Monoaminergic neurotransmitters are bimodal effectors of tau aggregation

Abstract

Neurotransmitters (NTs) mediate trans-synaptic signaling, and disturbances in their levels are linked to aging and brain disorders. Here, we ascribe an additional function for NTs in mediating intracellular protein aggregation by interaction with cytosolic protein fibrils. Cell-based seeding experiments revealed monoaminergic NTs as inhibitors of tau. Seeding is a disease-relevant mechanism involving catalysis by fibrils, leading to the aggregation of proteins in Alzheimer's disease and other neurodegenerative diseases. Chemotyping small molecules with varied backbone structures revealed determinants of aggregation inhibitors and catalysts. Among those identified were monoaminergic NTs. Dose titrations revealed bimodal effects indicative of fibril disaggregation, with aggregation catalysis occurring at low ratios of NTs and inhibited seeding ensuing at higher concentrations. Bimodal effects by NTs extend from in vitro systems to dopaminergic neurons, suggesting that pharmacotherapies that modify intracellular NT levels could shape the neuronal protein aggregation environment.

Fig. 1.. Tau inhibitors identified through phenol library screening.

(A) Fluorescent micrographs showing representative nonseeded tau biosensor cells without puncta (white arrow), or seeded by transfection with AD brain homogenate, which results in a green punctated appearance (orange arrows). Effects of tau seeding inhibitors and catalysts are shown in representative fluorescent micrographs, as indicated in the image header. (B) Heatmap from the MCE phenol library screening with seeding measured from tau biosensor cell assays. Values are expressed as the number of SDs seeding was for a given phenol above (green) or below (magenta) the mean level of seeding from two datasets that comprise biological replicates. Chemicals with seeding at the mean level are colored white in the heatmap. Note: Labels are only present for molecules on the extreme left and right sides of the heatmap. A complete table of molecules and AvZ values can be found in data S1. (C) Examples of chemicals and structures of seeding inhibitors and catalysts with related backbones identified by phenol screening, shown for herbacetin versus noricaritin, and gambogic acid versus garcinone C. (D) Effect of numbers of ArOHs on seeding. Chemicals with greater numbers of ArOHs exhibit a decreasing trend in AvZ values displaying a negative slope in the line of best fit prepared using ADMET Predictor software. Magenta box shows that a subset of chemicals with as few as one or two ArOHs can strongly inhibit seeding. (E) Aggregation catalysts are actually weak tau inhibitors. Garcinone C, garcinone D, and sciadopitysin increase seeding at 10 μM final concentration on cells, but inhibit seeding at 50 μM, which is consistent with results expected for inhibitors acting weakly as fibril disaggregants.

Fig. 2.. Dose-dependent effects of NT-like backbones from the MCE phenol library series.

(A) Biosensor seeding by AD brain homogenate pretreated with indicated amounts of dl-dopa or dl-m-Tyr. (B to G) Example images of cells. Nonseeded cells (white arrow) have no puncta. Transfecting with AD brain homogenate yields subcellular puncta (orange arrows). (H) Chemical structures of DA and chemically related analogs identified from the MCE phenol series. AvZ scores are listed for each respective phenol from library screening. (I) As in (G), except for analogs of serotonin. (J) Heatmap showing range of AvZ scores from the MCE phenol series for indole and monocyclic type tau inhibitors from the MCE library. Generally, both series, which relate to serotonin and DA, contain a preponderance of seeding inhibitors (pink), although phenols having catalytic effects on seeding (green) are also present.

Fig. 3.. Dose-dependent effects of NTs on tau seeding.

(A) Biosensor seeding by AD brain homogenate pretreated with indicated amounts of indicated NT. (B) SSA for NTs. SSA was conducted using ECFP keys in ADMET Predictor. Atoms depicted with red mesh indicate correlation to negative AvZ values (inhibited seeding). Atoms in green mesh indicate correlation with positive AvZ values (seeding catalysis). Atoms in gray mesh are considered to have negligible effects on seeding. (C) Semiquantitative analysis of key negative and positive keys from SSA of select MCE library compounds. Phenols having a higher fraction of negative keys correlate with more negative experimental AvZ scores. Spearman’s rank correlation was computed to assess correlation between the fraction of negative keys and AvZ. There was a negative correlation between the two variables, r = −0.8213, P < 0.0001.

Fig. 4.. Bimodal effects on seeding are a consequence of NT concentration and tau load.

(A) Seeding assays conducted using AD brain homogenates diluted to simulate differing tau loads. Treating AD brain homogenate containing a “high tau load” with ptau (1.1 ng/μl) shows a bimodal response to added DA with seeding catalyzed at low DA concentrations and dose-dependently inhibited at high DA concentrations (black bars and interpolated dotted line trace). The same treatment with diluted AD brain homogenate, labeled “low tau load,” containing ptau (0.11 ng/μl) eliminated low-dose catalytic effects of DA on seeding (red bars and interpolated trace). (B) IC₅₀s computed from curves shown for low and high tau load samples. (C) Dose effects of related NTs and metabolites using low tau load AD brain homogenate, as in (A). (D) Dose effects of NTs and metabolites from (C) except using high tau load AD brain homogenate.

Fig. 5.. Aromatic hydroxyls mediate fibril disaggregation by dopaminergic molecules.

(A) Results of qEM showing AD tau fibrils remaining after 24-hour treatment with 75 μM dopaminergic molecules. Fibrils were quantified from three sets of images, with N = 32 images per set. Error bars represent SDs from triplicate measures. l-Dopa and DA significantly reduce AD tau fibrils compared to the no inhibitor control, with highly significant P values (P < 0.0001). HVA led to a significant reduction in fibrils, though to a lesser extent, with P = 0.0006. (B to E) Representative EM images of fibrils without versus with treatment. Arrows point to representative tau fibrils. (F) Atomistic contributions toward tau inhibitor activity based on structure sensitivity analysis.

Fig. 6.. Effects of dopaminergic molecules on seeding in differentiated N2a dopaminergic biosensor cells expressing P301S 4R1N tau fused with YFP.

(A to C) Representative cell images showing seeding, and effects by DA and l-dopa. In all conditions with drug treatment, the final concentration was 50 μM on cells. Examples of cells with puncta are marked by orange arrows, and cells without puncta are marked by white arrows. (D) Quantification of puncta in control and seeded cells treated with DA and l-dopa. DA treatment significantly reduces puncta (P < 0.001), while l-dopa treatment also results in a statistically significant reduction (P < 0.01). (E) Dose-dependent effects of pretreatment with DA on tau seeding. Puncta were quantified from two arbitrary images per well, each for three technical replicates. Error bars show SDs.

Fig. 7.. Proposed biological mechanism illustrating how intracellular NTs foster or combat seeding, depending on concentration.

(A) In cells with sufficient concentrations of monoaminergic NTs and low tau loads, NTs (yellow) could disaggregate tau fibrils as they spontaneously form to keep seeding at bay. (B) In cells with low concentrations of monoaminergic NTs and/or high tau loads, monoaminergic NTs are unable to effectively disaggregate fibrils entirely to monomer. Resulting fractured fibrils are seeding-competent nuclei that catalyze tau seeding. Tau monomers are depicted by hexagons. NT concentrations and seeding levels are signified by gradient lines at the bottom. Images were created using Biorender.