The evolving mystery of why skeletal muscle is spared in seropositive
Alan S. Verkman *, Xiaoming Yao, Alex J. Smith
Departments of Medicine and Physiology, University of California, San Francisco, CA, USA
Autoantibodies against aquaporin-4 (AQP4) in seropositive neu-
romyelitis optica spectrum disorders (called here NMO) initiate
pathology in the central nervous system by binding to AQP4 on astro-
cytes. It has been puzzling why skeletal muscle, which also expresses
AQP4, is rarely affected in NMO, despite easy access of circulating
NMO autoantibody (AQP4-IgG) to AQP4 on skeletal muscle.
Rosito et al.  report evidence that differences in the
supramolecular organization of AQP4 in skeletal muscle versus brain
astrocytes are responsible for the sparing of skeletal muscle in NMO,
arguing that the size of AQP4 supramolecular clusters (called orthog-
onal arrays of particles, OAPs) is smaller in skeletal muscle than in
astrocytes resulting in reduced AQP4-IgG binding. This mechanism is
motivated by the observation that some AQP4-IgG autoantibodies
bind better to AQP4 OAPs than to separated AQP4 tetramers  and
that AQP4 OAPs are required for C1q binding and complement activa-
tion . Although the study of Rosito et al.  is a novel and earnest
attempt to solve the puzzle of skeletal muscle sparing in NMO, in our
opinion their explanation lacks theoretical plausibility and contradicts
The authors’ assertion that arrays are smaller in muscle than in
brain is based on biochemical analysis and super-resolution (STED)
microscopy. Non-denaturating gels showed minor differences in the
size of AQP4 aggregates between muscle and brain; however, the
ratio of M1-AQP4 to M23-AQP4 was 1:3 or less in both tissues and in
the range where large OAPs form, which can bind all AQP4-IgG
autoantibodies in heterologous systems . As OAPs in native tis-
sues are small (20-50 nm diameter) and tightly packed, detecting
individual arrays is a challenge even for super-resolution microscopy
and requires careful consideration of microscope sensitivity to cluster
size and density . Freeze-fracture electron microscopy, the gold
standard in studying AQP4 OAP size, has shown that OAPs in healthy
human skeletal muscle consist of 10-35 AQP4 tetramers spaced
6 nm apart , similar to the size of OAPs in brain ; Rosito et al.
, however, report cluster size an order of magnitude greater.
Even if OAPs were smaller in skeletal muscle than in astrocytes,
little difference in AQP4-IgG binding is expected for a ratio of M23-
AQP4 to M1-AQP4 more than 1, as has been shown experimentally
and by mathematical modelling . Conceptually, an AQP4-IgG
molecule bound anywhere within an AQP4 cluster away from the
cluster edge cannot sense cluster size. The same consideration
holds for complement activation. In the light of this, the data in
Figure 1 of Rosito et al.  showing minimal or non-speciﬁc
AQP4-IgG binding to skeletal muscle in seropositive NMO are per-
plexing, as other studies have shown that systemically administered
AQP4-IgG rapidly binds to rodent skeletal muscle  and that IgG
binding is found on skeletal muscle in humans with NMO myositis
. One potential explanation for this discrepancy might be loss of
AQP4 antigenicity for autoantibody binding in the non-ﬁxed frozen
muscle used by Rosito et al. .
If AQP4 cluster size does not account for sparing of skeletal mus-
cle in NMO, then what does? One possibility is complement inhibitor
protein CD59, which is strongly expressed in skeletal muscle and
other AQP4-expressing tissues in the periphery such as kidney and
stomach. We found marked skeletal muscle injury in AQP4-IgG
seropositive rats deﬁcient in CD59, with creatine phosphokinase
levels nearly 1000-fold over normal . Perhaps the very rare
patients with NMO myositis have CD59 polymorphisms. Another pos-
sibility, though speculative, is that unique environmental factors in
the central nervous system, such as the presence of microglia, a nar-
row extracellular space and the close proximity of AQP4 clusters to
blood vessels, may amplify subthreshold injury following AQP4-IgG
binding to astrocyte AQP4. Differences in astrocyte versus skeletal
muscle biology may also be involved.
The mystery continues!
Funding source: This study was supported by grants EY13574,
EB00415, DK101373 and DK72517 from the National Institutes of
Health and a grant from the Guthy-Jackson Charitable Foundation.
Conﬂict of interest
The authors declare that they have no conﬂict of interest to disclose.
*Correspondence to: Alan S. VERKMAN
ª 2018 The Authors.
Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
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Letter to the Editor
J. Cell. Mol. Med. Vol 22, No 3, 2018 pp. 2039-2040