Fig 2: Synaptic localization of tau in L66 using immunofluorescence. Tau immunoreactivity with the phosphorylation-independent antibody HT7 is localized in the neuronal soma (arrows), dendrites, and axons of neurons in hippocampal CA1 and CA3, cortex and hilus of the dentate gyrus. Co-localization of HT7-reactive tau and the synaptic marker synapsin-2, seen as yellow puncta in the merged images (double arrowhead), confirms the synaptic accumulation of transgenic tau. Synaptic staining of tau is evident mainly in striatum and, to a lesser extent, in cortex and stratum radiatum of the CA1. Green, tau; red, synapsin-2. Arrow, staining in the neuronal soma; double arrowhead, synaptic staining. Scale bars, 25 μm.

Fig 3: Quantification of synaptic markers and tau after subcellular fractionation in L66 mice. Densitometric quantification of synapsin-1 (A), PSD95 (B), Tau-46 reactive mouse tau at 50-55 kDa (C), as well as transgenic human tau at 62 kDa reactive with 7/51 (D), HT7 (E), and Tau-46 (F) was conducted with Image Laboratory using stain-free total protein loading for normalization. Data are expressed as mean ± S.E.

Fig 4: Tau immunohistochemistry in L66 and WT control mice. Tau immunoreactivity with phosphorylation-independent antibodies 7/51 (A), Tau46 (B), HT7 (C), and 27/499 (D), and with phosphorylation-specific antibodies pSer-202 (E), pSer-396 (F), and pSer-416 (G) show prominent intraneuronal staining in cortex and hippocampus in L66 brains. Phosphorylation-independent antibodies showed strong synaptic staining mainly in striatum in L66 (A–D). Phosphorylation-specific antibodies showed strong staining in cell bodies and axons in L66 (E–G). Black arrowhead, cytosolic staining in neurons; white arrowhead, axonal/dendritic staining; circle, synaptic staining. CA1, hippocampus subfield CA1; STRI, striatum. Scale bars, 100 μm. L66, line 66 tau transgenic mice.

Fig 5: Phosphorylation of tauC3 Ser416 is sufficient to inhibit interaction with CHIP.(A) Locations of serine and threonine phosphorylation sites identified on p.tauC3 by LC/MS-MS. Residue numbering is derived from 2N4R tau, while identified pS/pT sites are shown in yellow. (B) Cartoon depicting constructs used for mapping of inhibitory phosphorylation sites by NanoBiT live cell assays. (C) NanoBiT live cell assay for CHIP interactions with truncated tau constructs following treatment with okadaic acid (30 nM, 18 hours) or DMSO control. Data is shown as the difference between okadaic acid treated samples and vehicle control. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc analysis (ns = not significant, ***p<0.001, n = 3). (D) DSF curves for CHIP incubated with DMSO control or tauC3 peptides. Assay was performed in quadruplicate and melt curves were fit with a Boltzmann sigmoid. (E) Apparent melting temperatures (Tmapp) of CHIP in the absence or presence of 10-mer tau peptides as derived from (D). Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc analysis (****p<0.0001, n = 4). (F) Competition FP experiment showing displacement of fluorescent tracer from the CHIP TPR domain by various 10-mer tau peptides. Samples were performed in quadruplicate. (G) Inhibition constants for various tau peptides derived from (F). Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc analysis (***p<0.001, ****p<0.0001, n = 4). (H) Tau proteoforms binding to immobilized CHIP, as measured by ELISA. Assay was performed in triplicate and normalized to maximum absorbance @ 450 nM. (I) Dissociation constants derived from (H). Statistical significance was determined by unpaired student’s t-test (***p<0.001, n = 3).