U.S. patent application number 14/470667 was filed with the patent office on 2015-04-16 for materials and methods for evaluating and treating neuromyelitis optica (nmo).
The applicant listed for this patent is Mayo Foundation for Medical Education and Research. Invention is credited to Shannon Hinson, Vanda A. Lennon, Sean J. Pittock.
Application Number | 20150104813 14/470667 |
Document ID | / |
Family ID | 42099042 |
Filed Date | 2015-04-16 |
United States Patent
Application |
20150104813 |
Kind Code |
A1 |
Lennon; Vanda A. ; et
al. |
April 16, 2015 |
MATERIALS AND METHODS FOR EVALUATING AND TREATING NEUROMYELITIS
OPTICA (NMO)
Abstract
The invention provides prognostic methods for evaluating the
severity of NMO and NMO-associated diseases as well as methods of
treating NMO and NMO-associated diseases.
Inventors: |
Lennon; Vanda A.;
(Rochester, MN) ; Hinson; Shannon; (Rochester,
MN) ; Pittock; Sean J.; (Rochester, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mayo Foundation for Medical Education and Research |
Rochester |
MN |
US |
|
|
Family ID: |
42099042 |
Appl. No.: |
14/470667 |
Filed: |
August 27, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14044669 |
Oct 2, 2013 |
|
|
|
14470667 |
|
|
|
|
12573942 |
Oct 6, 2009 |
|
|
|
14044669 |
|
|
|
|
61104621 |
Oct 10, 2008 |
|
|
|
Current U.S.
Class: |
435/7.21 |
Current CPC
Class: |
A61K 39/395 20130101;
G01N 2800/52 20130101; G01N 2333/705 20130101; A61K 31/439
20130101; G01N 2800/285 20130101; G01N 33/564 20130101; A61K 45/06
20130101 |
Class at
Publication: |
435/7.21 |
International
Class: |
G01N 33/564 20060101
G01N033/564 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. NS049577 awarded by National Institutes of Health. The
government has certain rights in the invention.
Claims
1-12. (canceled)
13. A method comprising: administering an effective amount of an
antibody to an individual having NMO, wherein the antibody is an
anti-NMO antigen antibody having specific binding affinity for a
NMO antigenic polypeptide.
14. The method of claim 13, wherein the antibody comprises a
label.
15. The method of claim 14, wherein the label is an imaging
agent.
16. The method of claim 15, wherein the imaging agent is selected
from the group consisting of .sup.32P, .sup.99Tc, .sup.111In and
.sup.131I.
17. The method of claim 14, wherein the effective amount is an
amount of from about 0.1 mCi to about 50.0 mCi.
18. The method of claim 13, wherein the effective amount is an
amount of from about 0.01 mg to about 100 mg.
19. The method of claim 13, wherein the antibody is administered to
the individual parenterally.
20. The method of claim 13, wherein the antibody is administered to
the individual intravenously.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/044,669, filed Oct. 2, 2013, which is a continuation of U.S.
application Ser. No. 12/573,942, filed Oct. 6, 2009, which claims
priority under 35 U.S.C. .sctn.119(e) of U.S. Application No.
61/104,621, filed Oct. 10, 2008.
TECHNICAL FIELD
[0003] This disclosure generally relates to autoimmune
diseases.
BACKGROUND
[0004] Neuromyelitis optica (NMO) is currently the best defined
acquired inflammatory demyelinating disorder of the central nervous
system (CNS). NMO attacks optic nerves and spinal cord selectively
and repeatedly. Clinical, histopathological and immunobiological
observations support a pathogenic role for an IgG autoantibody
specific for the astrocytic water channel aquaporin-4 (AQP4), and
the severity of acute NMO is ameliorated by antibody-depleting
therapies.
[0005] In contrast to most inflammatory CNS demyelinating
disorders, tissue destruction in NMO is profound. In addition to
white matter lesions, NMO characteristically exhibits central
necrosis of spinal cord gray matter. Histopathological CNS lesions
lack AQP4 and show deposition of IgM and IgG and products of
complement activation in a vasculocentric pattern that coincides
with the normal distribution of AQP4.
[0006] Until recently, NMO was considered a rare and severe variant
of multiple sclerosis (MS). However, the advent of serological
testing for AQP4-IgG has revealed that NMO and its inaugural forms
are more common than previously recognized. They tend to be
misdiagnosed as MS, which lacks a specific biomarker.
SUMMARY
[0007] This disclosure provides prognostic methods for evaluating
the severity of NMO and NMO-associated diseases as well as methods
of treating NMO and NMO-associated diseases.
[0008] In one aspect, methods of providing a prognosis for an
individual that has NMO or a NMO-associated disease are provided.
Such methods typically include providing a biological sample from
the individual; and determining, in vitro, whether or not the
biological sample reduces cell surface expression of EAAT2 or
reduces uptake of extracellular glutamate compared to a biological
sample from an individual that does not have NMO or a
NMO-associated disease. Typically, a reduction in cell surface
expression of EAAT2 or a reduction in uptake of extracellular
glutamate correlates with a prognosis of the individual.
[0009] In another aspect, methods of providing a prognosis for an
individual that has NMO or a NMO-associated disease are provided.
Such methods generally include providing a biological sample from
the individual; contacting the biological sample with primary
astrocytes, a differentiated astrocyte-type cell, or a
non-astrocytic cell expressing a gene encoding aquaporin-4 or a
functional fragment thereof; and determining, in vitro, whether or
not the biological sample reduces cell surface expression of EAAT2
or reduces uptake of extracellular glutamate compared to a
biological sample from an individual that does not have NMO or a
NMO-associated disease. Generally, a reduction in cell surface
expression of EAAT2 or a reduction in uptake of extracellular
glutamate correlates with a prognosis of the individual.
[0010] Representative biological samples include serum, plasma,
cerebrospinal fluid (CSF), and immunoglobulins.
[0011] In still another aspect, methods of providing a prognosis
for an individual that has NMO or a NMO-associated disease are
provided. Such methods typically include providing a biological
sample from the individual; contacting the biological sample with
cells or tissues in the presence of aquaporin-4 polypeptides or
functional fragments thereof; and determining whether or not
complement is activated in the cells. Generally, an activation of
complement correlates with a prognosis of the individual. In one
embodiment, the aquaporin-4 polypeptides or functional fragments
thereof are expressed by the cells.
[0012] In one aspect, methods of treating an individual that has
NMO are provided. Such methods can include administering a
glutamate receptor antagonist to the individual. Representative
glutamate receptor antagonists include
1-amino-3,5-dimethyl-adamantane, 1-aminoadamantane,
(+)-3-methoxy-17-methyl-(9.alpha.,13.alpha.,14.alpha.)-morphinan,
17-methyl-9.alpha.,13.alpha.,14.alpha.-morphinan-3-ol,
2-(2-chlorophenyl)-2-methylamino-cyclohexan-1-one,
1-(1-phenylcyclohexyl)piperidine,
(.+-.)cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol
hydrochloride, and 6-(Dimethylamino)-4,4-diphenylheptan-3-one. Such
methods can further include administering a compound to the
individual that inhibits complement.
[0013] In still another aspect, methods of treating an individual
that has NMO are provided. Such methods typically include
administering a compound to the individual that inhibits
complement. Representative compounds that inhibit complement
include Compstatin, APT070 (MICROCEPT), soluble complement receptor
1 (sCR1), anti-CS antibody, eculizumab (SOLARIS.RTM.), and
substituted dihydrobenzofurans, spirobenzofuran-2(3H)-cycloalkanes,
and their open chain intermediates. Such methods can further
include administering a glutamate receptor antagonist to the
individual.
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the methods and compositions of
matter belong. Although methods and materials similar or equivalent
to those described herein can be used in the practice or testing of
the methods and compositions of matter, suitable methods and
materials are described below. In addition, the materials, methods,
and examples are illustrative only and not intended to be limiting.
All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
DESCRIPTION OF DRAWINGS
[0015] FIG. 1 is a graph showing the quantitation of membrane
cytotoxicity induced by NMO serum and active complement (30 minutes
at 37.degree. C.). Results were the average of seven independent
experiments each using an individual serum pool from 10-15
different NMO patients or from approximately 350 control patients
with miscellaneous disorders.
[0016] FIG. 2 are graphs that demonstrate that, in primary
astrocytes, NMO-IgG impaired glutamate uptake or complement
activation. FIG. 2A is a graph showing the quantitation of membrane
permeability after exposure to control or NMO serum. Increase in
permeability to propidium iodide (PI) >2 fold by NMO serum
required active complement (.DELTA.C'=inactivated complement). FIG.
2B is a graph showing the update of L-[.sup.3H]glutamate
(.+-.Na+-containing buffer) without human serum (open box) or in
control (shaded box) or NMO serum (hatched box). Excess unlabeled
glutamate (dark box) prevented L-[.sup.3H]glutamate uptake. NMO
serum reduced L-[.sup.3H]glutamate uptake by 50%. Experiments shown
in FIG. 2B were performed twice. All others were performed at least
3 times.
[0017] FIG. 3 demonstrates that expression of aquaporin proteins in
HEK-293 cells induced EAAT2 protein expression. Glutamate
transport: GFP-AQP4 cells took up approximately 3 fold more
L-[.sup.3H]glutamate than vector-transfected cells (note
Na+-dependence). All experiments were performed a minimum of two
times.
[0018] FIG. 4 shows a flow-chart showing pathways for serological
evaluation of patients.
DETAILED DESCRIPTION
[0019] NMO-IgG is a clinically validated serum biomarker that
distinguishes relapsing CNS inflammatory demyelinating disorders
related to neuromyelitis optica (NMO) from multiple sclerosis (MS).
This autoantibody targets astrocytic aquaporin-4 (AQP4) water
channels. Characteristic CNS lesions exhibit selective depletion of
AQP4, with and without associated myelin loss, focal vasculocentric
deposits of IgG, IgM and complement, prominent edema and
inflammation.
[0020] A marked reduction of the astrocytic Na+-dependent
excitatory amino acid transporter, EAAT2 (a homolog of rodent GLT-1
(Zeng et al., 2007, Mol. Cell. Neurosci., 34:34-9) in
AQP4-deficient regions of NMO patient spinal cord lesions is
described. Thus, binding of NMO-IgG to astrocytic AQP4 appears to
initiate several potentially neuropathogenic mechanisms: complement
activation and AQP4 and EAAT2 down-regulation. Since EAAT2 accounts
for >90% of glutamate uptake in the CNS, is critical for
clearing glutamate from excitatory synapses and is expressed
selectively in astrocytes, altered expression of EAAT2 can impair
glutamate homeostasis, resulting in over-stimulation of glutamate
receptors in neurons and oligodendrocytes. This disruption in
glutamate homeostasis may contribute indirectly to the pathobiology
of NMO or NMO-associated diseases. See, for example, Hinson et al.
2007, Neurology, 69:2221-31.
[0021] NMO and NMO-associated diseases encompass a number of
neurological disorders related to AQP4 autoimmunity. For example,
NMO diseases are represented by a spectrum of adult or pediatric
inflammatory CNS demyelinating disorders related to AQP4
autoimmunity involving longitudinally extensive transverse
myelitis, optic neuritis (e.g., relapsing optic neuritis) or
brainstem encephalitis. Representative NMO-associated diseases
include connective tissue disorders related to AQP4 autoimmunity
(e.g., Lupus or Sjogrens syndrome) and cancers related to AQP4
autoimmunity (e.g., breast).
Prognostics
[0022] The relationship described herein between aquaporin-4 and
EAAT2 and the associated loss of EAAT2 in NMO or NMO-associated
diseases can be used in the prognosis of an individual. For
example, the amount of reduction in cell surface expression of
EAAT2 or the amount of reduction in the uptake of extracellular
glutamate can be correlated with the severity of the NMO or
NMO-associated disease or the accompanying symptoms.
[0023] Well known in vitro bioassays can be used to evaluate
whether or not a reduction in cell surface expression of EAAT2 or
uptake of extracellular glutamate is observed. For example, a
biological sample from an individual can be contacted with primary
astrocytes, differentiated astrocyte-type cells, or non-astrocytic
cells expressing a gene encoding aquaporin-4 or a functional
fragment thereof, and either or both cell surface expression of
EAAT2 or the uptake of extracellular glutamate by the cells can be
evaluated. Cell surface expression can be evaluated using well
known immunoassays such as innumohistochemistry or Western blot
(Immunoassay, 1996, Diamandis & Christopoulos, Eds., Academic
Press), and glutamate uptake by a cell can be evaluated, for
example, using methods such as those described by Lin et al., 2001,
Nature, 410:84-8.
[0024] A reduction in the cell surface expression of EAAT2 or a
reduction in the uptake of extracellular glutamate (compared to the
cell surface expression of EAAT2 or the glutamate uptake that
occurs in the presence of a biological sample from an individual
that does not have NMO or a NMO-associated disease) can be used
prognostically. Typically, the amount of reduction in either or
both the cell surface expression of EAAT2 and the uptake of
extracellular glutamate is directly related to the severity of the
NMO or NMO-associated disease that the individual will
experience.
[0025] In addition, the involvement of complement activation in NMO
and NMO-associated diseases can be used in the prognosis of an
individual suffering from NMO or a NMO-associated disease. For
example, the extent or degree that complement is activated in the
presence of serum (or components therein) from an individual from
an individual that has NMO or a NMO-associated disease (compared to
the extent or degree that complement is activated in the presence
of serum (or components therein) from an individual that does not
suffer from NMO or a NMO-associated disease) can be correlated with
the severity of disease.
[0026] Methods for evaluating whether or not complement is
activated in the presence of a biological sample from an individual
are known in the art. Such methods include, without limitation,
exposing the biological sample to cells or tissues in the presence
of aquaporin-4 polypeptides (or functional fragments thereof), and
determining whether or not complement is activated in the cells.
For example, activation of complement by cells can be determined by
evaluating cells for propidium iodide uptake (Kasibhatla et al.,
2006, Cold Spring Harb. Protoc.; doi: 10.1101/pdb.prot4495) or
using immunoassays (e.g., immunohistochemistry) to detect the
deposition or accumulation of one or more components of complement
(e.g., C9neo assembly or C5 deposition).
[0027] Any number of biological samples can be used in the methods
described herein. For example, a biological sample can include,
without limitation, serum, plasma, and cerebrospinal fluid (CSF).
In addition, a biological sample can include immunoglobulins (e.g.,
engineered immunoglobulins or immunoglobulins that are secreted by
cultured B lymphocytes or plasma cells). Alternatively, under
autopsy conditions, biological samples can include tissue biopsies
such as from spinal cord and brain.
[0028] It would be understood by those of skill in the art that,
under conditions described herein in which aquaporin-4 polypeptides
or functional fragments are required, such polypeptides or
fragments can be naturally present in the cells (e.g., endogenously
expressed or native), or cells can be genetically engineered to
express aquaporin-4 polypeptides or functional fragments thereof.
In addition, aquaporin-4 polypeptides or functional fragments
thereof (e.g., purified aquaporin-4 or functional fragments
thereof) can be added exogenously to the cells, to the biological
sample, or to a combination of the two.
Thereapeutics
[0029] The relationship described herein between EAAT2 and/or
complement activation with NMO-IgG provides a number of novel
therapies for treating NMO or NMO-associated diseases. For example,
in some instances, an individual can be administered an effective
amount of an antagonist of glutamate receptors can be administered
to an individual. In some instances, the antagonists are specific
for glutamate receptors, and in other instances, the antagonists
are specific for glutamate receptors on neurons, oligodendrocytes
and astrocytes. An effective amount of an antagonist of glutamate
receptors is an amount that reduces or eliminates the neurological
effects caused by the accumulation of extracellular glutamate.
[0030] Antagonists or glutamate receptors, also referred to as
glutamate receptor blockers, include, without limitation,
1-amino-3,5-dimethyl-adamantane (MEMANTINEO), 1-aminoadamantane
(AMANTADINE.RTM.),
(+)-3-methoxy-17-methyl-(9.alpha.,13.alpha.,14.alpha.)-morphinan
(DEXTROMETHORPHAN.RTM.),
17-methyl-9.alpha.,13.alpha.,14.alpha.-morphinan-3-ol
(DEXTRORPHAN.RTM.),
2-(2-chlorophenyl)-2-methylamino-cyclohexan-1-one (KETAMINE.RTM.),
1-(1-phenylcyclohexyl)piperidine (PHENCYCLIDINE.RTM.),
(.+-.)cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol
hydrochloride (TRAMADOL.RTM.), and
6-(Dimethylamino)-4,4-diphenylheptan-3-one (METHADONE.RTM.).
Therapeutic agents also include, for example, agents that reduce
the extracellular glutamate concentration such as, without
limitation, soluble EAAT2 receptor or antibodies that specifically
recognize and bind to glutamate (e.g., anti-glutamate Ab).
[0031] In certain instances, an effective amount of a compound that
inhibits complement or the activation of complement can be
administered to an individual. Compounds that inhibit complement
are known in the art and include, without limitation, Compstatin,
APT070 (MICROCEPT), soluble complement receptor 1 (sCR1), anti-CS
antibody, eculizumab (SOLIRIS.RTM.), and substituted
dihydrobenzofurans, spirobenzofuran-2(3H)-cycloalkanes, and their
open chain intermediates (see, for example, U.S. Pat. No.
5,506,247).
[0032] In some instances, effective amounts of one or more
antagonists of glutamate receptors and one or more compounds that
inhibit complement can be administered to an individual. One or
more antagonists of glutamate receptors and one or more compounds
that inhibit complement can be administered simultaneously, or the
one or more antagonists of glutamate receptors and the one or more
inhibitors of complement can be administered sequentially. In
certain instances, an antagonist of glutamate receptors and a
compound that inhibits complement can be administered alternatively
to an individual.
[0033] Routes of administering compositions to an individual are
well known in the art and include, for example, parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., ingestion or
inhalation), transdermal (topical), transmucosal, and rectal
administration. A composition for administering to an individual
typically is formulated to be compatible with its intended route of
administration.
[0034] In accordance with the present invention, there may be
employed conventional molecular biology, microbiology, biochemical,
and recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. The invention
will be further described in the following examples, which do not
limit the scope of the methods and compositions of matter described
in the claims.
EXAMPLES
Part A
Example 1
Cell Lines and Transgenic Constructs
[0035] Major binding sites of NMO-IgG (and affinity-purified
AQP4-specific rabbit IgG) in sections of normal adult human and
mouse CNS tissues are where AQP4 is expressed in highest density,
namely in the dystroglycan complex of the highly polarized plasma
membrane of astrocytic foot processes. The reason AQP4 is readily
visualized at those sites immunohistochemically is because of its
ultrastructural organization at those sites in tightly packed
arrays. In preliminary Western blot studies (with protein
overloaded conditions), it was observed that the rat
oligodendroglial-astrocytic progenitor cell line (CG4), when
differentiated in vitro to astrocytic phenotype (stellate and
expressing prominent cytoplasmic GFAP intermediate filaments),
express minimal AQP4 immunoreactivity, and that two independent
human astrocytoma cell lines (HTB-14 and CRL-17; obtained from
ATCC) did not express detectable AQP4 by immunofluorescence or
Western blot. Therefore, as the initial system to investigate in
vitro the immunobiological consequences of IgG interacting with
extracellular epitopes of AQP4, transfected human embryonic kidney
cell line (HEK293) over-expressing full-length human AQP4 protein
(fused at its N-terminus with green fluorescent protein [GFP]) was
used. The parental HEK293 cell line does not express AQP4
constitutively, but it does express dystroglycan complex partner
proteins of AQP4, which assure stable AQP4 insertion in the plasma
membrane. Furthermore, the proximity of adjacent AQP4 homotetramers
in the transfected cell's membrane mimics the close packing of
epitopes in the highly polarized astrocytic foot process.
Example 2
Antibodies and Sera
[0036] Fluorochrome-conjugated goat IgGs were purchased from
Molecular Probes (Alex-Fluor 546; human, mouse or rabbit
IgG-specific, and Oregon Green; mouse IgG-specific) or Invitrogen
(Cy5; rabbit IgG-specific). Conjugated monoclonal mouse IgGs (FITC;
specific for human IgG subclasses, or Cy3; specific for glial
fibrillary acidic protein and sodium channel) were obtained from
Sigma, and unconjugated were obtained from BD Biosciences (Caspr1
and early endosome antigen-1 [EEA1]) and Dako, Denmark (human
CD138). Goat IgGs monospecific for human IgG (TRITC-conjugated) or
human IgM (FITC-conjugated) were from Southern Biotechnology.
Rabbit IgG specific for AQP4 residues 249-323 was obtained from
Sigma. Rabbit IgG specific for human C9neo was a gift from Dr. Paul
Morgan (Cardiff, UK). Deidentified patients' sera were obtained
from the Neuroimmunology Laboratory, Department of Laboratory
Medicine and Pathology, Mayo Clinic Rochester, Minn.
Example 3
Immunostaining
[0037] All final preparations of live cells and sections of mouse
tissue and autopsied human lymph node and brain tissues were
mounted in PROLONG.RTM. Gold DAPI antifade medium (Molecular
Probes). Fluorescent images were captured from a Zeiss LSM510
confocal microscope. Immunoperoxidase-stained sections were
counterstained with hematoxylin and photographed from an Olympus
DP-BSW microscope and DP70 digital camera system.
[0038] Live cells were exposed sequentially to 20% human serum or
rabbit anti-AQP4 IgG diluted 1:1000, secondary antibody (1:500) and
chilled 95% ethanol/5% acetic acid (15 minutes). Cells fixed in 10%
formalin were permeabilized, blocked, and incubated in primary and
secondary antibody probes. Tissues dissected from perfused mice
were post-fixed overnight in 4% paraformaldehyde, held 24 hours in
30% sucrose, frozen in OCT medium, sectioned (8 .mu.m) and
air-dried, incubated sequentially in 10% normal goat serum
containing 0.1% Triton X-100 (30 minutes), primary antibody (18
hours) and secondary antibody (1 hour; room temperature). Autopsied
human lymph node and brain tissues, fixed in 10% formalin and
embedded in paraffin, were deparaffinized, sectioned (5 .mu.m),
washed in bi-distilled water and steam-heated. Sections were
incubated with 10% normal goat serum (1 hour) and then overnight at
4.degree. C. with primary antibody (anti-CD138 at 1:50; anti-IgG at
1:750; anti-IgM at 1:200).
Example 4
Antigen Modulation Assay
[0039] Complement was inactivated in the serum of patients and
control subjects by incubating the serum for 30 minutes at
56.degree. C. Time-lapse photomicrographs of living cells were
captured at 15 minute intervals for 24 hours. Media containing
human serum were replaced at 16 hours with fresh media, with or
without cycloheximide (0.25 .mu.g/mL). Test sera were added to the
culture media for either 16 hours at 37.degree. C. or 1 hour at
4.degree. C. Cells were washed, exposed 30 minutes to Alexa-Fluor
546-labeled anti-human IgG (1:500), washed again, fixed in chilled
95% ethanol/5% acetic acid, rinsed in PBS, and stained with Hoescht
(1 .mu.g/mL).
Example 5
Complement Activation Assays
[0040] GFP-AQP4-HEK 293 cells were incubated for 2 hours on ice
with 20% heat-inactivated control or NMO patients' serum. Cells
were washed with chilled DMEM media and exposed to 20% fresh (or
heat-inactivated) normal human serum (as complement source) for 45
minutes at 37.degree. C. After rinsing and fixing (10% formalin, 4
minutes), cells were incubated sequentially at room temperature in
0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (1
minute), 10% normal goat serum and rabbit anti-human C9neo antibody
(30 minutes), washed and incubated in labelled anti-rabbit IgG. To
evaluate complement's effects on membrane integrity, test pools of
human serum were added to the cells at 4.degree. C. (for 1 hour).
After subsequent incubation for 90 minutes at 37.degree. C. (95%
air/5% CO.sub.2) in fresh or heat-inactivated Low-Tox-H rabbit
complement (Cedarlane Lab; 20% final concentration), the monolayer
was examined and photographed by transmitted bright field imaging.
To quantitate complement-mediated membranolytic activity by flow
cytometric analysis after sequential exposure to serum (30 minutes
at room temperature) and complement (37.degree. C. for 30 minutes),
the cells were lifted with trypsin, washed and suspended in chilled
buffer (10 mM Hepes/NaOH pH 7.4; 140 mM NaCl; 2.5 mM CaCl.sub.2)
containing 0.5 .mu.g propidium iodide (BD Biosciences) and held in
the dark for 15 minutes before analysis. Fold increase in membrane
permeability was determined by comparing the percentage of
propidium iodide permeable GFP-positive cells to propidium
iodide-permeable GFP-negative cells. In the absence of
antigen-specific activation of complement, the fold increase in
membrane permeability was less than 1.25 (125%).
Example 6
IgG Depletion and Purification
[0041] IgG was depleted from serum pools using protein G-agarose
beads (2 hours at 4.degree. C.). IgG-depleted serum was used at a
concentration of 20% in the complement assay. IgG eluted from
washed protein G beads in 0.1 M acetic acid was neutralized,
dialyzed against PBS and concentrated using Centricon YM-3 filter
devices (Millipore). IgG concentrations were determined by rate
nephelometry.
Example 7
Time-Lapse Photomicrographs
[0042] GFP-AQP4-HEK293 cells were seeded onto optical quality glass
bottom plates (MatTek) and exposed to 20% control or NMO patients'
serum. Time-lapse photomicrographs of the living cells were
captured at 15 minute intervals for 6 hours using a Zeiss LSM510
microscope stage held at 37.degree. C. in 95% air/5% CO.sub.2.
Z-series imaging was performed for each time point.
Example 8
NMO-IgG Initiates Endolysosomal Degradation of AQP4
[0043] A prerequisite for IgG to affect organ-specific
pathogenicity is its capacity to bind to specific epitopes
accessible on the surface of living target cells. When transfected
target cells expressing surface AQP4 were exposed to a pool of NMO
patients' sera (at 4.degree. C. to restrict membrane fluidity), IgG
bound to the plasma membrane in a linear pattern that co-localized
with GFP-AQP4. Serum IgG from control patients did not bind
detectably to the cell surface and a control rabbit IgG specific
for AQP4 cytoplasmic epitopes did not bind unless the plasma
membrane was permeabilized. NMO patients' serum IgG did not bind to
vector-transfected control HEK-293 cells. Thus, NMO patients' IgG
binds specifically to the extracellular domain of AQP4.
[0044] To evaluate the influence of this IgG on plasma membrane
AQP4, the distribution of GFP-AQP4 was recorded after exposure to
patients' serum at 37.degree. C. Control human serum had no effect
on GFP-AQP4, but NMO patients' sera containing AQP4-specific-IgG
resulted in rapid disappearance of GFP-AQP4 from the plasma
membrane. The phenomenon's temperature dependence is consistent
with modulation of surface antigens through intermolecular
cross-linking by bivalent IgG. Serial confocal images of living
cells held at 37.degree. C. for 16 hours in serum lacking AQP4-IgG
were indistinguishable from images of untreated cells with respect
to GFP-AQP4 fluorescence intensity and localization. Cells exposed
to NMO patients' serum underwent a striking redistribution within 5
minutes. The homotetrameric structure of AQP4, with multiple
repeated epitopes in the extracellular domain, and the proximity of
individual AQP4 homotetramers provide an optimal target for rapid
surface clearance from the plasma membrane by antigen-specific IgG.
Plasma membrane fading was accompanied by green fluorescent vesicle
accumulation in sub-plasmalemmal cytoplasm. After 20 minutes,
GFP-AQP4 aggregated in ring-like structures. At 5 hours, when
plasma membrane fluorescence was no longer detectable, these had
coalesced into large cytoplasmic aggregates. Removal of the
patients' serum at 16 hours resulted in rapid reappearance of
GFP-AQP4 in sub-plasmalemmal vesicles, presumably en route from the
endoplasmic reticulum. By 4 hours, GFP-AQP4 was visible in the
plasma membrane and, by 12 hours, it had attained normal surface
density. When cycloheximide was added at the time of withdrawing
patients' serum, no GFP-AQP4 was seen on the plasma membrane during
12 hours of observation. This implies that the "reversal" of the
autoantibody's effect was dependent on new protein synthesis.
[0045] The fate of GFP-AQP4 cleared from the plasma membrane after
exposure to NMO patients' serum was followed by probing with an
antibody specific for an early endosome antigen. In the absence of
serum, early endosomal vesicles did not colocalize with GFP-AQP4.
However, after 30 minutes exposure to NMO patients' serum, early
endosomal vesicles acquired green fluorescence. Early endosomal
vesicles did not acquire green fluorescence when cells were exposed
for 2 hours to control sera. These results indicate that
endocytosis initiated by the cross-linking of AQP4 by IgG binding
at the cell surface targets AQP4 to the endolysosomal pathway for
degradation.
Example 9
NMO-IgG Initiates Complement Activation
[0046] Activation of the classical complement cascade is another
potentially pathogenic outcome of autoantibody binding to AQP4.
This mechanism would increase the permeability of microvascular
endothelium, promote inflammatory cell infiltration, and possibly
inflict focal damage to astrocytic endfeet in central nervous
system regions enriched in AQP4. Surface membranes of transfected
cells were evaluated by indirect immunofluorescence for deposition
of the "C9neo" epitope created by the polymerizaton of multiple
perforin-like C9 molecules in assembling the terminal membrane
attack complex of complement. In the presence of NMO patients' sera
and active human complement, C9neo was clearly visualized in the
plasma membrane of HEK-293 cells expressing AQP4, but was not
visualized in the absence of disease-specific serum, in the
presence of inactive human complement, or in control cells
transfected with GFP-vector. These data indicate that NMO patients'
sera contain an AQP4-specific antibody capable of activating the
complement cascade. Reagents specific for human IgG subclasses
revealed that AQP4-specific serum IgG was, in all NMO-IgG positive
patients tested, exclusively IgG1, which is a major
complement-activating IgG subclass in humans.
[0047] Preliminary experiments indicated that membrane lysis
conditions had to be rigorously controlled to evaluate functional
evidence for antigen-specific complement activation by NMO-IgG on
membranes of living cells expressing AQP4 in high density. The
GFP-AQP4 transfected target cell line was a mixed population of
GFP-positive (AQP4+) and GFP-negative (AQP4-) cells. In the
presence of control human serum, phase microscopy revealed healthy
AQP4+ and AQP4- cells in an adherent monolayer. When the target
cells were pre-exposed to disease-specific patients' serum (on ice
for 2 hours) before adding complement for 90 minutes at 37.degree.
C. (conditions used for the C9neo visualization), all green cells
disintegrated rapidly. Therefore, a standardized commercial
preparation of rabbit complement, in limited concentration, was
used. The cells were pre-exposed to patient or control serum at
4.degree. C. for 1 hour before adding fresh or heat-inactivated
complement. In the presence of NMO patients' serum, adherent cells
that visibly expressed GFP were selectively lost. Provided that
complement was active and present more than 30 minutes, most AQP4+
cells were rounded and floating. Control human serum did not
visibly affect either adherent population regardless of complement
activity. Thus, the cytolytic activity of NMO patients' serum
depended on both complement activity and AQP4 expression in target
cells.
[0048] Quantitation of target cell membrane lesioning by complement
using flow cytometric analysis (indicated by permeability to the
red dye, propidium iodide) necessitated further attenuation of
complement activation. Therefore, cells were pre-exposed to
patients' (or control serum) at room temperature for 30 minutes
(rather than 1 hour at 4.degree. C.) to allow some reduction of
AQP4 antigen density by antigenic modulation before adding the
complement. Cells were then incubated with complement at 37.degree.
C. for only 30 minutes before harvesting for analysis of green and
red fluorescence intensities. Exposure to active or inactive
complement had no effect on either AQP4+ or AQP4- cells in the
presence of control patients' serum (FIG. 1). NMO patients' serum
had no effect on membrane integrity when complement was inactivated
regardless of whether or not AQP4 was expressed in the target
cells. However, with active complement, there was an average 5.6
fold (560%) increase in the membrane permeability of AQP4+ cells
compared with AQP4- cells (FIG. 1). Thus, the cytotoxicity of NMO
patients' serum depended on surface expression of AQP4 and the
availability of active complement.
Example 10
AQP4-Specific Autoantibodies are not IgM Class
[0049] Vasculocentric deposits of IgG, IgM and complement are a
hallmark of histopathological lesions found in central nervous
system tissue of NMO patients. Because pentameric IgM has five Fc
effector domains, it activates the complement cascade more
efficiently than IgG, which has only a single Fc domain. Therefore,
sera and cerebrospinal fluid of NMO patients were analyzed for
AQP4-specific IgM. By indirect immunofluorescence evaluation on
sections of AQP4-rich mouse tissues, none of 31 individual NMO
patients' sera nor 28 NMO patients' cerebrospinal fluid specimens
had detectable AQP4-specific IgM. The flow cytometry assay was used
to test for functional evidence of a complement-activating
AQP4-specific IgM in serum from patients who were either positive
or negative for AQP4 antibody of IgG class in the course of
clinical immunofluorescence evaluation. Only the AQP4 IgG-positive
sera (pools 1-4) were cytotoxic for AQP4+ cells. This cytotoxic
activity was lost when IgG was depleted. Recovery of AQP4-specific
complement activation restored by IgG from the original positive
serum pools was proportional to the % total recovery of IgG from
the Protein G-agarose beads.
[0050] To determine, immunohistochemically, the frequency of IgG
and IgM production in inflammatory central nervous system lesions
of NMO patients, accumulations of differentiated plasma cells were
investigated using fluorochrome conjugated anti-human IgG and IgM
class-specific reagents at limiting dilutions to avoid interference
from background extracellular IgG and IgM. The medullary lesion of
the illustrated patient contains abundant CD 138-positive plasma
cells and is in a region normally rich in AQP4. Plasma cells in the
medullary lesion stained brightly for cytoplasmic IgG, but
cytoplasmic IgM was not detected in any plasma cell. The
immunoglobulin class specificity of the detection antibody reagents
was confirmed on control sections of human lymph node tissue.
Clusters of plasma cells exhibited bright cytoplasmic IgG and
scattered plasma cells exhibited bright cytoplasmic IgM; very few
cells were dual-labeled. In summary, these data suggest that the
IgM deposited with IgG and complement products in AQP4-rich regions
of NMO patients' central nervous system tissues is not
AQP4-specific.
Example 11
AQP4-Enriched Astrocytic Processes Surround Nodes of Ranvier in
Spinal Cord and Optic Nerve
[0051] Involvement of myelin in the pathology of NMO remains to be
explained in the context of AQP4. Freeze-fracture studies in the
early 1970s defined "assemblies of particles" as an ultrastructural
characteristic of astrocytic endfeet surrounding axons at nodes of
Ranvier. These assemblies correspond to recently identified
orthogonal arrays formed by AQP4 homotetramers in transfected
cells. The proximity of AQP4-specific immunoreactivity in
astrocytic membranes to nodes of Ranvier was analyzed by examining
sections of mouse spinal cord and optic nerve tissues by confocal
fluorescence microscopy. Immune rabbit IgG (affinity purified on
AQP4 peptide) was used rather than NMO patient's serum because the
latter is a complex mixture of multiple autoantibodies, and is not
amenable to affinity purification because it binds only to native
AQP4 and not to denatured membrane-extracted AQP4 or synthetic AQP4
polypeptides. AQP4 was localized with respect to nodal regions by
simultaneously immunostaining sodium channels, which are densely
concentrated in the nodal axonal membrane (or Caspr1, an abundant
paranodal protein), and glial fibrillary acidic protein (GFAP), the
cytoplasmic intermediate filament of astrocytic processes. In both
spinal cord and optic nerve, AQP4 immunoreactivity was intense near
astrocytic elements and encircled periodic axonal segments
expressing sodium channel immunoreactivity. The concentric
paranodal AQP4 immunoreactivity was brighter than the background
mesh pattern of astrocytic AQP4 characteristic of central nervous
system white matter and was devoid of GFAP, which astrocytic foot
processes lack. The extracellular space surrounding astrocytic
processes is enlarged in the region of the axon segment bearing
sodium channels. The juxtaposition of intense AQP4 and Caspr1
immunoreactivities is consistent with extension of the astrocytic
foot process beyond the node of Ranvier into the paranode.
Part B
Example 1
Cell Lines and Transgenic Constructs
[0052] Primary astrocytes isolated from cerebral cortices of P1-3
rats, and the rat brain-derived O-2A progenitor cell line, CG-4,
were grown as previously described (Louis et al., 1992, J.
Neurosci. Res., 31:193-204; Garlin et al., 1995, J. Neurochem.,
64:2572-80). The GFP-AQP4 construct and stably transfected human
embryonic kidney cell lines (HEK-293) were described previously
(Lennon et al., 2005, J. Exp. Med., 202:473-7; Hinson et al., 2007,
Neurology, 69:2221-31). AQP5 was amplified from a human salivary
gland cDNA library, inserted into pEGFP-N1 vector (Clontech) and
transfected (Fugene 6) HEK-293 cells with the parent vector or
vector containing AQP5 transgenes. Stable clones were maintained in
DMEM supplemented with 10% bovine calf serum and antibiotics.
Example 2
Antibodies and Human Sera
[0053] Fluorochrome-conjugated goat IgGs were purchased from
Molecular Probes (Alex-Fluor 546; human, mouse or rabbit
IgG-specific, and Oregon Green; mouse IgG-specific) or Invitrogen
(Cy5; rabbit IgG-specific), and goat IgGs monospecific for human
IgG (TRITC-conjugated) or human IgM (FITC-conjugated) from Southern
Biotechnology. Rabbit IgG specific for: AQP4 (residues 249-323) was
purchased from Sigma, and rabbit IgG specific for EAAT1 and EAAT2
was purchased from Santa Cruz Biotechnologies (for Western blotting
and immunoprecipitation) or AbCam (for immunofluorescence). Mouse
monoclonal IgGs were purchased from AbCam (anti-EEA1), Sigma
(anti-GFAP conjugated to Cy3), Transduction Labs (anti-EAAT2) and
Santa Cruz Biotechnologies (anti-GFP). De-identified sera from NMO
and control patients were obtained, with Mayo Clinic IRB approval,
from the Neuroimmunology Laboratory, Department of Laboratory
Medicine and Pathology, Mayo Clinic Rochester, Minn.
Example 3
Immunostaining
[0054] Cell lines grown on glass coverslips were rinsed in PBS and
fixed in 4% PFA for 20 minutes at room temperature. After holding
30 minutes in 9% normal goat serum/0.1% triton X-100, the cells
were held at 4.degree. C. overnight in defined antibodies diluted
in 10% normal goat serum, then washed in PBS, and held 60 minutes
at room temperature in appropriate secondary antibody diluted in
10% normal goat serum. After washing in PBS and mounting on a slide
with PROLONG.RTM. Gold DAPI antifade medium (Molecular Probes),
fluorochrome-labeled cells were imaged using a Zeiss LSM510
confocal microscope.
[0055] Sections of archival CNS tissues derived from control and
NMO patients (5 .mu.m, formalin-fixed and paraffin-embedded) were
stained with haematoxylin and eosin (HE), Luxol-fast blue-periodic
acid-Schiff (LFB/PAS) or Bielschowsky silver impregnation.
Avidin-biotin-based immunohistochemistry was performed without
modification by incubating tissue sections 1 hour with 10% normal
goat serum, then holding overnight at 4.degree. C. with and without
primary antibodies.
Example 4
Antigen Modulation Assay
[0056] Cells were dispensed onto glass coverslips coated with
laminin (CG-4) or poly-L-lysine (HEK-293 and primary rat
astrocytes; Biocoat, BD Biosciences). After at least 48 hours,
control or NMO serum was added to 20% final concentration (with
complement inactivated by holding 30 minutes at 56.degree. C.) and
the cells were then processed for immunofluorescence analysis.
Example 5
Complement Membrane Lesioning
[0057] Growth medium of confluent primary astrocytes growing in
6-well plates was replaced with fresh medium containing 20% NMO or
control sera. After 15 minutes at room temperature, fresh or
heat-inactivated complement (Low-Tox-H rabbit complement; Cedarlane
Labs; final concentration 20%) was added and held for 40 minutes at
37.degree. C., then processed for flow cytometric analysis.
Example 6
Glutamate Uptake Assay
[0058] Na+-dependent radiolabeled glutamate uptake was measured
using primary astrocytes grown to confluence in 24-well dishes (Lin
et al., 2001, Nature, 410:84-8). After rinsing with 50 mM Tris-HCl
and 320 mM sucrose, pH 7.4, control wells were incubated for 15
minutes with 10 mM unlabeled L-glutamate, then L-[3H]glutamate (0.1
.mu.Ci, GE Healthcare, specific activity=55.0 .mu.Ci/nmol) was
added in 25 mM NaHCO.sub.3, 5 mM KCl, 1 mM KH.sub.2PO.sub.4, 1 mM
MgSO.sub.4, 2 mM CaCl.sub.2 and 555 mM D-glucose, pH 7.4, with or
without Na+ (Kreb's buffer containing 120 mM NaCl, or 120 mM
choline chloride, in 25 mM Tris, 5 mM KCl, 1 mM KH.sub.2PO.sub.4, 1
mM MgSO.sub.4, 2 mM CaCl.sub.2 and 555 mM D-glucose, pH 7.4). Both
buffers contained 40 .mu.M unlabeled glutamate. After 5 mins at
37.degree. C., the cells were transferred to 4.degree. C., washed
extensively with PBS, lysed in 0.1 M NaOH and uptake of
radiolabeled glutamate was measured using a Beckman LS 6000SC
scintillation counter. For all experimental conditions,
quadruplicate assays were performed and glutamate uptake was
calculated as counts/min/well, with and without Na+.
[0059] To assay glutamate uptake in GFP vector and GFP-AQP4 stable
HEK-293 cells, an alternative HEK-293-optimized protocol was used
(Jensen et al., 2004, Biochem. Pharmacol., 67:2155-27).
EAAT2-specific transport was defined as the difference in glutamate
uptake between GFP vector cells and GFP-AQP4 transfected cells.
Example 7
Isolation of RNA and RT-PCR Analysis
[0060] Total RNA was isolated from trizol-lysed cells, and double
stranded cDNA were generated using Superscript III First Strand
Synthesis kit with random hexamer primers (according to Invitrogen
protocols). To avoid amplifying contaminating genomic DNA, primer
pairs were used that anneal in adjacent exons: AQP4 (F5'-TGC ACC
AGG AAG ATC AGC ATC G-3' (SEQ ID NO:1) and R5'-CAG GTC ATC CGT CTC
TAC CTG-3' (SEQ ID NO:2)) and EAAT2 (F5'-GGT GGA AGT GCG AAT GCA
CGA CAG TCA TC-3' (SEQ ID NO:3) and R5'-CCT CGT CTG GCG GTG GTG CAA
CCA GGA C-3' (SEQ ID NO:4)). Beta-actin was amplified as a
control.
Example 8
Immunoprecipitation and Western Blot
[0061] Protocols were as previously described (Lennon et al., 2005,
J. Exp. Med., 202:473-477). Commercial antibodies (AQP4, EAAT1,
EAAT2 or GFP, 2 .mu.g/mL), or human sera (a high titer pool of NMO
patient or control patient sera, 30 tL/mL) were used as probes.
Immune complexes released from protein G-agarose were resolved by
electrophoresis (4 15% gradient polyacrylamide, room temperature).
Transblotted, blocked proteins were exposed for 1 hour to IgG
specific for: GFP (1:1,000), AQP4 (1:500), EAAT1 (1:200), actin
(1:2000) or EAAT2 (1:200), then probed with horseradish
peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG,
and detected bound IgG autoradiographically (SuperSignal West Pico
Chemiluminescence, Pierce).
Example 9
Statistical Analysis
[0062] Significance was calculated using Student's t-test
(2-tailed).
Example 10
NMO-IgG Binding to Primary Astrocytes Induces AQP4 Modulation and
Complement Activation
[0063] AQP4 distribution was monitored after applying NMO or
control serum to cerebral astrocytes. Serum containing NMO-IgG, but
not control serum, induced rapid down-regulation of surface AQP4.
AQP4 coalesced in cytoplasmic vesicles, reminiscent of those
observed in GFP-AQP4-transfected non-neural cells exposed to
NMO-IgG.
[0064] The complement-activating capacity of NMO-IgG binding to
astrocytic AQP4, determined by including complement with NMO or
control serum, was evaluated and the cells processed for flow
cytometric analysis of propidium iodide influx. In the presence of
NMO-IgG and active complement, plasma membrane permeability
increased approximately 2 fold (p<0.007; FIG. 2A). With
heat-inactivated complement, no discernible effect was observed
with control serum or NMO serum (FIG. 2A). Activation of early
complement components by IgG binding to the extracellular domain of
AQP4 in astrocytes would increase CNS microvascular endothelial
permeability and promote inflammatory cell infiltration. Assembly
of the final membrane attack complex might focally damage endfeet,
where AQP4 is expressed most highly. However, rich endowment of
astrocytic membranes with complement regulatory proteins, as has
been reported, may explain their relative resistance to
AQP4-IgG-dependent lysis in this study, in comparison to
AQP4-transfected HEK-293 cells.
Example 11
NMO-IgG Attenuates Na+-Dependent Glutamate Uptake in Astrocytes
[0065] Na+-dependent glutamate transport in astrocytes exposed to
NMO or control sera (FIG. 2B) was compared. Uptake of
L-[.sup.3H]glutamate was minimal (<200 counts/minute) in the
absence of Na+ or when excess unlabeled glutamate was present. In
the presence of Na+, L-[.sup.3H]glutamate uptake was increased to
approximately 3000 counts/minute in cells unexposed to serum or
exposed to control patient serum (FIG. 2B). These results are
consistent with the uptake of L-[.sup.3H]glutamate being
Na+-dependent and glutamate-specific. When serum containing NMO-IgG
was added in these experimental conditions, L-[.sup.3H]glutamate
uptake was reduced by approximately 50% (p<0.0003; FIG. 2B).
Example 12
NMO-IgG Down-Regulates Both EAAT2 and AQP4 From the Surface of
Differentiated Type-2 Astrocytic Cells
[0066] Attenuation of astrocytic L-[.sup.3H]glutamate uptake after
exposure to NMO-IgG parallels loss of AQP4 protein. To investigate
the possibility that EAAT2 protein may be lost secondary to loss of
surface AQP4, EAAT2-specific IgG was used to follow the fate of
EAAT2 after exposing astrocytes to NMO-IgG. Consistent with
previous reports, it was confirmed that EAAT1 and EAAT2 glutamate
transporter levels in primary rat astrocyte membranes are too low
to detect by immunofluorescence staining. Therefore, EAAT2
expression was investigated in the bipotential glial cell line,
CG-4, derived from 02-A progenitor cells in the developing rat CNS.
In prescribed culture conditions (Louis et al., 1992, supra), CG-4
cells differentiate into type-2 astrocytes. In
proliferation-promoting medium (containing bFGF and PDGF), the
cells (CG4-PM) lack GFAP intermediate filaments. However, in medium
with growth factors replaced by a high concentration of fetal
bovine serum, type-2 astrocyte differentiation was evident in the
cells (CG4-AM) by morphology and GFAP-immunoreactivity. In cells
grown 7 days in astrocyte differentiation medium, plasma membrane
expression of both AQP4 and EAAT2 was enhanced. However when NMO
serum was added to CG4-AM cells at day 7, both AQP4 and EAAT2 were
depleted from the plasma membrane. Addition of control sera in
identical conditions did not discernibly affect expression of
either AQP4 or EAAT2. Concomitant loss of both EAAT2 and AQP4
plausibly explains the reduced glutamate transport that was
observed in cultured astrocytes exposed to NMO-IgG.
Example 13
EAAT2 Expression is Up-Regulated When AQP4 Protein Expression is
Induced in Non-Neural Cells
[0067] AQP4 and EAAT2 are both enriched in the astrocytic endfoot
membrane. This finding that both AQP4 and EAAT2 are depleted from
plasma membranes of cultured astrocytes exposed to NMO-IgG is
consistent with the notion that EAAT2 expression depends on AQP4
expression. To further evaluate the relationship between AQP4 and
glutamate transport, the HEK-293 non-neural cell line were studied,
comparing EAAT2 expression in cells stably expressing GFP,
GFP-AQP4, or a non-neural AQP, AQP5-GFP. EAAT 1 was readily
detected in the plasma membranes of cell lines transfected with
either GFP vector or GFP-AQP4. EAAT2 was not detected in GFP vector
transfected cells, but EAAT2 was strikingly upregulated in the
plasma membrane of cells transgenically expressing either AQP4 or
AQP5.
[0068] To determine whether this observed increase in membrane
EAAT2 and functional glutamate transport might reflect an increase
in EAAT2 gene transcription or protein expression, EAAT2
transcripts were examined by RT-PCR. The three transfected HEK-293
lines expressed EAAT2 transcripts at similar levels. Western blot
analyses supported the immunofluorescence observations that
transgenic EAAT2 protein expression is upregulated when AQP4 or
AQP5 is expressed in HEK-293 cells by comparison with cells
transfected with vector alone. Expression of EAAT2 protein on the
surface of cells expressing AQP might be increased through
upregulated mRNA translation or, alternatively, through a
post-translational modification increasing EAAT2 protein stability
or trafficking to the plasma membrane. These complementary
observations accord with reports that EAAT2 protein expression is
restricted to astrocytes, despite ubiquitous expression of EAAT2
mRNA. It was concluded that restriction of EAAT2 expression to the
plasma membrane of astrocytes is determined by dependence on
astrocytic AQP expression.
[0069] These results confirmed that upregulated EAAT2 protein in
GFP-AQP4 transfected cells was functional by demonstrating that
GFP-AQP4 cells imported 2-3 fold more glutamate via the
Na+-dependent pathway relative to GFP vector cells (p<0.0002;
FIG. 3). It is noteworthy that glutamate transport in
Na+-containing buffer was unchanged in GFP vector cells compared to
GFP-AQP4 (FIG. 3). It was anticipated that constitutively expressed
EAAT1 in both cell lines would confer higher glutamate transport in
GFP vector cells in Na+-containing buffer than in buffer lacking
Na+. However, uptake of glutamate was unaffected in the presence of
Na+, suggesting that EAAT1 expressed constitutively in these cells
is not functional.
Example 14
Plasma Membrane Loss of EAAT2 Following Exposure to NMO Serum is
EAAT2-Selective and is Dependent on the Presence of Both AQP4
Protein and NMO-IgG
[0070] The data reported herein indicate that the concentration of
AQP4 protein in the plasma membrane and Na+-dependent glutamate
transport are both reduced by exposure of primary astrocytes to
NMO-IgG. These observations in differentiated CG-4 type-2
astrocytes suggest that the effect on glutamate transport in
primary astrocytes is due to plasma membrane loss of EAAT2. To
further investigate the association between AQP4 and EAAT2, the
effect of NMO and control sera on both EAAT1 and EAAT2 was
evaluated. Control serum did not appreciably affect localization or
expression of the EAAT2 transporter. However, serum containing
NMO-IgG induced rapid surface down-regulation of both GFP-AQP4 and
EAAT2. Higher magnification revealed apparent co-localization of
EAAT2 and AQP4 in early endosomal vesicles, to which AQP4
translocation after exposure to NMO serum was previously
demonstrated. The localization of AQP4 and the early endosome
antigen-1 marker was evaluated after exposing the cells to NMO
serum for 10 minutes. The white color that resulted when the images
were merged suggested partial co-localization of AQP4 and EAAT2 in
early endosomes. However, separate vesicles in close proximity or
overlapping in the z axis would yield the same result.
[0071] The possibility that NMO serum might contain EAAT2-specific
IgG in addition to AQP4 IgG was excluded by testing the effect of
NMO serum on plasma membrane expression of EAAT2 in HEK-293 cells
transfected with AQP5-GFP. Those cells express both EAAT1 and
EAAT2, but are devoid of AQP4. Exposure to NMO-IgG did not affect
the distribution of EAAT1 or EAAT2 in AQP5-GFP cells.
[0072] To investigate the specificity of EAAT2 down-regulation, the
effect of NMO-IgG on the EAAT1 glutamate transporter, which is
expressed constitutively in non-neural HEK-293 cells, was
evaluated. NMO patient sera did not appreciably affect EAAT1
expression in these cells, in contrast to the loss of EAAT2 from
the plasma membrane. These results imply a specific association
between AQP4 and EAAT2 that does not exist for EAAT1. The rapid
down-regulation of EAAT2 and AQP4 induced by NMO-IgG in
GFP-AQP4-expressing cells and co-localization of both proteins in
cytoplasmic endocytotic vesicles within 10 minutes is consistent
with a direct effect of IgG on a surface macromolecular complex.
The reduction in astrocytic glutamate transport accompanying AQP4
down-regulation after exposure to NMO-IgG further supports the idea
that AQP4 and EAAT2 are associated functionally in the plasma
membrane.
Example 15
AQP4 and EAAT2 Co-Immunoprecipitate
[0073] EAAT2 and EAAT1 are enriched in separate microdomains of the
astrocytic plasma membrane; EAAT2 is enriched in regions that
highly express AQP4. The data reported herein suggest that EAAT2
and AQP4 exist as a macromolecular complex. When exposed to
NMO-IgG, both are translocated from the plasma membrane to an
endolysosomal-targeted population of cytoplasmic vesicles. To
evaluate potential physical interaction, GFP-AQP4 transfected cells
was solubilized and the lysates were probed with EAAT2-IgG.
Addition of protein G-agarose captured both EAAT2 and AQP4. Similar
data was obtained using independent IgGs recognizing different
EAAT2 epitopes. As a specificity control, results obtained using
EAAT1-IgG as a probe for the cell lysates was compared. In contrast
to EAAT2-IgG, EAAT1-IgG did not pull down AQP4. These results
support the existence of AQP4 and EAAT2 as a macromolecular complex
independent of EAAT1.
Example 16
Co-Localization of AQP4 and EAAT2 in CNS Tissue
[0074] The observations in primary astrocytes, type-2
differentiated CG-4 cells and transfected non-neural HEK-293 cells
indicate that the interaction of NMO-IgG with AQP4 induces at least
three possible outcomes, each potentially pathogenic: complement
activation, down-regulation of AQP4, and coupled down-regulation of
the EAAT2 glutamate transporter. The immunohistochemical analysis
of non-pathologic human CNS tissue (both cortical and spinal cord)
revealed that EAAT2, but not EAAT1, normally co-localizes with AQP4
in gray matter astrocytes and that EAAT2 is most enriched in spinal
cord gray matter. These findings were reproducible.
Example 17
NMO Spinal Cord Lesions Lack Both AQP4 and EAAT2
[0075] Loss of AQP4 is a distinctive finding in both early and late
lesions of NMO. NMO spinal cord tissue of normal appearance
expresses normal levels of APQ4 and EAAT2 and lacks evidence of
complement deposition. Lesioned NMO spinal cord gray matter
contrasts to normal appearing gray matter by exhibiting markedly
reduced EAAT2, in addition to AQP4 loss and deposition of
complement activation products. Together, these findings are
consistent with the absence of EAAT2 staining being a biological
phenomenon within the NMO lesion. EAAT2 loss may partially account
for the destructive involvement of spinal cord gray matter which is
characteristic of NMO. Lesions in MS CNS tissues are typically not
necrotic. The marked loss of EAAT2 described herein parallels loss
of AQP4 in lesioned NMO spinal cord tissue and contrasts with the
increases in EAAT2 and AQP4 reported in both active and chronic MS
lesions. It was concluded that differences in regulation of
glutamate homeostasis further distinguish NMO from classical
MS.
Example 18
Concluding Remarks
[0076] The data presented herein from studies of living astrocytes,
patient and control sera, normal human spinal cord tissue and
spinal cord tissues from a patient with typical NMO, both
non-lesioned and lesioned, provide the first evidence supporting a
pathogenic role for NMO-IgG in disrupting glutamate transport.
Because astrocytes are relatively tolerant to increased glutamate
concentrations, disruption of glutamate homeostasis by NMO-IgG has
particular excitotoxic potential for neurons and oligodendrocytes.
A focal increase of extracellular glutamate levels secondary to
NMO-IgG-induced down-regulation of AQP4 may suffice to injure or
kill oligodendrocytes that express calcium-permeable glutamate
receptors. Oligodendrocytes in the spinal cord and optic nerve,
which are principal sites of demyelination in NMO, are particularly
sensitive to changes in glutamate concentration. Modest elevation
of extracellular glutamate concentration renders oligodendrocytes
additionally susceptible to Ig-independent complement attack. The
potential pathogenic sequelae demonstrated in this study for
NMO-IgG binding to AQP4-rich membranes in primary astrocytes are
both competing and cooperative. Depletion of AQP4 water channels in
the plasma membrane would disrupt water homeostasis and promote
edema. If complement were lacking, the consequences of impaired
glutamate transport would be particularly deleterious for
oligodendrocytes and neurons.
[0077] Outcomes of therapies directed at glutamate receptors have
been unimpressive for neurodegenerative conditions where glutamate
toxicity has been implicated in disease progression. However,
demonstrating that the major astrocytic glutamate transporter,
EAAT2, exists in a macromolecular complex with the AQP4 water
channel and is down-regulated by AQP4-specific autoantibodies that
are restricted to patients with the NMO-spectrum of CNS
inflammatory autoimmune demyelinating disorders has unanticipated
pathogenic implications for glutamate toxicity as a central
mechanism in a spectrum of disorders which are commonly mistaken
for MS. NMO is now recognized as a potentially reversible
IgG-mediated attack on astrocytic water channels. The results that
were obtained from studies of serum and spinal cord tissue of
patients with NMO hold promise for novel therapeutic strategies for
the management of NMO-spectrum disorders. For example, it might be
feasible to ameliorate tissue damage in both grey and white matter
if therapeutic upregulation of EAAT2 can be achieved in patients
whose neurological dysfunction is attributable to AQP4
autoimmunity.
Example 19
Prognostic Utility of Functional Assays for NMO/AQP4-IgG
[0078] Preliminary data (Table 1) revealed a lack of correlation
between serum levels of NMO-IgG (by immunofluorescence assay) and
attack severity and between NMO-IgG immunofluorescence titers and
complement activation quantified by flow cytometric analysis
comparison with cells exposed to healthy control subjects' serum.
In contrast, functional assays quantitating the outcome of NMO-IgG
binding to cells expressing AQP4 have revealed significant
correlation between the severity of an acute NMO attack and
activation of complement. For the purposes of these studies,
attacks were classified as "mild" when visual or limb impairment
was minimal or when complete recovery occurred (Patients 1-6), and
as "severe" when the attack resulted in residual blindness (from
optic neuritis) or paralysis (from spinal cord inflammation)
(Patients 7-12).
TABLE-US-00001 TABLE 1 Individuals Complement-dependent whose sera
NMO Attack lesioning of AQP4 + were tested Severity IF Titer HEK293
cells (%) Patients with acute attack 1 mild 1920 10 2 mild 240 15.9
3 mild 960 7.8 4 mild 960 19.1 5 mild 7680 12.3 6 mild 1920 19.2 7
severe 7680 59.8 8 severe 960 20.5 9 severe 1920 27.7 10 severe
3840 51.8 11 severe 15360 60.1 12 severe 7680 55.7 Healthy Controls
1 -- Negative 7.5% 2 -- Negative 8.2% 3 -- Negative 9.1% 4 --
Negative 8.0% 5 -- Negative 11.6% 6 -- Negative 7.7% 7 -- Negative
7.6% 8 -- Negative 9.9% 9 -- Negative 7.6% Multiple Sclerosis
Controls MS 1 -- Negative 9.2% MS 2 -- Negative 11.9% MS 3 --
Negative 10.5% MS 4 -- Negative 10.7% MS 5 -- Negative 12.5% MS 6
-- Negative 14.3% MS 7 -- Negative 13.2% MS 8 -- Negative 14.8% MS
9 -- Negative 14.3%
[0079] Measures of complement activation by NMO-IgG were
significantly (p<0.005) associated with attack severity
classification (mild or severe) but NMO-IgG titers were not
(p>0.05). Quantitative functional effects of patient serum on
AQP4-expressing cells can be assayed on cultured astrocytes,
astrocytic cell lines or transfected non-neural cells. Correlation
with clinical variables provides information pertinent to
anticipated outcome of attack (e.g., relapse risk, attack severity
(e.g., vision loss, paralysis), or extent of recovery).
[0080] These data support an algorithmic approach for the
serological evaluation of patients with inflammatory autoimmune
demyelinating disorders of the central nervous system (FIG. 4).
Seropositivity in initial immunochemical screening assays for
NMO-IgG supports the diagnosis of an NMO spectrum disorder. Further
testing of seropositive patients using functional assays (as
demonstrated by the complement activation assay described above;
and also by AQP4 and EAAT2 transporter modulating assays described
herein) is anticipated to provide prognostic information guiding
therapeutic decision making It is also observed that
AQP4-modulating assays are sufficiently sensitive to detect NMO-IgG
in some clinically proven cases that are negative by immunochemical
assays.
[0081] It is possible that diverse serological effects may occur in
individual patients. For example, one patient's disease severity
may correlate well with measures of AQP4 complement activation
while another may correlate with measures of AQP4/EEAT2
downregulation. In future clinical practice, the choice of
treatment for individual patients with NMO spectrum disorders may
be determined by the individual's serological profile in functional
assays of AQP4/NMO-IgG. For example, patients whose sera are highly
complement activating may be ideal candidates for complement
inhibitory therapies, or patients whose EAAT2 levels are
significantly reduced may be ideal candidates for glutamate
receptor antagonist therapies.
[0082] It is to be understood that, while the methods and
compositions of matter have been described herein in conjunction
with a number of different aspects, the foregoing description of
the various aspects is intended to illustrate and not limit the
scope of the methods and compositions of matter. Other aspects,
advantages, and modifications are within the scope of the following
claims.
[0083] Disclosed are methods and compositions that can be used for,
can be used in conjunction with, can be used in preparation for, or
are products of the disclosed methods and compositions. These and
other materials are disclosed herein, and it is understood that
combinations, subsets, interactions, groups, etc. of these methods
and compositions are disclosed. That is, while specific reference
to each various individual and collective combinations and
permutations of these compositions and methods may not be
explicitly disclosed, each is specifically contemplated and
described herein. For example, if a particular composition of
matter or a particular method is disclosed and discussed and a
number of compositions or methods are discussed, each and every
combination and permutation of the compositions and the methods are
specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed.
Sequence CWU 1
1
4122DNAArtificial SequenceOligonucleotide 1tgcaccagga agatcagcat cg
22221DNAArtificial SequenceOligonucleotide 2caggtcatcc gtctctacct g
21329DNAArtificial SequenceOligonucleotide 3ggtggaagtg cgaatgcacg
acagtcatc 29428DNAArtificial SequenceOligonucleotide 4cctcgtctgg
cggtggtgca accaggac 28
* * * * *