Gi protein, respectively. Note the changes in the
phosphorylation patterns occurring in Gi proteins from AIS patient,
showing increased phosphorylation and distinct regulation by kinase
and phosphatase inhibitors tested. The results presented in FIG. 21
were obtained as described above and relate to cells isolated from
other patients: human osteoblast cultures isolated from AIS patient
(panel A, case 37 of Table 1; panel B, case 29 of Table 1) treated
overnight in different conditions: 1-6-11) untreated; 2-7-12) with
melatonin; 3-9-13) with Na.sub.3VO.sub.4, a tyrosine phosphatase
inhibitor; 4-10-14) with genistein, a tyrosine kinase inhibitor;
and 5-11-15) with herbimycine, another tyrosine kinase inhibitor.
Lanes 1-5 with anti-Gi1; lanes 6-10 with anti-Gi2 and lanes 11-115
with anti-Gi3. Lanes 16-18 correspond to purified recombinant Gi1-3
proteins respectively and were used as control for antibody
specificity. Note the changes in the phosphorylation patterns
occurring in Gi proteins in both AIS patient, showing a predominant
coupling with phosphorylated Gi proteins.
[0075] Expression analysis by RT-PCR did not show any significant
variation in Gi mRNA levels encoding for the three Gi proteins
present in human osteoblast (data not shown), although it cannot be
excluded that such variation might occur at the protein level.
[0076] The affinities of the three Gi proteins to the MT1 and MT2
receptors enabling them to be associated and pre-coupled to these
receptors differ. The Gi3 has the strongest affinity to these
receptors in absence or presence of melatonin, followed by Gi2 and
then Gi1. In both conditions, only a weak interaction of Gi1
protein was detected with both receptor subtypes. Interestingly, in
absence of melatonin, 2 forms of Gi3 and Gi2 proteins were detected
suggesting that one of these forms could be phosphorylated (FIG.
12). Interestingly, overnight treatment of the cells with melatonin
or genistein (a tyrosine kinase inhibitor) completely abolished the
presence of both phosphorylated forms in MT1 or MT2 immune
complexes. This suggests that a tyrosine phosphorylation regulates
indirectly Gi proteins functions through the activation a
downstream unknown serine kinase.
[0077] It cannot be ruled-out that changes in Gi proteins affinity
for GTP and Gpp(NH)p could be triggered by post-translational
modifications of Gi proteins involving serine residues
phosphorylation. Phosphorylation of Gi proteins at their N-terminus
is well known to block the formation of functional heterotrimers
with G.beta. and G.gamma. subunits preventing the inhibition of
adenylyl cyclase activity either in presence of melatonin or
Gpp(NH)p.
Example 10
Clinical Characteristics of AIS Patients and Control Subjects from
which Myoblasts were Isolated
[0078] The clinical characteristics of the examined AIS and control
subjects are shown in Table 1 (cases 33, 22 and 8 in Table 1).
Example 11
Study Design for Myoblast Assays
[0079] The melatonin signal transduction pathway functionality was
investigated in myoblasts from patients with clinically
well-defined AIS (n=3, namely cases 33, 22 and 8 in Table 1) and
compared with age- and gender-matched subjects presenting or not a
scoliosis (Table 1).
Example 12
Isolation of Human Myoblasts
[0080] Myoblasts were obtained intraoperatively from normal and AIS
patients and enzymatically dispersed, incubated, separated and put
into cultures according to methods known in the art.
Example 13
Assay for Detecting Melatonin-Signaling Pathway Impairment in AIS
Myoblasts--cAMP Accumulation Using Melatonin as Known Agonist of
the Melatonin-Signaling Pathway
[0081] Preliminary tests performed with skeletal myoblasts from
showed the effect of increasing concentrations of melatonin
(10.sup.-11 to 10.sup.-5M) on forskolin-stimulated adenylyl cyclase
activity in myoblasts isolated from AIS patients (cases 8, 22 and
23 in Table 1). These results (FIG. 15) show the incapacity of
melatonin to inhibit cAMP accumulation induced by forskolin
although only a treatment with a supra pharmacological dose of
melatonin (10.sup.-5M) is able in 2 cases to inhibit cAMP
accumulation in myoblasts.
[0082] The functionality of melatonin signaling is assessed by
investigating the ability of Gi proteins to inhibit stimulated
adenylyl cyclase activity in intact skeletal myoblasts. Cells
prepared from patients with AIS and control subjects are seeded in
quadruplet on 24-wells plate (1.times.10.sup.5 cells/well) and
incubated either with dimethyl sulphoxide (DMSO, Sigma.TM.) or
forskolin (10.sup.-5M, Sigma.TM.) to stimulate the cAMP formation.
To obtain the inhibition curve of cAMP production, melatonin is
added to the forskolin-containing samples in concentrations ranging
from 10.sup.-11M to 10.sup.-5M in a final volume of 1 ml of DMEM
media with 0.5% bovine serum albumin (BSA). After a 30-minute
incubation at 37.degree. C., the cells are lysed and the sample
centrifuged at 4.degree. C. The cAMP content is determined in 200
.mu.l aliquot of the supernatant using an enzyme immunoassay kit
(Amersham-Pharmacia Biosciences).
Example 14
Assay for Detecting Melatonin-Signaling Pathway Impairment in AIS
Osteoblasts--cAMP Accumulation Using GTP or Gpp(NH)p as Known
Agonists of the Melatonin-Signaling Pathway
[0083] The functionality of Gi proteins is assessed by
investigating their ability to inhibit adenylyl cyclase activity in
myoblasts isolated from patients with AIS and control subjects. To
obtain inhibition curve of cAMP production, the non-hydrolysable
analogue of GTP, Gpp(NH)p (guanilyl 5'-imidophosphate, Sigma.TM.)
is added to the forskolin-containing samples in concentrations
ranging from 10 nM to100 .mu.M. Protein determination is made by
the method of Bradford using BioRad.TM. protein assay reagents
(BioRad.TM.) with BSA as standard. All assays are performed in
duplicate.
[0084] Basal cAMP levels are obtained from untreated cells, while
cells tested with forskolin alone corresponds to the induced
levels. Standard curve for sensitivity and quantification is
performed with standards provided by the manufacturer of respective
assays. (23)
Example 15
Statistical Analysis for Assays with Myoblasts
[0085] Results from the cAMP accumulation assays are given as the
mean.+-.SEM. An analysis of variance (ANOVA), followed by Fishers
protected least significant difference (PLSD) procedure for
post-hoc comparison is used to verify the significance between 2
means.
Example 16
Assay for Determining Mel Receptors Function and Distribution in
Myoblasts: Radioligand Binding and IHC Assays
[0086] Cellular localization and distribution of MT1 and MT2
melatonin receptors are determined on histological sections of
human skeletal muscles obtained intraoperatively during spine
surgeries and on skeletal myoblast cultures generated in parallel
from patients with AIS and control subjects.
[0087] In order to determine whether or not melatonin receptors
density or function could be affected in skeletal myoblasts from
patients with AIS, total binding assays are conducted using the
radioligand 2-[.sup.125I] iodomelatonin (Amersham-Pharmacia
Biosciences). This approach is also useful with the primary cell
cultures to determine the effects of melatonin pre-treatment on
receptor density and function ((47; 48)). Briefly, cells are washed
with phosphate buffered saline (PBS), lifted in buffer, and
pelleted by centrifugation. The cells are resuspended in Tris (50
mM, pH 7.4) buffer and then added to tubes containing 500 pM of
2-[.sup.125I] iodomelatonin in the absence (total) or presence
(non-specific) of melatonin (1 .mu.M) in a final reaction volume of
0.26 ml. Cells are then incubated for 1 h at room temperature and
harvested by filtration over glass filters (Millipore.TM.)
pre-soaked in 10% polyethylenimine (Sigma.TM.) and counted in a
gamma counter. All reactions are run in duplicate. The data is
expressed as femtomoles of receptor per milligram of protein.
Protein determination is made by the method of Bradford using the
BioRad.TM. protein assay reagents (BioRad.TM.).
[0088] IHC experiments are performed with polyclonal antibodies
reacting specifically with either the MT1 or MT2 receptor subtypes
(kind gift from Dr Debora Angeloni and Prof Franco Fraschini,
University of Milan, Italy) using a confocal microscope. In order
to assess whether melatonin or estrogens could modify the cellular
localization and/or distribution of MT1 or MT2 subtype, IHC
experiments are performed with primary cell cultures treated with a
physiological dose of melatonin (10.sup.-9M) or estradiol
(10.sup.-10M).
[0089] Negative control for IHC is generated by omitting the
primary antibody and by competition with specific blocking peptide.
Positive controls are provided for IHC experiments and binding
assays using stably transfected C2C12 myoblastic cells expressing
constitutively MT1 or MT2 receptor. Specificity of each antibody
has been already tested with human osteoblasts. In binding assays
with [.sup.125I] iodomelatonin, subtraction of non-specific binding
obtained in presence of melatonin from the total binding generated
with the radioligand alone determines the specific binding.
However, MT1 and MT2 can bind this radioligand with almost the same
affinity. Alternatively, the addition of luzindole (10 .mu.M), a
MT2 antagonist, could reveal indirectly the contribution of
individual receptors in the total binding of 2-[.sup.125I]
iodomelatonin. All assays are performed in duplicate. Data obtained
in total binding assays with the radioligand is analysed by
Student's unpaired t-test. Significance is defined as P<0.05,
and data will be analysed with StatView.TM. and Statistica.TM.
softwares.
[0090] It cannot be ruled-out at this stage that scoliotic patients
could display a distinct distribution of melatonin receptors.
However, reduced receptor binding in situ could indicate potential
interference by an unknown factor (calmodulin, estrogens etc.),
that could be easily correlated at least for the estrogens by
similar assay in vitro. A marked reduction of
2-[.sup.125I]iodomelatonin binding in scoliotic sections could be
caused by either a reduction in the number of a specific receptor
subtype or a by a mutation reducing the affinity of this receptor.
It is unlikely that the presence of serum in the in vitro binding
assay may interfere with this assay since 10% FBS should contain
less than 10.sup.-11M of melatonin.
Example 17
Assay for Determining Gi Protein Coupling to Individual Melatonin
Receptors and Phosphorylation State in Myoblasts
[0091] Muscle cells are grown to confluence in 10 cm tissue culture
dishes, rinsed once with ice-cold PBS, and scraped off their
plastic support. After sedimentation, the cell pellets are
resuspended in 2 ml of buffer A (5 mM Tris-HCl pH 7.4/2 mM
EDTA/protease inhibitors cocktail) and subsequently disrupted by
sonication. Then, membranes are sedimented by centrifugation
450.times.g/5 min at 4.degree. C. and the supernatant added on the
top of 9 ml 35% sucrose cushion. Membranes will be sedimented by
ultracentrifugation at 150,000.times.g/90 min. Purified membrane
fraction sediment at the bottom of the sucrose cushion. Membrane
fractions are resuspended in 1 ml of buffer B (50 mM Tris-HCl pH
7.4/5 mM MgCl.sub.2) and incubated with or without ligand
(melatonin) for 1 h at 25.degree. C. For ligand-stimulated samples,
all subsequent steps are performed in the continued presence of
ligand. Thereafter, membranes are centrifuged at 18,000.times.g/30
min at 4.degree. C. and washed once in 1 ml buffer C (75 mM
Tris-HCl pH 7.4/12 mM MgCl.sub.2/2 mM EDTA/protease inhibitors
cocktail) and then resuspended in the same buffer containing 1%
Triton X-100 (V/V), and agitated for 3 h at 4.degree. C.
Non-solubilized membrane proteins are removed by centrifugation at
18,000.times.g/30 min at 4.degree. C. Immunoprecipitation of
solubilized melatonin receptors are performed with gentle agitation
overnight at 4.degree. C. with antibodies (1:40) reacting
specifically with either human MT1 or MT2 subtypes (kind gifts of
Dr Debora Angeloni and Pr Franco Fraschini, Milan University,
Italy), followed by a 6 h incubation at 4.degree. C. with 50 .mu.l
of Protein-A agarose suspension (Sigma.TM.) to immunoprecipitate by
centrifugation the individual melatonin receptor. G proteins are
dissociated from immune complexes by treatments with Gpp(NH)p (0.1
mM) for 1 h at 37.degree. C. and are separated by 12% SDS-PAGE and
transferred to nitrocellulose membranes. Immunoblot are carried-out
in TBST buffer containing 5% skim milk with commercially available
antibodies recognizing individual Gi proteins (Gi1-3, Santa Cruz)
and reactive bands are visualized using enhanced chemiluminescence.
In parallel, similar assays are performed with purified membranes
from cells treated overnight or less with physiological doses of
melatonin, estradiol or with different kinase and phosphatase
inhibitors such as tyrosine kinase inhibitors, tyrosine phosphatase
inhibitors and PKC specific inhibitors. Additional immunodetection
is performed with antibodies reacting against Gz proteins, a
related Gi family member.
Example 18
Assay for Detecting Melatonin-Signaling Impairment in
Osteoblasts--Proliferation Assay
[0092] An assay was performed to compare the proliferation of
osteoblasts of normal subjects with that of scoliotic patients. In
FIG. 6, Panel A represents time-courses experiments (triplicates)
of [3H]-thymidine incorporation (cpm values in abscise axis) in
human osteoblasts generated from bone specimens isolated from
normal (NS) subject (F1/female 17 years old) and scoliotic patients
(AIS M2/male 18 years old and F1/female, 17 years old) stimulated
by a physiological dose of melatonin (10.sup.-9M) used as known
agonist of the melatonin-signaling pathway. This assay showed that
normal osteoblasts growth rate increases linearly in response to a
physiological dose of melatonin (10.sup.-9M) while those from
scoliotic patients did not respond to melatonin.
Example 19
Assay for Detecting Melatonin-Signaling Impairment in
Lymphocytes--cAMP Accumulation Using Melatonin as Known Agonist of
the Melatonin-Signaling Pathway
[0093] Melatonin inhibition assays of cAMP accumulation assays
induced by forskolin have been performed in vitro on human
lymphocytes isolated from control subjects using 10 ml of blood or
less. Anticoagulant-treated blood was layered on the
Ficoll-Paque.TM. solution and centrifuged for a short period of
time. Differential migration during centrifugation resulted in the
formation of layers containing different blood cells. Because of
their lower density, the lymphocytes were found at the interface
between the plasma and the Ficoll-Paque.TM. with other slowly
sedimenting particles (platelet and monocytes). The lymphocytes
were then recovered from the interface and subjected to a short
washing step with a balanced salt solution to remove any platelets,
Ficoll-Paque.TM. and plasma. Then, the cells were counted and used
to perform the cAMP assays described in Examples above. As is known
from the literature, the lymphocytes have melatonin receptors at
their surface. Results could be obtained in 3 h or less with this
particular assay. This assay is advantageously rapid as compared to
assays using osteoblasts (at least a month) because they do not
require culture time.
Example 20
Assay for Detecting Melatonin-Signaling Impairment in Osteoclasts
Derived from Monocytesllymphocytes
[0094] Other functional assays with melatonin using osteoclasts
derived from monocytes/lymphocytes fraction isolated from
peripheral blood of AIS patients and control subjects are
performed. Primary osteoclasts are derived from the peripheral
blood of patients and non-scoliotic subjects using 10 ml of blood
or less. Anticoagulant-treated blood is layered on the
Ficoll-Paque.TM. solution and centrifuged for a short period of
time. Differential migration during centrifugation results in the
formation of layers containing different blood cells. Because of
their lower density, the lymphocytes are found at the interface
between the plasma and the Ficoll-Paque.TM. with other slowly
sedimenting particles (platelet and monocytes). The lymphocytes are
then recovered from the interface and subjected to a short washing
step with a balanced salt solution to remove any platelets,
Ficoll-Paque.TM. and plasma. The cells are then counted and seeded
at high density (1.times.10.sup.6 cells per cm.sup.2) onto
artificial bone or dentin matrix in .alpha.-MEM with 10% FBS and
antibiotics. After a few days, cells that remain adherent will
start to differentiate into osteoclasts, forming large
multinucleate cells after 15-20 days. Addition of melatonin
(10.sup.-9M to 10.sup.-7M) inhibits osteoclasts resorption
activity, which is visualized by the absence of resorption pit in
the bone matrix (i.e. absence of holes or fewer holes on the
surface).
[0095] Different approaches (RT-PCR, immunohistochemistry) have
demonstrated the presence of both melatonin receptor subtypes (MT1
and MT2) at the surface of human osteoclasts from normal subjects
and from AIS patient (see FIGS. 16 and 17).
[0096] It was also evidenced that inhibitory activity of melatonin
is mediated in osteoclasts through the MT2 receptor since the
addition of luzindole, a MT2 specific antagonist prevents or
reduces the inhibitory effect of melatonin on osteoclasts
resorption activity (See FIG. 18). It is reasonably predicted that
melatonin does not affect resorbing activity of osteoclasts
isolated from animals with AIS or any related syndrome causing
spinal deformities contrasting with the results observed in normal
human osteoclasts.
Example 21
Melatonin-Signaling Pathway Modulated by Estradiol
[0097] A method of screening of the present invention was performed
and identified estrogen as one compound able to modulate the
melatonin-signaling pathway impairment in AIS patients. Experiments
were performed showing the effect of increasing concentrations of
melatonin (10.sup.-11 to 10.sup.-5M) used as known agonist of
melatonin-signaling pathway on forskolin-stimulated adenylyl
cyclase activity in osteoblasts from two patients with AIS (group 3
see Table 2) treated or not with a physiological dose of estradiol
(10.sup.-19M). Results illustrated in panels A and B of FIG. 19
correspond to AIS patient numbered 13 and 29 in Table 1,
respectively. It is apparent from this figure that the treatment
with a physiological dose of estrogen (oe) is sufficient to further
block the inhibitory effect of melatonin in scoliotic patients
belonging to the AIS group 3 (see table 2).
Example 22
Evaluation of Bone Mineral Density in Scoliotic and Control
Chicken
[0098] Non-invasive analyses were performed with a DEXA bone
densitometer (PixiMus.TM. II GE Lunar) and showed a significant
decrease in bone mineral density (BMD) in both vertebrae and femur
of all chicken although no difference was observed between those
exhibiting a scoliosis and those without scoliosis (FIGS. 11-12).
Note that in our surgical conditions the rate of scoliosis in
pinealectomized chicken was about 50% although non-scoliotic
pinealectomized chicken showed a similar BMD than the controls
(sham or intact chicken). In FIG. 11, the chickens were exhibiting
a scoliosis 7-days post-pinealectomy, while in FIG. 12, they were
exhibiting a scoliosis 21-days post-pinealectomy. Treatment with
melatonin, 3 mg/kg/day ip, increased BMD in treated animals.
Treatment with melatonin, 3 mg/kg/day ip, increased BMD in treated
animals.
[0099] Histological analysis indicated that decreased BMD occurred
particularly at the cortical bone level (not shown).
[0100] Interestingly, EMG measurement of paraspinal musculature
activity using intra-muscular electrodes revealed a 75% bi-lateral
increase in muscular tone in scoliotic pinealectomized chicken at
rest when compared to sham or non-scoliotic pinealectomized groups
(FIG. 13). EMG analysis was performed with implanted electrodes
21-days post-pinealectomy. EMG activities were recorded in active
chicken and compared between sham and scoliotic pinealectomized
chicken. Determination of EMG activity at rest in paraspinal
musculature of pinealectomized chicken.
[0101] Similar EMG analysis in active chicken showed an
asymmetrical activity increased by 30% on the left side of
paraspinal musculature of scoliotic chicken, corresponding to the
spine deformation curve (left sided in 99% of scoliotic chicken,
FIG. 14). No such effect was observed with non-scoliotic
chicken.
[0102] Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be
modified without departing from the spirit and nature of the
subject invention as defined in the appended claims.
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