Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation (2022)
Figure 6

Figure 6. Tau limits NSF-dependent associative learning and mediates pathologic inhibition of NSF in tau-transgenic mouse models and in AD brain lysates

(A) Experimental schematic of contextual fear conditioning (CFC) paradigm. Tau+/+ or tau-/- mice were stereotaxically (STX) injected with AAV.syn-EGFP or co-injected with AAV.syn-EGFP and AAV.syn-hTau (= tau-/-.AAVtau) and implanted with bilateral hippocampal cannulas. TAT-NSF or scramble control peptide (TAT-Scr) was bilaterally infused after cued fear conditioning. Memory was tested 24 hours later.

(B) Examples of sagittal sections of tau-/- and AAV.syn-hTau-injected tau-/- (tau-/-.AAVtau) mice showing localized AAV-mediated expression of eGFP or tau and position of infusion cannulas ending in the hippocampus (dashed line). GluA1, immunolabelling for GluA1 AMPA receptor subunit. Scale bar, 50 μm.

(C) Cued fear conditioning memory in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr. Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included. Note that infusion of TAT-NSF lowers memory performance to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. (n = 2-5 biological replicates)

(D) Novel object preference (fold preference over familiar object) in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr (n = 2-5 biological replicates). Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included (n = 2 for GA). Note that infusion of TAT-NSF lowers object discrimination to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice.

(E) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from cortical lysates of indicated genotypes, including from tau-transgenic mice (ALZ17, Tau58/2) (n = 5-8 biological replicates). IgG, control immunoprecipitate with non-specific IgG from tau+/+ lysate.

(F) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from brain lysates of healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer's disease (AD) (n = 3-4 biological replicates). IgG, control immunoprecipitate.

(G) Graphical schematic summarising BioID labelling and mechanism of tau interaction with NSF. Biotinylation proximity proteomics identified synaptic vesicle associated proteins within the tau protein interactome, including NSF ATPase. The presence of tau in dendrites inhibits NSF-dependent regulation of AMPARs during LTP, limiting performance of mice in CFC trials. In AD models, amyloid-β (Aβ) enhances inhibition of NSF primarily through tau.

Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.

Extended View Figures and Figure Legends

Extended View Figure EV1. Proximity labelled tau interactomes in adult mouse brain link cytoskeletal, synaptic, and synaptic vesicle-associated proteins. Related to Figure 3.

Experimental schematic of neuronal interactome labelling in vivo in adult tau-/- (6 months).

Immunoblot of hippocampal lysates from tau-/- mice expressing BioID2-tau or BioID2 control (n = 3 biological replicates) probed for myc-tagged BioID2 and BioID2-tau. Controls: lysates from (1) BioID2- or (2) BioID2-tau-expressing and (3) non-transduced mouse brain. Loading, glyceraldehyde-3-phosphate dehydrogenase (Gapdh). *, unspecific band. Relative quantification confirmed similar expression levels of BioID2 and BioID2-tau. Means ± S.E.M. ns, not significant (Student's t-test).

Tau interactome in adult tau-/- mouse brain transduced to express BioID2-tau or BioID2 control (n = 6 biological replicates). Venn diagram shows overlapping and unique interacting proteins identified by biotinylation. Protein ontology (PO) enrichment (false discovery rate (FDR)) for highest ranking categories in 1 biological processes, 2 cell component, 3 molecular function, 4 KEGG pathway, 5 Reactome pathway, 6 PFAM protein domain are shown. Note most enriched functional annotation clusters: 2 cell projection and 4 endocytosis.

Proximity labelled tau interactome network in adult tau-/- mouse brain (n = 6 biological replicates). Known functional associations and novel BioID2 identified interactions are integrated into network of significantly more abundant and exclusive interactors in BioID2-tau. Network properties and enrichment scores are listed (STRING v11.5). Novel association identified by BioID2 (yellow lines) are partially omitted for clarity.

Functional ontology clusters mapped onto neuronal tau interactome network in (D) for cell component: cell projection and for KEGG: endocytosis. Additional associations with synaptic localization/function and synaptic vesicle regulation described in literature are mapped, respectively.

Extended View Figure EV2. Proximity ligation-identified tau interactors map onto microtubule and actin cytoskeleton and associated processes.

(A, B) BioID2-identified tau interactors mapped onto cytoskeletal complexes and involving (A) microtubule and microtubule associated factors and (B) actin and actin-modulating factors. Additional key process/pathway/complex components are included that were not identified by BioID2 are displayed.

Extended View Figure EV3. Characteristics of tau interaction with and inhibition of synaptic ATPase NSF.

(A) Co-immunoprecipitation (Co-IP) of tau and NSF. V5-tagged wildtype tau (tauWT) or tauT205E variant and FLAG-tagged NSF were expressed in heterologous 293T cells and immunoprecipitated from cell lysates using anti-V5 antibody. Immunoblots were probed with antibodies specific for tau and NSF. GAPDH, input loading control (n = 6 biological replicates). Quantification shows similar levels of NSF bound to tauWT and tauT205E (n = 6).

(B) Co-IP of NSF and tau. V5-tagged wildtype tau and FLAG-tagged NSF were expressed in heterologous 293T cells and immunoprecipitated from cell lysates using anti-NSF antibody. Immunoblots were probed with antibodies specific for tau and NSF. GAPDH, input loading control (n = 4 biological replicates).

(C) Co-incubation of recombinant human tau protein with NSF reduces maximal NSF ATPase rate of activity. See also Figure 4G-H. (n = 3 technical replicates)

(D) NSF affinity for ATP was lowest when co-incubated with soluble - but not heat-denatured - tau, and with TAT-NSF. (n = 3 technical replicates)

(E) Maximal ATPase activity of NSF remained high despite high concentrations of added heat-denatured tau (dTau, 24 nM) or albumin (dAlb, 8nM). Data were fitted with the Michaelis-Menten kinetic model to determine kinetic constants. (n = 2 technical replicates)

(F) Bar graph representation of maximal ATPase activity from E for kcat and Km values of NSF, see Appendix Figure S4. (n = 2 technical replicates)

(G) NSF ATPase activity in presence of full-length tau (16 nM) or indicated tau deletion or truncation variants (16 nM). Phosphate release from NSF activity (70 nM) was assessed alone or in the presence of recombinant tau protein (16 nM) or heat-denatured tau (dTau, 8 nM) or albumin (Alb, 16 nM). The design of recombinant tau variants is represented in Figure 4C. Data were fitted to Michaelis-Menten model to determine rate constants (R2 = 0.842 - 0.933 for all conditions). (n = 3 technical replicates)

(H) Rate constants derived from kinetic data in G. (n = 3) ATP hydrolysis is not significantly lower in NSF incubated with tau truncation and deletion variants, while full-length tau incubation with NSF results in significantly lower ATPase activity. (n = 3 technical replicates)

Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.

Extended View Figure EV4. Regulation of GluA surface and dendritic expression by tau and NSF.

(A-C) Endogenous NSF expression remained stable in tau+/+ and tau-/- cortical neurons across treatment conditions. See Figure 5A-B. (A) Biological replicate immunoblots used to quantify and compare surface GluA1 levels across MK801 (50 μM, 3 min) and/or bicuculline (30 mM, 10 min) treatments. Identical symbols beside immunoblots are from the same experiment. Surface proteins were enriched with streptavidin (SA) beads and resolved on SDS-PAGE for immunodetection of GluA1 levels. Input was loaded for detection of total cellular GluA1, NSF, tau, and βIII-tubulin. Input and SA-enrichment from non-surface biotinylated tau+/+ or tau-/- neurons were included as negative controls. (B-C) Densitometric quantification of NSF and tau levels. (B) No statistically significant difference in endogenous NSF levels between conditions. (C) Endogenous tau expression confirmed in tau+/+ neurons. (n = 3 biological replicates)

(D) Tau expression levels were consistent with reported genotypes and conditions at DIV15. Related to Figure 5C. Tau+/+, tau-/- neurons and tau-/- neurons expressing human tau (tau-/-.AAVtau). Confocal images show immunofluorescent staining for NSF (cyan), tau (magenta), filamentous F-actin (yellow). Scale bar, 5 μm.

(E) Cell surface GluA1 and total NSF immunostaining in primary mouse hippocampal neurons (DIV15) treated with bicuculline (Bic, 30 μM, 10 min). Representative confocal images of tau+/+ and tau-/- neurites are shown, from n = 2 mice per genotype. Cyan indicates NSF and magenta indicates surface GluA1. Scale bar, 3 μm.

(F) Reduced neurite surface GluA1 in Bic-treated tau+/+, yet not tau-/- neurons. (n = 11 neurites from 3 biological replicates per condition).

(G) Lower neurite NSF in tau-/- compared to tau+/+ of Veh controls. Higher levels of NSF in neurites of tau-/- compared to tau+/+ neurons treated with bicuculline. (n = 21 neurites from 3 biological replicates per condition).

(H) Effect of NSF inhibition on dendritic GluA2 localization in cultured neurons upon chemically-induced LTP/LTD. Tau+/+ primary neurons (DIV20) were treated with TAT-Scr control or NSF-inhibiting peptide TAT-NSF before induction of chemical long-term potentiation (cLTP) or chemical long-term depression (cLTD) models for 30 min. Cells were fixed 120 min post-cLTP/cLTD induction, and immunolabelled for Map2 and surface GluA2. Signal intensity threshold-based binary maps for surface GluA2 and Map2 traces were used to map signals of surface GluA2 to dendritic shaft or proxy to synaptic localization. aCSF, artificial cerebrospinal fluid control solution. Scale bar, 3 μm.

(I) Quantification of dendritic GluA2 signals relative to binary Map2 signal map in primary neurons upon inhibition of NSF and induction of cLTP or cLTD. (n = 10-12 cells from 2 experiments)

(J-K) Effect of NSF inhibition on signal overlap of somatic NSF and GluA2 localization in cultured neurons upon chemically-induced LTP/LTD. Tau+/+ primary neurons (DIV20) were treated with TAT-Scr control or NSF-inhibiting peptide TAT-NSF before induction of chemical long-term potentiation (cLTP) or chemical long-term depression (cLTD) models for X min. Cells were fixed 120 min post-cLTP/cLTD induction, and immunolabelled for NSF and GluA2. Somatic signals for NSF and GluA2 showed partial overlap in TAT-Scr-treated cells in aCSF and cLTP conditions. Scale bar, 10 μm. (K) Co-distribution (R-coloc.) of NSF and GluA2 signals in J. (n = 3 biological replicates)

Data information: Values are means ± S.E.M. Adjusted p-values: ****p < 0.0001, **p< 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.

Extended View Figure EV5. PSD-95-GluA1/2 complexes in object recognition and inhibition of NSF ATPase activity by pathologic tau from mice and from human MCI and AD tissue.

(A) Experimental schematic of novel object recognition tests. Tau+/+ or tau-/- mice were stereotaxically (STX) injected with AAV.syn-EGFP or co-injected with AAV.syn-EGFP and AAV.syn-hTau (= tau-/-.AAVtau) and implanted with bilateral hippocampal cannulas. TAT-NSF or scramble control peptide (TAT-Scr) was bilaterally infused after cued fear conditioning. Recognition memory was tested 6 hours later by exposure to a familiar and a novel object in the same test environment.

(B) Co-immunoprecipitation of GluA1 and GluA2 AMPAR subunits with PSD-95 from hippocampus of mice with indicated genotype and peptide/AAV treatment after object recognition paradigm. Immunoblots of co-immunoprecipitates of hippocampal lysates of tau+/+, tau-/-, or tau-/- mice upon AAV injection (+ tau), cannula implantation and object recognition testing. Immunoblots were probed for tau, NSF, PSD-95, GluA1, and GluA2. Gapdh, loading control. (n = 1).

(C) Densitometric quantification of synaptic GluA1 and GluA2 in co-precipitates with PSD-95 from B.

(D) Immunoblots of ATPase assay of recombinant NSF upon incubation with tau immunoprecipitated (tau5) from cortical lysates of indicated genotypes, including from tau+/+, tau-/-, P301S tau transgenic TAU58/2, wildtype tau transgenic ALZ17, APP-transgenic mice (APP23), APP knockout (App-/-), and APP23 transgenic mice on tau knockout background (APP23.tau-/-). Positive control for immunoblot ((+)Control) is recombinant tau and NSF.

(E) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau-5) from cortical lysates of indicated genotypes, including from tau-transgenic mice (ALZ17, Tau58/2) (n = 4-6 biological replicates). IgG, control immunoprecipitate with non-specific IgG from tau+/+ lysate.

(F) Immunoblot of Immunoprecipitated Aβ (D54D2) from cortical lysates from APP-transgenic mice (APP23) and non-transgenic control (non-tg) was probed for Aβ (6E10). †, monomeric Aβ; ‡†, tri-/tetrameric Aβ.

(G) ATPase assay of recombinant NSF upon incubation with immunoprecipitated Aβ (D54D2) from cortical lysates from APP-transgenic mice (APP23) and non-transgenic control (non-tg) (n = 4-7 biological replicates). IgG, control immunoprecipitate with non-specific IgG from non-transgenic lysate. (z)

(H) ATPase assay of recombinant NSF upon incubation with oligomerised synthetic Aβ (n = 3 technical replicates). NSF (70 nM) ATPase activity in presence of albumin (16 nM) or oligomerized synthetic Aβ1-42 (25 or 50 nM) measured at indicated ATP concentrations. Data were fitted to Michaelis-Menten model to determine rate constants (R2 = 0.886 - 0.972 for all conditions).

(I) Rate constants derived from ATPase activity in G. (n = 3 technical replicates) ATP hydrolysis is not significantly lower in NSF incubated with oligmerized synthetic Aβ1-42.

Data information: Values are means ± S.E.M. Adjusted p-values: ***p < 0.001, ns, not significant, ANOVA with Tukey's test.