## 6 Molecular Mechanism of NMOSD
### AQP4 Dependent Pathology
Most NMOSD patients are seropositive for circulating AQP4 immunoglobulin G (AQP4-IgG) antibodies, which target various extracellular epitopes of the AQP4 water channel [8; 14]. AQP4-IgG autoantibodies possess a polyclonal ability that recognizes and binds to various epitopes and domains of the AQP4 channel [118]. Anti-AQP4-IgG produced by the plasma cells in peripheral blood enters the CNS through endothelial transcytosis and a permeable BBB and binds with the AQP4 channel. The binding of AQP-IgG with AQP4 activates the complement factors leading to astrocytic injuries through CDC and ADCC. The CDC and ADCC induce further inflammatory signals downstream and the migration of macrophages, neutrophils, and eosinophils to the inflammation site, resulting in BBB interruptions, loss of myelin, and neuronal injury [116; 119]. Increased levels of IL-6 are correlated with increased anti-AQP4-IgG and glial fibrillary acidic protein and increased damage in NMOSD [120]. AQP4 crosses the BBB, and the Fc domain of AQP4-IgG binds to the AQP4 in the astrocyte end feet, thereby employing complement activation and causing complement-dependent cytotoxicity (CDC). CDC is activated by AQP4 binding with C1q complement protein. The formation of OAPs by clustering AQP4 results in adding more AQP4-IgGs, which enhances the multivalent binding of C1q onto the grouped AQP4-IgGs over the OAPs [42]. The binding of AQ4-IgG causes endocytosis of AQP4 and reduces water transport across the plasma membrane [120]. To develop memory for AQP4 antigen, B cells require AQP4-specific T cells [53]. AQP4-IgG-generating plasma cells were originally developed by the recognition of T cells with AQP4, which led to the expansion of B cell differentiation into plasmablasts [121]. Identifying the subsets of B cells that produce AQP4-IgG helps in targeted immunotherapies. Antibody-secreting cells (ASCs) are the proliferative plasmablasts that emerge from bone marrow. Initially they are naive B cells, later developing antigen memory, acquiring CD 27, and switching the IgD/IgM expression into IgG expression on its surface [119; 122]. [PERSON] et al. studied the capacity of B cells to produce AQP4-IgGs. The circulating B cells produce AQP4 antibodies in the absence of antigens. Few other factors induce AQP4-IgGs without the cytokines. Hence, it was found that antigens are not necessary for AQP4-IgG production [121]. The production cycle and availability of AQP4-IgGs in the peripheral nervous system and the CNS, and their function in the serum and CNS, are described in Figure 2.  
The binding of AQP4-IgG to AQP4 induces endocytosis, which results in the loss or internalization of the excitatory amino acid transporter 2 (EAT2) [123]. The internalization of glutamate receptors reduces the astrocytic enzyme glutamate synthase activity, causing glutamate accumulation [123]. More accretion of glutamate stimulates the production of various immunological factors, chemokines, cytokines, and complement factors [124]. The production of immunological components activates immune cells such as eosinophils,  
Figure 2: The AQP4-IgG antibodies are produced by antibody-secreting cells (ASCs). Precursors of ASCs secreted from bone marrow as naive B cells. Naive B cells acquire memory antigen CD 27 on their surface along with AQP4 antigen-specific T cells and switch their IgM/IgD expression into IgG. Thus, developed antibody-secreting plasma differentiates into short, long-lived (these are tissue-resident plasma cells that produce constant IgGs), and peripheral circulating B cells secreting AQP4-IgG antibodies with other cell surfaces markers such as CD 19, CD 27, and CD 38 on its surface. Thus, produced AQP4-IgGs are available in the serum and enter the CNS, thereby inducing cytokine production, developing the inflammation cycle, and promoting T cells to generate more ASCs. In the CNS, the AQP4-IgGs bind with AQP4 present in the end feet of astrocytes and initiate NMOSD injury and lesions [109; 121; 122]. (Figure created using BioRender.com; accessed on 7 February 2023).