ADNP/NAP dramatically increase microtubule end-binding protein–Tau interaction: a novel avenue for protection against tauopathy

Abstract

Activity-dependent neuroprotective protein (ADNP), vital for brain formation and cognitive function, is mutated in autism and linked to neurodegenerative/psychiatric diseases. An eight-amino-acid peptide snippet of ADNP, NAP (NAPVSIPQ), identified as a smallest active fragment, includes the SxIP microtubule (MT) end-binding protein (EB) association motif, and enhances ADNP-EB3 interaction. Depletion of EB1 or EB3 abolishes NAP protection against zinc intoxication. Furthermore, NAP enhances Tau-MT interaction, and Tau regulates the localization and function of EB1 and EB3 in developing neuronal cells. Here, we asked how NAP (ADNP) enhances Tau-MT interactions and whether this is mediated by EBs. We showed, for we believe the first time, that NAP augmented endogenous EB1 comet density in the N1E-115 neuroblastoma neuronal model. This finding was substantiated by cell transfection with fluorescent EB1 and live cell imaging. NAP increased comet amounts, length and speed. At the molecular level, NAP enhanced EB3 homodimer formation, while decreasing EB1-EB3 heterodimer content and driving EB1- and EB3-Tau interactions (dramatic 20-fold increases), leading to recruitment of EB1/EB3 and Tau to MTs under zinc intoxication. Our previous results showed that while NAP protected neuronal-like cells against oxidative stress, it did not protect NIH3T3 fibroblasts. Here, NAP did not protect NIH3T3 cells against zinc intoxication, unless these cells were transfected with Tau. Interestingly, other MT associated proteins (MAPs) may replace Tau, thus, EB-Tau (MAPs) interaction is identified as a novel target for endogenous ADNP neuroprotection, and a future target for drug development, with NAP as a prototype.

NAP (davunetide) preferential interaction with dynamic 3-repeat Tau explains differential protection in selected tauopathies

“…NAP also protects against the accumulation of pathologically modified, hyperphosphorylated Tau (14, 34–36). We have previously suggested the mechanism of NAP protective activity on MT-mediated cellular processes through the involvement of Tau and MT end-binding proteins (EBs) (16, 29). Specifically, NAP contains an ADNP association site (SIP) a signature motif for direct interaction with the EB1 and the EB3 proteins (29), which in turn bind to MTs (37) and Tau (17).…”

“…Specifically, NAP contains an ADNP association site (SIP) a signature motif for direct interaction with the EB1 and the EB3 proteins (29), which in turn bind to MTs (37) and Tau (17). Furthermore, Tau has been identified as a regulator of EB’s action, and localization on MTs in developing neuronal cells (17) and NAP increases Tau-EB1/3 association (16). Tau is essential for the establishment of MT dynamic instability and axonal transport, while EB1 is more prevalent in neuronal axons (38) and EB3 in dendritic spines (39).…”

“…Tau is essential for the establishment of MT dynamic instability and axonal transport, while EB1 is more prevalent in neuronal axons (38) and EB3 in dendritic spines (39). Formerly, it has been shown that expression of Tau and EB1/3 proteins are required for NAP-dependent neuronal survival (16, 29). Here, we added details to the understanding of the molecular mechanism underlying MT-related activity of NAP.…”

“…NAP does not interact with MT proteins from various cancer cell lines or fibroblasts (27), which may not express any MT-associated proteins with properties that are similar to Tau3R, and NAP does not affect cell division (48). Furthermore, NAP does not protect cells from fibroblast origin unless those cells are transfected with Tau3R-expressing plasmid (16). However, NAP protects MT organization in mature neurons and glia (26, 27, 49).…”

“…We have previously shown that the expression of activity-dependent neuroprotective protein (ADNP), a protein vital for brain formation (11, 12)), is correlated with Tau3R expression (13) and Adnp+/- mice exhibit tauopathy features - significant increase in phosphorylated Tau, prevented by treatment of ADNP-derived peptide NAP (NAPVSIPQ) (14) as well as and tangle-like structures. Our cell culture results have indicated that NAP enhances Tau-MT interaction in the face of zinc intoxication (15) and NAP protective activity requires Tau expression (16). We have further revealed that NAP-Tau association is mediated by direct interaction of NAP and Tau with MT end-binding proteins (EBs) (15, 17).…”