U.S. patent application number 15/004078 was filed with the patent office on 2016-06-02 for compositions and methods for treating alzheimer`s disease and related disorders and promoting a healthy nervous system.
The applicant listed for this patent is Icahn School of Medicine at Mount Sinai. Invention is credited to Giulio Maria Pasinetti.
Application Number | 20160151301 15/004078 |
Document ID | / |
Family ID | 36228394 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160151301 |
Kind Code |
A1 |
Pasinetti; Giulio Maria |
June 2, 2016 |
Compositions and methods for treating alzheimer`s disease and
related disorders and promoting a healthy nervous system
Abstract
The present invention relates generally to methods of inhibiting
the onset and progression of Alzheimer's disease (AD), mild
cognitive impairment (MCI), and related neurodegenerative disorders
involving amyloidosis. The methods involve administering a
composition comprising a therapeutically effective amount of
D-pinitol to individuals who are at risk of developing the disease
or have one or more symptoms of the disease. The invention also
relates to methods of treating or preventing Alzheimer's disease or
MCI that involve the use of such compositions. The invention
further provides methods to promote the general health of the
nervous system through administration of active ingredient
compositions. The invention further relates to animal and cellular
systems for the study of AD, MCI, and related disorders, and the
identification of reagents useful in treating amyloidosis. The
invention further provides methods to inhibit AD or MCI in an
individual based on reducing caloric intake by the individual. The
invention further relates to a method of treating or inhibiting an
amyloidogenic disorder by caloric restriction.
Inventors: |
Pasinetti; Giulio Maria;
(New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Icahn School of Medicine at Mount Sinai |
New York |
NY |
US |
|
|
Family ID: |
36228394 |
Appl. No.: |
15/004078 |
Filed: |
January 22, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14550189 |
Nov 21, 2014 |
9254273 |
|
|
15004078 |
|
|
|
|
13486439 |
Jun 1, 2012 |
8921347 |
|
|
14550189 |
|
|
|
|
11255780 |
Oct 21, 2005 |
8193250 |
|
|
13486439 |
|
|
|
|
60621331 |
Oct 22, 2004 |
|
|
|
Current U.S.
Class: |
514/729 |
Current CPC
Class: |
A61K 31/045 20130101;
A61K 31/047 20130101; A61K 31/075 20130101; A61K 9/0053
20130101 |
International
Class: |
A61K 31/047 20060101
A61K031/047; A61K 9/00 20060101 A61K009/00 |
Claims
1-77. (canceled)
78. A method of ameliorating a symptom of cognitive impairment,
which method comprises administering a therapeutically effective
amount of a composition comprising D-pinitol to an individual
suffering from cognitive impairment.
79. The method of claim 1, wherein the symptom of cognitive
impairment comprises a learning or memory deficit.
80. The method of claim 1, wherein the cognitive impairment is mild
cognitive impairment.
81. The method of claim 1, which further comprises a reduction in
the caloric intake by the individual.
82. The method of claim 1, wherein the reduction comprises a
reduction in the intake of carbohydrates.
83. The method of claim 1, wherein said individual has been
identified as being at risk of developing Alzheimer's disease.
84. The method of claim 6, wherein said individual has been
identified as being at risk of developing Alzheimer's disease due
to the presence of mild cognitive impairment.
85. The method of claim 6, wherein said individual has been
identified as being at risk of developing Alzheimer's disease due
to the presence of at least one .epsilon.4 allele or the absence of
an .epsilon.2 allele at the apolipoprotein E locus.
86. The method of claim 6, wherein said individual is provided with
D-pinitol in an amount and for a sufficient period of time to
ameliorate a symptom of Alzheimer's disease.
87. The method of claim 6, wherein said individual has been
identified as being at risk of developing Alzheimer's disease due
to a genetic factor.
88. The method of claim 6, wherein said individual has been
identified as being at risk of developing Alzheimer's disease due
to family history.
89. The method of claim 1, wherein said individual is provided with
D-pinitol in an amount and for a sufficient period of time to
achieve a concentration of D-pinitol in blood or plasma of about 1
to 19 .mu.M.
90. The method of claim 1, wherein said D-pinitol is provided at a
daily dose of over 10 mg/kg.
91. The method of claim 1, wherein the composition is administered
orally.
92. The method of claim 1, wherein the individual has a symptom of
Non-Insulin Dependent Diabetes Mellitus (NIDDM).
93. The method of claim 1, wherein the cognitive impairment is
associated with .beta.-amyloid accumulation.
Description
[0001] This application claims priority to U.S. Patent Application
Ser. No. 60/621,331 filed on Oct. 22, 2004 and incorporated herein,
by reference, in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to methods of
inhibiting the onset and progression of Alzheimer's disease (AD),
mild cognitive impairment (MCI), and related neurodegenerative
disorders involving amyloidosis. The methods involve administering
a composition comprising a therapeutically effective amount of
D-pinitol to individuals who are at risk of developing the disease
or have one or more symptoms of the disease. The invention also
relates to methods for treating or preventing Alzheimer's disease
or MCI that involve the use of such compositions. The invention
further provides methods to promote the general health of the
nervous system through administration of active ingredient
compositions. The invention further provides methods to inhibit or
treat AD or MCI in an individual based on reducing caloric intake
by the individual. The invention further relates to animal and
cellular systems for the study of AD, MCI, and related disorders,
and the identification of reagents useful in treating
amyloidosis.
BACKGROUND OF THE INVENTION
Dementia and the Prevalence of Alzheimer's Disease
[0003] Dementia is a neurodegenerative disorder that affects as
many as 10% of individuals over 65 years of age, and more than 35%
of those over 85 (Hofman et al. Int. J. Epidemiol. 1991;
20:736-748; Jorm and Jolley. Neurology 1998; 1:728-733; Lobo et al.
Neurology 2000; 4Suppl5:S4-S9). Dementia is a complex syndrome and
is typically progressive or chronic. The spectrum of this disorder
is broad-sweeping and encompasses multiple cognitive functions,
including learning, memory, thinking, calculation, language,
judgment, comprehension, and spatial and temporal recognition. In
addition, the syndrome can be preceded or accompanied by disruption
in emotional states, including emotional control, social behavior,
and motivation.
[0004] Of the diseases leading to dementia, Alzheimer's Disease
(AD) is the most common, accounting for greater than half the cases
in people over 65 years of age. First recognized nearly a century
ago (Alzheimer. Algemeine Zeitschrift fur Pschiatrie. 1907;
64:146-148). Alzheimer's disease (AD) is a devastating disorder,
affecting nearly 2-3 million individuals in the United States and
about 15 million people world-wide (including all races and ethnic
groups). Moreover, because advanced age in the biggest risk factor
for AD, the prevalence of this disorder doubles every 5 years
beyond age 65 (National Institute on Aging: Prevalence and costs of
Alzheimer's disease. Progress Report on Alzheimer's Disease. NIH
Publication No. 99,3616, November 1998; Polvikoski et al. Neurology
2001; 56:1690-1696).
Pathology of Alzheimer's Disease
[0005] The pathological hallmarks of clinical AD are confined to
the central nervous system (CNS) and comprise two principal
features: amyloid deposits and neurofibrillary tangles (NFT).
Amyloidosis--the deposition of amyloid--occurs through the vascular
and neuronal architecture of the CNS.
[0006] Although amyloidosis occurs to a limited extent in probably
all individuals, especially with advanced age, it is aberrantly
high in AD and related disorders. The main molecular component of
amyloid is .beta.-amyloid, a highly hydrophobic peptide, derived
from processing of the amyloid precursor protein (APP; See FIG. 6).
This peptide aggregates into filaments in an anti-.beta.-pleated
sheet structure. Elevated expression disrupts the structure and
function of neuronal and glial cells, ultimately resulting in
extensive cell death in regions of the brain underlying higher
cognitive functions, (Francis et al. J. Neurol. Neurosurg.
Psychiatry 1999; 66:137-147).
[0007] These pathological hallmarks, however, reflect terminal
points of a lengthy disease process. Consequently, post-mortem
brain studies have yielded little insight into the molecular and
biochemical components involved in the initiation and progression
of this disease. Underscoring this issue is the molecular
complexity of amyloid, which comprises many additional proteins,
including al-anti-chymotrypsin (Abraham et al. Cell 52:487-501,
1988), cathepsin D (Cataldo et al. Brain Res. 1990; 513:181-192)
and apolipoprotein E (apoE) (Namba et al. Brain Res. 1991;
541:163-166; Wisniewski and Frangione. Neurosci. Lett. 1992;
135:235-238; Strittmatter et al. Proc. Nat. Acad. Sci. USA 1993;
90:1977-1981).
Etiology of Alzheimer's Disease
[0008] Although amyloidosis is a conserved feature of AD, the
underlying etiology is highly complex and the vast majority of
instances likely involves multiple genetic and environmental
determinants. AD has been divided into two categories: Familial AD
(FAD) and Sporadic AD. FAD is associated with mutations in least
three known loci: presenilin 1 (PS1), presenilin 2 (PS2) and
amyloid precursor protein (APP) genes. Studies of FAD have
underscored the importance of amyloid .beta.-peptide (A.beta.) in
AD, but are limited in scope: FAD only accounts for a small
fraction of AD cases (less than 5%), has a much earlier age onset
(often in the 40's) and follows clear patterns of inheritance (Bird
et al. Ann. Neurol. 1989; 25:12-25; Heston and White. Behavior
Genet. 1978; 8:315-331; Pericak-Vance et al. Exp. Neurol. 1988;
102:271-279).
[0009] In contrast Sporadic AD can reflect a genetic etiology, but
it is not a tight linkage. The .epsilon.4 allele of the
apoliprotein E locus, for example, increases the risk of developing
AD; however, its presence does not guarantee AD, nor does its
absence preclude it. (Consensus report of the Working Group.
Neurobiology of Aging 1998; 19:109-116).
[0010] The vast majority of AD cases, therefore, will likely
comprise a complex etiology, implicating multiple genetic and
environmental factors. In light of such constraints, many
investigations have focused on identifying biological markers that
correlate with different stages of AD. Candidates for such markers
have included cerebrospinal fluid profiles, peripheral tissue
markers, pharmacologic and neuroendocrine probes, and behavioral
and biochemical correlates. (See, e.g., Neurobiology of Aging 1998;
19:109-116 for a review).
[0011] Nevertheless, such biological markers are not likely to
provide a cure-all for at least two reasons. First, the extended
time course of AD poses the inescapable challenge of separating
normal changes during aging, such as impairments of cognitive
abilities, from pathogenic ones. Second, dietary, environmental,
and genetic factors are not easily controlled with this approach,
presenting the additional challenge of resolving diagnostic effects
within a heterogeneous background. At present, AD remains
incurable. Nor is there available a treatment that effectively
prevents AD or reverses its symptoms.
[0012] Thus, there is a need in the art to develop new approaches
to identify candidate compounds effective to inhibit and treat
symptoms or clinical manifestations of AD and related disorders.
The present invention meets this need by disclosing new mechanistic
and temporal links between the induction of an insulin-resistant
Type 2 diabetes state--a known risk factor for AD--and the
subsequent appearance of cognitive defects and .beta.-amyloid
pathologies in the brain. In turn, these findings reveal specific
and unexpected uses for D-pinitol--an insulin sensitizing
compound--that include inhibiting AD pathology.
SUMMARY OF THE INVENTION
[0013] In one embodiment, the invention provides a method of
inhibiting a symptom of Alzheimer's disease in an individual by
administering a therapeutically effective amount of a composition
comprising D-pinitol. In a further embodiment, the method provides
a composition that is pharmaceutically acceptable. In another
aspect, the method comprises administering the composition prior to
the onset of Alzheimer's disease. In yet another aspect, the method
comprises administering the composition to an individual who is
asymptomatic and at risk of developing Alzheimer's disease, with or
without NIDDM; who has a genetic predisposition to Alzheimer's
disease; who has a genetic factor that denotes increased risk of
Alzheimer's disease, wherein the genetic factor may also comprise
at least one .epsilon.4 allele or absence of an .epsilon.2 allele
at the apolipoprotein E locus; who bears a biological marker that
denotes increased risk of Alzheimer's disease; who has a behavioral
state that denotes increased risk of Alzheimer's disease, and which
may further comprise mild cognitive impairment (MCI); who is an age
of 20 or older and prior to onset of clinical symptoms of
Alzheimer's disease; who is an age of 40 or older and prior to
onset of clinical symptoms of Alzheimer's disease; or who is over
about 60 years old and free of clinical symptoms of Alzheimer's
disease. The invention further provides a method of inhibiting a
symptom of Alzheimer's disease in an individual by administering a
therapeutically effective amount of a composition comprising
D-pinitol, wherein the symptom is a cognitive impairment, which may
further comprise a learning or memory deficit.
[0014] In another embodiment, the invention also provides a method
of treating a symptom of Alzheimer's disease in an individual by
administering a therapeutically effective amount of a composition
comprising D-pinitol. In a further embodiment the method provides a
composition that is pharmaceutically acceptable. In another aspect,
the method comprises administering the composition prior to the
onset of Alzheimer's disease. In yet another aspect, the method
comprises administering the composition to an individual who is
asymptomatic and at risk of developing Alzheimer's disease, with or
without NIDDM; who has a genetic predisposition to Alzheimer's
disease; who has a genetic factor that denotes increased risk of
Alzheimer's disease, wherein the genetic factor may also comprise
at least one 64 allele or absence of an .epsilon.2 allele at the
apolipoprotein E locus; who bears a biological marker that denotes
increased risk of Alzheimer's disease; who has a behavioral state
that denotes increased risk of Alzheimer's disease, and which may
further comprise mild cognitive impairment (MC1); who is an age of
20 or older and prior to onset of clinical symptoms of Alzheimer's
disease; who is an age of 40 or older and prior to onset of
clinical symptoms of Alzheimer's disease; or who is over about 60
years old and free of clinical symptoms of Alzheimer's disease. The
invention further provides a method of treating a symptom of
Alzheimer's disease in an individual by administering a
therapeutically effective amount of a composition comprising
D-pinitol, wherein the symptom is a cognitive impairment, which may
further comprise a learning or memory deficit.
[0015] In another embodiment, the invention provides a method of
inhibiting amyloidosis in an individual by administering to the
individual a therapeutically effective amount of a composition
comprising D-pinitol. In a further aspect of this method, the
individual has been diagnosed with Alzheimer's disease or is at
risk of developing Alzheimer's disease. In another aspect of this
method, the composition can be administered prior to the onset of
Alzheimer's disease and may also be pharmaceutically acceptable. In
yet another aspect, the method can be used to inhibit only that
class of amyloidoses that are .beta.-amyloidoses.
[0016] In still another embodiment, the invention provides a method
of inhibiting the activity of .gamma.-secretase in an individual by
administering to the individual a therapeutically effective amount
of a composition comprising D-pinitol. In a related aspect of the
method, the individual can be diagnosed with Alzheimer's disease or
be at risk of developing Alzheimer's disease. The method can
further comprise administering the composition prior to the onset
of Alzheimer's disease. In still another aspect, the invention
provides a method of inhibiting the activity of .gamma.-secretase
in an individual by administering to the individual a
therapeutically effective amount of a composition comprising
D-pinitol that is also pharmaceutically acceptable.
[0017] In a different embodiment, the invention provides a method
of treating a symptom of Alzheimer's disease in an individual by
reducing the daily caloric intake of the individual. More
specifically, the method can comprise reducing caloric intake of
carbohydrates only, and if desired, by thirty percent.
[0018] In an alternative embodiment, the invention provides a
method of preventing a symptom of Alzheimer's disease in an
individual by reducing the daily caloric intake of the individual.
In another aspect, the method may comprise reducing caloric intake
of carbohydrates only, and if desired, by thirty percent.
[0019] In a different embodiment, the invention provides a method
of treating a symptom of amyloidosis in an individual by reducing
the daily caloric intake of the individual. In another aspect, the
method may comprise reducing caloric intake of carbohydrates only,
and if desired, by only thirty percent. In still another aspect,
the amyloidosis is .beta.-amyloidosis only.
[0020] In a different embodiment, the invention provides a method
of inhibiting a symptom of amyloidosis in an individual by reducing
the daily caloric intake of the individual. In another aspect, the
method may comprise reducing caloric intake of carbohydrates only,
and if desired, by only thirty percent. In still another aspect,
the amyloidosis is .beta.-amyloidosis only.
[0021] In yet another embodiment, the invention comprises a method
of promoting healthy nervous system function in a mammal by
administering a composition comprising D-pinitol to the mammal. In
a further aspect, the mammal can be asymptomatic or not have a
condition of insulin resistance, hyperlipidemia, or dyslipidemia.
In yet another aspect, the mammal is a human. In still another
aspect, the nervous system function can be a cognitive ability,
which can further comprise learning or memory.
[0022] In yet another embodiment, the invention comprises a method
of promoting healthy nervous system function in a mammal by
administering a composition comprising D-pinitol to the mammal. In
a further aspect, the mammal can be asymptomatic or not have a
condition of insulin resistance, hyperlipidemia, or dyslipidemia.
In still another aspect, the function of the nervous system may be
function of the brain or even function of a sub-region of the
brain, such as the hippocampus.
[0023] In another embodiment, the invention provide a method of
studying the role of NIDDM in AD neuropathology by placing an
animal on a diet sufficient to induce NIDDM-like insulin
resistance, wherein the insulin resistance is associated with a
symptom of Alzheimer's disease. In a further aspect, the animal may
be a mammal, or more specifically a mouse, or even more
specifically, the Tg2576 strain of mice.
[0024] In a related aspect of the invention, the symptom of
Alzheimer's disease can be amyloidosis or an impairment of
cognitive function generally or alternatively, just learning or
memory.
[0025] In another embodiment, the invention provides a cell-free
system for studying enzymatic cleavage of the amyloid precursor
protein by .alpha.-, .beta.-, or .gamma.-secretase under conditions
which allow the amyloid precursor protein to remain bound to its
membrane under physiological conditions.
[0026] In still another embodiment, the invention provides a method
of using the cell-free system to study amyloidosis by incubating
the extract under physiological conditions and quantifying activity
of a .alpha.-, .beta.-, and .gamma.-secretase.
[0027] In yet another embodiment, the invention provides a method
of using the cell-free system to quantify activity of a .alpha.-,
.beta.-, and .gamma.-secretase, by incubating the extract under
physiological conditions.
[0028] In still another embodiment, the invention provides a method
of using the cell-free system to screen for compound useful for
preventing or treating an amyloidogenic disorder, by incubating the
extract under physiological conditions in the presence of the
compound; and quantifying activity of .alpha.-, .beta.-, and
.gamma.-secretase.
[0029] The methods and pharmaceutical compositions of the invention
include an effective amount of D-pinitol used in combination with a
drug approved for treatment of Alzheimer's disease, such as
donepezil (Aricept.RTM.), galantamine (Reminyl.RTM.), tacrine
hydrochloride (Cognex.RTM.), and rivastigmine tartrate
(Exelon.RTM.), and a pharmaceutically acceptable carrier.
[0030] The methods and pharmaceutical compositions of the invention
include an effective amount of D-pinitol used alone or in
combination with caloric restriction for prevention or treatment of
mild cognitive impairment (MCI).
[0031] In preferred embodiments, the methods and pharmaceutical
compositions of the invention provide a plasma or blood
concentration of 10 micromolar D-pinitol, plus or minus 90%, 50%,
30%, 10%, 5% or 3%.
[0032] The above features and many other attendant advantages of
the invention will become better understood by reference to the
following detailed description when taken in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 depicts data showing that a high-fat diet induces
insulin resistance in Tg2576 mice. Female Tg2576 (Hsiao et al.
Science 1996; 274:99-102) mice were exposed to either the high-fat
diet (Tg insulin-resistant group) or a standard rodent laboratory
diet (Tg control group), starting at 3-4 months of age. Animals
were then assessed for indexes of NIDDM-like insulin resistance at
9 months of age. (A) ELISA detection of serum insulin content. (B)
Body weight. (C) ependymal fat-pad weight measurements. (D)
Post-prandial intraperitoneal glucose tolerance response in which
blood glucose content was monitored over time following an i.p.
injection of glucose (2 g/kg BW). In A-D, values represent
means.+-.SEM (standard error of the mean) n=5-6 per group; *
P<0.05 vs. control group (2-tailed Student's t test).
[0034] FIG. 2 depicts data showing that diet-induced insulin
resistance promotes amyloid burden in Tg2576 mice. (A) ELISA
detection of A.beta..sub.1-40 and A.beta..sub.1-42 peptide content
and 22C11-immunopositive holo-APP content in the hippocampal
formation. (B) Stereological cerebral cortical (neocortex and
hippocampal formation) 6E10-immunopositive amyloid plaque volume
and burden (amyloid plaque volume as a percentage of cerebral
cortex volume). In A-B, values represent means.+-.s.e.m., n=5-6 per
group; * P<0.05, ** P<0.01, *** P<0.001 vs. control group
(2-tailed Student's t test).
[0035] FIG. 3 depicts data showing that insulin resistance promotes
.gamma.-secretase activity in the brain of Tg2576 mice. (A)
Increased .gamma.-secretase activity in cerebral cortex (left
panel) assessed by generation of .gamma.-CTF from membrane
associated APP (normalized to holo-APP in the membrane
preparation). Parallel western blot analysis of holo-APP from total
brain extract (right panel) confirmed that the diet-induced insulin
resistance did not influence holo-APP expression; (A inset),
representative cleavage products generated from membrane-bound APP;
as expected, negative controls (no incubation) yielded no
CTF-.gamma. cleavage product. (B-C) Scatter plot analysis of
.gamma.-secretase activity as a function of
pS.sup.21-GSK-3.alpha./GSK-3.beta. (B) and
pS.sup.9-GSK-3.alpha./GSK-3.beta. (C). Straight line represents
best linear regression fit.
[0036] FIG. 4 depicts data showing that insulin-resistant Tg2576
mice exhibit impaired ability to learn in a Morris spatial water
maze task. (A) Tg2576 control mice improved their latencies during
the learning phase of testing (repeated measures ANOVA P<0.001,
F.sub.7,28=5.04), while insulin-resistant Tg2576 mice were impaired
in their ability to learn the task and displayed consistently
longer latencies throughout testing relative to control Tg2576 mice
(two-way ANOVA P<0.0017, F.sub.1,72=10.76 for diet treatment).
(B) Tg2576 control mice displayed a significant bias for the former
platform location shown in (C), suggesting that their superior
performance in the learning phase coincided with acquisition and
retention of the spatial location of the escape platform. (D)
Insulin-resistant Tg2576 mice swam in a random manner depicted in
(C), during the probe trial, further suggesting that diet-induced
insulin resistance led to an impaired ability to learn this task.
(E) During probe trial, both groups exhibited equivalent latencies,
suggesting that deficits observed in insulin-resistant Tg2576 mice
were unlikely to have arisen from impaired swimming or visual
ability.
[0037] FIG. 5 depicts data showing that pinitol treatment reduced
insulin-resistance mediated promotion of .beta.-amyloidosis in
Tg2576 AD mice.
[0038] FIG. 6 depicts two representations of the .beta.-amyloid
precursor protein (.beta.APP) protein, emphasizing different
features pertinent to the invention. (A) C-terminal region of
.beta.APP, showing the membrane spanning region, sites of cleavage
by .alpha.-, .beta.-, and .gamma.-secretases, and the sequence of
.beta.-amyloid. (B) Diagrammatic representation of in vitro
.gamma.-secretase assay based on generation of the P7 (.gamma.-CTF)
fragment from the amyloid precursor protein (APP) (Sastre et al.
EMBO Rep. 2001; 2:835-841).
[0039] FIG. 7 depicts data showing that D-pinitol does not
influence .alpha.- and .beta.-secretase activity, but has a robust
inhibitory effect on .gamma.-secretase activity in H4-APPswe cells.
(A) .alpha.-secretase. (B) .beta.-secretase. (C) .gamma.-secretase
(A.sub.1-40). (D) .gamma.-secretase (A.sub.1-42).
[0040] FIG. 8 depicts data showing that anti-amyloidogenic activity
of D-pinitol is associated with selective reduction of
.gamma.-secretase-mediated APP cleavage in H4-APPswe cells. (A) APP
(B) NOTCH
[0041] FIG. 9 depicts data showing the change in metabolic indexes
in response to low carbohydrate CR dietary regimen relative to AL
(ad libitum) feeding. (A) Mean body weight in response to CR versus
AL-feeding over time. Arrow indicates initiation of CR treatment
regimen. (B) Ependymal fat pad weight assessed at the time of
sacrifice. (C) Mean blood glucose content over time in AL and CR
mice in response to IGTT. In (A), two-way ANOVA; p<0.0001,
F1,139=150.5, for dietary regimen; p<0.0249, F13,139=2.0 for
time and p<0.0080, F13,139=2.32 for interaction. In (B),
two-tailed t-test, p<0.02, AL versus CR. In (C), two-way ANOVA;
P<0.0001, F1,50=20.32 for dietary regimen; P<0.0001,
F4,50=30.32 for glycemic content over time and P<0.03,
F4,50=3.02 for interaction. Values represent mean.+-.SEM, n=5-7
mice per group.
[0042] FIG. 10 depicts data showing that CR attenuates AD-type
amyloid neuropathology in the brain of Tg2576 mice.
Twelve-month-old Tg2576 mice were assessed for indices of AD-type
amyloid burden in the neocortex and hippocampus in response to 9
months CR or AL dietary regimens (see Examples). (A) Stereological
assessment of neocortical and hippocampal A.beta.-amyloid plaque
burden (thioflavin-S positive volume as percent of regional volume)
(Bussiere et al. Neuroscience 2002; 112:75-91). (B) Representative
thioflavin-S positive A.beta. amyloid plaque neuropathology in the
neocortex (CTX) and the hippocampal subicular (S) region in the AL
(left panel) and CR mice (right panel). (C) Assessment of
neocortical and hippocampal A.beta.1-40 and A.beta.1-42 peptide
concentrations in CR and AL-fed control Tg2576 mice. (D-E) IP-MS
spectra of neocortical A.beta. peptide profiles of CR and AL-fed
control mice detected after immunoprecipitation with 4G8 (D) and
6E10 (E) antibodies. The A.beta. IP-MS spectra from 4G8 IP was
normalized to the internal standard A.beta.12-28 while the A.beta.
IP-MS spectra from 6E10 was plotted using a relative scale to the
peak intensity of A.beta.1-40 in the AL-fed control group and
referenced to their relationship in the 4G8 IP to compare the
A.beta. fragment peptides. In (D) and (E) MS peaks corresponding to
A.beta. peptides are indicated with the A.beta. peptide sequence
number. Peaks labeled as 1-402+ and Insulin2+ represent doubly
protonated A.beta.1-40 peptide and doubly protonated insulin
molecular ions, respectively; A.beta.12-28 was added during the IP
procedure and used as internal standard (int. std.) ions as
previously reported (Wang et al. J. Biol. Chem. 1996;
271:31894-31902). Bar graphs represent group mean+SEM, n=5-7
animals per group; *p<0.01, ** p<0.005, 2-tailed t-test, CR
versus AL groups. In (B), arrowheads point toward thioflavin-S
positive A.beta. amyloid plaques; length bar=30 .mu.M.
Abbreviations: CR, caloric restriction; AL, ad-libitum; in (B),
CTX, neocortex; S, subiculum; CC corpus collosum.
[0043] FIG. 11 depicts data showing that there is no detectable
change in total full length-APP and CTF-.gamma. concentrations in
the brain of Tg2576 mice in response to CR relative to AL-feeding.
(A) Assessment of steady state total full-length APP concentrations
in neocortical and hippocampal samples relative to .beta.-actin
immunoreactive signal; in (A) inset, representative immunoblot of
full-length APP and .beta.-actin immunoreactivities. (B) Assessment
of APP CTF-.gamma. cleavage product concentrations; in (B) inset,
representative CTF-.gamma. and full-length APP immunoreactive
signal from the same immunoblot. Bar graph represents group
mean+SEM, n=5-7 mice per group.
[0044] FIG. 12 depicts data showing that CR promotes
.alpha.-secretase activity in the brain of Tg2576 mice relative to
AL-fed control mice. Parallel aliquots of neocortical tissue used
for assessment of A.beta. peptide concentrations were used in the
assessment of indices of .alpha.-secretase activity in response to
CR, relative to AL feeding. (A) Assessment of changes in
neocortical sAPP.alpha. concentration expressed as percent of
full-length APP immunoreactivity; in (A) inset, representative
sAPP.alpha. and full-length APP immunoreactive signal in the same
sample. (B) Assessment of APP CTF-.alpha. cleavage product
concentrations; in (B) inset, representative CTF-.alpha. (and
CTF-.beta.) and full-length APP immunoreactive signal from the same
immunoblot. In (C), assessment of mature and proform ADAM10
concentrations in the same neocortical specimen relative to
.beta.-actin immunoreactive signal; in (C) inset, representative
immunoblot of proform and mature forms of ADAM10 protein species
and .beta.-actin immunoreactivities. In (D), fluorometric
assessment of .alpha.-secretase activity. Bar graphs represent
group mean+SEM, n=5-7 mice per group; * p<0.02, ** p<0.01,
two-tailed t-test, AL versus CR group.
[0045] FIG. 13 depicts data showing that there are no detectable
changes in neocortical ADAM17 (TACE), ADAM9, BACE, and
.beta.-secretase activity in response to CR relative to AL-feeding.
(A-C) assessment of proform and mature ADAM17 (TACE), ADAM9, and
BACE1 concentrations, relative to .beta.-actin immunoreactive
signal, respectively. In (A-C) insets, representative ADAM17,
ADAM9, and BACE1 immunoreactivities and .beta.-actin immunoreactive
signal in the same sample. In (C), .beta.-secretase was assessed
fluorometrically in the parallel neocortical frozen tissue samples
used to assess BACE immunoreactive material. Bar graphs represent
means+SEM, n=5-7 mice per group.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0046] The present invention is based, in part, on the discovery
that controlled dietary treatment of Tg2576 transgenic mice
recapitulates the hallmarks of human non-insulin-dependent
diabetes-mellitus (NIDDM) (e.g., insulin resistance, glucose
intolerance, and increased body weight). The induction of NIDDM is
accompanied by the premature appearance in the brain of
pathological hallmarks associated with human Alzheimer's disease
(AD), including aberrant accumulation of .beta.-amyloid plaques,
increased .gamma.-secretase activity, and decreased
insulin-degrading enzyme (IDE) activity. Moreover, the AD
neuropathology is further accompanied by premature cognitive
deficits, including specific disruptions of learning and
memory.
[0047] In addition, the present invention is based on the discovery
that molecules useful for controlling insulin resistance in NIDDM
can inhibit or preclude appearance of biochemical, anatomical, and
cognitive pathologies associated with AD. In particular, early
treatment with D-pinitol reduces .beta.-amyloidosis and restores
signal transduction by both the insulin-receptor and glycogen
synthase kinase-3.
[0048] Further, the present invention is based on the generation of
a novel cell-free system, allowing for the first time, analysis of
secretase-mediated cleavage of APP under physiological conditions
that retain APP in its native membrane-bound conformation. In this
system, D-pinitol inhibits .beta.-amyloid with exquisite
specificity, inhibiting the activity of .gamma.-secretase but not
that of .alpha.- and .beta.-secretase.
[0049] Accordingly, the present invention provides a number of uses
for D-pinitol. These include uses for the prevention, treatment,
and management for diseases associated with .beta.-amyloid
accumulation. In addition to Alzheimer's, these disorders include,
but are not limited to, MCI, Down's Syndrome, cerebral amyloid
angiopathy, thrombolysis-related intracerebral haemorrhage, and
other diseases involving beta-amyloid deposition. In particular,
the present application provides methods for inhibiting the onset
and progression of AD as well as methods of treating AD symptoms
and AD disease. In another embodiment, the present invention
provides methods based on the use of D-pinitol to inhibit
amyloidosis and gamma-secretase activity in the nervous system,
preferably the brain. More generally, the present invention
provides methods for using D-pinitol compositions to treat MCI or
to promote a healthy nervous system through its neuroprotective
effects on neural structure and function.
[0050] The invention also provides animal systems for analyzing the
mechanistic relationship between NIDDM and AD-type diseases. In
addition, the present invention provides cell-free compositions
that preserve the membrane-bound configuration of APP under
physiological conditions. The invention provides uses of the
cell-free system in analyzing the processing of APP and identifying
modulatory compounds that target amyloidosis.
[0051] The present invention is also based, in part, on the finding
that caloric restriction attenuates .beta.-amyloid neuropathology
in a mouse model of Alzheimer's disease. Accordingly, the invention
provides methods to inhibit or treat AD and related disorders in an
individual based on reducing caloric intake.
DEFINITIONS
[0052] As used herein, "D-pinitol" takes its ordinary meaning in
the art.
[0053] As used herein, "mild cognitive impairment" or "MCI" takes
its ordinary meaning in the art.
[0054] As used herein, an "individual" is preferably a mammal and
more preferably a human, but can be any animal, including a
laboratory animal in the context of a clinical trial or screening
or activity experiment. Thus, as can be readily appreciated by one
of ordinary skill in the art, the methods of the present invention
are particularly suited to administration to any animal,
particularly a mammal, and including, but not limited to domestic
animals, wild animals and research animals.
[0055] As used herein, the term "inhibit" means preventing or
delaying the onset or progression of one or more clinical symptoms
of AD. Common early symptoms of Alzheimer's include confusion,
disturbances in short-term memory, problems with attention and
spatial orientation, personality changes, language difficulties,
unexplained mood swings.
[0056] The term "therapeutically effective amount" is used herein
to mean an amount or dose of d-pinitol that is effective to
ameliorate, delay, or prevent any of the foregoing symptoms,
behaviors or events associated with Alzheimer's disease or related
neurodegenerative disorders. Alternatively, a therapeutically
effective amount is sufficient to cause an improvement in a
clinically significant condition in the individual.
[0057] As used herein, the phrase "pharmaceutically acceptable"
refers to molecular entities and compositions that are "generally
regarded as "safe," e.g., that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government of listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans.
[0058] The term "treat" is used herein to mean to relieve or
alleviate at least one symptom of a disease in a subject. Within
the meaning of the present invention, the term "treat" also denotes
to arrest, delay the onset (i.e., the period prior to clinical
manifestation of a disease) and/or reduce the risk of developing or
worsening a disease. For example, it is known that there is a
familial pattern of AD, whereby first-degree biological relatives
of children with AD is more common than in the general
population.
[0059] The term promote as used herein means to have any positive
effect or benefit. As used, the term is relative and does not
necessarily mean to enhance or improve. For example, a composition
that maintains a normal rate of neuronal loss with age could still
be deemed as having a promoting effect.
[0060] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable, or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water or aqueous saline solutions and aqueous
dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E.W. Martin.
[0061] The terms "about" and "approximately" shall generally mean
an acceptable degree of error or variation for the quantity
measured given the nature or precision of the measurements.
Typical, exemplary degrees of error or variation are within 20
percent (%), preferably within 10%, and more preferably within 5%
of a given value or range of values. For biological systems, the
term "about" refers to an acceptable standard deviation of error,
preferably not more than 2-fold of a give value. Numerical
quantities given herein are approximate unless stated otherwise,
meaning that the term "about" or "approximately" can be inferred
when not expressly stated.
Molecular Biology
[0062] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. The general
genetic engineering tools and techniques discussed herein,
including transformation and expression, the use of host cells,
vectors, expression systems, etc., are well known in the art. (See,
e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual 3rd
ed. (2001), Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, New York; Ausubel et al. eds. Current Protocols in
Molecular Biology (2005), John Wiley and Sons, Inc.: Hoboken, N.J.;
Bonifacino et al. eds. Current Protocols in Cell Biology (2005),
John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds.
Current Protocols in Immunology (2005), John Wiley and Sons, Inc.:
Hoboken, N.J.; Coico et al. eds. Current Protocols in Microbiology
(2005), John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al.
eds. Current Protocols in Protein Science (2005), John Wiley and
Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. Current Protocols
in Pharmacology (2005), John Wiley and Sons, Inc.: Hoboken,
N.J.).
Therapeutic Methods
[0063] Disorders treatable by the compositions and methods of the
invention include, but are not limited to, Alzheimer's disease,
MCI, Down's Syndrome, cerebral amyloid angiopathy, and cerebral
hemorrhage, as well as other diseases involving beta-amyloid
deposition in other organs.
[0064] In particular, the present invention advantageously provides
a method of inhibiting the onset of the symptoms of Alzheimer's
disease or related disorders. In the case of AD, these symptoms may
range in degree from mild or moderate to severe (clinically
diagnosable Alzheimer's disease). The method comprises
administering to an individual at risk of developing the disease a
D-pinitol composition in an amount sufficient to prevent or delay
onset of the symptoms.
[0065] The present invention also relates to methods for treating
an individual who manifests one or more symptoms of Alzheimer's
disease or a related neurodegenerative disorder, such as MCI. Such
symptoms may be mild, moderate, or severe. The method comprises
administering to an individual displaying such symptoms a
therapeutically effective amount of D-pinitol, alone or in
combination with another effective agent, which is sufficient to
alleviate or slow the progression of one or more symptoms, or to
alleviate or slow the progression of the disease.
[0066] The concentration of D-pinitol depends on the desired dosage
and administration regimen. In one preferred embodiment, the
concentration of D-pinitol in blood or plasma is 10
micromolar.+-.50%.
[0067] The route of administration may be oral, parenteral,
transmucosal, intranasal, rectal, vaginal, or transdermal. Oral is
preferred. Parental routes include intravenous, intra-arteriole,
intramuscular, intradermal, subcutaneous, intraperitoneal,
intraventricular, intrathecal, and intracranial administration.
[0068] The pharmaceutical composition may also include other
biologically active substances in combination with the
compound.
[0069] The pharmaceutical composition can be added to a retained
physiological fluid such as blood or synovial fluid. For CNS
administration, a variety of techniques are available for promoting
transfer of the therapeutic agent across the blood brain barrier,
including disruption by surgery or injection, co-administration of
a drug that transiently opens adhesion contacts between CNS
vasculature endothelial cells, and co-administration of a substance
that facilitates translocation through such cells.
[0070] In another embodiment, the therapeutic agent can be
delivered in a controlled release manner. For example, a
therapeutic agent can be administered using intravenous infusion
with a continuous pump, in a polymer matrix such as
poly-lactic/glutamic acid (PLGA), in a pellet containing a mixture
of cholesterol and the active ingredient (SilasticR.TM.; Dow
Corning, Midland, Mich.; see U.S. Pat. No. 5,554,601), by
subcutaneous implantation, or by transdermal patch.
[0071] Preferably, the optimal therapeutically effective amount
should be determined experimentally, taking into consideration the
exact mode of administration, the indication toward which the
administration is directed, the subject involved (e.g., body
weight, health, age, sex, etc.), and the preference and experience
of the physician or veterinarian in charge.
[0072] The effective agent may be combined with a pharmaceutically
acceptable carrier, which term includes any and all solvents,
dispersion media, coatings, anti-oxidants, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions and
methods described herein is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0073] The formulation may be prepared for use in various methods
for administration. The formulation may be given orally, by
inhalation, applied topically or may be injected, e.g.
intravascular, intratumor, subcutaneous, intraperitoneal,
intramuscular, etc.
[0074] The dosage of the therapeutic formulation will vary widely,
depending upon the nature of the disease, the frequency of
administration, the manner of administration, the clearance of the
agent from the host, and the like. The initial dose may be larger,
followed by smaller maintenance doses. The dose may be administered
as infrequently as weekly or biweekly, or fractionated into smaller
doses and administered daily, semi-weekly, etc. to maintain an
effective dosage level. In some cases, oral administration will
require a higher dose than if administered intravenously.
[0075] The effective agent of the invention can be incorporated
into a variety of formulations for therapeutic administration. More
particularly, the complexes can be formulated into pharmaceutical
compositions by combination with appropriate, pharmaceutically
acceptable carriers or diluents, and may be formulated into
preparations in solid, semi-solid, liquid or gaseous forms, such as
tablets, capsules, powders, granules, ointments, solutions,
suppositories, injections, inhalants, gels, microspheres, and
aerosols.
[0076] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
D-Pinitol
[0077] Pinitol and derivatives and metabolites thereof were known
to be useful prior to the invention in certain nutritional and
medicinal compositions, such as for diabetes mellitus and its
chronic complications, but not AD or MCI. Pinitol and its
derivatives or metabolites are available from a number of natural
sources (such as, for example, pine needles, chick peas,
Bougainvillea leaves, alfalfa, soy beans and other legumes) and
synthetic processes. Use of D-pinitol for the treatment of
disorders associated with resistance to insulin is described, for
example, in WO 96/29063.
Methods of Promoting a Healthy Nervous System
[0078] The present invention relates further to a general method of
promoting a healthy nervous system by administration of active
ingredient compositions comprising D-pinitol. Such methods promote
normal structure and function of the nervous system, which is
critical for cognitive processes, such as learning and memory, as
well as emotional well-being.
[0079] The compositions of the present invention are intended for
administration to a mammal, in particular a human being, in a
suitable dosage form as is known in the art. Suitable dosage forms
known in the art include parenteral, enteral, and especially oral.
Oral solid and liquid dosage forms are particularly preferred.
[0080] Oral solid dosage forms are well known in the art and
include tablets, caplets, gel caps, capsules, and edible food
items. Oral solid dosage forms can be made with one or more
pharmaceutically acceptable excipients. Pharmaceutically acceptable
excipients assist or make possible the formation of a dosage form
for a bioactive material and include diluents, binding agents,
lubricants, glidants, disintegrants, coloring agents, and
flavorants and nutrients. An excipient is pharmaceutically
acceptable if, in addition to performing its desired function, it
is non-toxic, well tolerated upon ingestion, and does not interfere
with absorption of bioactive materials.
[0081] Tablets can be made by well-known compression techniques
using wet, dry, or fluidized bed granulation methods. For example,
the ingredients are mixed in a blender. Useful blenders include the
twin-shell type, the planetary mixer type, and the high-speed
high-shear type; all of which are known to the skilled artisan. The
blended combination is sieved and dried to a granulate. The
granulate is then compressed into tablets using a tableting press
as is known in the art. Preferably, the granulate is sieved before
the compression to make sure that the granulate has the desired
particle size.
D-Pinitol, a Natural Insulin Sensitizer Agent: Implications for AD
Treatment
[0082] The insulin sensitizer agent D-pinitol, which is
structurally related to the phosphatidylinositol phosphates that
participate in insulin-mediated stimulation of glucose transport
(Holman and Kasuga. Diabetologia 1997; 40:991-1003; White.
Diabetologia 1997; 40(Suppl2):S2-17), may beneficially influence
AD-type amyloid neuropathology through inhibition of
.gamma.-secretase activity in the brain resulting in reduced
generation of amyloidogenic A.beta..sub.1-42 and A.beta..sub.1-40
peptides. D-pinitol is found in high concentrations in pine tree
components (Asiegbu. FEMS Microbiol. Ecol 2000; 33:101-110) and is
present at about 1% dry weight in soy (Davis et al. Diabetes Care
2000; 23:1000-1005). In Indonesia, about 2 million metric tons of
soy were consumed in 1994 by a total population of 200 million
(Davis et al. Diabetes Care 2000; 23:1000-1005). This converts to
an average intake of 25-30 grams of soy per day per person or about
250-300 mg of pinitol, which is about 4 mg/kg/day in a 70 kg
individual. This daily dose range of pinitol is therefore
considered safe as evidenced by centuries of soy intake. In a like
manner, a serving containing over 100 grams (about 1/4 pound) of
soy per day, which is a reasonable portion for some individuals,
would provide about 1 gram of pinitol or a dose level of over 10
mg/kg in a 70 kg individual. While crude soy preparations are not
substantially pure and may not provide the most readily or rapidly
assimilated form of pinitol, it is obvious that pinitol consumption
through soy is substantial. Thus, by history of use, animal and
human clinical scientific studies, D-pinitol is non-toxic and safe
in man.
[0083] D-Pinitol has a demonstrated blood glucose lowering and
insulin function enhancing effect. Thus, dietary supplementation of
pinitol may be recommended to help maintain normal metabolic
function (Davis et al. Diabetes Care 2000; 23:1000-1005; Lamer.
IUBMB Life 2001; 51:139-148; Bates et al. Br. J. Pharmacol. 2000;
130:1944-1948). D-pinitol also has been identified in putative
insulin mediator fractions that have hypoglycemic activity, and
appears to act downstream in the insulin-signaling pathway to mimic
the effects of insulin (Lamer. IUBMB Life 2001; 51:139-148; Bates
et al. Br. J. Pharmacol. 2000; 130:1944-1948). Administration of
D-pinitol has been shown to lower blood glucose concentration in
streptozotocin-induced diabetic rats and in normal rats given
glucose (Bates et al. Br. J. Pharmacol. 2000; 130:1944-1948; Lamer
et al. J. Med. Chem. 2003; 46:3283-3291; Ortmeyer et al.
Endocrinology 1993; 132:646-65). This isomer also increased the
rate of glucose disappearance in insulin-resistant and
hyperinsulinemic monkeys (Ortmeyer et al. Endocrinology 1993;
132:646-651; Ortmeyer et al. Endocrinology 1993; 132:640-645),
suggesting that the D-pinitol itself might improve glucose
metabolism. Consistent with this evidence, supporting the
feasibility of the proposed studies, we found in preliminary
studies that treatment with safe doses of D-pinitol in the drinking
water (80 mg/kg body weight, per day) promotes cognitive functions
in Tg2576 mice that model AD neuropathology and memory
impairment.
Cell-Free Systems, Screening Methods, Assays, and Animal
Systems
[0084] The present invention further provides cell-free systems and
methods for identifying a candidate compound useful for modulating
the production of .beta.-amyloid. These systems are described in
detail in the Examples which follow.
[0085] The methods of the present invention can be used to identify
a candidate compound useful to treat a condition that can be
treated by modulating the levels of beta-amyloid.
[0086] The test compound can be, without limitation, a small
organic or inorganic molecule, a polypeptide (including an
antibody, antibody fragment, or other immunospecific molecule), an
oligonucleotide molecule, a nucleic acid molecule, or a chimera or
derivative thereof.
[0087] Furthermore, the present invention relates to the use of an
animal model of studying the impact of NIDDM on Alzheimer's
disease. In addition, the present invention provides a cell-free
system that faithfully carries out .gamma.-secretase-mediated
cleavage of the APP protein under physiological conditions.
EXAMPLES
[0088] The following Example(s) are understood to be exemplary
only, and do not limit the scope of the invention or the appended
claims.
Example 1
Confirmation of Diet-Induced NIDDM-Like Insulin Resistance in
Tg2576 Mice
Background
[0089] Type II diabetes (a non-insulin dependent form of diabetes
mellitus (NIDDM) is characterized by glucose intolerance, obesity,
and hyperinsulinemia. Studies of the proposed mechanistic link
between NIDDM and AD first required confirmation of diet-induced
NIDDM-like insulin resistance in the AD susceptible transgenic
mouse strain Tg2576.
Materials and Methods
[0090] Female Tg2576 mice (Hsiao et al. Science 1996; 274:99-102)
were exposed to either a high-fat diet (Tg insulin-resistant group)
or a standard rodent laboratory diet (Tg control group), starting
at 3-4 months of age. At 9 months of age, animals were then
assessed for indexes of NIDDM-like insulin resistance, including
insulin content, body weight, fat measurements, and glucose
tolerance.
Results and Discussion
[0091] FIG. 1 shows that administration of a high fat diet, as
described in the Materials and Methods, gives rise to the hallmarks
of NIDDM: increased levels of serum insulin (FIG. 1A); increased
body weight (FIG. 1B); increased weight of the ependymal fat-pad
(FIG. 1C); and increased levels of plasma glucose (FIG. 1D).
Example 2
Diet-Induced NIDDM-Like Insulin Resistance Promotes AD-Type Amyloid
Burden in Tg2576 Mice
Background
[0092] As shown in FIG. 1, dietary conditions in insulin-resistant
Tg2576 mice recapitulated the cardinal features of human-NIDDM,
including hyperinsulinemia, increased body weight, and glucose
intolerance, relative to the normoglycemic control mouse group. The
present study addressed the hypothesis that these altered metabolic
conditions might also causally promote AD-type amyloidosis in
Tg2576 mice.
Materials and Methods
[0093] Nine-month old insulin-resistant and normoglycemic Tg2576
female mice were assessed for indexes of AD-type amyloid burden in
the brain, as shown in FIG. 2.
Results and Discussion
[0094] Five months of exposure to a high-fat diet resulting in
NIDDM-like insulin resistance conditions promoted a greater than
two-fold elevation in amyloidogenic A.beta..sub.1-40 and
A.beta..sub.1-42 peptide content in the hippocampal formation of
9-month old Tg 2576 mice, relative to normoglycemic Tg2576 control
mice (P<0.05) (FIG. 2A). No detectable alteration in
22C11-immunoreactive total holo-APP steady-state level was found in
the hippocampal formation of insulin-resistant relative to
normoglycemic Tg2576 mice (FIG. 2A). Moreover, consistent with the
evidence that diet-induced NIDDM-like insulin resistance promoted
amyloidogenic A.beta. peptide accumulation in the hippocampal
formation, we found a .about.2 fold increase in cerebral cortical
AD-type amyloid (6E10-immunopositive) plaque burden (plaque volume
as percentage of the total cerebral cortical volume; P<0.05)
(FIG. 2B), relative to normoglycemic Tg2576 mice. The increased
volumetric cerebral cortical amyloid plaque burden in the
insulin-resistant Tg2576 mice was characterized by larger AD-type
(6E10 immunoreactive) amyloid plaque volume (P<0.001) rather
than increased number of AD-type amyloid plaques (FIG. 2B). Thus,
it is possible that diet-induced insulin resistance exacerbates
amyloidosis through mechanisms involved in A.beta. peptide
generation.
[0095] Subsequent experiments addressed the molecular mechanisms
through which NIDDM-like insulin resistance promoted AD-type
amyloidosis. The results revealed that dietary conditions leading
to NIDDM-like insulin resistance in Tg2576 mice is associated with
decreased IR signaling in the brain (data not shown; Ho et al.
FASEB J. 2004; 18: 902-904). Moreover, the NIDDM-like insulin
resistance in the brain resulted in altered regulation of AKT/PKB
and GSK-3.alpha. and GSK-3.beta. phosphorylation (data not shown).
This evidence suggests that diet induced NIDDM-like insulin in
Tg2576 mice is associated with decreased AKT/PKB activity leading
to GSK-3 activation. More specifically, insulin resistance
conditions may specifically promote GSK-3.alpha. and GSK-3.beta.
activities through attenuation of AKT/PKB mediated
pS.sup.21-GSK-3.alpha. and pS.sup.9-GSK-3.beta. phosphorylation in
the brain.
Example 3
NIDDM-Like Insulin Resistance Promotes .gamma.-Secretase Activity,
which Correlates with pS.sup.21-GSK-3.alpha. and
pS.sup.9-GSK-3.beta. Phosphorylation
Background
[0096] Based on the evidence that NIDDM-like insulin resistance
results in activation of GSK-3.alpha. and GSK-3.beta. activities in
the brain of Tg2576 mice disclosed above and on recent studies
suggesting that activation of GSK-3 (GSK-3.alpha.) can promote
A.beta. peptide generation (Phiel et al. Nature 2003; 423:435-439),
subsequent experiments addressed the role of NIDDM-like insulin
resistance on .gamma.-secretase activity in the brain and its
relationship with GSK-3.alpha. and GSK-3.beta. activities.
Materials and Methods
[0097] The activity of .gamma.-secretase was measured by an
established enzymatic assay (Shearman et al. Biochemistry 2000;
39:8698-8704), which is based on detection of the .about.7 kDa
carboxy terminal fragment (CTF)-.gamma. formation from
membrane-bound APP.
Results and Discussion
[0098] Insulin-resistant Tg2576 mice exhibited .about.2 fold
increased generation of cerebral cortical CTF-.gamma. (P<0.05),
in the absence of changes in steady-state holo-APP (22C11
immunopositive) content compared to normoglycemic Tg2576 mice (FIG.
3A). Most interestingly, the level of pS.sup.21-GSK-3.alpha. and
pS.sup.9-GSK-3.beta. phosphorylation (relative to total
GSK-3.alpha. and GSK-3.beta.) in the cerebral cortex positively
correlated with the generation of brain C-terminal fragment
(CTF)-.gamma. cleavage product of APP, an index of
.gamma.-secretase activity, in the brain of insulin-resistant
Tg2576 mice (pS.sup.21-GSK-3.alpha.: Pearson r=-0.9011, P<0.02;
pS.sup.9-GSK-3.beta.: Pearson r=-0.9206, P<0.01) (FIGS.
3B-C).
[0099] This evidence supports the hypothesis that the activation of
GSK-3.alpha. and GSK-3.beta. (reflected by decreased
pS.sup.21-GSK-3.alpha. and pS.sup.9-GSK-3.beta. phosphorylation)
may be a mechanism through which insulin resistance may promote the
generation of A.beta. peptides in the brain. This evidence suggests
that in Tg2576 mice, dietary conditions leading to NIDDM-like
insulin resistance may result in altered IR signaling in the brain
and eventually "unleash" GSK-3.alpha. and GSK-3.beta. activity
capable of promoting .gamma.-secretase activity and the generation
of amyloidogenic A.beta. peptides. Further support derives from the
analysis of 12-month old insulin resistant- and control
normoglycemic-Tg2576 mice for indices of IR signaling and GSK-3
activity in the cerebral cortex. Compared with controls, the
insulin-resistant mice showed a .about.2-fold reduction of
Y.sup.1162/1163-IR.beta. autophosphorylation (an index of reduced
IR.beta. activation) coinciding with a decrease in PI3K-(p85) and
pS.sup.473-AKT (data not shown). This is indicative of reduced
PI3K-AKT signaling in the brain of insulin-resistant versus
normogylcemic Tg2576 mice. Compared to normoglycemic controls, the
brain of insulin-resistant Tg2576 mice also showed decreased
pS.sup.21-GSK-3.alpha. and pS.sup.9-GSK-3.beta. phosphorylation
(indicative of GSK-3 activation) coinciding with increased
.gamma.-secretase activity (data not shown).
Example 4
Diet-Induced NIDDM-Like Insulin Resistance Accelerates Cognitive
Impairment in Insulin-Resistant Tg2576 Mice Relative to
Normoglycemic Tg2576 Mice
Background
[0100] Based on the previous results showing that diet-induced
NIDDM-like insulin resistance coincided with promotion of cortical
AD-type amyloidosis, and the existing evidence that AD type
amyloidosis correlates with cognitive impairment in Tg2576 mice
(Kawarabayashi et al. J. Neurosci. 2001; 21:372-381; King and
Arendash. Physiol Behav. 2002; 15:627-642), the present studies
addressed the possibility that the increased AD-type amyloidosis
might have accelerated cognitive impairment in 9-month Tg2576 mice,
which are otherwise not behaviorally impaired at this age (King and
Arendash. Physiol Behav. 2002; 15:627-642).
Materials and Methods
[0101] In this study, nine-month old Tg2576 female mice (n=5-6 per
group) were trained for eight days in a hidden platform version of
the water maze. Twenty-four hours following the learning phase, a
probe trial was conducted wherein the platform was removed.
Results and Discussion
[0102] Nine-month old insulin-resistant Tg2576 mice exhibited
impaired ability to learn in a Morris spatial maze task relative to
control age-gender matched normoglycemic Tg2576 mice. In
particular, we found that insulin-resistant Tg2576 mice exhibited
longer escape latencies during a hidden platform-learning phase of
testing (which is an index of hippocampal memory dysfunction
severely affected in AD) compared to control Tg2576 mice fed
standard rodent laboratory diet (FIG. 4A). Moreover, this apparent
impairment in learning coincided with a lack of navigation bias
(FIGS. 4B-D) for the former platform location in a probe trial,
wherein the escape platform was removed 24 h following the learning
phase.
[0103] This evidence suggests that diet-induced NIDDM-like insulin
resistance induced a deficit in spatial learning in Tg2576 mice in
the absence of changes to baseline visual ability (assessed in a
cued version of the swim task; FIG. 4E) or motor activity (not
shown). The study provided the basis to further explore the role of
D-pinitol in this setting.
Example 5
Treatment of Tg2576 Mice with D-Pinitol Reduces
Insulin-Resistance-Mediated Promotion of .beta.-Amyloidosis in the
Brain
Background
[0104] Given that insulin resistance promotes .beta.-amyloid
accumulation in Tg2576 mice that model AD type neuropathology (FIG.
2; Ho et al. FASEB J. 2004; 18:902-904), the present experiments
tested whether treatment of Tg2576 mice with insulin sensitizing
agent D-pinitol might ameliorate the .beta.-amyloid pathology in
different regions of the brain.
Materials and Methods
[0105] This study assessed A.beta. peptide contents in the brain
and in the periphery (serum) of 12-month old female Tg2576 mice in
response to chronic treatment, initiating at 4 month of age, with a
high-fat (HF) diet or HF diet plus pinitol (N=3-4 per group).
Gender-age-matched Tg2576 mice maintained on standard rodent
laboratory diet (LF diet) served as controls. High-fat diet and
standard laboratory diet containing equal % of cholesterol per
g/diet were purchased from Research Diet, Inc. (New Brunswick,
N.J.) (Ho et al, FASEB J 2004; 18:902-204). Pinitol (Humanetics
Corp.) was provided by incorporation into drinking water (600 mg
pinitol/liter water), which is available ad libitum. Based on the
assumption that an adult mouse drinks 5-ml of water per day, and
that the body weight of an adult mouse is 30 gm, we estimated each
mouse received 100 mg pinitol per kg body weight per day.
Twelve-month old mice were sacrificed by decapitation. Blood was
collected, clotted in room temperature for 15 min and centrifuged
at 3,000.times.g for 20 min to separate the serum. Serum specimens
were collected and stored at -80.degree. C.
[0106] For quantitative assessment of A.beta. peptides (Xiang et
al. Gene Expr. 2002, 10:271-278), frozen pulverized brain tissues
were homogenized in 5.0 M guanidine buffer, diluted (1:10) in
phosphate-buffered saline containing 0.05% (v/v) Tween-20 and 1 mM
Pefabloc protease inhibitors (Roche Biochemicals, Indianapolis,
Ind.) and centrifuged for 20 min at 4.degree. C. Total
A.beta..sub.1-40 or A.beta..sub.1-42 was measured by sandwich ELISA
(BioSource, Camarillo, Calif.) according to the manufacturer's
instructions. Serum specimens were assessed directly using the same
ELISA according to the manufacturer's instructions.
Results and Discussion
[0107] The results show amelioration of .beta.-amyloidogenesis in
all regions of the brain tested (FIG. 5). Moreover, the
anti-amyloidogenic activity of D-pinitol in Tg2576 mice is also
associated with the restoration in the brain of (i) insulin
receptor signal transduction; and (ii) GSK-3 activity. Indeed,
D-pinitol treatment at 80 mg/kg/day D-pinitol in the drinking water
beginning at six months of age increased
y.sup.1162/1163-phosphorylated IR.beta. (but not total IR.beta.) as
well as PI3K-p85.alpha. and S.sup.473-AKT in 12-month old Tg2576
mice, compared to insulin resistant and control Tg2576 mice.
Example 7
D-Pinitol Selectively Inhibits .gamma.-Secretase Activity in
Cultured Cells
Background
[0108] The observation that D-pinitol beneficially influences
cognitive impairment in NIDDM-like insulin resistant TG2576 mice
prompted exploration of the potential role of D-pinitol in
mechanisms associated with generation of amyloidogenic A.beta.
peptides in an in vitro model of amyloidosis. The generation of
A.beta. from APP requires at least two proteolytic enzymes referred
to as .beta.- and .gamma.-secretase. .beta.-secretase mediates the
N-terminal cleavage of APP producing C-terminal fragment
(CTF)-.beta., which is the immediate precursor for intra-membranous
cleavage by .gamma.-secretase. The .gamma.-secretase cut liberates
A.beta. from the membrane and generates an intracellular APP
CTF-.gamma. fragment, which represents the carboxyl terminal
counterpart of A.beta. (see FIG. 6). A third proteolytic enzyme,
.alpha.-secretase, may be responsible for breaking down A.beta.
peptides.
[0109] The activity of .gamma.-secretase is of great importance for
two reasons: firstly, this enzyme (in conjunction with
.beta.-secretase) is essential to A.beta. generation; and secondly
.gamma.-secretase cleavage determines the amyloidogenicity of the
A.beta. peptide to be formed (i.e. A.beta.1-38, A.beta.1-40,
A.beta.1-42). For these reasons, .gamma.-secretase inhibition is
currently thought to be a major therapeutic target for A.beta.
lowering strategies.
Materials and Methods
Cell-Based Secretase Assays
[0110] In this study, cultured human glioma H4-cells with
constitutive APP.sub.751 expression were treated with increasing
concentrations of pinitol, and then cellular .alpha.- and
.beta.-secretase activity as well as A.beta. peptide contents in
the conditioned media were assessed in response to pinitol
treatment over time. Growth and maintenance of H4-APP.sub.751 cells
were as described in Qin et al. (J. Biol. Chem. 2002;
278:50970-50977). Confluent cell cultures were treated with
increasing concentration of pinitol. At 24 and 48 hours after
pinitol treatment, cells were harvested for secretase activity
assays and conditioned media were collected for measurement of
A.beta. peptide contents. The .alpha.-secretase (FIG. 7A) and
.beta.-secretase (FIG. 7B) activities in pinitol-treated cells were
measured using commercially available kits (R&D Systems).
Harvested cells were homogenized in supplied buffers and homogenate
was added to a secretase-specific APP peptide conjugated to the
reporter molecules EDANS and DABCYL. In the uncleaved form, the
fluorescent emissions from EDANS are quenched by the physical
proximity of the DABCYL moiety, which exhibits maximal absorption
at the same wavelength (495 nm). Cleavage of the peptide by the
secretase physically separates the EDANS and DABCYL, allowing the
release of a fluorescent signal. The level of .alpha.-secretase
(FIG. 7A) or .beta.-secretase (FIG. 7B) enzymatic activity is
proportional to the fluorometric reaction in the homogenate. These
data depict the content of A.beta..sub.1-40 (FIG. 7C) and
A.beta..sub.1-42 (FIG. 7D) peptides in the conditioned culture
media in response to pinitol treatment over time. A.beta. peptides
were measured by ELISA as described above. In FIG. 7, each value
represents Mean.+-.SEM; n=4 cultures per assay; * P<0.05 vs.
control (no pinitol treatment) group (2-tailed Student's t
test).
In-Vitro Model of Amyloidosis
[0111] For assessment of .gamma.-secretase activity, cell
monolayers were rinsed twice with ice-cold PBS on ice, scraped from
tissue culture dishes and centrifuged (1,500 rpm, 10 min, 4.degree.
C.). Cell pellets were then resuspended (0.5 ml/10 cm dish) in
homogenization buffer (10 mM MOPS pH 7.0, 10 mM potassium chloride,
1.times. complete protease inhibitor (PI) (Roche Molecular
Biochemicals, Germany) and homogenized by passing cell suspensions
through a 23-gauge needle 10 times. Homogenates were then
centrifuged (2,500 rpm, 15 min at 4.degree. C.) to remove unbroken
cells and nuclei. The supernatant (membrane and post-nuclear
supernatant) was then centrifuged (14,000 rpm, 20 min, 4.degree.
C.) and rinsed in homogenization buffer. Membranes were then
resuspended (0.5 ml/10 cm dish) in assay buffer (150 mM sodium
citrate pH 6.4; 1.times.PI) and incubated at (2 hr) at 37.degree.
C. in 25 .mu.l of incubation buffer per assay sample to allow for
generation of the CTF-.gamma. cleavage product; negative control
samples were maintained on ice. CTF-.gamma. cleavage products (as
well as CTF-.alpha. and CTF-.beta.) were resolved
electrophoretically in 10-20% Tris-Tricine gels (Bio Rad
Laboratories, CA), and identified using an anti APP polyclonal C8
antibody. Immunoreactivities were visualized autoradiographically
using a chemiluminescence detection kit (SuperSignal; Pierce
Biotechnology, Rockford, Ill.). A.beta.1-40 was also assayed by
ELISA in the tissue culture preparations used for .gamma.-secretase
activity. Bar graphs represent Mean.+-.SEM; n=4 *P<0.01 vs.
vehicle treated cells (PBS).
Results and Discussion
[0112] Dose-dependent studies of D-pinitol administration in
cultured cells showed no effect on .alpha.- or .beta.-secretase
activity (FIGS. 8A-B). In contrast, these studies did reveal a
robust effect on .gamma.-secretase activity, leading to a
dose-dependent inhibition of both A.beta..sub.1-40 and
A.beta..sub.1-42 production (FIGS. 8C-D).
[0113] The selective effect of D-pinitol on .gamma.-secretase
activity was further analyzed with a powerful in vitro amyloidosis
system developed by the inventors. Unlike conventional cell-free
assays, this in vitro .gamma.-secretase assay detects enzymatic
cleavage under physiologic conditions, wherein APP and/or
CTF-.gamma. are cleaved while membrane bound. The .about.7 kDa
CTF-.gamma. APP fragment is readily segregated away from other CTFs
generated by .alpha.- and .beta.-secretase (CTF-.alpha. and
CTF-.beta., respectively) (see FIG. 6). Quantification of
CTF-.gamma. provides a direct assessment of .gamma.-secretase
activity. As shown in FIG. 8A, the formation of the .about.7 kDa
CTF-.gamma. APP fragment in H4-APPswe cells was blocked by
treatment with D-pinitol in a dose-dependent manner, supporting the
specificity of D-pinitol on .gamma.-secretase activity. In
contrast, d-chiro-inositol (DCI), a metabolite of D-pinitol, showed
no inhibitory effect on .beta.-amyloid formation in the in vitro
system.
[0114] As independent corroboration of these findings, formation of
the .about.7 kDa CTF-.gamma. APP was also blocked in a dose
dependent fashion in extracts obtained from CHO-APPswe cells, and
this reduction coincided with a corresponding reduction of
A.beta.1-40 peptide in the same tissue culture (data not shown).
Furthermore, control studies, based on the LDL assay, revealed no
detectable cell toxicity following D-pinitol treatment at all
concentration tested (at 50-250 .mu.M) as assessed by the LDH assay
(not shown).
[0115] The selectivity of D-pinitol action was further addressed by
examining its effect on cleavage of the signaling molecule NOTCH.
The "gamma-secretase" complex, consisting of presenilins (PS),
nicastrin (NCT), APH-1 and PEN-2, catalyzes the intramembraneous
proteolysis of truncated Notch derivatives to generate NICD,
respectively. In contrast to its effect on APP, D-pinitol had no
inhibitory effect on cleavage of NOTCH (FIG. 8B). In accord with
these observations, D-pinitol also had no inhibitory effect on
cleavage of NOTCH in the neocortex of Tg2576 transgenic mice,
compared to insulin-resistant and control Tg2576 mice (data not
shown).
Example 8
Caloric Restriction Attenuates .beta.-Amyloid Neuropathology in a
Mouse Model of Alzheimer's Disease
Background
[0116] Recent prospective studies indicate that increased caloric
intake is a risk factor for Alzheimer's disease (AD). While there
is evidence supporting a potential neuroprotective role of caloric
restriction (CR) in the brain (Cummings. N. Engl. J. Med. 2004;
315:56-67; Anson et al. Proc. Natl. Acad. Sci. USA 100; 6216-6220),
there is no information whether a CR regimen may influence AD. A
fundamental problem in AD is the aberrant generation of
amyloidogenic .beta.-amyloid peptides in the brain leading to
abnormal deposition of neuritic plaques, which is a
neuropathological landmark in AD (Cummings. N. Engl. J. Med. 2004;
351:56-67). Based on this consideration, this study used the mouse
model of AD-type amyloidosis (Hsiao et al. Science 1996;
274:99-102) to test the hypothesis that CR may beneficially
influence AD through mechanisms that prevent A.beta. generation and
eventually, neuritic plaque deposition in the brain.
Material and Methods
Tg2576 Mice and Diets
[0117] Three-month-old female Tg2576 mice (Hsiao et al. Science
1992; 274:99-102) (Taconic, Inc. Germantown Inc.) were randomly
assigned to a CR or an ad-libitum (AL) dietary regimen. CR was
achieved by feeding Tg2576 mice 70% of the calories consumed by the
pair-controlled AL animals, as previously reported (Anson et al.
Proc. Natl. Acad. Sci. USA. 2003; 100:6216-6220) with a few
modifications. At 10 AM each day, CR mice were provided with a food
allotment equal to 70% of the average daily caloric intake in the
AL-fed group. Food consumption in the CR-fed group was adjusted
weekly, based on the average daily caloric intake among the AL-fed
mice. Components of the modified AL and CR diets (AL diet
#D03020305, CR diet #D03081901; Research Diet, Inc. New Brunswick,
N.J.) are further described in Table 1. Tap water was available to
both the CR and AL-fed groups. At 12 months of age, the mice were
anesthetized with the general inhalation anesthetic
1-chloro-2,2,2-trifluoroethyl difluoromethyl ether (Baxter
Healthcare, Deerfield, Ill.) and sacrificed by decapitation. Brain
specimens were harvested and hemi-dissected, with one hemisphere
post-fixed in 4% paraformaldehyde (24 hr) for morphological studies
(see below). Hippocampus and neocortex were dissected from the
opposite hemisphere in each case. Brain samples were then rapidly
frozen and pulverized in liquid nitrogen, then stored at
-80.degree. C. for subsequent ELISA, immunoprecipitation-mass
spectrometry (IP-MS) studies for A.beta. peptide assessment,
western blot studies and .alpha.- and .beta.-secretase activity
assays. Ependymal fat-pad was collected and weighted at the time of
sacrifice. To assess glucose utilization as an index of CR, glucose
tolerance was assessed using an intraperitoneal glucose tolerance
test (IGTT), as previously described in Ho et al. (FASEB J. 2004;
18:902-904) two weeks before sacrifice. Briefly, mice were given a
single dose of glucose administered intraperitoneally (2 g/kg body
weight) postprandially, and blood was collected from a tail vein
periodically over a 2-hr period. Blood glucose content was assessed
using the OneTouch LifeScan System (LifeScan, Milpitas, Calif.)
according to manufacturer's instructions. All animal studies were
conducted following protocols approved by the Mount Sinai School of
Medicine Institutional Animal Use Committee.
Stereologic Assessment of Alzheimer's Disease-Type Amyloid Burden
in Tg2576 Mice
[0118] Freshly harvested mouse brain hemispheres were
immersion-fixed overnight in 4% paraformaldehyde, then sectioned in
the coronal plane on a Vibratome at a nominal thickness of 50
.mu.m. Every 20th section was selected from a random start position
and processed for thioflavin-S staining as previously described
(Vallet et al. Acta Neuropathol. (Berl) 1992; 83:170-178). Sections
adjacent to those used for thioflavin-S staining were stained with
cresyl violet to clarify the cytoarchitecture. This resulted in two
adjacent series of 14 evenly spaced sections each. All stereologic
analyses were performed using a Zeiss Axiophot photomicroscope
equipped with a Zeiss motorized stage and MSP65 stage controller, a
high resolution Zeiss ZVS-47E digital camera, and a Macintosh G3
computer running the custom designed software NeuroZoom (Nimchinsky
et al. J. Comp. Neurol. 2000; 416:112-125). The amyloid burden was
estimated using the Cavalieri principle with a small size grid
(50.times.50 .mu.m) for point counting according to the method
described by Bussiere et al. (Neuroscience 2002; 112:75-91). This
procedure provides an unbiased estimate of the fractional volume of
the region of interest occupied by amyloid plaques, and the amyloid
burden was calculated in percentage from the neocortical or
hippocampal volume itself. The number of amyloid plaques was
estimated by systematically counting all the thioflavin-S amyloid
plaques present in the series of sections at low magnification
(10.times.). Estimates of plaque volume were obtained using a
systematic random sampling procedure at intermediate magnification
(40.times.) as previously described in Bussiere et al.
(Neuroscience 2002; 112:75-91).
A.beta. ELISA Assay
[0119] For quantitative assessment of brain A.beta. peptides,
frozen pulverized tissue was homogenized in 5.0 M guanidine buffer,
diluted (1:10) in phosphate-buffered saline containing 0.05% (v/v)
Tween-20 and 1 mM Pefabloc protease inhibitors (Roche Biochemicals,
Indianapolis, Ind.) and centrifuged for 20 min at 4.degree. C.
Total A.beta.1-40 or A.beta.1-42 was quantified by sandwich ELISA
(BioSource, Camarillo, Calif.) as previously reported (Ho et al.
FASEB J. 2004:18; 902-904).
Assessment of Amyloid Precursor Protein (APP) Processing
(sAPP.alpha., Full-Length APP, ADAM10, ADAM17, ADAM 9, and
BACE1)
[0120] For western blot analysis of sAPP.alpha., frozen pulverized
tissue was homogenized in 0.5 ml of 20 mM Tris.HCl buffer (2 mM
EGTA, I mM EDTA, I mM benzamide, 1 mM dithiothreitol and 1 mM PMSF.
PH 7.4) and centrifuged (100,000.times.g for I hr). The
supernatants were collected and protein content was determined by
the Bradford method (Bio-Rad Laboratories, Hercules, Calif.) and
boiled. Proteins were resolved electrophoretically by SDS-PAGE and
transferred to Immobilon-P transfer membranes (Millipore
Corporation, MA) and immunoreacted with 6E10 antibody (1:1,000
dilution, Senetek, St. Louis, Mo.) raised against amino acid
residues 1-17 of the A.beta. domain of APP, a site that constitutes
the C-terminus of the sAPP.alpha. peptide sequence. For western
blot analysis of full-length APP, ADAMs, and BACE1, frozen
pulverized tissue was lysed in RIPA buffer (150 mM NaCl, 1% Nonidet
P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris, pH 8.0)
containing protease inhibitors. Lysates were then centrifuged
(10,000.times.g for 10 min) and supernatants collected. Following
this step, protein content determination, electrophoresis and
membrane transfer were carried out as described for sAPP.alpha.
assessment. Monoclonal 22C11 antibody (1:1,000 dilution, Chemicon
International, Temecula, Calif.) raised against amino acid residues
60-100 of the amino-terminal epitope of human APP was used to
assess total full-length APP. Polyclonal anti-ADAM10 and anti-ADAM9
antibodies (1:500 dilution respectively, Chemicon International,
Temecula, Calif.), polyclonal TACE antibody (1:1,000 dilution,
Santa Cruz Biotechnology, CA) and polyclonal BACE (M-83) (1:200
dilution, Santa Cruz Biotechnology, CA) were used for detection of
the concentrations of ADAM10, ADAM9, TACE (ADAM17) and BACE1,
respectively. .beta.-actin immunoreactivity (1:3,000 dilution,
Sigma, St. Louis, Mo.) was used to control for selectivity of
changes. Immunoreactivities were visualized by fluorescence
autoradiography using enhanced chemiluminescence detection
(SuperSignal Chemiluminescent detection kit, Pierce, Rockford,
Ill.) and quantified densitometrically.
Immunoprecipitation-Mass Spectrometry (IP-MS)
[0121] IP-MS analysis of AP peptides was performed as previously
described (Wang et al. J. Biol. Chem. 1996; 271:31894-31902).
Formic acid (5M) extracts were neutralized with 2 M Tris to a final
pH of 8.0 in the presence of protease inhibitors as previously
described (Wang et al. J. Biol. Chem. 1996; 271:31894-31902).
Synthetic A.beta.12-28 (20 nM) was added as an internal standard
and AP peptides were immunoprecipitated with the monoclonal
antibody 4G8 and 6E10 (Senetek, St. Louis, Mo.) sequentially using
Protein A/G-Plus agarose beads. A.beta. peptides were analyzed by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry using a Voyager-DE STR mass spectrometer (Applied
Biosystems). Each mass spectrum was averaged from 1000 measurements
and calibrated with bovine insulin as an internal calibrant.
Relative peak intensities of A.beta. peptide to internal standard
were used for quantitation.
Secretase Activity Assays
[0122] .alpha.- and .beta.-secretase activities were assessed using
commercially available kits (R&D Systems, Minneapolis, Minn.).
Briefly, brain samples were homogenized in supplied buffers. The
homogenate was then added to a secretase-specific APP peptide
conjugated to the reporter molecules EDANS and DABCYL. In the
uncleaved form, the fluorescent emissions from EDANS are quenched
by the physical proximity of the DABCYL moiety, which exhibits
maximal absorption at the same wavelength (495 nm). Cleavage of the
peptide by the secretase physically separates the EDANS and DABCYL,
allowing for the release of a fluorescent signal. The level of
secretase enzymatic activity is proportional to the fluorometric
reaction in the homogenate (R & D Systems).
Statistical Analysis
[0123] All values are expressed as means.+-.SEM. Differences among
means were analyzed using two-way ANOVA, while differences between
means were assessed by two-tailed Student t test as indicated in
Results and Discussion. In all analyses, the null hypothesis was
rejected at the 0.05 level, calculated by the Prism Stat software
program (GraphPad Software, Inc., San Diego, Calif.).
Results and Discussion
Chronic Treatment of Tg2576 Mice with a Caloric Restriction Dietary
Regimen Resulted in Significantly Lower AD Amyloid Neuropathology
in the Brain
[0124] To test the hypothesis that CR can modulate amyloidosis,
3-month-old Tg2576 mice, which develop AD-type amyloid
neuropathology by 8 to 10 months of age (Hsiao et al. Science 1996;
274:99-102; Kawarabayashi et al. J. Neurosci. 2001; 21:372-381)
were maintained for 9 months with a daily low carbohydrate diet
resulting in a 30% lower caloric intake compared to that consumed
by age- and gender-matched control Tg2576 mice fed ad-libitum (AL)
with a standard laboratory rodent diet.
[0125] Nutrient composition in the CR diet was adjusted so that CR
was achieved by selectively reducing the carbohydrate content of
the diet while consumption of protein, fat, cholesterol, vitamins,
and minerals was identical to that of AL-fed Tg2576 mice (Table
1).
TABLE-US-00001 TABLE 1 Nutrient composition in ad-libitum (AL) and
caloric restriction (CR) diets. Animals on the CR diet consumed
amounts of proteins, fat, cholesterol, and micronutrients identical
to those consumed by those on the AL diet. CR animals, however,
consumed 42% less carbohydrates, accounting for a 30% decreases in
total caloric intake. Ad-libitum diet Caloric restriction diet
nutrient composition nutrient composition (per 1 g diet) (per 0.71
g diet) Total calories 3.8 2.7 (30% caloric restriction) Protein
(g) 0.19 0.20 Carbohydate (g) 0.67 0.39 Fat (g) 0.04 0.04 Vitamin
mix (mg) 9.48 9.48 Mineral mix (mg) 9.48 9.48 Cholesterol (mg) 0.20
0.20 Cellulose (mg) 47.38 47.38 Dicalcium phosphate (mg) 12.32
12.32 Calcium carbonate (mg) 5.21 5.21 Potassium citrate (mg) 15.64
15.64 Choline bitartrate (mg) 1.90 1.90
[0126] This dietary regimen resulted in body weight stabilization
over the 9-month study period among CR Tg2576 mice relative to the
AL-fed group (FIG. 9A), and coincided with .about.3-fold lower
ependymal fat pad weight (FIG. 9B), and improved glucose tolerance
response as determined by an intraperitoneal glucose tolerance test
(FIG. 9C). These physiological adaptations in the CR Tg2576 mice
relative to AL-fed controls are consistent with clinical evidence
that low carbohydrate CR considerably improves abnormal glucose
control and obesity (Meckling et al. J. Clin. Endocrinol. Metab.
2004; 89:2717-2723; Yancy et al. Ann. Int. Med. 2004; 140:769-777;
Stern et al. Ann. Int. Med. 2004; 140:778-785), which are risk
factors not only for diabetes but also for AD (Gustafson et al.
Arch. Intern. Med. 2003; 163:1524-1528; Vanhanen and Soininen.
Curr. Opin. Neurol. 1998; 11:673-677; Kuusisto et al. BMJ 1997;
315:1045-1049).
[0127] When Tg2576 mice were examined for AD-type neuropathology at
12 months of age, we found that 9-month CR treatment almost
completely prevented cortical and hippocampal AD-type amyloid
plaque development (FIGS. 10A-B) relative to animals in the AL-fed
group. To further evaluate the anti-amyloidogenic role of CR in the
brain of Tg2576 mice, studies were carried out to explore APP
processing and A.beta. peptide generation using immunoprecipitation
(IP)-mass spectrometry (IP-MS) (Wang et al. J. Biol. Chem 1996;
271:31894-31902). Consistent with the aforementioned ELISA
evidence, using 4G8 antibody for A.beta. IP, the results confirmed
decreased levels of A.beta..sub.1-40 and A.beta..sub.1-42 in the
same neocortical samples used for the A.beta. ELISA assay (FIGS.
10C-D). In addition, a relative proportional reduction in
A.beta..sub.1-37, A.beta..sub.1-38, and A.beta..sub.1-39 peptide
content was also observed in the neocortex of the CR group compared
to the AL-fed control group. This evidence, together with our
observation that the concentration of the .about.7 kDa carboxy
terminal fragment (CTF)-.gamma. cleavage product of APP, an index
of .gamma.-secretase activity, was unchanged in the neocortex of
the CR group relative to AL-fed controls (FIGS. 11A-B), is
consistent with the possibility that .gamma.-secretase activity was
not involved in the CR-associated anti-amyloidogenic activity.
The Anti-Amyloidogenic Role of CR is Associated with Selective
Promotion of Anti-Amyloidogenic .alpha.-Secretase Activity.
[0128] To further identify A.beta. carboxy-termini peptide
fragments that would have been otherwise undetected in the 4G8
IP-MS studies, additional A.beta. IP-MS studies were carried out
using 6E10 antibody. Consistent with the 4G8 IP-MS spectra, the
results showed decreased levels of A.beta.1-40 and A.beta.1-42 as
well as A.beta.1-37, A.beta.1-38, and A.beta.1-39 peptides, in the
CR group relative to AL-fed control animals (FIG. 10E), In
addition, the results reveal major elevation in A.beta.1-16 peptide
fragment concentration in the neocortex of the CR group that was
not detected in the AL-fed controls (FIG. 10E). Because
.alpha.-secretase can cleave APP, eventually resulting in the
generation of AP C-termini fragments ending at the AA residue
leucine16 of A.beta. (Lammich et al. Proc. Natl. Acad. Sci. USA
1999; 96:3922-3927; Lichtenthaler et al. J. Clin. Invest. 2004;
113:1384-1387; Wang et al. J. Biol. Chem. 1991; 226:16960-16864),
further studies were carried out to explore the role of CR in
.alpha.-secretase activity in the brain.
[0129] Cleavage of APP by .alpha.-secretase releases the
amino-terminal extracellular domain known as soluble
.alpha.-secretase amyloid precursor protein (sAPP.alpha.) domain
coinciding with elevation in membrane-bound
.alpha.-secretase-cleaved APP carboxy-terminal fragment
(CTF)-.alpha. (Wang et al. J. Biol. Chem. 1996; 271:31894-31902).
Studies were therefore carried out to explore the regulation of
sAPP.alpha. and CTF-.alpha. cleavage products of APP in the brain
as indices of .alpha.-secretase activity in response to CR.
Interestingly, CR in Tg2576 mice resulted in a >2-fold elevation
in concentration of neocortical sAPP.alpha. (FIG. 12A) and
membrane-associated CTF-.alpha. (FIG. 12B) relative to AL-fed
control Tg2576 mice. The increase in CTF-.alpha.was somewhat less
(.about.4.6-fold) presumably because of further cleavage of
CTF-.alpha. by .gamma.-secretase. Compared with the CTF-.alpha.
fragment, the abundance of CTF-.beta. signal was at the limit of
detection in the neocortex of both CR and AL-fed Tg2576 mice,
preventing exact quantification (FIG. 12B).
CR May Influence .alpha.-Secretase Activity in the Brain, in Part
by Selectively Promoting the Generation of Mature, Catalytically
Active ADAM Species.
[0130] In light of recent evidence indicating that the proteinase
ADAM10 (a disintegrin and metalloproteinase) may act as an
.alpha.-secretase (Postina et al. J. Clin. Invest. 2004;
113:1456-1464), further studies were carried out to explore the
regulation of ADAM10 expression in the brains of Tg2576 mice in
response to CR, relative to AL-fed controls. Both mature (62 kDa)
and proform (90 kDa) ADAM10 species were detected in the neocortex
of the AL-fed control animals, confirming previous evidence
(Postina et al. J. Clin. Invest. 2004; 113:1456-1464) (FIG. 12C).
The 62 kDa mature ADAM10 protein species is known to act as an
.alpha.-secretase in vitro and to cleave A.beta.-derived peptides
at leucine.sub.16 (Lichtenthaler et al. J. Clin. Invest. 2004;
113:1384-1387). Strikingly, CR diet regimen resulted in a 30%
elevation of neocortical mature ADAM10 species concentration (FIG.
12C), coinciding with a commensurate elevation in neocortical
.alpha.-secretase activity (assessed fluorimetrically (Burns et al.
J. Neurosci. 2003; 23:5645-5649)) compared to AL-fed control mice
(FIG. 12D). No detectable change in proform ADAM10 species
concentration was noted in the neocortex of the CR group relative
to the AL-fed control group (FIG. 12C). In addition to ADAM10, two
other members of the ADAM proteinase family, namely ADAM17 (TACE)
and ADAM9, can act as .alpha.-secretase in various cell lines (20;
25-27).
[0131] In parallel studies, CR regimen in Tg2576 mice did not
change the concentration of the proform or the mature form of
either the ADAM17 (FIG. 13A) or the ADAM9 (FIG. 13B) species,
relative to AL-fed control Tg2576 mice. Similarly, there was no
detectable change in the neocortical concentration of the BACE1
species or of .beta.-secretase activity in response to CR relative
to the AL-fed control group (FIG. 13C).
[0132] These studies support the hypothesis that low carbohydrate
CR may prevent AD-type amyloid neuropathology through mechanisms
that influence .alpha.-secretase activity in the brain, possibly by
promoting the generation of mature, catalytically active ADAM10
species. Since .alpha.-secretase proteolysis of the APP sequence
within the A.beta. peptide would preclude the generation of
amyloidogenic A.beta. peptides, the studies here suggest that CR
may provide an attractive anti-amyloidogenic strategy by promoting
.alpha.-secretase activity in the brain. Moreover, because AD-type
amyloid neuropathology is undetectable in the brain of 3-month-old
Tg2576 mice (Kawarabayashi et al. J. Neurosci. 2001; 21:372-381),
the age when our mice were initially exposed to the CR dietary
regimen, the studies here support the possibility that CR, by
promoting .alpha.-secretase activity preventively, possibly through
mechanisms involving ADAM10 maturation, may result in reduced
AD-type amyloid neuropathology. This possibility is supported by
recent evidence showing that decreased .alpha.-secretase activity
in the brain of dominant negative ADAM10-APP(v717) double
transgenic mice develop more amyloid neuropathology than controls
(Postina et al. J. Clin. Invest. 2004; 113:1456-1464).
[0133] Among the three ADAM family members exhibiting
.alpha.-secretase activity, ADAM10 has many properties of a
physiologically relevant .alpha.-secretase: it cleaves APP-derived
peptides at the main .alpha.-secretase cleavage site between
position 16 and 17 of the AP region and exhibits .alpha.-secretase
activity in cultured cells (Lammich et al. Proc. Natl. Acad. Sci.
USA 1999; 96:3922-3927). Moreover, the mature ADAM10 species may
also act as an efficient .alpha.-secretase in the brain of
ADAM10-APP.sub.(v717) double transgenic mice (Postina et al. J.
Clin. Invest. 2004; 113:1456-1464). Our evidence therefore
indicates increased brain repair activities as a consequence of
sAPP.alpha. neurotrophic function.
[0134] Current strategies to treat AD are aimed at preventing
formation of amyloidogenic A.beta. peptides. Therefore, .beta.- and
.gamma.-secretases that generate A.beta. peptides by sequential
cleavage of the amyloid precursor protein (APP) or proteases
responsible for degrading released A.beta. peptides are obvious and
central targets for development of therapeutic reagents (Cummings.
N. Engl. J. Med. 2004; 351:56-67). It has, however, been difficult
to find safe, selective .beta.- and .gamma.-secretase inhibitors,
mainly because of the influence of these inhibitors on other
cellular substrates (Cummings. N. Engl. J. Med. 2004; 351:56-67).
Thus, the evidence provided here showing that CR may positively
influence .alpha.-secretase, possibly through mechanisms that may
involve the generation of mature, catalytically active ADAM10
species in the brain, provides a basis of potential novel
preventative measures aimed at delaying the onset of AD
neuropathology. It is also possible, however, that CR might also
influence other mechanisms, eventually resulting in decreased
amyloid deposition in the brain by promoting .alpha.-site cleavage
of APP by other proteases (e.g. plasmin (Ledesma et al. EMBO Rep.
2003; 4:1190-1196)) or degradation of released A. In addition,
since .alpha.-secretase cleavage of APP releases sAPP.alpha., which
is well known for its neuroprotective properties (Lichtenthaler et
al. J. Clin. Invest. 2004; 113:1384-1387), the results here suggest
that promoting low carbohydrate CR dietary regimen may also result
in increased brain repair activities as a consequence of
sAPP.alpha. neurotrophic function.
[0135] In conclusion, consistent with the evidence that caloric
intake may be a risk factor for AD (Luchsinger. Arch. Neurol. 2002;
59:1258-1263; Hendrie et al. JAMA 2001; 285:739-747; Mattson et al.
Physiol. Rev. 2002; 82:637-672; Mattson. Nature 2004; 430:631-639),
this study for the first time offers a rational basis for a
potential future preventative measure aimed at delaying the onset
of AD amyloid neuropathology via control of dietary intake.
[0136] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0137] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
Sequence CWU 1
1
1142PRTHomo sapiens 1Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu
Val Arg His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly
Ser Lys Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val
Val Ile Ala 35 40
* * * * *