Abrogating tau binding to heat shock proteins alleviates pathology in a PL-SF tau aggregation model.

A) Representative immunoblots (RIPA fraction) of QBI-293 cells transfected with 2N4R WT, P301L, S320F, or P301L-S320F tau. B) Immunoblot quantification of total tau, p-tau (AT8 and AT100), and cleaved tau (TauC3) in RIPA fraction. N=4 per condition; C) Immunocytochemistry quantification of QBI-293 cells transduced with WT, P301L, S320F, or PL-SF tau labeled with AT8, TauC3, or ThioS. N=48 FOVs per condition. *= p-value significant by one-way ANOVA. Representative images found in Supplemental Figure 1. D) Representative immunoblots (RIPA fraction) of QBI-293 cells transfected with 2N4R P301L-S320F tau or P301L-S320F 4Δ (deletion of I277/278, I308/V309 residues). *AT100 was run on a separate blot with the exact same lysates to improve detection. E) Immunoblot quantification of total tau, p-tau (AT8 and AT100), and cleaved tau (TauC3) in RIPA soluble fractions. N=3 per condition. F) Immunocytochemistry quantification of QBI-293 cells transduced with 2N4R PL-SF or PL-SF 4Δ tau labeled with AT8, TauC3, or ThioS. N=16 FOVs per condition. *= p-value significant by unpaired Student’s t-test. Representative images found in Supplemental Figure 3. G) Co-immunoprecipitation of QBI-293 cells transfected with WT, P301L, PL-SF, or PL-SF 4Δ tau. Tau5 antibody was used to IP tau and immunoblots were probed with total tau (rabbit polyclonal) and Hsp70, Hsc70, and Hsp90 antibodies. H) Representative immunoblots of QBI-293 cells transfected with 2N4R tau variants and probed with acetyl-tubulin and total alpha-tubulin antibodies. Note: Acetyl tubulin and alpha tubulin blots were run separately with the same lysates to improve detection. I) Quantification of acetyl-tubulin across all 2N4R tau variants. -- line signifies separate sets of transfections in panel I and J. N=4 per condition; *= p-value significant by one-way ANOVA. Control= empty plasmid vector (pcDNA3.1). J) Quantification of in vitro Thioflavin T aggregation assay using purified protein from PLSF and PLSF 4Δ. N=4 replicates per condition. *= significant by unpaired student t-test at 12hr timepoint. Coomassie (right) shows equal protein loading into reactions.

Development and characterization of a PL-SF tau aggregation model in primary cortical neurons.

A) Representative RIPA-soluble immunoblots of DIV10 primary cortical neurons transduced with lentiviruses expressing 2N4R WT, P301L, S320F, P301L-S320F, or PL-SF 2Δs (R2 and R3) and 4Δ tau variants. B) Quantification of AT8, AT100, AT180, and total tau immunoblots in primary cortical neurons (RIPA fraction). N=3 per condition; *= p-value significant by one-way ANOVA. C) Representative SDS-soluble immunoblots of DIV10 primary cortical neurons transduced with lentiviruses expressing 2N4R WT, P301L, S320F, P301L-S320F, or PL-SF 2Δ and 4Δ tau variants. N=2 for control, WT, and P301L; N=3 for other variants. D) Representative immunocytochemistry images of 2N4R tau variants labeled with AT8 (p-tau), MC1 (conformational tau), ThioS (aggregated tau) in GFP. Scale bar= 125 µm. E) Quantification of the number of AT8, MC1, and ThioS inclusions in PL-SF and PL-SF 2Δ (R2 and R3) and 4Δ variants. N=12-24 FOVs per condition. *= significant by one-way ANOVA. F, G) Representative immunocytochemistry images of PL-SF tau displaying colocalization between F) AT8 (p-tau) or G) MC1 (conformational tau) with ThioS (aggregated tau). Scale bar= 50um. H) Representative immunocytochemistry of PL-SF GFP colocalized with T49 tau (mouse-specific). Scale bar= 125 µm. Control= empty viral vector (pUltra).

Hsp70 exacerbates tau hyperphosphorylation and aggregation.

A) Representative immunoblots of 2N4R PL-SF tau co-transduced with HSP70, HSC70, and HSP90 in DIV10 primary cortical neurons (RIPA fraction). B) Quantification of AT8 (p-tau), total tau (rabbit polyclonal), or Hsp70, Hsc70, Hsp90 (FLAG-tagged) in the RIPA fraction. N=6 per condition. *= p-value significant by one-way ANOVA. C) Representative immunoblots of 2N4R PL-SF tau co-transduced with HSP70, HSC70, and HSP90 in DIV10 primary cortical neurons (SDS fraction). D) Quantification of AT8 (p-tau), total tau (rabbit polyclonal) in the SDS fraction. N=6 per condition. ns= p-value is not significant by one-way ANOVA. E) Representative immunocytochemistry images of primary cortical neurons co-transduced 2N4R PL-SF tau and HSP70, HSC70, or HSP90 labeled with AT8, MC1, Tau12 (RFP) or ThioS (GFP). Scale bar= 125 µm. F) Quantification of MC1, AT8, and ThioS positive inclusions in primary cortical neurons co-transduced 2N4R PL-SF tau and either HSP70, HSC70, or HSP90. n=12 FOVs per condition. *= p-value significant by one-way ANOVA. Control= empty viral vector (pUltra).

Reducing Hsp70 can suppress tau hyper-phosphorylation and aggregation.

A) Representative immunoblots of 2N4R PL-SF tau co-transduced with a shRNA against HSP70 in DIV10 primary cortical neurons (RIPA fraction). B) Quantification of AT8 (p-tau), total tau (rabbit polyclonal) and endogenous Hsp70 and Hsc70 in the RIPA fraction. N=3. C) Representative immunoblots of 2N4R PL-SF tau co-transduced a shRNA against HSP70 in DIV10 primary cortical neurons (SDS fraction). D) Quantification of AT8 (p-tau), total tau (rabbit polyclonal) in the SDS fraction. N=3. *= p-value significant by unpaired Student’s t-test. Control= empty viral vector (pUltra).

Mature tau aggregates cause functional deficits in neurons that are reversed by abrogating tau-Hsp binding.

A-C) Quantification of neuronal activity comparing PL-SF to PL-SF 4Δ across 53 days of multi-electrode array recording starting at DIV18 following initial pre-recording. A) Mean firing rate-activity B) Number of network bursts-connectivity C) Area under normalized cross-correlation-synchrony. N=4 replicate wells per condition. *= p-value significant by one-way ANOVA. D-F) Representative raster and network burst plots at DIV54 (day 36 of recording) comparing empty control (pUltra) to PL-SF and PL-SF 4Δ. D) Number of network bursts across electrodes over time (pink lines) E) number of spikes across electrodes over time F) Average number of spikes per network burst over time. G) Representative immunoblot of isolated post synaptic density (PSD) in DIV18 primary cortical neurons transduced with lentiviruses expressing P301L, PL-SF, or PL-SF 4Δ. Immunoblot was probed with PSD95, AT8 (p-tau), and rabbit polyclonal tau (total tau). H) Representative 20X immunocytochemistry images of primary cortical neurons comparing empty vector control (pUltra) to PL-SF and PL-SF 4Δ tau variants at DIV29 and DIV62. Neurons were labeled with ThioS (aggregated tau), MC1 (conformational tau), AT8 (p-tau), and Tau12 (total tau) in GFP. Tau12 was co-labeled with GFAP (RFP) to label astrocytes. Scale bar= 125 µm. I) Representative immunocytochemistry images of primary cortical neurons comparing PL-SF and PL-SF 4Δ at DIV29. Neurons were labeled with endogenous Hsp70 (GFP), MC1 or AT8 (RFP), and DAPI (BFP). Scale bar= 50 µm. Control= empty viral vector (pUltra).

Abrogating tau-Hsp binding suppresses tau pathology in three-dimensional organotypic hippocampal slice cultures.

Rat hippocampal slices were processed for immunocytochemistry after 9 weeks in culture following lentiviral infection of eGFP empty vector control, wild-type tau, PL-SF tau, and PL-SF 4Δ. A, B) Immunocytochemistry of the entire hippocampal slice following labeling of reveal sufficient expression of PL-SF and PL-SF 4Δ lentiviruses (GFP). Left-AT8 staining; Right-MC1 staining. CA1, CA3, and DG regions are labeled in white font. White boxes denote magnified images in panel C, D. Scale bar= 500 µm. C, D) Representative immunocytochemistry images of C) AT8 and D) MC1 labeled in RFP for empty vector control, wild-type tau, PL-SF tau, and PL-SF 4Δ taken from the dentate gyrus (DG) region of the hippocampal slice culture (inset white boxes from A, B). Scale bar= 100 µm.

HSP70 is upregulated in human AD brain and intercalates within tau aggregates.

A) Hi-Salt soluble immunoblots from control (n=5) and AD (n=6) cortical lysates probed for Hsp70, Hsc70, and GAPDH. B) Quantification of hi-salt immunoblots. *= p-value significant by unpaired Student’s t-test. C) Urea soluble immunoblots from control (n=4) and AD (n=5) probed for Hsp70 and Hsc70. D) Quantification of urea immunoblots. *= p-value significant by unpaired Student’s t-test. Overexposed Hsp70 AD sample in urea was excluded from quantification. E) Representative 63X confocal single-plane z-stacks from AD human hippocampus stained with AT8 (p-tau) and Hsp70. White arrow label neuritic plaques and white arrowheads label NFTs.

Primers used for vector construction

Accelerated PL-SF aggregation compared to single tau mutants in immortalized cell lines.

A) Representative immunocytochemistry images from QBI-293 cells expressing empty vector (pcDNA) or WT, PL, SF, and PL-SF tau variants. Cells were labeled with AT8 (p-tau) or TauC3 (C-terminal cleaved tau) with rabbit polyclonal tau (total tau) and ThioS with Tau12 (human tau). Quantification of images is found in Figure 1C. Control= empty plasmid vector (pcDNA3.1). Scale bar= 125 µm.

Immunoblotting analysis of a panel of P301 and S320 single and combination mutants.

A-G) Quantification of AT8, MC1, TauC3, and ThioS immunocytochemistry in QBI-293 cells expressing various P301 and S320 mutants alone or in combination. Panels E-G are subsets of data from A-C to perform additional comparisons among combinatorial mutants. Representative images not shown. Error bars= SEM. *= significant difference by one-way ANOVA. H) Representative immunoblots from QBI-293 cells expressing wild-type, P301S/S320F, P301T/S320F, and P301L/S320F tau variants. RIPA (soluble) and urea (insoluble) fractions were analyzed by immunoblotting for p-tau epitopes (AT8, AT180, AT270, p-396) and total tau (rabbit polyclonal). I, J) Quantification of RIPA soluble and urea (insoluble) fractions. Error bars= SEM. *= significant difference by one-way ANOVA.

Abrogating Hsp binding via the PL-SF 4Δ variant suppresses the accumulation of pathological tau.

Representative immunocytochemistry images from QBI-293 cells expressing tau PL-SF and PL-SF 4Δ tau variants. Cells were probed with AT8 (p-tau), TauC3 (C-terminal cleaved tau) with rabbit polyclonal tau (total tau), and ThioS with Tau12 (human tau). Quantification of images is found in Figure 1F. Control= empty plasmid vector (pcDNA3.1). Scale bar= 125 µm.

PLSF containing the β-structure breaking mutations, I277P/I308P, prevents tau aggregation similar to PLSF 4Δ.

A) Representative RIPA immunoblot of primary neurons transduced with empty control, PLSF 4Δ-eGFP, PLSF I277P/I308P (2IP), or PLSF-eGFP and probed for p-tau (AT8) and total tau (rabbit polyclonal). B) Quantification of AT8 and total tau in RIPA fraction. C) Representative SDS immunoblot of primary neurons transduced with empty control, PLSF 4Δ-eGFP, PLSF I277P/I308P (2IP), or PLSF-eGFP and probed for p-tau (AT8) and total tau (rabbit polyclonal. D) Quantification of SDS fraction. *= significant difference by one-way ANOVA.

PL-SF aggregates emerge similarly in either wild-type or tau knockout neurons lacking endogenous mouse tau.

A) RIPA (soluble) immunoblot of wild-type tau and tau knockout neurons probed with p-tau (AT8, AT180, AT100) and total tau (rabbit polyclonal). B) SDS (insoluble) immunoblot of wild-type tau and tau knockout neurons probed with p-tau (AT8) and total tau (rabbit polyclonal). C) Representative immunoblotting images of wild-type, P301L, S320F, and P301L/S320F tau variants in wild-tau-expressing or tau knockout neurons. Neurons were labeled with AT8 (GFP) and rabbit polyclonal total tau (RFP), MC1 (GFP), or ThioS (GFP) and Tau12 (RFP). Scale bar= 125 µm. Control= empty viral vector (pUltra).

Overexpression of HSP70 in P301L-expressing neurons does not promote tau phosphorylation or aggregation.

A) RIPA (soluble) immunoblot of primary neurons following 1-week of lentiviral co-transduction with P301L tau and HSP70. Blot was probed with p-tau (AT8), total tau (rabbit polyclonal), and M2 FLAG (Hsp70-FLAG). B) SDS (insoluble) immunoblot of primary neurons was unable to detect insoluble tau species with total tau antibody (rabbit polyclonal). Control= empty viral vector (pUltra).

HSP90 depletion does not alter phosphorylated or aggregated PL-SF tau.

A) RIPA (soluble) immunoblot of primary neurons probed with p-tau (AT8) and Hsp90 following 1-week of lentiviral HSP90 knockdown with two shRNAs (called TRC91 and TRC94). B) SDS (insoluble) immunoblot of primary neurons probed with p-tau (AT8) and total tau (rabbit polyclonal) following 1-week of lentiviral HSP90 knockdown. C) Quantification of AT8 and Hsp90 in the RIPA fraction. Error bars= SEM. D) Quantification of AT8 and total tau (rabbit polyclonal) in SDS fraction. *Note: Only TRC94, and not TRC91, HSP90 shRNA was able to reduce Hsp90 expression.

Hsp70 foci accumulation within neuritic plaques and neurofibrillary tangles in human AD brain

A) Representative stitched widefield images of a human AD patient hippocampus labeled with AT8 (GFP) and Hsp70 (RFP). AT8/Hsp70 positive plaques are labeled with white arrowheads. AT8/Hsp70 positive tangles are labeled with white arrows. * = a single AT8 positive tangle that is absent for Hsp70 foci.