Neuromyelitis optica (NMO) is an autoimmune demyelinating disease associated with recurrent episodes of optic neuritis and transverse myelitis, often resulting in permanent blindness and/or paralysis. The discovery of autoantibodies (AQP4-IgG) that target aquaporin-4 (AQP4) has accelerated our understanding of the cellular mechanisms driving NMO pathogenesis. AQP4 is a bidirectional water channel expressed on the plasma membranes of astrocytes, retinal Müller cells, skeletal muscle, and some epithelial cells in kidney, lung and the gastrointestinal tract. AQP4 tetramers form regular supramolecular assemblies at the cell plasma membrane called orthogonal arrays of particles. The pathological features of NMO include perivascular deposition of immunoglobulin and activated complement, loss of astrocytic AQP4, inflammatory infiltration with granulocyte and macrophage accumulation, and demyelination with axon loss. Current evidence supports a causative role of AQP4-IgG in NMO, in which binding of AQP4-IgG to AQP4 orthogonal arrays on astrocytes initiates complement-dependent and antibody-dependent cell-mediated cytotoxicity and inflammation. Immunosuppression and plasma exchange are the mainstays of therapy for NMO optic neuritis. Novel therapeutics targeting specific steps in NMO pathogenesis are entering the development pipeline, including blockers of AQP4-IgG binding to AQP4 and inhibitors of granulocyte function. However, much work remains in understanding the unique susceptibility of the optic nerves in NMO, in developing animal models of NMO optic neuritis, and in improving therapies to preserve vision.

Background

Neuromyelitis optica (NMO), an autoimmune inflammatory disease of the central nervous system, is often associated with retinal abnormalities including thinning of the retinal nerve fiber layer and microcystic changes. Here, we demonstrate that passive transfer of an anti-aquaporin-4 autoantibody (AQP4-IgG) produces primary retinal pathology.

Methods

AQP4-IgG was delivered to adult rat retinas by intravitreal injection. Rat retinas and retinal explant cultures were assessed by immunofluorescence.

Results

Immunofluorescence showed AQP4-IgG deposition on retinal Müller cells, with greatly reduced AQP4 expression and increased glial fibrillary acidic protein by 5 days. There was mild retinal inflammation with microglial activation but little leukocyte infiltration and loss of retinal ganglion cells by 30 days with thinning of the ganglion cell complex. Interestingly, the loss of AQP4 was complement independent as seen in cobra venom factor-treated rats and in normal rats administered a mutated AQP4-IgG lacking complement effector function. Exposure of ex vivo retinal cultures to AQP4-IgG produced a marked reduction in AQP4 expression by 24 h, which was largely prevented by inhibitors of endocytosis or lysosomal acidification.

Conclusions

Passive transfer of AQP4-IgG results in primary, complement-independent retinal pathology, which might contribute to retinal abnormalities seen in NMO patients.

Purpose

The epithelium lining the ocular surface, which includes corneal and conjunctival epithelia, expresses the prosecretory chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the proabsorptive epithelial sodium channel (ENaC). Here, methodology was established to measure the millivolt (mV) potential differences at the ocular surface, called ocular surface potential difference (OSPD), in human subjects produced by ion transport.

Methods

OSPD was measured in human subjects in which a fluid-filled measuring electrode contacted a fluid pool created by eversion of the lateral lower eyelid, with a reference electrode placed subcutaneously in the forearm. Through the use of a high-impedance voltmeter, OSPD was measured continuously over 10 to 15 minutes in response to a series of perfusate fluid exchanges.

Results

Baseline OSPD (± SEM) in six normal human subjects was -21.3 ± 3.6 mV. OSPD depolarized by 1.7 ± 0.6 mV following the addition of the ENaC inhibitor amiloride, hyperpolarized by 6.8 ± 1.5 mV with a zero chloride solution, and further hyperpolarized by 15.9 ± 1.6 mV following CFTR activation by isoproterenol. The isoproterenol-induced hyperpolarization was absent in two cystic fibrosis subjects lacking functional CFTR. OSPD measurement produced minimal epithelial injury.

Conclusions

Our results establish the feasibility and safety of OSPD measurement in humans and demonstrate robust CFTR activity, albeit minimal ENaC activity, at the ocular surface. OSPD measurement may be broadly applicable to investigate fluid transport mechanisms and test drug candidates to treat ocular surface disorders.

Translational relevance

To the best of our knowledge, this is the first measurement of the electrical potential generated by the ocular surface epithelium in human subjects, offering a new approach to study ocular surface function and health.

Purpose

Pharmacological activation of ocular surface cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels is a potential pro-secretory approach to treat dry eye disorders. We previously reported the discovery of aminophenyl-1,3,5-triazines, one of which, N-methyl-N-phenyl-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine (herein called CFTRact-K267), fully activated human wildtype CFTR with EC50 ∼ 30 nM and increased tear volume for 8 hours in mice. Here, functional and pharmacological studies of CFTRact-K267 were done in adult New Zealand white rabbits.

Methods

CFTR chloride conductance was measured in vivo by ocular surface potential differences and in ex vivo conjunctiva by short-circuit current. Tear volume was measured by the Schirmer tear test II and CFTRact-K267 pharmacokinetics and tissue distribution by liquid chromatography/mass spectrometry. Toxicity profile was studied for 28 days with twice-daily topical administration.

Results

Electrophysiological measurements in vivo and in ex vivo conjunctiva demonstrated CFTR activation by CFTRact-K267. A single topical dose of 3 nmol CFTRact-K267 increased tear production by >5 mm for 9 hours by the Schirmer tear test, with predicted therapeutic concentrations maintained in tear fluid. No tachyphylaxis was seen following 28-day twice-daily administration, and changes were not observed in corneal surface integrity or thickness, intraocular pressure, or ocular histology. At day 28, CFTRact-K267 was concentrated in the cornea and conjunctiva and was not detectable in blood or peripheral organs.

Conclusions

These studies support the development of CFTRact-K267 as a pro-secretory therapy for dry eye disorders.