Aquaporin 4 and neuromyelitis optica

Review
Aquaporin 4 and neuromyelitis optica
Marios C Papadopoulos, A S Verkman
Neuromyelitis optica is an infl ammatory demyelinating disorder of the CNS. The discovery of circulating IgG1 Lancet Neurol 2012; 11: 535-44
antibodies against the astrocyte water channel protein aquaporin 4 (AQP4) and the evidence that AQP4-IgG is Academic Neurosurgery Unit,
involved in the development of neuromyelitis optica revolutionised our understanding of the disease. However, St George's, University of
important unanswered questions remain--for example, we do not know the cause of AQP4-IgG-negative disease, London, London, UK
(Dr M C Papadopoulos MD); and
how astrocyte damage causes demyelination, the role of T cells, why peripheral AQP4-expressing organs are Department of Medicine and
undamaged, and how circulating AQP4-IgG enters neuromyelitis optica lesions. New drug candidates have emerged, Department of Physiology,
such as aquaporumab (non-pathogenic antibody blocker of AQP4-IgG binding), sivelestat (neutrophil elastase University of California San
inhibitor), and eculizumab (complement inhibitor). Despite rapid progress, randomised clinical trials to test new Francisco, San Francisco, CA,
USA (Prof A S Verkman MD)
drugs will be challenging because of the small number of individuals with the disorder.
Correspondence to:
Dr Marios C Papadopoulos,
Introduction
AQP4-IgG seropositivity is higher in patients with Academic Neurosurgery Unit,
The coexistence of optic neuritis with myelitis was relapsing disease, optic neuritis, or longitudinally Room 1.122 Jenner Wing,
recognised in the early 19th century by several phys-
extensive transverse myelitis than in those with St George's, University of
London, London SW17 0RE, UK
icians, such as the Frenchman Antoine Portal, the monophasic disease.7-9 The percentage of patients with mpapadop@sgul.ac.uk
Italian Giovanni Battista Pescetto, the Englishman demyelinating disease that fulfi l the present diagnostic
Lockhart Clarke, and the Scotsman John Abercrombie.1-3
criteria is low (1-2%) in white people from Europe,
Abercrombie described intractable vomiting in some of North America, or Australia, and high (20-48%) in
these patients, indicating involvement of the medulla. In
people from the West Indies and Asia.8
1894, the French neurologist Eugene Devic and his
The prevalence of neuromyelitis is estimated at
student Fernand Gault reviewed published cases of optic
0*3-4*4 per 100
000 individuals,8 equivalent to
neuritis with myelitis, after which the disease became 900-13 200 people in the USA and 180-2640 in the UK.
known as neuromyelitis optica or Devic's disease.1-3
This prevalence is likely to increase as AQP4-IgG testing
Neuromyelitis optica was thought to be a variant of becomes wide
spread and AQP4-IgG assay sensitivity
multiple sclerosis, but in 2004, a circulating IgG auto-
improves. The median age at presentation is 39 years.8
antibody was reported in patients with neuromyelitis Women are most commonly aff ected, accounting for
optica that was absent in those with multiple sclerosis.4
roughly 85% of cases.8 Occur
rence of relapse might
Within a year, the astrocyte water channel protein increase in the last trimester of pregnancy and post
aquaporin 4 (AQP4) was identifi ed as its target.5 Here, partum,10 and safe prophylactic treatment might be
we review advances in understanding of the pathogenesis
appropriate during late pregnancy and breastfeeding.
of neuromyelitis optica and their implications for clinical
Transmission of neuromyelitis optica from mother to fetus
practice. We explain the structure, function, and has not been reported, possibly because little AQP4 is
biological roles of AQP4, and the cellular eff ects of expressed in the fetal CNS before formation of the blood-
AQP4-IgG binding. We then summarise the evidence brain barrier.11 An intriguing association between the
that AQP4-IgG causes disease and discuss major disorder and myasthenia gravis has been noted, with
unanswered questions. We refer readers elsewhere6-8 for
neuromyelitis optica often developing decades after
reviews with a clinical focus, including diagnosis and myasthenia.12,13
present treatments.
About 3% of patients with neuromyelitis optica have
relatives with the disease, but the underlying genetic
Epidemiology
susceptibility is complex.14 Some HLAs are associated
A defi nitive diagnosis of neuromyelitis optica can be with increased risk of neuromyelitis, such as DRB1*0301
made when optic neuritis, myelitis, and at least two of in white people and people with one white and one black
three supportive criteria (MRI evidence of a contiguous
parent,15 and DPB1*0501 in people from Asia.16 The HLA
spinal cord lesion in 3 segments; brain MRI not associations are diff erent in multiple sclerosis compared
diagnostic of multiple sclerosis; and AQP4-IgG with neuromyelitis optica.15,16 Genetic variations in the
seropositivity) are present.7 This scheme allows for a AQP4 sequence do not substantially account for
diagnosis of neuromyelitis optica in some patients with
neuromyelitis optica susceptibility.17,18
brain lesions who would not be thought to have the
disorder, and when detectable AQP4-IgG is absent.
AQP4: the target of antibodies
The spectrum of neuromyelitis optica disorders Structure, function, and cellular localisation
encom passes defi nitive neuromyelitis optica, as well as AQP4 was originally cloned from rat lung in 1994, on
limited forms, such as AQP4-IgG-positive, longitudinally
the basis of its homology with other aquaporins.19
exten sive, transverse myelitis, or recurrent or bilateral A high-resolution x-ray structure shows that AQP4
AQP4-IgG-positive optic neuritis. The frequency of monomers consist of six helical, membrane-spanning
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Review
Both isoforms are expressed in astrocytes and co-mingle
A
to form heterotetramers. M23-AQP4 forms OAPs be-
B
cause of intermolecular N-terminus interactions with
M6
residues just downstream of Met-23; M1-AQP4 does not
M2 M7
M4
M5
form OAPs on its own and restricts the size of OAPs
when coexpressed with M23-AQP4.25,29,30 Both M1-AQP4
and M23-AQP4 function as bidirectional, water-selective
M8
M3
channels that enable water fl ow across cell membranes
M1
100 nm
in response to osmotic gradients.
AQP4 is most strongly expressed in the CNS, but is
C
also present in epithelial cells in the kidney (collecting
V G G L G V T T VHG
duct), the stomach (parietal cells), airways, glands, and
V
S E N PL
S
N
G
W
skeletal muscle.31 AQP4 is expressed in astrocytes in the
P
L
E
M1 G
P
T
N
M2 V
M4 P
M5
M6
N M8
brain, spinal cord, and optic nerve, and is particularly
A
G
T I N W
L V T
H
L V M D
L G H G
A V I M
W I
concentrated at pial and ependymal surfaces in contact
S G V S
Y
I
Y L
S L
I
I N Y T G
W
C
G
L
A P G F S
F V L L
M3
I I G A
V E L A F L H
I P G V
with the CSF.32,33 At the cell level, AQP4 expression is
S L G F
S
I L M A
A
I I
F T
I
I
G A
A
I N P
G L C
V A I G
M N P A R
V
polarised to foot processes of astrocytes in contact with
T M
A
Q
A E F L
L
H
V
I T A Q
V F S F G I
M7
A G A L
F C Q V
M A V T
A F I T
blood vessels.32,33 AQP4 is also expressed in so-called
T V A K
G
L
G
Y F V S
H
Y
I
E
S G V
V A L A
Y 250
C T
I A K
S C
S
V
supportive cells--similar to astrocytes--in sensory
Q A F W
D
T
S
K I
S
G
F
organs such as Muller cells in the retina.34 Pathological
W
T
C
V
K
G
K L R R K L E V D P
K F A
R
changes associated with neuromyelitis optica mostly
V
T D V
E
M 23
A F S K A A Q Q T K G
occur in the spinal cord and optic nerve, and to a lesser
C S R E S I
P
S
Q S R N D E V E M Y
V
extent in the brain, with a notable absence of
P G C K G W R
E T E D L I L K P G V V
abnormalities in peripheral AQP4-expressing tissues.
1 M S D G A A A R
H
S S D K G K K E D G R D I D I V
G
N-terminus
E V L S S V C-terminus
Biological roles
Knowledge of the biological roles of AQP4 is important
Figure 1: The astrocyte water channel AQP4
because AQP4 expression is reduced in neuromyelitis
(A) High-resolution crystal structure of human AQP4 (Protein Data Bank reference 3GD8) showing eight
optica lesions as a result of astrocyte cytotoxicity. Available
membrane-associated helical segments, labelled M1-M8. (B) Freeze-fracture electron microscopy of AQP4
information about AQP4 physiology comes mainly from
orthogonal arrays of particles in plasma membranes of Chinese hamster ovary cells transfected with M23-AQP4.
(C) Protein sequence of human AQP4, showing Met-1 and Met-23 translation inhibition sites (black), residues
AQP4 knockout mice because people with loss-of-
that form intermolecular N-terminus associations to produce orthogonal arrays of particles (pink), residues
function mutations in AQP4 have not been identifi ed. At
preventing formation of orthogonal arrays of particles by M1-AQP4 (green), cysteine residues involved in
baseline, AQP4 knockout mice have normal appearance,
palmitolyation-regulated assembly of orthogonal arrays of particles (blue), and the C-terminus PDZ domain (red).
survival, growth,35 and neuromuscular function,36 but
partial impairment in visual,34 auditory,37 and olfactory38
domains and two short helical segments surrounding a signalling, as deduced from electrophysiological or
narrow aqueous pore (fi gure 1),20 similar to other behavioural studies, or both.
aquaporins. Additionally, as with other aquaporins,
Several phenotypes of AQP4 knockout mice have been
AQP4 monomers assemble as tetramers; however, identifi ed after various stresses have been imposed.
AQP4 tetramers uniquely further aggregate in cell Outside of the CNS, AQP4 knockout mice have a mild
plasma membranes to form supramolecular assembles reduction in maximum urine concentration, which is
called orthogonal arrays of particles (OAPs). OAPs were related to reduced water permeability in the renal inner
originally visualised in membranes by freeze-fracture medullary collecting duct.35 In the brain, mice without
electron microscopy (fi gure 1).21 AQP4 was identifi ed as AQP4 show reduced cytotoxic (cell swelling) brain
the major OAP protein from the appearance of OAPs in oedema after water intoxication or ischaemic stroke,39
AQP4-transfected cells22 and their absence in mice that increased vasogenic (leaky vessel) brain oedema in
did not have AQP4.23 Advanced optical microscopy brain tumours,40 and increased ventricular enlargement
methods have elucidated OAP dynamics24 and regulatory
in obstructive hydro cephalus.41 These phenotypes can be
mechanisms.25,26 Of relevance to neuromyelitis optica, explained by bidirectional impairment in water
most AQP4-IgGs bind preferentially to OAPs;9,27 OAPs transport with AQP4 defi ciency, with reduced water
seem to be crucial for complement-dependent cyto-
entry into the brain in cytotoxic oedema and impaired
toxicity through multivalent C1q binding to clustered removal of excess interstitial water in vasogenic oedema.
AQP4-IgG.28
In the spinal cord, swelling is reduced and neurological
AQP4 is present in two major isoforms produced by outcome is improved in AQP4 knockout mice in a
alternative splicing: a relatively long (M1) isoform with model of cytotoxic oedema (transient cord com-
translation initiation at Met-1, and a shorter (M23) pression),42 but worsened in a model of vasogenic
isoform with translation initiation at Met-23 (fi gure 1).
oedema (contusion injury).43
536
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Review
Phenotype studies31,44-46 have implicated AQP4 in such as natural killer cells, to cause cell death (antibody-
neuroexcitation, astrocyte migration, and neuro-
dependent cell-mediated cytotoxicity). Although one
infl ammation. AQP4 knockout mice have in
creased investigation with frog oocytes suggested that AQP-IgG
seizure duration,44 impaired glial scarring,45 and attenu-
binding causes partial inhibition of AQP4 water
ated experimental autoimmune encephalo
myelitis.46
permeability,48 another with cultured rat cortical
Impaired AQP4 water permeability in astrocytes is astrocytes47 and other work53 showed no eff ect: the large
probably the cause of these phenotypes. Cellular size of AQP4-IgG compared with AQP4 precludes
mechanisms of AQP4-dependent astrocyte migration, binding of more than one antibody per AQP4 tetramer.
neurosensory signalling, and infl ammation have been
Initial studies with AQP4-transfected HEK293 cells48,54
reviewed previously.31
showed that AQP4-IgG binding causes AQP4 internal-
isation; however, there was little or no internalisation in
AQP4-IgG binding to AQP4
mouse cortical astrocyte cultures or mouse brain in vivo
Several studies9,27,47,48 have shown that more binding of after binding.55 In mice and in vitro, AQP4-IgG binding to
AQP4-IgG in the serum of patients with neuromyelitis
AQP4 causes complement-dependent cytotoxicity when
optica occurs with transfected cells expressing M23-
complement is present,54,56 and antibody-dependent cell-
AQP4 than with M1-AQP4. This diff erence might be mediated cytotoxicity when natural killer cells are
caused by AQP4-IgG preferentially binding to OAPs, present.57-59 Complement-dependent cytotoxicity is probably
because M23-AQP4 forms OAPs and M1-AQP4 does the major mechanism by which neuromyelitis optica
not. Binding specifi city has been investigated with a develops.56,60-62
two-colour quantitative fl uorescence ratio imaging
assay of AQP4-transfected astrocyte cells stained with Mechanisms of pathogenesis
AQP4-IgG and a C-terminus anti-AQP4 antibody.27 The
Overview
researchers used serum from control patients and Substantial evidence is available that AQP4-IgG initiates
those with neuromyelitis optica, and monoclonal formation of a neuromyelitis optica lesion once it enters
recombinant AQP4-IgGs derived from clonally the CNS (fi gure 2). Complement-mediated astrocyte
expanded plasma cells from the CSF of individuals damage occurs fi rst, followed by granulocyte infi ltration,
with seropositive neuromyelitis optica.49 Diff erent oligodendrocyte death, and ultimately neuronal cell
monoclonal and serum AQP4-IgGs showed wide death. Because the lesions are characterised by necrosis
variation in binding to M1-AQP4 or M23-AQP4, from of the major CNS cell types,63 the clinical defi cits are
similar binding to both isoforms to exclusive binding often severe and recovery is poor.8 AQP4-IgM is found in
to M23-AQP4. The affi
nity of the tightest binding 10% of serum samples positive for AQP4-IgG, but its role
AQP4-IgG was 44 nM.27
in the disease remains unknown.64
Measurements in cells expressing M23-AQP4 with
OAP-disrupting mutations suggested that the diff erential Evidence that AQP4-IgG is pathogenic
binding of AQP4-IgG to M1 or M23 is due to OAP AQP4-IgG binds to AQP4 on the plasma membrane
assembly; studies27 with purifi ed Fab fragments suggested
of non-permeabilised cells, such as AQP4-transfected
that the AQP4 epitope undergoes a structural change on HEK293 and Chinese hamster ovary cells, and mouse
array assembly. Some reports50,51 have shown that specifi c cortical astrocyte cultures.54-56 Because AQP4-IgG is
extracellular epitopes of AQP4 might be associated with an IgG1-isotype antibody, bound AQP4-IgG causes
AQP4-IgG binding; however, the data are diffi
cult to complement-dependent cytotoxicity. Exposure of AQP4-
interpret because polyclonal AQP4-IgG was studied, and transfected HEK293 and Chinese hamster ovary cells,
the AQP4-IgG binding site probably includes three-
and mouse cortical astrocytes to AQP4-IgG and com-
dimensional epitopes that are specifi c to AQP4-IgG and plement causes plasma membrane deposition of the
are potentially modifi ed by point mutations in AQP4 complement membrane attack complex and cyto-
extracellular loops. Although clinical AQP4-IgG assays toxicity.54,56 AQP4-IgG also causes antibody-dependent
give semi-quantitative titres to one AQP4 isoform or to cell-mediated cytotoxicity of mouse cortical astrocyte
both isoforms,52 measurement of concentration-
cultures when natural killer cells are present in the
dependent binding to M1-AQP4 and M23-AQP4 absence of complement.57,59 These fi ndings provide
separately might provide useful information about how evidence for AQP4-IgG pathogenicity at the cell level.
to monitor and manage clinical neuromyelitis optica.
Evidence from clinical observations6,65 and histological
and animal studies61-63 also shows that AQP4-IgG causes
Cellular eff ects of AQP4-IgG binding to AQP4
neuromyelitis optica lesions. AQP4-IgG is identifi ed in
Antibodies can have several eff ector functions when most patients with the disease (68-91%) and is highly
bound to their target: modifi cation of target function; specifi c (85-99%).65 Generally, AQP4-IgG serum
target intern
alisation, reducing surface expression;
concentrations increase before a relapse,65 and a
complement activation to cause cell death (complement-
decrease in plasma AQP4-IgG by plasmapheresis
dependent cytotoxicity); and activation of eff ector cells, reduces disease activity.6
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A Normal CNS
B Complement activation; astrocyte damage
C Granulocyte entry; astrocyte death
Astrocyte
Oligodendrocyte
Membrane attack
AQP4
complex
Dying astrocyte
Complement
Neuron
CNS
Possible entry
Blood-brain
?
Tight junction
?
barrier
route
Blood vessel
Blood
Degranulating
AQP4-IgG
Eosinophil
neutrophil
F Reactive gliosis
E Macrophage entry; neuron death
D Oligodendrocyte damage; axon loss
Dying oligodendrocyte
Damaged axon
Microglia
AQP4
Reactive astrocytes
Dying
neuron
Myelin
fragments
Cellular
debris
Macrophage
Figure 2: Formation of neuromyelitis optica lesion
(A) Normal CNS with AQP4-IgG in the serum. (B) At the start of lesion formation, AQP4-IgG binds to AQP4 on foot processes of astrocytes, activates complement, and
causes deposition of membrane attack complexes. (C) Cytokines (eg, interleukin 17, interleukin 8, and granulocyte colony-stimulating factor) recruit neutrophils and
eosinophils into perivascular spaces; neutrophils degranulate to cause astrocyte death. Loss of astrocytes leads to oligodendrocyte death, which causes axon
degeneration (D), and neuronal death (E). Infi ltrating macrophages (and possibly microglia) phagocytose cellular and myelin debris. (F) Mature lesions are characterised
by pan-necrosis (complete tissue necrosis), and widespread infi ltration by macrophages; AQP4-positive reactive astrocytes are confi ned around the lesion.
Human neuromyelitis optica lesions show vasculo-
which are characteristically seen in allergic and parasitic69
centric deposition of immunoglobulin and activated rather than antibody-mediated diseases, is intriguing. In
complement components, suggesting humoral im munity
neuromyelitis optica, the eosinophils might stimulate
against a perivascular antigen.61-63 The substantial loss of the humoral immune response by producing type 2
AQP4 and the astrocyte marker glial fi brillary acidic cytokines, and might contribute to CNS damage by
protein (GFAP) in lesions is consistent with a humoral secreting eosinophil-derived neurotoxin and generating
response targeting AQP4 and causing astrocyte death. free radicals.69
The presence of a few AQP4-negative or GFAP-positive
Lesions similar to those reported in neuromyelitis optica
astrocytes in lesions suggests that loss of AQP4 precedes have been produced in rodents by passive transfer of
astrocyte death.61-63 Occasionally, AQP4 and GFAP are AQP4-IgG.46,56,70,71 Intraperitoneal injection of AQP4-IgG
lost, but myelin is preserved,61-63 supporting the notion exacerbates CNS infl ammation in rats with experimental
that astrocyte death precedes myelin breakdown.
autoimmune encephalomyelitis49,70 or those pretreated
Granulocytes (neutrophils and eosinophils) and with Freund's adjuvant.71 Although these studies provide
macrophages predominate in human neuromyelitis evidence that AQP4-IgG is pathogenic, the pre-existing
optica lesions, with a paucity of T lymphocytes63 and infl ammation confounds further inter
pretation. Direct
natural killer cells (unpublished). Most neutrophils are evidence that AQP4-IgG is pathogenic in vivo comes from
degranulated (fi gure 2),66 with evidence from mouse intracerebral injection of AQP4-IgG and human
models66,67 and case reports68 implicating neutrophils complement in mice, which reproduces the characteristic
early in lesion development. The presence of eosinophils,
histological features of human neuromyelitis optica
538
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lesions, such as loss of AQP4 and GFAP, infl ammatory monoclonal anti-4-integrin antibody, in some patients
cell infi ltration, loss of myelin, and perivascular deposition
with neuromyelitis optica78,79 suggests that macrophages
of activated comple ment.56 In these experiments, human potentially have a benefi cial role in the disorder. 4-
complement is injected because AQP4-IgG does not Integrin is expressed in human macrophages but not in
activate mouse complement eff ectively. The cell infi ltrate neutrophils,80 and so would inhibit macrophage but not
consists of granulocytes and macrophages with few neutrophil entry into lesions.
T lymphocytes, similar to human lesions. In key control
Demyelination, a characteristic feature of human and
experiments, no lesions are produced when complement mouse neuromyelitis optica, is not a primary event.
is injected with IgG from individuals without neuro-
Mouse experiments show that myelin loss is a result of
myelitis optica or with AQP4-IgG-depleted neuromyelitis astrocyte damage81 and leads to axonal degeneration56
optica serum, when complement is inhibited or not (fi gure 2). In humans63 and mouse models,56 neuronal
administered, or when complement and AQP4-IgG are necrosis follows axonal degeneration (fi gure 2).
injected in AQP4-defi
cient mice. The intracerebral
injection mouse model does not reproduce clinical signs Unanswered questions and controversies
of neuromyelitis optica because of the site of injection. An
What causes AQP4-IgG-negative disease?
ex-vivo model of neuromyelitis optica has been described,72
Whether AQP4-IgG-negative neuromyelitis optica
in which exposure of cultured mouse spinal cord slices or
diff ers from AQP4-IgG-positive disease is debated.
optic nerve to AQP4-IgG and complement causes loss of Reports show that AQP4-IgG-positive neuromyelitis
AQP4, GFAP, and myelin.
optica diff ers clinically and epidemiologically from
seronegative disease (strong predominance in women;
Origin of lesions
frequent association with coexisting autoimmunity;
AQP4-IgG is more than 500 times more concentrated in more severe clinical attacks; higher spinal cord lesion
plasma than in CSF, which has been taken to suggest that
load; and more frequent relapse)65,82 and that the HLA
AQP4-IgG forms peripherally and enters the susceptibility profi le of AQP4-IgG-negative disorder
CNS secondarily.73 However, plasma cells that secrete could be more similar to that of multiple sclerosis than of
AQP4-IgG have been detected in the CSF,49 and could AQP4-positive neuromyelitis optica.15
potentially lodge in CNS tissue and constitute a
Another possibility is that AQP4-IgG is present in
pathogenically relevant source of AQP4-IgG production. some patients with seronegative disease, but present
Circulating AQP4-IgG alone is insuffi
cient to produce assays are not sensitive enough to detect it. The
neuromyelitis optica lesions, because serial plasma proportion of patients with AQP4-IgG-seronegative
samples obtained from patients in remission often have neuromyelitis optica who have AQP4-IgG in the CSF is
AQP4-IgG, as do serum samples taken before disease low.73,83 With improvements in assay sensitivity, serum
becomes clinically apparent.65
AQP4-IgG has been detected in an increasing number of
Substantial evidence from rodent models56,58,66,70
patients with apparently AQP4-IgG-negative disease.84
suggests that, once in the CNS, AQP4-IgG causes Some patients with AQP4-IgG-negative neuromyelitis
astrocyte cytotoxicity, initially by complement activation
optica might have antibodies against antigens other than
and then by a cascade of infl ammatory events such as AQP4, perhaps against other astrocyte targets. IgG1
granulocyte infi
ltration and then macrophage antibodies that can mediate complement-dependent
infi ltration, causing further disruption of the blood-
cytotoxicity against myelin oligodendrocyte glycoprotein
brain barrier (fi gure 2). The central involvement of have been detected in some patients with AQP4-IgG-
astrocyte damage has prompted assays for astrocytic negative disease, and in some with recurrent optic
markers, such as CSF GFAP, for monitoring of disease neuritis, longitudinally extensive transverse myelitis,
progression and response to treatment.74 Studies in multiple sclerosis, or lupus.85 Together, these fi ndings
humans62,63,66,67,75,76 support a key role for neutrophils and suggest that AQP4-IgG-negative disease is probably a
eosinophils in neuromyelitis optica. Granulocytes are heterogeneous group that could include some patients
abundant within lesions,62,63,66 and patients often have with multiple sclerosis, some who are AQP4-IgG-
granulocytes66,76 and high concentrations of granulocyte positive, some with other autoantibodies, and perhaps
colony-stimulating factor in their CSF,76 and a serum some whose disease is not antibody mediated.
granulocyte cytokine signature.67
The role of macrophages, which abundantly infi ltrate How does AQP4-IgG enter the CNS?
lesions (fi gure 2), is unclear. In multiple sclerosis, Unlike other CNS autoimmune diseases, neuromyelitis
activated macrophages cause axonal damage by optica is notable for the absence of intrathecal antibody
phagocytosing myelin and secreting proinfl ammatory synthesis.73,75 Serum AQP4-IgG might be present for
cytokines, free radicals, glutamate, and metallopro-
years before onset of the disorder.86 Disease in the brain
teases.77 Macrophages could scavenge the cellular debris could start with the movement of AQP4-IgG from the
produced by astrocyte cytotoxicity and granulocyte periphery into the circumventricular organs, which do
infi ltration.77 The detrimental eff ect of natalizumab, a not have a blood-brain barrier but express AQP4. The
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539

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involvement of the area postrema--causing nausea, absence of components of the neuroinfl ammatory
vomiting and hiccups87--and of the posterior hypo-
cascade in these tissues (eg, microglia) and confi nement
thalamus--causing inappropriate antidiuretic hormone of infl
ammatory cells and cytokines to the small
secretion--supports this notion.88
extracellular space in the CNS.
The reason why pathological changes occur in the
optic nerves and spinal cord is unclear. The blood-brain
How does astrocyte damage cause myelin loss?
barrier might not be fully developed in the prelaminar Ablation of reactive astrocytes in mice by genetic
portion of optic nerve89 and root entry zones in the manipulation causes substantial leucocyte infi ltration
spinal cord,90 allowing easier access of circulating IgG and myelin loss after traumatic CNS injury,100 suggesting
than elsewhere in the CNS. Perhaps an additional important interactions exist between astrocytes and
factor, such as an infection, transiently disrupts the oligodendrocytes. Gap junctional connections101 could
blood-brain barrier preferentially in the optic nerve or propagate the astrocyte damage signals to oligo-
spinal cord, allowing AQP4-IgG access, as suggested by
dendrocytes. Additionally, AQP4 might co-localise with
reports that 25-30% of exacerbations are preceded by the excitatory aminoacid transporter EAAT2 in astrocytes,
infections.91,92 Downregulation of the neonatal Fc and EAAT2 downregulation in neuromyelitis optica
receptor93 and disruption of the AQP4-rich interfaces might cause glutamate excitotoxicity and oligodendrocyte
between the peripheral and central nervous systems94
damage.102 However, the involvement of EAAT2 in
have not been investigated. The population of neuromyelitis optica pathogenesis has been questioned
circulating plasmablasts increases before an episode of because little or no internalisation of EAAT2 occurs in
neuromyelitis optica and they migrate into the CNS,95
primary astrocyte cultures, glutamate uptake is unaf-
raising the possibility of AQP4-IgG production by fected by AQP4-IgG exposure, and intracerebral injection
plasmablasts in the CNS. Exposure of plasmablasts to of AQP4-IgG in mice in vivo does not cause internalisation
interleukin-6 (IL-6), which is high in serum and CSF of
of AQP4 or AQP4-IgG.55
patients with neuromyelitis optica, promotes AQP4-
Another possibility is that reactive astrocytes initiate a
IgG production.95 Therefore, research into inhibition of
positive-feedback cycle, resulting in widespread damage
IL-6 receptors with tocilizumab in neuromyelitis optica
to several cell types, such as oligodendrocytes. Any CNS
might be worthwhile.
lesion generates AQP4-expressing reactive astrocytes in
the surrounding CNS, which migrate towards the
Does AQP4-IgG damage peripheral AQP4-expressing
lesion.45,103 This fi nding also applies in neuromyelitis
organs?
optica, in which many AQP4-expressing reactive
Although peripheral AQP4-expressing organs do not astrocytes surround lesions in people61,63 and mice.56 In
have the equivalent of the blood-brain barrier and were CNS lesions not associated with the disorder, reactive
shown in a mouse model96 to rapidly bind circulating astrocytes enter the lesions to form a glial scar, but in
AQP4-IgG, no substantial peripheral organ damage neuromyelitis optica only a few scattered reactive
occurs in neuromyelitis optica. One study97 showed that astrocytes are present in the lesions.61 We suggest that the
serum creatine kinase concentrations were raised in less
reactive astrocytes are promptly destroyed as they enter a
than 0*5% of patients, indicating possible skeletal neuromyelitis optica lesion, because of exposure to
muscle damage. Potential explanations for the absence of
AQP4-IgG and complement. Thus, the entry of reactive
substantial abnormalities are a higher expression of M1-
astrocytes in such a lesion might sustain damage, leading
AQP4 than of M23-AQP4 in the peripheral nervous to oligodendrocyte damage and demyelination.
system, the minimal importance of peripheral AQP4-
expressing cells for tissue function compared with What roles do T cells have?
astrocytes for CNS function,35,36,97 the low AQP4 expression
T cells are implicated in neuromyelitis optica, because
in the peripheral nervous system compared with the AQP4-IgG is a T-cell-dependent immunoglobulin
CNS,98 and the unique extracellular micro environment subclass (IgG1). T cells are probably involved in the
in some peripheral organs (eg, gastric acidity reducing peripheral immune response, including breaking
AQP4-IgG binding). However, these proposed tolerance (when the immune system becomes responsive
explanations are not supported by the known bio-
to self antigens) and antibody production. Indirect
chemistry and physiology of AQP4.96
evidence from models of experimental autoimmune
Another possibility is reduced expression of com-
encephalomyelitis has suggested that T cells could cause
plement regulator proteins (CD46, CD55, CD59)99 in neuromyelitis optica infl ammation to be targeted to the
astrocyte foot processes compared with peripheral optic nerve and spinal cord.70 Findings that suggest that
AQP4-expressing cells, causing AQP4-expressing cells T cells are probably not involved directly in lesion
in the peripheral nervous system to have increased formation include: the high plasma versus low CSF
resistance to complement-dependent cytotoxicity. The AQP4-IgG titre;104 the paucity of T cells in human63 and
absence of pathological changes in peripheral organs of mouse105 lesions; the harmful eff ect of natalizumab
patients with neuromyelitis optica might also refl ect an (which inhibits CNS entry of leucocytes, including
540
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Review
T cells, but not neutrophils) in some patients with
neuromyelitis optica;78,79 the ability to produce similar
Complement
Natural killer cell
Plasmapheresis
Immunomodulation eg, rituximab
lesions in nude (T-cell-depleted) mice;105 and the
Inhibit
complement
development of widespread lesions in patients after
C1q
inhibition of T-cell entry into the CNS with fi ngolimod.106
Complement-dependent
Antibody-dependent
AQP4-IgG
production
Th17 cells, a subgroup of T helper cells, might be involved
cytotoxicity
cell-mediated
cytotoxicity
in the recruitment of granulocytes into early lesions by
Block AQP4-IgG by
AQP4-IgG
B cell
T cell
binding to AQP4
inducing release of cytokines, such as CXCL8, from
non-T cells.67 The generation of AQP4-specifi c T cells107
AQP4 OAPs
Cytotoxicity
Cell-specific
might help to clarify the role of T cells in rodent models
inhibition eg,
Disrupt AQP4
of neuromyelitis optica.
neutrophil
OAPs
Macrophage
Eosinophil
protease
Degradation
Synthesis
inhibitors
Treatment strategies
Present treatments
Reduce AQP4
Soluble
expression
Oligodendrocyte
Acute neuromyelitis optica is treated with methyl-
inflammatory
damage
Neutrophil
factors
Microglia
prednisolone and, in resistant cases, plasmapheresis.6
Maintenance treatments are corticosteroids, azathi-
Astrocyte
Demyelination
General immunosuppression
oprine, rituximab, mitoxantrone, cyclophosphamide, and
Present treatments
In clinical trials
In preclinical development
Theoretical
mycophenolate.6 Treatment regimens vary widely,
ranging from corticosteroids alone to complex drug Figure 3: Potential targets for treatment
combinations. The rarity of neuromyelitis optica has OAP=orthogonal array of particles.
precluded large-scale, randomised trials to rationalise
treatment.
of AQP4-IgG in the sera of patients with neuromyelitis
Interferon beta--the most commonly used therapy optica. Additionally, it prevented cytotoxicity in AQP4-
for multiple sclerosis--is acknowledged to be generally expressing cell cultures, in spinal cord slice cultures ex
harmful in neuromyelitis optica. It increases relapses vivo, and in mice receiving intraparenchymal AQP4-IgG
and AQP4-IgG antibody titre,108,109 probably by exacer-
and complement in vivo. Aquaporumab, like other
bating Th17-mediated infl ammation and demyelin-
AQP4-IgG antibodies, does not alter AQP4 water
ation,67 and by increasing the amounts of B-cell permeability (unpublished).
activating factor.110 Reports78-80 have suggested that
In an alternative approach, high-throughput screening
natalizumab, which is also used for treating multiple identifi ed several small-molecule drugs and natural
sclerosis, could be harmful in some patients with products that bind to AQP4 and reduce AQP4-IgG
neuromyelitis optica. In these cases natalizumab did binding.111 The challenge in blocker treatment will be
not control or indeed increased disease activity,78,79
maintenance of therapeutic blocker concentrations in
possibly by inhibiting macrophage but not neutrophil the CNS to chronically inhibit binding.
entry into lesions.80 Present treatments are associated
Neutrophils are important causes of CNS injury in
with potential long-term side-eff ects because they do early neuromyelitis-optica-like lesions in mice.66,67 Live
not target the cause of neuromyelitis optica, but produce
neutrophils and purifi
ed neutrophil elastase also
general immuno suppression.
exacerbated these lesions in an ex-vivo spinal cord
model.72 Sivelestat, a potent neutrophil elastase inhibitor,
Emerging treatments
reduced the lesions in mice and ex vivo by inhibiting
New treatments for neuromyelitis optica have been neutrophil entry into the lesion and tissue damage
proposed that target specifi c components of disease produced by neutrophil elastase. Sivelestat is licensed in
pathogenesis (fi gure 3). Because AQP4-IgG binding to Japan for acute respiratory distress syndrome, but is not
AQP4 is probably the initiating event in neuromyelitis approved in Europe or the USA. Assessment of sivelestat
optica, a blocker strategy has been introduced in which a
as a short-term corticosteroid-sparing agent to treat acute
non-pathogenic antibody or small molecule binds to exacerbations of neuromyelitis optica is warranted.
AQP4, preventing the binding of pathogenic AQP4-IgG.57
In a proof-of-concept study,57 investigators generated a Future directions
non-pathogenic, high-affi
nity monoclonal antibody Improved animal models are needed to further elucidate
(called aquaporumab) from a recombinant monoclonal neuromyelitis optica pathogenesis and to test new
neuromyelitis optica antibody with tight binding and treatments. The next generation of models might use
slow washout (derived from clonally expanded active immunisation to study the initial immune
plasmablasts in the CSF of patients) by mutation of its Fc
response against AQP4, the generation of AQP4-IgG,
region to eliminate eff ector func tions of complement-
and its entry into the CNS. Although rodents have been
mediated cytotoxicity and antibody-dependent cell-
used for passive transfer, they might not be an appropriate
mediated cytotoxicity.57 Aquaporumab prevented binding species for active immunisation. The amount of brain
www.thelancet.com/neurology Vol 11 June 2012
541

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Review
3
Jarius S, Wildemann B. An early case of neuromyelitis optica: on a
Search strategy and selection criteria
forgotten report by Jacob Lockhart Clarke, FRS. Mult Scler 2011;
17: 1384-86.
References for this report were identifi ed through searches of
4
Lennon VA, Wingerchuk DM, Kryzer TJ, et al. A serum
autoantibody marker of neuromyelitis optica: distinction from
PubMed; the ECTRIMS website; and the websites of Annals of
multiple sclerosis. Lancet 2004; 364: 2106-12.
Neurology, Neurology, Brain, and Multiple Sclerosis. The search
5
Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG
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marker of optic-spinal multiple sclerosis binds to the aquaporin-4
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Collongues N, de Seze J. Current and future treatment approaches
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References were chosen on the basis of originality and
7
Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF,
relevance to the broad scope of this Review.
Weinshenker BG. Revised diagnostic criteria for neuromyelitis
optica. Neurology 2006; 66: 1485-89.
8
Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ,
tissue made up of glial cells is 90% in people and 65% in
Weinshenker BG. The spectrum of neuromyelitis optica.
Lancet Neurol 2007; 6: 805-15.
mice.112 Mice also have lower neutrophil counts than do 9 Mader S, Lutterotti A, Di Pauli F, et al. Patterns of antibody binding
people (5-10% vs 60-70%), and a poorer responsive
to aquaporin-4 isoforms in neuromyelitis optica. PLoS One 2010;
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5: e10455.
10 Bourre B, Marignier R, Zephir H, et al. Neuromyelitis optica and
Although many anecdotal reports and open-label
pregnancy. Neurology 2012; 78: 875-79.
studies suggest that immunosuppressive treatments 11 Gomori E, Pal J, Abraham H, et al. Fetal development of membrane
and plasmapheresis are eff ective in neuromyelitis
water channel proteins aquaporin-1 and aquaporin-4 in the human
brain. Int J Dev Neurosci 2006; 24: 295-305.
optica, few well controlled clinical trials have been 12 Jarius S, Paul F, Franciotta D, et al. Neuromyelitis optica
done. The small number of patients and their highly
spectrum disorders in patients with myasthenia gravis: ten new
variable clinical course (partly a result of the present
aquaporin-4 antibody positive cases and a review of the literature.
Mult Scler 2011; published online Dec 19.
diagnostic system) will remain major challenges in
DOI:10.1177/1352458511431728.
trials. New treatments targeting AQP4-IgG binding to 13 McKeon A, Lennon VA, Jacob A, et al. Coexistence of myasthenia
AQP4 are promising. Results of an open-label trial
gravis and serological markers of neurological autoimmunity in
(NCT00904826)
of the complement inhibitor
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14 Matiello M, Kim HJ, Kim W, et al. Familial neuromyelitis optica.
eculizumab in refractory neuromyelitis optica are
Neurology 2010; 75: 310-15.
eagerly awaited, not withstanding the drug's high cost 15 Zephir H, Fajardy I, Outteryck O, et al. Is neuromyelitis optica
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000 per patient per year) and risk of
associated with human leukocyte antigen? Mult Scler 2009;
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meningococcal meningitis and other infections. The 16 Wang H, Dai Y, Qiu W, et al. HLA-DPB1 0501 is associated with
repurposing of existing drugs, such as the neutrophil
susceptibility to anti-aquaporin-4 antibodies positive neuromyelitis
inhibitor sivelestat and the IL-6 receptor inhibitor
optica in southern Han Chinese. J Neuroimmunol 2011; 233: 181-84.
tocilizumab, for treatment of neuromyelitis optica also 17 Matiello M, Schaefer-Klein JL, Hebrink DD, Kingsbury DJ,
Atkinson EJ, Weinshenker BG. Genetic analysis of aquaporin-4 in
warrants consideration. In the long term, targeted
neuromyelitis optica. Neurology 2011; 77: 1149-55.
immunomodulation--perhaps tolerising treatment--
18 Crane JM, Rossi A, Gupta T, Bennett JL, Verkman AS. Orthogonal
might be suffi
ciently advanced to warrant possible use
array formation by human aquaporin-4: examination of
neuromyelitis optica-associated aquaporin-4 polymorphisms.
in neuromyelitis optica.
J Neuroimmunol 2011; 236: 93-98.
Contributors
19 Hasegawa H, Ma T, Skach W, Matthay MA, Verkman AS. Molecular
MCP and ASV searched for published work and wrote the Review.
cloning of a mercurial-insensitive water channel expressed in
selected water-transporting tissues. J Biol Chem 1994; 269: 5497-500.
Confl icts of interest
20 Ho JD, Yeh R, Sandstrom A, et al. Crystal structure of human
The University of California San Francisco and the University of
aquaporin 4 at 1*8 A and its mechanism of conductance.
Colorado have fi led a joint US provisional patent application for
Proc Natl Acad Sci USA 2009; 106: 7437-42.
aquaporumab antibody treatment for neuromyelitis optica, of which
21 Wolburg H, Wolburg-Buchholz K, Fallier-Becker P, Noell S,
ASV is a named co-inventor. The intellectual property rights are owned
Mack AF. Structure and functions of aquaporin-4-based orthogonal
by the University of California and the University of Colorado. MCP
arrays of particles. Int Rev Cell Mol Biol 2011; 287: 1-41.
declares that he has no confl icts of interest.
22 Yang B, Brown D, Verkman AS. The mercurial insensitive water
channel (AQP-4) forms orthogonal arrays in stably transfected
Acknowledgments
Chinese hamster ovary cells. J Biol Chem 1996; 271: 4577-80.
MCP and ASV were supported by the Guthy-Jackson Charitable
23 Verbavatz JM, Ma T, Gobin R, Verkman AS. Absence of
Foundation; ASV was also supported by the National Institutes of
orthogonal arrays in kidney, brain and muscle from transgenic
Health. The funding sources were not involved in writing the
knockout mice lacking water channel aquaporin-4. J Cell Sci 1997;
manuscript or the decision to submit for publication. We thank
110: 2855-60.
B Weinshenker (Mayo Clinic) and S Zamvil (University of California
24 Crane JM, Van Hoek AN, Skach WR, Verkman AS. Aquaporin-4
San Francisco) for critical review of this report.
dynamics in orthogonal arrays in live cells visualized by quantum
dot single particle tracking. Mol Biol Cell 2008; 19: 3369-78.
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