Fungally Derived Isoquinoline Demonstrates Inducer-Specific Tau Aggregation Inhibition

Abstract

The microtubule-associated protein tau promotes the stabilization of the axonal cytoskeleton in neurons. In several neurodegenerative diseases, such as Alzheimer's disease, tau has been found to dissociate from microtubules, leading to the formation of pathological aggregates that display an amyloid fibril-like structure. Recent structural studies have shown that the tau filaments isolated from different neurodegenerative disorders have structurally distinct fibril cores that are specific to the disease. These "strains" of tau fibrils appear to propagate between neurons in a prion-like fashion that maintains their initial template structure. In addition, the strains isolated from diseased tissue appear to have structures that are different from those made by the most commonly used in vitro modeling inducer molecule, heparin. The structural differences among strains in different diseases and in vitro-induced tau fibrils may contribute to recent failures in clinical trials of compounds designed to target tau pathology. This study identifies an isoquinoline compound (ANTC-15) isolated from the fungus Aspergillus nidulans that can both inhibit filaments induced by arachidonic acid (ARA) and disassemble preformed ARA fibrils. When compared to a tau aggregation inhibitor currently in clinical trials (LMTX, LMTM, or TRx0237), ANTC-15 and LMTX were found to have opposing inducer-specific activities against ARA and heparin in vitro-induced tau filaments. These findings may help explain the disappointing results in translating potent preclinical inhibitor candidates to successful clinical treatments.

Figure 1

(A) Chemical structure of the isoquinoline ANTC-15 (7-methyl-3-nonylisoquinoline-6,8-diol). (B) Chemical structure of the phenothiazine LMTX (TRx0237, leuco-methylthionine mesylate salt).

Figure 2

Initial inhibition assay of 200 μM ANTC-15 and LMTX against ARA-induced tau filaments. Sandwich ELISA using the TOC1 capture antibody and TNT1 capture antibody (A and B, respectively) normalized against the 1% DMSO no-compound control (no compound = 100). Samples were compared to the no-compound control using a Tukey’s multiple-comparison test (p values of all comparisons can be found in Table S1): *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. ns denotes no significant difference. Representative transmission electron micrographs at 5000× magnification of (C) the no-compound control, (D) the no-ARA monomer control, (E–H) ANTC-15, and (I–L) LMTX at concentrations of 200, 100, 25, and 3 μM (from left to right, respectively). The scale bar in panel L represents 500 nm for all images.

Figure 3

(A and B) Initial inhibition assay of 200 μM compound of ANTC-15 and LMTX against heparin (Hep)-induced tau filaments. Sandwich ELISA using the TOC1 capture antibody and TNT1 capture antibody (A and B, respectively) normalized against the 1% DMSO no-compound control (no compound = 100). Samples were compared to no-compound controls using a Tukey’s multiple-comparison test (p values of all comparisons can be found in Table S1): *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. ns denotes no significant difference. Representative transmission electron micrographs at 5000× magnification of (C) the no-compound control, (D) the no-heparin monomer control, (E–H) ANTC-15, and (I–L) LMTX at concentrations of 200, 100, 25, and 3 μM (from left to right, respectively). The scale bar in panel L represents 500 nm for all images.

Figure 4

Disassembly assay of 200 μM ANTC-15 and LMTX against (A and B) ARA-induced tau filaments and (C and D) heparin-induced tau filaments. Sandwich ELISA using the TOC1 capture antibody (left) and TNT1 capture antibody (right) normalized against the 1% DMSO no-compound control (no compound = 100). Samples were compared to the no-compound control using a Tukey’s multiple-comparison test (p values of all comparisons can be found in Table S2): *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. ns denotes no significant difference.

Figure 5

ANTC-15 dose dependence: (A, C, and E) inhibition of ARA filaments and (B, D, and F) disassembly of ARA preformed filaments (PFF). Inhibition and disassembly of both TOC1 (○) and TNT (●) reactive species as shown by a sandwich ELISA at different concentrations of ANTC-15 (A and B). Inhibition and disassembly of the average number of filaments as determined by TEM at different concentrations of ANTC-15 (C and D). Inhibition and disassembly of total filament length as determined by TEM at different concentrations of ANTC-15 (E and F).

Figure 6

Tubulin polymerization assay. Tubulin (Tb) at a final concentration of 2 mg/mL was incubated at 37 °C with or without an inhibitor compound at a concentration of 40 μM in the presence of 0.5 μM tau. Data were then normalized against a Taxol (10 μM) positive control and fit to a Finke–Watzky polymerization curve.