Amyloid plaques and neurofibrillary tangles formed from amyloid-β (Aβ) peptide and Tau protein are two hallmark lesions of Alzheimer’s disease (AD).1, 2 Non-receptor Src tyrosine kinase, Fyn, uniquely interconnects the above two proteins associated with AD pathologies.3, 4 Fyn kinase gets activated by Aβ via cellular prion protein (PrPC) receptor and is also known to interact with, phosphorylated Tau at the Y18 site, and co-localize with Tau protein.5, 6, 7 Previous studies reported that Tau/Fyn knockout in mice produces a converging phenotype showing a protective effect in the AD mouse model. In addition, studies have also demonstrated that mice expressing a truncated form of Tau that inhibits Tau-Fyn interaction are protected against behavioral deficits in the AD model.4, 8, 9, 10 Thus far, studies highlight that the Fyn-Tau interaction is critical for Aβ oligomer toxicity.11

Neuronal cell loss in several neurological conditions, such as Alzheimer’s, Huntington’s, amyotrophic lateral sclerosis, and Parkinson’s disease is believed to be due to abnormal NMDA receptor activity, involving both synaptic and extrasynaptic NMDARs.12, 13, 14, 15, 16 The neuronal loss occurs because of massive activation of NMDARs leading to excessive rise in cytoplasmic Ca2+.17 Fyn kinase acts as a point of convergence for two important signaling pathways: 1. Aβ signaling reaches Tau via Fyn. 2. It is a central hub for NMDAR signaling and phosphorylates its GluN2B subunit.18, 19, 20 But targeting of Fyn kinase to the postsynaptic density protein (PSD), a larger multiprotein complex assembly (1.5MDa) beneath the postsynaptic membrane of excitatory synapses is mediated through the Tau interaction.8, 10, 21, 22 Furthermore, Tau knockout mice showed a marked reduction in the localization of Fyn to the post-synapse, GluN2B phosphorylation, and the interaction of postsynaptic NMDAR with PSD-95. Since Tau interaction with Fyn is mediated through PXXP motifs, studying the effect of PXXP motif with/without AD-specific phosphorylation could provide an in-depth understanding of NMDAR regulation.

Fyn–Tau interaction is mediated through the SH3 (Src homology 3) domain in Fyn and proline-rich Pro-X-X-Pro motifs (PXXP; “X” – denotes non-specific amino acid residue) in Tau protein, respectively.3, 4, 8 SH3 domain is a conserved protein module composed of approximately 60 residues in several proteins. It mediates protein–protein interactions by binding to PXXP motifs in the target proteins.23, 24, 25, 26, 27 Tau consists of seven PXXP motifs localized in a proline-rich domain (PRD; 150–240 residues). Six PXXP motifs exist in partially overlapping tandem sequences (P176-P179-P182, P200-P203-P206, and P213-P216-P219), and the seventh exists as a separate motif at P233-P236.28 Fyn is a multidomain protein comprised of SH2, SH3, and kinase domains, and it binds Tau through one or more PXXP motifs (Figure 1A).

Tau’s physiological role in synaptic and pathological function is regulated by the phosphorylation/hyperphosphorylation.29, 30 Among Tau isoforms, PRD and C-terminus tail regions are highly conserved and known to undergo extensive hyperphosphorylation under AD-specific conditions. Among 85 identified potential Tau phosphorylation sites, about ∼ 50% of sites are primarily residing in the PRD alone. More specifically, the regions encompassing Tau PXXP motifs are AD-specific hyperphosphorylation ‘hotspots,’ and they might regulate its binding to the Fyn-SH3 domain. Here, we investigated the consequence of AD-specific Tau phosphorylation in Fyn-SH3 domain interactions.

Fyn-SH3 domain interaction with Tau is extensively studied for its role in AD pathology and NMDAR signalling.8, 10, 21, 30 Nevertheless, a structural understanding of how the Fyn-SH3 domain interacts with the Tau PXXP motifs at a molecular level is not available. Likewise, hyperphosphorylation of Tau is considered a key pathological feature and a primary driver behind fibrillar deposits in several tauopathies, including AD.29 So far studies have shown how hyperphosphorylation at the Tau microtubule-binding domain reduces binding affinity for its primary ligand tubulin.31 However, the consequence of AD-specific phosphorylation encompassing PXXP motifs’ interaction with the SH3 domain needs to be studied. Here we combined structural and biophysical approaches to determine Fyn-SH3 domain -Tau (207–221) peptide consisting of 5th and 6th tandem PXXP motifs and studied the effect of AD-specific phosphorylation in the above interaction. Subsequently, we investigated whether the above Tau peptide has any role in remodeling NMDAR current using whole-cell patch clamp electrophysiology.