Fig 2: Initial screen identified parental MAPT-targeting sequence ASO-000013(A and B) In vitro ASO-tiling and toxicity screen using hTau mouse primary neuronal cultures. Quantitative analysis of Tau protein levels after ASO treatments. ASO-targeting sites along MAPT transcript are shown on the x axis (A). Calcium oscillations (CaOsc) and ß-??? tubulin levels were evaluated for all ASOs used in tiling experiment (B). Each spot represents an ASO. Forty-seven ASOs (shown in green) showed low in vitro toxicity (tubulin >70% UTC and CaOsc >75% UTC) and good target knockdown efficiency (>80% UTC, B). (C) Thirteen out of 47 ASOs were selected for further evaluation using hTau mice. Assessment of in vivo acute tolerability and relative MAPT expression for each selected ASO. Each spot represents an ASO. ASO (shown in green) was selected as a parental MAPT-targeting sequence ASO-000013. (D) Alternative splicing of the MAPT gene generates six tau isoforms, named after the number of N-terminal repeats (0 N, 1 N, and 2 N) and C-terminal microtubule-binding domain repeats (3R and 4R). The first two exons (-1, 1) encode the 5' untranslated region (UTR) and exon 14 encodes the 3' UTR. The exons 4a, 6, and 8 are only part of the mature tau transcript in the peripheral nervous system. The target RNA sequence of ASO-000013 in exon 14 of tau mRNA is highlighted in green. The surrounding sequence in human, Cyno monkey, mouse, and rat show high conservation across different species. Nucleotides in red differ from the human sequence.

Fig 3: Inhibition of calpain-2 or c-Abl attenuates the formation of tau oligomers in brain following TBI. (A) IHC with an anti-tau oligomer antibody (T22) in cortical and hippocampal CA1 areas of coronal brain sections 24 h after TBI or sham surgery. Vehicle (5% DMSO in PBS), NA101 (0.3 mg/kg) or Nilotinib (25 mg/kg) was injected i.p. 1 h after TBI. Scale bar = 100 µm. (B) Quantification of IHC results. Three coronal brain sections (Bregma −1.58 mm, −1.94 mm and −2.30 mm) from each brain were imaged. MFIs in cortical and CA1 areas from each section were measured in ImageJ. N = 3 for Sham, C2I and Nilotinib. N = 5 for Vehicle. **p < 0.01, ****p < 0.0001 Sham vs. Vehicle, #p < 0.05 C2I vs. Vehicle, ##p < 0.01 Nilotinib vs. Vehicle. Two-way ANOVA followed by Bonferroni test. (C) ELISA analysis of brain homogenates using Tau-5 and T22 antibodies 24 h after TBI or sham surgery. Vehicle (5% DMSO in PBS), NA101 (0.3 mg/kg) or Nilotinib (25 mg/kg) was injected i.p. 1 h after TBI. The ratios of tau oligomers to total tau were analyzed. N = 4 (animals). ***p < 0.001 sham vs. vehicle, #p < 0.05 vehicle vs. C2I. ##p < 0.01 vehicle vs. Nilotinib. One-way ANOVA followed by Bonferroni test. (D) Levels of Tau monomers and oligomers and Serine/Threonine phosphorylation of tau at S202, T205, T231 and S416 in ipsilateral cortical homogenates collected 24 h after sham (lane 1) or TBI (lane 2) or TBI plus NA101 injection (lane 3) were analyzed with WB of non-reducing SDS-PAGE (NuPAGE 4–12% Bis-Tris gel). Full-length blots are presented in Supplementary Figure 2. (E) Quantification of tau oligomers (boxed area) as detected by WB. N = 3 (animals). ****p < 0.0001 sham vs. TBI. ###p < 0.001 TBI vs. TBI + NA101. Two-way ANOVA followed by Bonferroni test. (F) Post-mortem hippocampal samples from 4 AD patients and 4 age-matched controls (N) were probed with antibodies against PTPN13 and actin in WB. Full-length PTPN13 at 275 kDa and a P13BP at approximately 210 kDa were detected. (G) Quantification of the Western blots. The ratio of P13BP to full-length PTPN13 was significantly higher in AD patients than that in controls. N = 4. *p < 0.05. Two-tailed t-test.

Fig 4: Changes in levels and membrane distribution of key autophagosome and endosome-lysosome fusion proteins in mice overexpressing SREBF2 are prevented by in vivo treatment with 2-hydroxypropyl-ß-cyclodextrin resulting in improved MAPT and Aß clearance. (A) Western blot analysis of the indicated proteins in endosomes-lysosomes isolated from 7-mo-old WT and transgenic mice. (B) Immunoblot analysis of VAMP7 and VTI1B distribution into soluble fractions and DRMs isolated from endosomes-lysosomes of 7-mo-old WT and the indicated transgenic mice. Immunoblots with FLOT1 identified DRMs mainly in fractions 1 and 2. Int-I and Int-II: intermediate fraction I and II, Sol.: soluble fraction. (C) Expression levels of ATG14 and STX17 in autophagosomes isolated from 7-mo-old WT and the indicated mutant mice. To induce autophagy WT and SREBF2 mice were treated with rapamycin (RM, 5 mg/kg) for 24 h. LC3B protein levels were analyzed as a marker of autophagosomes. ATG14 and STX17 were normalized to the corresponding LC3B (I and II) bands. (D to H) Mice were treatment with HP-ß-CD (4 g/kg) for 10 wk. To induce autophagy WT and SREBF2 mice were treated with rapamycin (RM, 5 mg/kg) 24 h prior sacrifice. (D) Expression levels of RAB7A and the indicated SNARE proteins in endosomes-lysosomes. LAMP1 was used as a marker of lysosomes. Densitometric values of the bands representing the specific protein immunoreactivity were normalized with the values of the corresponding LAMP1 bands. (E) Immunoblot analysis of VAMP7 and VTI1B distribution into soluble fractions and DRMs isolated from endosomes-lysosomes (EL). Immunoblots with FLOT1 identified DRMs mainly in fractions 1 and 2. Int-I and Int-II: intermediate fractions I and II, Sol.: soluble fraction. (F) Expression levels of ATG14 and STX17 in autophagosomes. LC3B was used as a marker of autophagosomes. ATG14 and STX17 were normalized to the corresponding LC3B (I and II) bands. (G) Expression levels of endogenous MAPT in autophagosomes (AP) and endosomes-lysosomes (EL). LC3B and CTSD (intermediate, 45 kDa; and mature form, 34 kDa) levels were used as autophagosome and lysosome markers, respectively. MAPT values were normalized to the corresponding LC3B (I and II) and CTSD (mature form) bands. (H) Representative immunoblot showing that HP-ß-CD treatment prevents the accumulation of Aß in autophagosomes isolated from APP-PSEN1-SREBF2 mice. In all western blots, densitometric values of the bands representing specific protein immunoreactivity were first normalized to Ponceau S staining to adjust for protein loading. *P< 0.05 and **P< 0.01; n=3. See Figure S18 for uncropped blots.

Fig 5: Raising tau isoform-specific antibodies.(A) Schematic representation of the exon structure of the MAPT locus that encodes murine tau. Alternative splicing of exons 2, 3, and 10 generates the three isoforms 0N4R, 1N4R, and 2N4R that are present in the adult murine brain. The scheme shows the location of the epitopes that were used to raise specific antibodies for 0N, 1N and 2N murine tau, as well as for total murine tau (M), without cross-reactivity with human tau. (B) Western blot analysis of RAB-soluble tau extracts obtained from brains of 2-months old wild-type (WT) mice, with stripes probed separately with Tau5, M (total mouse tau) and the murine tau isoform-specific antibodies 01, 2N, and 2N reveals their specificity. Tau knock-out (KO) tissue was included as negative control.