U.S. patent application number 12/508908 was filed with the patent office on 2010-11-18 for modulation of the neuroendocrine system as a therapy for motor neuron disease.
This patent application is currently assigned to University of Piitsburgh of the Commonwealth System of Higher Education. Invention is credited to Robert P. Bowser.
Application Number | 20100292149 12/508908 |
Document ID | / |
Family ID | 36615374 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100292149 |
Kind Code |
A1 |
Bowser; Robert P. |
November 18, 2010 |
Modulation of the Neuroendocrine System as a Therapy for Motor
Neuron Disease
Abstract
The invention provides a method for treating amyotrophic lateral
sclerosis (ALS) in a subject. The method comprises administering to
the nervous system of the subject a composition comprising a
thyroxine protein or a therapeutic fragment or pharmacologic mimic
thereof and a pharmaceutically acceptable carrier. The invention
also provides a method for treating ALS in a subject that comprises
administering to the subject a transthyretin protein, 7B2 protein,
a cystatin C protein, a neuroendocrine protein, a cysteine protease
inhibitor, or an inhibitor of an enzyme that processes a 7B2
protein. In addition, the invention provides methods for
determining the susceptibility of a subject to developing ALS and
for determining the progression of ALS in a subject.
Inventors: |
Bowser; Robert P.;
(Cranberry Township, PA) |
Correspondence
Address: |
Pabst Patent Group LLP
1545 PEACHTREE STREET NE, SUITE 320
ATLANTA
GA
30309
US
|
Assignee: |
University of Piitsburgh of the
Commonwealth System of Higher Education
|
Family ID: |
36615374 |
Appl. No.: |
12/508908 |
Filed: |
July 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11294161 |
Dec 2, 2005 |
7572596 |
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12508908 |
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60632450 |
Dec 2, 2004 |
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Current U.S.
Class: |
514/10.9 ;
435/7.92; 436/86; 436/94; 514/11.1; 514/13.5; 514/17.7; 514/18.1;
514/44R |
Current CPC
Class: |
A61K 38/1709 20130101;
Y10T 436/143333 20150115; A61K 38/22 20130101; A61K 38/31 20130101;
A61K 48/00 20130101; G01N 2800/28 20130101; A61K 38/2271 20130101;
A61K 38/095 20190101; Y10T 436/24 20150115; A61P 25/28 20180101;
G01N 2333/8139 20130101; A61K 38/57 20130101; Y10T 436/25 20150115;
G01N 2333/76 20130101; G01N 33/6896 20130101 |
Class at
Publication: |
514/10.9 ;
514/13.5; 514/44.R; 514/17.7; 514/18.1; 514/11.1; 435/7.92; 436/86;
436/94 |
International
Class: |
A61K 38/11 20060101
A61K038/11; A61K 38/17 20060101 A61K038/17; A61K 31/7105 20060101
A61K031/7105; A61P 25/28 20060101 A61P025/28; A61K 38/31 20060101
A61K038/31; G01N 33/53 20060101 G01N033/53; G01N 33/00 20060101
G01N033/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made in part with Government support
under Grant Number ES013469 awarded by the National Institute of
Environmental Health Sciences. The Government may have certain
rights in this invention.
Claims
1. A method for treating amyotrophic lateral sclerosis (ALS) in a
subject, which method comprises administering to the nervous system
of the subject a composition comprising a thyroxine protein or a
therapeutic fragment or pharmacologic mimic thereof and a
pharmaceutically acceptable carrier.
2. A method for treating ALS in a subject, which method comprises
administering to the subject a transthyretin protein.
3. The method of claim 2, wherein the transthyretin protein is
administered to the subject through a gene transfer vector
comprising a nucleic acid sequence encoding the transthyretin
protein operably linked to a promoter.
4. The method of claim 2, wherein the transthyretin protein is
administered to the subject through a composition comprising the
transthyretin protein or a therapeutic fragment thereof and a
pharmaceutically acceptable carrier.
5. A method for treating ALS in a subject, which method comprises
administering to the subject a 7B2 protein.
6. The method of claim 5, wherein the 7B2 protein is administered
to the subject through a gene transfer vector comprising a nucleic
acid sequence encoding the 7B2 protein operably linked to a
promoter.
7. The method of claim 5, wherein the 7B2 protein is administered
to the subject through a composition comprising the 7B2 protein or
a therapeutic fragment thereof and a pharmaceutically acceptable
carrier.
8. A method for treating ALS in a subject, which method comprises
administering to the subject an inhibitor of an enzyme that
processes a 7B2 protein.
9. The method of claim 8, wherein the enzyme is selected from the
group consisting of furin and carboxypeptidase E.
10. A method for treating ALS in a subject, which method comprises
administering to the subject a cystatin C protein.
11. The method of claim 10, wherein the cystatin C protein is
administered to the subject through a gene transfer vector
comprising a nucleic acid sequence encoding the cystatin C protein
operably linked to a promoter.
12. The method of claim 5, wherein the cystatin C protein is
administered to the subject through a composition comprising the
cystatin C protein or a therapeutic fragment thereof and a
pharmaceutically acceptable carrier.
13. A method for treating ALS in a subject, which method comprises
administering to the subject a composition comprising a cysteine
protease inhibitor and a pharmaceutically acceptable carrier.
14. A method for treating ALS in a subject, which method comprises
administering to the subject a composition comprising a
neuroendocrine protein and a pharmaceutically acceptable
carrier.
15. The method of claim 11, wherein the neuroendocrine protein is
selected from the group consisting of neuropeptide Y, somatostatin,
galanin, and vasopressin.
16. A method for determining the susceptibility of a subject to
developing ALS, which method comprises (a) obtaining a sample from
the subject, (b) isolating from the sample a transthyretin protein,
and (c) determining the amino acid or nucleotide sequence of the
protein, wherein a protein or nucleic acid molecule encoding a
variant of the transthyretin protein indicates that the subject is
susceptible to developing ALS.
17. The method of claim 16, wherein the sample is selected from the
group consisting of cerebrospinal fluid, blood serum, plasma,
urine, and tissue.
18. A method for determining progression of ALS in a subject, which
method comprises: (a) obtaining a sample from the subject, (b)
isolating from the sample a transthyretin protein, (c) analyzing
the transthyretin protein from the sample by mass spectroscopy, and
(d) determining a mass spectral profile for the sample, wherein the
presence of a variant of a wild type transthyretin protein in the
sample indicates progression of ALS in the animal.
19. The method of claim 18, wherein the sample is selected from the
group consisting of cerebrospinal fluid, blood serum, plasma,
urine, and tissue.
20. A method for determining progression of ALS in a subject, which
method comprises: (a) obtaining a sample from the subject, (b)
isolating from the sample a transthyretin protein, (c) analyzing
the transthyretin protein levels from the sample, and (d) comparing
the protein levels to transthyretin protein levels obtained from
the same subject at an earlier time, wherein a change in the
protein levels indicates progression of ALS in the subject.
21. The method of claim 20, wherein the sample is selected from the
group consisting of cerebrospinal fluid, blood serum, plasma,
urine, and tissue.
22. The method of claim 20, wherein the change is a decrease in the
levels of transthyreitn protein.
23. The method of claim 20, wherein the change is an increase in
the levels of transthyreitn protein.
24. The method of claim 20, wherein the samples are analyzed by
mass spectroscopy.
25. The method of claim 20, wherein the samples are analyzed using
immunological techniques.
26. The method of claim 20, wherein the samples are analyzed by
ELISA.
27. The method of claim 20, wherein the samples are analyzed by
immunoblot.
28-36. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 60/632,450, filed Dec. 2,
2004.
BACKGROUND OF THE INVENTION
[0003] Amyotrophic lateral sclerosis (ALS), also known as Lou
Gehrig's disease or motor neuron disease (MND), is one of several
neurodegenerative diseases of the central nervous system. ALS is
the most common adult onset motor neuron disease, affecting one in
every 20,000 individuals, with an average age of onset of 50-55
years. ALS is characterized by rapidly progressive degeneration of
motor neurons in the brain, brainstem, and spinal cord (Cleveland,
2001). The median survival of patients from time of diagnosis is
five years.
[0004] ALS exists in both sporadic and familial forms. Familial ALS
(FALS) comprises only 5-10% of all ALS cases. Over the last decade,
a number of basic and clinical research studies have focused on
understanding the familial form of the disease, which has led to
the identification of eight genetic mutations related to FALS.
Transgenic mice expressing point mutants of the Cu/Zn superoxide
dismutase-1 (SOD1) gene develop an age-dependent progressive motor
weakness similar to human ALS due to a toxic gain of function
(Rosen, 1993; Rosen, 1994; Borchelt, 1994).
[0005] These genetic mutations, however, do not explain sporadic
ALS (SALS). The pathogenesis of SALS is multifactorial. A number of
different model systems, including SOD1 transgenic mice, in vitro
primary motor neuron cultures or spinal cord slice cultures, in
vivo imaging studies, and postmortem examination of tissue samples,
have been utilized to understand the pathogenesis of ALS
(Subramaniam, 2002); Nagai, 2001; Menzies, 2002; Kim, 2003;
Ranganathan, 2003). Although these studies have yielded therapeutic
targets and several clinical trials, there are no drugs that delay
disease onset or prolong longterm survival of ALS patients.
Riluzole (Rilutek.RTM., Aventis), a glutamate antagonist, currently
is the only FDA-approved medication available to treat ALS.
Riluzole, however, extends life expectancy by only a few months
(Miller, 2003). Creatine and atocopherol have shown some efficacy
in relieving the symptoms of ALS in SOD1 transgenic mice, but
exhibit minimal efficacy in human ALS patients (Groeneveld, 2003;
Desnuelle, 2001).
[0006] Studies have been performed which have identified early
protein biomarkers for ALS, using mass spectrometry based
proteomics of cerebrospinal fluid (CSF) and spinal cord samples of
human subjects (see U.S. patent application Ser. No. 10/972,732,
published as US 2005-0148026 A1, the disclosure of which is
incorporated herein). For example, three neuroendocrine proteins
(transthyretin, 7B2, and cystatin C) that exhibit alterations early
in the disease pathogenesis in humans were identified in a
proteomics analysis.
[0007] Transthyretin regulates thyroid function and retinoic acid
signaling in the brain by binding T4 and retinol-binding protein,
respectively, and binds other proteins including the amyloid beta
peptide (A.beta.) to help sequester and prevent amyloid deposition
(Palha, 2002; Bernstein, 2002; Tsuzuki, 2000). Retinoic acid
signaling is an important component of neural plasticity and
regeneration, and absence of retinoids can induce motor neuron
disease in rats and decreased retinoid signaling has been observed
in sporadic ALS patients (Mey, 2004; Corcoran, 2002).
[0008] Transthyretin (TTR) is a protein made by motor neurons in
the spinal cord and choroid plexus cells that line the ventricles
of the nervous system and produce the CSF. Transthyretin regulates
thyroid function and retinoic acid signaling in the brain by
binding T4 and retinol-binding protein, respectively, and binds
other proteins including the amyloid beta peptide (A.beta.) to help
sequester and prevent amyloid deposition (Palha, 2002; Bernstein,
2002; Tsuzuki, 2000). T4 is a critical component of thyroid
function and can also function to impede cell cycle progression and
induce cell differentiation. Direct administration of T4 into a
transgenic model for multiple sclerosis enhances remyelination and
is neuroprotective for axonal pathology (Fernandez et al, 2004).
Retinoic acid is a known antioxidant, and oxidative injury is one
proposed mechanism for motor neuron cell death in ALS. Decreased
levels of TTR also have been associated with increased brain levels
of metals such as lead in the nervous system and metal toxicity
induced neurodegeneration (Zheng et al., 2001). TTR also has been
recently reported to have direct neuroprotective functions in
neurons. The addition of TTR to neurons was found to be protective
to the addition of the A.beta. peptide that accumulates during
Alzheimer's disease (AD) (Stein et al., 2004). Injection of
inhibitory antibodies to TTR into a transgenic animal model for AD
results in the loss of neuroprotective functions of TTR and
acceleration of the disease process (Stein et al., 2004). Thus, the
loss of TTR function hastened disease pathogenesis in an animal
model of a neurodegenerative disease. Finally, genetic variants of
TTR cause transthyretin-associated hereditary amyloidosis (ATTR) in
which amyloid accumulates in various tissues and organs (Bergen et
al, 2004). ATTR is most common between the third and seventh
decades of life, similar to that of ALS. During ALS, it is likely
that reduced CSF levels of TTR result in altered transport of T4
and retinol/vitamin A, thus altering thyroid function and
increasing oxidative stress. It is also probable that reduced TTR
levels in motor neurons decrease neuroprotective functions and
increase oxidative stress in motor neurons, thus enhancing
neurodegeneration. In addition, TTR genetic variants may increase
susceptibility of individuals to develop ALS or hasten disease
pathogenesis.
[0009] Transthyretin (TTR) variants can be identified by mass
spectrometry and verified by gene sequencing. For the mass
spectrometry, TTR first is purified from a human sample by affinity
chromatography using anti-TTR antibody. The purified TTR is reduced
using tris(2-caroxyethyl)phosphine (TCEP) and then analyzed by mass
spectrometry to resolve differences in the protein mass indicative
of a polymorphism in the TTR gene resulting in an altered amino
acid. The most common variants (denoted as wildtype amino acid,
location within the protein, and mutant amino acid) are Va130Met
and Gly6Ser, though over 100 variants are known (see, e.g.,
Connors, 2003). Specific mass shifts observed by mass spectrometry
are indicative of gene polymorphisms. Samples containing such
variant proteins can be analyzed by DNA sequencing to confirm the
mass spectrometry results. TTR genetic variants may increase
susceptibility to developing ALS due to environmental
exposures.
[0010] Recent studies have shown that transthyretin functions as a
neuroprotective protein and injection of transthyretin protein
reduces amyloid deposition and cognitive decline in an animal model
of Alzheimer's disease (Stein, 2004). Transthyretin protein levels
are reduced by exposure to heavy metals and other toxins (Zheng,
Toxicol. Sci., 2001; Zheng, Microscopy Res. & Tech., 2001).
Environmental exposure to lead and other heavy metals has been
proposed as a risk factor in the etiology of ALS (Vinceti, 1997;
Kamel, 2002). Genetic variants of transthyretin decrease protein
function and cause a familial form of polyneuropathy (Benson,
1985). ALS subjects that have transthyretin genetic variants that
decrease transthyretin function will make the individual at
increased risk for developing ALS, thus suggesting that
transthyretin is a novel risk or susceptibility factor for ALS. It
is likely that the transthyretin plays a role in motor neuron
survival and genetic polymorphisms and/or decreased expression
levels make neurons more susceptible to injury or toxin exposure.
This could help explain the increased incidence of ALS in Gulf War
Veterans deployed to specific active zones noted above. TTR protein
may also directly participate in motor neuron degeneration by
generating extracellular or intracellular toxic protein aggregates.
TTR fibrils have been observed in familial polyneuropathies induced
by TTR mutations (Sousa, 2003). In vitro studies have shown that
the toxic form of TTR is the non-fibrillar protein aggregates and
that fully formed TTR containing fibrils is non-toxic (Sousa, 2001;
Sousa, 2002). Therefore the presence of non-fibrillar TTR protein
aggregates in ALS spinal cord tissue will be interpreted as
directly contributing to motor neuron degeneration. These
experiments provide novel data implicating TTR and TTR functional
pathways in motor neuron survival and highlight new pathogenic
mechanisms for ALS. Reduced TTR function, either by genetic
polymorphisms or post-translational modifications that alter
protein function or by reduced expression levels during ALS, may
directly induce motor neuron cell loss since the loss of retinoid
signaling can induce motor neuron disease in rodents (Corcoran,
2002).
[0011] The second neuroendocrine protein is 7B2 (Martens, 1989). It
has been determined that 7B2 protein alterations occur during ALS.
More specifically, in ALS subjects increased levels of a
carboxy-terminal fragment of 7B2 referred to as 7B2CT were
observed. 7132 is a neuroendocrine secretory protein that functions
as a chaperone for the proprotein convertase 2 protein (PC2). 7B2
binds to PC2 in the endoplasmic reticulum and facilitates its
transport to other compartments of the secretory pathway where it
is proteolytically matured and activated (Mbiday, 2001). PC2
participates in the production and secretion of numerous hormones
and neuropeptides, including neuropeptide Y, somatostatin, galanin,
and vasopressin. A prior study has shown expression of 7B2 in motor
neurons and in the spinal cord. 7132 also acts as a chaperone
protein for the maturation and secretion of insulin-like growth
factor 1 (IGF-1), which is currently in trials as a therapy for ALS
(Chaudhuri, 1995). 7B2 is processed by the enzyme furin to form a
carboxy-terminal fragment called 7B2CT that functions as an
inhibitor of PC2 activation. Carboxypeptidase E cleaves 7B2CT for
its degradation. Furin and/or carboxypeptidase E activity may also
be altered in ALS subjects. Therefore increased levels of 7B2CT in
the CSF of ALS subjects may indicate that PC2 activation is reduced
in multiple cell types of the nervous system, including motor
neurons. This implies that maturation and secretion of many
neuroendocrine peptides, growth factors and hormones is reduced in
ALS.
[0012] The third neuroendocrine protein identified is cystatin C
(Abrahamson, 1990). Cystatin C has been identified using mass
spectrometry as a diagnostic biomarker for ALS. CSF and lumbar
spinal cord tissue samples from ALS subjects exhibit less cystatin
C than control subjects. Cystatin C is a secreted protein that
functions both as a cysteine protease inhibitor and can function as
an autocrine or paracrine factor in neurogenesis of neural stem
cells. Mutations in the cystatin C gene cause a rare disease called
hereditary brain amyloid angiopathy, and increased levels of
cystatin C have been found in other neurodegenerative diseases
including Alzheimer's disease, ischemia, and Creutzfeldt-Jakob
disease (CID). Decreased levels of cystatin C in the CSF of ALS
subjects or altered post-translational modifications to cystatin C
suggest decreased levels of protease inhibitors, which may
contribute to disease pathogenesis.
[0013] Despite the identification of early protein biomarkers for
ALS, there remains a need, however, for improved methods for
identifying therapeutic targets of ALS, and improved methods of
diagnosing and monitoring the progress of the disease.
BRIEF SUMMARY OF THE INVENTION
[0014] The invention provides a method for treating amyotrophic
lateral sclerosis (ALS) in a subject.
[0015] The invention also provides a method for determining the
susceptibility of a subject to developing ALS. The method comprises
(a) obtaining a sample from the subject, (b) isolating from the
sample a nucleic acid molecule encoding the transthyretin protein,
and (c) determining the amino acid or nucleotide sequence of the
protein, wherein a protein or nucleic acid molecule encoding a
variant of the transthyretin protein indicates that the subject is
susceptible to developing ALS.
[0016] In addition, the invention provides a method for determining
progression of ALS in a subject. The method comprises (a) obtaining
a sample from the subject, (b) isolating from the sample a
transthyretin protein, (c) analyzing the transthyretin protein from
the sample by mass spectroscopy, and (d) determining a mass
spectral profile for the sample,
wherein the presence of a variant of a wild type transthyretin
protein in the sample indicates progression of ALS in the
animal.
[0017] The invention also provides a method for determining
progression of ALS in a subject. The method comprises (a) obtaining
a sample from the subject, (b) isolating from the sample a
transthyretin or cystatin C protein, (c) analyzing the
transthyretin or cystatin C protein levels from the sample, and (d)
comparing the protein levels to transthyretin or cystatin C protein
levels obtained from the same subject at an earlier time, wherein a
change in the protein levels indicates progression of ALS in the
subject.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0018] FIG. 1 presents data demonstrating transthyretin mass shifts
in ALS patients. Mass spectral analysis of CSF from healthy and
disease controls are shown in the top 4 panels, and mass spectral
analysis from ALS subjects are shown in the bottom 3 panels. The
mass region containing TTR is shown for each sample (mass region
between 13,500 to 14,500 Daltons). The control subjects (both
healthy and neurologic disease controls) exhibit similar TTR
profiles, whereas the ALS subjects exhibit mass shifts and the
presence of TTR mass doublets that were observed in approximately
30% of ALS subjects. Molecular mass (Da) is shown for the major TTR
peaks for the healthy and other neurologic disease control subjects
(top panel). TTR protein peaks in ALS patients often exhibit a
30-Da mass shift (13810.fwdarw.13841; 13901.fwdarw.13930) as shown
in the third panel from the bottom.
[0019] FIG. 2 presents data demonstrating transthyretin mass shifts
in ALS patients. Immunoprecipitated transthyretin (TTR) was treated
with tris(2-carboxyethyl)phosphine (TCEP) and examined by mass
spectrometry using an ABI Voyager/DE mass spectrometer. Control
subjects (bottom panels, labeled control 1 and control 2) exhibit a
mass peak of 13742 Daltons (Da) and a minor shoulder at
approximately 13732 Da. For ALS patients (top panels, labeled ALS1
and ALS2), a TTR doublet with a second peak of approximately 13772
Da (30 Da mass shift) was observed. This additional TTR peak was
seen in 9 of 30 ALS patients whereas only 2 of 20 control subjects
exhibited a similar TTR protein doublet. This mass shift is
consistent with an amino acid substitution resulting from a genetic
polymorphism.
[0020] FIG. 3 presents data demonstrating the presence of TTR
(transthyretin) monomer in ALS via immuno-SELDI-TOF-MS. Panel A
depicts a representative SELDI spectra of purified human TTR
protein (positive control), control CSF, and ALS CSF samples.
Purified anti-TTR antibody was bound to the protein chip surfaces
and individual samples were added to measure the level of TTR
protein. Seven control and seven ALS subjects were tested using
these antibody containing chips. The arrow indicates the presence
of the 13.78 kDa TTR monomer. The two-fold difference in the
relative peak intensity values for the 13.78 kDa peak between
control and ALS subjects was significantly significant (p=0.02).
Panel B depicts a ciphergen-based software assisted gel view of the
spectra represented in panel A.
[0021] FIG. 4 presents data demonstrating an overall increase in
the total level of TTR in the CSF of ALS patients relative to
control subjects. TTR ELISA data from 10 ALS and 8 control subjects
is shown. The total level of TTR in ALS subjects is significantly
increased over that present in control subjects. The bar represents
the mean average for all samples within the group.
[0022] FIG. 5 depicts an immunoblot analysis of TTR and cystatin C.
Panel (a) is an immunoblot of TTR protein in control and ALS CSF.
50 .mu.g of control (lanes 1-4) or ALS (lanes 5-8) CSF were probed
for transthyretin (TTR). Purified human TTR (50 .mu.g) was used as
a positive control (lane "+"). The arrows on the right indicate the
monomeric and homotetrameric forms of TTR protein. Panel (b) is an
immunoblot of cystatin C protein in control and ALS CSF. 25 .mu.g
of total CSF protein from control (lanes 1-4) or ALS (lanes 5-8)
subjects were probed for cystatin C. Purified human cystatin C (10
.mu.g) was used as a positive control (lane "+"). Molecular weight
markers indicated in kDa are listed to the left of the positive
control lanes in (a) and (b). Panel (c) is a densitometric
quantification of TTR and cystatin C proteins from panels (a) and
(b) using NIH 1.58 software. The relative abundance was expressed
per amount of CSF protein loaded per gel lane and the height of the
bars corresponds to the average value for each protein in control
and ALS subjects +/- standard error mean (SEM). Single factor ANOVA
was performed with a confidence interval of 95% to compare the
protein levels of controls and ALS. The p values for TTR and
cystatin C were 0.03 and 0.04, respectively. Asterisks indicate the
statistical significance.
[0023] FIG. 6 depicts an immunohistochemical analysis of a cross
section of lumbar spinal cord tissue. Lumbar spinal cord tissue
sections from 7 control and 7 ALS subjects were used to determine
TTR immunoreactivity in motor neurons using anti-TTR antibody. In
control subjects, TTR was present within the cytoplasm of motor
neurons, often in a speckled pattern (left panel). In ALS spinal
cord we observed decreased levels of TTR in surviving motor neurons
(right panel). Both panels are at 10.times. magnification. Insets
in both panels are high magnification image (40.times.) of a motor
neuron. Therefore decreased levels of TTR are present in surviving
motor neurons in ALS subjects.
[0024] FIG. 7 presents data demonstrating decreases in the mass
spectral peak for either cystatin C or transthyretin over a 12 or
15-month time frame within individual ALS subjects.
[0025] FIG. 8 presents data demonstrating alterations in
transthyretin protein mass spectral peaks within an ALS patient
over time. The data was obtained using a zinc coated Ciphergen
IMAC-30 Protein Chip and analyzed on the Ciphergen mass
spectrometer.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In one aspect, the present invention provides a method for
treating amyotrophic lateral sclerosis (ALS) in a subject or for
prophylaxis thereof within the subject. In accordance with this
aspect, an anti-ALS-effective amount of a composition comprising
either a thyroxine protein, a transthyretin protein, a 7B2 protein,
an inhibitor of an enzyme that processes a 7B2 protein, a cystatin
C protein, cysteine protease inhibitor, a neuroendocrine protein,
or combinations thereof (or, in some embodiments, a gene transfer
vector encoding the same) is administered to the subject in an
amount, at a location, and for a time course sufficient to treat
ALS within the subject Preferably, the desired factor is
administered to the nervous system of the subject, which is
particularly preferred where the factor is a thyroxine.
[0027] The "subject" can be any suitable animal, but preferably is
a mammal, such as a mouse, rat, monkey, or human. It is
contemplated that the aforementioned inventive method can be used
to treat ALS in animal models of the disease, in which case the
subject is a non-human animal (e.g., a mouse, rat, monkey, dog,
etc.). In a preferred embodiment, the subject is a human suffering
from ALS.
[0028] For treatment of ALS in a subject, following introduction of
the composition into the subject in accordance with the inventive
method, the subject's condition is monitored to assess the severity
of ALS. Suitable application of the inventive method will result in
slowing of the progression of ALS and, in preferred embodiments,
result in plateauing of the progress of the disease. Indeed, in
more preferred embodiments, application of the inventive method
will result in a reduction of the symptoms associated with ALS in
the subject or even in substantial or complete remission of ALS.
Thus, while the inventive method can lead to a cure of ALS in some
subjects, any degree of improvement in the prognosis of the subject
following application of the inventive method is considered to be
successful application. Moreover, it is to be understood that the
inventive method can be used as monotherapy or adjunctively in
combination with other therapeutic agents (e.g., riluzole) or
therapeutic methods.
[0029] For prophylaxis, following introduction of the composition
into the subject, the subject is suitably monitored to assess the
development of ALS and/or continued risk of developing ALS.
Successful prophylaxis can be measured by the absence of ALS in the
subject for longer than the initial assessment of risk had
predicted.
[0030] The desired factor (e.g., protein, therapeutic fragment, or
pharmacologic mimic, or gene transfer vector) can be prepared by
methods known to those of ordinary skill in the art. For example,
the protein or fragment can be synthesized using solid phase
peptide synthesis techniques (e.g., Fmoc). Alternatively, the
factor can be synthesized using recombinant DNA technology (e.g.,
using bacterial or eukaryotic expression systems). Accordingly, to
facilitate such methods, where the factor is a protein or fragment
thereof, the invention provides genetic vectors (e.g., plasmids)
comprising a sequence encoding the factor, as well as host cells
comprising such vectors. Methods for solid state protein synthesis
and recombinant protein synthesis are well-known in the art. For
example, "Molecular Cloning, A Laboratory Manual" (Sambrook et al.,
3d Ecition, Cold Spring Harmor Press), is a well-known reference
detailing many suitable techniques for recombinant production of
polypeptides. Accordingly, the invention provides the protein or
fragment in recombinant form. Alternatively a protein or fragment
can be isolated from any species as described herein. In addition,
the pharmacological mimic can be prepared as described above, or it
can be synthesized using appropriate organic synthesis methods,
which are well known to those of ordinary skill in the art.
[0031] However it is made, a protein, fragment, or mimic can be
isolated and/or purified (or substantially isolated and/or
substantially purified). Accordingly, the invention provides the
protein, fragment, or mimic in substantially isolated form (i.e.,
substantially isolated from other polypeptides or impurities). The
protein, fragment, or mimic can be isolated from other proteins as
a result of solid phase protein synthesis, for example.
Alternatively, the protein, fragment, or mimic can be substantially
isolated from other proteins after cell lysis from recombinant
production. Standard methods of protein purification (e.g., HPLC)
can be employed to substantially purify the protein, fragment, or
mimic. Thus, a preparation of the protein, fragment, or mimic
preferably is at least 90% free of other proteins and/or
contaminants, and more preferably is at least about 95% free of
other proteins and/or contaminants (such as at least about 97% or
98% free of other proteins and/or contaminants). In a most
preferred embodiment, the invention provides a preparation of the
protein, fragment, or mimic that is greater than 99% free of other
proteins and/or contaminants (e.g., greater than 99.5% or even
99.9% or even 99.99% free of other proteins).
[0032] In another embodiment, the invention provides a preparation
of the protein, fragment, or mimic in a number of formulations,
depending on the desired use. For example, where the protein,
fragment, or mimic is substantially isolated (or even nearly
completely isolated from other proteins), it can be formulated in a
suitable medium solution for storage (e.g., under refrigerated
conditions or under frozen conditions). Such preparations can
contain protective agents, such as buffers, preservatives,
cryprotectants (e.g., sugars such as trehalose), etc. The form of
such preparations can be solutions, gels, etc., and the protein
can, in some embodiments, be prepared in lyophilized form.
Moreover, such preparations can include other desired agents, such
as small molecules or even other proteins or peptides, if desired.
Indeed, the invention provides such a preparation comprising a
mixture of different embodiments of the protein, fragment, or mimic
(e.g., a plurality of species as described herein). Technology for
preparing such compositions (e.g., lyophilization, preparation of
protein solutions, etc.), is within the state of the art.
[0033] In accordance with this aspect of the inventive method, the
composition administered to the subject can comprise, consist of,
or consist essentially of a desired protein or a therapeutic
fragment or pharmacologic mimic thereof, and preferably also a
pharmaceutically acceptable carrier. Any carrier which can supply
the protein, fragment, or mimic without destroying the protein,
fragment, or mimic within the carrier is a suitable carrier, and
such carriers are well known in the art. The composition can be
formulated for parenteral, oral, or topical administration. For
example, a parenteral formulation could consist of a prompt or
sustained release liquid preparation, dry powder, emulsion,
suspension, or any other standard formulation. An oral formulation
of the composition could be, for example, a liquid solution, such
as an effective amount of the composition dissolved in diluents
(e.g., water, saline, juice, etc.), suspensions in an appropriate
liquid, or suitable emulsions. An oral formulation could also be
delivered in tablet form, and could include excipients, colorants,
diluents, buffering agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible excipients. A
topical formulation could include compounds to enhance absorption
or penetration of the active ingredient through the skin or other
affected areas, such as dimethylsulfoxide and related analogs. The
composition could also be delivered topically using a transdermal
device, such as a patch, which could include the composition in a
suitable solvent system with an adhesive system, such as an acrylic
emulsion, and a polyester patch.
[0034] In one embodiment of this aspect of the invention, the
method comprises, consists of, or consists essentially of
administering to the nervous system of the subject a composition
comprising a thyroxine protein or a therapeutic fragment or
pharmacologic mimic thereof. Preferably, the composition also
includes a pharmaceutically acceptable carrier. The composition can
comprise, consist or, or consist essentially of any protein
identified or characterized as a thyroxine protein. The protein can
be synthetic or can be isolated from any species (e.g., human). In
a preferred embodiment, the amino acid sequence of the protein is
identical or substantially similar (e.g. at least about 80%, at
least about 90%, or at least about 95% identical) to the full
length human thyroxine protein. For example, the protein may
contain substitutions of one or more amino acid residues for an
amino acid other than the indicated residue. The substitution can
be, but need not be, a conservative substitution. Conservative
substitutions are well known in the art and can be amino acid
replacements that preserve the structure and functional properties
of proteins, such as the substitution of one or more amino acids by
similar amino acids. For example, a conservative substitution can
be the substitution of an amino acid for another amino acid within
the same general class (e.g., an acidic amino acid, a basic amino
acid, or a neutral amino acid). In another embodiment, the
composition comprises, consists or, or consists essentially of a
therapeutic fragment of a thyroxine protein. A therapeutic fragment
is any fragment that has an effect on a subject with ALS as
described above. The fragment can have any suitable amino acid
sequence and can be of any length. For example, the fragment can
have an amino acid sequence identical to a portion of the human
protein, or it can be substantially similar to a portion of the
human protein (e.g., at least about 80%, at least about 90%, or at
least about 95% identical). In another embodiment the composition
comprises, consists or, or consists essentially of a pharmacologic
mimic of a thyroxine protein. A pharmacological mimic can be any
molecule or composition that simulates the role of a thyroxine
protein (e.g. thyroid function) or the effect of a thyroxine
protein on a subject with ALS.
[0035] In another embodiment of this aspect, the method comprises,
consists of, or consists essentially of modulation of the
neuroendocrine system to increase endogenous transthyretin gene
expression or to directly add transthyretin to provide
neuroprotective functions in ALS patients. Preferably, the method
comprises administering to the subject a composition comprising a
transthyretin protein or a therapeutic fragment or pharmacologic
mimic thereof. Preferably, the composition also includes a
pharmaceutically acceptable carrier. The inventive method is
performed substantially as described above. Alternatively, the
protein can be administered to a subject using a gene transfer
vector. Any suitable vector can be used such as viral (e.g.,
adenovirus or lentivirus) or non-viral vectors, and such vectors
are well known in the art. The transthyretin protein is encoded by
a nucleic acid sequence and can be linked to a promoter. A promoter
can be any element which drives production of the protein.
[0036] The transthyretin protein can be any protein identified or
characterized as a transthyretin protein. The protein can be
synthetic or can be isolated from any species (e.g., human). In a
preferred embodiment, the amino acid sequence of the protein is
identical or substantially similar (e.g. at least about 80%, at
least about 90%, or at least about 95% identical) to the full
length human transthyretin protein. For example, the protein may
contain substitutions of one or more amino acid residues for an
amino acid other than the indicated residue. The substitution can
be, but need not be, a conservative substitution. The nucleic acid
sequence of the protein can be any appropriate sequence given the
redundancy of the genetic code. In another embodiment, the protein
is a therapeutic fragment of a transthyretin protein. A therapeutic
fragment is any fragment that has an effect on a subject with ALS
as described above. The fragment can have any suitable amino acid
sequence and can be of any length. For example, the fragment can
have an amino acid sequence identical to a portion of the human
protein, or it can be substantially similar to a portion of the
human protein (e.g., at least about 80%, at least about 90%, or at
least about 95% identical). In another embodiment the protein or
composition is a pharmacologic mimic of a transthyretin protein. A
pharmacological mimic can be any molecule or composition that
simulates the role of a transthyretin protein (e.g. thyroxine
transport) or the effect of a transthyretin protein on a subject
with ALS.
[0037] In another embodiment of this aspect of the invention, the
method comprises administering to the subject a 7B2 protein or a
therapeutic fragment or pharmacologic mimic thereof. Preferably,
the composition also includes a pharmaceutically acceptable
carrier. The inventive method is performed substantially as
described above. The 7B2 protein can be any protein identified or
characterized as a 7B2 protein (an example of which is disclosed in
embl locus HSA290438, accession AJ290438.1; see also accession no.
CAB90397, SEQ ID NOs: 1 and 2). The protein can be synthetic or can
be isolated from any species (e.g., human). In a preferred
embodiment, the amino acid sequence of the protein is identical or
substantially similar (e.g. at least about 80%, at least about 90%,
or at least about 95% identical) to the full length human 7B2
protein. For example, the protein may contain substitutions of one
or more amino acid residues for an amino acid other than the
indicated residue. The substitution can be, but need not be, a
conservative substitution. The nucleic acid sequence of the protein
can be any appropriate sequence given the redundancy of the genetic
code. In another embodiment, the protein is a therapeutic fragment
of a 7B2 protein. A therapeutic fragment is any fragment that has
an effect on a subject with ALS as described above. The fragment
can have any suitable amino acid sequence and can be of any length.
For example, the fragment can have an amino acid sequence identical
to a portion of the human protein, or it can be substantially
similar to a portion of the human protein (e.g., at least about
80%, at least about 90%, or at least about 95% identical). In
another embodiment the protein or composition is a pharmacologic
mimic of a 7B2 protein. A pharmacological mimic can be any molecule
or composition that simulates the role of a 7132 protein (e.g.
proprotein convertase 2 transport) or the effect of a 7B2 protein
on a subject with ALS.
[0038] In another embodiment of this aspect of the invention, the
inventive method comprises administering to the subject an
inhibitor of an enzyme that processes a 7B2 protein. The inventive
method is performed substantially as described above. The inhibitor
can be any molecule that decreases or abolishs the activity of an
enzyme that processes a 7B2 protein, such as a competitive or
non-competitive inhibitor. The enzyme can be any enzyme involved in
the prossessing (e.g., synthesis, modification, or transport) of a
782 protein. For example, the inhibitor can be furin,
carboxypeptidase E, or a combination thereof (see Paquet, 1994;
Zhu, 1996).
[0039] In another embodiment of this aspect of the invention, the
inventive method comprises administering to the subject a cystatin
C protein or a therapeutic fragment or pharmacologic mimic thereof.
Preferably, the composition also includes a pharmaceutically
acceptable carrier. The inventive method is performed substantially
as described above. The cystatin C protein can be any protein
identified or characterized as a cystatin C protein (see, e.g.,
embl locus HSCST3G, accession X52255.1, embl locus HSCYSTC1,
accession X61681.1, embl locus HSCYSTCR, accession X05607.1, locus
HUMCYS3A1 accession M27889.1, UniProtKB/Swiss-Prot:P01034, SEQ ID
NOs: 3 and 4). The protein can be synthetic or can be isolated from
any species (e.g., human). In a preferred embodiment, the amino
acid sequence of the protein is identical or substantially similar
(e.g. at least about 80%, at least about 90%, or at least about 95%
identical) to the full length human cystatin C protein. For
example, the protein may contain substitutions of one or more amino
acid residues for an amino acid other than the indicated residue.
The substitution can be, but need not be, a conservative
substitution. The nucleic acid sequence of the protein can be any
appropriate sequence given the redundancy of the genetic code. In
another embodiment, the protein is a therapeutic fragment of a
cystatin C protein. A therapeutic fragment is any fragment that has
an effect on a subject with ALS as described above. The fragment
can have any suitable amino acid sequence and can be of any length.
For example, the fragment can have an amino acid sequence identical
to a portion of the human protein, or it can be substantially
similar to a portion of the human protein (e.g., at least about
80%, at least about 90%, or at least about 95% identical). In
another embodiment the protein or composition is a pharmacologic
mimic of a cystatin C protein. A pharmacological mimic can be any
molecule or composition that simulates the role of a cystatin C
protein (e.g., cysteine protease inhibition, autocrine or paracrine
factors in neurogenesis of neural stem cells) or the effect of a
cystatin C protein on a subject with ALS.
[0040] In another embodiment of this aspect of the invention, the
inventive method comprises administering to the subject a
composition comprising a cysteine protease inhibitor and a
pharmaceutically acceptable carrier. The inhibitor can be any
molecule that decreases or abolishs the activity of a cysteine
protease, such as a competitive or non-competitive inhibitor.
Cysteine proteases (e.g., cathepsins B, C, H, L, and S; cystatin F,
etc.) are a class of enzymes involved in the formation and
hydrolysis of the peptide amide bond and are well known in the art.
Cysteine proteases play a role in mammalian cellular turnover and
apoptosis. The mechanism of action of a cysteine protease involves
attack of the nucleophilic thiol of an enzyme's cysteine residue on
the carbonyl of the scissile amide bond of a bound substrate. The
covalent intermediate that is formed is subsequently hydrolyzed to
generate an amine and a carboxylic acid while also regenerating the
free enzyme.
[0041] In another embodiment of this aspect of the invention, the
inventive method comprises administering to the subject a
composition comprising a neuroendocrine protein and a
pharmaceutically acceptable carrier. A neuroendocrine protein is
any protein that is expressed in or secreted by neural or endocrine
tissues. The protein can be, for example, neuropeptide Y,
somatostatin, galanin, or vasopressin.
[0042] In another aspect, the present invention also provides a
method for determining the susceptibility of a subject to
developing ALS. The method comprises (a) obtaining a sample from
the subject, (b) isolating from the sample a transthyretin protein
or nucleic acid encoding a transthyretin protein, and (c)
determining sequence of transthyretin protein or nucleic acid
encoding transthyretin protein obtained from the sample, wherein a
varient transthyretin protein or nucleic acid molecule encoding a
variant of the transthyretin protein indicates that the subject is
susceptible to developing ALS.
[0043] The term "sample", as used herein refers to biological
material isolated from an animal. The animal can be any suitable
animal, but preferably is a mammal, such as a mouse, rat, monkey,
or human. It is contemplated that the aforementioned inventive
method can be used to diagnose ALS in animal models of the disease,
in which case the subject is a non-human animal (e.g., a mouse,
rat, monkey, dog, etc.). In a preferred embodiment, the subject is
a human. The sample can contain any suitable biological material,
but preferably comprises cells obtained from a particular tissue or
biological fluid. The sample can be isolated from any suitable
tissue or biological fluid. In this respect, the sample can be
blood, blood serum, plasma, urine, or spinal cord tissue. In that
ALS affects the central nervous system, the sample preferably is
isolated from tissue or biological fluid of the central nervous
system (CNS) (i.e., brain and spinal cord). In a preferred
embodiment of the invention, the sample is isolated from
cerebrospinal fluid (CSF). CSF from ALS patients has been used for
biochemical assays that have identified changes in the levels of
glutamate, glutamine synthetase, transglutaminase activity,
.gamma.-aminobutyric acid, and various markers of oxidative injury
(see, e.g., Spreux-Varoquaux, 2002; Shaw, 2000; Smith, 1998).
[0044] The sample can be obtained in any suitable manner known in
the art, such as, for example, by biopsy, blood sampling, urine
sampling, lumbar puncture (i.e., spinal tap), ventricular puncture,
and cisternal puncture. In a preferred embodiment of the invention,
the sample is obtained by lumbar puncture, which also is referred
to as a spinal tap or cerebrospinal fluid collection. Lumbar
puncture involves insertion of a spinal needle, usually between the
3rd and 4th lumbar vertebrae, into the subarachnoid space where CSF
is collected. In instances where there is lumbar deformity or
infection which would make lumbar puncture impossible or
unreliable, the sample can be collected by ventricular puncture or
cisternal puncture. Ventricular puncture typically is performed in
human subjects with possible impending brain herniation.
Ventricular puncture involves drilling a hole in the skull and
inserting a needle directly into the lateral ventricle of the brain
to collect CSF. Cisternal puncture involves insertion of a needle
below the occipital bone (back of the skull), and can be hazardous
due to the proximity of the needle to the brain stem.
[0045] A transthyretin protein or nucleic acid in the sample can be
separated by any suitable method known in the art. Suitable methods
include, for example, centrifugation, ion exchange chromatography,
reversed-phase liquid chromatography, and gel electrophoresis
(e.g., one-dimensional or two-dimensional gel electrophoresis).
Amino acid and nucleic acid sequencing methods are well known in
the art and any suitable method can be utilized (e.g., Edman
degradation, Sanger method). A variant of the transthyretin protein
is any transthyretin protein which has a protein or nucleic acid
sequence that differs from the wild type transthyretin protein of
the species from which the sample was obtained. The difference
between the wild type protein and the protein isolated from the
sample can be a difference in one or more nucleotides or amino
acids. For example, amino acid 30 of the wild type protein is
valine, but amino acid 30 of the protein from the sample is
methionine (e.g., Connors, 2003). Such a difference indicates that
the subject is susceptible to developing ALS.
[0046] In another embodiment, the present invention provides a
method for determining progression of ALS in a subject, which
method comprises (a) obtaining a sample from the subject, (b)
isolating from the sample a transthyretin protein, (c) analyzing
the transthyretin protein from the sample by mass spectroscopy, and
(d) determining a mass spectral profile for the sample, wherein the
presence of a variant of a wild type transthyretin protein in the
sample indicates progression of ALS in the subject.
[0047] Once the proteins in the sample are separated as described
above, the inventive method comprises analyzing the proteins in the
sample by mass spectroscopy. In mass spectroscopy, a substance is
bombarded with an electron beam having sufficient energy to
fragment the molecule. The positive fragments that are produced
(cations and radical cations) are accelerated in a vacuum through a
magnetic field and are sorted on the basis of mass-to-charge ratio
(m/z). Since the bulk of the ions produced in the mass spectrometer
carry a unit positive charge, the value m/z typically is equivalent
to the molecular weight of the fragment. Any suitable mass
spectroscopy method can be used in connection with the inventive
method. Examples of suitable mass spectroscopy methods include
matrix-assisted laser desorption/ionization mass spectroscopy
(MALDI), matrix-assisted laser desorption/ionization-time of flight
(MALDI-TOF) mass spectroscopy, plasma desorption/ionization mass
spectroscopy (PDI), electrospray ionization mass spectroscopy
(ESI), and surface enhanced laser desorption/ionization-time of
flight (SELDI-TOF) mass spectroscopy. In time-of-flight (TOF)
methods of mass spectroscopy, charged (ionized) molecules are
produced in a vacuum and accelerated by an electric field produced
by an ion-optic assembly into a free-flight tube or drift time. The
velocity to which the molecules may be accelerated is proportional
to the square root of the accelerating potential, the square root
of the charge of the molecule, and inversely proportional to the
square root of the mass of the molecule. The charged molecules
travel down the TOF tube to a detector. Mass spectroscopy methods
are further described in, for example, International Patent
Application Publication No. WO 93/24834, U.S. Pat. No. 5,792,664,
U.S. Patent Application Publication No. 2004/0033530 A1, and
Hillenkamp et al., Matrix Assisted UV-Laser Desorption/Ionization:
A New Approach to Mass Spectroscopy of Large Biomolecules,
Biological Mass Spectroscopy, Burlingame and McCloskey, eds.,
Elsevier Science Publ., pp. 49-60 (1990), the disclosures of which
are incorporated herein.
[0048] In a preferred embodiment of the invention, the proteins in
the sample are analyzed by SELDI-TOF mass spectroscopy. Surface
enhanced desorption/ionization processes refer to those processes
in which the substrate on which the sample is presented to the
energy source plays an active role in the desorption/ionization
process. In this respect, the substrate (e.g., a probe) is not
merely a passive stage for sample presentation. Several types of
surface enhanced substrates can be employed in a surface enhanced
desorption/ionization process. In one embodiment, the surface
comprises an affinity material, such as anion exchange groups or
hydrophilic groups (e.g., silicon oxide), which preferentially bind
certain classes of molecules. Examples of such affinity materials
include, for example, silanol (hydrophilic), C.sub.8 or C.sub.16
alkyl (hydrophobic), immobilized metal chelate (coordinate
covalent), anion or cation exchangers (ionic) or antibodies
(biospecific). The sample is exposed to a substrate bound adsorbent
so as to bind analyte molecules according to the particular basis
of attraction. When the analytes are biomolecules (e.g., proteins),
an energy absorbing material (e.g., matrix) typically is associated
with the bound sample. A laser is then used to desorb and ionize
the analytes, which are detected with a detector. For SELDI-TOF
mass spectroscopy, the mass accuracy for each protein peak is
+/-0.2%. SELDI-TOF mass spectroscopy systems are commercially
available from, for example, Ciphergen Biosystems, Inc. (Fremont,
Calif.). Surface enhanced desorption/ionization methods are
described in, e.g., U.S. Pat. Nos. 5,719,060, 6,294,790, and
6,675,104, and International Patent Application Publication No. WO
98/59360, the disclosures of which are incorporated herein.
[0049] One of ordinary skill in the art will appreciate that the
output of a mass spectroscopy analysis is a plot of relative
intensity as a function of the mass-to-charge ratio (m/z) of the
proteins in the sample, which is referred to as a "mass spectral
profile" or "mass spectrum." The mass spectral profile, which
typically is represented as a histogram depicting protein "peaks,"
serves to establish the molecular weight and structure of the
compound being analyzed. Thus, the inventive method further
comprises determining a mass spectral profile for the sample. The
most intense peak in the spectrum is termed the base peak, and all
other peaks are reported relative to the intensity of the base
peak. The peaks themselves are typically very sharp, and are often
simply represented as vertical lines.
[0050] The ions that are formed by fragmentation of the proteins in
the sample during mass spectroscopy are the most stable cations and
radical cations formed by the protein molecules. The highest
molecular weight peak observed in a spectrum typically represents
the parent molecule less an electron, and is termed the molecular
ion (M+). Generally, small peaks are also observed above the
calculated molecular weight due to the natural isotopic abundance
of .sup.13C, .sup.2H, etc. Many molecules with especially labile
protons do not display molecular ions. For example, the highest
molecular weight peak in the mass spectrum of alcohols occurs at an
m/z one less than the molecular ion (m-1). Fragments can be
identified by their mass-to-charge ratio, but it is often more
informative to identify them by the mass which has been lost. For
example, loss of a methyl group will generate a peak at m-15, while
loss of an ethyl will generate a peak at m29.
[0051] After the mass spectral profile for the sample has been
determined, the profile is then compared to that of a wild type
transthyretin protein from the same species from which the sample
was obtained. A mass shift in the mass spectral profile of the
sample, when compared with the wild type mass spectral profile,
indicates diagnosis or progression of ALS in the source of the
sample.
[0052] In another embodiment, the invention provides a method for
determining progression of ALS in a subject. The method comprises
(a) obtaining a sample from the subject, (b) isolating from the
sample a transthyretin protein, (c) analyzing the transthyretin
protein levels from the sample, and (d) comparing the protein
levels to transthyretin protein levels obtained from the same
subject at an earlier time, wherein a change in the protein levels
indicates progression of ALS in the subject.
[0053] Once the protein has been isolated, the amount of protein in
the sample can be determined using any acceptable technique, such
as mass spectroscopy and immunological techniques (e.g.,
immunoblotting, ELISA), which are well known in the art.
[0054] The protein levels described above can be compared to any
protein levels obtained from the same subject at any point in time
which is earlier than the time at which the protein levels were
obtained. The determination of the progression of ALS in a subject
can be made if a sample obtained from the subject, when compared
with a sample obtained from the same animal at an earlier time,
comprises either lower or higher levels of transthyretin protein.
For example, a sample taken from the CSF of a subject may have
higher levels of TTR, whereas a sample taken from spinal cord or
brain tissue may have lower levels of TTR.
[0055] In another embodiment, the invention provides a method for
determining progression of ALS in a subject. The method comprises
(a) obtaining a sample from the subject, (b) isolating from the
sample a cystatin C protein, (c) analyzing the cystatin C protein
levels from the sample, and (d) comparing the protein levels to
cystatin C protein levels obtained from the same subject at an
earlier time, wherein a change in the protein levels indicates
progression of ALS in the subject. The method can be performed
substantially as described above. Once the protein has been
isolated, the amount of protein in the sample can be determined
using any acceptable technique, such as mass spectroscopy and
immunological techniques (e.g., immunoblotting, ELISA), which are
well known in the art.
[0056] The protein levels described above can be compared to any
protein levels obtained from the same subject at any point in time
which is earlier than the time at which the protein levels were
obtained. The determination of the progression of ALS in a subject
can be made if a sample obtained from the subject, when compared
with a sample obtained from the same animal at an earlier time,
comprises either lower or higher levels of cystatin C protein. For
example, a sample taken from the CSF of a subject may have higher
levels of cystatin C, whereas a sample taken from spinal cord or
brain tissue may have lower levels of cystatin C.
EXAMPLES
General Procedures
Subjects
[0057] Subjects for the studies described in this application
included Caucasian and African American males and females between
the ages of 21-85. Samples were obtained from subjects at both the
University of Pittsburgh School of Medicine (Pittsburgh, Pa.) and
Massachusetts General Hospital (Boston, Mass.). Cerebrospinal fluid
(CSF) was obtained by lumbar puncture, immediately centrifuged at
1500 rpm for 5 min at 4.degree. C. to remove cellular debris,
aliquoted, frozen at -80.degree. C. and thawed on ice prior to use.
2D-Quant kits (Amersham, USA) were used to determine protein
concentrations (0.06 .mu.g/.mu.l to 0.6 .mu.g/.mu.l for each CSF
sample). University of Pittsburgh Institutional review board (IRB)
and Massachusetts General Hospital IRB approved informed consent
for this procedure.
ELISA
[0058] ELISA was used to quantify the amount of TTR protein present
in the CSF of ALS and control subjects. Dilute human prealbumin in
1.times.PBS was used to create a standard curve. Samples of 200 uL
were diluted 1:2000 in 1.times.PBS. 50 uL of the standard solutions
and sample solutions were dispensed in triplicate to 96 well plate,
in addition to 50 uL of wash buffer in triplicate. The wells were
allowed to dry overnight on an Eppendorf Thermomixer set to 37 C
and 300 rpm. After the wells were completely dried, they were
washed at least 3 times with 0.1% Tween-20 in 1.times.PBS. Washing
is done by using a squirt bottle and forcefully filling each well
with about 300 uL of the wash solution. Each well is aspirated
after washing. The top of the plate was dried by inverting it and
blotting against a clean paper towel. The wells were then blocked
using SuperBlock blocking buffer in TBS (Pierce) with 0.05%
Tween-20 (Sigma Aldrich). 300 uL of the solution was added to each
well and then the plate was completely emptied by inversion. This
step was repeated 2-3 additional times. The wells were then washed
as described at least 3 times. Rabbit anti-human prealbumin (TTR)
antibody was diluted to 1:1000 in the wash solution and 50 uL was
added to each well. The plate was covered with a disposable plate
sealer and incubated at room temperature for 1 hour. The plate
sealer was removed and the wells were aspirated and then washed 3
times as described. Goat anti-rabbit Ig-HRP human adsorbed antibody
was diluted to 1:5000 in the wash solution. 50 uL was added to each
well. The plate was then covered with a disposable plate sealer and
incubated at room temperature for 1 hour. The plate sealer was
removed and the wells were aspirated and then washed 5 times as
described. 50 uL of 3,3',5,5'-Tetramethylbenzidine (TMB) Liquid
Substrate System for ELISA was dispended to each well and a blue
color was allowed to develop (10-15 minutes). 50 uL of 1M HCl was
added to each well to stop color development. The plate was read
with a plate reader at 450 nm.
Sample Preparation for Mass Spectroscopy
[0059] CSF samples were immunoprecipitated using TTR antibody and
mass spectrometric analysis was performed. 50 .mu.L of protein A/G
beads were added to each sample, which were then spun briefly to
form a pellet. Excess liquid was removed and the beads were washed
with 250 .mu.L of PBS. The beads were spun again and the PBS was
removed. The wash was then repeated. After the second PBS wash was
removed, 400 .mu.L of PBS and 10 .mu.L of the TTR antibody were
added. Each sample was then rotated (at a speed of about 2S) at
4.degree. C. for 1 hour. The samples were then spun briefly, the
supernatant was removed, and 500 .mu.L of 0.5% NP-40 in PBS was
added. The samples were then rotated at 4.degree. C. for about 10
min. The samples were spun briefly, the supernatant was removed,
and the detergent wash was repeated. 120 .mu.L of the sample was
mixed with 400 .mu.L 0.5% NP-40 in PBS and then vortexed briefly.
After the supernatant was removed from the beads, the
sample/detergent mixture was returned to its corresponding tube,
and rotated overnight at 4.degree. C. The samples were spun
briefly, and the supernatant was removed. The beads were washed
with 500 .mu.l, of 0.5% NP-40 in PBS, and the mixture was rotated
in the cold room for about 10 min. Each tube was spun, the
supernatant was removed, and the detergent wash was repeated. After
the supernatant was removed, each sample was washed twice with PBS.
The beads were washed with 100 mM Hepes (pH 7.5) to remove salt
from the PBS. The bound proteins were eluted with 15 .mu.L of 0.1%
TFA in 50% ACN. 10 .mu.l., of 100 mmol/L TCEP was added to each
sample. The samples were incubated for 20 min. at 55.degree. C.
Mass Spectroscopy
[0060] Mass spectroscopy performed in immunoprecipitation
experiments or experiments incorporating ion exchange
pre-fractionation of the samples was performed using SELDI
ProteinChip.degree. technology (Ciphergen Biosystems, Inc.,
Fremont, Calif.). A gold chip was washed with water, and then
rinsed with a mild detergent (RBS 35). The chip was rinsed again
with water, then with methanol, and placed on a heat block at
37.degree. C. to dry. The matrix was prepared by adding
approximately 1.0 mL of 0.1-0.5% TFA in 50% ACN to a small amount
of sinapic acid. The solution was vortexed and the mixture was spun
down to form a pellet. The supernatent was removed and 5 .mu.L of
the eluted protein mixture was added to 5 .mu.l, of supernatant
matrix (1:1 ratio). 2 .mu.L of the protein/matrix mixture was
spotted onto its corresponding well of the chip. After drying, an
additional 2 .mu.L of sample was added to each well. Once dry,
samples were analyzed.
[0061] In addition, three separate experimental runs for each
ProteinChip were performed. Therefore, each sample was analyzed in
duplicate within each experiment, and each experiment was repeated
three times. For each experiment, one CSF sample was used as an
internal standard to compare peak intensities from four selected
m/z peaks to measure variability of the mass spectra. The
coefficient of variance (CV) for these selected peaks was less than
25%.
[0062] External calibration of the Protein Chip Reader was
performed using the Ciphergen All-in-One peptide/protein standard
mix containing peptides ranging from 1000 Da to 20 kDa. The dried
chips were immediately loaded into the calibrated Chip Reader using
optimal laser intensity and detector sensitivity with a mass
deflector setting of 1000 Da for low mass range (2-20 kDa) and
10,000 kDa for high mass range (20 kDa-80 kDa). These settings were
kept constant for all the chips of every experiment. The
mass/charge (m/z) ratios were determined using time of flight (TOF)
analysis.
[0063] Mass spectroscopy was also performed using strong anion
exchange surface (Q10) and IMAC ProteinChips.COPYRGT. (Ciphergen
Biosystems, Inc., Palo Alto, Calif.). Q10 chips were placed in a
bioprocesser (Ciphergen Biosystems, inc., Palo Alto, Calif.) and
equilibrated with 200 .mu.L of 100 mM Hepes pH 7.3 (titrated with
NH.sub.4OH) for ten minutes on a micromix shaker (set at 20/7).
Hepes was removed and 100 .mu.L of each sample was applied to the
wells. The IMAC Protein Chips were first treated with 100 mM zinc
sulfate followed by washing with 50 mM sodium acetate prior to
addition of sample. All samples were done in duplicate. A control
CSF sample was applied to a different spot of each
ProteinChip.RTM.. The arrays were then incubated for 30 minutes at
room temperature on a micromix (set at 20/7). The CSF was removed
and 200 .mu.l of Hepes pH 7.3 (titrated with NH.sub.4OH) was added
to each well. The arrays were placed on the micromix for ten
minutes (set at 20/7). After ten minutes the Hepes was removed and
200 uL of Hepes was added to the arrays, which were then shaken for
another 10 minutes. The ProteinChips.degree. were then removed from
the bioprocesser and, using a squirt bottle, rinsed quickly five
times with HPLC water pH 7.3 (titrated with NH.sub.4OH). Any excess
water was blotted off with a Kimwipe.RTM. and the ProteinChips.RTM.
were left to dry. Once dry, the ProteinChips.RTM. were placed on a
heat block (set at 40.degree. C.) and 1.5 .mu.L of Sinapinic Acid
(SPA) (Fluka) in 50% v/v acetonitrile and 0.3% v/v trifluoroacetic
acid was added twice to each spot. The Q10 arrays were then read in
a Ciphergen PBS IIC Chip reader system containing an autoloader
(Ciphergen Biosystems). Spectra were generated using a laser
intensity range of 190 and a detector sensitivity range of 8-9 with
a mass deflector setting of 1000 Da for the low mass range (1-20
kDa). These settings were kept constant for all chips analyzed in
an experiment. Two mass spectra were obtained for each sample: one
for the mass range of 1-20 kDa, and a second for the mass range
from 20-160 kDa. For each Q10 ProteinChip array, a standard CSF
sample was loaded onto one spot to measure chip-to-chip
variability. The coefficient of variance (COY) for four-selected
m/z signals was less than 30% across all chip arrays. External
calibration of the spectrometer was performed using the "7-in-1"
peptide mix from Ciphergen Biosystems [(vasopressin (1084.247 Da),
somatostatin (1637.903 Da), porcine dynorphin A (2147.5 Da), human
ACTH (2933.5 Da), bovine insulin B chain (3495.941 Da), human
recombinant insulin (5807.633 Da), and hirudin (7033.614 Da)].
Human SOD1 (15591.4 Da) was also added to this mix.
[0064] Protein profile comparisons were made after normalization of
each spectrogram to total ion current, and raw spectral data
consisted of 37,000 mass peak values for each individual sample.
All samples within each sample set were prepared and analyzed at
the same time within the same experiment. Spectra were analyzed
using Ciphergen ProteinChip software (Version 3.2.1). Statistical
analysis of all mass peaks was performed using the nonparametric
Mann-Whitney test on the maximal intensity of each peak. Peak
labeling was performed using second-pass peak selection with a
signal to noise ratio of 1.5. Significance threshold was set of
p<0.05.
[0065] All CSF samples were analyzed by mass spectroscopy for the
presence of hemoglobin peaks (15.1 and 15.9 kDa mass peaks). The
presence of these distinctive peaks denotes the presence of blood
contamination. Such samples were not used and eliminated from
further analysis.
Example 1
[0066] This example demonstrates different TTR spectral patterns in
ALS and control subjects.
[0067] FIG. 1 shows results obtained from the Ciphergen Protein
chip reader. The CSF from 30 ALS and 15 healthy control or disease
control (multiple sclerosis) subjects was analyzed for the presence
or absence of TTR mass alterations. TTR was immunoprecipitated
using an antibody specific to human transthyretin as described
above. Proteins eluted from the anti-TTR coated beads was directly
added to gold coated Protein Chips (Ciphergen) and read on the mass
spectrometer. A series of 4-5 mass spectral peaks are typically
observed for transthyretin. A characteristic TTR pattern is shown
for both healthy and disease control subjects (top 4 panels).
However, 30% of ALS subjects exhibited split mass spectral peaks
for transthyretin (bottom 3 panels). This suggests either altered
post-translational modifications to transthyretin or genetic
polymorphisms of transthyretin causing altered amino acid
composition of the protein occurs in sporadic ALS patients.
Example 2
[0068] This example demonstrates mass spectral shifts of TTR in ALS
patients using MALDI-TOF-MS.
[0069] TTR was immunoprecipitated from CSF samples from 30 ALS and
20 healthy or disease control subjects. This high resolution mass
spectrometer was used to define any mass changes in TTR with higher
resolution as compared to the Ciphergen mass spectrometer used in
Example 1. To determine if mass spectral shifts may be due to
genetic polymorphisms, transthyretin was immunoprecipitated from
each CSF sample and then the reduced the transthyretin using
tris(2-carboxyethyl)phosphine (TCEP) for 20 min. Reduction of TTR
was necessary to resolve the protein mass peaks such that one can
identify mass shifts induced by amino acid substitutions caused by
genetic polymorphisms. The reduced TTR protein was then analyzed by
mass spectrometry using an ABI Voyager/DE instrument. Within the
control subjects, transthyretin is seen as a single major protein
peak of 13742 Da. A small shoulder was also observed in all
samples. A transthyretin doublet was observed in 9 of 30 (30%)
sporadic ALS patients (FIG. 2). The second peak was approximately
30 Da increased mass than the native protein. Both transthyretin
peaks were observed in these subjects, suggesting that if the
additional peak is due to a genetic polymorphism then the ALS
patients are heterozygotic for this polymorphism. The increased
molecular mass is consistent with the expected mass shift due to a
major transthyretin genetic polymorphism present in the general
population. The TTR mass shift was observed in 2 of 20 (10%)
control subjects. Therefore, a threefold increase was observed in
this specific transthyretin mass shift in the ALS population. This
result was further confirmed by the immuno-SELDI-TOF-MS TTR data
shown in FIG. 3. A specific polymorphism has been identified, which
appears to be increased in sporadic ALS patients and may be a risk
factor for the disease (Goodall, 2005). This is one example of a
genetic polymorphism that may contribute to sporadic ALS.
Polymorphisms in the transthyretin gene are proposed to constitute
another genetic risk factor for ALS.
Example 3
[0070] This example demonstrates increased levels of TTR in the CSF
of ALS subjects.
[0071] Levels of transthyretin (TTR) in the CSF of control and ALS
subjects were examined using sandwich ELISA. The results
demonstrated an overall increase in the total level of TTR in the
CSF of ALS patients relative to control subjects (FIG. 4). This is
due to that fact that individual mass spectral TTR peaks exhibit
alterations that result in decreased overall peak intensity values
though the total level of TTR protein within the CSF has increased.
This data is also contradictory to TTR western blot data using
human spinal cord tissue (data not shown), further suggesting that
TTR levels may decrease within the spinal cord and brain tissue
while increasing within the CSF.
Example 4
[0072] This example demonstrates reduced levels of TTR and cystatin
C in ALS subjects.
[0073] Immunoblotting for cystatin C and transthyretin was
performed using a separate cohort of recently diagnosed ALS and
control subjects (FIG. 5). 25 or 50 .mu.g of CSF protein from
controls (n=17) and ALS (n=17) subjects were electrophoresced on a
10-20% Tris-Tricine Ready Gels (BioRad Laboratories, USA) and
transferred to a PVDF membrane. The control group included 6
healthy subjects, 2 multiple sclerosis, 2 lyme disease, 2 normal
pressure hydrocephalus, 1 dementia, 1 epilepsy, 1 myopathy, 1
meningitis, and 1 neurofibromatosis subjects. The primary
antibodies were used at a dilution of 1:500. For protein
confirmation we loaded 10 .mu.g of purified human cystatin C
protein (Calbiochem, USA) or 50 .mu.g of transthyretin (Biodesign,
USA) into separate gel lanes. These results demonstrate that the
level of the 13.8 kDa TTR monomer is significantly reduced in the
CSF of ALS subjects as compared to control CSF, The immunoblot also
revealed the presence of a 55 kDa peak that represents the
homotetramer form of TTR. The level of homotetramer was also
increased in control subjects. In addition, the level of cystatin C
was reduced in the CSF of ALS subjects as compared to CSF from
control subjects. Although not wishing to be bound by any
particular theory, it is likely that the ELISA analysis (Example 3)
identified a more complete set of TTR proteins due to the use of
multiple, less specific antibodies, thus resulting in an increase
in the total level of TTR in the CSF of ALS patients. The antibody
utilized in the immunoblot, however, was specific to the TTR
biomarker, the levels of which decrease in the CSF of ALS patients.
Therefore, although total TTR levels increase in the CSF of ALS
patients, the TTR biomarker levels decrease.
Example 5
[0074] This example demonstrates decreased levels of TTR in the
motor neurons of ALS subjects.
[0075] Immunohistochemistry for transthyretin by light microscopy
was performed with paraffin embedded lumbar spinal cord sections
from archived post-mortem tissues from the University of Pittsburgh
ALS Tissue Bank. Neuropathologic assessment confirmed the clinical
diagnosis of ALS or the lack of any central nervous system
abnormalities within the control subjects. Healthy controls (n=8)
and ALS (n=16) cases were probed with anti-rabbit polyclonal
antibodies (DAKO, Denmark) at a concentration of 1:300. All
sections were immunostained simultaneously and examined in a coded
manner by two independent investigators. Controls included tissue
sections lacking either primary or secondary antibody. The results
demonstrate decreased levels of TTR in surviving motor neurons in
ALS subjects (FIG. 6). Thus, TTR levels are decreased in the motor
neurons of ALS subjects.
Example 6
[0076] This example demonstrates alterations in transthyretin (FIG.
8) and cystatin C (FIG. 7) protein levels (13.4 kDa peak) in 3 ALS
subjects over a 12-month timeframe.
[0077] In FIG. 7, patient 1 exhibits a linear decrease in cystatin
C levels over a 12-month time frame. Patient 2 fails to exhibit
cystatin C alterations, indicating that not every ALS patient can
be followed for disease progression by this single marker. Patient
3 also exhibits continued decreased levels of the full-length
cystatin C protein peak over a 12-month period. Samples were
analyzed using Q10 Protein Chips and the Ciphergen mass
spectrometer.
[0078] FIG. 8 shows the fluctuation in TTR levels in a patient over
a 15 month time frame. The TTR peaks reside between 13.8-14.3 kDa
region of these spectra. At Time 0 months the patient has already
been diagnosed with ALS and therefore this TTR signature is already
abnormal when compared to a healthy control subject. At 6 months
there are additional alterations to the first mass peak doublet at
approximately 13.8 kDa. At both 12 and 15 months there are
additional decreases in TTR peak intensity values that correlate
with clinical measures of disease progression.
[0079] All references, including publications, patent applications,
and patents, cited herein, including the following bibliography,
are hereby incorporated by reference to the same extent as if each
reference were individually and specifically indicated to be
incorporated by reference and were set forth in its entirety
herein.
[0080] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein is
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0081] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
BIBLIOGRAPHY
[0082] Abrahamson et al., "Structure and expression of the human
cystatin C gene.", Biochem J. 1; 268(2), 287-94 (1990). [0083]
Bensonet al., "Identification of carriers of a variant plasma
prealbumin (transthyretin) associated with familial amyloidotic
polyneuropathy type I", J Clin Invest 75, 71-75 (1985). [0084]
Bergen et al., "Identification of transthyretin variants by
sequential proteomic and genomic analysis", Clin Chem.,
50:1544-1552 (2004). [0085] Bernstein et al., "Transthyretin: Its
response to malnutrition and stress injury. Clinical usefulness and
economic implications", Clin Chem Lab Med 40, 1344-1348 (2002).
[0086] Borchelt et al., "Superoxide dismutase 1 with mutations
linked to familial amyotrophic lateral sclerosis possesses
significant activity", Proc. Natl. Acad. Sci. USA, 91, 8292-8296
(1994). [0087] Chaudhuri et al., "The neuroendocrine protein 7B2
acts as a molecular chaperone in the in vitro folding of human
insulin-like growth factor-1 secreted from yeast", Biochem Biophys
Res Comm., 211:417-425 (1995). [0088] Cleveland et al., "From
Charcot to Lou Gehrig: deciphering selective motor neuron death in
ALS", Nat. Rev. Neurosci., 2, 806-19 (2001). [0089] Connors et al.,
"Tabulation of human transthyretin (TTR) variants", Amyloid, 10,
160-184 (2003). [0090] Corcoran et al., "Absence of retinoids can
induce motoneuron disease in the adult rat and a retinoid defect is
present in motoneuron disease patients", J Cell Sci 115, 4735-4741
(2002). [0091] Desnuelle et al., Amyotrophic Lateral Sclerosis
& Other Motor Neuron Disorders, 2, 9-18 (2001). [0092]
Fernandez et al., "Thyroid hormone administration enhances
remyelination in chronic demyelinating inflammatory disease", Proc.
Natl. Acad. Sci. USA, 101:16363-16368 (2004). [0093] Goodall, et
al., "Association of the H63D polymorphism in the hemochromatosis
gene with sporadic ALS", Neurology, 65, 934-937 (2005). [0094]
Groeneveld et al., "A randomized sequential trial of creatine in
amyotrophic lateral sclerosis", Annals of Neurology, 53, 437-45
(2003). [0095] Kamel et al. "Lead exposure and amyotrophic lateral
sclerosis", Epidemiology 13, 311-319 (2002). [0096] Kim et al.,
"PARP expression is increased in astrocytes but decreased in motor
neurons in the spinal cord of sporadic ALS patients", J.
Neuropathol. Exp. Neural., 62, 88-103 (2003). [0097] Martens et
al., "The novel pituitary polypeptide 7B2 is a highly-conserved
protein coexpressed with proopiomelanocortin ", Eur. J Biochem.,
181, 75-70 (1989). [0098] Mbikay et al., "Neuroendocrine secretory
protein 7B2: structure, expression and functions", Biochem J.,
357:329-342 (2001). [0099] Menzies et al., "Mitochondrial
dysfunction in a cell culture model of familial amyotrophic lateral
sclerosis", Brain, 125, 1522-1533 (2002). [0100] Mey et al.,
"Retinoic acid signaling in the nervous system of adult
vertebrates", Neuroscientist 10, 409-421 (2004). [0101] Miller et
al., Amyotrophic Lateral Sclerosis& Other Motor Neuron
Disorders, 4, 191-206 (2003). [0102] Nagai et al., "Rats expressing
human cytosolic copper-zinc superoxide dismutase transgenes with
amyotrophic lateral sclerosis: associated mutations develop motor
neuron disease", J. Neurosci., 21, 9246-9254 (2001). [0103] Palha,
"Transthyretin as a thyroid hormone carrier: Function revisited",
Clin Chem Lab Med 40, 1292-1300 (2002). [0104] Paquet et al., "The
neuroendocrine precursor 7B2 is a sulfated protein proteolytically
processed by a ubiquitous furin-like convertase", J Biol. Chem.,
29; 269(30):19279-85 (1994). [0105] Ranganathan et al.,
"Alterations in G(1) to S phase cell-cycle regulators during
amyotrophic lateral sclerosis", Am. J. Pathol., 162, 823-835
(2003). [0106] Ranganathan et al., "Proteomic profiling of
cerebrospinal fluid identifies biomarkers for amyotrophic lateral
sclerosis", J Neurochem, 9, 1461-1471 (2005). [0107] Rosen et al.,
"Mutations in Cu/Zn superoxide dismutase gene are associated with
familial amyotrophic lateral sclerosis", Nature, 362, 59-62 (1993).
[0108] Rosen et al., "A frequent ala 4 to val superoxide
dismutase-1 mutation is associated with a rapidly progressive
familial amyotrophic lateral sclerosis", Hum Mol Genet, 3, 981-987
(1994). [0109] Shaw et al., Amyotrophic Lateral Sclerosis &
Other Motor Neuron Disorders, 1, Suppl. 2, 861-67 (2000). [0110]
Smith et al., "Presence of 4-hydroxynonenal in cerebrospinal fluid
of patients with sporadic amyotrophic lateral sclerosis", Ann.
Neurol., 44, 696-699 (1998). [0111] Sousa et al.,
"Neurodegeneration in familial amyloid polyneuropathy: from
pathology to molecular signaling", Progr Neurobiol 71, 385-400
(2003). [0112] Sousa et al., "Deposition of transthyretin in early
stages of familial amyloidotic polyneuropathy: evidence for
toxicity of nonfibrillar aggregates", Am J Pathol 159, 1993-2000
(2001). [0113] Sousa et al., "Evidence for early cytotoxic
aggregates in transgenic mice for human transthyretin Leu55Pro", Am
J Pathol 161, 1935-1948 (2002). [0114] Spreux-Varoquaux et al.,
"Glutamate levels in cerebrospinal fluid in amyotrophic lateral
sclerosis: a reappraisal using a new HPLC method with coulometric
detection in a large cohort of patients", Journal of the
Neurological Sciences, 193, 73-78 (2002). [0115] Stein et al.,
"Neutralization of transthyretin reverses the neuroprotective
effects of secreted amyloid precursor protein (APP) in A.beta.Psw
mice resulting in tau phosphorylation and loss of hippocampal
neurons: Support for the amyloid hypothesis", J Neurosci 24,
7707-7717 (2004). [0116] Subramaniam et al., "Mutant SOD1 causes
motor neuron disease independent of copper chaperone-mediated
copper loading", Nat. Neurosci., 5, 301-307 (2002). [0117] Tsuzuki
et al., "Transthyretin binds amyloid beta peptides, Abetal-42 and
Abetal-40 to form complex in the autopsied human kidney-possible
role of transthyretin for Abeta sequestration", Neurosci Lett 281,
171-174 (2000). [0118] Vinceti et al., "Lead, cadmium, and selenium
in the blood of patients with sporadic amyotrophic lateral
sclerosis", Ital J Neurol Sci 18, 87-92 (1997). [0119] Zheng et
al., "Transthyretin, thyroxine, and retinol-binding protein in
human cerebrospinal fluid: effect of lead exposure", Toxicol Sci
61, 107-114 (2001). [0120] Zheng, "Toxicology of choroid plexus:
Special reference to metal-induced neurotoxicities", Microscopy Res
& Tech 52, 89-103 (2001). [0121] Zhu et al., "Internal cleavage
of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential
mechanism for its inactivation", Proc Nad Acad Sci, 14;
93(10):4919-24 (1996).
Sequence CWU 1
1
41636DNAHomo sapiensmisc_feature(1)..(636)7b2 cDNA 1gcaactgttt
tgtttaatac ctttctgcaa tgatgctctg cctagcatgg aaacttaaga 60caaaagcaac
ctcctaagga ttctttgtta caaccccttt ctgggtggtc ctcgaaccac
120aacctggagt ggttgcatca ttataattgt attatcacaa ttgccgattg
tagcctatca 180gtatacattt ggtcttttat ctgcaggttg acaatggtct
ccaggatggt ctctaccatg 240ctatctggcc tactgttttg gctggcatct
ggatggactc cagcatttgc ttacagcccc 300cggacccctg accgggtctc
agaagcagat atccagaggc tgcttcatgg tgttatggag 360caattgggca
ttgccaggcc ccgagtggaa tatccagctc accaggccat gaatcttgtg
420ggcccccaga gcattgaagg tatttactgt gttctgatgg tttgaagttt
ccgttagcat 480tttaaataat atatttgcac acttctcaca acaaccttat
aagtagatgc ctttattatc 540ctattttttt cagaggagga agctaatttt
taagagactc tactttctca tggttttccc 600tttcttcttc tacacagtgt
ctacatactt acatat 6362211PRTHomo sapiensMISC_FEATURE(1)..(211)7b2
2Met Val Ser Arg Met Val Ser Thr Met Leu Ser Gly Leu Leu Phe Trp1 5
10 15Leu Ala Ser Gly Trp Thr Pro Ala Phe Ala Tyr Ser Pro Arg Thr
Pro 20 25 30Asp Arg Val Ser Glu Ala Asp Ile Gln Arg Leu Leu His Gly
Val Met 35 40 45Glu Gln Leu Gly Ile Ala Arg Pro Arg Val Glu Tyr Pro
Ala His Gln 50 55 60Ala Met Asn Leu Val Gly Pro Gln Ser Ile Glu Gly
Gly Ala His Glu65 70 75 80Gly Leu Gln His Leu Gly Pro Phe Gly Asn
Ile Pro Asn Ile Val Ala 85 90 95Glu Leu Thr Gly Asp Asn Ile Pro Lys
Asp Phe Ser Glu Asp Gln Gly 100 105 110Tyr Pro Asp Pro Pro Asn Pro
Cys Pro Val Gly Lys Thr Asp Asp Gly 115 120 125Cys Leu Glu Asn Thr
Pro Asp Thr Ala Glu Phe Ser Arg Glu Phe Gln 130 135 140Leu His Gln
His Leu Phe Asp Pro Glu His Asp Tyr Pro Gly Leu Gly145 150 155
160Lys Trp Asn Lys Lys Leu Leu Tyr Glu Lys Met Lys Gly Gly Glu Arg
165 170 175Arg Lys Arg Arg Ser Val Asn Pro Tyr Leu Gln Gly Gln Arg
Leu Asp 180 185 190Asn Val Val Ala Lys Lys Ser Val Pro His Phe Ser
Asp Glu Asp Lys 195 200 205Asp Pro Glu 2103482DNAHomo
sapiensmisc_feature(1)..(482)cystatin C cDNA 3ttccggaaaa gggagtgcag
ggcccggggg ggtggggcgg cgaaggcggg aagggataaa 60accgcagtcg ccggcctcgc
ggggctcacg gcctcgcctc ggtatcgccg cgggtcctct 120ctatctagct
ccagcctctc gcctgcgccc cactccccgc gtcccgctcc tagccgacca
180tggccgggcc cctgcgcgcc ccgctgctcc tgctggccat cctggccgtg
gccctggccg 240tgagccccgc gaccggctcc agtcccggca agccgccgcg
cctggtggga ggccccatgg 300acgccagcgt ggaggaggag ggtgtgcggc
gtgcactgga ctttgccgtc ggcgagtaca 360acaaagccag caacgacatg
taccacagcc gcgcgctgca ggtggtgcgc gcccgcaagc 420aggtgcgtgc
cgccccccgc aggtccgaag ccccggcccc gccgtcccag cctccccccg 480cg
4824146PRTHomo sapiensMISC_FEATURE(1)..(146)cystatin C 4Met Ala Gly
Pro Leu Arg Ala Pro Leu Leu Leu Leu Ala Ile Leu Ala1 5 10 15Val Ala
Leu Ala Val Ser Pro Ala Thr Gly Ser Ser Pro Gly Lys Pro 20 25 30Pro
Arg Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly 35 40
45Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys Ala Ser
50 55 60Asn Asp Met Tyr His Ser Arg Ala Leu Gln Val Val Arg Ala Arg
Lys65 70 75 80Gln Ile Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu
Leu Gly Arg 85 90 95Thr Thr Cys Thr Lys Thr Gln Pro Asn Leu Asp Asn
Cys Pro Phe His 100 105 110Asp Gln Pro His Leu Lys Arg Lys Ala Phe
Cys Ser Phe Gln Ile Tyr 115 120 125Ala Val Pro Trp Gln Gly Thr Met
Thr Leu Ser Lys Ser Thr Cys Gln 130 135 140Asp Ala145
* * * * *