Neuromyelitis optica pathogenesis and aquaporin 4 (1)
Neuromyelitis optica (NMO)
NMO is a devastating disease affecting primarily young women (relapsing NMO) but either sex can develop monophasic NMO, and NMO rarely occurs in adolescents. The disease principally attacks the optic nerves and spinal cord causing blindness and paralysis. An autoantibody (IgG1) that binds Aquaporin 4 (AQP4) has been found in a high percentage (~75%) of NMO patients (NMO-IgG). Other CNS antigens such as the Kir4.1 present on astrocytes might be targets for autoantibodies in those NMO patients. Other autoantibodies have been found in NMO patient sera and CSF, including antinuclear antibodies, SS antibodies, anti-myelin oligodendrocyte glycoprotein (MOG) antibodies, in addition to antibodies against extra- or intracellular antigens (myelin basic protein, S100β, CPSF-73, RNF-141, and myosin light chain. These latter autoantibodies likely represent a response to neo-antigen liberated from dead cells and thus are not the initial cause of NMO but could be involved in the pathogenesis of recurrent disease via a type III hypersensitivity reaction.
AQP4 a transmembrane protein important for CNS function
AQP4 is a type III transmembrane protein (intracellular n- and c-termini) that regulates water entry into and out of specific cells in the brain and interfaces with blood vessels within the neuropil and around the ventricles. AQP4 is the pre- dominant water channel in the brain of humans and rodents. The expression pattern of AQP4 protein is most well characterized for rats which express AQP4 in multiple tissues and in specific cell populations in particular tissues. AQP4 is also expressed in situ in LPS-activated microglial cell (monocytic origin) membranes.
Different isoforms of AQP4
Aquaporin 4 exists as two major isoforms (M1 and M23) in rat and human astrocytes and some epithelial tissues. The M1 and M23 isoforms differ by 23 amino acids at the N-terminus depending on which methionine is used to start translation. AQP4 has been proposed to have three extracellular loops (A, C, and E) that connect the 6 alpha helices that span the membrane. Loop E is more intramembranous and involved in water transport. AQP4-specific antibodies in NMO patients that bind extracellular AQP4 support the notion that parts of AQP4 are accessible on the outside of the cell. A recent study indicated AQP4 was internalized following overnight culture at 37°C with NMO-IGG serum. The presence of cysteine residues and thus the potential post translational changes (palmitoylation) in M1 residues 13 and 17 of the N-terminus of AQP4 prevents formation of AQP4 arrays.
AQP4 homotetramers and arrays
While AQP4 cannot associate with other AQPs, it can form heterotetramers of M1 and M23. M1 tetramers, which are the same size as M23, are over- whelmingly (95%) found as single tetramers. Quater- nary interactions are possible between AQP4 molecules within tetramers or between AQP4 tetramers on apposing membranes thus potentially limiting access of some AQP4 regions to AQP4-specific antibodies.
AQP4 structure, immunogenicity, and B cell epitopes
Expression of AQP4 on the basolateral cell membranes versus apical cell membrane could also moderate the availability of AQP4 targets for immune system effectors, especially AQP4-spe- cific antibodies.
Optic nerve and AQP4 distribution
ON with anti-AQP4-specific antibodies might ini- tially be more prevalent than LETM in NMO is that the tis- sues of the optic nerve are more sensitive to volume changes induced by AQP4 dysfunction than other areas of the CNS.
Spinal cord and AQP4 distribution
The cervical cord is closest to the cervical lymph nodes where priming AQP4-specific responses is likely to occur. One animal model proposes that the cervical lymph nodes play a major role in the transverse myelitis associated with EAE.
Ependyma and AQP4 distribution
When anti-AQP4 antibodies are detected in the CSF of NMO patients, the concentration is less than 1:500 compared to the serum.
Potential AQP4 B cell epitopes and relationship to pathogenesis
Resolution of the AQP4 structure provides evidence that both small defined B cell epitopes and extended conformation epitopes are possible based on exposed amino acids. The surface area of the top of the AQP4 molecule is on the order of 35 × 30 Å2, a size that could represent a conformational epitope with contributions from several exposed loops of AQP4.
Loop C, the longest loop, contains many non-polar residues and the structure. The other loops, A and E are smaller and contain several residues that are hydrophilic and typical partners for H-bonding or salt bridge interactions that are important for antibodies binding affinity. In mice and rats, Loop A has 62S, 63E, and 64N, whereas in humans the residues are 62T, 63E, and 64K. Thus the binding of human NMO-IgG to murine AQP4 suggests at least three interpreta- tions: 1) that Loop A is not a B cell epitope, 2) the changes between rodents and humans do not affect antibody binding if the loop A sequence is a B cell epitope, or 3) the sequence changes do affect binding of anti-AQP4 antibodies, but other AQP4 epitopes are still available and antibodies in polyclonal sera have enough reactivity to AQP4 to be scored as positive for binding if one AQP4 epitope is compromised. The second interpretation would suggest more defined linear sequences as B cell epitopes that would be expressed in the A or E loop structures.
A monoclonal antibody to rat AQP4 peptides, IgM of low affinity, was made to residues 206–231 (VRGASMNPARSFGPAVIMGNWENHWI) which form part of the water channel helix and Loop E. The AQP4 crystal model suggests that only the N- and C-termini of the pep- tide 206–231 are surface exposed and thus residues able to form a B cell epitope might be limiting.
In the MOG-mouse TCR transgenic model of ON a related question is apparent. Interestingly, treatments to induce changes in the BBB increase the manifestation of ON, but not of EAE, suggesting that the access of MOG-activated T cells to the spinal cord or brain is not sufficient to cause EAE.
NMO pathogenesis
the patho- genic anti-AQP4 antibody binds to the extracellular com- ponent of the AQP4 protein and induces a reversible internalization of the AQP4-IgG complex. the potential pathogenicity of NMO-IgG antibodies which is probably limited by the isotype, as IgM and IgA have more limited access to tissue parenchyma than IgG.
HLA-restriction, AQP-4 specific T cells, and NMO
A MHC class II linkage (HLA, DPA1*0202; DPB1 0501; reactive with myelin basic pro- tein) has been identified in patients (90%) with Asian MS that some consider NMO; however the high expression of this HLA type in Asians tempers the initial interpretation of a causal link between HLA type and disease. The severity of debilitation of NMO patients correlates with the increased frequency of Vβ 7 and Vβ 13 [one of the chains forming the antigen specific T cell receptor (TCR)] expressing T cells in CSF of NMO patients.
Priming of AQP4-specific B and T cells
Bacterial and viral infection can damage respiratory epi- thelial cells which express AQP4, thus providing condi- tions to enhance antigen presentation by mature dendritic cells as well as obviating the influence of regulatory T cells which could otherwise play a role in the typical non-path- ogenic responsive to AQP4.Infection and autoimmunity can result in events that alter the BBB and thus permit access of AQP4-specific antibod- ies and T cells to the CNS. Some cytokines, universal reg- ulators of cells of the immune system, can affect the BBB as well as the expression of glial cell-associated AQP4. Interleukin 1β and interferon β up-regulate AQP on rat astrocytes, whereas interferon γ(IFN-γ) but not Interleukin 1β or TNF-α increases expression of human astro- cyte AQP4 in vivo.
Potential pathogenic role of AQP4-specific antibodies
The presence of IgM, IgG, and complement cascade prod- ucts in the pathology of NMO optic nerve and spinal cord may represent non-specific antibodies as well as anti- AQP4-specific antibodies.
Autoimmune disorders and NMO
Sjögren’s Syndrome is an autoimmune dis- ease characterized by inadequate tear and saliva produc- tion and a lymphocyte infiltration of exocrine glands, especially the lacrimal and salivary glands, which express AQP 5 on the apical membrane and AQP4 on lateral membranes. Sjögren’s Syndrome patients typi- cally have autoantibodies that bind the nuclear antigens and RNA-binding proteins, Ro52, Ro60, and La48. In SLE, immune complexes deposition can affect the skin, joints, kidneys, lungs, nervous system, serous membranes and/or other organs of the body whereby inflammation damages membranes (lungs, kidney, nervous system) which also can contain AQP4. Autoantibodies associated with SLE typically bind ssDNA or dsDNA, DNA-associated proteins (PCNA and histones), snRNAs, ribosomal P, and phospholipids. Myasthenia gravis is an autoimmune disease that features antibodies that bind the acetylcholine receptor in muscle motor neural endplates thereby causing weakness (ocular, bulbar, limb), and respiratory distress. Mycobacterium and Mycoplasma sp. express that have similar residues to human AQP4.
Animal models of NMO-like diseases
Currently there are no animal models that feature AQP4 as the autoantigen that induces NMO-like pathology. It is important to initiate development of an AQP4-based ani- mal model of NMO to determine whether AQP4 is the autoantigen required for NMO, either due to AQP4-spe- cific T and B cells, specific AQP4 antibody or both cellular and soluble effectors. High titer antiserum in mice to the rat 206–231 sequence of the E Loop of AQP4 was established. Pooled sera from the AQP4-peptide immunized mice bind the M1 human isoform and the rat M23 isoform of AQP4 in HEK cells. Similarly, an AQP4 peptide (QTKGSYMEVEDNRSQVETED, amino acids 272–291) from rat AQP4’s cytoplasmic domain, that is predicted to be an autoimmune T cell target, was immunogenic in rats. High titers of autoantibodies to extra- and intracellular AQP4 peptides in Lewis rats were induced although it has been more difficult in C57 mice.
Approachable questions based on a small animal NMO model
Expression of class II molecules is a potential issue that might mediate the choice or rats over mice, as rats express class II on non-professional antigen presenting cells in a similar manner to humans. An AQP4-based animal model needs to be developed to address these issues and determine if AQP4-specific antibodies represent a novel means to cause CNS immunopa- thology similar to NMO disease in humans.