U.S. patent application number 17/580689 was filed with the patent office on 2022-05-05 for inhibition of let7i as a means to enhance the protective effect of progesterone against stroke.
The applicant listed for this patent is UNIVERSITY OF NORTH TEXAS HEALTH SCIENCE CENTER. Invention is credited to TRINH NGUYEN, MEHARVAN SINGH, CHANG SU.
Application Number | 20220135975 17/580689 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220135975 |
Kind Code |
A1 |
SINGH; MEHARVAN ; et
al. |
May 5, 2022 |
INHIBITION OF LET7I AS A MEANS TO ENHANCE THE PROTECTIVE EFFECT OF
PROGESTERONE AGAINST STROKE
Abstract
The subject invention provides methods of treating neurological
disease or disorder, such as brain injuries, such as stroke,
traumatic brain injury (TBI), or other ischemic events that cause
brain injury by inhibiting or down-regulating Let-7i activity or
function. The disclosed methods may have the potential to extend
the "window of opportunity" for treatment of such injuries and
enhance the effectiveness of existing therapeutics.
Inventors: |
SINGH; MEHARVAN; (BENBROOK,
TX) ; SU; CHANG; (LUCAS, TX) ; NGUYEN;
TRINH; (HUMBLE, TX) |
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Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF NORTH TEXAS HEALTH SCIENCE CENTER |
Fort Worth |
TX |
US |
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Appl. No.: |
17/580689 |
Filed: |
January 21, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16639139 |
Feb 14, 2020 |
11230711 |
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PCT/US2018/046456 |
Aug 13, 2018 |
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17580689 |
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62544994 |
Aug 14, 2017 |
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International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713; A61K 38/18 20060101
A61K038/18 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under
AG027956 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method of treating a neurological disease or disorder
comprising administering one or more antagonist of Let-7i or a
composition comprising said Let-7i antagonist to a subject having a
neurological disease or disorder.
2. The method according to claim 1, wherein the neurological
disease or disorder is selected from: severance of nerves or nerve
damage, severance of cerebrospinal nerve cord (CNS), damage to
brain or nerve cells, traumatic brain injury, spinal cord injury,
hypoxia, ischemia, brain injury, diabetic neuropathy, peripheral
neuropathy, aging, neurodegenerative disease, or peripheral nerve
injury.
3. The method according to claim 2, wherein said neurological
disease or disorder is aging of the nervous system or an
age-associated neurodegenerative disease.
4. The method according to claim 3, wherein said aging of the
nervous system comprises one or more of the following: (a) changes
in memory, (b) alterations of language function, (c)
visual-perceptual changes, (d) slowing of reaction time, and/or (f)
decreased balance and coordination.
5. The method according to claim 1, wherein said antagonist of
Let-7i is an antisense oligonucleotide, siRNA, shRNA, or
interfering RNA that down-regulates Let-7i activity or
function.
6. The method according to claim 1, said method further comprising
the administration of progesterone or a composition thereof to said
subject.
7. The method according to claim 1, said method further comprising
the administration of BDNF or a composition thereof to said
subject.
8. The method according to claim 6, wherein said antagonist of
Let-7i or composition thereof and/or said progesterone or a
composition thereof is/are administered separately, concurrently,
or as a single composition.
9. The method according to claim 7, wherein said antagonist of
Let-7i or a composition thereof and/or said BDNF or composition
thereof is/are administered separately, concurrently, or as a
single composition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 16/639,139, filed Feb. 14, 2020, now U.S. Pat. No. 11,230,711,
which is the U.S. National Stage Application of International
Patent Application No. PCT/US2018/046456, filed on Aug. 13, 2018,
which claims the benefit of U.S. Provisional Application Ser. No.
62/544,994, filed Aug. 14, 2017, the disclosure disclosures of
which is are hereby incorporated by reference in its their
entirety, including all figures, tables and amino acid or nucleic
acid sequences.
[0003] The Sequence Listing for this application is labeled
"Seq-List.txt" which was created on Aug. 13, 2018 and is 1 KB. The
entire content of the sequence listing is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0004] Stroke is the fourth leading cause of death and a major
cause of disability in the US [14], costing approximately $34
Billion annually (according to the Center for Disease Control). The
risk of ischemic stroke dramatically increases with age. Of note,
the incidence of ischemic stroke is relatively rare among
pre-menopausal women [1]. Although middle aged women have a lower
risk of stroke than men, stroke becomes more prevalent in
post-menopausal women compared to men of the same age [1]. With
increasing age, circulating gonadal hormone levels decline in both
males and females, however, such age-associated decreases are much
more dramatic in women, and is a function of the menopause. While
much attention has been placed on the loss of estrogen following
the menopause, it is worth noting that the levels of P4 also
decline precipitously. As such, the increased risk for stroke in
postmenopausal women may be due to a decline in not just estrogen
levels, but that of P4 as well. In fact, growing literature has
suggested that P4 is protective, and is (neuro)protective in a
variety of experimental models of stroke [2-4]. However, the
underlying mechanisms for P4's protective effects remain unclear.
It is this incomplete information that limits our understanding of
why P4's beneficial effects were equivocal in the latest Phase III
clinical trial of P4 efficacy in treating traumatic brain injury,
despite numerous other studies (both preclinical and clinical) that
demonstrate its positive efficacy. We suggest that a better
understanding of the factors that influence the expression of key
mediators (e.g., receptors) of P4 is critical to advancing the
development of effective P4-based neuroprotectants.
[0005] It is also worth pointing out that the literature associated
with P4's protective effects has focused on a direct effect of P4
on neurons. The notion that glia may be an equally important target
underlying P4's protective effects on the brain has only been
studied minimally. Indeed, astrocytes have been considered as an
important component in the post-ischemic recovery, as these cells
are critical for regeneration and remodeling of neural circuits
following stroke [9].
[0006] A known mediator of P4's neuroprotective action is
brain-derived neurotrophic factor (BDNF) [15]. A deficit in BDNF
has been linked to stroke pathophysiology [16, 17]. In the central
nervous system (CNS), BDNF also has an established role in
promoting neuronal differentiation, survival, synaptic plasticity
[6-8] and synaptogenesis [18-20]. Synaptogenesis occurring in the
penumbra is known to strongly contribute to enhanced functional
recovery from stroke [21-24]. Based on these observations, it is
plausible that the P4/BDNF signaling-mediated enhanced
synaptogenesis and neuroprotection may contribute to P4's
protective effects during post stroke brain repair. We recently
reported that P4 elicits the release of BDNF from primary
astrocytes via a putative membrane progesterone receptor consisting
of progesterone-receptor-membrane-component-1 (Pgrmc1) [10]. Our
results suggest that conditioned medium derived from P4-treated
astrocytes, when applied to primary cortical neurons, increases the
expression of synaptic markers in these neural cells and enhances
their survival against oxidative stress. Our data support the model
whereby P4 elicits its (neuro)protective effects through a
mechanism that involves Pgrmc1-dependent BDNF release from
glia.
[0007] Currently, knowledge regarding the regulation of Pgrmc1 in
brain and the consequence of such regulation is limited. Studies
from our lab demonstrate that the miRNA, let-7i, negatively
regulates expression of both Pgrmc1 and BDNF in glia, leading to
suppression of P4-induced BDNF release from glia and attenuation of
P4's beneficial effects on neuroprotection and synaptogenesis in
the ischemic brain. Furthermore, the increased expression of let-7i
with stroke may explain why post stroke therapy may not be so
effective. As there remains a significant need for treatments of
brain injuries, such as stroke and traumatic brain injury (TBI),
down-regulation of let-7i may have the potential to extend the
"window of opportunity" for treatment of such injuries.
BRIEF SUMMARY OF THE INVENTION
[0008] The neuroprotective effects of P4 have been reported since
1996 [25], however, knowledge of what governs the protective
effects of P4 is still largely lacking. Further, a heavy emphasis
has been placed on P4's "genomic" mechanism(s) of action, elicited
via the classical progesterone receptor (PR), and that too, focused
on neurons. However, evidence from emerging literature as well as
from our own recent studies have highlighted the critical role of
glia, both as a site of local P4 synthesis and as a mediator of
P4's pro-survival functions in CNS [26-28]. The subject invention
provides methods of treating neurological disease or disorder, such
as brain injuries, such as stroke, traumatic brain injury (TBI), or
other ischemic events that cause brain injury by inhibiting or
down-regulating Let-7i activity or function. The disclosed methods
may have the potential to extend the "window of opportunity" for
treatment of such injuries and enhance the efficacy of existing
treatments for such injuries.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication, with color drawing(s), will be provided by the Office
upon request and payment of the necessary fee.
[0010] FIGS. 1A-1B: Conditioned medium derived from P4 (10
nM)-treated astrocytes (P4-ACM, 18 hrs) and BDNF (50 ng/ml, 18 hrs)
increased expression of Synaptophysin and Gap-43 in primary
cortical neurons. FIG. 1A represents immune-staining of Gap43
(green) and Synaptophysin (red). FIG. 1B represents qRT-PCR
analysis of Gap43 (left) and Synaptophysin (right) mRNA levels.
Data are presented as a percentage of control (non-treated group)
(*:p.ltoreq.0.05, **:p.ltoreq.0.01).
[0011] FIGS. 2A-2B: Over-Expression of Let-7i down-regulated Pgrmc1
and BDNF mRNA in primary astrocytes. Cells were transfected with
let-7i or let-7f mimic or a negative control 48 hrs prior to RNA
isolation for qRT-PCR. Quantification of Pgrmc1 and BDNF mRNA
levels was normalized to GAPDH (FIG. 2A). Quantification of let-7
levels was normalized to U6 snRNA (FIG. 2B). Data are presented as
a percentage of control (***:p.ltoreq.0.001, n.s:
non-significant).
[0012] FIG. 3: Let-7i attenuates P4-induced BDNF release from
primary cortical astrocytes, measured by in-situ BDNF ELISA
(***:p.ltoreq.0.001, n.s: non-significant).
[0013] FIG. 4: Age-related decrease of Pgrmc1 expression correlated
with decrease of BDNF in mouse hippocampus. mRNA levels were
measured by qRT-PCR (young: 6-mo old; middle-aged: 12-mo old; old:
24-mo old. Data presented as a percentage of young group
(*:p.ltoreq.0.05, **:p<0.01, n.s: non-significant).
[0014] FIGS. 5A-5B: A decrease of Pgrmc1 expression correlates with
an increase of Let-7i in cortex at day 7 post-stroke. Total RNA was
measured by qRT-PCR. Pgrmc1 mRNA was normalized to GAPDH (FIG. 5A).
Let-7i expression was normalized to U6 snRNA (FIG. 5B)
(*:p.ltoreq.0.05).
[0015] FIG. 6: Ischemic injury is greatly reduced in animals
receiving P4 and the Let-7i inhibitor.
[0016] FIG. 7: Functional recovery (grip strength) is greatly
enhanced in animals receiving ICV injections of the Let-7i
inhibitor.
[0017] FIGS. 8A-8B: Oxygen-Glucose Deprivation (OGD) results in an
increase in let-7i expression and suppresses progesterone
(P4)-induced BDNF release from primary cortical astrocytes. Primary
cortical astrocytes were exposed to one-hour of OGD. Immediately
after re-instatement of normal oxygen and glucose concentrations,
these cells were either mock transfected (control) or transfected
with the let-7i antagomir. 12 hours later, expression of let-7i was
evaluated (FIG. 8A) (n=4). n.s: not significant, ****P<0.0001
compared to mock transfected control (mock). (FIG. 8B) Quantitation
of BDNF release measured by BDNF in situ ELISA (n=4). n.s: not
significant compared to DMSO group. Data are presented as
mean.+-.SEM.
[0018] FIG. 9: let-7i prevents progesterone (P4)-induced
neuroprotection against oxygen-glucose-deprivation (OGD).
Conditioned-media derived from hormone or control-treated
astrocytes were applied to primary cortical neurons (DIV 14) after
one-hour exposure to OGD. BDNF (50 ng/ml) was directly added to
neurons after OGD to serve as positive control. Neuronal viability
was measured by CellTiter-Glo viability assay (n=5). n.s: not
significant, ***P<0.001 and **P<0.01 compared to normoxia.
Data are presented as the mean.+-.SEM.
[0019] FIGS. 10A-10C. let-7i inhibits progesterone (P4) induces
synaptophysin (SYP) expression in primary cortical neurons. (FIG.
10A) Representative confocal images of primary cortical neurons
(DIV 14) immunostained with synaptophysin (SYP, green) and DAPI
(blue). (60x, Scale bars=30.mu.m). (FIG. 10B) Quantification of
average number of SYP puncta per neuron (n=3). n.s: not
significant, ***P<0.001 compared to mock transfected +DMSO
group. (FIG. 10C) Representative immunoblots probed for SYP and
quantification graph of relative SYP protein ratio to Gapdh (n=4).
n.s: not significant, ****P<0.0001, ***P<0.001 compared to
mock transfected+DMSO group. Data are presented as the
mean.+-.SEM.
[0020] FIGS. 11A-11E: Combined treatment with progesterone (P4) and
the let-7i inhibitor reversed ischemia-induced suppression of
Pgrmc1 and BDNF expressions in the penumbra. (FIG. 11A)
Representative immunoblots probed for Pgrmc1, pro- and mature-BDNF.
(FIG. 11B) Quantitation graph of relative Pgrmc1 protein ratio to
Gapdh (n=4-5 per group). (FIG. 11C) Quantitation graph of relative
pro-BDNF protein ratio to Gapdh (n=4-5 per group). (FIG. 11D)
Quantitation graph of relative mature BDNF protein ratio to Gapdh
(n=4-5 per group). (FIG. 11E) Quantitation graph of relative let-7i
expression in ischemic brain (n=4-5 per group). n.s: not
significant, ** P<0.01 and *P<0.05 compared to sham, and
#P<0.05 compared to P4+ scrambled. Data are presented as the
mean.+-.SEM.
[0021] FIGS. 12A-12B: Co-administration of let-7i antagomir
(anti-let-7i) and progesterone (P4) reduces ischemic injury. (FIG.
12A) Representative images of serial coronal brain sections stained
with triphenyltetrazolium chloride (TTC). (FIG. 12B) Quantification
of infarct sizes of TTC-stained images (n=4 per group). n.s: not
significant, ***P<0.001 and **P<0.01 compared to
cholesterol+scrambled group. Data are presented as the mean
.+-.SEM.
[0022] FIG. 13: Co-administration of let-7i antagomir (anti-let-7i)
and progesterone (P4) enhances recovery of motor function/grip
strength following stroke. Results of wire suspension test at day
3, 7 and 14 post stroke (n=15-20 per group). n.s: not significant,
***P<0.001 and ** P<0.01 compared to sham, ###P<0.001,
##P<0.01 compared to P4+ scrambled, and $$P<0.01 compared to
cholesterol+scrambled. Data are presented as the mean .+-.SEM.
[0023] FIGS. 14A-14D: Inhibition of let-7i enhances progesterone
(P4)'s effect on the expression of synaptophysin in the penumbra.
(FIG. 14A) Representative confocal images of penumbra region
staining for synaptophysin (SYP, red) and DAPI (blue). (60.times.,
Scale bars=30 .mu.m). (FIG. 14B) Quantification of average relative
SYP puncta presents in each field (n=3 per group). n.s: not
significant and ***P<0.001 compared to sham, ###P<0.001 and
##P<0.01 compared to P4+ scrambled. (FIG. 14C) Representative
immunoblots probed for SYP protein. (FIG. 14D) Quantification graph
of Syp signal, expressed as a ratio to Gapdh (n=4-5 per group).
n.s: not significant, ***P<0.001 and **P<0.01 compared to
sham, ##P<0.01 and #P<0.05 compared to P4+ scrambled.
DETAILED DISCLOSURE OF THE INVENTION
[0024] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. As used herein, the singular forms "a", "an" and
"the" are intended to include the plural forms as well, unless the
context clearly indicates otherwise. Furthermore, to the extent
that the terms "including", "includes", "having", "has", "with", or
variants thereof are used in either the detailed description and/or
the claims, such terms are intended to be inclusive in a manner
similar to the term "comprising."
[0025] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, preferably up
to 10%, more preferably up to 5%, and more preferably still up to
1% of a given value.
[0026] As used herein, the "Let-7i" sequence comprises:
mmu-let-7i-5p MIMAT0000122 5' UGAGGUAGUAGUUUGUGCUGUU 3' (SEQ ID NO:
1). The let-7i-5p sequence is identical for both the human
(MIMAT0000415) and murine (MIMAT0000122) miRNA. The full length
human and murine let-7i sequences, including the stem loop can be
obtained at the miRBase database (mirbase.org) as accession numbers
MI0000434 (human, SEQ ID NO: 2) and MI0000138 (murine, SEQ ID NO:
3).
[0027] By "antisense oligonucleotides" or "antisense compound" is
meant an RNA or DNA molecule that binds to another RNA or DNA
(target RNA, DNA). For example, if it is an RNA oligonucleotide it
binds to another RNA target by means of RNA-RNA interactions and
alters the activity of the target RNA. An antisense oligonucleotide
can upregulate or downregulate expression and/or function of a
particular polynucleotide. The definition is meant to include any
foreign RNA or DNA molecule which is useful from a therapeutic,
diagnostic, or other viewpoint. Such molecules include, for
example, antisense RNA or DNA molecules, interference RNA (RNAi),
short hairpin RNA (shRNA), and silencing RNA (siRNA). Inhibitory
oligonucleotides and vectors for delivering inhibitory
oligonucleotides for Let-7i are commercially available from vendors
such as Vigene Biosciences, Inc. (Rockville, Md. 20850 USA),
OriGene Technologies, Inc. (Rockville, Md. 20850 USA), and Santa
Cruz Biotechnology, Inc. (Dallas, Tex. 75220 USA).
[0028] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or mimetics thereof. The term
"oligonucleotide", also includes linear or circular oligomers of
natural and/or modified monomers or linkages, including
deoxyribonucleosides, ribonucleosides, substituted and
alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked
nucleic acids (LNA), phosphorothioate, methylphosphonate, and the
like. Oligonucleotides are capable of specifically binding to a
target polynucleotide by way of a regular pattern of
monomer-to-monomer interactions, such as Watson-Crick type of base
pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or
the like.
[0029] The oligonucleotide may be "chimeric", that is, composed of
different regions. In the context of this invention "chimeric"
compounds are oligonucleotides, which contain two or more chemical
regions, for example, DNA region(s), RNA region(s), PNA region(s)
etc. Each chemical region is made up of at least one monomer unit,
i.e., a nucleotide in the case of an oligonucleotides compound.
These oligonucleotides typically comprise at least one region
wherein the oligonucleotide is modified in order to exhibit one or
more desired properties. The desired properties of the
oligonucleotide include, but are not limited, for example, to
increased resistance to nuclease degradation, increased cellular
uptake, and/or increased binding affinity for the target nucleic
acid. Different regions of the oligonucleotide may therefore have
different properties. The chimeric oligonucleotides of the present
invention can be formed as mixed structures of two or more
oligonucleotides, modified oligonucleotides, oligonucleosides
and/or oligonucleotide analogs as described above.
[0030] The oligonucleotide can be composed of regions that can be
linked in "register", that is, when the monomers are linked
consecutively, as in native DNA, or linked via spacers. The spacers
are intended to constitute a covalent "bridge" between the regions
and have in preferred cases a length not exceeding about 100 carbon
atoms. The spacers may carry different functionalities, for
example, having positive or negative charge, carry special nucleic
acid binding properties (intercalators, groove binders, toxins,
fluorophores, etc.), being lipophilic, inducing special secondary
structures like, for example, alanine containing peptides that
induce alpha-helices.
[0031] As used herein "BDNF" and "Brain derived neurotrophic
factor" are inclusive of all family members, mutants, alleles,
fragments, species, coding and noncoding sequences, sense and
antisense polynucleotide strands, etc. As used herein, the terms
"Brain derived neurotrophic factor", "Brain-derived neurotrophic
factor" and BDNF, are considered the same in the literature and are
used interchangeably in the present application.
[0032] "Progesterone" includes all natural forms of progesterone as
well as chemically synthesized analogs of progesterone.
[0033] As used herein, the term "oligonucleotide specific for" or
"oligonucleotide which targets" refers to an oligonucleotide having
a sequence (i) capable of forming a stable complex with a portion
of the targeted gene (in this case let-7i), or (ii) capable of
forming a stable duplex with a portion of a mRNA transcript of the
targeted gene. Stability of the complexes and duplexes can be
determined by theoretical calculations and/or in vitro assays.
Exemplary assays for determining stability of hybridization
complexes and duplexes are described in the Examples below.
[0034] RNA interference "RNAi" is mediated by double stranded RNA
(dsRNA) molecules that have sequence-specific homology to their
"target" nucleic acid sequences (in this case let-7i). In certain
embodiments of the present invention, the mediators are 5-25
nucleotide "small interfering" RNA duplexes (siRNAs). The siRNAs
are derived from the processing of dsRNA by an RNase enzyme known
as Dicer. siRNA duplex products are recruited into a multi-protein
siRNA complex termed RISC (RNA Induced Silencing Complex). Without
wishing to be bound by any particular theory, a RISC is then
believed to be guided to a target nucleic acid (suitably mRNA),
where the siRNA duplex interacts in a sequence-specific way to
mediate cleavage in a catalytic fashion. Small interfering RNAs
that can be used in accordance with the present invention can be
synthesized and used according to procedures that are well known in
the art and that will be familiar to the ordinarily skilled
artisan. Small interfering RNAs for use in the methods of the
present invention suitably comprise between about 1 to about 50
nucleotides (nt). In examples of non-limiting embodiments, siRNAs
can comprise about 5 to about 40 nt, about 5 to about 30 nt, about
10 to about 30 nt, about 15 to about 25 nt, or about 20-25
nucleotides.
[0035] Selection of appropriate oligonucleotides is facilitated by
using computer programs that automatically align nucleic acid
sequences and indicate regions of identity or homology. Such
programs are used to compare nucleic acid sequences obtained, for
example, by searching databases such as GenBank or by sequencing
PCR products. Comparison of nucleic acid sequences from a range of
species allows the selection of nucleic acid sequences that display
an appropriate degree of identity between species. In the case of
genes that have not been sequenced, Southern blots are performed to
allow a determination of the degree of identity between genes in
target species and other species. By performing Southern blots at
varying degrees of stringency, as is well known in the art, it is
possible to obtain an approximate measure of identity. These
procedures allow the selection of oligonucleotides that exhibit a
high degree of complementarity to target nucleic acid sequences in
a subject to be controlled and a lower degree of complementarity to
corresponding nucleic acid sequences in other species. One skilled
in the art will realize that there is considerable latitude in
selecting appropriate regions of genes for use in the present
invention.
[0036] The term "nucleotide" covers naturally occurring nucleotides
as well as non-naturally occurring nucleotides. It should be clear
to the person skilled in the art that various nucleotides which
previously have been considered "non-naturally occurring" have
subsequently been found in nature. Thus, "nucleotides" includes not
only the known purine and pyrimidine heterocycles-containing
molecules, but also heterocyclic analogues and tautomers thereof.
Illustrative examples of other types of nucleotides are molecules
containing adenine, guanine, thymine, cytosine, uracil, purine,
xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine,
7-deazaguanine, N4,N4-ethanocytosin,
N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,
5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,
pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin,
isocytosine, isoguanin, inosine and the "non-naturally occurring"
nucleotides described in Benner et al., U.S. Pat. No. 5,432,272.
The term "nucleotide" is intended to cover every and all of these
examples as well as analogues and tautomers thereof. Especially
interesting nucleotides are those containing adenine, guanine,
thymine, cytosine, and uracil, which are considered as the
naturally occurring nucleotides in relation to therapeutic and
diagnostic application in humans. Nucleotides include the natural
2'-deoxy and 2'-hydroxyl sugars, e.g., as described in Komberg and
Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) as
well as their analogs.
[0037] "Analogs" in reference to nucleotides includes synthetic
nucleotides having modified base moieties and/or modified sugar
moieties (see e.g., described generally by Scheit, Nucleotide
Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997)
Nucl. Acid. Res., 25(22), 4429-4443, Toulme, J. J., (2001) Nature
Biotechnology 19:17-18; Manoharan M., (1999) Biochemica et
Biophysica Acta, 1489:117-139; Freier S. M., (1997) Nucleic Acid
Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery &
Development, 3: 203-213, Herdewin P., (2000) Antisense &
Nucleic Acid Drug Dev., 10:297-310); 2'-O, 3'-C-linked [3.2.0]
bicycloarabinonucleosides. Such analogs include synthetic
nucleotides designed to enhance binding properties, e.g., duplex or
triplex stability, specificity, or the like.
[0038] As used herein, "hybridization" means the pairing of
substantially complementary strands of oligomeric compounds. One
mechanism of pairing involves hydrogen bonding, which may be
Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,
between complementary nucleoside or nucleotide bases (nucleotides)
of the strands of oligomeric compounds. For example, adenine and
thymine are complementary nucleotides which pair through the
formation of hydrogen bonds. Hybridization can occur under varying
circumstances.
[0039] An antisense compound is "specifically hybridizable" when
binding of the compound to the target nucleic acid interferes with
the normal function of the target nucleic acid to cause a
modulation of function and/or activity, and there is a sufficient
degree of complementarity to avoid non-specific binding of the
antisense compound to non-target nucleic acid sequences under
conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or
therapeutic treatment, and under conditions in which assays are
performed in the case of in vitro assays.
[0040] As used herein, the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a compound of the invention will hybridize to its target
sequence, but to a minimal number of other sequences. Stringent
conditions are sequence-dependent and will be different in
different circumstances and in the context of this invention,
"stringent conditions" under which oligomeric compounds hybridize
to a target sequence are determined by the nature and composition
of the oligomeric compounds and the assays in which they are being
investigated. In general, stringent hybridization conditions
comprise low concentrations (<0.15 M) of salts with inorganic
cations such as Na+ or K+ (i.e., low ionic strength), temperature
higher than 20.degree. C.-25.degree. C. below the Tm of the
oligomeric compound:target sequence complex, and the presence of
denaturants such as formamide, dimethylformamide, dimethyl
sulfoxide, or the detergent sodium dodecyl sulfate (SDS). For
example, the hybridization rate decreases 1.1% for each 1%
formamide. An example of a high stringency hybridization condition
is 0.1. times. sodium chloride-sodium citrate buffer (SSC)/0.1%
(w/v) SDS at 60.degree. C. for 30 minutes.
[0041] "Complementary," as used herein, refers to the capacity for
precise pairing between two nucleotides on one or two oligomeric
strands. For example, if a nucleobase at a certain position of an
antisense compound is capable of hydrogen bonding with a nucleobase
at a certain position of a target nucleic acid, said target nucleic
acid being a DNA, RNA, or oligonucleotide molecule, then the
position of hydrogen bonding between the oligonucleotide and the
target nucleic acid is considered to be a complementary position.
The oligomeric compound and the further DNA, RNA, or
oligonucleotide molecule are complementary to each other when a
sufficient number of complementary positions in each molecule are
occupied by nucleotides which can hydrogen bond with each other.
Thus, "specifically hybridizable" and "complementary" are terms
which are used to indicate a sufficient degree of precise pairing
or complementarity over a sufficient number of nucleotides such
that stable and specific binding occurs between the oligomeric
compound and a target nucleic acid.
[0042] It is understood in the art that the sequence of an
oligomeric compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, an
oligonucleotide may hybridize over one or more segments such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure, mismatch or hairpin
structure). The oligomeric compounds of the present invention
comprise at least about 70%, or at least about 75%, or at least
about 80%, or at least about 85%, or at least about 90%, or at
least about 95%, or at least about 99% sequence complementarity to
a target region within the target nucleic acid sequence to which
they are targeted. For example, an antisense compound in which 18
of 20 nucleotides of the antisense compound are complementary to a
target region, and would therefore specifically hybridize, would
represent 90 percent complementarity. In this example, the
remaining non-complementary nucleotides may be clustered or
interspersed with complementary nucleotides and need not be
contiguous to each other or to complementary nucleotides. As such,
an antisense compound which is 18 nucleotides in length having 4
(four) non-complementary nucleotides which are flanked by two
regions of complete complementarity with the target nucleic acid
would have 77.8% overall complementarity with the target nucleic
acid and would thus fall within the scope of the present invention.
Percent complementarity of an antisense compound with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art. Percent homology, sequence identity or
complementarity, can be determined by, for example, the Gap program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, Madison, Wis.), using
default settings, which uses the algorithm of Smith and Waterman
(Adv. Appl. Math., 1981, Vol. 2, pp. 482-489).
[0043] As used herein, the term "Thermal Melting Point (Tm)" refers
to the temperature, under defined ionic strength, pH, and nucleic
acid concentration, at which 50% of the oligonucleotides
complementary to the target sequence hybridize to the target
sequence at equilibrium. Typically, stringent conditions will be
those in which the salt concentration is at least about 0.01 to 1.0
M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short
oligonucleotides (e.g., 10 to 50 nucleotide). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide.
[0044] The terms "mammal", "patient" or "subject" covers warm
blooded mammals that are typically under medical care (e.g., humans
and domesticated animals). Examples include feline, canine, equine,
bovine, and human, as well as only human.
[0045] "Treating" or "treatment" covers the treatment of a
disease-state in a mammal, and includes: (a) preventing the
disease-state from occurring in a mammal, in particular, when such
mammal is predisposed to the disease-state but has not yet been
diagnosed as having it; (b) inhibiting the disease-state, e.g.,
arresting it development; and/or (c) relieving the disease-state,
e.g., causing regression of the disease state until a desired
endpoint is reached. Treating also includes the amelioration of a
symptom of a disease (e.g., lessen the pain or discomfort), wherein
such amelioration may or may not be directly affecting the disease
(e.g., cause, transmission, expression, etc.).
[0046] In general, methods of administering compounds, including
nucleic acids, are well known in the art. In particular, the routes
of administration already in use for nucleic acid therapeutics,
along with formulations in current use, provide preferred routes of
administration and formulation for the nucleic acids described
above. Compositions disclosed herein can be administered by a
number of routes including, but not limited to: oral, intravenous,
intracranial, intracerebro-ventricular, intraperitoneal,
intramuscular, transdermal, subcutaneous, topical, sublingual, or
rectal means. The disclosed compositions can also be administered
via liposomes or other nanoparticles (e.g., packaged microsomes).
Such administration routes and appropriate formulations are
generally known to those of skill in the art.
[0047] Accordingly, the subject invention provides methods of
treating neurological disease or disorder comprising administering
an antagonist of Let-7i to a subject having a neurological disease
or disorder. In various embodiments, the neurological disease or
disorder is selected from: severance of nerves or nerve damage,
severance of cerebrospinal nerve cord (CNS) or CNS damage, damage
to brain or nerve cells, traumatic brain injury, spinal cord
injury, stroke, hypoxia, ischemia, brain injury, diabetic
neuropathy, aging, neurodegenerative disease (such as Alzheimer's
disease, Parkinson's disease or dementia), peripheral neuropathy,
or peripheral nerve injury. Antagonists of Let-7i include antisense
oligonucleotides, siRNA, shRNA, or interfering RNA that
down-regulate or inhibit Let-7i activity or function. The
inhibition of Let-7i function or activity can be mediated by
degradation of the Let-7i miRNA when antisense oligonucleotides,
siRNA, shRNA, or interfering RNA specifically hybridize with
Let-7i. In various additional embodiments, the disclosed methods of
treatment can, optionally, include the method the administration of
progesterone or a composition thereof to said subject. The subject
method can also further comprise the administration of BDNF to said
subject. Antagonists of Let-7i, progesterone, BDNF and compositions
thereof can be administered to a subject as independent
compositions sequentially (e.g., a composition comprising one of
more Let-7i antagonist, a composition comprising progesterone
and/or a composition comprising BDNF) or as a combined composition
(i.e., a compositions comprising one or more antagonist of Let-7i,
progesterone, and/or BDNF).
[0048] Where the subject invention is used to treat signs of aging
in a subject, for example, cognitive, behavioral and functional
consequences of aging in the nervous system are to be treated.
These include, and are not limited to: (a) changes in memory, (b)
alterations of language function, (c) visual-perceptual changes,
(d) slowing of reaction time, and/or (e) decreased balance and
coordination. One or more of these consequences of aging in the
nervous system may be treated.
[0049] Following are examples which illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
General Methods
[0050] Generation of Primary Neuron- or Glia-enriched Cultures: The
use of animals for the purpose of generating primary cultures was
approved by the Institutional Animal Care and Use Committee at the
University of North Texas Health Science Center. All mice will be
handled according to the Guide for the Care and Use of Laboratory
Animals. Primary cultures of cortex and hippocampal neurons will be
prepared from neonatal murine pups (C57BL/6 mice, Jackson
Laboratory) as described by Sarkar et al. with modifications [22,
41]. Briefly, cortical tissues isolated from newborn mice
(postnatal days 2-4, mixed gender) will be then dissociated with
trypsin and DNase I for 10 min at 37.degree. C., and wash twice
with Neurobasal-A medium containing B-27 and further dissociated by
gentle titration using a graded series of fine polished Pasteur
pipettes. After centrifugation at 200.times.g for 3 min at
4.degree. C., dissociated cells will be resuspended in
Neurobasal-A/B-27 medium, passed through a cell strainer with 70
.mu.m mesh, and plated at 1.0.times.105 cells/cm2 on culture dishes
precoated with poly-D-lysine. The culture dishes were kept at
37.degree. C. in humidified 95% air and 5% CO2. For primary
neuron-enriched culture, the initial culture medium was replaced
after 5 h; subsequently, half of the medium was changed every 3
days. At day in vitro (DIV) 3, 1-.beta.-arabinofuranosylcytosine
(AraC) was added to a final concentration of 5 .mu.M to prevent
glial proliferation. Treatments of the primary neuronal cultures
started at DIV 14. For glial-enriched cultures, confluent mixed
glial cultures were placed on the shaker for 48 hrs to dislodge
microglia, resulting in cultures enriched with astrocyte
population.
[0051] Quantitative RT-PCR (microRNA): Total RNA was isolated from
primary astrocytes and mouse brains using the MiRNeasy Mini Kit
(QIAGEN, Valencia, Calif.) according to the manufacturer's
instructions. Concentrations of extracted RNA were determined using
absorbance values at 260 nm. The purity of RNA was assessed by
ratios of absorbance values at 260 and 280 nm (A260/A280 ratios of
1.9-2.0 were considered acceptable). Total RNA (10 ng) was reverse
transcribed into cDNA in a total volume of 15 .mu.l using the
High-Capacity DNA Archive Kit (Roche Applied Science, Indianapolis,
Ind.) according to the manufacturer's instructions. The reaction
mixture contained water, 2.times. quantitative PCR Master Mix
(Eurogentec, Freemont, Calif.), and 20.times. Assay-On-Demand for
each target gene. A separate reaction mixture was prepared for the
endogenous control, U6. The reaction mixture was aliquoted in a
96-well plate, and cDNA added to give a final volume of 20 .mu.l.
Each sample was analyzed in triplicate. The comparative cycle
threshold (Ct) method (2-.DELTA..DELTA.Ct) was used to calculate
the relative changes in target gene expression.
[0052] BDNF Immuno Assay In situ: To define the amount of
endogenous BDNF released, we will modify the ELISA in situ protocol
developed by Promega. A 96-well Nunc MaxiSorp surface polystyrene
flat-bottom immunoplate was precoated with an anti-BDNF monoclonal
antibody [diluted 1:1,000 in coating buffer (25 mM sodium
bicarbonate and 25 mM sodium carbonate, pH 9.7)]. After rinsing off
unbound antibody with TBS-T buffer [20 mM Tris-HCl (pH 7.6), 150 mM
NaCl and 0.05% (v/v) Tween-20] and blocking the plate to minimize
nonspecific binding, the culture media was added to the plate for 2
hrs to equilibrate the cell growth environment. Primary astrocytes
were then plated, and after a period of time to ensure cell
attachment to the plate, the appropriate treatments were applied.
BDNF standards, ranging in concentration from 1.95 to 500 pg/ml,
was added in parallel wells. At the end of hormone treatment, cells
were carefully washed with TBST. The plate was then incubated with
the polyclonal anti-human BDNF antibody. The amount of specifically
bound polyclonal antibody was then detected through the use of the
anti-IgY-horseradish peroxidase (HRP) tertiary antibody (final
concentration=0.5 .mu.g/mL), which when exposed to the chromogenic
substrate (TMB reagent; Promega), changes color in proportion to
the amount of BDNF present in the sample. The color intensity was
quantified by measuring the absorbance at 450 nm with a Viktor3
ELISA plate reader (Perkin Elmer). Only values within the linear
range of the standard curve, and above the lowest standard, were
considered valid. BDNF levels were normalized to protein and are
reported as a percentage of vehicle control. This method allowed
detection of as little as 2 pg/ml BDNF release in control cultures
to .about.250 pg/ml in P4-treated cultures.
[0053] Ovariectomy: Mice will receive bilateral ovariectomy (OVX)
using a dorsal approach under isoflurane anesthesia. A small cut is
made through skin and abdominal muscles in left and right lateral
abdomen. The arteries to left and right ovaries will be ligated,
and ovaries will be cut. The muscles and skin will be sutured with
4-0 Vicryl absorbable suture.
[0054] Implantation of flash-fused steroid pellets: Fused steroid
pellets will be made using the flash-melt method described by Ratka
and Simpkins [40]. The pellets (containing P4 or control) will be
implanted subcutaneously into abdominal area.
[0055] Transient middle cerebral artery occlusion (MCAo): MCAo will
be used to induce transient focal cerebral ischemia (as previously
described [41]). Briefly, under isoflurane anesthesia, a mid line
incision will be made on the neck. Common carotid artery (CCA),
external carotid artery (ECA) and internal carotid artery (ICA)
will be dissected from the connective tissue. A silicon coated 6-0
nylon monofilament will be inserted into the left ECA and advanced
till it occludes the origin of MCA. The MCA will be occluded for 60
minutes and then reperfusion attained by withdrawing the
suture.
[0056] ICV (intracerebroventricular) antagomir injections: ICV
injections will be performed as described by Sananbenesi et al.
[42]. In brief, mice will be anaesthetized and affixed with a
cannula ipsilateral to the side of surgery (coordinates from
Bregma: AP1/4_0.4 mm, L1/4_1.15 mm, V1/4_2.0 mm). Mice will receive
a 0.5 uL infusion of Let-7i-silencing antagomir (Exiqon, Vedbaek,
Denmark) or scrambled antagomir (5 ug), in artificial cerebrospinal
fluid (Harvard Apparatus).
[0057] The pole test: Animals will be trained 2 days before MCAo
procedure and will be tested on day 3, 7, and 14 post stroke.
Training will be achieved by placing animals facing downward on the
pole and allow them to descend. After repeating this training 5
times, animals will then be trained in the regular turning and
descending procedure. Mice will be placed on the rod facing upward.
Normally, animals will turn around and start descending themselves.
Those that do not, however, can be encouraged to turn by gently
pushing to a side. After each trial, mice will be allowed to
explore the cage for 15 s and then returned to their home cage. An
interval of at least 5 min will be allowed between trials. Mice
will tested 3 trials and average performance is recorded.
[0058] The wire hanging test: Animals will be trained 2 days before
MCAo procedure and will be tested on day 3, 7,and 14 post stroke.
Animals will be allow to suspend their bodies on a single wire
stretched between 2 posts 50 cm above the ground. Between the
posts, a soft pillow will be placed to avoid injury in case of a
fall. Training will be achieved simply with several rounds of
habituation and trials. In the actual testing phase, mice will be
tested 3 times and average performance is recorded as final
values.
[0059] Statistical Analysis: We anticipate a minimal sample size
(i.e., "n") of 4 per group in aim 1 studies and an "n" of 13 per
group in aim 2 studies. This number of sample size is based on the
following parameters: Detecting a minimal effect size of 20%,
setting alpha=0.05, and a desired power of 80% or greater. Data
(densitometric analysis for Western blotting, or numerical data
from cell viability assays and from synapse quantification assays)
will be analyzed using analyses of variance (ANOVA) followed by
analysis of differences between individual groups using Tukey's
post-hoc tests. Relative abundance of miRNA and mRNA transcripts
will be evaluated using the 2^(-.DELTA..DELTA.Ct) method [43].
Resulting data will be analyzed using Dunnett's test to compare
fold change in the experimental groups relative to the control
group.
[0060] Both purified BDNF and conditioned medium derived from
P4-treated astrocytes increased the expression of synaptic markers
in neurons: Synaptogenesis has been considered as an important
mechanism for functional recovery after stroke [21, 29, 30]. P4 has
been shown to induce synaptogenesis in various brain locations,
including cortex and hippocampus [4, 31, 32]. Although the
underlying mechanism remains unclear, one potential mediator for
P4-induced synaptogenesis is BDNF [33]. Our preliminary data showed
that both conditioned media derived from P4-treated mouse primary
astrocyte cultures and purified recombinant BDNF increased the
expression of synaptophysin (a presynaptic terminal marker, usually
overexpressed during the neuronal remodeling [34]) and GAP43 (a
synaptic marker that is mainly synthesized during axonal outgrowth
during neuronal development and regeneration [7]) in primary
cortical neurons (FIGS. 1A-1B). Increased expressions of both
markers have been linked to P4-induced synaptogenesis following
stroke [4]. In conjunction with our previous work demonstrating
that P4-induced BDNF release from glia is dependent on Pgrmc1[10],
this data supports that P4-induced increase in expression of
synaptic markers is mediated, at least in part, by the
Pgrmc1-dependent release of BDNF from glia.
[0061] Overexpression of let-7i decreased Pgrmc1 and BDNF mRNA in
primary astrocytes: An in silico analysis, using three prediction
software programs (miRDB, TargetScan and microRNA.org), revealed
putative Let-7 binding sites in the 3'-UTR of Pgrmc1 and BDNF that
were conserved in rat, mouse and human sequences. The Let-7 family
of miRNAs includes multiple evolutionarily conserved members
(Let-7a, b, c, d, e, f, g, i; miR-98) that can exert similar
functions [35]. Since it has been reported that miRNA Let-7i
directly binds to the 3'-untranslated terminal region (UTR) of
Pgrmc1 mRNA, thereby repressing Pgrmc1 expression in a peripheral
(non-CNS) cell type [36], we chose to focus on let-7i in this study
and used another let-7 family member, let-7f, as a control for
specificity. Our data show that in primary cortical astrocytes, an
overexpression of the let-7i mimic (synthetic double-stranded
miRNA-like RNA fragment), but not the let-7f mimic, resulted in
decreased Pgrmc1 and BDNF mRNA levels (FIGS. 2A-2B), supporting the
notion that Let-7i negatively regulates Pgrmcl/BDNF system in
glia.
[0062] Overexpression of let-7i attenuated P4-induced BDNF release
from primary astrocytes: FIG. 3 demonstrates that overexpression of
let-7i abolished P4-induced BDNF release from primary cortical
astrocytes. We previously showed that P4 triggered significant
release from glia by acting via Pgrmcl [10]. When considering data
in both FIGS. 2A-2B and 3, they support our experimental model that
states that let-7i inhibits P4-induced BDNF release from glia by
down-regulating expression of both Pgrmcl and BDNF.
[0063] Expression of Pgrmc1 and BDNF decrease as the function of
age in mouse brain: Current literature lacks information regarding
the effects of age on the expression of Pgrmc1 within the brain.
Interestingly, we found an age-associated decrease of Pgrmc1 and
BDNF mRNA in mouse hippocampus (FIG. 4). The decline in Pgrmc1
level was noted in middle-aged mice, and preceded the decrease of
BDNF in old animals. Since Pgrmc1 is required for P4-induced BDNF
release from glia, decrease of Pgrmc1 expression during normal
aging may dampen P4's neuroprotective effect. Moreover, such a
decline in Pgrmc1 (and BDNF) may also explain the increased risk
for stroke in older individuals.
[0064] A decreased expression of Pgrmcl correlates with an
increased expression of let-7i in the cerebral cortex following an
experimentally-induced ischemic stroke: To determine the potential
involvement of let-7i in stroke, we induced an ischemic stroke in
C57/B16 female mice using the method of middle cerebral artery
occlusion (MCAo), then we examined expression of the miRNA in the
cerebral cortex 7 days post stroke. FIG. 5 shows that, compared to
sham group, expression of let-7i increases by about 60% in stroked
animals, which correlated with a decreased in Pgrmc1 expression.
Data from FIGS. 2A-2B and 5A-5B support our conclusion that let-7i
represses expression of Pgrmc1. The data also support our use of
the MCAo method as a suitable model to study the regulation of
let-7i/Pgrmc1/BDNF axis in ischemic stroke.
[0065] Our data show that let-7i negatively regulates expression of
Pgrmc1 and BDNF in primary astrocytes and there is an inverse
correlation between let-7i and BDNF/Pgrmc1 in the ischemic brain.
Therefore, we anticipated that BDNF and Pgrmc1 will be elevated
following intracerebro-ventricular (ICV) injection of anti-let-7i,
relative to the scrambled control. P4 is known to reduce infarct
size, reverse functional deficits, and induce synaptogenesis in
experimental stroke models [3, 37]. Therefore, we predicted that
mice exposed to P4 will show an increase in synaptogenesis in the
penumbra, smaller ischemic lesion and hence, a positive functional
recovery (demonstrated by measures of motor function, to include
the wire-hanging test). ICV injection of the Let-7i antagomir under
conditions of stroke was thus, expected to reverse the suppression
of glial Pgrmc1/BDNF pathway, thereby, contributing to an enhanced
P4-induced upregulation of synaptogenesis, smaller ischemic lesion
and enhanced motor function. The scientific literature suggests
that synaptogenesis in the penumbra significantly increases within
hours of stroke and can last for several weeks [23, 24, 38].
Therefore, we examined a window of 0-14 day post MCAo to monitor
synaptogenesis both acute and intermediate time points. We used a
published protocol for the delivery of microRNA to the central
nervous system [39], to ensure that anti-let-7i ICV injection
result in sufficient miRNA knock-down that would lead to an
observable effect on synaptogenesis and neuroprotection.
[0066] FIG. 6 shows the effect of co-administration of the Let-7i
antagomir and P4 on the stroke-induced lesion size. Areas of
damaged/dead cells appear white, whereas live tissue appears red,
as a function of metabolism of the TTC stain. Compared to animals
that were not subject to sham surgery (i.e., all aspects of the
surgery were conducted, except the occlusion of middle cerebral
artery--1.sup.st column of sections representing rostral (top most)
to caudal (bottom most) aspects of the brain), the induction of
stroke (2.sup.nd column of sections) showed obvious ischemic
damage. P4 had no statistically significant effect. Remarkably, the
co-application of Let-7i and P4 led to a near complete protection
from the ischemic stroke.
[0067] FIG. 7 shows the functional recovery in the same four groups
of animals depicted in FIG. 6. Functional recovery of motor
function, as defined by an assessment of grip strength, revealed
that the combination of both the Let-7i antagomir and P4 led to
complete functional recovery 7 days post treatment.
EXAMPLE 2
Materials and Methods
[0068] Primary cultures: Dissociated cortical neurons were prepared
and maintained as previously described (44). Briefly, cortices were
removed from neonatal mouse brains (postnatal day 2-4, mixed
gender) and dissociated with 0.25% trypsin. Cortical neurons were
then plated on glass coverslip or plastic culture dishes coated
with poly-D-lysine (Sigma). The culture medium used was Neurobasal
(ThermoFisher Scientific), supplemented with Glutamax and B27
serum-free supplement (ThermoFisher Scientific). At day in vitro
(DIV) 3, 5 .mu.M final concentration of
1-.beta.-arabinofuranosylcytosine (45) (Sigma) was added to the
neuronal cultures to prevent glial proliferation. Half of the
medium was replaced with fresh medium every four days. For
viability assay, cortical neurons were plated onto 96-well plates
(Corning) at the concentration of 1.2.times.10.sup.5
cells/cm.sup.2. For immunocytochemistry, cortical neurons were
plated onto 12 mm glass coverslip (Neuvitro) at the density of
4.times.10.sup.4 cells/cm.sup.2. Treatments of primary cortical
neurons started at DIV12.
[0069] Primary cortical astrocytes were prepared and maintained as
previously described (46), with some modifications. Briefly,
cortices of post-natal day 2-4 mouse pups were dissociated with
0.25% trypsin and plated onto 75 cm.sup.2 tissue culture flask. The
culture medium used was Dulbecco's modified Eagle's medium (DMEM)
(ThermoFisher Scientific), supplemented with 10% fetal bovine serum
(FBS) (GE Healthcare Life Sciences) and 10000U/ml
Penicillin-Streptomycin (ThermoFisher Scientific). After reaching
confluence, mixed glial cultures were placed on the shaker for 48 h
to dislodge microglia, resulting in cultures enriched with
astrocyte population.
[0070] Treatment of primary cultures: To determine the miRNA
regulation of downstream targets in primary cortical astrocytes,
miRNA mimics and inhibitors were transfected into these cells for
48 hrs. After transfection, total RNA and proteins were isolated
for gene and protein expression analysis. Mock transfection was
used as the control for these experiments.
[0071] To study the effect of miRNA on P4-induced BDNF release from
astrocytes, BDNF in-situ ELISA were performed. Expression of miRNA
was first manipulated by transfection as described above. 24 h
after transfection, 10 nM P4 was added to primary cortical
astrocytes for additional 24 h without changing media containing
transfection complexes. Vehicle controls were performed in parallel
such that control cultures were exposed to 0.1% dimethylsulfoxide
(DMSO). The 10 nM concentration of P4 used in studies described
here was chosen because it has been reported to elicit a maximal
release of BDNF from astrocytes (10).
[0072] In experiments evaluating the effect of miRNA on P4-induced
neuroprotection and the synaptogenic marker, synaptophysin, we
first transfected miRNA mimic and inhibitor into primary cortical
astrocytes for 24 h. Afterward, P4 (10 nM) was added to these
cultures for additional 24 h to generate
P4-treated-astrocytes-derived-conditioned-media (P4-ACM). In
parallel, treatment of 0.1% DMSO was performed to generate
DMSO-treated-astrocytes-derived-conditioned-media (DMSO-ACM), which
served as vehicle controls. Before applying to primary neurons,
these conditioned media were filtered through a 10 kD cut-off
column to eliminate residual P4 and miRNA mimic or inhibitor. In
neuroprotection assay, astrocytes-conditioned-media were added to
primary cortical neurons with prior exposure to one hour of
oxygen-glucose-deprivation (OGD), an in-vitro model of
ischemic-like insult. Based on our experience, 1 h of OGD was
enough to induce 50% neuronal cell death. BDNF (50 ng/ml) was
directly added to different groups after OGD to serve as positive
control. Neuronal cultures exposed to normoxia were used as the
control for these data sets. 24 h after the applications of BDNF or
conditioned-media, CellTiter-Glo Luminescent cell viability assay
(Promega) was performed to measure neuroprotection. In synaptogenic
marker measurement assay, BDNF and
astrocytes-derived-conditioned-media were directly added to primary
cortical neurons for 24 hrs. Synaptophysin expression and number of
synaptophysin puncta in these neuronal cultures were assessed by
immunocytochemistry, followed by confocal imaging and analyzed
using ImageJ (National Institutes of Health) software (47).
[0073] Transfection: Transfection of miRNA mimics and inhibitors
was performed using the Hiperfect transfection reagent (Qiagen)
according to manufacturer's instructions. Cells were transfected
with miRNA mimics and inhibitors for 48 h. This duration was chosen
since it resulted in an optimal effect on targets-of-interest.
Synthetic miRNA mimics (Syn-mmu-let-7i-5p, Syn-mmu-let-7f-5p) and
inhibitors (Anti-mmu-let-7i-5p, Anti-mmu-let-7f-5p) were purchased
from Qiagen.
[0074] Quantitative RT-PCR: Total RNA was isolated from primary
cortical astrocytes and mouse brains using the MiRNeasy Mini Kit
(Qiagen) according to the manufacturer's instructions.
Concentrations of extracted RNA were determined using absorbance
values at 260 nm. The purity of RNA was assessed by ratios of
absorbance values at 260 and 280 nm (A260/A280 ratios of 1.9-2.0
were considered acceptable).
[0075] For miRNA expression measurements, total RNA (10 ng) was
reverse transcribed into cDNA in a total volume of 15 .mu.l using
the microRNA cDNA Archive Kit (ThermoFisher Scientific) according
to the manufacturer's instructions. The reaction mixture contained
water, 2x quantitative PCR Master Mix (Eurogentec), and 20.times.
Assay-On-Demand for each target gene. A separate reaction mixture
was prepared for the endogenous control, U6. The reaction mixture
was aliquoted in a 96-well plate, and cDNA added to give a final
volume of 20 .mu.l. Each sample was analyzed in triplicate. The
comparative cycle threshold (Ct) method (2.sup..DELTA..DELTA.Ct)
was used to calculate the relative changes in target miRNA
expression.
[0076] For mRNA expression measurements, total RNA (1.6 .mu.g) was
reverse transcribed into cDNA in a total volume of 20 .mu.l using
the High-Capacity cDNA Archive Kit (ThermoFisher Scientific)
according to the manufacturer's instructions. The reaction mixture
contained water, 2.times. quantitative PCR Master Mix (Eurogentec),
and 20.times. Assay-On-Demand for each target gene. A separate
reaction mixture was prepared for the endogenous control, GAPDH.
The reaction mixture was aliquoted in a 96-well plate, and cDNA (30
ng RNA converted to cDNA) was added to give a final volume of 30
.mu.l. Each sample was analyzed in triplicate.
[0077] The comparative cycle threshold (Ct) method
(2.sup.-.DELTA..DELTA.Ct) was used to calculate the relative
changes in target gene expression.
[0078] PCR primers were purchased as Assay-On-Demand from
ThermoFisher Scientific. The assays were supplied as a 20 mix of
PCR primers (900 nM) and TaqMan probes (200 nM). The let-7i
(002221), U6 (001973), BDNF (Mm00432069_ml), GAP-43
(Mm00500404_ml), GAPDH (Mm99999915_gl), PSD-95 (Mm00492193_ml),
Pgrmc1 (Mm00443985_ml) and SYP (Mm00436850_ml) assays contain FAM
(6-carboxy-fluorescein phosphoramidite) dye label at the 5' end of
the probes and minor groove binder and nonfluorescent quencher at
the 3' end of the probes.
[0079] CellTiter-Glo Luminescent cell viability assay (Promega):
This assay uses the level of adenosine triphosphate (48) as an
indicator of metabolically active cells and is directly
proportional to the number of living cells (49, 50). The assay was
performed according to manufacture's instruction. In brief, cell
plate was first equilibrated to room temperature for 30 minutes. A
volume of the kit reagent equal to the volume of cell culture
present was then added to each well. The plate was then placed on
an orbital shaker for 2 minutes to induce cell lysis, followed by
10 minutes of incubation at room temperature. Luminescence was
recorded using a plate reader.
[0080] BDNF Immuno Assay In situ: To determine the amount of
endogenous BDNF released with P4 treatment, we performed ELISA in
situ assay, as previously described (10). In brief, a 96-well Nunc
MaxiSorp surface polystyrene flat-bottom immunoplate was precoated
with an anti-BDNF monoclonal antibody [diluted 1:1,000 in coating
buffer (25 mM sodium bicarbonate and 25 mM sodium carbonate, pH
9.7). After blocking nonspecific binding, primary cortical
astrocytes were then plated, followed by appropriate treatments
application. BDNF standards, ranging in concentration from 1.95 to
500 pg/ml, was added to parallel wells. At the end of hormone
treatment, cells were carefully washed with TBST. The plate was
then incubated with the polyclonal anti-human BDNF antibody. The
amount of specifically bound polyclonal antibody was then detected
through the use of the anti-IgY-horseradish peroxidase (HRP)
tertiary antibody, which when exposed to the chromogenic substrate
(TMB reagent, Promega), changes color in proportion to the amount
of BDNF present in the sample. The color intensity was quantified
by measuring the absorbance at 450 nm with a Viktor3 ELISA plate
reader (Perkin Elmer). Only values within the linear range of the
standard curve, and above the lowest standard, were considered
valid. This method allowed detection of as little as 2 pg/ml BDNF
release in control cultures to .about.250 pg/ml in P4-treated
cultures.
[0081] Oxygen-glucose Deprivation (OGD): OGD was performed
according to an established protocol, as described elsewhere, with
minor modifications (51). Briefly, primary cortical neurons were
carefully washed five times with Hank's balanced salt solution
(HBSS, ThermoFisher Scientific) to remove residual glucose.
Glucose-free DMEM (ThermoFisher Scientific) was then added to the
cultures, and the plates were transferred into a hypoxic chamber
(0.1% oxygen) for 1 h. At the end of hypoxia, glucose-free DMEM was
replaced with regular maintaining media. Reoxygenation was
initiated by transferring the cells to normoxic 5% CO2 cell culture
incubator.
[0082] Western blotting: Primary cortical astrocytes and mouse
brains were lysed with RIPA lysis buffer containing protease and
phosphatase inhibitors, as previously described (44). After
homogenization, samples were centrifuged at 45,000 rpm for 30 min
at 4.degree. C. and supernatants were collected. Total protein
concentrations were determined using the Bio-Rad DC protein assay
kit (Bio-Rad Laboratories). Cell lysates were separated by SDS-PAGE
and transferred onto polyvinylidene fluoride membrane (Bio-Rad
Laboratories) by electroblotting. Membranes were blocked with 5%
skim milk in tris-buffered saline containing 0.2% Tween 20 (TBS-T)
for lh at room temperature, followed by overnight incubations of
primary antibodies at 4.degree. C. The following primary antibodies
were used: rabbit polyclonal anti-PSD 95 (1:1000, ab18258, Abcam),
rabbit polyclonal anti-Synaptophysin (1:1000, ab14692, Abcam),
rabbit monoclonal anti-GAP43 (1:200000, ab75810, Abcam), rabbit
monoclonal anti-GAPDH (1:1000, 14C10, Cell Signaling), rabbit
polyclonal anti-BDNF (1:300, sc546, Santa Cruz) and goat polyclonal
anti-Pgrmc1 (1:500, ab48012, Abcam). After washing three times with
TBS-T, membranes were incubated with anti-goat IgG or anti-rabbit
IgG conjugated with horseradish peroxidase (Millipore) for 1 hr at
room temperature. After triple washes with TBS-T, immunoreactive
bands were visualized with the ECL detection system (ThermoFisher
Scientific) and were captured using a luminescent image analyzer
(Alpha Innotech). Densitometric analysis was conducted using ImageJ
(National Institutes of Health) software (47).
[0083] Immunofluorescence: The cortical neurons were fixed in 4%
paraformaldehyde (45) for 15 min, followed by incubation in 0.2%
Triton X-100 in Tris-buffered saline (TB S) for 15 min at room
temperature for permeabilization. Cultures were then blocked with
5% donkey serum/1% bovine serum albumin (BSA) in TBS for 1 h at
room temperature and incubated with rabbit monoclonal
anti-Synaptophysin (1:500, ab32127, Abcam) for 48 h at 4.degree. C.
After extensive rinsing with TBS-Tween 20, cultures were incubated
with Alexa Fluor 647-conjugated secondary antibody (1:500, Jackson
ImmunoResearch Laboratories) for 2 h at room temperature. After
extensive washing with TBS to remove unbound secondary antibody,
the coverslips were mounted onto glass slides (VWR Scientific)
using Vectashield mounting medium with DAPI (Vector Laboratories).
The slides were observed under a confocal fluorescence microscope
(FV1200, Olympus) with a 60.times. objective.
[0084] Mouse brains were fixed in 4% PFA overnight at 4.degree. C.
and subsequently cryoprotected in 30% sucrose solution. The brains
were then sectioned into 40-.mu.m thick coronal slices and
subjected to immunostaining using an established protocol described
elsewhere, with some modifications (52). In brief, brain sections
were blocked in 5% donkey serum/1%BSA/TBS solution for 2h at room
temperature. In staining using mouse primary antibody, sections
were subsequently blocked in F(ab) fragment donkey anti-mouse IgG
(50 ug/ml, Jackson ImmunoResearch Laboratories) for 2 h at room
temp to reduce background caused by secondary antibody binding to
endogenous mouse IgG in the tissue. After blocking step, brain
sections were then incubated in primary antibody solution at
4.degree. C. for 72 h. Primary antibodies used were as follow:
mouse monoclonal anti-NeuN (1:500, ab104224, Abcam); rabbit
polyclonal anti-GFAP (1:1000, ab7260, Abcam); rabbit monoclonal
anti-Synaptophysin (1:500, ab32127, Abcam) and goat polyclonal
anti-Pgrmc1 (1:200, ab48012, Abcam). Alexa Fluor 647, Alexa Fluor
594 or Rhodamine Red-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories) were used at 1:500 dilution. After
immunostaining, sections were mounted onto microscope slides with
Vectashield mounting medium (Vector Laboratories) and observed
under a confocal fluorescence microscope (FV1200, Olympus) with a
63.times. objective.
[0085] Mice and treatments: All procedures with animals were
reviewed and approved by the Institutional Animal Care and Use
Committee of the University of North Texas Health Science Center.
All institutional and federal guidelines for the care and the use
of animals were followed. Female C57BL/6J mice (18-week-old) were
purchased from Jackson Laboratory. Animals were habituated to
housing conditions one week before experiments.
[0086] All mice were first ovariectomized to deplete endogenous
ovarian hormone levels. Two weeks after ovariectomy (OVX), P4
pellets were subcutaneously implanted into these animals to
replenish their progesterone levels. In parallel, different groups
received cholesterol pellet implantations to serve as vehicle
control. One week after pellet implantation, stroke was induced in
these mice using middle cerebral artery occlusion (MCAo) procedure.
In parallel, different groups received sham operation (non-stroke).
30 min after MCAo, 5 .mu.g of either scrambled or let-7i inhibitor
was injected into each animal brain via intracerebroventricular
(ICV) injection. Experimental groups included sham-operated mice
with cholesterol pellet implantation (sham), stroked mice with
cholesterol pellet implantation and scrambled ICV injection
(cholesterol+scrambled), stroked mice with P4 pellet implantation
and scrambled ICV injection (P4+ scrambled), and stroked mice with
P4 pellet implantation and let-7i inhibitor ICV injection (P4
+anti-let-7i).
[0087] Ovariectomy: Bilateral ovariectomy (OVX) was performed using
a dorsal approach under isoflurane anesthesia, as described
elsewhere (53). Briefly, small incisions were made bilaterally to
expose ovaries. The arteries adjacent to ovaries were ligated
before ovaries removal. Incisions were then closed using 4-0 Vicryl
absorbable suture.
[0088] Transient middle cerebralaArtery occlusion (MCAo): MCAo was
performed to induce transient focal cerebral ischemia, as
previously described (54). In brief, mice were anesthetized with
isoflurane inhalation. A mid-line incision was made on the neck.
Left common carotid artery (CCA), external carotid artery (55) and
internal carotid artery (ICA) were dissected from the connective
tissue. The left MCA was occluded by a 6-0 monofilament suture
(Doccol Corporation) introduced via internal carotid artery. After
45 minutes occlusion, the suture was withdrawn for reperfusion. In
sham-operated animals, monofilament was advanced to MCA region and
withdraw immediately without MCA occlusion.
[0089] Intracerebroventricular (ICV) injection: 5 .mu.g of either
scrambled or let-7i inhibitor (GE Healthcare Dharmacon) was
suspended in 0.5 .mu.L of PBS and injected into lateral ventricles
using a stereotaxic instrument, as previously described, with minor
modifications (56). In brief, the solution was injected using a
5-uL Hamilton syringe attached to the Ultra Micro Pump UMP3 system
(World Precision Instruments) at a flow rate of 0.2 .mu.l/min.
Coordinates used for ICV injection were AP -0.58 mm, ML+1.2 mm, DV
-2.1 mm.
[0090] Assessment of brain tissue damage:
2,3,5-Triphenyltetrazolium chloride (TTC) staining: TTC staining
was performed to assess ischemic injury among groups, as described
in an established protocol (57). Briefly, 24 h after MCAo, mouse
brains were harvested and sectioned into 2-mm thick coronal
sections. These sections were immersed in 2% TTC solution for 30
min at 37.degree. C. and then fixed in 10% formalin. The stained
slices were photographed and subsequently measured for the surface
area of the slices and the ischemic lesion (Image-Pro Plus 3.0.1,
Silver Springs, Md., U.S.A.). Imaged of stained sections were
captured and infarct sizes were analyzed using ImageJ (National
Institutes of Health) software (47).
[0091] Functional recovery assessment: wire suspension test: In
ordered to assess motor function recovery with different
treatments, wire suspension test, a test of grip strength and
endurance, was used, as described elsewhere (58). In brief, mice
were allowed to suspend their bodies on a single wire that was
elevated above a padded platform. The latency for animals to fall
off the wire was recorded. Mice were trained two days prior to MCAo
to establish a baseline across groups. Training was achieved with
several rounds of habituation and trials. In the actual testing
phase, each mouse was tested 3 times, and average performance was
taken as final values. Performances of these mice was evaluated at
day 3, 7 and 14 post stroke.
[0092] Synaptophysin (SYP) optical density analysis and puncta
quantification: For experiments using primary cortical neurons,
mounted coverslips were imaged using a confocal fluorescence
microscope (FV1200, Olympus) with a 63.times. objective. Healthy
cells that were at least two cell diameters from their nearest
neighbor were identified and selected at random by eye by DAPI
fluorescence. Ten non-overlapping fields per sample were imaged.
Quantification of SYP immunoreactivity (IR) was performed using
ImageJ (National Institutes of Health) software (47). Average IR
was calculated by dividing total IR value by the number of cells
presented in the captured image. Synaptophysin puncta
quantification was analyzed with a custom plug-in (written by Barry
Wark; available upon request from c.eroglu@cellbio.duke.edu) for
ImageJ program. The details of this imaging and quantification
method can be found in a previous publication (59).
[0093] To quantify SYP fluorescence intensity and number of puncta
in mouse brain, three independent coronal brain sections per animal
were stained with SYP. 5-.mu.m confocal scans were performed
(optical section width, 0.33 .mu.m; 15 optical sections each) at
63.times. magnification, as previously described (60). Maximum
projections of three consecutive optical sections corresponding to
1-.mu.m sections were analyzed by using the ImageJ puncta analyzer
option to quantify for numbers of SYP puncta (.gtoreq.5 optical
sections per brain section and .gtoreq.15 total images per brain).
Average SYP puncta density per imaged area was calculated for each
treatment group.
[0094] Statistical Analysis: In vitro data obtained from no fewer
than three independent experiments (where each independent
experiment consisted of between 5-8 replicates), and in vivo data
obtained from at least 4 animals per group (as many as 20 animals
per group for the functional recovery/motor function tests) were
analyzed using an analysis of variance (ANOVA), followed by
appropriate post hoc analyses for the assessment of group
differences, and presented as a bar graph depicting the
mean.+-.S.E.M, using the GraphPad Software (San Diego, Calif.). The
parameters used to inform sample size considered the following:
detecting an effect size of at least 30%, .alpha.=0.05, the
variance of the endpoint measured, and achieving a statistical
power of at least 0.8.
[0095] Let-7i antagomir inhibits oxygen-glucose-deprivation (OGD)
induced increase in let-7i expression: Oxygen glucose deprivation
(OGD), used in the primary cortical astrocytes as an in vitro model
of ischemia, revealed an increase in let-7i expression.
Importantly, the data also verified the effectiveness of the let-7i
antagomir to attenuate the OGD-induced increase in let-7i
expression (FIG. 8A). The data in FIG. 8B demonstrate that OGD
(which increases let-7i expression) compromised the ability of
progesterone (P4)-induced BDNF release from primary cortical
astrocytes, similar to what was noted when let-7i was specifically
over-expressed.
[0096] Let-7i represses progesterone (P4)'s neuroprotection and its
enhancement on synaptogenesis: To investigate the role of let-7i in
P4's neuroprotective effects, we manipulated miRNA expression in
primary cortical astrocytes, then treated them with either vehicle
(DMSO) or P4, following which astrocyte-derived conditioned media
(ACM) was collected. The conditioned media was then applied to
primary cortical neurons (days in vitro (DIV)14) that had been
exposed to oxygen-glucose deprivation (OGD). The neurons were then
assessed for cell viability to ascertain if conditioned media from
P4-treated astrocytes elicited greater neuroprotection relative to
neurons treated with conditioned media from DMSO-treated astrocytes
(FIG. 9). We found that conditioned media collected from P4-treated
astrocytes conferred similar neuroprotection as seen in the
positive control group (consisting of direct administration of BDNF
(50 ng/ml) to the neuronal cultures). However, conditioned media
collected from P4-treated astrocytes that overexpressed let-7i
failed to promote the protection of neurons from OGD.
[0097] Next, we determined if conditioned media from the different
experimental groups represented in FIGS. 10A-10C resulted in
changes in expression of synaptophysin, a presynaptic marker
closely linked to synaptogenesis (4). We observed that conditioned
media derived from P4-treated astrocytes (P4-ACM) resulted in a
robust increase in SYP (green) immunofluorescence (FIG. 10A),
relative to neurons treated with conditioned media from
DMSO-treated, and mock-transfected astrocytes. Quantitative
analysis revealed that P4-ACM significantly increased both SYP
protein level (FIG. 10C) and the number of SYP puncta (FIG. 10B).
The same observations were seen in the positive control group
(consisting of direct application of BDNF (50 ng/ml) to the primary
neuronal cultures). Application of conditioned media collected from
P4-treated astrocytes that overexpressed let-7i (group label:
let-7i+P4), however, failed to elicit the increase in synaptophysin
expression.
[0098] Combined treatment of progesterone (P4) and let-7i
inhibition alleviate ischemia-induced suppression of Pgrmc1 and
BDNF expressions in the penumbra of the ischemic brain: We next
determined the expression of let-7i in the middle cerebral artery
occlusion model of ischemic stroke, focusing on changes in the
penumbra. Assessments of let-7i expression were conducted at
different time points--2, 7 and 14 days following stroke.
Representative images of immunoblots probed for Pgrmc1, along with
pro-and mature-BDNF, are shown in FIG. 11A. We found that compared
to sham (non-stroked controls), ischemic injury resulted in an
up-regulation of let-7i expression (FIG. 11E), starting at day 7
and remained elevated up to 14 days following stroke. P4 treatment
alone (P4+a control sequence for let-7i (scrambled)) did not
attenuate the stroke-induced increase in Let-7i. As expected,
ischemia-induced-increase in let-7i expression was repressed in the
group receiving combined treatment P4 and let-7i inhibition
(P4+anti-7i) (FIG. 11E). Importantly, along with upregulating
let-7i level, ischemia also resulted in a reduction of Pgrmcl
protein level observed at day 7 and day 14 (FIG. 11B). P4 treatment
alone did not restore Pgrmc1 level at either of the two time
points. Combined treatment (P4+anti-let-7i), however, reversed
ischemia-induced suppression of Pgrmcl protein levels. Furthermore,
expression of mature BDNF was reduced as a consequence of stroke at
the 14 days post stroke evaluation period (FIG. 11D), while
pro-BDNF levels (FIG. 11C) remained unchanged across all time
points and all treatments. Compared to sham, the treatment of P4
alone was able to maintain the same level of mature BDNF, even at
14 days post stroke. Remarkably, combined treatment
(P4+anti-let-7i) led to a robust increase in expression of mature
BDNF observed at day 7 and day 14.
[0099] Combined treatment of progesterone (P4) and let-7i
inhibition reduces ischemic injury and enhances functional
recovery: To examine the effect of P4 with or without the let-7i
antagomir on the extent of ischemic injury, we utilized
2,3,5-Triphenyltetrazolium chloride (TTC) staining to visualize the
size of the ischemic lesion. Representative images of TTC stained
are shown in FIG. 12A. Quantification of relative infarct size
(FIG. 12B) revealed that the combined treatment (P4+anti-let-7i)
significantly reduced ischemic injury; whereas P4 treatment alone
did not.
[0100] Motor function (grip strength) was also evaluated using the
wire suspension test. Results (FIG. 13) showed that compared to the
vehicle group (DMSO+scrambled), treatment of P4 led to a partial
recovery of motor function, observed on day 7 and day 14.
Interestingly, the combined treatment of P4 and the let-7i
antagomir resulted in a rapid, but partial, motor function recovery
as early as 3 days post-treatment. By day 7, combined treatment led
to complete functional recovery, and the improvement was still
evident at day 14. Results from FIGS. 12A-12B and FIG. 13 support
our hypothesis that let-7i inhibition enhances P4's neuroprotective
effects that importantly, enhances functional recovery.
[0101] Inhibition of let-7i enhances progesterone (P4)'s effect on
a synaptogenic marker: Synaptic plasticity in the ischemic penumbra
region has long been known to influence the functional recovery
after stroke (21, 23, 38). Therefore, to determine whether
synaptogenesis occurring in the penumbra could be a factor
contributing to functional recovery observed in FIG. 13, we
extended our in vitro findings, to evaluate the expression of
synaptophysin (SYP), a synaptogenic marker, in the penumbra of
stroked mice. To do so, we performed immunofluorescence to
visualize SYP expression (red) (FIG. 14A) and quantified the
relative number of SYP puncta, which is an indication of potential
synapses (FIG. 14B). In parallel, Western blot analysis was
performed to evaluate total SYP protein levels. Representative
immunoblots probed for SYP are shown in FIG. 14C, and its relative
quantification of protein level is depicted in FIG. 14D. Results
revealed that ischemia resulted in a sustained downregulation of
synaptophysin puncta (FIG. 14B) in the penumbra at day 2,7 and 14
post-stroke. In addition, ischemic injury led to decreased SYP
protein level at day 2 and 14. There was a transient increase in
SYP expression at day 7, which could be due to a compensatory
response to the ischemic injury. P4 treatment alone led to a
delayed, but sustained, restoration in SYP total protein
expression, observed at day 7 and day 14. With regards to the
number of SYP puncta, the positive effect of P4 was only evident at
day 14 post-treatment. Interestingly, at day 7 and 14, combined
treatment (P4+anti-let-7i) resulted in significantly higher
expression of SYP, compared to sham controls and P4 treatment
alone. This combined treatment also led to a complete restoration
of synaptophysin puncta at day 7, an effect that was further
enhanced at day 14. Taken together, these findings indicate that P4
induces synaptogenesis in the penumbra of ischemic brain and that
let-7i inhibition further enhances this beneficial function of
P4.
[0102] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
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Sequence CWU 1
1
3122RNAHomo sapiensmisc_feature(1)..(22)Mus musculus sequence is
identical 1ugagguagua guuugugcug uu 22284RNAHomo sapiens
2cuggcugagg uaguaguuug ugcuguuggu cggguuguga cauugcccgc uguggagaua
60acugcgcaag cuacugccuu gcua 84385RNAMus musculus 3cuggcugagg
uaguaguuug ugcuguuggu cggguuguga cauugcccgc uguggagaua 60acugcgcaag
cuacugccuu gcuag 85
* * * * *