U.S. patent application number 12/040739 was filed with the patent office on 2009-02-05 for truncated fragments of alpha-synuclein in lewy body disease.
This patent application is currently assigned to Elan Pharmaceuticals, Inc.. Invention is credited to John P. Anderson, Tamie J. Chilcote, Jason Goldstein, Donald Walker.
Application Number | 20090035217 12/040739 |
Document ID | / |
Family ID | 36203707 |
Filed Date | 2009-02-05 |
United States Patent
Application |
20090035217 |
Kind Code |
A1 |
Chilcote; Tamie J. ; et
al. |
February 5, 2009 |
Truncated Fragments of Alpha-Synuclein in Lewy Body Disease
Abstract
The application identifies novel fragments of alpha-synuclein in
patients with Lewy Body Disease (LBD) and transgenic animal models
thereof. These diseases are characterized by aggregations of
alpha-synuclein. The fragments have a truncated C-terminus relative
to full-length alpha-synuclein. Some fragments are characterized by
a molecular weight of about 12 kDa as determined by SDS gel
electrophoresis in tricine buffer and a truncation of at least ten
contiguous amino acids from the C-terminus of natural
alpha-synuclein. The site of cleavage preferably occurs after
residue 117 and before residue 126 of natural alpha-synuclein. The
identification of these novel fragments of alpha-synuclein has a
number of application in for example, drug discovery, diagnostics,
therapeutics, and transgenic animals.
Inventors: |
Chilcote; Tamie J.; (San
Francisco, CA) ; Goldstein; Jason; (Burlingame,
CA) ; Anderson; John P.; (San Francisco, CA) ;
Walker; Donald; (Pleasant Hill, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Elan Pharmaceuticals, Inc.
South San Francisco
CA
|
Family ID: |
36203707 |
Appl. No.: |
12/040739 |
Filed: |
February 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11194115 |
Jul 29, 2005 |
7358331 |
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12040739 |
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10969335 |
Oct 19, 2004 |
7306945 |
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11194115 |
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10850570 |
May 19, 2004 |
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10969335 |
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11194115 |
Jul 29, 2005 |
7358331 |
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10850570 |
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10969335 |
Oct 19, 2004 |
7306945 |
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11194115 |
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PCT/US04/15836 |
May 19, 2004 |
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10969335 |
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60471929 |
May 19, 2003 |
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60471929 |
May 19, 2003 |
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Current U.S.
Class: |
424/9.1 ;
424/139.1; 435/219; 435/29; 436/86; 514/1.1; 530/387.3; 530/387.9;
800/3; 800/9 |
Current CPC
Class: |
G01N 2800/28 20130101;
A01K 2227/706 20130101; A61P 25/28 20180101; A61K 38/00 20130101;
A61P 25/00 20180101; C07K 16/18 20130101; A01K 67/0275 20130101;
A61P 25/16 20180101; A61P 43/00 20180101; A01K 2267/0356 20130101;
A01K 2227/105 20130101; G01N 2800/2821 20130101; G01N 2800/2835
20130101; C12Q 1/6883 20130101; G01N 33/6896 20130101; C12N 15/8509
20130101; C07K 2317/34 20130101; C12Q 2600/156 20130101; G01N
2800/2814 20130101; C07K 2317/33 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
424/9.1 ; 436/86;
800/3; 800/9; 435/29; 530/387.9; 530/387.3; 514/12; 424/139.1;
435/219 |
International
Class: |
A61K 49/00 20060101
A61K049/00; G01N 33/00 20060101 G01N033/00; A01K 67/027 20060101
A01K067/027; A61K 38/00 20060101 A61K038/00; C12N 9/50 20060101
C12N009/50; A61P 25/00 20060101 A61P025/00; A61K 39/395 20060101
A61K039/395; C12Q 1/02 20060101 C12Q001/02; C07K 16/18 20060101
C07K016/18 |
Claims
1. A method of screening for an agent having a pharmacological
activity useful for treating a Lewy Body Disease (LBD) comprising:
contacting the agent with phosphorylated alpha-synuclein or a
phosphorylated fragment thereof, wherein the fragment is
characterized by presence of at least 100 contiguous amino acids of
intact alpha-synuclein and a deletion of 1-11 contiguous amino
acids from the C-terminus of intact alpha-synuclein; determining
the rate or extent of aggregation of the alpha synuclein or
fragment of alpha-synuclein, wherein a reduction in the rate or
extent of aggregation relative to a control lacking the agent
indicates the agent has the pharmacological activity.
2. The method of claim 1, wherein the agent is contacted with
intact alpha synuclein or SN1-133 or SN1-135 numbered according to
SEQ ID NO:1.
3. The method of claim 1, wherein the fragment of alpha-synuclein
bears a mutation associated with a hereditary LBD.
4. The method of claim 3, wherein the mutation is an A53T
mutation.
5. The method of claim 1, further comprising conducting a trial in
a human having a LBD or an animal model of LBD to determine whether
the agent treats or inhibits a symptom of the LBD.
6. A method of screening for an agent having a pharmacological
activity useful for treating a Lewy Body Disease (LBD) comprising:
contacting the agent with a fragment of alpha synuclein, which is
SN1-115, SN1-133 or SN1-135, with residues numbered according to
SEQ ID NO:1; determining the rate or extent of aggregation of the
alpha synuclein or fragment of alpha-synuclein, wherein a reduction
in the rate or extent of aggregation relative to a control lacking
the agent indicates the agent has the pharmacological activity.
7. The method of claim 6, wherein the fragment of alpha-synuclein
bears a mutation associated with a hereditary LBD.
8. The method of claim 37 wherein the mutation is an A53T
mutation.
9. The method of claim 6, further comprising conducting a trial in
a human having a LBD or an animal model of LBD to determine whether
the agent treats or inhibits a symptom of the LBD.
10. A method of screening an agent for a pharmacological activity
useful in treating a LBD, comprising contacting a cell expressing
alpha-synuclein and processing the alpha-synuclein into a fragment
with an agent, wherein the fragment is SN1-115, SN1-133 or SN1-135
numbered according to SEQ ID NO:1; determining a level of the
fragment in the cell relative to a baseline level in the same cell
type in the absence of the agent, a reduction in the level of the
fragment relative to the baseline indicating the agent has the
pharmacological activity useful in treating a LBD.
11. The method of claim 10, wherein the fragment of alpha-synuclein
bears a mutation associated with a hereditary LBD.
12. The method of claim 11, wherein the mutation is an A53T
mutation.
13. The method of claim 10, further comprising conducting a trial
in a human having a LBD or an animal model of LBD to determine
whether the agent treats or inhibits a symptom of the LBD.
14. The method of claim 13, wherein the Lewy Body disease is
Parkinson's disease or Diffuse Lewy Body Disease (DLBD).
15. A method of screening for an agent having a pharmacological
activity useful for treating a LBD, comprising: contacting a
transgenic animal having a transgene expressing a fragment of
alpha-synuclein, wherein the fragment is SN1-115, SN1-133 or
SN1-135 numbered according to SEQ ID NO:1; determining a level of
aggregated forms of the fragment in the brain of the transgenic
animal relative to a baseline level of aggregated forms of the
fragment in a comparable transgenic animal in the absence of the
agent, a reduction in the level of the aggregated forms fragment
relative to the baseline indicating the agent has a pharmacological
activity useful in treating a LBD.
16. The method of claim 15, wherein the fragment of alpha-synuclein
bears a mutation associated with a hereditary LBD.
17. The method of claim 16, wherein the mutation is an A53T
mutation.
18. The method of claim 15, wherein the transgenic animal is a
mouse.
19. The method of claim 15, wherein the transgenic animal is a
Drosophila.
20. The method of claim 15, further comprising conducting a trial
in a human having a LBD or an animal model of LBD to determine
whether the agent treats or inhibits a symptom of the LBD.
21. The method of claim 20, wherein the LBD is Parkinson's disease
or DLBD.
22. A method of screening an agent for a pharmacological activity
useful for treating a LBD, comprising: contacting a transgenic
animal having a transgene expressing alpha-synuclein and processing
the alpha-synuclein into a fragment with an agent, wherein the
fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ
ID NO:1; determining a level of the fragment in a neuronal cell
relative to a baseline level in the absence of the agent, a
reduction in the level of the fragments relative to the baseline
indicating the agent has the pharmacological activity useful for
treating the LBD.
23. The method of claim 22, wherein the fragment of alpha-synuclein
bears a mutation associated with a hereditary LBD.
24. The method of claim 23, wherein the mutation is an A53T
mutation.
25. The method of claim 22, wherein the transgenic animal is a
mouse.
26. The method of claim 22, wherein the transgenic animal is a
Drosophila.
27. The method of claim 22, further comprising conducting a trial
in a human having a LBD or an animal model of LBD to determine
whether the agent treats or inhibits a symptom of the LBD.
28. The method of claim 27, wherein the LBD is Parkinson's disease
or DLBD.
29. A transgenic animal having a genome comprising a transgene
comprising a promoter operably linked to a nucleic acid segment
encoding a fragment of alpha-synuclein wherein the fragment is
SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO:1;
wherein expression of the fragment in the transgenic animal
disposes the animal to develop at least one characteristic of a
LBD. Screening any animal or cell
30. The transgenic animal of claim 29, wherein the promoter is a
PDGF promoter.
31. The transgenic animal of claim 29, wherein the at least one
characteristic is an impairment of motor function.
32. The transgenic animal of claim 29, wherein the at least one
characteristic is an impairment of cognitive function.
33. The transgenic animal of claim 29 that is a mouse.
34. The transgenic animal of claim 29 that is a Drosophila.
35. A method of detecting presence or susceptibility to an LBD in a
patient, comprising detecting a level of a fragment of
alpha-synuclein in body fluid, wherein the fragment is SN1-115,
SN1-133 or SN1-135 numbered according to SEQ ID NO:1; a level
greater than a baseline level in undiseased individuals indicating
presence or susceptibility to LBD.
36. An antibody that specifically binds to a fragment of
alpha-synuclein, wherein the fragment is SN1-115, SN116-140,
SN1-133, SN1-135, SN134-140 or SN136-140 numbered according to SEQ
ID NO:1; without specifically binding to full-length alpha
synuclein.
37. The antibody of claim 36 that is a human antibody.
38. The antibody of claim 36 that is a humanized antibody.
39. The antibody of claim 36 that is a monoclonal antibody.
40. The antibody of claim 36 having human isotype IgG1.
41. The antibody of claim 36 designated 12C6 or 7G8.
42. A method of diagnosing presence of or susceptibility to LBD,
comprising administering to a patient an antibody that specifically
binds to SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID
NO:1 without specifically binding to full length alpha-synuclein;
determining a level of binding of the antibody in the patients,
wherein a higher level of binding relative to a base line level in
undiseased individuals indicates presence or susceptibility to the
LBD.
43. A method of effecting treatment or prophylaxis of a LBD,
comprising administering to a patient suffering from or at risk of
a LBD, an effective regime of phosphorylated alpha synuclein or a
phosphorylated fragment of alpha-synuclein, wherein the fragment is
characterized by presence of at least 100 contiguous amino acids of
intact alpha-synuclein and a deletion of 1-11 contiguous amino
acids from the C-terminus of intact alpha-synuclein; and thereby
effecting treatment or prophylaxis of the LBD.
44. The method of claim 43, wherein the fragment is SN1-133 or
SN1-135.
45. The method of claim 43, further comprises administering an
adjuvant that augments an immune response comprising antibodies to
the fragment.
46. The method of claim 43, wherein the fragment is linked to a
carrier forming a fusion protein, wherein the carrier augments an
immune response comprising antibodies to the fragment.
47. A method of effecting treatment or prophylaxis of a LBD,
comprising administering to a patient suffering from or at risk of
a LBD, an effective regime of a fragment of alpha-synuclein,
wherein the fragment is SN1-115, SN1-133 or SN1-135 numbered
according to SEQ ID NO:1; or a subfragment of any of these
including at least five continuous residues starting from the
C-terminus of one of the fragments, and thereby effecting treatment
or prophylaxis of the LBD.
48. A method of effecting treatment or prophylaxis of a LBD
comprising administering to a patient suffering from or at risk of
a LBD an effective regime of an antibody that specifically binds to
a fragment of alpha-synuclein, wherein the fragment is SN1-115,
SN1-133 or SN1-135, with residues numbered according to SEQ ID NO:1
without binding to intact alpha-synuclein, whereby the antibody
effects prophylaxis or treatment of the disease.
49. A method of purifying a protease that cleaves intact
alpha-synuclein to form a fragment, wherein the fragment is
SN1-115, SN1-133 or SN1-135 with residues numbered according to SEQ
ID NO:1; comprising identifying an inhibitor of the protease;
contacting the inhibitor with a cellular or tissue extract
containing the protease, whereby the protease binds to the
inhibitor; and releasing the protease from the inhibitor.
50. The method of claim 49, wherein the inhibitor is a peptide of
alpha-synuclein comprising a contiguous segment of at least 5
residues of intact alpha-synuclein between positions 129-139, or
111-119.
51. The method of claim 49, wherein at least one of the residues is
a transition state analog.
52. A monoclonal antibody designated 12C6 or 7G8 or a chimeric or
humanized form thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 10/969,335, filed Oct. 19, 2004, which is a
continuation-in-part of U.S. application Ser. No. 10/850,570 filed
May 19, 2004, which claims the benefit under 35 USC .sctn.119(e) of
U.S. Application No. 60/471,929, filed May 19, 2003, each of which
are incorporated by reference in its entirety for all purposes.
U.S. application Ser. No. 10/969,335, filed Oct. 19, 2004, is also
a continuation-in-part of PCT/US0401/15836 filed May 19, 2004,
published as WO 05/013889, which claims the benefit under 35 USC
.sctn. 119(e) of US Application No. 60/471,929, filed May 19, 2003,
each incorporated by reference in its entirety for all
purposes.
BACKGROUND
[0002] Lewy body diseases (LBDs) are characterized by degeneration
of the dopaminergic system, motor alterations, cognitive
impairment, and formation of Lewy bodies (LBs). (McKeith et al.,
Clinical and pathological diagnosis of dementia with Lewy bodies
(DLB): Report of the CDLB International Workshop, Neurology (1996)
47:1113-24). LBDs include Parkinson's disease, Diffuse Lewy body
disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), and
combined PD and Alzheimer's disease (AD) and the syndromes
identified as multiple system atrophy (MSA). Dementia with Lewy
bodies (DLB) is a term coined to reconcile differences in the
terminology of LBDs. Disorders with LBs continue to be a common
cause for movement disorders and cognitive deterioration in the
aging population (Galasko et al., Clinical-neuropathological
correlations in Alzheimer's disease and related dementias. Arch.
Neurol. (1994) 51:888-95). Although their incidence continues to
increase creating a serious public health problem, to date these
disorders lack approved treatments (Tanner et al., Epidemiology of
Parkinson's disease and akinetic syndromes, Curr. Opin. Neurol.
(2000) 13:427-30). The cause for LBD's is controversial and
multiple factors have been proposed to play a role, including
various neurotoxins and genetic susceptibility factors.
[0003] AD, PD, and DLBD are the most commonly found
neurodegenerative disorders in the elderly. Recent epidemiological
studies have demonstrated a close clinical relationship between AD
and PD, as about 30% of Alzheimer's patients also have PD. Compared
to the rest of the aging population, patients with AD are thus more
likely to develop concomitant PD. Furthermore, PD patients that
become demented usually have developed classical AD. Although each
neurodegenerative disease appears to have a predilection for
specific brain regions and cell populations, resulting in distinct
pathological features, PD, AD, and DLBD also share common
pathological hallmarks. Patients with familial AD, Down syndrome,
or sporadic AD develop LBs on the amygdala, which are the classical
neuropathological hallmarks of PD. Additionally, each disease is
associated with the degeneration of neurons, interneuronal synaptic
connections and eventually cell death, the depletion of
neurotransmitters, and abnormal accumulation of misfolded proteins,
the precursors of which participate in normal central nervous
system function. Biochemical studies have confirmed a link between
AD, PD and DLB.
[0004] In recent years, new hope for understanding the pathogenesis
of LBD has emerged. Specifically, several studies have shown that
the synaptic protein alpha-synuclein plays a central role in PD
pathogenesis since: (1) this protein accumulates in LBs
(Spillantini et al., Nature (1997) 388:839-40; Takeda et al., J.
Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett.
(1997) 239:45-8), (2) mutations in the alpha-synuclein gene
co-segregate with rare familial forms of parkinsonism (Kruger et
al., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science
(1997) 276:2045-7) and, (3) its overexpression in transgenic mice
(Masliah et al., Science (2000) 287:1265-9) and Drosophila (Feany
et al., Nature (2000) 404:394-8) mimics several pathological
aspects of PD. Thus, the fact that accumulation of alpha-synuclein
in the brain is associated with similar morphological and
neurological alterations in species as diverse as humans, mice, and
flies suggests that this molecule contributes to the development of
PD.
[0005] The neuritic plaques that are the classic pathological
hallmark of AD consist essentially of amyloid beta (A.beta.)
peptide, an amino acid proteolytic product of the amyloid precursor
protein (APP), and NAC, a 35 amino acid proteolytic fragment of
alpha-synuclein. Both A.beta. and NAC were first identified in
amyloid plaques as proteolytic fragments of their respective
full-length proteins, for which the full-length cDNAs were
identified and cloned. (Iwai A., Biochim. Biophys. Acta (2000)
1502:95-109); Masliah et al., AM. J Pathol (1996) 148:201-10; Ueda
et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6).
[0006] Alpha-synuclein is part of a large family of proteins
including beta- and gamma-synuclein and synoretin. Alpha-synuclein
is expressed in the normal state associated with synapses and is
believed to play a role in neural plasticity, learning and memory.
Mutations in human (h) alpha-synuclein that enhance the aggregation
of alpha-synuclein have been identified (Ala30Pro and Ala53Thr) and
are associated with rare forms of autosomal dominant forms of PD.
The mechanism by which these mutations increase the propensity of
alpha-synuclein to aggregate are unknown.
SUMMARY OF THE CLAIMED INVENTION
[0007] The invention provides methods of screening for an agent
having a pharmacological activity useful for treating a Lewy Body
Disease (LBD). The method involves contacting the agent with a
fragment of alpha-synuclein, wherein the fragment is characterized
by presence of at least 100 contiguous amino acids of intact (i.e.,
full-length) alpha-synuclein and a deletion of 1-25 contiguous
amino acids from the C-terminus of intact alpha-synuclein; and
determining the rate or extent of aggregation of the fragment of
alpha-synuclein, wherein a reduction in the rate or extent of
aggregation relative to a control lacking the agent indicates the
agent has the pharmacological activity.
[0008] Optionally, the fragment has a C-terminus at a residue
within residues 115 and 125 of intact alpha-synuclein with residues
numbered according to SEQ ID NO:1. Preferred fragments include
alpha-synuclein SN1-115, SN1-116, SN1-117, SN1-118, SN1-119,
SN1-120, SN1-121, SN1-122, SN1-123, SN1-124, and SN1-125. SN1-115,
SN1-119, SN1-122, SN1-133 and SN1-135 are particularly preferred.
Optionally, the fragment of alpha-synuclein is 1-X, wherein X is
130-139. Optionally, the fragment of alpha-synuclein bears a
mutation associated with a hereditary LBD, such as an A53T
mutation. Optionally, the method involves an additional step of
conducting a trial in a human having a LBD or an animal model of
LBD to determine whether the agent treats or inhibits a symptom of
the LBD.
[0009] The invention further provides methods of screening for an
agent having a pharmacological activity useful for treating a Lewy
Body Disease (LBD). The methods involve contacting the agent with
phosphorylated alpha-synuclein or a phosphorylated fragment
thereof, wherein the fragment is characterized by presence of at
least 100 contiguous amino acids of intact alpha-synuclein and a
deletion of 1-11 contiguous amino acids from the C-terminus of
intact alpha-synuclein; and determining the rate or extent of
aggregation of the alpha synuclein or fragment of alpha-synuclein,
wherein a reduction in the rate or extent of aggregation relative
to a control lacking the agent indicates the agent has the
pharmacological activity. Optionally, the agent is contacted with
intact alpha synuclein or SN1-133 or SN1-135 numbered according to
SEQ ID NO:1. Optionally, intact alpha synuclein or the fragment of
alpha-synuclein bears a mutation associated with a hereditary LBD.
Optionally, the mutation is an A53T mutation. Optionally, the
method further comprises conducting a trial in a human having a LBD
or an animal model of LBD to determine whether the agent treats or
inhibits a symptom of the LBD.
[0010] The invention further provides methods of screening an agent
for a pharmacological activity useful in treating a LBD (e.g.,
Parkinson's disease or DLBD). These methods comprise contacting a
cell expressing alpha-synuclein and processing the alpha-synuclein
into a fragment with an agent. The fragment is characterized by
presence of at least 100 contiguous amino acids of intact
alpha-synuclein and a deletion of 1-25 contiguous amino acids from
the C-terminus of intact alpha-synuclein. One then determines a
level of the fragment in the cell relative to a baseline level in
the same cell type in the absence of the agent, a reduction in the
level of the fragment relative to the baseline indicating the agent
has the pharmacological activity useful in treating a LBD.
Optionally, the fragment of alpha-synuclein has a C-terminus at a
residue between 115 and 125 of intact alpha-synuclein. Preferred
fragments are SN1-115, SN1-116, SN1-117, SN1-118, SN1-119, SN1-120,
SN1-121, SN1-122, SN1-123, SN1-124, and SN1-125 of alpha synuclein.
SN1-115, SN1-119, SN1-122, SN1-133 and SN1-135 are particularly
preferred. Optionally, the fragment of alpha-synuclein is 1-X,
wherein X is 130-139. Optionally, the fragment of alpha-synuclein
bears a mutation associated with a hereditary LBD, such as an A53T
mutation. The cell can be a human cell, a neuronal cell, a
dopaminergic cell or a nondopaminergic cell. Optionally, the cell
is a PC12 or Sy5Y cell. Optionally, the method involves a step of
conducting a trial in a human having a LBD or an animal model of
LBD to determine whether the agent treats or inhibits a symptom of
the LBD.
[0011] The invention further provides methods of screening for an
agent having a pharmacological activity useful for treating a LBD
(e.g., Parkinson's disease or DLBD). The methods involve contacting
a transgenic animal expressing a fragment of alpha-synuclein,
wherein the fragment is characterized by presence of at least 100
contiguous amino acids of intact alpha-synuclein and a deletion of
1-25 contiguous amino acids from the C-terminus of intact
alpha-synuclein; and determining a level of aggregated forms of the
fragment in the brain of the transgenic animal relative to a
baseline level of aggregated forms of the fragment in a comparable
transgenic animal in the absence of the agent, a reduction in the
level of the aggregated forms fragment relative to the baseline
indicating the agent has a pharmacological activity useful in
treating a LBD. Optionally, the fragment of alpha-synuclein has a
C-terminus at a residue between 115 and 125 of intact
alpha-synuclein. Preferred fragments include SN1-115, SN1-116,
SN1-117, SN1-118, SN1-119, SN1-120, SN1-121, SN1-122, SN1-123,
SN1-124, and SN1-125 of alpha synuclein. SN1-115, SN1-119, SN1-122,
SN133 and SN135 are particularly preferred. Optionally, the
fragment of alpha-synuclein is 1-X, wherein X is 119-139.
Optionally, the fragment of alpha-synuclein bears a mutation
associated with a hereditary LBD, such as an A53T mutation.
Optionally, the transgenic animal is a rodent. The transgenic
animal can also be a Drosophila. Optionally, the method involves
conducting a trial in a human having a LBD or an animal model of
LBD to determine whether the agent treats or inhibits a symptom of
the LBD.
[0012] The invention further provides methods of screening an agent
for a pharmacological activity useful for treating a LBD (e.g.,
Parkinson's disease or DLBD). The methods involve contacting a
transgenic animal expressing alpha-synuclein and processing the
alpha-synuclein into a fragment with an agent, wherein the fragment
is characterized by presence of at least 100 contiguous amino acids
of intact alpha-synuclein and a deletion of 1-25 contiguous amino
acids from the C-terminus of intact alpha-synuclein; and
determining a level of the fragment in a neuronal cell relative to
a baseline level in the absence of the agent, a reduction in the
level of the fragments relative to the baseline indicating the
agent has the pharmacological activity useful for treating the LBD.
Optionally, the fragment of alpha-synuclein has a C-terminus at a
residue between 115 and 125 of intact alpha-synuclein. Preferred
fragments include SN1-115, SN1-116, SN1-117, SN1-118, SN1-119,
SN1-120, SN1-121, SN1-122, SN1-123, SN1-124, and SN1-125 of alpha
synuclein. SN1-115, SN1-119, SN1-122, SN133 and SN135 are
particularly preferred. Optionally, the fragment of alpha-synuclein
is 1-X, wherein X is 130-139. Optionally, the fragment of
alpha-synuclein bears a mutation associated with a hereditary LBD,
such as an A53T mutation. Optionally, the transgenic animal is a
rodent, mouse or Drosophila. Optionally, the method involves a step
of conducting a trial in a human having a LBD or an animal model of
LBD to determine whether the agent treats or inhibits a symptom of
the LBD.
[0013] The invention further provides a transgenic animal having a
genome comprising a transgene comprising a promoter operably linked
to a nucleic acid segment encoding a fragment of alpha-synuclein
wherein the fragment is characterized by presence of at least 100
contiguous amino acids of intact alpha-synuclein and a deletion of
1-25 contiguous amino acids from the C-terminus of intact
alpha-synuclein; wherein expression of the fragment in the
transgenic animal disposes the animal to develop at least one
characteristic of a LBD. Optionally, the fragment of
alpha-synuclein is selected from the group consisting of SN1-115,
SN1-116, SN1-117, SN1-118, SN1-119, SN1-120, SN1-121, SN1-122,
SN1-123, SN1-124, and SN1-125. SN1-115, SN1-119, SN1-122, SN133 and
SN135 are particularly preferred. Optionally, the fragment of
alpha-synuclein is 1-X, wherein X is 130-139. Optionally, the
promoter is a PDGF promoter. Optionally, at least one
characteristic is an impairment of motor function. Optionally, at
least one characteristic of the transgenic animal is an impairment
of cognitive function. Optionally, the transgenic animal is a
rodent, mouse or Drosophila.
[0014] The invention further provides methods of detecting presence
or susceptibility to an LBD in a patient. The methods involve
detecting a level of a fragment of alpha-synuclein in cerebrospinal
fluid, wherein the fragment is characterized by presence of at
least 100 contiguous amino acids of intact alpha-synuclein and a
deletion of 1-25 contiguous amino acids from the C-terminus of
intact alpha-synuclein. A change in level, usually an increase,
relative to the baseline level in undiseased individuals indicating
presence or susceptibility to LBD.
[0015] The invention further provides an antibody that specifically
binds to a fragment of alpha-synuclein, wherein the fragment is
characterized by presence of at least 100 contiguous amino acids of
intact alpha-synuclein and a deletion of 1-25 contiguous amino
acids from the C-terminus of intact alpha-synuclein; without
specifically binding to full-length alpha synuclein. Preferred
fragments include SN1-115, SN1-116, SN1-117, SN1-118, SN1-119,
SN1-120, SN1-121, SN1-122, SN1-123, SN1-124, and SN1-125 of alpha
synuclein. SN1-115, SN1-119, SN1-122, SN133 and SN135 are
particularly preferred. Optionally, the fragment is 1-X, wherein X
is 130-139. Optionally, the antibody is a human, humanized,
chimeric antibody. Optionally, the antibody is monoclonal.
Optionally, the antibody has human isotype IgG1.
[0016] The invention further provides methods of diagnosing
presence or susceptibility to LBD. The methods involve
administering to a patient an antibody that specifically binds to a
fragment of alpha-synuclein having a free C-terminus at residues
115-135 without specifically binding to full length synuclein; and
determining a level of binding of the antibody in the patients,
wherein a higher level of binding relative to a base line level in
undiseased individuals indicates presence or susceptibility to the
LBD. Preferably the antibody specifically binds to the free
C-terminus of a fragment selected from the group consisting of
SN1-115, SN1-19, SN1-122, SN133 and SN135.
[0017] The invention further provides methods of effecting
treatment or prophylaxis of a LBD, comprising administering to a
patient suffering from or at risk of a LBD, an effective regime of
a fragment of alpha-synuclein, wherein the fragment is
characterized by presence of at least 100 contiguous amino acids of
intact alpha-synuclein and a deletion of 1-25 contiguous amino
acids from the C-terminus of intact alpha-synuclein, and thereby
effecting treatment or prophylaxis of the LBD. Optionally, the
fragment of alpha-synuclein is SN1-115, SN1-116, SN1-117, SN1-118,
SN1-119, SN1-120, SN1-121, SN1-122, SN1-123, SN1-124, and SN1-125
of alpha synuclein. SN1-115, SN1-119, SN1-122, SN133 and SN135 are
particularly preferred. Optionally, the fragment is 1-X, wherein X
is 130-139. Optionally, the method further comprises administering
an adjuvant that augments an immune response comprising antibodies
to the fragment. Optionally, the fragment is linked to a carrier
forming a fusion protein, wherein the carrier augments an immune
response comprising antibodies to the fragment.
[0018] The invention further provides methods of effecting
treatment or prophylaxis of a LBD. The method involves
administering to a patient suffering from or at risk of a LBD an
effective regime of an antibody that specifically binds to a
fragment of alpha-synuclein, wherein the fragment is selected from
the group consisting of SN1-115, SN1-116, SN1-117, SN1-118,
SN1-119, SN1-120, SN1-121, SN1-122, SN1-123, SN1-124, and SN1-125
and 1-X, wherein X is 130-139, without binding to intact
alpha-synuclein, whereby the antibody effects prophylaxis or
treatment of the disease. SN1-115, SN1-19, SN1-122, SN133 and SN135
are particularly preferred.
[0019] The invention further provides methods of effecting
treatment or prophylaxis of a LBD. The methods comprise
administering to a patient suffering from or at risk of a LBD, an
effective regime of phosphorylated alpha synuclein or a
phosphorylated fragment of alpha-synuclein, wherein the fragment is
characterized by presence of at least 100 contiguous amino acids of
intact alpha-synuclein and a deletion of 1-10 contiguous amino
acids from the C-terminus of intact alpha-synuclein; and thereby
effecting treatment or prophylaxis of the LBD. Optionally, the
fragment is SN1-133 or SN1-135. Optionally, the method further
comprises administering an adjuvant that augments an immune
response comprising antibodies to the fragment. Optionally, the
fragment is linked to a carrier forming a fusion protein, wherein
the carrier augments an immune response comprising antibodies to
the fragment.
[0020] The invention further provides a method of isolating a
protease that cleaves intact alpha-synuclein to form a fragment,
wherein the fragment is characterized by presence of at least 100
contiguous amino acids of intact alpha-synuclein and a deletion of
1-25 contiguous amino acids from the C-terminus of intact
alpha-synuclein. SN1-115, SN1-119, SN1-122, SN133 and SN135 are
particularly preferred. The method involves identifying an
inhibitor of the protease; contacting the inhibitor with a cellular
or tissue extract containing the protease, whereby the protease
binds to the inhibitor; and releasing the protease from the
inhibitor. Optionally, the inhibitor is a peptide of
alpha-synuclein comprising a contiguous segment of at least 5
residues and up to 20 residues of intact alpha-synuclein between
positions 111 and 130. Alternatively the peptide is an inhibitor
comprising least 5 residues of intact alpha-synuclein between
positions 129 and 139. Optionally, the peptide comprises a
contiguous segment of at least 5 residues between positions 118 and
122. Optionally, the peptide comprises a contiguous segment of at
least four residues between positions 114 and 117. Optionally, at
least one of the residues is a transition state analog.
[0021] The invention further provides a monoclonal antibody that
specifically binds to an epitope within residues 109-120 of alpha
synuclein. Optionally, the monoclonal antibody is chimeric,
humanized or human.
[0022] The invention further provides a monoclonal antibody that
specifically binds to an epitope within residues 115-123 of alpha
synuclein.
[0023] The invention further provides a monoclonal antibody that
specifically binds to a discontinuous epitope within residues 43-51
and 58-65 of alpha synuclein. Optionally, the antibody is chimeric,
humanized or human.
[0024] The invention further provides an end-specific monoclonal
antibody that specifically binds to isolated full-length
alpha-synuclein having a free C-terminus without specifically
binding to a fusion protein comprising alpha synuclein having a
C-terminus linked to a second polypeptide. Optionally, the antibody
is chimeric, humanized or human.
[0025] The invention further provides methods of detecting presence
or susceptibility to a Lewy body disease in a patient. The methods
involves determining a level of alpha-synuclein phosphorylated at
position 129 or phosphorylated or nitrated at position 125 of
alpha-synuclein in a sample from a brain of the patient, an
elevated level relative to the mean level in a population of
undiseased individuals indicating the patient has or is susceptible
to a Lewy body disease. The invention provides other methods of
detecting presence or susceptibility to a Lewy body disease in a
patient by determining a level of ubiquitinated alpha synuclein in
a sample from a brain of the patient, an elevated level relative to
the mean level in a population of undiseased individuals indicating
the patient has or is susceptible to a Lewy body disease.
Optionally, levels of both phosphorylated alpha-synuclein and
ubiquitinated alpha-synuclein can be detected.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIGS. 1A and B show a Western blot of various extracts from
the cortex and hippocampus of a transgenic mouse (B) and a matched
control (A) with a polyclonal antibody that binds to an epitope
within SN115-122.
[0027] FIG. 2 shows a Western blot with the same antibody as FIGS.
1A and B to compare the level of the truncated form of
alpha-synuclein in Triton-X100 extractions of the cortex and
hippocampus mice of 3 months and 12 months in age.
[0028] FIGS. 3A and B shows a Western blot with a different
antibody termed 12C1 (a monoclonal binding to epitope at amino
acids 43-51 and 58-65) of a Triton extracts from the brain of a
transgenic mouse three months old (B) compared with an aged matched
control (A).
[0029] FIG. 4 shows a further Western blot using the same antibody
as FIG. 3 on a Triton extract from the brain of transgenic mice of
three and twelve months of age.
[0030] FIGS. 5 A, B, C, D, E show Western blots with four different
antibodies (B, C, D, E) and an epitope map (A) of the binding sites
of the antibodies to various extracts from the brains of transgenic
mice.
[0031] FIGS. 6 A, B, C shows Tris extracts of the brain of a
patient with Lewy body disease probed with three different
antibodies (A, B, C), subject to 2-D gel electrophoresis and
subjected to Western blotting. All 2D gels in this document are
shown with acidic proteins on the left, more basic proteins on the
right.
[0032] FIG. 7A, B, C, D shows additional blots of Tris extracts of
the brain of a patient with Lewy body disease with four antibodies
(A, B, C, D) of additional specificities.
[0033] FIG. 8 summarizes the sites of cleavage relative to the
epitopes bound by antibodies used in the Western blotting.
[0034] FIGS. 9A, B compares the Tris soluble proteins (A) with
proteins extracted from Lewy bodies (B) by 2D electrophoresis and
Western blotting.
[0035] FIGS. 10A, B, C, D show the immunoblots of proteins from
Lewy bodies reprobed with various C-terminal antibodies.
[0036] FIGS. 11A, B show Western blots of various extracts of an
undiseased and Contursi patient probed with an antibody recognizing
either total alpha synuclein (A) or specific for phospho-129 alpha
synuclein (B).
[0037] FIG. 12 Extracted ion chromatogram of C-terminal peptide of
SN1-122.
[0038] FIG. 13: Extracted ion chromatogram of C-terminal peptide of
SN1-119.
[0039] FIGS. 14 A, B, C, D, E and F: 2D immunoblot with antibody
recognizing total synuclein. Dashes mark positions of four rows of
C-truncated synuclein species. FIGS. 14A, B, C, E, and F show
different preparations from different patients and FIG. 14D is a
control.
[0040] FIGS. 15 A, B: 2D immunoblots comparing ELADW101 (B), which
is end specific for SN1-119 with an antibody to total alpha
synuclein (A). Asterisks indicate spots which react with both
antibodies. These spots are identified as SN1-119.
[0041] FIGS. 16A and B respectively show labeling of Lewy bodies
and neuritis with the SN1-119 end-specific polyclonal antibody
ELADW-101.
[0042] FIGS. 17A and B are controls from a normal individual
stained with ELADW-101.
[0043] FIGS. 18A and B are brain sections from a DLBD patient
stained with SN1-119 end-specific monoclonal antibody 12C6.
DEFINITIONS
[0044] The term "agent" is used to describe a compound that has or
may have a pharmacological activity. Agents include compounds that
are known drugs, compounds for which pharmacological activity has
been identified but which are undergoing further therapeutic
evaluation, and compounds that are members of collections and
libraries that are to be screened for a pharmacological
activity.
[0045] A "pharmacological" activity means that an agent exhibits an
activity in a screening system that indicates that the agent is or
may be useful in the prophylaxis or treatment of a disease. The
screening system can be in vitro, cellular, animal or human. Agents
can be described as having pharmacological activity notwithstanding
that further testing may be required to establish actual
prophylactic or therapeutic utility in treatment of a disease.
[0046] In the context of molecular weight determinations based on
gel electrophoresis, the term "about" indicates the standard
deviation of molecular weight expected due to experimental error in
repetitions of the method under the same conditions. The molecular
weight determination of 12 kDa for certain fragments of
alpha-synuclein applies to determinations using a tricine
buffer.
[0047] The phrases "specifically binds" refers to a binding
reaction which is determinative of the presence of the protein in
the presence of a heterogeneous population of proteins and other
biologics. Thus, under designated conditions, a specified ligand
binds preferentially to a particular protein and does not bind in a
significant amount to other proteins present in the sample. A
molecule such as antibody that specifically binds to a protein
often has an association constant of at least 10.sup.6 M.sup.-1 or
10.sup.7 M.sup.-1, preferably 10.sup.8 M.sup.-1 to 10.sup.9
M.sup.-1, and more preferably, about 10.sup.10 M.sup.-1 to
10.sup.11 M.sup.-1 or higher. A variety of immunoassay formats may
be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immunoreactive with a protein. See, e.g., Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for a description of immunoassay formats and conditions
that can be used to determine specific immunoreactivity.
[0048] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0049] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., supra).
[0050] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al., J. Mol.
Biol. 215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al., supra.).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. For identifying whether a nucleic acid or polypeptide
is within the scope of the invention, the default parameters of the
BLAST programs are suitable. The BLASTN program (for nucleotide
sequences) uses as defaults a word length (W) of 11, an expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a word length
(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring
matrix. The TBLATN program (using protein sequence for nucleotide
sequence) uses as defaults a word length (W) of 3, an expectation
(E) of 10, and a BLOSUM 62 scoring matrix. (see Henikoff &
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
[0051] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g. Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0052] For purposes of classifying amino acids substitutions as
conservative or non-conservative, amino acids are grouped as
follows: Group I (hydrophobic side chains): norleucine, met, ala,
val, leu, ile; Group II (neutral hydrophilic side chains): cys,
ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic
side chains): asn, gln, his, lys, arg; Group V (residues
influencing chain orientation): gly, pro; and Group VI (aromatic
side chains): trp, tyr, phe. Conservative substitutions involve
substitutions between amino acids in the same class.
Non-conservative substitutions constitute exchanging a member of
one of these classes for a member of another.
[0053] Therapeutic agents of the invention are typically
substantially pure from undesired contaminant. This means that an
agent is typically at least about 50% w/w (weight/weight) purity,
as well as being substantially free from interfering proteins and
contaminants. Sometimes the agents are at least about 80% w/w and,
more preferably at least 90 or about 95% w/w purity. However, using
conventional protein purification techniques, homogeneous peptides
of at least 99% w/w can be obtained.
[0054] The term "antibody" or "immunoglobulin" is used to include
intact antibodies and binding fragments thereof. Typically,
fragments compete with the intact antibody from which they were
derived for specific binding to an antigen fragment including
separate heavy chains, light chains Fab, Fab' F(ab').sub.2, Fabc,
and Fv. Fragments are produced by recombinant DNA techniques, or by
enzymatic or chemical separation of intact immunoglobulins. The
term "antibody" also includes one or more immunoglobulin chains
that are chemically conjugated to, or expressed as, fusion proteins
with other proteins. The term "antibody" also includes bispecific
antibody. A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0055] The term "adjuvant" refers to a compound that when
administered in conjunction with an antigen augments the immune
response to the antigen, but when administered alone does not
generate an immune response to the antigen. Adjuvants can augment
an immune response by several mechanisms including lymphocyte
recruitment, stimulation of B and/or T cells, and stimulation of
macrophages.
[0056] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0057] Competition between antibodies is determined by an assay in
which the immunoglobulin under test inhibits specific binding of a
reference antibody to a common antigen, such as alpha-synuclein.
Numerous types of competitive binding assays are known, for
example: solid phase direct or indirect radioimmunoassay (RIA),
solid phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay (see Stahli et al., Methods in Enzymology
9:242-253 (1983)); solid phase direct biotin-avidin EIA (see
Kirkland et al., J. Immunol. 137:3614-3619 (1986)); solid phase
direct labeled assay, solid phase direct labeled sandwich assay
(see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Press (1988)); solid phase direct label RIA using I-125
label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solid
phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552
(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J.
Immunol. 32:77-82 (1990)). Typically, such an assay involves the
use of purified antigen bound to a solid surface or cells bearing
either of these, an unlabelled test immunoglobulin and a labeled
reference immunoglobulin. Competitive inhibition is measured by
determining the amount of label bound to the solid surface or cells
in the presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. Antibodies identified by
competition assay (competing antibodies) include antibodies binding
to the same epitope as the reference antibody and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope
bound by the reference antibody for steric hindrance to occur.
Usually, when a competing antibody is present in excess, it will
inhibit specific binding of a reference antibody to a common
antigen by at least 50 or 75%.
[0058] Epitope co-ordinates are approximate (+2 amino acids). Not
every amino acid within an epitope is necessarily required for
binding.
[0059] Compositions or methods "comprising" one or more recited
elements may include other elements not specifically recited. For
example, a composition that comprises alpha-synuclein peptide
encompasses both an isolated alpha-synuclein peptide and
alpha-synuclein peptide as a component of a larger polypeptide
sequence.
[0060] Unless otherwise apparent from the context, each embodiment,
element, step or feature of the invention can be used in
combination with any other.
DETAILED DESCRIPTION OF THE INVENTION
[0061] I. General
[0062] The invention is premised in part on the identification of
novel fragments of alpha-synuclein in patients with Lewy Body
Disease (LBD) and transgenic animal models thereof. These diseases
are characterized by aggregations of alpha-synuclein. The fragments
have a truncated C-terminus relative to full-length
alpha-synuclein. Some fragments are characterized by a molecular
weight of about 12 kDa (corresponding to SN1-119), 12.5 kDa
(corresponding to SN1-122), 13.5 kDa (which fragment is found only
in patients with LBD) and 15 kDa (probably corresponding to SN1-133
or SN1-135) as determined by SDS gel electrophoresis in tricine
buffer and a truncation of at least ten contiguous amino acids from
the C-terminus of natural alpha-synuclein. The site of cleavage
preferably occurs after residue 115 and before residue 136 of
natural human alpha-synuclein. Particularly preferred sites of
cleavage are between residues 115 and 116, 119 and 120, between
residues 122 and 123, between residues 132 and 133 and between
residues 135 and 136. The identification of these novel fragments
of alpha-synuclein has a number of application in for example, drug
discovery, diagnostics, therapeutics, and transgenic animals.
[0063] The invention is further premised in part on the result that
phosphorylation synuclein partitions more to the particulate (Lewy
body enriched fraction) relative to the soluble cytosolic fraction
in patients with synucleinopathic disease relative to controls.
Phosphorylation occurs at position 129 of alpha synuclein. Although
an understanding of mechanism is not required for practice of the
invention, it is proposed that phosphorylation of alpha synuclein
drives subsequent processing to truncated forms (i.e., cleavages
between residues between residues 132 and 133 and between residues
135 and 136) and aggregation of alpha synuclein. The invention
further shows that small amounts of alpha synuclein in insoluble
fractions from patients with synucleinopathic disease are
ubiquitinated at lysine residues 6, 10, 12, 21, 23, 32 and 34.
Ubiquitination is known to have a role in degradation of proteins
(see, e.g., Cierchanover, EMBO J. 17, 7151-7160 (1998)).
Ubiquitination also renders alpha synuclein prone to aggregation
and changes its intracellular path from proteosomes to lysosomes.
Thus, ubiquitination can both increase degradation of alpha
synuclein and promote its aggregation. Modulation of ubiquitination
can therefore be useful in treating synucleinopathic disease.
[0064] The invention provides several methods for screening agents
for activity useful in treating LBDs. Some methods identify agents
that inhibit the cleavage reaction that generates the novel
fragments of the invention. Other method identify agents that
inhibit aggregation of the products of the cleavage reaction. Such
inhibitors are useful for treatment of LBD's. Inhibitors of the
cleavage reaction are also useful for affinity purification of the
protease responsible for the cleavage reaction.
[0065] The invention also provides transgenic animal models and
cells expressing fragments of alpha-synuclein as described above.
The transgenic animal models and cells are disposed to develop
characteristics of Lewy body disease, including Lewy bodies
containing aggregations of the fragments. The animal models and
cells can be used in the screening methods described above.
[0066] The invention further provides end-specific antibodies that
specifically bind to fragments of alpha-synuclein without
specifically binding to intact alpha-synuclein per se. These
antibodies are useful for in vivo imaging of alpha-synuclein
aggregations and also in methods of treatment. The novel
alpha-synuclein fragments can also be used in methods of treatment,
optionally, in combination with an adjuvant.
[0067] II. Alpha-Synuclein Fragments
[0068] Human alpha-synuclein is a peptide of 140 amino acids having
the following amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 1) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA
GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA
ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
(Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6);
GenBank accession number: P37840). The protein has three recognized
domains, a KTKE repeat domain covering amino acids 1-61, a NAC
(Non-amyloid component) domain running from about amino acids
60-95, and a C-terminal acidic domain running from about amino acid
98 to 140.
[0069] Some novel fragments of the invention have C-terminal
truncations of at least ten contiguous amino acids, preferably at
least 15 contiguous amino acids, and optionally at up to 20, 22, 23
or 25 amino acids. The fragments include all or substantially all
(i.e., at least 100 contiguous residues from alpha-synuclein other
than the deletion). Some fragments also have relatively short
truncations at the N-terminus of up to 20 amino acids, such as
deletions of residues 1-4, 1-6, 1-10 and 1-12. Some fragments have
N-terminal deletions of residues 1-23, 1-38 or 1-45. Preferred
fragments are SN1-115, SN1-116, 1-117, SN1-118, SN1-119, SN1-120,
SN1-121, SN1-122, SN1-123, SN1-124, SN1-125, SN1-126, SN1-127,
SN1-128, SN1-129 and SN1-130. Particularly preferred fragments are
SN1-115, SN1-119, SN1-120, SN1-121, SN1-122, SN1-123, SN1-124 and
SN1-125. Especially preferred fragments are SN1-115, SN1-119,
SN1-122 SN1-133 and SN1-135. The cleavage reaction preferably
occurs at a peptide bond between amino acid residues 115 and 116,
or 118 and 136, e.g., particularly between residue 119 and 120 or
residues 122 and 123 or residues 133-134 or residues 135-136.
[0070] The C-terminal fragments resulting from cleavage are also
included in the invention and can be used in the methods described
below. These fragments include SN116-140, SN117-140, SN118-140,
SN119-140, SN119-140, SN120-140, SN121-140, SN122-140, SN123-140,
SN124-140, SN125-140, SN126-140, SN1-127-140, SN128-140, SN129-140,
SN 130-140 and SN131-140. Preferred fragments are SN116-140,
SN120-140, SN123-140, SN 134-140 and SN136-140.
[0071] Other fragments of the invention include N-terminal
fragments of alpha-synuclein of about 6 to 7 kDa (as determined by
SDS electrophoresis) or 50-80 amino acids. Other fragments of the
invention include N-terminal fragments of alpha-synuclein that are
free of 1-10 amino acids from the C-terminus of intact
alpha-synuclein, i.e., SN 1-X, wherein X is 130-139. Some fragments
are characterized by specific binding to antibodies ELADW43 (free
N-terminus) and 5C12 (111-118) and lack of specific binding to 8A5
(free C-terminus), LB509 (115-123) and ELADW47 (115-122). Some
fragments are characterized by specific binding to ELADW43 (intact
N-terminus) and 5C12 (111-118), LB509 (115-123) and ELADW47
(115-122) and lack of specific binding to 8A5 (free C-terminus).
Some fragments are characterized by specific binding to ELADW43
(free N-terminus) and 5C12 (111-118), LB509 (115-122) and ELADW47
(118-123) and 8A5 (free C-terminus) and lack of specific binding to
ELADW43 (free N-terminus).
[0072] Some fragments or full-length alpha synuclein are
phosphorylated at position 125 or 129 or nitrated at the tyrosine
residue occupying position 125 of alpha synuclein. Fragments
retaining amino acid serine 125 or full-length alpha synuclein can
also be phosphorylated at this position. Detection of enhanced
phosphorylation or nitration at position 125 or phosphorylation at
position 129 in a patient relative to the mean in a population of
undiseased individuals is an indication of a Lewy body disease.
Detection can be performed using an antibody specific for
alpha-synuclein phosphorylated or nitrated at position 125. A level
is considered enhanced if greater than the mean plus one standard
deviation in a population of undiseased individuals.
[0073] The invention also provides isolated peptides of up to five
or ten contiguous residues of alpha synuclein containing at least
one of the above-mentioned ubiquitination sites. These peptides can
be used to compete with sites in the full-length alpha-synuclein
for ubiquitination or as immunogens to generate antibodies that
block ubiquitination of full-length alpha-synuclein.
[0074] The fragments of the invention are distinct from the
non-A.beta. component of Alzheimer's disease amyloid (NAC)
previously reported. This fragment consisting of at least 28 amino
acids residues (residues 60-87) and optionally 35 amino acid
residues (residues 61-95). See Iwai, et al., Biochemistry,
34:10139-10145); Jensen et al., Biochem. J. 310 (Pt 1): 91-94
(1995); GenBank accession number S56746.
[0075] Unless otherwise apparent from the context, reference to
alpha-synuclein or its fragments includes the natural human amino
acid sequence indicated above, or fragments thereof, as well as
analogs including allelic, species and induced variants (e.g.,
E83Q, A90V, A76T). Amino acids of analogs are assigned the same
numbers as corresponding amino acids in the natural human sequence
when the analog and human sequence are maximally aligned. Analogs
typically differ from naturally occurring peptides at one, two or a
few positions, often by virtue of conservative substitutions. Some
natural allelic variants are genetically associated with hereditary
LBD. The term "allelic variant" is used to refer to variations
between genes of different individuals in the same species and
corresponding variations in proteins encoded by the genes. Allelic
variants include E46K, A30P and A53T (the first letter indicates
the amino acid in SEQ ID NO:1, the number is the codon position in
SEQ ID NO:1, and the second letter is the amino acid in the allelic
variant). Analogs can include any combination of allelic variants.
The A53T variation is associated with enhanced levels of
phosphorylation at position 129 of alpha synuclein in an individual
having the mutation relative to the norm of phosphorylation in
undiseased individuals who lack the mutation. Analogs exhibit at
least 80 or 90% sequence identity with natural peptides. Some
analogs also include unnatural amino acids or modifications of N or
C terminal amino acids at one, two or a few positions. For example,
the natural glutamic acid residue can be replaced with iso-aspartic
acid. Examples of unnatural amino acids are D, alpha,
alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid,
4-hydroxyproline, gamma-carboxyglutamate,
epsilon-N,N,N-trimethyllysine, epsilon-N-acetyllysine,
O-phosphoserine, N-acetyl serine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, omega-N-methylarginine,
.beta.-alanine, ornithine, norleucine, norvaline, hydroxyproline,
thyroxine, gamma-amino butyric acid, homoserine, citrulline, and
isoaspartic acid. Analogs typically specifically bind to a
polyclonal antibody population generated against natural human
alpha-synuclein, and each end of an analog of a specific fragment
of a natural human alpha synuclein also specifically bind to a
monoclonal antibody that is end specific for the respective end of
the natural fragment. The invention also provides D-peptides, in
which D-amino acids can be substituted for corresponding natural
L-amino acids of alpha-synuclein at most or all positions. A
fragment designated in the form SNx-y means a fragment of alpha
synuclein that begins at amino acid X and ends at amino acid Y, and
contains each amino acid between X and Y. Such a fragment can (but
need not) be linked to a heterologous polypeptide but not to other
amino acids of human alpha synuclein such that the fragment begins
before X or ends after Y. Residues in a fragment are numbered
according to SEQ ID NO:1 when the fragment is maximally aligned
with SEQ ID NO:1 as described above using default parameters.
[0076] Alpha-synuclein, its fragments, and analogs can be
synthesized by solid phase peptide synthesis or recombinant
expression, or can be obtained from natural sources. Automatic
peptide synthesizers are commercially available from numerous
suppliers, such as Applied Biosystems, Foster City, Calif.
Recombinant expression can be in bacteria, such as E. coli, yeast,
insect cells or mammalian cells. Procedures for recombinant
expression are described by Sambrook et al., Molecular Cloning: A
Laboratory Manual (C.S.H.P. Press, NY 2d ed., 1989).
[0077] III. Lewy Body Diseases
[0078] Lewy Body Disease (LBD) is characterized by degeneration of
the dopaminergic system, motor alterations, cognitive impairment,
and formation of Lewy bodies (LBs). (McKeith et al., Clinical and
pathological diagnosis of dementia with Lewy bodies (DLB): Report
of the CDLB International Workshop, Neurology (1996) 47:1113-24).
Lewy Bodies are spherical protein deposits found in nerve cells.
Their presence in the brain disrupts the brain's normal function
interrupting the action of chemical messengers including
acetylcholine and dopamine. Lewy Body diseases include Parkinson's
disease (including idiopathic Parkinson's disease (PD)), Diffuse
Lewy Body Disease (DLBD) also known as Dementia with Lewy Bodies
(DLB), Combined Alzheimer's and Parkinson disease and multiple
system atrophy (MSA). DLBD shares symptoms of both Alzheimer's and
Parkinson's disease. DLBD differs from Parkinson's disease mainly
in the location of Lewy Bodies. In DLBD Lewy Bodies form mainly in
the cortex. In Parkinson's disease, they form mainly in the
substantia nigra. Other Lewy Body diseases include Pure Autonomic
Failure, Lewy body dysphagia, Incidental LBD, Inherited LBD (e.g.,
mutations of the alpha-synuclein gene, PARK3 and PARK4) and
Multiple System Atrophy (e.g., Olivopontocerebellar Atrophy,
Striatonigral Degeneration and Shy-Drager Syndrome).
[0079] IV. Transgenic Animals and Cells
[0080] The invention provides transgenic animals having a genome
comprising a transgene comprising a nucleic acid segment encoding a
C-terminal truncated form of alpha-synuclein as described above.
Preferred truncated forms are SN1-115, SN1-119, SN1-122, SN1-133
and SN1-135. The transgene is preferably present in all or
substantially of the somatic and germline cells of the transgenic
animal. The nucleic acid segment encoding the C-terminal truncated
form of alpha-synuclein is operably linked to one or more
regulatory segments that allow the truncated form of
alpha-synuclein to be expressed in neuronal cells of the animal.
Promoters such as the rat neuron specific enolase promoter, human
beta-actin gene promoter, human platelet derived growth factor B
(PDGF-B) chain gene promoter, rat sodium channel gene promoter,
mouse myelin basic protein gene promoter, human copper-zinc
superoxide dismutase gene promoter, and mammalian POU-domain
regulatory gene promoter can be used. The PDGF promoter is
particularly suitable. Optionally, an inducible promoter is used.
The mouse metallothionine promoter, which can be regulated by
addition of heavy metals such as zinc to the mouse's water or diet,
is suitable. Such transgenic animals can be produced by the same
general approaches described by (Masliah et al., Am. J. Pathol.
(1996) 148:201-10 and Feany et al., Nature (2000) 404:394-8)) for
transgenic animals with full-length alpha-synuclein or U.S. Pat.
No. 5,811,633 (for transgenic animals with a mutant form of APP).
Optionally, transgenic animals bearing a transgene expressing a
truncated alpha-synuclein protein can be crossed with other
transgenic models of neurogenic disease, such as models of
Alzheimer's disease. For example, transgenic animals bearing a
transgene expressing a truncated alpha-synuclein protein can be
crossed with transgenic animals bearing a transgene expressed APP
with a FAD mutation as described by e.g., Games et al., Nature 373,
523 (1995) McConlogue et al., U.S. Pat. No. 5,612,486, Hsiao et
al., Science 274, 99 (1996); Staufenbiel et al., Proc. Natl. Acad.
Sci. USA 94, 13287-13292 (1997); Sturchler-Pierrat et al., Proc.
Natl. Acad. Sci. USA 94, 13287-13292 (1997); Borchelt et al.,
Neuron 19, 939-945 (1997)). The procedure for performing such a
cross is described by e.g., Masliah et al., PNAS USA 98:12245-12250
(2001), which reports a cross between transgenic mice expressing a
full length alpha-synuclein with PDAPP mice as described by Games
et al Transgenic animals of the invention are preferably rodents,
such as mice or rats, or insects, such as Drosophila. Transgenic
animals can be produced by introduction of a transgene at the
germline stage in which case all or substantially all (except for
rare loss through somatic mutation) of the cells of the transgenic
animal include the transgene integrated into the genome. Transgenes
can be introduced by microinjection, nuclear transfer or viral
infection into cells or animals. Lentiviruses are particularly
suitable for the latter. Alternatively, transgenes can be
introduced by viral infection into the brain of the animal. Such
transgenes are not part of the germline of recipient animals but
can be targeted to regions of the brain responsible for disease
(e.g., the substantia nigra). Such animal models incorporate an
alpha synuclein into the genome of brain cells and are disposed to
develop at least one characteristic of synucleinopathic disease.
Lentiviruses provide a suitable vehicle for so introducing an alpha
synuclein transgene into the brain (see Brain Pathology 13, 364-372
(2003); Bjorklund, Trends Neurosci. 26, 386-92 (2003), Lotharius et
al., J. Biol. Chem. 277, 38884-94 (2002), Zhou et al., Brain
Research 866, 33-43 (2000)).
[0081] The expression of truncated forms of alpha-synuclein in
animal models gives rise to animals disposed to develop at least
one characteristic of a Lewy Body disease. Such characteristics
include increased levels of intracellular deposits of
alpha-synuclein, increased formation of Lewy bodies, and impaired
cognitive and motor functions relative to normal nontransgenic
animals of the same species. Such transgenic animals are useful for
screening agents for pharmacological activity in treating Lewy Body
disease.
[0082] The invention also provides cells transformed with truncated
alpha-synuclein which form inclusion bodies containing aggregated
truncated alpha-synuclein. The transformed cells are preferably
neuronal cells, such as GT1-7 neuronal cells (Hsue et al. Am. J.
Pathol. 157:401-410 (2000)), PC12 cells or SY5Y neuroblastoma
cells. PEAK cells can also be used. The cells are preferably human
cells. A vector comprising a segment encoding a truncated form of
alpha-synuclein operably linked to one or more regulatory sequences
that ensure expression of the truncated expression is transfected
into the cells. Transfected cells can be used to screen agents for
activity in clearing alpha-synuclein inclusions.
[0083] V. Screening Methods
[0084] The invention provide several screening methods to identify
agents having a pharmacological activity useful in treating a LBD.
The methods include screens that can be performed in vitro, in
cells or transgenic animals, and which test a variety of parameters
as an indication of activity. Agents determined to have an activity
in these screens can be retested in secondary screens of animal
models of LBD or in clinical trials to determine activity against
behavioral or other symptoms of these diseases.
[0085] 1. In Vitro
[0086] In vitro assays are performed to test the capacity of an
agent to inhibit aggregation of truncated forms of alpha-synuclein,
particularly SN1-115, SN1-119, SN1-122, SN1-133 and SN1-135. The
basis format for analyzing in vitro aggregation of alpha-synuclein,
albeit in the context of full-length alpha-synuclein, is described
by (Wood, J. Biol. Chem. 274, 19509-19512 (1999)). Truncated
fragments can be phosphorylated for performing the assay. The assay
can also be performed with full-length phosphorylated alpha
synuclein. Phosphorylation is preferably at position 129. Synuclein
can be phosphorylated in vitro using a serine kinase. In the
present methods, the assay is performed in the presence of an agent
being tested. The rate or extent of aggregation of alpha-synuclein
in the presence of an agent is determined and compared with the
rate or extent of aggregation of alpha-synuclein in a
contemporaneous or historical control in which the agent was
omitted. A reduction in the rate or extent of aggregation in the
presence of the agent relative to the control indicates that the
agent has activity in inhibiting aggregation of truncated forms of
alpha-synuclein. This activity is potentially useful in treating or
preventing Lewy Body diseases.
[0087] 2. Cellular Assays
[0088] Some cellular assays are performed on cells transfected with
nucleic acids encoding truncated forms of alpha-synuclein as
described above (particularly SN1-115, SN1-119, SN1-122, SN1-133
and SN1-135), optionally with a hereditary variation, such as
Ala30Pro or Ala53Thr. Cells can also bear mutations in other genes
associated with Parkinson's disease, such as leucine rich repeat
kinase PARK8. Such cells are contacted with an agent under test,
and the rate of extent of aggregation of the truncated
alpha-synuclein is measured. The rate of extent of aggregation of
alpha-synuclein is then compared to that of similarly transfected
control cells in the absence of the agent. Aggregation can be
monitored by immunohistochemical analysis, light microscopy,
sedimentation, or by gel analysis. Gel analysis can detect
formation of dimmers, trimers or higher oligomers as well as
inability of synuclein to enter gels due to a high level of
oligomerization. A reduction in the rate or extent of aggregation
in the presence of the test agent relative to the control indicates
the agent has activity has a pharmacological activity in inhibiting
aggregation of truncated forms of alpha-synuclein. This activity is
potentially useful in treating or preventing Lewy Body
diseases.
[0089] Other cellular assays are performed on cells transfected
with nucleic acids encoding full-length alpha-synuclein, optionally
with a hereditary variation, such as Ala30Pro or Ala53Thr. Cells
can also have mutations in other genes associated with Parkinson's
disease such as leucine rich repeat kinase, PARK8. Similar assays
can also be performed on cells naturally expressing alpha
synuclein. Such cells are contacted with an agent under test and
the rate or extent of formation of truncated forms of
alpha-synuclein (particularly SN1-115, SN1-119, SN1-122, SN1-133
and SN1-135) and/or phosphorylated or nitrated forms of synuclein
is/are measured. The presence of these forms can be detected by
Western blotting using one or more antibodies to alpha-synuclein.
End specific antibodies (i.e., antibodies that bind to a truncated
form without binding to full length alpha-synuclein) are
particularly useful for this analysis. Collections of antibodies
having different epitope specificities can also be used. For
example, presence of truncated forms of alpha-synuclein can be
shown by presence of bands when blotted with antibodies recognizing
an epitope N-terminal of an amino acid segment defined
approximately by amino acids 115-125 or 118-135 (particularly
SN1-115, SN1-119, SN1-122, SN1-133, and SN1-135) of intact
alpha-synuclein, and, and lack of bands when blotted with an
antibody recognizing an epitope C-terminal of this region. The rate
or extent of formation of truncated forms of alpha-synuclein and/or
phosphorylated or nitrated forms in the presence of agent is
compared with that of comparable control cells in the absence of
agent. A reduction in the rate or extent of formation of truncated
forms of alpha-synuclein in the presence of the test agent relative
to the control indicates that the agent has a pharmacological
activity that inhibits processing of alpha-synuclein to its
truncated forms. This activity is useful for treating or preventing
LBD.
[0090] 3. Transgenic Animal Assays
[0091] Transgenic animals have a transgene expressing a truncated
form of alpha-synuclein as described above (particularly SN1-115,
SN1-119, SN1-122, SN1-133 or SN1-135), optionally with a hereditary
variation, such as Ala30Pro or Ala53Thr. Transgenic animals can
also bear mutations in other genes associated with Parkinson's
disease such as leucine rich repeat kinase, PARK8. Such an animal
is contacted with an agent under test, and the rate of extent of
aggregation of the truncated form of alpha-synuclein is measured
compared with that in a contemporaneous or historical control. The
control is usually a similar transgenic animal of the same species
that has not been exposed to the agent. Aggregation of
alpha-synuclein in a transgenic animal can be monitored by Western
blotting or immunohistochemistry as described in the examples.
Alternatively or additional, activity of the agent in such
transgenic animals can be determined from behavioral
characteristics such as motor or cognitive characteristics, as
described in the Examples. In such assays, pharmacological activity
of the agent is shown by improved motor or cognitive
characteristics (i.e., decrease impairment of such characteristics)
relative to a comparable control transgenic animal not exposed to
the agent.
[0092] Other assays are performed on transgenic animals having a
transgene expressing a full-length form of alpha-synuclein,
optionally with a hereditary variation, such as Ala30Pro or Ala53,
or mutations in other genes associated with Parkinson's disease
such as leucine rich repeat kinase, PARK8. Similar assays can be
performed on nontransgenic animals expressing endogenous alpha
synuclein. Such animals are contacted with an agent under test, and
the rate or extent of appearance of truncated forms of
alpha-synuclein (particularly SN1-115, SN1-119, SN1-122, SN1-133 or
SN1-135) is detected, optionally with a hereditary variation, such
as Ala30Pro or Ala53Th. Such forms can be detected using Western
blotting or immunohistochemical analysis using appropriate
anti-alpha-synuclein antibodies (as described for the cellular
assays). The rate of extent of appearance of truncated forms of
alpha-synuclein and/or phosphorylated or nitrated forms is compared
with the rate or extent of appearance of such forms in a
contemporaneous or historical control constituting a comparable
transgenic animal that has not been exposed to the agent. A
reduction in the rate or extent of appearance of the truncated
forms of alpha-synuclein in the animal exposed to the test agent
relative to the control indicates that agent has activity in
inhibiting processing of full-length alpha-synuclein to truncated
forms.
[0093] 4. Agents to be Screened
[0094] Agents to be screened include antibodies to alpha-synuclein,
peptides of alpha-synuclein, drugs known or suspected to have
activity in treating a LBD, natural products, and combinatorial
libraries. Preferred peptides of alpha-synuclein are relatively
short peptides of 30, 25, 20 10, 5 or fewer amino acid including
amino acids 114-117, 117-126, 118-125, 117-120, 120-124, 130-136,
132-138, 131-135, 132-134, 133-137, 134-136 of alpha-synuclein.
Optionally, an amino acid immediately on the N-terminal side of the
cleavage site that generates C-terminal truncated forms of
alpha-synuclein is replaced with a transition state analog amino
acid that forms a nonhydrolizable bond between the two amino acids
flanking the cleavage site, e.g., between residues 115-116,
119-120, 122-123, 133-134 and 135-136 of alpha synuclein. Examples
of analogs are transition state analogs are statine,
hydroxyethelene, hydroxyethelamine, AHPPA, ACHPA, and derivatives
thereof. One or more amino acids of a natural alpha-synuclein
sequence can also be substituted with other natural amino
acids.
[0095] Natural products to be screened can also be obtained from
the National Cancer Institute's Natural Product Repository,
Bethesda, Md. Random libraries of peptides or other compounds can
also be screened for suitability. Combinatorial libraries can be
produced for many types of compounds that can be synthesized in a
step-by-step fashion. Such compounds include polypeptides,
beta-turn mimetics, polysaccharides, phospholipids, hormones,
prostaglandins, steroids, aromatic compounds, heterocyclic
compounds, benzodiazepines, oligomeric N-substituted glycines and
oligocarbamates. Large combinatorial libraries of the compounds can
be constructed by the encoded synthetic libraries (ESL) method
described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia
University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO
95/30642 (each of which is incorporated herein by reference for all
purposes). Peptide libraries can also be generated by phage display
methods. See, e.g., Devlin, WO 91/18980. Combinatorial libraries
and other compounds can initially be screened for suitability by
determining their capacity to bind to alpha-synuclein.
[0096] VI. Toxicity Assays
[0097] Analogous strategies to those described in the screening
assays can be used to determine whether existing drugs, foods,
environmental toxins, and other compounds exert toxic effects via
promotion of alpha-synuclein processing, phosphorylation or
aggregation. Such assays are performed in the same manner as the
screening assays. Toxic activity is indicated by the opposite
result to pharmacological activity in the screening assays.
[0098] VII. Isolation of Protease
[0099] Processing of full-length alpha-synuclein to the truncated
forms of the invention is effected by a protease. The protease can
be purified using an inhibitor identified by the screening methods
discussed above. A preferred inhibitor is a peptide of
alpha-synuclein of e.g., up to 20 contiguous amino acids from SEQ
ID NO:1 including residues 114-117, 111-126, 113-126, 113-119,
117-121 or 120-125, or 130-136, 132-138, 131-135, 133-134, 133-137,
or 135-136, in which a residue N-terminal to the cleavage site
(e.g., between residues 115-116, 119-120, 122-123, 133-134 and
135-136) has been replaced by a transition state analog. Such an
inhibitor is used as an affinity purification reagent to purify the
protease from extracts of brain cells. Such cells can be obtained
from cadaver of a normal individual or one who has suffered from a
LBD disease. Levels of protease may be elevated in the latter. The
protease can be assayed by presenting it with an alpha-synuclein
substrate and monitoring formation of cleavage products.
End-specific antibodies described below are useful for detection of
cleavage products. The substrate can be, for example, the natural
human form of alpha-synuclein described above, a fragment thereof
containing residues flanking both sides of the cleavage site, or a
mutant form thereof in which the mutation is associated with a
hereditary form of LBD. Optionally, the C-terminus of the substrate
can be immobilized to the solid phase, and the N-terminus to a
label. Cleavage of the substrate releases the label to the liquid
phase. The liquid phase can readily be separated from the solid
phase, and the amount of label quantified as a measure of
proteolytic activity.
[0100] VIII. End-Specific Antibodies
[0101] The invention provides end-specific antibodies. Such
antibodies specifically bind to a truncated form of alpha-synuclein
(at the C-terminus), preferably a form selected of the group
consisting of SN1-115, SN1-116, SN1-117, SN1-118, SN1-119, 1-120,
1-121, 1-122, 1-123, 1-124, 1-125, 1-126 without specifically
binding to full-length alpha-synuclein. Preferred antibodies are
end-specific for SN1-115, SN1-119, SN1-122, SN1-133 and SN1-135.
Such antibodies are useful for in vivo imaging of alpha-synuclein
deposits, as therapeutic agents (see below), and for detecting
cleavage products resulting from proteolytic cleavage of
alpha-synuclein in the screening methods described above.
End-specific antibodies are also provided to corresponding
C-terminal fragments, e.g., 116-140, 117-140, 118-140, 119-140,
120-140, 121-140, 122-140, 123-140, 124-140, 125-140, 126-140,
134-140 and 136-140. Preferred fragments are 116-140, 120-140,
123-140, 134-140 and 136-140. The end-specific antibodies recognize
the N-terminus of these fragments such that they specifically bind
to the fragment without specifically binding to full-length alpha
synuclein.
[0102] Preferred end specific antibodies are ELADW-101 (polyclonal)
and 12C6 (monoclonal) specific for the C-terminus of SN1-119, and
ELADW-105 (polyclonal) and 7G8 (monoclonal) specific for the
C-terminus of SN1-122. The monoclonals are mouse monoclonals
expressed by hybridomas produced by conventional methods.
[0103] Such antibodies can be generated by immunizing a laboratory
animal with alpha-synuclein or a fragment thereof to induce
antibodies, and screening the resulting antibodies to identify
those having the desired binding specificity. Optionally,
immunization can be performed with relatively short peptides of
less than 20 amino acids, usually 7 or 8 amino acids that include
the C-terminus of the truncated fragments of the invention (e.g.,
SN 99-118, SN106-115, SN 110-119, SN-113-122, SN126-133, SN128-135.
Optionally, such short peptides are linked to a carrier that helps
elicit an immune response. For example, the peptide CGGDMPVD which
corresponds to amino acids SN 115-119 with a CGG linker is useful
for generating antibodies such as ELADW-101 and 12C6, and the
peptide CGGVDPDN which corresponds to amino acids 118-122 with a
CGG linker is useful for generating antibodies ELADW-105 and
7G8.
[0104] Optionally, specific binding to a labeled or immobilized
truncated fragment can be performed in competition with unlabelled
full-length alpha-synuclein. Optionally, large libraries of
antibodies can be screened simultaneously using the phage display
technique.
[0105] The production of non-human monoclonal antibodies, e.g.,
murine, guinea pig, primate, rabbit or rat, can be performed as
described by Harlow & Lane, Antibodies, A Laboratory Manual
(CSHP NY, 1988) (incorporated by reference for all purposes).
Complete Freund's adjuvant followed by incomplete adjuvant is
preferred for immunization of laboratory animals. Rabbits or guinea
pigs are typically used for making polyclonal antibodies. Mice are
typically used for making monoclonal antibodies. Binding can be
assessed, for example, by Western blot or ELISA. The smallest
fragment to show specific binding to the antibody defines the
epitope of the antibody. Alternatively, epitope specificity can be
determined by a competition assay is which a test and reference
antibody compete for binding to alpha-synuclein. If the test and
reference antibodies compete, then they bind to the same epitope or
epitopes sufficiently proximal that binding of one antibody
interferes with binding of the other.
[0106] Chimeric and humanized antibodies have the same or similar
binding specificity and affinity as a mouse or other nonhuman
antibody that provides the starting material for construction of a
chimeric or humanized antibody. Chimeric antibodies are antibodies
whose light and heavy chain genes have been constructed, typically
by genetic engineering, from immunoglobulin gene segments belonging
to different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments, such as IgG1 and IgG4. Human isotype IgG1 is
preferred. In some methods, the isotype of the antibody is human
IgG1. IgM antibodies can also be used in some methods. A typical
chimeric antibody is thus a hybrid protein consisting of the V or
antigen-binding domain from a mouse antibody and the C or effector
domain from a human antibody.
[0107] Humanized antibodies have variable region framework residues
substantially from a human antibody (termed an acceptor antibody)
and complementarity determining regions substantially from a
mouse-antibody, (referred to as the donor immunoglobulin). See,
Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989), WO
90/07861, U.S. Pat. No. 5,693,762, U.S. Pat. No. 5,693,761, U.S.
Pat. No. 5,585,089, U.S. Pat. No. 5,530,101, and Winter, U.S. Pat.
No. 5,225,539 (each of which is incorporated by reference in its
entirety for all purposes). The constant region(s), if present, are
also substantially or entirely from a human immunoglobulin. The
human variable domains are usually chosen from human antibodies
whose framework sequences exhibit a high degree of sequence
identity with the murine variable region domains from which the
CDRs were derived. The heavy and light chain variable region
framework residues can be derived from the same or different human
antibody sequences. The human antibody sequences can be the
sequences of naturally occurring human antibodies or can be
consensus sequences of several human antibodies. See Carter et al.,
WO 92/22653. Certain amino acids from the human variable region
framework residues are selected for substitution based on their
possible influence on CDR conformation and/or binding to antigen.
Investigation of such possible influences is by modeling,
examination of the characteristics of the amino acids at particular
locations, or empirical observation of the effects of substitution
or mutagenesis of particular amino acids.
[0108] Human antibodies against alpha-synuclein are provided by a
variety of techniques described below. Some human antibodies are
selected by competitive binding experiments, or otherwise, to have
the same epitope specificity as a particular mouse antibody.
Techniques for producing human antibodies include the trioma
methodology of Oestberg et al., Hybridoma 2:361-367 (1983);
Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat.
No. 4,634,666 (each of which is incorporated by reference in its
entirety for all purposes), use of non-human transgenic mammals
having transgenes encoding at least a segment of the human
immunoglobulin locus as described by, e.g., Lonberg et al.,
WO93/1222, U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S.
Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No.
5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S.
Pat. No. 5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No.
5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14,
826 (1996), Kucherlapati, WO 91/10741 (each of which is
incorporated by reference in its entirety for all purposes) and
phage display methods see, e.g., Dower et al., WO 91/17271 and
McCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat.
No. 5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242,
U.S. Pat. No. 5,733,743 and U.S. Pat. No. 5,565,332 (each of which
is incorporated by reference in its entirety for all purposes).
[0109] The heavy and light chain variable regions of chimeric,
humanized, or human antibodies can be linked to at least a portion
of a human constant region. The choice of constant region depends,
in part, whether antibody-dependent complement and/or cellular
mediated toxicity is desired. For example, isotopes IgG1 and IgG3
have complement activity and isotypes IgG2 and IgG4 do not. Choice
of isotype can also affect passage of antibody into the brain.
Human isotype IgG1 is preferred. Light chain constant regions can
be lambda or kappa. Antibodies can be expressed as tetramers
containing two light and two heavy chains, as separate heavy
chains, light chains, as Fab, Fab' F(ab').sub.2, and Fv, or as
single chain antibodies in which heavy and light chain variable
domains are linked through a spacer.
[0110] In another embodiment, monoclonal antibodies specifically
binding to an epitope within residues 109-120, or 115-122, of alpha
synuclein, or a discontinuous epitope within residues 43-51 and
58-65, or end-specific to the C-terminus of alpha-synuclein are
also provided, including humanized, chimeric and human forms
thereof. An end-specific antibody to the C-terminus of
alpha-synuclein can be recognized by capacity to specifically bind
to alpha-synuclein as a free protein without specifically binding
to alpha synuclein as a component of a fusion protein when the
C-terminus of alpha-synuclein is linked to a second peptide. These
antibodies can be screened for therapeutic activity, and if
positive results are obtained, can be used in therapeutic methods.
The antibodies can also be used in detecting fragments of
alpha-synuclein as described above.
[0111] IX. Diagnostics
[0112] The invention provides methods of in vivo imaging LBs in a
patient. Such methods are useful to diagnose or confirm diagnosis
of a Lewy Body disease of PD or susceptibility thereto. For
example, the methods can be used on a patient presenting with
symptoms of dementia. If the patient has LBs, then the patient is
likely suffering from a Lewy Body disease. The methods can also be
used on asymptomatic patients. Presence of abnormal deposits of
amyloid indicates susceptibility to future symptomatic disease. The
methods are also useful for monitoring disease progression and/or
response to treatment in patients who have been previously
diagnosed with a Lewy Body disease.
[0113] The methods work by administering an end-specific antibody
as described above that binds to alpha-synuclein in the patient and
then detecting the antibody after it has bound. If desired, a
clearing response can be avoided by using antibody fragments
lacking a full length constant region, such as Fabs. In some
methods, the same antibody can serve as both a treatment and
diagnostic reagent.
[0114] Diagnostic imaging can also be performed using an antibody
specific for phosphorylated alpha synuclein, such as the 11A5
(phosphor-specific) or 5C12 (binds to phosphorylated and
nonphosphorylated forms of alpha synuclein) monoclonal described in
copending application Ser. No. 10/984,192. Presence of
phosphorylated alpha synuclein associated with deposits of alpha
synuclein is an indication of synucleinopathic disease or
susceptibility thereto. Presence of ubiquitinated alpha-synuclein
is also a marker of disease. Such can be detected using a two step
assay in which alpha-synuclein is precipitated with a first
antibody to alpha-synuclein, and the amount of ubiquitinated
alpha-synuclein is detected using an antibody to ubiquitin.
[0115] Diagnostic reagents can be administered by intravenous
injection into the body of the patient, or directly into the brain
by intracranial injection or by drilling a hole through the skull.
The dosage of reagent should be within the same ranges as for
treatment methods. Typically, the reagent is labeled, although in
some methods, the primary reagent with affinity for alpha-synuclein
is unlabelled and a secondary labeling agent is used to bind to the
primary reagent. The choice of label depends on the means of
detection. For example, a fluorescent label is suitable for optical
detection. Use of paramagnetic labels is suitable for tomographic
detection without surgical intervention. Radioactive labels can
also be detected using PET or SPECT.
[0116] Diagnosis is performed by comparing the number, size and/or
intensity of labeled loci to corresponding base line values. The
base line values can represent the mean levels in a population of
undiseased individuals. Base line values can also represent
previous levels determined in the same patient. For example, base
line values can be determined in a patient before beginning
treatment, and measured values thereafter compared with the base
line values. A decrease in values relative to base line signals a
positive response to treatment.
[0117] End-specific antibodies are also useful to determine whether
truncated forms of alpha-synuclein are present in cerebrospinal
fluid or other body tissues or fluids. Presence of such forms at
significantly changed, usually greater, levels (i.e., greater or
less than mean plus one standard deviation) in a patient relative
to the normal level in a population of undiseased individuals is
indicative of presence or susceptibility to a LBD.
[0118] X. Methods of Treatment
[0119] The invention provides several methods of preventing or
treating Lewy Body disease in patients suffering from or at risk of
such disease. Therapeutic agents include the truncated forms of
alpha-synuclein described above, particularly SN1-115, SN-1-119,
SN1-122, SN1-133 and SN1-135 and fragments thereof effective to
induce antibodies, end-specific antibodies as described above, and
inhibitors of aggregation of truncated fragments of alpha-synuclein
or proteolytic processing of alpha-synuclein as described above.
Optionally, the fragments are phosphorylated particularly at
position 129. Other agents include full-length phosphorylated alpha
synuclein, preferably at position 129, agents that inhibit
phosphorylation of alpha synuclein, or which promote removal of
phosphorylated alpha synuclein by ubiquitination or otherwise, or
which promote or inhibit ubiquitination. General approaches for
administering agents to patients suffering or at risk of LBD are
described in copending application U.S. Ser. No. 60/423,012 filed
Nov. 1, 2002, and PCT US00/15239 filed Jun. 1, 2000, and
PCT/US03/34527, filed Oct. 31, 2003, each of which are incorporated
by reference in their entirety for all purposes, including all
references cited therein.
[0120] Patients amenable to treatment include individuals at risk
of disease of a LBD but not showing symptoms, as well as patients
presently showing symptoms. Therefore, the present methods can be
administered prophylactically to individuals who have a known
genetic risk of a LBD. Such individuals include those having
relatives who have experienced this disease, and those whose risk
is determined by analysis of genetic or biochemical markers.
Genetic markers of risk toward PD include mutations in the
alpha-synuclein or Parkin, UCHL1, and CYP2D6 genes; particularly
mutations at positions 30 and 53 of the alpha-synuclein gene.
Individuals presently suffering from Parkinson's disease can be
recognized from its clinical manifestations including resting
tremor, muscular rigidity, bradykinesia and postural
instability.
[0121] In some methods, the patient is free of clinical symptoms or
risk factors any amyloidogenic disease other than one characterized
by Lewy bodies. In some methods, the patient is free of clinical
symptoms or risk factors of any disease characterized by
extracellular amyloid deposits. In some methods, the patient is
free of diseases characterized by amyloid deposits of A.beta.
peptide. In some methods, the patient is free of clinical symptoms
and risk factors of Alzheimer's disease. In some methods, the
patient has concurrent Alzheimer's disease and a disease
characterized by Lewy bodies. In some methods, the patient has
concurrent Alzheimer's and Parkinson's disease.
[0122] In asymptomatic patients, treatment can begin at any age
(e.g., 10, 20, 30). Usually, however, it is not necessary to begin
treatment until a patient reaches 40, 50, 60 or 70. Treatment
typically entails multiple dosages over a period of time. Treatment
can be monitored by assaying antibody, or activated T-cell or
B-cell responses to a therapeutic agent (e.g., a truncated form of
alpha-synuclein peptide) over time. If the response falls, a
booster dosage is indicated.
[0123] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of a LBD in regime comprising an amount and
frequency of administration of the composition or medicament
sufficient to eliminate or reduce the risk, lessen the severity, or
delay the outset of the disease, including physiological,
biochemical, histologic and/or behavioral symptoms of the disease,
its complications and intermediate pathological phenotypes
presenting during development of the disease. In therapeutic
applications, compositions or medicates are administered to a
patient suspected of, or already suffering from such a disease in a
regime comprising an amount and frequency of administration of the
composition sufficient to cure, or at least partially arrest, the
symptoms of the disease (physiological, biochemical, histologic
and/or behavioral), including its complications and intermediate
pathological phenotypes in development of the disease. An amount
adequate to accomplish therapeutic or prophylactic treatment is
defined as a therapeutically- or prophylactically-effective dose. A
combination of amount and dosage frequency adequate to accomplish
therapeutic or prophylactic treatment is defined as a
therapeutically or prophylactically-effective regime. In both
prophylactic and therapeutic regimes, agents are usually
administered in several dosages until a sufficient immune response
has been achieved. Typically, the immune response is monitored and
repeated dosages are given if the immune response starts to
wane.
[0124] In some methods, administration of an agent results in
reduction of intracellular levels of aggregated alpha-synuclein. In
some methods, administration of the agent results in a reduction in
levels of C-terminal truncated forms of alpha-synculein. In some
methods, administration of an agent results in improvement in a
clinical symptom of a LBD, such as motor or cognitive function in
the case of Parkinson's disease. In some methods, reduction in
intracellular levels of aggregated alpha-synuclein or improvement
in a clinical symptom of disease is monitored at intervals after
administration of an agent.
[0125] Effective doses of the compositions of the present
invention, for the treatment of the above described conditions vary
depending upon many different factors, including means of
administration, target site, physiological state of the patient,
whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
Usually, the patient is a human but nonhuman mammals including
transgenic mammals can also be treated. Treatment dosages need to
be titrated to optimize safety and efficacy.
[0126] In some methods, the agent is a truncated fragment of
alpha-synuclein or a fragment thereof capable of inducing
antibodies to alpha-synuclein. The amount of such a fragment
depends on whether adjuvant is also administered, with higher
dosages being required in the absence of adjuvant. The amount of a
fragment for administration sometimes varies from 1-500 .mu.g per
patient and more usually from 5-500 .mu.g per injection for human
administration. Occasionally, a higher dose of 1-2 mg per injection
is used. Typically about 10, 20, 50 or 100 .mu.g is used for each
human injection. The mass of fragment also depends on the mass
ratio of immunogenic epitope within the fragment to the mass of
fragment as a whole. Typically, 10.sup.-3 to 10.sup.-5 micromoles
of immunogenic epitope are used for microgram of fragment. The
timing of injections can vary significantly from once a day, to
once a year, to once a decade. On any given day that a dosage of
immunogen is given, the dosage is greater than 1 .mu.g/patient and
usually greater than 10 .mu.g/patient if adjuvant is also
administered, and greater than 10 .mu.g/patient and usually greater
than 100 .mu.g/patient in the absence of adjuvant. A typical
regimen consists of an immunization followed by booster injections
at time intervals, such as 6 week intervals. Another regimen
consists of an immunization followed by booster injections 1, 2 and
12 months later. Another regimen entails an injection every two
months for life. Alternatively, booster injections can be on an
irregular basis as indicated by monitoring of immune response.
[0127] Truncated fragments of alpha-synuclein can also be
administered in the form of nucleic acids encoding the fragments
operably linked to one or more regulatory elements to ensure
expression of the truncated fragment of alpha-synuclein. Doses for
nucleic acids encoding immunogens range from about 10 ng to 1 g,
100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300 .mu.g DNA per
patient. Doses for infectious viral vectors vary from 10-100, or
more, virions per dose.
[0128] Some methods involve passive immunization with an
end-specific antibody. In such methods, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 1 mg/kg body weight or
10 mg/kg body weight or within the range of 1-10 mg/kg or, in other
words, 70 mg or 700 mg or within the range of 70-700 mg,
respectively, for a 70 kg patient. An exemplary treatment regime
entails administration once per every two weeks or once a month or
once every 3 to 6 months. In some methods, two or more monoclonal
antibodies with different binding specificities are administered
simultaneously, in which case the dosage of each antibody
administered falls within the ranges indicated. Antibody is usually
administered on multiple occasions. Intervals between single
dosages can be weekly, monthly or yearly. Intervals can also be
irregular as indicated by measuring blood levels of antibody to
alpha-synuclein in the patient. In some methods, dosage is adjusted
to achieve a plasma antibody concentration of 1-1000 .mu.g/ml and
in some methods 25-300 .mu.g/ml. Alternatively, antibody can be
administered as a sustained release formulation, in which case less
frequent administration is required. Dosage and frequency vary
depending on the half-life of the antibody in the patient. In
general, human antibodies show the longest half life, followed by
humanized antibodies, chimeric antibodies, and nonhuman antibodies.
The dosage and frequency of administration can vary depending on
whether the treatment is prophylactic or therapeutic. In
prophylactic applications, a relatively low dosage is administered
at relatively infrequent intervals over a long period of time. Some
patients continue to receive treatment for the rest of their lives.
In therapeutic applications, a relatively high dosage at relatively
short intervals is sometimes required until progression of the
disease is reduced or terminated, and preferably until the patient
shows partial or complete amelioration of symptoms of disease.
Thereafter, the patent can be administered a prophylactic
regime.
[0129] Therapeutic agents can be administered by parenteral,
topical, intravenous, oral, subcutaneous, intraarterial,
intracranial, intrathecal, intraperitoneal, intranasal or
intramuscular means for prophylactic and/or therapeutic treatment.
The most typical route of administration of an immunogenic agent is
subcutaneous although other routes can be equally effective. The
next most common route is intramuscular injection. This type of
injection is most typically performed in the arm or leg muscles. In
some methods, agents are injected directly into a particular tissue
where deposits have accumulated, for example intracranial
injection. Intramuscular injection or intravenous infusion is
preferred for administration of antibody. In some methods,
particular therapeutic antibodies are injected directly into the
cranium. In some methods, antibodies are administered as a
sustained release composition or device, such as a Medipad.TM.
device. Small molecules that act by inhibiting protease processing
of alpha-synuclein can be administered intravenously if the small
molecules pass through the blood brain barrier sufficiently for
therapeutic or prophylactic efficacy or directly into the cranium
otherwise.
[0130] Agents of the invention can optionally be administered in
combination with other agents that are at least partly effective in
treatment of LBD. Agents of the invention can also be administered
in conjunction with other agents that increase passage of the
agents of the invention across the blood-brain barrier.
[0131] Immunogenic agents are sometimes administered in combination
with an adjuvant. A variety of adjuvants can be used in combination
with a peptide, such as alpha-synuclein, to elicit an immune
response. Preferred adjuvants augment the intrinsic response to an
immunogen without causing conformational changes in the immunogen
that affect the qualitative form of the response. Preferred
adjuvants include aluminum hydroxide and aluminum phosphate, 3
De-O-acylated monophosphoryl lipid A (MPL.TM.) (see GB 2220211
(RIBI ImmunoChem Research Inc., Hamilton, Mont., now part of
Corixa). Stimulon.TM. QS-21 is a triterpene glycoside or saponin
isolated from the bark of the Quillaja Saponaria Molina tree found
in South America (see Kensil et al., in Vaccine Design: The Subunit
and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY,
1995); U.S. Pat. No. 5,057,540), (Aquila BioPharmaceuticals,
Framingham, Mass.). Other adjuvants are oil in water emulsions
(such as squalene or peanut oil) optionally in combination with
immune stimulants, such as monophosphoryl lipid A (see Stoute et
al., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and
killed mycobacteria. Another adjuvant is CpG (WO 98/40100).
Alternatively, alpha-synuclein can be coupled to an adjuvant.
However, such coupling should not substantially change the
conformation of alpha-synuclein so as to affect the nature of the
immune response thereto. Adjuvants can be administered as a
component of a therapeutic composition with an active agent or can
be administered separately, before, concurrently with, or after
administration of the therapeutic agent.
[0132] A preferred class of adjuvants is aluminum salts (alum),
such as alum hydroxide, alum phosphate, alum sulfate. Such
adjuvants can be used with or without other specific
immunostimulating agents such as MPL or 3-DMP, QS-21, polymeric or
monomeric amino acids such as polyglutamic acid or polylysine.
Another class of adjuvants is oil-in-water emulsion formulations.
Such adjuvants can be used with or without other specific
immunostimulating agents such as muramyl peptides (e.g.,
N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-
-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
N-acetylglucsaminyl-N-acetylmuramyl-L-A1-D-isoglu-L-Ala-dipalmitoxy
propylamide (DTP-DPP) Theramide.TM.), or other bacterial cell wall
components. Oil-in-water emulsions include (a) MF59 (WO 90/14837),
containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally
containing various amounts of MTP-PE) formulated into submicron
particles using a microfluidizer such as Model 110Y microfluidizer
(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene,
0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP,
either microfluidized into a submicron emulsion or vortexed to
generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant
system (RAS), (Ribi ImmunoChem, Hamilton, Mont.) containing 2%
squalene, 0.2% Tween 80, and one or more bacterial cell wall
components from the group consisting of monophosphoryllipid A
(MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),
preferably MPL+CWS (Detox.TM.).
[0133] Another class of preferred adjuvants is saponin adjuvants,
such as Stimulon.TM. (QS-21, Aquila, Framingham, Mass.) or
particles generated there from such as ISCOMs (immunostimulating
complexes) and ISCOMATRIX. Other adjuvants include RC-529, GM-CSF
and Complete Freund's Adjuvant (CFA) and Incomplete Freund's
Adjuvant (IFA). Other adjuvants include cytokines, such as
interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-12, IL13, and
IL-15), macrophage colony stimulating factor (M-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), and
tumor necrosis factor (TNF). Another class of adjuvants is
glycolipid analogues including N-glycosylamides, N-glycosylureas
and N-glycosylcarbamates, each of which is substituted in the sugar
residue by an amino acid, as immuno-modulators or adjuvants (see
U.S. Pat. No. 4,855,283). Heat shock proteins, e.g., HSP70 and
HSP90, may also be used as adjuvants.
[0134] An adjuvant can be administered with an alpha-synuclein
fragment as a single composition, or can be administered before,
concurrent with or after administration of the alpha-synuclein
fragment. The alpha-synuclein fragment and adjuvant can be packaged
and supplied in the same vial or can be packaged in separate vials
and mixed before use. The alpha-synuclein fragment and adjuvant are
typically packaged with a label indicating the intended therapeutic
application. If the alpha-synuclein fragment and adjuvant are
packaged separately, the packaging typically includes instructions
for mixing before use. The choice of an adjuvant and/or carrier
depends on the stability of the immunogenic formulation containing
the adjuvant, the route of administration, the dosing schedule, the
efficacy of the adjuvant for the species being vaccinated, and, in
humans, a pharmaceutically acceptable adjuvant is one that has been
approved or is approvable for human administration by pertinent
regulatory bodies. For example, Complete Freund's adjuvant is not
suitable for human administration. Alum, MPL and QS-21 are
preferred. Optionally, two or more different adjuvants can be used
simultaneously. Preferred combinations include alum with MPL, alum
with QS-21, MPL with QS-21, MPL or RC-529 with GM-CSF, and alum,
QS-21 and MPL together. Also, Incomplete Freund's adjuvant can be
used (Chang et al., Advanced Drug Delivery Reviews 32, 173-186
(1998)), optionally in combination with any of alum, QS-21, and MPL
and all combinations thereof.
[0135] Agents of the invention are often administered as
pharmaceutical compositions comprising an active therapeutic agent,
i.e., and a variety of other pharmaceutically acceptable
components. See Remington's Pharmaceutical Science (15th ed., Mack
Publishing Company, Easton, Pa., 1980). The preferred form depends
on the intended mode of administration and therapeutic application.
The compositions can also include, depending on the formulation
desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent is selected so as not to affect the biological activity of
the combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0136] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized Sepharose.TM., agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0137] For parenteral administration, agents of the invention can
be administered as injectable dosages of a solution or suspension
of the substance in a physiologically acceptable diluent with a
pharmaceutical carrier that can be a sterile liquid such as water
oils, saline, glycerol, or ethanol. Additionally, auxiliary
substances, such as wetting or emulsifying agents, surfactants, pH
buffering substances and the like can be present in compositions.
Other components of pharmaceutical compositions are those of
petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, and mineral oil. In general, glycols such
as propylene glycol or polyethylene glycol are preferred liquid
carriers, particularly for injectable solutions. Antibodies can be
administered in the form of a depot injection or implant
preparation which can be formulated in such a manner as to permit a
sustained release of the active ingredient. An exemplary
composition comprises monoclonal antibody at 5 mg/mL, formulated in
aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl,
adjusted to pH 6.0 with HCl. Compositions for parenteral
administration are typically substantially sterile, substantially
isotonic and manufactured under GMP conditions of the FDA or
similar body.
[0138] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced adjuvant effect, as
discussed above (see Langer, Science 249, 1527 (1990) and Hanes,
Advanced Drug Delivery Reviews 28, 97-119 (1997). The agents of
this invention can be administered in the form of a depot injection
or implant preparation which can be formulated in such a manner as
to permit a sustained or pulsatile release of the active
ingredient.
[0139] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications. For
suppositories, binders and carriers include, for example,
polyalkylene glycols or triglycerides; such suppositories can be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0140] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins
(See Glenn et al., Nature 391, 851 (1998)). Co-administration can
be achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein. Alternatively, transdermal delivery can be achieved
using a skin path or using transferosomes (Paul et al., Eur. J.
Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta
1368, 201-15 (1998)).
EXAMPLES
1. Detecting Truncated Forms of Alpha-Synuclein in a Transgenic
Animal
[0141] Transgenic mice having a nucleic acid encoding intact
alpha-synuclein operably linked to a PDFG promoter were analyzed at
6 weeks, 3 months and 12 months old. The animals were euthanized
and the cortex and hippocampus tissue from four mice (2 male/2
female) was pooled. The tissue was homogenized in TBS (250 mM
NaCl), and spun at 150,000.times.g for 15 minutes. The pellet was
then extracted with 1% Triton-X 100 for 30 min at 4 degrees and
spun as before. The resulting pellet was then extracted with 1% SDS
for 30 min at 25 degrees and spun as before. Finally, the pellet
was extracted with 8 M Urea/1% SDS. This procedure resulted in four
extracts which will be referred to as Tris, Triton, SDS, and Urea
extracts in the description that follows.
[0142] FIGS. 1A and B show a Western blot of extracts from a
transgenic mouse and a matched control with antibody ELADW-47. This
antibody is a polyclonal that binds to an epitope within SN115-122
(but does not necessarily require each amino acid for some binding
to occur). The antibody preferentially binds the human form of
alpha-synuclein but also binds the mouse form to a lesser extent.
FIGS. 1A and B shows an alpha-synuclein band at 14 kDa for both the
control mouse and the transgenic mouse. The band is stronger for
the transgenic mouse than the control. For the different extracts,
the band is most intense in the Triton extract. This extract
solubilizes membrane bound alpha-synuclein and possibly Lewy
body-like inclusions. The Tris and particularly the Triton
extractions of the transgenic mouse (but not the control) show a
band at about 12 kDa in a tricine buffer. This is a truncated form
of alpha-synuclein. The molecular weight of the band corresponds to
a length of about 115-120 amino acids.
[0143] FIG. 2 shows a Western blot with the same antibody as FIGS.
1A and B to compare the level of the truncated form of
alpha-synuclein in mice of 3 months and 12 months in age. The
Figure shows that the truncated form appears more strongly in the 3
month old mice. Again, the truncated band does not appear in the
control mice. The more intense appearance of the truncated form of
alpha-synuclein early in development of the transgenic mice
indicates that the truncated form of alpha-synuclein has a role
early in the pathogenesis of Lewy Body disease.
[0144] FIGS. 3A and B show a Western blot with a different antibody
termed 12C1 (binds epitope at amino acids 43-51 and 58-65,
monoclonal, IgG1 k). This antibody binds equally to mouse and human
forms of alpha-synuclein at an epitope including amino acids 43-51
and 58-65. FIG. 3 shows the truncated band of 12 kDa in the Triton
extract of the transgenic mice. The same band appears much more
faintly in the Triton extract of the control mice. Thus, processing
of alpha-synuclein to a truncated form occurs in both normal mice
and transgenic mice, but more strongly in the latter. The greater
extent of processing in the transgenic mice may be due to
processing of the human alpha-synuclein directly, or may be due to
the presence of human alpha-synuclein driving mouse alpha-synuclein
down a path that is used to a lesser extent in nontransgenic
mice.
[0145] FIG. 4 shows a further Western blot using the same antibody
as FIG. 3. This gel shows two additional bands of molecular weights
approximately 6 or 7 kDa. The 7 kDa band appears more strongly in
the transgenic mice than control mice. The 6 kDa band appears only
in the transgenic mouse, and then only in the 3 mo sample. The 6 or
7 kDa bands are indicative of shorter N-terminal fragments of
alpha-synuclein of length about 50-80 amino acids.
[0146] FIGS. 5A, B, C, D, E shows Western blots with four different
antibodies and epitope maps of the binding sites of the antibodies.
ELADW-44 is a polyclonal that binds only to the human form of
alpha-synuclein (i.e., not the mouse form) It binds to at epitope
at amino acids 103-105. ELADW-47 is a polyclonal that binds
preferentially to the human form but also binds the mouse form. It
binds to an epitope at amino acids 115-122. ELADW-48 is a
polyclonal that binds the human and mouse forms equally. It binds
to an epitope between amino acids 131 and 140. 8A5 is a monoclonal
that binds to the human and mouse forms equally. It binds to the
C-terminus of alpha-synuclein. FIGS. 5A-E shows that of these four
antibodies, only ELADW-47 generated a 12 kDa band indicative of a
truncated form of alpha-synuclein. The result that ELADW48 did not
give rise to this band is of assistance in mapping the site of
cleavage. Because ELADW-47 did bind and ELADW-48 did not, the site
of cleavage is bordered by the N-terminal end of the ELADW-47
epitope and the C-terminal amino acid of the ELADW48 epitope.
Further, because some amino acids from the ELADW-47 epitope must be
present to allow binding and some of the ELADW-48 epitope must be
absent to prevent binding, the cleavage site is further confined to
a region approximately within amino acids 118-135. When this data
is considered with the size of the truncated fragment (about
115-120 amino acids) then the probable site of cleavage is around
amino acids 118-121. The lack of binding by the C-terminal antibody
8A5 is consistent with this cleavage site. The lack of binding by
the antibody ELADW-44, however, requires further comment. The lack
of cleavage can be explained if a truncated form of human
alpha-synuclein resulting from cleavage adapts a different
conformation to intact alpha-synuclein preventing binding of
ELADW-44. Alternatively, the truncated form of alpha-synuclein
present in transgenic mice to a greater extent than in normal mice
represents a form of mouse alpha-synuclein. In this case, the
greater amount of the truncated form in the transgenic mouse would
be due to the presence of the human alpha-synuclein driving more of
the mouse alpha-synuclein down a processing path that leads to
truncated alpha-synuclein relative to the situation in a control
mouse.
2. Detecting Truncated Forms of Alpha-Synuclein in the Brain of a
Patient with DLBD
[0147] This example compares alpha-synuclein species in LBs to
those in the remaining soluble and particulate protein fractions of
a DLBD brain. LBs and soluble protein were prepared from the cortex
of a single DLBD patient (see Jensen et al., J. Biol. Chem. 275
21500-21507 (2000)). Tissue was homogenized in Tris/sucrose (0.32
mM)/EDTA (5 mM) and protease inhibitors buffer. The homogenate was
spun at 1000.times.g. The supernatant was subjected to a further
spin at 150,000.times.g. The supernatant from this spin was used to
prepare a Tris soluble fraction of proteins. The pellet from the
1000.times.g was resuspended and used to prepare a Lewy body
fraction. Lewy bodies were purified by immunoprecipitation on
magnetic beads bearing anti-synuclein antibodies. The precipitate
was then extracted with 7 M Urea/2 M Thiourea/4% CHAPS. The
unextracted material was reextracted with Urea/Thiourea/CHAPS. The
extracts from this step and the previous extraction were then
pooled and analyzed by 2D PAGE and immunoblot. The unextracted
residue was subject to further extraction with 90% formic acid.
This extract was stored diluted to 9% formic acid. The extract was
then analyzed by SDS PAGE and RP-HPLC. Little or no synuclein was
found in this last extract, and what was present resembled the
material extracted by Urea/Thiourea/CHAPS, indicating that the
Urea/Thiourea/CHAPS gave a comprehensive extraction.
[0148] Synuclein species were resolved on 2-D gels and detected on
Western blots. All 2D Western blots are shown with more acidic
proteins on the left, more basic on the right. Multiple
alpha-synuclein species, including phosphorylated and truncated
species, were present in both LBs and the soluble brain fraction.
The predominant truncations were in the C-terminal region of
alpha-synuclein at approximately amino acids 118-125. An additional
larger fragment cleaved close to the C-terminus was also observed.
No beta or gamma-synuclein was detected in the LBs despite being
found in the soluble protein fraction. The alpha-synuclein in the
LB preparation differed from that in the soluble fraction in that
it had additional C-terminal cleavages, and that overall the
truncated alpha-synuclein species were enriched in the LBs relative
to the soluble protein fraction. In addition, multiple
alpha-synuclein species of higher molecular mass, approximately
25-35 kDa, were detected only in the LB preparation. These include
ubiquitinated species, as identified by us and by others (Tofaris
et al. J. Biol. Chem. 278(45): 44405-44411, 2003). The C-terminally
truncated fragments are of the same size as those observed in the
transgenic mouse model of Example 1 indicating a role in disease
pathogenesis.
[0149] FIGS. 6A, B, C shows Tris extracts probed with different
antibodies, subject to 2-D gel electrophoresis and subjected to
Western blotting. The dark spots present toward the left of the
charts represent full-length alpha-synuclein. The most notable
feature is four spots in the Syn-1 chart that are absent in the 8A5
chart. These four spots represent truncated forms of
alpha-synuclein that are unable to bind the 8A5 antibody because of
the lack of a C-terminal amino acid. These truncations correspond
approximately to forms of SN between 1-118 and 1-125. Several
additional spots are seen underneath and adjacent to the full
length alpha-synuclein spots. The spots underneath the full length
spots probably represent minor truncations from the C-terminus
(i.e., synuclein 1-X, wherein X is 130-139), since they react with
antibodies to phosphorylated S129 but not with 8A5. The spot
adjacent the full-length spots but to the right represent a minor
deletion from the N-terminus (due to lack of this spot in the blot
with ELADW43).
[0150] FIGS. 7A, B, C, D shows blots with additional antibodies.
The four spots are present with 5C12 (111-118). Two of the spots
are present with ELADW47 (115-122) and the spots are absent with
LB509 (115-123). The spots may differ from each other both in
molecular weight and in the presence or absence of
posttranslational modification, such as nitration or
phosphorylation. The shift of these fragments towards a basic pH,
relative to the full length synuclein, is consistent with removal
of a portion of the acidic C-terminal sequence. These results fix
the sites of cleavage to within about amino acids 120-125 of
alpha-synuclein. Also notable are several spots running slight
below (lower molecular weight) or to the left (lower pH) than the
unmodified synuclein spots. These likely may represent forms of
synuclein that have undergone a small extent of N-terminal
truncation and/or different posttranslational modification relative
to the main spots. Note that some spots visualized by ELADW43 and
8A5 in the blots of soluble protein are beta synuclein,
particularly the prominent spot to the left and slightly above the
full length alpha.
[0151] FIG. 8 summarizes the sites of cleavage relative to the
epitopes bound by antibodies used in the Western blotting.
[0152] FIGS. 9A, B compare the Tris soluble proteins with proteins
extracted from Lewy bodies by 2D electrophoresis and Western
blotting. The Tris blot on the left shows four spots at lower
molecular weight representing truncated forms of alpha-synuclein
(probably in the 1-120 to 1-125 amino acid range). These are of
relatively low intensity compared to the spots representative of
full length alpha-synuclein. The blot of proteins from Lewy bodies
shows more spots representative of truncated forms of alpha
synuclein in the 1-120 to 1-125 range. However, these are of
greater intensity relative to the spots representative of full
length alpha synuclein. Also, apparent are two spots migrating
faster than full-length alpha synuclein but slower than the
collection of spots at the bottom of the blot. These spots probably
represent truncations in the range 1-X wherein X is 130-139 amino
acids. As above, these spots react with antibodies to
phosphorylated S129 but not with 8A5.
[0153] FIGS. 10A, B, C, D show the immunoblots of proteins from
Lewy bodies reprobed with various C-terminal antibodies. All spots
appear with Syn-1 (91-96) and 5C12 (111-118). With ELADW47, the
spot running at the fastest speed and most basic position in the
Syn-1 and 5C12 blots is missing. In the LB509 blot, the 12 kD spots
corresponding to those in the Tris soluble samples are missing or
faint, although the row of spots just above them ("tier 3") still
react. The absence or reduced intensity of certain spots in the
ELADW47 and LB509 blots indicates that these spots represent
truncated forms of alpha-synuclein and are consistent with cleavage
occurring approximately between amino acids 120 and 125.
3. Detecting Aggregated Alpha-Synuclein in a Transgenic Animal
[0154] Transgenic animals are euthanized and brains are removed for
neurochemical and neuropathological analysis Briefly, the right
hemibrain is frozen and homogenized for determinations of
aggregated and non-aggregated human alpha-synuclein
immunoreactivity by Western blot (Masliah et al., Science (2000)
287:1265). The left hemibrain is fixed in 4% paraformaldehyde,
serially sectioned in the vibratome for immunocytochemistry and
ultrastructural analysis.
[0155] Brain sections are immunostained with a rabbit polyclonal
antibody against human alpha-synuclein (1:500). After an overnight
incubation at 4.degree. C., sections are incubated with
biotinylated anti-rabbit secondary antibody followed by Avidin
D-Horseradish peroxidase (HRP) complex (1:200, ABC Elite, Vector).
The reaction is visualized with 0.1% 3,3-diaminobenzidine
tetrahydrochloride (DAB) in 50 mM Tris-HCl (pH 7.4) with 0.001%
H.sub.2O.sub.2 and sections are then mounted on slides under
Entellan. Levels of immunoreactivity are semi quantitatively
assessed by optical densitometry using the Quantimet 570C. These
sections are also studied by image analysis to determine the
numbers of alpha-synuclein immunoreactive inclusions and this
reliable measure of alpha-synuclein aggregation acts as a valuable
index of the anti-aggregation effects (Masliah et al. Science
(2000) 287:1265).
[0156] Analysis of patterns of neurodegeneration is achieved by
analyzing synaptic and dendritic densities in the hippocampus,
frontal cortex, temporal cortex and basal ganglia utilizing
vibratome sections double-immunolabeled for synaptophysin and
microtubule-associated protein 2 (MAP2) and visualized with LSCM.
Additional analysis of neurodegeneration is achieved by determining
tyrosine hydroxylase (TH) immunoreactivity in the caudoputamen and
substantia nigra (SN) as previously described (Masliah, et al.
(2000)). Sections will be imaged with the LSCM and each individual
image is interactively thresholded such that the TH-immunoreactive
terminals displaying pixel intensity within a linear range are
included. A scale is set to determine the pixel to .mu.m ratio.
Then, this information is used to calculate the % area of the
neuropil covered by TH-immunoreactive terminals. These same
sections are also utilized to evaluate the numbers of TH neurons in
the SN.
4. Analysis of Alpha-Synuclein in LBD Patients
[0157] To determine which species of .alpha.-synuclein are enriched
in or unique to disease tissue, we have examined brain samples from
patients with multiple system atrophy (MSA) and a familial
Parkinson's disease mutation (A53T; Contursi kindred). Particulate
fractions of MSA and Contursi brain were prepared by homogenizing
brain tissue in 50 mM Tris, 140 mM NaCl and 1% Triton respectively
Age matched, control patients ("normals") were prepared identically
to the disease brain. Samples were analyzed on western blots of 1-D
gels and by ELISA as described below, and also on 2-D gels. Part of
the particulate fraction was analyzed. The rest was spun. The
supernatant was also analyzed. The pellet was extracted in 7 M
urea. The supernatant from this extraction was analyzed. The pellet
was further extracted in 7 M urea/1% SDS. The supernatant was
analyzed. Western blots using an antibody to detect total
alpha-synuclein or to specifically alpha-synuclein phosphorylated
at position 129 are shown in FIGS. 11 A and B.
[0158] The synuclein fractionated differently from the particulate
fraction of the Contursi brain versus control brain. Most of the
synuclein in the particulate fraction of the normal brain was
soluble after homogenization in tris buffered sucrose but almost
all of the synuclein in the Contursi brain required urea plus SDS
for solubilization suggesting a massive amount of Lewy bodies in
this patient. The synuclein in the Contursi patient was strikingly
different from that in the control patient in the amount of ser 129
phosphorylation. Only a small amount of phosphorylated
.alpha.-synuclein was detected in the control patient (left
tracks), whereas the Contursi patient (right tracks) had an
extremely large amount of phospho-synuclein by comparison on
western blots. Thus, the insolubility of synuclein in the Contursi
brain was associated with a large increase in synuclein
phosphorylation at ser 129. The .alpha.-synuclein in the Contursi
patient also differed from that in normal brain in the distribution
of C-terminal truncations. C-terminally truncated .alpha.-synuclein
were observed in both control and Contursi particulate brain
fraction However, all detectable truncations were highly insoluble
(urea/SDS extract) in the Contursi patient, whereas those in the
control brain were soluble (tris buffered sucrose extract). The
enrichment of the C-terminally truncated synuclein in a LB-enriched
fraction of a Contursi patient is in agreement with our finding of
C-terminally truncated synuclein enrichment in DLBD LBs. The MSA
brain was also enriched in phospho (ser 129)-.alpha.-synuclein
revealed C-terminal truncation and an abundance of phosphorylation
and other acidic modifications also seen in LBs. High levels of
phospho (ser 129) were also seen in the brain of a DLBD patient
relative to an undiseased control.
5. Identification of Truncated Synuclein Species from DLBD Brain by
LC-MS/MS
[0159] To generate an enriched pool of .alpha.-synuclein, a
Urea/Thiourea/CHAPS solubilized particulate fraction of DLBD brain
was first purified by anion exchange chromatography. The resulting
fractions were analyzed by Western Blot and separated into crude
pools enriched in truncated, full length, or phospho-synuclein. The
truncated pool was further purified by affinity chromatography
(5C12 antibody conjugated to Sepharose). Next, individual fractions
were individually concentrated and separated by capillary HPLC.
[0160] The three major peaks from one fraction (designated C6) were
subsequently digested with trypsin and analyzed by LC-MS/MS to
determine the identity and composition of the protein present in
the sample. Peak 1 was analyzed and .alpha.-synuclein was
identified with sequence coverage spanning amino acids 1-97. Since
the sequence coverage ends at the C-terminus with a lysine residue,
which is a trypsin cleavage site, additional downstream peptides
may be present.
[0161] Peak 2 from fraction C6 was analyzed and found to contain
.alpha.-synuclein sequence from positions 11-97. Again, since
tryptic fragments on both the N and C termini are absent; the exact
composition of the species is not able to be determined. Also, the
majority of the protein present in this fraction is not synuclein,
but a form of myelin basic protein that co-purified with this
synuclein pool.
[0162] Analysis of Peak 3 identified two different synuclein
species, both beginning at amino acid 1, with one terminating at
position 119D and the other at position 122N. Both of these
truncated forms were identified using a database search designed to
detect truncated C-terminal tryptic peptides. The database search
results listing the truncated C-terminal peptide fragments are
shown below (Table 1) along with their respective extracted ion
chromatograms (FIGS. 12 and 13) which illustrate the intensity of
the precursor (unfragmented) ion signals for these peptides when
compared to baseline level.
TABLE-US-00002 TABLE 1 Database search results for Fraction-C6 Peak
3 Reference Scan(s) Sequence MH+ Charge XCorr #2
alpha_synuclein(C122) DQLGKNEEGAPQEGILEDMPVDPDN. 2710.22 2 4.53
2690-2694 (SEQ. ID NO: 2) 2344-2679 KDQLGKNEEGAPQEGILEDMPVDPDN.
2838.31 3 2.38 (SEQ. ID NO: 3) #3 alpha_synuclein(C119)
KDQLGKNEEGAPQEGILEDMPVD. 2512.19 2 1.94 2675 (SEQ. ID NO: 4)
[0163] The search identified the 122N truncation twice, finding two
different tryptic sequences for the 122N species, with one peptide
being the result of a missed tryptic cleavage of the N-terminal
lysine residue. Also, the Xcorr, or cross correlation score, for
the first listed sequence of the 122N variant is very high. Three
additional truncated forms SN1-115, SN1-133 and SN1-35 have also
been found in DLBD brains. Truncations were identified by matching
MS/MS peptide fragmentation spectra from the truncated C-terminal
a-synuclein peptides against a theoretical spectrum generated by
TurboSequest Mass spectral analysis software (licensed from
ThermoElectron, Inc). Both forms were phosphorylated at Ser129. The
greater the Xcorr value, the higher the confidence in the match,
which lends more support to the data.
[0164] FIGS. 14 A-F show immunoblots of 2D gels of extracts of Lewy
body preparations from DLB patients P48 and P52 and MSA patient P2
(strictly speaking, the MSA preparation contains glial cortical
inclusions) prepared as above compared with a control. The probe is
antibody to total alpha synuclein. The identifying numbers (A/1,
U/1, etc.) refer to different Lewy body preparations, done on
different days and using different cortical regions. Different
preparations from diseased patients are shown in FIGS. 14 A, B, C,
E and F. The control is shown in FIG. 14D. No synuclein
modifications were consistently present or absent in the MSA
preparation compared to those from DLB, so the two pathologies will
be discussed together. Most preparations have the same groups of
adducts migrating above full-length synuclein monomer although the
relative amount of each group varies. The C-truncated synuclein
between 16 and 12 kD can be grouped into four tiers, ranging from
full-length in the first tier to the 12 kD fragment in the fourth.
The relative amounts of the different truncations vary. P52U/2 and
P28U/1 have none of the DLB-specific tier 3 detectable. P48 has
very little C-truncated material; its tier 3 spots are barely
visible (although present), and the tier 4 is not much more
intense, if at all, than the soluble proteins. FIG. 15B shows the
results of reprobing a 2D immunoblot of the P52U/2 Lewy body
preparation with ELADW101. A previous probe of the same blot with
the monoclonal Syn-1 is shown to the left for comparison (FIG.
15A). Three asterisks mark spots that overlay each other on the two
probes. The P28U/1 blot was also reprobed with identical results.
The polyclonal antibody ELADW101, directed against termination at
Asp119, specifically reacts with the 3 most prominent spots in the
12 kD fourth row of truncated species The multiple spots suggest
different forms of SN1-119 differing by charge modifications, e.g.
phosphorylation, of the remaining sequence.
[0165] Free-floating, cortical brain sections from a normal and
DLBD patient were probed with the neo-epitope specific antibody
ELADW-101. The results are shown in FIGS. 16A and B (diseased
brain) and FIGS. 17A and B (controls). The boxed regions labeled A
and B are shown magnified in the right panels. A typical LB and LN
are labeled with arrows. Only faint synaptic staining is seen in
the normal brain. The same experiment was performed with
neo-epitope specific antibody 12C6. The results are shown in FIGS.
18A and B. The boxed regions labeled A and B are shown magnified in
the right panels. Typical LB and LNs are labeled with arrows. In
panel B, an arrow indicates granular cytoplasmic staining by 12C6.
This staining is not seen in normal brain. These results show that
the truncated alpha synuclein fragment SN1-119 is enriched in
patients with Lewy Body disease.
5. Behavioral Analysis in a Transgenic Animal
[0166] For locomotor activity, mice are analyzed for 2 days in the
rotarod (San Diego) Instruments, San Diego, Calif.), as previously
described (Masliah, et al. (2000)). On the first day mice are
trained for 5 trials: the first one at 10 rpm, the second at 20 rpm
and the third to fifth at 40 rpm. On the second day, mice are
tested for 7 trials at 40 rpm each. Mice are placed individually on
the cylinder and the speed of rotation is increased from 0 to 40
rpm over a period of 240 sec. The length of time mice remain on the
rod (fall Latency) is recorded and used as a measure of motor
function.
[0167] Mice are tested for cognitive ability in the Morris Water
maze (Morris, Learn Motivat. 12; 239-260 (1981)). In this
procedure, the animal is placed in a circular pool filled with
water, with an escape platform submerged just below the surface of
the water. A visible marker is placed on the platform so that the
animal can find it by navigating toward a proximal visual cue.
Alternatively, a more complex form of the test in which there are
no formal cues to mark the platform's location will be given to the
animals. In this form, the animal must learn the platform's
location relative to distal visual cues. The length of time the
animal remains in the water is inversely related to its cognitive
ability.
6. Analysis of Aggregated Alpha-Synuclein Fragments in a Cell
Line
[0168] GT1-7 neuronal cells (Hsue et al. Am. J. Pathol. 157:401-410
(2000)) are transfected with a pCR3.1-T expression vector
(Invitrogen, Carlsbad, Calif.) expressing a truncated fragment of
alpha-synuclein as described above murine alpha-synuclein and
compared with cells transfected with expression vector alone Cells
transfected with vector alone have a fibroblastic appearance while
cells transfected with alpha-synuclein are rounded, with inclusion
bodies at the cell surface visible via both light and confocal
scanning microscopy. Transfected GT1-7 cells can be used to screen
agents for activity in clearing synuclein inclusions.
[0169] The above examples are illustrative only and do not define
the invention; other variants will be readily apparent to those of
ordinary skill in the art. The scope of the invention is
encompassed by the claims of any patent(s) issuing herefrom. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the issued claims along with their
full scope of equivalents. All publications, references, and patent
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were so individually
denoted.
Sequence CWU 1
1
111140PRTHomo sapiens 1Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala
Lys Glu Gly Val Val1 5 10 15Ala Ala Ala Glu Lys Thr Lys Gln Gly Val
Ala Glu Ala Ala Gly Lys 20 25 30Thr Lys Glu Gly Val Leu Tyr Val Gly
Ser Lys Thr Lys Glu Gly Val 35 40 45Val His Gly Val Ala Thr Val Ala
Glu Lys Thr Lys Glu Gln Val Thr 50 55 60Asn Val Gly Gly Ala Val Val
Thr Gly Val Thr Ala Val Ala Gln Lys65 70 75 80Thr Val Glu Gly Ala
Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95Lys Asp Gln Leu
Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110Leu Glu
Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120
125Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135
140225PRTHomo sapiens 2Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro
Gln Glu Gly Ile Leu1 5 10 15Glu Asp Met Pro Val Asp Pro Asp Asn 20
25326PRTHomo sapiens 3Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala
Pro Gln Glu Gly Ile1 5 10 15Leu Glu Asp Met Pro Val Asp Pro Asp Asn
20 25423PRTHomo sapiens 4Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly
Ala Pro Gln Glu Gly Ile1 5 10 15Leu Glu Asp Met Pro Val Asp
2058PRTHomo sapiens 5Lys Asn Glu Glu Gly Ala Pro Gln1 5631PRTHomo
sapiens 6Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu
Ala Tyr1 5 10 15Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro
Glu Ala 20 25 3078PRTMus musculus 7Lys Gly Glu Glu Gly Val Pro Gln1
5831PRTMus musculus 8Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro
Gly Ser Glu Ala Tyr1 5 10 15Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp
Tyr Glu Pro Glu Ala 20 25 30911PRTArtificialEpitope bound by
antibody as depicted in Figure 8 9Asp Met Pro Val Asp Pro Asp Asn
Glu Ala Tyr1 5 10108PRTArtificialImmunogenic peptide 10Cys Gly Gly
Asp Met Pro Val Asp1 5118PRTArtificialImmunogenic peptide 11Cys Gly
Gly Val Asp Pro Asp Asn1 5
* * * * *
References