Table 3 Box: GVB immunogenicity

In the neuropathological literature on proteins found in GVBs, a noteworthy discrepancy in GVB immunogenicity is apparent. Typically, phosphorylation-dependent antibodies recognize GVBs, whereas phosphorylation-independent antibodies targeting the same protein do not. A comprehensive list of proteins detected in GVBs till the year 2016 using either phospho-specific or generic antibodies has been published previously [65]. In the novel experimental GVB model in cultured neurons, tau is detected in GVBs using a phosphorylation-dependent antibody, but not using an antibody-independent approach that employs the overexpression of fluorescent protein-tagged tau [143]. Therefore, it is possible that only a protein fragment containing the epitope accumulates in GVBs, which is a plausible explanation in view of the identification of GVBs as proteolytically active compartment (see GVB identity). Alternatively, some phosphorylation-dependent antibodies may bind non-specifically to phospho-epitopes that are immunological mimics of their phosphorylated target protein. Recently, it was published that the necroptosis-related proteins RIPK1 and RIPK3 could be detected in GVBs by generic, phosphorylation-independent antibodies only after dephosphorylation of the human brain tissue [68]. This indicates that for some proteins, phosphatase treatment may be necessary to reveal their GVB localization. Therefore, caution is warranted in the interpretation of the functional implications of GVB localization for proteins that have so far solely been detected using phospho-specific antibodies, until the presence of the total protein is confirmed by immunolabeling or using direct fluorescence in experimental models. Even though the functional significance is unclear, phospho-specific antibodies – such as pPERK, peIF2α, pIRE1α and pTDP-43 – consistently identify GVBs in the human and mouse brain and in cultured mouse neurons and are therefore convenient and reliable GVB markers (Table 1). In conclusion, a clear distinction can be made between immunolabeling for the purpose of GVB detection or for the functional interpretation of the immunopositive signal.