Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE: MeCP2 ISOFORM-SPECIFIC ANTIBODY FOR DETECTION OF
ENDOGENOUS EXPRESSION OF MeCP2E2
TECHNICAL FIELD
The present disclosure relates generally to compositions and methods for
detecting and/or
monitoring abnormalities associated with over-expression and under-expression
of Methyl CpG
Binding Protein 2. More particularly, the present disclosure relates to
antibodies or antigen-
binding fragments thereof which specifically bind to the MeCP2E2 isoform of
Methyl CpG
Binding Protein 2, to methods for preparing the antibodies, to compositions
containing such
antibodies, and to use of the antibodies and/or compositions for detecting the
MeCP2E2 isoform
of Methyl CpG Binding Protein 2.
BACKGROUND
Mutation or altered expression of the X-linked Methyl CpG Binding Protein 2
(MECP2)
gene leads to a wide spectrum of neurodevelopmental disorders including Rett
Syndrome.
MeCP2 is a multifunctional epigenetic factor that is involved in multiple
nuclear events
including transcriptional repression, transcriptional activation, RNA
splicing, and chromatin
compaction. MeCP2 was first discovered as a repressor protein that binds to
methylated DNA at
the 5-methylcytosine (5mC) residues. However, recent studies have shown that
MeCP2 also
binds to 5-hydroxymethylcytosine (5hmC), presumably as an activator (Mellen et
al., 2012,
MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in
the nervous
system. Cell 151:1417-1430). While 5mC is a hallmark of inactive genes
(Delcuve et al., 2009,
Epigenetic control. J. Cell. Physiol. 219:243-250), 5hmC is generally
associated with active
genes (Mellen et al., 2012). Currently, it is unclear how, as a single
protein, MeCP2 provides so
many different nuclear functions.
In mice and humans, alternative splicing of the Mecp2/MECP2 gene leads to the
generation of two protein isoforms, MeCP2E1 and MeCP2E2. MeCP2E1 contains a
unique 21
amino acid sequence at its N-terminus, whereas the N-terminus of MeCP2E2
includes 9
exclusive amino acids (Kriaucionis et al., 2004, The major form of MeCP2 has a
novel N-
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terminus generated by alternative splicing. Nucleic Acids Res. 32:1818-1823).
Other than their
N-terminal regions, MeCP2 isoforms are similar and share the same functional
domains,
including the Methyl Binding Domain (MBD) and the Transcriptional Repression
Domain
(TRD) (Zachariah et al., 2012, Linking epigenetics to human disease and Rett
syndrome: the
emerging novel and challenging concepts in MeCP2 research. Neural Plasticity
2012:415825).
Previous studies indicate differential properties of MeCP2E1 and MeCP2E2
regarding their
interacting protein partners, impact on neuronal survival, role during
embryonic development,
and sensitivity to different drugs.
Distinct transcript expression patterns have been reported for Mecp2/MECP2
isoforms in
brain, with higher expression of Mecp2e1 than of Mecp2e2 (Dragich et al.,
2007, DOerential
distribution of the MeCP2 splice variants in the postnatal mouse brain. J.
Com.p Neurol.
501:526-542). However, comparative analysis of MeCP2 isoforms at the protein
levels in any
system has not been reported to date, due to the lack of specific anti-MeCP2E2
antibodies.
SUMMARY
The exemplary embodiments of the present disclosure relate to antibodies that
selectively
bind to the MeCP2E2 isoform of the MeCP2 protein, to compositions comprising
the anti-
MeCP2E2 antibodies, to methods for producing the anti-MeCP2E2 antibodies and
compositions
comprising the anti-MeCP2E2 antibodies, and to use of the anti-MeCP2E2
antibodies and
compositions for detection of and monitoring of the over-expression and/or
under-expression of
MeCP2E2.
One exemplary embodiment of the present disclosure pertains to methods of
preparing
anti-MeCP2E2 antibodies or antigen-binding fragments that do not bind to or
otherwise engage
the MeCP2E1 isoform of the MeCP2 protein. The anti-MeCP2E2 antibodies are
generated by a
synthetic peptide that consists of a sequence of twelve amino acids selected
from the N-terminus
of the anti-MeCP2E2 isoform. Alternatively, the anti-MeCP2E2 antibodies are
generated by a
synthetic peptide that consists of a sequence of eleven amino acids selected
from the N-terminus
of the anti-MeCP2E2 isoform.
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Another exemplary embodiment of the present disclosure pertains to
compositions that
include the foregoing antibodies or antigen-binding fragments thereof.
Another exemplary embodiment of the present disclosure pertains to foregoing
isolated
anti-MeCP2E2 antibodies or antigen-binding fragments thereof packaged in
lyophilized form, or
packaged in an aqueous medium.
Another exemplary embodiment of the present disclosure pertains to kits for
detecting
over-expression of MeCP2E2 or under-expression of MeCP2E2 for diagnosis,
prognosis or
monitoring. The kits include the foregoing isolated anti-MeCP2E2 antibody or
antigen-binding
fragment thereof labelled with a selected compound, and one or more compounds
for detecting
the label. Preferably the label is selected from the group consisting of a
fluorescent label, an
enzyme label, a radioactive label, a nuclear magnetic resonance active label,
a luminescent label,
and a chromophore label.
Another exemplary embodiment of the present disclosure pertains to methods for
detecting an over-expression of MeCP2E2 or an under-expression of MeCP2E2, in
a sample
from a mammalian subject. The methods include contacting the sample with any
of the foregoing
antibodies or antigen-binding fragments thereof which specifically bind to an
extracellular or a
N-terminal domain of MeCP2E2, for a time sufficient to allow the formation of
a complex
between the antibody or antigen-binding fragment thereof and MeCP2E2, and
detecting the
MeCP2E2-antibody complex or MeCP2E2-antigen-binding fragment complex. The
presence of a
complex in the sample is indicative of the presence in the sample of MeCP2E2
or a cell
expressing MeCP2E2.
In another aspect, the invention provides other methods for diagnosing a
MeCP2E2-
mediated disease or disorder in a mammalian subject. The methods include
administering to a
subject suspected of having or previously diagnosed with MeCP2E2-mediated
disease an amount
of any of the foregoing antibodies or antigen-binding fragments thereof which
specifically bind
to an extracellular or a N-terminal domain of MeCP2E2 antigen. The method also
includes
allowing the formation of a complex between the antibody or antigen-binding
fragment thereof
and MeCP2E2, and detecting the formation of the MeCP2E2-antibody complex or
MeCP2E2-
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antigen-binding fragment antibody complex to the target epitope. The presence
of a complex in
the subject is indicative of the presence of a MeCP2E2-mediated disease or
disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
The present disclosure will be described in conjunction with reference to the
following
drawings, in which:
Fig. 1 shows the alignment of MeCP2E1 Isoform 1 amino acid sequences between
human, mouse, and rat;
Fig. 2 is a Western blot gel to detect MeCP2E1 expression in control non-
transfected
(NT), MECP2E1 transfected (El -T), MECP2E2 transfected (E2-T), and MECP2E1 pre-
incubated with the antigenic peptide. Anti-MYC labelling was used as a
positive control while
GAPDH labelling was used as a loading control;
Figs. 3(A)-3(D) are micrographs showing detection of MeCP2E1 by
immunofluorescence
in NIH3T3 cells transduced with MECP2E1 (Figs 3(A), 3(C)), NI113T3 cells
transduced with
MECP2E2 (Fig. 3(B)), and non-transduced NI113T3 cells (Fig. 3(D)) (Scale bars
represent 10
lm);
Fig. 4 shows the alignment of MeCP2E2 Isoform 2 amino acid sequences between
human, mouse, and rat;
Figs. 5(A) and 5(B) show negative controls for immunofluorescence detection of
5(A)
MeCP2E2 and C-MYC in non-transduced NIH3T3 cells, and 5(B) absence of signals
in primary
omission controls with Rhodamine Red X (RRDX) and FITC in MECP2E2 transduced
NIH3T3
cells (scale bars represent 10 p.m);
Fig. 6 is a Western blot analysis for detection of MeCP2E2 expression in
control non-
transfected (NT), MECP2E1 transfected (El -T), MECP2E2 transfected (E2-T), and
E2-T pre-
incubated with E2 antigenic peptide. Anti-MYC labelling was used as a positive
control;
Fig. 7 shows a Western blot analysis with Phoenix cell extracts from non-
transfected cells
(NT), and MECP2E2 transfected cells (E2-T), probed with the anti-MeCP2E2
antibody after pre-
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incubation with increasing concentrations of peptide (0%, 0.1%, 1%, and 5%, of
peptide
compared to the amount of antibody used);
Figs. 8(A), 8(C), 8(E), 8(G) are micrographs showing detection by
immunofluorescence
staining of MeCP2E2 in NI113T3 cells transduced with a MECP2E1 retroviral
vector, while
Figs. 8(B), 8(D), 8(F), 8(11) are micrographs showing detection by
immunofluorescence staining
of MeCP2E2 in NIH3T3 cells transduced with a MECP2E2 retroviral vector (scale
bars
represent 20 m);
Fig. 9(A) is a Western blot gel showing Detection of MeCP2E1 in the nuclear
extracts
from adult mouse brain but not in the cytoplasmic extracts, while Fig. 9(B) is
a Western blot gel
showing Detection of MeCP2E2 in the nuclear extracts from adult mouse brain
but not in the
cytoplasmic extracts. Increasing amounts of nuclear and cytoplasmic protein
extracts were used.
and the membranes were re-probed with GAPDH as a loading control;
Fig. 10 shows a Western blot analysis of MeCP2E1 in the isolated nuclear
extracts from
the whole brain at the indicated developmental time points. Nuclear extracts
from null brain were
used as negative controls while ACTIN was used as a loading control (E =
embryonic days; P =
postnatal days; null = mecp2tm1.1Bird y/-
brain tissue; N = 3 SEM);
Fig. 11 is a chart showing quantitative RT-PCR with specific primers to detect
Mecp2e1
and Mecp2e2 transcripts. Total RNA from null brain was used as the negative
control (E ¨
embryonic days; P = postnatal days; null = mecp2tmLiBird Y/ brain tissue; N =
3 SEM; significant
differences are indicated at P<0.001).;
Fig. 12 shows the results of a Pearson's correlation analysis for the
indicated Mecp2
transcripts and MeCP2 protein levels;
Fig. 13 is a schematic representation of the MECP2E1 retroviral vector with a
C-MYC
tag (Retro-EF 1 a-E1) and the MECP2E2 retroviral vector with a C-MYC tag
(Retro-EF 1 a-E2)
retroviral vectors with C-MYC tag that were used for transfection of Phoenix
cells (shown in
Figs. 2, 6) and transduction of NIH3T3 cells (shown in Figs. 3, 8);
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Figs. 14(A), 14(C), 14(E) are micrographs showing detection by
immunohistochemistry
of endogenous MeCP2E2 in the CA1 region of adult mouse hippocampus from wild
type
mecp2tm1.1Bird y/+ mice, while Figs. 14(B), 14(D), 14(F) are micrographs
showing absence of
detection of endogenous MeCP2E2 in the CA1 region of adult mouse hippocampus
from null
(mecp2tm 1Bird y/-) Mecp2 mice (scale bars represent 20 [im);
Figs. 15(A)-15(E) are micrographs of controls to verify the specificity of
anti-MeCP2E2
detection by immunohistochemistry in the adult mouse brain wherein 15(A) are
views of primary
omission, 15(B) are views of anti-MeCP2E2 incubation with IgY and pre-
incubation of the
newly generated anti-MeCP2E2 antibody with the antigenic peptide MeCP2E2 shown
in 15(C),
15(D) are views of pre-incubation of the newly generated anti-MeCP2E2 antibody
with the
antigenic peptide MeCP2E1, 15(E) are views of anti-MeCP2E2 incubation with a
peptide against
the C-terminus of MeCP2 (scale bars represent 10 [im);
Fig. 16 is a Western blot analysis to detect endogenous MeCP2E2 expression in
the WT
adult mouse brain and its absence in Mecp2 null mice brain, GAPDH was used as
a loading
control;
Figs. 17(A)-17(C) are micrographs of confocal images of MeCP2E1 in WT adult
mouse
brain hippocampus CA1 region, while 17(D) is a chart showing the signal
intensity profile of
MeCP2E1 and DAPI co-localization indicating MeCP2E1 detection at the DAPI-rich
heterochromatin regions of nuclei (scale bar represents 2 gm);
Fig. 18(A)-18(C) are micrographs of confocal images of MeCP2E2 in WT adult
mouse
brain hippocampus CAI region, while 18(D) is a chart showing the signal
intensity profile of
MeCP2E2 and DAPI co-localization indicating MeCP2E2 detection at the DAPI-rich
heterochromatin regions of nuclei (scale bar represents 2 pm);
Fig. 19 shows a Western blot analysis of MeCP2E2 in the isolated nuclear
extracts from
the whole brain at the indicated developmental time points. Nuclear extracts
from null brain were
used as negative controls while ACTIN was used as a loading control (E =
embryonic days; P =
tm1.-
postnatal days; null = Mecp2 1Bird y/
brain tissue; N = 3 SEM);
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Figs. 20(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform in neurons from the CA1 hippocamus region of adult male mouse brain,
while Figs.
20(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform
(scale bar
represents 20 gm);
Figs. 21(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform in a single neuron nucleus from the CA1 hippocamus region of adult
male mouse brain,
while Figs. 21(B) are micrographs of immunofluorescence expression of the
MeCP2E2 isoform
in a single neuron nucleus from the CA1 hippocamus region of adult male mouse
brain (scale bar
represents 2 p.m);
Figs. 22(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform in GFAP-positive astrocytes from the CA1 hippocamus region of adult
male mouse
brain, while Figs. 22(B) are micrographs of immunofluorescence expression of
the MeCP2E2
isoform in GFAP-positive astrocytes from the CA1 hippocamus region of adult
male mouse
brain (scale bar represents 20 gm);
Figs. 23(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform
in CNPase-positive oligodendrocytes from the CA1 hippocamus region of adult
male mouse
brain, while Figs. 23(B) are micrographs of immunofluorescence expression of
the MeCP2E2
isoform in CNPase-positive oligodendrocytes from the CA1 hippocamus region of
adult male
mouse brain (scale bar represents 20 p.m);
Figs. 24(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform in a single astrocyte nucleus from the CA1 hippocamus region of adult
male mouse
brain, while Figs. 24(B) are micrographs of immunofluorescence expression of
the MeCP2E2
isoform in a single astrocyte nucleus from the CA1 hippocamus region of adult
male mouse
brain (scale bar represents 2 gm);
Figs. 25(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform
in a single oligodendrocyte nucleus from the CA1 hippocamus region of adult
male mouse brain,
while Figs. 25(B) are micrographs of immunofluorescence expression of the
MeCP2E2 isoform
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in a single oligodendrocyte nucleus from the CA1 hippocamus region of adult
male mouse brain
(scale bar represents 2 um);
Figs. 26(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform
in neurons from the CA1 hippocamus region of adult female mouse brain, while
Figs. 26(B) are
micrographs of immunofluorescence expression of the MeCP2E2 isoform (scale bar
represents
20 um);
Figs. 27(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform
in a single neuron nucleus from the CA1 hippocamus region of adult female
mouse brain, while
Figs. 27(B) are micrographs of immunofluorescence expression of the MeCP2E2
isoform in a
single neuron nucleus from the CA1 hippocamus region of adult female mouse
brain (scale bar
represents 2 um);
Figs. 28(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform
in GFAP-positive astrocytes from the CA1 hippocamus region of adult female
mouse brain,
while Figs. 28(B) are micrographs of immunofluorescence expression of the
MeCP2E2 isoform
(scale bar represents 20 um);
Figs. 29(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform
in CNPase-positive oligodendrocytes from the CA1 hippocamus region of adult
female mouse
brain, while Figs. 29(B) are micrographs of immunofluorescence expression of
the MeCP2E2
isoform in CNPase-positive oligodendrocytes from the CA1 hippocamus region of
adult female
mouse brain (scale bar represents 20 pm);
Figs. 30(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform
in a single astrocyte nucleus from the CA1 hippocamus region of adult female
mouse brain,
while Figs. 30(B) are micrographs of immunofluorescence expression of the
MeCP2E2 isoform
in a single astrocyte nucleus from the CA1 hippocamus region of adult female
mouse brain
(scale bar represents 2 um);
Figs. 31(A) are micrographs of immunofluorescence expression of the MeCP2E1
isoform in a single oligodendrocyte nucleus from the CA1 hippocamus region of
adult mouse
brain, while Figs. 31(B) are micrographs of immunofluorescence expression of
the MeCP2E2
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isoform in a single oligodendrocyte nucleus from the CA1 hippocamus region of
adult mouse
brain (scale bar represents 2 pm);
Figs. 32(A, A) - 32(A, D) are micrographs of immunohistochemical detection of
the
expression of the MeCP2E1 isoform in the whole hippocampus (32(A, A)), in the
CA2 region of
the hippocampus (32(A, B)), in the CA3 region of the hippocampus (32(A, C)),
and the dentate
gyms region of the hippocampus (32(A, D)), while Figs. 32(B, Al) - 32(B, D1)
are micrographs
of immunohistochemical detection of the expression of the MeCP2E2 isoform in
the whole
hippocampus (32(B, Al)), in the CA2 region of the hippocampus (32(B, B1)), in
the CA3 region
of the hippocampus (32(B, Cl)), and the dentate gyrus region of the
hippocampus (32(B, D1))
(scale bars represent 200 pin in A, Al; scale bars represent 20 pm in B, Bl,
C, Cl, D, D1);
Figs. 33(A, A) - 33(A, C) are micrographs of immunohistochemical detection of
the
expression of the MeCP2E1 isoform in the olfactory bulb region of the brain
(33(A, A)), in the
striatum region of the brain (33(A, B)), and in the cortex region of the brain
(33(A, C)), while
Figs. 33(B, Al) - 33(B, Cl) are micrographs of immunohistochemical detection
of the
expression of the MeCP2E2 isoform in the olfactory bulb region of the brain
(33(B, Al)), in the
striatum region the brain (33(B, B1)), and in the cortex region of the brain
(33(B, Cl)) (scale
bars represent 20 pm);
Figs. 34(A, A) - 34(A, C) are micrographs of immunohistochemical detection of
the
expression of the MeCP2E1 isoform in the thalamus region of the brain (34(A,
A)), in the brain
stem region of the brain (34(A B)), and in the cerebellum region of the brain
(34(A, C)), while
Figs. 34(B,A1) - 34(B,C1) are micrographs of immunohistochemical detection of
the expression
of the MeCP2E2 isoform in the thalamus region of the brain (34(B, Al)), in the
brain stem
region the hippocampus (34(B, B1)), and in the cerebellum region of the brain
(34(B, Cl)) (scale
bars represent 20 p.m);
Figs. 35(A)-35(D) are micrographs of immunohistochemical detection of the
expression
of the MeCP2E1 isoform and the MeCP2E2 isoform in the molecular layer (m1),
the Purkinje
cell layer (pep, and granule cell layer (gcl) of the cerebellum (the scale bar
represents 20 m);
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Figs. 36(A)-36(C) are micrographs showing colocalization of the expression of
the
MeCP2E1 isoform and the MeCP2E2 isoform in the molecular layer of the
cerebellum (36(A)),
in the Purkinje cell layer of the cerebellum (36(B)), and in the granule cell
layer of the
cerebellum (36(C)) (the scale bar represents 2 }im);
Fig. 37 is a chart and a Western gel showing the expression of the MeCP2E1
isoform in
adult mouse brain regions. Whole WT Mecp2 and null Mecp2 adult brains were
used as controls,
while ACTIN used as the loading control (OB = olfactory bulb; STR = striatum;
CTX = cortex;
HIPP = hippocampus; THAL = thalamus; BS = brain stem; CERE = cerebellum; N = 3
SEM);
Fig. 38 is a chart showing the results of quantitative RT-PCR to detect
transcript levels of
-- Mecp2 isoforms in adult mouse brain regions (OB = olfactory bulb; STR =
striatum; CTX =
cortex; HIPP = hippocampus; THAL = thalamus; BS = brain stem; CERE =
cerebellum; N =
3 SEM; significant differences between the two isoforms are indicated with
P<0.0001****,
P<0.001***, P<0.01** or P<0.05*);
Fig. 39 shows the results of a Pearson's correlation analysis for indicated
Mecp2
-- transcripts and MeCP2 protein levels;
Fig. 40 is a chart and a Western gel showing the expression of the MeCP2E2
isoform in
adult mouse brain regions. Whole WT Mecp2 and null Mecp2 adult brains were
used as controls,
while ACTIN used as the loading control (OB = olfactory bulb; STR = striatum;
CTX = cortex;
HIPP = hippocampus; THAL = thalamus; BS = brain stem; CERE = cerebellum; N = 3
SEM);
and
Fig. 41(A) is a chart showing a semi-quantitative representation of MeCP2E I
and
MeCP2E2 levels in WT Mecp2 and null Mecp2 adult brain (N = 3 SEM; significant
differences
between the two isoforms are indicated with P<0.001***), while Fig. 41(B) is a
chart showing a
Quantitative RT-PCR to detect transcript levels of Mecp2 isoforms in WT Mecp2
and null
-- Mecp2 adult mouse whole brains. (N = 3 SEM; significant differences between
the two
isoforms are indicated with P<0.0001****).
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DETAILED DESCRIPTION
The exemplary embodiments of the present disclosure pertain to antibodies that
selectively bind to the MeCP2E2 isoform of the MeCP2 protein, to compositions
comprising the
anti-MeCP2E2 antibodies, to methods for producing the anti-MeCP2E2 antibodies
and
compositions comprising the anti-MeCP2E2 antibodies, and to use of the anti-
MeCP2E2
antibodies and compositions for detection of and monitoring of the over-
expression and/or
under-expression of MeCP2E2.
Accordingly, one exemplary embodiment of the present disclosure pertains to
methods of
preparing anti-MeCP2E2 antibodies or antigen-binding fragments that do not
bind to or
otherwise engage the MeCP2E1 isoform of the MeCP2 protein. The anti-MeCP2E2
antibodies
can be generated by a peptide that consists of the sequence of twelve amino
acids set forth in
SEQ ID NO:10. Alternatively, the anti-MeCP2E2 antibodies can be generated by a
peptide that
consists of the sequence of eleven amino acids set forth in SEQ ID NO:11.
Another exemplary embodiment of the present disclosure pertains to
compositions that
include the foregoing antibodies or antigen-binding fragments thereof.
Another exemplary embodiment of the present disclosure pertains to foregoing
isolated
anti-MeCP2E2 antibodies or antigen-binding fragments thereof packaged in
lyophilized form, or
packaged in an aqueous medium.
Another exemplary embodiment of the present disclosure pertains to kits for
detecting
over-expression of MeCP2E2 or under-expression of MeCP2E2 or relative protein
expression to
MeCP2E1 for diagnosis, prognosis or monitoring a disease or a disorder in a
subject. The kits
include the foregoing isolated anti-MeCP2E2 antibody or antigen-binding
fragment thereof
labelled with a selected compound, and one or more compounds for detecting the
label.
Preferably the label is selected from the group consisting of a fluorescent
label, an enzyme label,
a radioactive label, a nuclear magnetic resonance active label, a luminescent
label, and a
chromophore label.
Another exemplary embodiment of the present disclosure pertains to methods for
detecting an over-expression of MeCP2E2 or an under-expression of MeCP2E2 or
relative
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protein expression to MeCP2E1, in a sample collected from a mammalian subject.
The methods
include contacting the sample with any of the foregoing antibodies or antigen-
binding fragments
thereof which specifically bind to an extracellular or a N-terminal domain of
MeCP2E2, for a
time sufficient to allow the formation of a complex between the antibody or
antigen-binding
-- fragment thereof and MeCP2E2, and detecting the MeCP2E2-antibody complex or
MeCP2E2-
antigen-binding fragment complex. The presence of a complex in the sample is
indicative of the
presence in the sample of MeCP2E2 or a cell expressing MeCP2E2.
Another exemplary embodiment pertains to methods for diagnosing a MeCP2E2-
mediated disease or disorder in a mammalian subject. The methods include
administering to a
-- subject suspected of having or previously diagnosed with MeCP2E2-mediated
disease an amount
of any of the foregoing antibodies or antigen-binding fragments thereof which
specifically bind
to an extracellular or a N-terminal domain of MeCP2E2 antigen. The method also
includes
allowing the formation of a complex between the antibody or antigen-binding
fragment thereof
and MeCP2E2, and detecting the formation of the MeCP2E2-antibody complex or
MeCP2E2-
-- antigen-binding fragment antibody complex to the target epitope. The
presence of a complex in
the subject is indicative of the presence of a MeCP2E2-mediated disease or
disorder.
Another exemplary embodiment pertains to use of the anti-MeCP2E2antibodies or
antigen-binding fragments thereof, and/or use of the compositions that include
the foregoing
antibodies or antigen-binding fragments thereof, and/or use of the kits,
and/or use methods for
-- detecting and/or diagnosing Rett's syndrome in a mammalian subject.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
the disclosure
belongs. Certain terms are discussed in the specification to provide
additional guidance to the
practitioner in describing the methods, uses and the like of embodiments of
the disclosure, and
-- how to make or use them. It will be appreciated that the same thing may be
said in more than one
way. Consequently, alternative language and synonyms may be used for any one
or more of the
terms discussed herein. No significance is to be placed upon whether or not a
term is elaborated
or discussed herein. Recital of one or a few synonyms or equivalents does not
exclude use of
other synonyms or equivalents, unless it is explicitly stated. Use of examples
in the specification,
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including examples of terms, is for illustrative purposes only and does not
limit the scope and
meaning of the embodiments of the disclosure herein. Although any methods and
materials
similar or equivalent to those described herein can be used in the practice or
testing of the
present disclosure, the preferred methods and materials are now described.
To facilitate understanding of the disclosure, the following definitions are
provided.
The word "comprise" or variations such as "comprises" or "comprising" will be
understood to imply the inclusion of a stated integer or groups of integers
but not the exclusion
of any other integer or group of integers.
As used herein, the word "complexed" means attached together by one or more
linkages.
The term "a cell" includes a single cell as well as a plurality or population
of cells.
Administering an agent to a cell includes both in vitro administrations and in
vivo
administrations.
The term "subject" as used herein includes all members of the animal kingdom,
and
specifically includes humans.
The term "about" or "approximately" means within 20%, preferably within 10%,
and
more preferably within 5% of a given value or range.
The term "homologous" in all its grammatical forms and spelling variations
refers to the
relationship between proteins that possess a "common evolutionary origin,"
including
homologous proteins from different species. Such proteins (and their encoding
genes) have
sequence homology, as reflected by their high degree of sequence similarity.
This homology is
greater than about 75%, greater than about 80%, greater than about 85%. In
some cases the
homology will be greater than about 90% to 95% or 98%.
"Amino acid sequence homology" is understood to include both amino acid
sequence
identity and similarity. Homologous sequences share identical and/or similar
amino acid
residues, where similar residues are conservative substitutions for, or
"allowed point mutations"
of, corresponding amino acid residues in an aligned reference sequence. Thus,
a candidate
polypeptide sequence that shares 70% amino acid homology with a reference
sequence is one in
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which any 70% of the aligned residues are either identical to, or are
conservative substitutions of,
the corresponding residues in a reference sequence.
The term "polypeptide" refers to a polymeric compound comprised of covalently
linked
amino acid residues. Amino acids are classified into seven groups on the basis
of the side chain
R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH)
group, (3) side chains
containing sulfur atoms, (4) side chains containing an acidic or amide group,
(5) side chains
containing a basic group, (6) side chains containing an aromatic ring, and (7)
proline, an imino
acid in which the side chain is fused to the amino group. A polypeptide of the
disclosure
preferably comprises at least about 14 amino acids.
The term "protein" refers to a polypeptide which plays a structural or
functional role in a
living cell.
The term "corresponding to" is used herein to refer to similar or homologous
sequences,
whether the exact position is identical or different from the molecule to
which the similarity or
homology is measured. A nucleic acid or amino acid sequence alignment may
include spaces.
Thus, the term "corresponding to" refers to the sequence similarity, and not
the numbering of the
amino acid residues or nucleotide bases.
The term "derivative" refers to a product comprising, for example,
modifications at the
level of the primary structure, such as deletions of one or more residues,
substitutions of one or
more residues, and/or modifications at the level of one or more residues. The
number of residues
affected by the modifications may be, for example, from 1, 2 or 3 to 10, 20,
or 30 residues. The
term derivative also comprises the molecules comprising additional internal or
terminal parts, of
a peptide nature or otherwise. They may be in particular active parts,
markers, amino acids, such
as methionine at position ¨1. The term derivative also comprises the molecules
comprising
modifications at the level of the tertiary structure (N-terminal end, and the
like). The term
derivative also comprises sequences homologous to the sequence considered,
derived from other
cellular sources, and in particular from cells of human origin, or from other
organisms, and
possessing activity of the same type or of substantially similar type. Such
homologous sequences
may be obtained by hybridization experiments. The hybridizations may be
performed based on
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nucleic acid libraries, using, as probe, the native sequence or a fragment
thereof, under
conventional stringency conditions or preferably under high stringency
conditions.
The term "antibody" as used herein refers to a glycoprotein comprising at
least two
heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
Each heavy chain
is comprised of a heavy chain variable region (abbreviated herein as HCVR or
VH) and a heavy
chain constant region. The heavy chain constant region is comprised of three
domains, CH1, CH2
and CH3. Each light chain is comprised of a light chain variable region
(abbreviated herein as
LCVR or VL) and a light chain constant region. The light chain constant region
is comprised of
one domain, CL. The VH and VL regions can be further subdivided into regions
of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions
that are more conserved, termed framework regions (FR). Each VH and VL is
composed of three
CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the
following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and
light chains
contain a binding domain that interacts with an antigen. The constant regions
of the antibodies
may mediate the binding of the immunoglobulin to host tissues or factors,
including various cells
of the immune system (e.g., effector cells) and the first component (Cl q) of
the classical
complement system.
The term "antigen-binding fragment" of an antibody as used herein, refers to
one or more
portions of an antibody that retain the ability to specifically bind to an
antigen (e.g., MeCP2E2
isoform of the MeCP2 protein). It has been shown that the antigen-binding
function of an
antibody can be performed by fragments of a full-length antibody. Examples of
binding
fragments encompassed within the term "antigen-binding fragment" of an
antibody include (i) a
Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI
domains; (ii) a
F(ab1)2 fragment, a bivalent fragment comprising two Fab fragments linked by a
disulfide bridge
at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains;
(iv) a Fv fragment
consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et
al., (1989) Nature 341:544-546) which consists of a VH domain; and (vi) an
isolated
complementarity determining region (CDR). Furthermore, although the two
domains of the Fv
fragment, V and VH, are coded for by separate genes, they can be joined, using
recombinant
methods, by a synthetic linker that enables them to be made as a single
protein chain in which
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the VL and VH regions pair to form monovalent molecules (known as single chain
Fv (scFv); see
e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.
NatL Acad. Sci. USA
85:5879-5883). Such single chain antibodies are also intended to be
encompassed within the
term "antigen-binding portion" of an antibody. These antibody fragments are
obtained using
conventional procedures, such as proteolytic fragmentation procedures, as
described in J.
Goding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (N.Y.
Academic Press
1983). The fragments are screened for utility in the same manner as are intact
antibodies.
An "isolated antibody", as used herein, is intended to refer to an antibody
which is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated
antibody that specifically binds to MeCP2E2 isoform of the MeCP2 protein and
is substantially
free of antibodies that specifically bind antigens other than the MeCP2E2
isoform). As used
herein, "specific binding" refers to antibody binding to a predetermined
antigen. Typically, the
antibody binds with an affinity that is at least two-fold greater than its
affinity for binding to a
non-specific antigen (e.g., BSA, casein) other than the predetermined antigen
or a closely-related
antigen.
The term "complementarity determining regions" as used herein refers to the
regions
within antibodies where these proteins complement an antigen's shape. The
acronym CDR is
used herein to mean "complementarity determining region".
The antibodies of the present disclosure may be polyclonal antibodies and can
be
produced by a variety of techniques well known in the art. Procedures for
raising polyclonal
antibodies are well known. For example anti-MeCP2E2 polyclonal antibodies may
be raised by
administering a synthetic peptide (e.g., SEQ ID NO:10, or alternatively, SEQ
ID NO:11)
subcutaneously to New Zealand white rabbits which have first been bled to
obtain pre-immune
serum. The synthetic peptide can be injected at a total volume of 100 IA per
site at six different
sites, typically with one or more adjustments. The rabbits are then bled two
weeks after the first
injection and periodically boosted with the same antigen three times every six
weeks. A sample
of serum is collected 10 days after each boost. Polyclonal antibodies are
recovered from the
serum, preferably by affinity chromatography using the synthetic peptide to
capture the antibody.
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This and other procedures for raising polyclonal antibodies are disclosed in
E. Harlow, et. al.,
editors, Antibodies: A Laboratory Manual (1988).
An embodiment of the present disclosure relates to a method of detecting cells
or portions
thereof in a biological sample (e.g., histological or cytological specimens,
biopsies, whole blood
samples, separated blood cells, and the like) wherein the MeCP2E2 is
overexpressed. This
method involves providing an antibody or an antigen-binding binding fragment
thereof, probe, or
ligand, which specifically binds to an a peptide having a sequence with at
least 90% to SEQ ID
NO:10, preferably at least about 95% identical, more preferably at least about
97% identical, still
more preferably at least about 98% identical, and most preferably is at least
about 99% identical.
This method also involves providing an antibody or an antigen-binding binding
fragment thereof,
probe, or ligand, which specifically binds to an a peptide having a sequence
with at least 90% to
SEQ ID NO:11, preferably at least about 95% identical, more preferably at
least about 97%
identical, still more preferably at least about 98% identical, and most
preferably is at least about
99% identical. The anti-MeCP2E2 antibody is bound to a label that permits the
detection of the
cells or portions thereof upon binding of the anti-MeCP2E2 antibody to the
cells or portions
thereof. The biological sample is contacted with the labeled anti-MeCP2E2
antibody under
conditions effective to permit binding of the anti-MeCP2E2 antibody to the
extracellular domain
or N-terminal domain of MeCP2E2 of any of the cells or portions thereof in the
biological
sample. The presence of any cells or portions thereof in the biological sample
is detected by
detection of the label. In one preferred form, the contact between the anti-
MeCP2E2 antibody and
the biological sample is carried out in a living mammal and involves
administering the anti-
MeCP2E2 antibody to the mammal under conditions that permit binding of the
anti-MeCP2E2
antibody to MeCP2E2 of any of the cells or portions thereof in the biological
sample. Again, such
administration can be carried out by any suitable method known to one of
ordinary skill in the art.
In addition, the anti-MeCP2E2 antibodies of the present disclosure can be used
in
immunofluorescence techniques to examine human tissue, cell and bodily fluid
specimens. In a
typical protocol, slides containing cryostat sections of frozen, unfixed
tissue biopsy samples or
cytological smears are air dried, formalin or acetone fixed, and incubated
with the monoclonal
antibody preparation in a humidified chamber at room temperature. The staining
pattern and
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intensities within the sample are then determined by fluorescent light
microscopy and optionally
photographically recorded.
As yet another alternative, computer enhanced fluorescence image analysis or
flow
cytometry can be used to examine tissue specimens or exfoliated cells, i.e.,
single cell
preparations from aspiration of tissues or organs using the anti-MeCP2E2
antibodies of this
disclosure. The percent MeCP2E2 positive cell population, alone or in
conjunction with
determination of other attributes of the cells (e.g., DNA ploidy of these
cells), may, additionally,
provide very useful prognostic information by providing an early indicator of
disease progression.
The method of the present disclosure can be used to screen patients for
diseases or
disorders associated with the over-expression of MeCP2E2 or under-expression
of MeCP2E2 or
changes in MeCP2E2 protein expression relative to MeCP2E1. Alternatively, it
can be used to
identify the recurrence of such diseases or disorders, particularly when the
disease or disorder is
localized in a particular biological material of the patient.
Also within the scope of the disclosure are kits comprising the compositions
of the
disclosure and instructions for use. Kits containing the antibodies or antigen-
binding fragments
thereof of the present disclosure can be prepared for in vitro diagnosis,
prognosis and/or
monitoring of the over-expression of MeCP2E2 or the under-expression of
MeCP2E2 or its
protein expression relative to MeCP2E1 by the immunohistological,
immunocytological and
immuno serological methods described above. The components of the kits can be
packaged either
in aqueous medium or in lyophilized form. When the antibodies or antigen-
binding fragments
thereof are used in the kits in the form of conjugates in which a label moiety
is attached, such as
an enzyme or a radioactive metal ion, the components of such conjugates can be
supplied either in
fully conjugated form, in the form of intermediates or as separate moieties to
be conjugated by the
user or the kit.
A kit may comprise a carrier being compartmentalized to receive in close
confinement
therein one or more container means or series of container means such as test
tubes, vials, flasks,
bottles, syringes, or the like. A first of said container means or series of
container means may
contain one or more anti-MeCP2E2 antibodies or antigen-binding fragments
thereof or
MeCP2E2. A second container means or series of container means may contain a
label or linker-
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label intermediate capable of binding to the primary anti-MeCP2E2 antibodies
(or fragment
thereof).
The present disclosure will be further elaborated in the following examples.
However, it
is to be understood that these examples are for illustrative purposes only,
and should not be used
to limit the scope of the present disclosure in any manner.
EXAMPLES
Example 1: Generation of polyclonal and monoclonal MeCP2E1 isoform specific
antibodies
An isoform-specific antibody that was generated in a different species was
required to
enable double-labelling of the MeCP2E1 and MeCP2E2 isoforms in a single
organism..
Accordingly, the strategy disclosed by Zachariah et al. (2012, Novel MeCP2
isoform-specific
antibody reveals the endogenous MeCP2E1 expression in murine brain, primary
neurons and
astrocytes. PloS ONE 7, e49763) was followed as taught for the production of
polyclonal
MeCP2E1 antibodies in rabbits.
Fig. 1 shows the alignment of MeCP2E1 Isoform 1 amino acid sequences between
human, mouse, and rat, and a peptide sequence "GGGEEERLEEKS" (SEQ ID NO:4;
shaded
section in Fig. 1) selected from the N-terminus for use as an antigen for
generating polyclonal
MeCP2E1 antibodies in rabbits. The IgY molecules were purified from rabbit
blood and anti-
MeCP2E1-specific immunoglobulins were isolated by peptide affinity
purification.
An additional "C" residue was inserted at the N-terminal end of the sequence
"GGGEEERLEEKSC" (SEQ ID NO:6). The additional C (underlined) was used for
conjugation
with BSA (Bovine Serum Albumin) and KLH (Keyhole limpet hemocyanin) for
antibody
purification. The generated antibodies were tested against MeCP2E1 peptide by
ELISA during
production, and were also tested by WB (Western Blot) and immunofluorescence
(IF) studies in
transfected Phoenix cells and transduced NIH3T3 cells with MECP2E1 retroviral
vectors
carrying human MECP2E1 cDNA. The overexpressed MeCP2E1 has a MYC tag and we
confirmed the detection of similar signals with C-MYC antibody. Importantly, C-
MYC also
detects MeCP2E2 (the other MeCP2 isoform that has a MYC tag). However, the
anti-MeCP2E1
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antibody disclosed herein does not cross-react with the MeCP2E2 isoform.
The specificity and sensitivity of the anti-MeCP2E1 antibody was initially
verified by
Western blotting. Probing protein extracts from non-transfected, MeCP2E1-
transfected and
MeCP2E2-transfected phoenix cells, the affinity purified anti-MeCP2E1 detected
specific bands
at approximately 75 kDa in MeCP2E1-transfected extracts (Fig. 2, lane 2). No
signal was
detected in non-transfected cells (Fig. 2, lane 1), nor in transfected cells
with MECP2E2 (Fig. 2,
lane 3). The presence of exogenous MeCP2 in the transfected cells with either
Retro-EF la-El or
Retro-EF 1 a-E2 was verified by immunolabelling with an anti-C-MYC antibody
(Fig. 2, lanes 5-
6), with no detectable signal in non-transfected cells (Fig. 2, lane 4).
Furthermore, pre-incubation
of the anti-MeCP2E1 antibody with the antigenic peptide before probing the
membranes with
MECP2E1 transfected cell lysate (Fig. 2, lane 7) completely abrogated the
detection of
exogenous MeCP2E1. Immunofluorescence staining with the anti-MeCP2E1 antibody
revealed
the expression of MeCP2 in the DAPI-rich heterochromatic foci within the
NIH3T3 cells
transduced with MECP2E1 (Fig. 3(A)), but no signal was detected in the MECP2E2
transduced
cells (Fig. 3(B)) confirming that the anti-MeCP2E1 antibody does not cross-
react with the
overexpressed MECP2E2. In both MECP2E1 and MECP2E2 overexpressed cells,
incubation
with an anti-C-MYC antibody resulted in detectable signals indicating that the
transduced protein
is properly expressed in both cases. As expected, no signals were detected in
primary omission
experiments using Retro-EF 1 a-E1 transduced cells with the same secondary
antibody (Fig. 3(C).
The absence of endogenous MECP2E2 expression was confirmed in the non-
transduced NIH3T3
cells by using the anti-MeCP2E2 antibody (Fig. 3(D)), thus confirming that the
anti-MeCP2E1
antibody disclosed herein does not cross-react with the MeCP2E2 isoform.
Additionally, mouse monoclonal antibodies were generated against MeCP2E1 with
the
same strategy and the corresponding clones were selected based on positive
ELISA readings. It
was confirmed that these monoclonal antibodies detect exogenous MeCP2E1 by WB
and IF.
The data generated in this study shows that inclusion or exclusion of an extra
amino acid
at the N-terminal part of the peptide does not affect the specificity of the
generated antibody and
does not cause any cross-reactivity with the other MeCP2E2 isoform. Therefore,
both peptides:
"GGGEEERLEEKS" (SEQ ID NO:4) or "GGGEEERLEEK" (SEQ ID NO:5) can be
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successfully used for generating polyclonal or monoclonal antibodies against
MeCP2E1 protein
isoform.
Example 2: Generation of polyclonal and monoclonal MeCP2E2 isoform specific
antibodies
Fig. 4 shows the alignment of MeCP2E2 Isoform 2 amino acid sequences between
human, mouse, and rat, and a peptide sequence "VAGMLGLREEKS" (SEQ ID NO:10;
shaded
section in Fig. 4) selected from the N-terminus for use as an antigen for
generating polyclonal
MeCP2E1 antibodies in chickens. The IgY molecules were purified from chicken
egg yolks and
anti-MeCP2E1-specific immunoglobulins were isolated by peptide affinity
purification.
Equivalent anti-MeCP2E2 antibodies have also been generated in rabbit and show
similar
specificity for detecting MeCP2E2 protein isoform.
An additional "C" residue was inserted at the N-terminal end of the sequence
"VAGMLGLREEKSC" (SEQ ID NO:12). The additional C (underlined) was used for
conjugation with BSA (Bovine Serum Albumin) and KLH (Keyhole limpet
hemocyanin) for
antibody purification. The generated antibodies were tested against MeCP2E2
peptide by ELISA
during production, and were also tested by WB (Western Blot) and
immunofluorescence (IF)
studies in transfected Phoenix cells and transduced NIH3T3 cells with MECP2E2
retroviral
vectors carrying human MECP2E2 cDNA. The overexpressed MeCP2E2 has a MYC tag
and we
confirmed the detection of similar signals with C-MYC antibody. Importantly, C-
MYC also
detects MeCP2E1 (the other MeCP2 isoform that has a MYC tag). However, the
anti-MeCP2E2
antibody disclosed herein does not cross-react with the MeCP2E1 isoform.
The specificity of the anti-MeCP2E2 antibody by Western blot (WB) and
immunofluorescence (IF) experiments at various stages of the antibody
production and after IgY
purification. For validations by WB, the affinity purified antibody was tested
using protein
extracts from Phoenix cells transfected with either Retro-EF1a-E1 or Retro-EF
1 a-E2 (Fig. 6), in
parallel to non-transfected control cells following the method taught by
Rastegar et al. (2009,
MECP2 isoform-specific vectors with regulated expression for Rett syndrome
gene therapy. PloS
ONE 4:e6810).
Western blot analysis with the anti-MeCP2E2 antibody yielded a specific band
at the
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expected molecular weight (approximately75 kDa) in MECP2E2-transfected cells
(Fig. 6, lane
3). In contrast, no signal was detected in non-transfected cells (Fig. 6, lane
1), nor in transfected
cells with MECP2E1 (Fig. 6, lane 2). Importantly, pre-incubation of the anti-
MeCP2E2 antibody
with the antigenic peptide used to generate the antibody (peptide competition)
eliminated the
detection of signal in the MECP2E2 transfected cells (Fig. 6, lane 7). The
specificity and
sensitivity of this newly developed anti-MeCP2E2 antibody was verified by pre-
incubation of
antibody with increasing concentrations of the antigenic peptide before
probing the membranes
with MECP2E2 transfected cell lysates (Fig. 7, lanes 2-4). The presence of
exogenous MeCP2 in
the
transfected cells with either Retro-EF 1 a-E1 or Retro-EF 1 a-E2 was
verified by
immunolabelling with an anti-C-MYC antibody (Fig. 6, lanes 5-6), with no
detectable signal in
non-transfected cells (Fig. 6, lane 4).
Further verification of the specificity of the custom-made anti-MeCP2E2
antibody using
IF, revealed the localization of MeCP2E2 in the DAPI-rich heterochromatic foci
within the
NIH3T3 cells transduced with MECP2E2 (Fig. 8(B)). No signal was detected in
the MECP2EI
transduced cells (Fig. 8(A)). C-MYC labelling confirmed the successful
transduction of both
MeCP2E1 and MeCP2E2 within the tested samples (Figs, 8(C), 8(D)). The absence
of
endogenous MECP2E2 expression was verified in non-transduced NIH3T3 cells
using the anti-
MeCP2E2 antibody (Fig. 5(A)). No signal could be observed in primary omission
experiments
using Retro-EF 1 a-E1 transduced cells with the same secondary antibodies
(Fig. 5(B)).
Additionally, mouse monoclonal antibodies were generated against MeCP2E2 with
the
same strategy using the amino acid sequence set forth in SEQ ID NO:11 and the
corresponding
clones were selected based on positive ELISA readings. It was confirmed that
these monoclonal
antibodies detect exogenous MeCP2E2 by WB and IF.
The data shows that inclusion or exclusion of an extra amino acid at the N-
terminal part
of the peptide does not affect the specificity of the generated antibody and
does not cause any
cross-reactivity with the other MeCP2E1 isoform. Therefore, both peptides:
"VAGMLGLREEKS" (SEQ ID NO:10) or "VAGMLGLREEK" (SEQ ID NO: 11) can be
successfully used for generating polyclonal or monoclonal antibodies against
MeCP2E2 protein
isoform.
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Example 3: Generation of MECP2E1/E2 transfected/transduced cells
Retro-EF 1 a-E1 (expressing MECP2E1) and Retro-EF 1 a-E2 (expressing MECP2E2)
vectors were transfected into Phoenix retroviral packaging cells (Kinsella et
al., 1996, Episomal
vectors rapidly and stably produce high-titer recombinant retrovirus. Hum.
Gene. Ther .7:1405-
1413.) following the method taught by Rastegar et al. (2009, MECP2 isoform-
specific vectors
with regulated expression for Rett syndrome gene therapy. PLoS One 4: e6810)
to generate (i)
infectious retroviral MECP2E1 vectors with a C-terminal C-Myc tag particles,
and (ii) retroviral
MECP2E2 vectors with a C-terminal C-Myc tag particles. Culture supernatants
containing viral
particles were harvested at 48 hours post-transfections. The transfected
phoenix cells were
collected and lysed for protein extraction, and the retroviral particles were
used to transduce
NIH3T3 mouse fibroblasts following the method taught by Rastegar et al.
(2009). The
transduced cells were fixed with 4% paraformaldehyde for imrnunofluorescent
studies, 48 hours
after transduction. NIH2T3 cells, Phoenix cells, and MECP2 vectors were
obtained from The
Hospital for Sick Children (Toronto, ON, CA).
Example 4: Quantitative Real Time PCR (qRT-PCR)
Total RNA from brain regions and brains at developmental stages were extracted
using
RNEASY Mini Kit (RNEASY is a registered trademark of Qiagen GmbH, Hilden,
Fed. Rep.
Ger.) and converted to cDNA using SUPERSCRIPT III Reverse Transcriptase
(SUPERSCRIPT
is a registered trademark of Life Technologies Corp., Carlsbad, CA, USA)
following the
methods taught by Barber et al. (2013, Dynamic expression of MEIS1
homeoprotein in E14.5
forebrain and differentiated forebrain-derived neural stem cells. Annals of
Anatomy /
Anatomischer Anzeiger: official organ of the Anatomische Gesellschaft),
Kobrossy et al. (2006,
Interplay between chromatin and trans-acting factors regulating the Hoxd4
promoter during
neural differentiation. J. Biol. Chem. 281:25926-25939), and
Nolte et al. (2006,
Stereospecificity and PAX6 function direct Hoxd4 neural enhancer activity
along the antero-
posterior axis. Devel. Biol. 99:582-593). Quantitative RT-PCR was carried out
using SYBR
Green-based RT2 qPCR Master Mix (SYBR is a registered trademark of Molecular
Probes Inc.,
Eugene, OR, USA) in a Fast 7500 Real-Time PCR machine (Applied Biosystems,
Foster City,
CA, USA) following the method taught by Barber et al. (2013, Dynamic
expression of MEIS1
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homeoprotein in E14.5 forebrain and differentiated forebrain-derived neural
stem cells. Ann. of
Anat. Available on line). Transcript levels of Mecp2e1 and Mecp2e2 were
examined using gene
specific primers (Table 1). PCR program for Mecp2 consisted of initial
denaturation at 95 C for
3 mm followed by 35 cycles of 1 mm at 95 C, 30 sec at 60 C, and 45 sec at 72
C, with a final
extension step at 72 C for 10 mm. The threshold cycle values (Ct) for each
gene was normalized
against the housekeeping gene Gapdh to obtain ACt values for each sample.
Relative
quantification of gene expression was carried out by calculating 2-Act of each
sample. Analysis
was performed using MICROSOFT EXCEL 2010 and GraphPad Prism 6.0 (MICROSOFT
and EXCEL are registered trademarks of Microsoft Corp., Redmond, WA, USA). Two-
Way
ANOVA was used to calculate significant differences between different brain
regions.
Table 1: List of primers used for qRT-PCR
Gene SEQ ID NO: Direction Sequence (5' to 3')
SEQ ID NO:13 Forward AGG AGA GAC TGG AGG AAA AGT
Mecp2 e 1
SEQ ID NO:14 Reverse CTT AAA CTT CAG TGG CTT GTC TCT G
SEQ ID NO:15 Forward CTC ACC AGT TCC TGC TTT GAT GT
Mecp2e2
SEQ ID NO:16 Reverse CTT AAA CTT CAG TGG CTT GTC TCT G
SEQ ID NO:17 Forward AAC GAC CCC TTC ATT GAC
Gapdh
SEQ ID NO:18 Reverse TCC ACG ACA TAC TCA GCA C
Example 5: Immunofluorescence and immunohistochemistry
Detection of immunofluorescence (IF) antibodies in cultured NI113T3 cells was
carried
out following the method taught by Zachariah et al. (2012, Novel MeCP2 isoform-
specific
antibody reveals the endogenous MeCP2E1 expression in murine brain, primary
neurons and
astrocytes. PloS ONE 7, e497630) using the primary antibodies listed in Table
2 and the
secondary antibodies listed in Table 3. Briefly, cultured NIH3T3 cells on
coverslips were washed
with phosphate buffered saline (PBS, GIBCO) and fixed in 4% formaldehyde.
Fixed cells were
then permeabilized with 2% NP40 in PBS for 10 min, followed by preblocking
with 10% normal
goat serum (NGS, Jackson Immunoresearch Laboratories Inc.) in PBS for lb.
Primary antibodies
were diluted in PBS with 10% NGS and the cells were incubated in primary
antibodies overnight
at 4 C followed by three washes with PBS. Secondary antibodies diluted in 10%
NGS were
added to the cells for 1 h, followed by three washes with PBS. Coverslips were
mounted on glass
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slides with PROLONG Gold antifade (PROLONG is a registered trademark of
Molecular
Probes Inc., Eugene, OR, USA) containing 2 jig/m1 4',6-diamidino-2-
phenylindole (DAPI)
(Calbiochem, EMD Millipore) counter-stain.
Table 2: Primary antibodies
Primary Antibody Application & dilution Description
Source
MeCP2 (C-terminal) IHC 1:300 Rabbit polyclonal Millipore, 07-
013
MeCP2 (C-terminal) WB 1:100 Mouse monoclonal Abcam, Ab50005
MeCP2E1 WB 1:100, IHC: 3mg/m1 Chicken polyclonal Custom-
made 3
WB 1:100, IF 1:200,
MeCP2E2 Chicken polyclonal Custom-made
IHC: 1mg/m1
WB 1:1000,
MeCP2E1 Rabbit polyclonal Custom-made
IHC: 3mg/m1
GAPDH WB 1:500 Rabbit polyclonal Santa Cruz, Sc
25778
Beta-ACTIN WB 1:2000 Mouse monoclonal Sigma Aldrich,
A2228
C-MYC WB 1:1500, IF 1:200 Rabbit polyclonal Santa
Cruz, Sc789
C-MYC IF 1:200 Mouse monoclonal Invitrogen,
21280
GFAP IHC 1:500 Mouse monoclonal Invitrogen,
421262
NEUN IHC 1:400 Mouse monoclonal Millipore,
Mab377
CNPase IHC 1:5000 Mouse monoclonal Covance, SMI-
91R
Table 3: Secondary antibodies
Application
Secondary AntibodySource
and dilution
Rhodamine Red-X conjugated goat anti mouse IgG IF 1:400 Jackson
Immunoresearch, 115-259-146
Dylight 649 conjugated goat anti chicken IgY IF 1:400 Jackson
Immunoresearch, 103-485-155
FITC-Conjugated Affinipure goat anti rabbit IgG IF 1:400 Jackson
Immunoresearch, 111-095-144
Rhodamine Red-X conjugated goat anti chicken IgY IHC 1:400 Jackson
Immunoresearch, 103-295-155
Alexa 488 goat anti rabbit IHC 1:1000 Invitrogen, A11034
Alexa 488 goat anti mouse IHC 1:1000 Invitrogen, A11017
Alexa 488 goat anti chicken IHC 1:1000 Invitrogen, A11042
Peroxidase-Affinipure sheep anti-mouse IgG WB 1:7500 Jackson
ImmunoResearch 115-035-174
Peroxidase-AffiniPure donkey anti-rabbit IgG WB 1:7500 Jackson
ImmunoResearch 711-036-152
Peroxidase-AffiniPure goat anti-chicken IgY WB 1:5000 Jackson
ImmunoResearch 103-035-155
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Immunohistochemistry (IHC) experiments for adult murine brain were carried out
following the method taught by Zachariah et al. (2012). Briefly, brain tissues
were fixed in ice-
cold freshly de-polymerized paraformaldehyde (PFA) (0.16 M sodium phosphate
buffer, pH 7.4
with PFA). Subsequently, tissue blocks were incubated in cryoprotectant (25 mM
sodium
phosphate buffer, pH 7.4, 10% sucrose, 0.04% NaN3) at 4 C for approximately 24
h.
Cryosections were processed onto gelatinized slides and stored at -20 C.
Prior to IHC
experiments, tissues were permeabilized with 0.3% Triton X-100 Iris-buffered
saline (TBS-Tr)
(50 mM Tris-HC1, pH 7.6, containing 1.5% NaC1) solution. The slides were then
pre-blocked
with normal goat serum (NGS) in TBS-Tr and incubated with appropriate primary
antibodies
diluted inTBS-Tr/serum overnight at 4 C. Secondary antibodies were diluted in
TBS-Tr/serum
and applied, followed by washes using Tris-HC1 buffer (50 mM, pH 7.4).
Coverslips were
prepared after incubation with 0.2 g/m1 DAPI counterstain, washes with Tris-
HC1, and
application of PROLONG Gold antifade (Life Technologies Inc.).
Immunolabelled signals were detected using a ZEISS AXIO Observer Z1 inverted
microscope and LSM710 Confocal microscope (Carl Zeiss Canada Ltd, Toronto, ON,
CA;
ZEISS and AXIO are registered trademarks of Carl Zeiss AG Corp., Oberkochen,
Fed. Rep.
Ger.). Images were obtained using AXIO VISION 4.8 (AXIO VISION is a
registered trademark
of Carl Zeiss AG Corp.), Zen Blue 2011, Zen Black 2009 and Zen Black 2011
softwares (Carl
Zeiss Canada Ltd) and assembled using ADOBE PHOTOSHOP CS5 and ADOBE
ILLUSTRATOR CS5 (ADOBE, PHOTOSHOP, and ILLUSTRATOR are registered
trademarks of Adobe Systems Inc., San Jose, CA, USA).
Example 6: Western blotting
Total cell extracts were prepared and Western blotting (WB) was done following
the
methods taught by Rastegar et al. (2009). 2 jig of total protein extracts from
transfected cells or
20-80 lig of nuclear or cytoplasmic proteins were loaded into each lane and
were subjected to
WB analysis. Nuclear extracts were prepared with the NE-PER (NE-PER is a
registered
trademark of Pierce Biotechnology Inc., Rockford, IL, USA) Nuclear and
Cytoplasmic
Extraction Kit (Thermo Scientific Inc., Waltham, MA, USA)) according to the
manufacturer's
instructions. All probed membranes were subjected to a second WB with an anti-
ACTIN
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antibody as a loading control. Quantification of detected MeCP2 or MeCP2E1
bands was done
with ADOBE PHOTOSHOP CS5 software and all bands were normalized to ACTIN
signals.
Student's t-test was used to analyze the significance of MeCP2 protein levels
between samples.
For peptide incubation experiments, increasing amounts of peptide antigen (as
compared to the
antibody concentration) was pre-incubated with the antibody for 3-5 hours at 4
C before probing
the membrane. Primary antibodies and secondary antibodies used in these
experiments are listed
in Tables 2 and 3, respectively.
Example 7: MeCP2E2 expression displays a later onset than MeCP2E1 during brain
development
It is known that the temporal expression profile of MeCP2 increases during the
course of
mouse brain development and follows neuronal maturation (Jung et at., 2003,
The expression of
methyl CpG binding factor MeCP2 correlates with cellular differentiation in
the developing rat
brain and in cultured cells. J. Neurobiol. 55:86-96; Shahbazian et al., 2002,
Insight into Rett
syndrome: MeCP2 levels display tissue- and cell-specific differences and
correlate with
neuronal maturation. Hum. Molec. Gen. 11, 115-124). Accordingly, anti-MeCP2E1
antibodies
produced as disclosed in Example 1 and anti-MeCP2E2 antibodies produced as
disclosed in
Example 2 were used to detect and assess the expression of MeCP2E1 and MeCP2E2
during
brain development before and after birth. The studies included brain tissues
from embryonic day
(E) 14, E18, postnatal day (P) 1, P7, P21, and P28. Expression of both
Mecp2/MeCP2 isoforms
at the transcript and protein levels was studied by qRT-PCR and WB
experiments, respectively.
MeCP2E1 expression was assessed using the anti-MeCP2E1 antibody. Nuclear
extracts were
prepared from dissected whole brain tissues at the aforementioned
developmental time points for
WB experiments. Because MeCP2 is a nuclear protein, the first step was to
confirm whether
MeCP2E1 could be detected specifically in the nuclear fractions of the
samples. Therefore,
increasing concentrations of brain nuclear and cytoplasmic extracts were
subjected to WB
analysis with anti-MeCP2E1 antibody.
As expected, MeCP2E1 was only detected in the nuclear fractions, and not in
cytoplasmic
fractions (Fig. 9(A)) confirming that MeCP2E1 is a nuclear protein in the
adult brain. Further
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WB experiments using developmental brain samples indicated that MeCP2E1 is
detected as early
as E14.
A gradual increase was observed in MeCP2E1 expression levels, which reached a
plateau
between P7 to P21, and subsequent decline at P28 that was not statistically
significant (Fig. 11;
Table 4). Surprisingly, Mecp2e1 transcripts were significantly higher at E14
and E18, with
noticeable decline after birth (Fig. 11). In all of the protein and transcript
analysis studies
disclosed herein, Mecp2 null mouse brain (Mecp2"11Bird YID were used as
controls because this
mouse model is reported to lack Mecp2/MeCP2 transcript and protein (Guy et
al., 2001, A mouse
Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.
Nature Genetics
27:322-326).
Pearson's correlation analysis (r) was performed between MeCP2E1 protein and
Mecp2e1 transcript levels, and indicated no significant relation between
Mecp2e1/MeCP2E1 at
the analyzed time points (r=0.44) (Fig. 12).
Next, a similar analysis of Mecp2e2/MeCP2E2 expression was performed at the
same
selected developmental time points using the chicken polyclonal anti-MeCP2E2
antibody
disclosed in Example 2. The specificity of this anti-MeCP2E2 antibody was
validated by
multiple techniques including WB and IF in transfected Phoenix cells (for WB)
and transduced
NIH3T3 cells (for IF) with Retro-EF 1 a-E1 or Retro-EF 1 a-E22 (Fig. 13).
Control studies
included non-transfected Phoenix and non-transduced NIH3T3 cells (Figs. 5(A),
5(B), 7, 8(A),
8(B)). The anti-MeCP2E2 antibody produced as disclosed in Example 2 showed
positive signals
in WT adult mouse hippocampus CA1 region in brain by IHC experiments (Figs.
14(A), 14(C),
14(E)), but as expected, MeCP2E2 signals were not detected in the null mice
brain
(me cp2m1.113irdy/-) (Figs. 14(B), 14(D), 14(F)). Further control experiments
showed that
MeCP2E2 signals were eliminated by MeCP2E2-antigenic peptide, but not when the
antibody
was pre-incubated with MeCP2E1-specific or a MeCP2 C-terminal-specific peptide
(Abeam
PLC, San Francisco, CA, USA) (Figs 15(A), 15(B), 15(C)). Additional controls
with primary
omission and IgY incubation did not result in any detectable signal, as
expected (Figs. 15(D),
15(E)). WB analysis with the anti-MeCP2E2 antibody produced a clear band at
about 75kDa in
WT brain nuclear extracts that was absent in the null mice brain (Fig. 16).
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Table 4: Differences in expression of Mecp2/MeCP2 isoforms during brain
development
MeCP2E1 MeCP2E2
Mecp2e1 Mecp2e2
AGE MD SIG P MD SIG P MD SIG P
MD SIG
E14 vs. E18 -0.2647 **** <0.0001 -0.1089 ns
0.2642 -0.00262 ns 0.7446 -0.00158 ns 0.9979
E14 vs. P1 -0.3697 **** <00001 -0.4495 ****
<00001 -0.00192 ns 0.9787 -0.0051 * 0.0162
E14 vs. P7 -0.5155 **** < 0.0001 -0.7871 **** <
0.0001 0.004596 * 0.0424 -0.00472 * 0.0335
E14 vs. P21 -0.4578 **** < 0.0001 -
0.7829 **** < 0.0001 0.009475 **** < 0.0001 -0.00075 ns >
0.9999
E14 vs. P28 -0.3498 **** <0.0001 -
0.4899 **** <0.0001 0.009832 **** <0.0001 1.57E-05 ns
> 0.9999
E14 vs. Null 0.5253 **** <0.0001 0.00697 ns >
0.9999 0.01573 **** <0.0001 0.004131 **** <0.0001
E18 vs. P1 -0.105 ns 0.3166 -0.3407 **'""`
<0.0001 0.000703 ns >0.9999 -0.00353 * 0.0347
E18 vs. P7 -0.2508 **** < 0.0001 -0.6782 ****
< 0.0001 0.007218 *** 0.0002 -0.00315 * 0.0254
E18 vs. P21 -0.1931 ** 0.0016 -0.6741 ****
<0.0001 0.0121 **** <0.0001 0.000828 ns > 0.9999
E18 vs. P28 -0.0851 ns 0.668 -0.3811 **** <0.0001
0.01245 **** <0.0001 0.001591 ns 0.9976
1.)
E18 vs. NULL 0.79 **** <00001 0.1158 ns
0.1862 0.01835 **** <00001 0.005706 **** <00001
1.)
0
P1 vs. P7 -0.1458 * 0.0333 -0.3375 ****
<0.0001 0.006515 *** 0.001 0.000379 ns > 0.9999
P1 vs. P21 -0.0881 ns 0.6105 -0.3334 ****
<0.0001 0.01139 **** <0.0001 0.004354 *** 0.001 1.)
0
P1 vs. P28 0.0199 ns >0.9999 -0.0404 ns
0.9998 0.01175 **** <0.0001 0.005117 *** 0.0005
P1 vs. NULL 0.895 **** <0.0001 0.4565 ****
<0.0001 0.01765 **** <0.0001 0.009232 **** <0.0001
0
P7 vs. P21 0.0577 ns 0.9837 0.00413 ns >
0.9999 0.004878 * 0.0249 0.003975 ** 0.0086
P7 vs. P28 0.1657 ** 0.0096 0.2971 **** <
0.0001 0.005235 * 0.0125 0.004737 ** 0.0031
P7 vs. NULL 1.041 **** <0.0001 0.794 ****
<0.0001 0.01113 **** <0.0001 0.008853 **** <0.0001
P21 vs. P28 0.108 ns 0.2748 0.293 **** <0.0001
0.000357 ns > 0.9999 0.000763 ns > 0.9999
P21 vs. NULL 0.9831 **** < 0.0001 0.7899 **** <
0.0001 0.006255 *** 0.0009 0.004878 **** < 0.0001
P28 vs. NULL 0.8751 **** <0.0001 0.4969 ****
<0.0001 0.005898 *** 0.00034 0.004116 **** <0.0001
vs = versus
MD = mean difference
SIG = significance
P = P value
Bonferroni's multiple comparisons test (P<0.05 was considered to be
statistically significant) (N=3)
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The MeCP2E2 signal from the WT mouse brain was only present in the nuclear
extracts and not
cytoplasmic extracts (Fig. 11(B)), indicating that similar to MeCP2 (total)
and MeCP2E1,
MeCP2E2 is also a nuclear protein. Similar to MeCP2E1 (Figs. 17(A)-17(D)),
confocal analysis
of a single nucleus from adult mouse hippocampus showed that MeCP2E2 is also
enriched in the
DAPI-rich chromocenters (Figs. 18(A)-18(D)).
After confirming that the newly developed anti-MeCP2E2 antibody specifically
detects
endogenous MeCP2E2, MeCP2E2 expression levels during mouse brain development
were
assessed. WB analysis of nuclear extracts from selected developmental time
points indicated that
MeCP2E2 has a delayed onset of protein expression compared to MeCP2E1, with
the earliest
detection at E18 (Fig. 19). From El 8 onwards, MeCP2E2 showed a similar
expression pattern
when compared to MeCP2E1, albeit at lower levels (Fig. 19; Table 5). On the
other hand,
Mecp2e2 transcript levels increased prenatally until birth with highest levels
at P1 -P7, and
significantly declined by P28 (Fig. 10; Table 5). Comparison of
Mecp2ellMecp2e2 transcript
levels indicated significantly higher Mecp2e1 levels from E14 until birth;
however no significant
difference in transcript expression was detected between P7-P28 (Fig. 11).
Pearson's correlation
analysis showed no significant relation between Mecp2e2/MeCP2E2 transcript and
protein
(r=0.58) at these selected developmental time points (Fig. 12).
Taken together, the results disclosed herein confirm that in the adult mouse
brain,
MeCP2E2 is a nuclear protein that is enriched in the chromocenters of the
hippocampus CA1
nuclei, and that while the onset of expression of MeCP2E1 and MeCP2E2
significantly differs
prenatally during mouse brain development, their expression overlap after
birth. Additionally,
these data indicate there is no significant correlation between Mecp2/MeCP2
isoform-specific
transcript and protein expression levels during mouse brain development.
Example 8: MeCP2 protein isoforms show similar cell type-specific patterns in
brain
hippocampus of both male and female adult mice.
It is known that MeCP2 is expressed in the three main brain cell types
including neurons,
astrocytes and oligodendrocytes (Rastegar et at., 2009, MECP2 isoform-specific
vectors with
regulated expression for Rett syndrome gene therapy. PloS ONE 4:e6810; Ballas
et al., 2009,
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Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic
morphology. Nat.
Neurosci. 12:311-317). Because consistent and uniform expression patterns were
detected for
both MeCP2E1 and MeCP2E2 in the nuclei of hippocampal cells in the CA1 region
(Figs.
17(A)-17(D), 18(A)-18(D)), further assessments were made of MeCP2E1 and
MeCP2E2
-- expression in neurons, astrocytes and oligodendrocytes in this region.
In male mouse brain, IF co-labelling of MeCP2E1 with a neuronal nuclei marker
NeuN
showed that the majority of MeCP2E1-labelling was localized to NeuN + positive
cells in the
hippocampus CA1 layer (Fig. 20(A)). Similarly to MeCP2E1, MeCP2E2 was also
detected in the
majority of neuronal nuclei in the hippocampus CA1 layer of male mice (Fig.
20(B)). Confocal
-- analysis showed similar nuclear patterns for MeCP2E1 and MeCP2E2 in NeuN+
nuclei (Fig.
21(A), 21(B)). Immunofluorescence detection of MeCP2E1 and MeCP2E2 occurred
less
frequently in DAPI counterstained NeuN- cells indicating the possibility for
expression of
MeCP2 isoforms in non-neuronal cells. Therefore, IF co-labelling experiments
were performed
with anti-MeCP2E1 or anti-MeCP2E2 antibodies in combination with an astrocyte
marker
-- (GFAP; Figs. 22(A), 22(B)), or an oligodendrocyte marker (CNPase; Figs.
23(A), 23(B)).
Abundant labelling for each cell type-specific marker was detected in the CA1
of male brain.
However, co-localization of MeCP2 isoforms with glial cell markers was not
easily determined
by regular microscopy. Therefore, confocal microscopy imaging analysis was
performed, and
MeCP2E1 and MeCP2E2 signals were detected in both GFAP+ astrocytes (Figs.
24(A), 24(B))
-- and CNPase+ oligodendrocytes (Figs. 25(A), 25(B)) in the hippocampus CA1
layer of adult male
brain.
Immunofluorescence labelling similar to that of male for MeCP2E1 and MeCP2E2
was
detected in the hippocampus CA1 layer of female mouse brain. IF co-labelling
of MeCP2E1 with
a neuronal nuclei marker NeuN also showed the majority of MeCP2E1 and MeCP2E2
signals to
-- be localized to NeuN + positive cells in the hippocampus CA1 layer (Figs.
26(A), 26(B)).
Confocal analysis showed similar nuclear patterns for MeCP2E1 and MeCP2E2 in
NeuN+ nuclei
(Figs. 27(A), 27(B)). Also detected was abundant labelling for each cell type-
specific marker in
the CA1 of female brain (Figs. 28(A), 28(B), 29(A), 29(B)). Confocal
microscopy imaging
analysis was performed and MeCP2E1 and MeCP2E2 signals were detected in GFAP+
astrocytes
-- (Figs. 30(A), 30(B)) and CNPase+ oligodendrocytes (Figs.31(A), 31(B)) in
the hippocampus
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V86625CA 32
CA1 layer of adult female brain. Also compared were distributions of MeCP2
isoforms in the
CA2, CA3 and dentate gyrus regions (DG) of male hippocampus (Figs. 32(A-A),
32(A-A1).
Higher magnification of CA2, CA3 and DG in mouse hippocampus sections showed
no obvious
differences for MeCP2E1 and MeCP2E2 labelling in these regions (Figs. 32(A-
B,C,D), 32(A1-
B1,C1,D1)) similar to what was observed in the CA1 region. Nuclear labelling
was evident in
other hippocampus layers surrounding the pyramidal and dentate regions as seen
in the low
magnification tiled images (Figs. 32(A), 32(B)).
It is known that Mecp2e1 and Mecp2e2 transcripts are differentially
distributed
throughout different mouse brain regions (Dragich et al., 2007, Differential
distribution of the
MeCP2 splice variants in the postnatal mouse brain. J. Comp. Neurol. 501:526-
542)., and that
the pattern of distribution of MeCP2E1 in the adult mouse brain is similar to
the distribution
pattern total MeCP2 (Zachariah et al., 2012). However, comparative analysis of
MeCP2E1 and
MeCP2E2 endogenous protein expression and localization in different brain
regions has not been
reported to date. IHC experiments in different regions of the adult mouse
brain, i.e. the olfactory
bulb, striatum, cortex, hippocampus, thalamus, brain stem and cerebellum were
performed to
assess the spatial expression of the two MeCP2 isoforms. As was also observed
in the
hippocampus, labelling for both MeCP2 isoforms in the other brain regions was
abundant with
no obvious differences in staining patterns in the majority of studied regions
(Figs. 33(A), 33(B),
34(A), 34(B)). The most intense signals detected in the olfactory bulb by anti-
MeCP2 isoform-
specific antibodies were in the mitral cell layer, presumably in mitral cells
(Figs. 33(A-A), 33(B-
Al). Similar staining intensities were also observed in the nuclei localized
within the inner and
outer plexiform layers. Lower levels of staining intensities were observed for
both MeCP2
isoforms in the granule cell layer. Interestingly, while MeCP2E1 and MeCP2E2
labelling was
observed in the same juxtaglomerular nuclei of the olfactory bulb, some nuclei
were devoid of
labelling for both isoforms.
Similar IF labelling patterns of MeCP2 isoforms were observed in
dorsal/ventral and
medial regions of the striatum (Figs. 33(A-B), 33(B-B1)). IF labelling of
MeCP2E1 and
MeCP2E2 was detected in all layers of rostral to caudal cerebral cortex as
shown in layers 5-6
(Figs. 33(A-C), 33(B-C1)). Likewise, positive labelling for both MeCP2
isoforms was observed
throughout the thalamus of mouse brain, including medial areas (Figs. 34(A-A),
34(B-A)) and
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the dorsal region underlying the ventral hippocampus (Figs. 34(A-A), 34(B-
A1)), as well as
throughout regions of the brain stem, including the medial vestibular nucleus
(Figs. 34(A-B),
34(B-B1)).
Interestingly, distribution of MeCP2E1 was different from the distribution of
MeCP2E2
in the cerebellum (Figs.34(A-C), 34(B-C1)), where under low magnification;
detection of
MeCP2E2 was the greater of the two isoforms in the granule cell layer of the
cerebellum. To
further confirm the differential levels of MeCP2E1 and MeCP2E2 signals, IHC
double labelling
was performed for both MeCP2E1 using rabbit polyclonal anti-MeCP2E1 antibodies
and anti-
MeCP2E2 antibodies in sections of male mouse cerebellum. Confocal microscopy
of double IF-
labelling with rabbit anti-MeCP2E1 and chicken anti-MeCP2E2 antibodies in
mouse cerebellum
supported the single-labelling data and confirmed differential detection
levels of MeCP2E1 and
MeCP2E2 in the granule cell layer of mouse cerebellum (Figs. 35(A)-35(D)).
Using confocal
microscopy, it was observed that in the cerebellum sub-regions of molecular
layer, Purkinje cell
and granule cell layer, MeCP2E1 and MeCP2E2 signals were co-localized with
each other at the
chromocenters (Figs. 36(A), 36(B), 36(C)).
Taken together, these data demonstrate an overall similar cell type-specific
distribution of
MeCP2 isoforms between CA1 region of mouse male and female brain. Although
MeCP2E1 and
MeCP2E2 signals are mostly identical throughout brain regions, differential
abundance of
MeCP2E1 and MeCP2E2 exists at least in the granule cell layer of the
cerebellum as seen in
male brain. Furthermore, both MeCP2 isoforms were detected in all three neural
cell types
examined in the present study.
Example 9: Mecp2/MeCP2 isoforms show differential abundance in adult murine
brain
regions
Since highly similar distribution and localization of MeCP2 isoforms were
observed by
IHC within all the brain regions except for the cerebellum, the next step was
to quantify the
abundance of MeCP2 isoforms in different brain regions by WB. Nuclear extracts
were for these
experiments because the previous WB, IHC and IF studies disclosed herein
showed the nuclear
localization of both MeCP2 isoforms. Expression analysis of MeCP2E1 protein
levels showed
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uniform expression levels across different brain regions that were analyzed
(Fig. 37; Table 5).
Similar expression profile was seen with Mecp2e1 transcripts in all the
studied brain regions
(Fig. 38; Table 5). Pearson's correlation analysis revealed a statistically
significant correlation
between Mecp2e//MeCP2E1 transcripts and protein (r---0.91, P<0.01) (Fig. 39).
In contrast to the
results obtained with MeCP2E1, MeCP2E2 showed a differential expression
pattern across
different brain regions with significantly higher expression in the olfactory
bulb and the
cerebellum compared to other regions (Fig. 40; Table 5). Brain stem showed the
lowest
expression of MeCP2E2 compared to other examined regions. Mecp2e2 transcript
levels were
also differentially expressed in different brain regions with significant
differences between the
cortex and thalamus, and cortex and brain stem (Fig. 40; Table 5).
Correlation analysis between MeCP2E2 protein and Mecp2e2 transcript levels
revealed a
statistically significant correlation between Mecp2e2/MeCP2E2
P<0.05). As positive
and negative controls for the aforementioned analysis, Whole Mecp2 WT and null
adult brains
(adult mice at 6 weeks of age) were used as the positive and negative controls
respectively for
analysis of Mecp2/MeCP2 isoform-specific expression. As expected, the
expression levels of
MeCP2E1 were significantly higher (2.8-fold) than that of MeCP2E2 in the WT
whole brain,
whereas neither isoform was detected in the nuclear extracts of null mouse
brain (Fig. 41(A)).
Similarly, higher Mecp2e1 transcript levels were detected in the WT brain (2.6-
fold), relative to
lower Mecp2e2 levels, while no transcripts were detected in the null brain
(Fig. 41(B)). These
observations further confirm that MeCP2E1 is the major isoform in the adult
mouse brain.
Taken together, these results demonstrate that Mecp2/MeCP2 isoforms are
different with
respect to their distribution and expression levels in the adult mouse brain
regions.
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Table 5:
Differences in expression of Mecp2/MeCP2 isoforms in different regions
of the brain
MeCP2E1 I MeCP2E2
Mecp2e1 Mecp2e2
REGION MD SIG P MD SIG P MD
SIG P MD SIG P
WB vs. NULL 0.9703 **** <0.0001 0,3291 ****
<0.0001 0.00785 **** <0.0001 0.002907 * 0.0422
WB vs. OB -0.07358 ns 0.997 -0.5935 ****
<0.0001 0.001652 ns 0.8598 -0.00198 ns 0.5525
WB vs. STR -0.08335 ns 0.9804 -0.3884 ****
<0.0001 -0.000085 ns > 0.9999 -0.0022 ns 0.3449
WB vs. CTX -0,04608 ns > 0.9999 -0.1679 ns 0.0638
0.000652 ns > 0.9999 -0.00397 *** 0.001
WB vs. HIPPO -0.03852 ns >0.9999 -0.3517 ****
<0.0001 0.001999 ns 0.5294 -0.00112 ns 0.9993
WB vs. THAL -0.05912 ns > 0.9999 -0.0257 ns >
0.9999 -0.00083 ns > 0.9999 -0.00084 ns >0.9999
WB vs. BS -0.08505 ns 0.9745 0.1816 * 0.0302
0.001533 ns 0.9312 0.00059 ns > 0.9999
WB vs. CERE -0.07208 ns 0.9979 -0.6069 ****
<0.0001 0.001866 ns 0.6671 -0.00212 ns 0.4079
OB vs. STR -0.00977 ns > 0.9999 0.2051 ** 0.0078
-0.001738 ns 0.7902 -0.00022 ns > 0.9999
OB vs. CTX 0.0275 ns > 0.9999 0.4256 ****
<0.0001 -0.001 ns > 0.9999 -0.00199 ns 0.5356
OB vs. HIPPO 0.03507 ns > 0.9999 0.2418 *** 0.0009
0.00034 ns > 0.9999 0.000854 ns > 0.9999 0
4)
OB vs. THAL 0.01447 ns > 0.9999 0.5678 ****
<0.0001 -0.002482 ns 0.1596 0.001139 ns 0.9991
0
OB vs. BS -0.01147 ns > 0.9999 0.7751 ****
<0.0001 -0.00011 ns > 0.9999 0.002566 ns 0.1244 N.)
co
OB vs. CERE 0.0015 ns > 0.9999 -0.0134 ns >
0.9999 0.000213 ns > 0.9999 -0.00015 ns >0.9999
w
N.)
STR vs. CTX 0.03727 ns > 0.9999 0.2205 **
0.0031 0.000737 ns > 0.9999 -0.00177 ns 0.7573 0
ko
STR vs. HIPPO 0.04483 ns > 0.9999 0.0367 ns > 0.9999
0.002085 ns 0.4441 0.001073 ns 0.9997 ....I
STR vs. THAL 0.02423 ns > 0.9999 0.3627 ****
<0.0001 -0.000744 ns > 0.9999 0.001358 ns 0.9843 N.)
0
STR vs. BS -0.0017 ns > 0.9999 0.57 **** <0.0001
0.001619 ns 0.8827 0.002785 ns 0.0628
w
'
STR vs. CERE 0.01127 ns >0.9999 -0.2185 ** 0,0035
0.001951 ns 0.5783 7.19E-05 ns >0.9999
CTX vs. HIPPO 0.00756 ns >0.9999 -0.1838 * 0.0267
0.001347 ns 0.986 0.002847 ns 0.0514 0
1
CTX vs. THAL -0.01303 ns >0.9999 0.1422 ns 0.2286
-0.001482 ns 0.9526 0.003132 * 0.0197 w
1-,
CTX vs. BS -0.03897 ns > 0.9999 0.3495 ****
<0.0001 0.000881 ns > 0.9999 0.004559 *** 0.0008
CTX vs. CERE -0.026 ns > 0.9999 -0.439 ****
<0.0001 0.001214 ns 0.9972 0.001846 ns 0.6871
HIPPO vs. THAL -0.0206 ns > 0.9999 0.326 ****
<0.0001 -0.002829 ns 0.0544 0.000285 ns > 0.9999
HIPPO vs. BS -0.04653 ns > 0.9999 0.5333 ****
<0.0001 -0.000465 ns > 0.9999 0.001713 ns 0.8122
HIPPO vs. CERE -0.03357 ns >0.9999 -0.2552 ***
0.0004 -0.000133 ns > 0.9999 -0.001 ns > 0.9999
THAL vs. BS -0.02593 ns > 0.9999 0.2073 ** 0.0069
0.002363 ns 0.2233 0.001427 ns 0.9698
THAL vs. CERE -0.01297 ns >0.9999 -0.5812 ****
<0.0001 0.002696 ns 0.0835 -0.00129 ns 0.9929
BS vs. CERE 0.01297 ns > 0.9999 -0.7885 ****
<0.0001 0.000332 ns > 0.9999 -0.00271 ns 0.079
vs = versus MD = mean differences
SIG = significance P = P value
Bonferroni's multiple comparison's test (P<0.05 was considered to be
statistically significiant)
N = 3
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SUMMARY:
The studies and related data disclosed herein report a number of comparative
analyses of
Mecp2/MeCP2 isoform-specific expression during mouse brain development and in
different
brain regions of young adult mice at 6 weeks of age. MeCP2 isoforms show
significant increase
at the protein levels during the early postnatal mouse development (P1 -P7).
This time period has
been reported to coincide with the onset of neuronal maturation and
synaptogenesis in several
brain regions (Jung et al., 2003, The expression of methyl CpG binding factor
MeCP2 correlates
with cellular differentiation in the developing rat brain and in cultured
cells. J. Neurobiol.
55:86-96; Shahbazian et al., 2002, Insight into Rett syndrome: MeCP2 levels
display tissue- and
cell-spec(ic differences and correlate with neuronal maturation. Hum. Mol.
Gen. 11:115-124).
Thus, the possibility of both MeCP2 isoforms contributing to these processes
cannot be ruled
out. It is noteworthy that the later onset of MeCP2E2 protein expression, as
compared to the
onset of MeCP2E1 expression, might reflect the developmental pattern of a
regional, neuronal or
cellular subtype in the brain. This is important in light of the knowledge
that MeCP2 dysfunction
affects different regions of the brain to different extents, suggesting that
MeCP2E2 may
contribute to normal function of specific types of neurons or other brain cell
types. Moreover, the
absence of a significant correlation between Mecp2/MeCP2 transcript and
protein expression of
the two isoforms during brain development suggest possible post-
transcriptional regulation of
Mecp2 isoforms during development.
Recent studies have shown that MeCP2 expression levels are critical to
maintain, and
higher or lower levels than normal in different brain regions correlate with
specific behavioural
impairments (Wither et al., 2013, Regional MeCP2 expression levels in the
female MeCP2-
deficient mouse brain correlate with specific behavioral impairments. Exp.
Neurol. 239:9-59).
Moreover, deletion of Mecp2 in neurons in specific brain-regions is associated
with RTT
phenotypes (Adachi et al., 2009, MeCP2-mediated transcription repression in
the basolateral
amygdala may underlie heightened anxiety in a mouse model of Rett syndrome. J.
Neurosci.
29:4218-4227; Wu et al., 2009, MeCP2 function in the basolateral amygdala in
Rett syndrome.
J. Neurosci. 29:9941-9942; Gemelli et al., 2006, Postnatal loss of methyl-CpG
binding protein 2
in the forebrain is sufficient to mediate behavioral aspects of Rett syndrome
in mice. Biol. Psych.
VAN_LAW\ 1320991\4
CA 02832097 2013-10-31
V86625CA 37
59:468-476). This reinforces the requirement for precise levels of MeCP2
expression for normal
brain function, as both higher or lower levels of MeCP2 expression (compared
to normal) results
in neurological dysfunction.
The WB data disclosed herein show that both MeCP2 isoforms are present in the
adult
mouse brain, with MeCP2E1 showing more uniform expression levels in different
brain regions
compared to MeCP2E2 and confirm that, similar to MeCP2, MeCP2E1 is also a
nuclear protein.
Therefore, the influence of cellular size and also nuclear to cytoplasmic
ratio which might not be
the same in different cell types or regions of the brain, was eliminated by
the use of nuclear
extracts. Although the significance of the uniform nuclear distribution of
MeCP2E1 in brain
remains to be elucidated, it may be hypothesized to relate to a specialized
MeCP2E1 nuclei
structural function in different brain regions. The data disclosed herein
regarding the MeCP2E2
expression patterns suggest that it may contribute to MeCP2 brain region-
specific functions or
target genes.
Interestingly, semi-quantitative WB analysis showed similar overall protein
expression
levels of MeCP2 isoforms in the adult mouse cerebellum, but further IHC
characterization
revealed differential localization of MeCP2 isoforms in sub regions of the
cerebellum. The data
disclosed herein indicate that MeCP2E2 is the more abundant isoform in the
granule cell layer of
the cerebellum compared to MeCP2E1. Thus, these data confirm that at the
protein levels,
MeCP2 isoforms are also differentially localized in this part of the brain.
The detected
differentiation distribution of MeCP2 isoforms in the cerebellum might be
helpful to understand
the contribution of individual MeCP2 isoforms in cerebellar functions and gene
expression,
The anti-MeCP2E2 antibody produced with an antigen comprising a peptide with a
sequence of twelve amino acids (e.g., SEQ ID NO:10) or alternatively with an
antigen
comprising a peptide with a sequence of eleven amino acids (e.g., SEQ ID
NO:11) from the N-
terminus of MeCP2E2 as disclosed herein, provides novel avenues for
understanding brain
region and/or cell type-specific expression of MeCP2E2 that will provide vital
insights for the
efficient design of future gene therapy approaches. The data further indicate
that in adult mice
brain, MeCP2E2 signals overlap with DAPI-rich heterochromatin regions in the
nucleus. The
data disclosed herein show that both MeCP2 isoforms are expressed in three
major brain cell
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V86625CA 38
types; neurons, astrocytes and oligodendrocytes of both male and female adult
mouse.
Furthermore, it is noted that transcript and protein expression of Mecp2/MeCP2
isoforms
significantly correlate with each other in different regions of the adult
brain, while such
correlations do not exist in the developing brain. The generated and validated
anti-MeCP2E2
antibody will have important applications for future diagnosis, prognosis or
understanding the
mechanism of MeCP2-associated diseases.
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