Amyloids are self-assembled protein aggregates that represent a major hallmark of many neurologic and systemic diseases. Among the common features of amyloids is the presence of a high density of multiple binding sites for small molecule ligands, making them an attractive target for design of multimeric binding agents. Here, we demonstrate that noncovalent, intermolecular interactions between a 1:1 mixture of oppositely charged benzothiazole molecules enhances their binding to two different amyloid aggregates: Alzheimer's-related amyloid-β (Aβ) peptides or Parkinson's-related α-synuclein (αS) proteins. We show that this mixture leads to positively cooperative binding to amyloid targets, with up to 10-fold enhancement of binding compared to the uncharged parent compound. The observed enhancement of amyloid binding using noncovalent interactions was similar in magnitude to a benzothiazole dimer to aggregated Aβ. These results represent a novel strategy for designing amyloid-targeting molecules with enhanced affinity, which could aid in the development of new diagnostic or treatment strategies for amyloid-associated diseases.

The tetra(ethylene glycol) derivative of benzothiazole aniline, BTA-EG4, is a novel amyloid-binding small molecule that can penetrate the blood-brain barrier and protect cells from Aβ-induced toxicity. However, the effects of Aβ-targeting molecules on other cellular processes, including those that modulate synaptic plasticity, remain unknown. We report here that BTA-EG4 decreases Aβ levels, alters cell surface expression of amyloid precursor protein (APP), and improves memory in wild-type mice. Interestingly, the BTA-EG4-mediated behavioral improvement is not correlated with LTP, but with increased spinogenesis. The higher dendritic spine density reflects an increase in the number of functional synapses as determined by increased miniature EPSC (mEPSC) frequency without changes in presynaptic parameters or postsynaptic mEPSC amplitude. Additionally, BTA-EG4 requires APP to regulate dendritic spine density through a Ras signaling-dependent mechanism. Thus, BTA-EG4 may provide broad therapeutic benefits for improving neuronal and cognitive function, and may have implications in neurodegenerative disease therapy.

Our recent study demonstrated that an amyloid-β binding molecule, BTA-EG4, increases dendritic spine number via Ras-mediated signaling. To potentially optimize the potency of the BTA compounds, we synthesized and evaluated an amyloid-β binding analog of BTA-EG4 with increased solubility in aqueous solution, BTA-EG6. We initially examined the effects of BTA-EG6 on dendritic spine formation and found that BTA-EG6-treated primary hippocampal neurons had significantly increased dendritic spine number compared to control treatment. In addition, BTA-EG6 significantly increased the surface level of AMPA receptors. Upon investigation into the molecular mechanism by which BTA-EG6 promotes dendritic spine formation, we found that BTA-EG6 may exert its effects on spinogenesis via RasGRF1-ERK signaling, with potential involvement of other spinogenesis-related proteins such as Cdc42 and CDK5. Taken together, our data suggest that BTA-EG6 boosts spine and synapse number, which may have a beneficial effect of enhancing neuronal and synaptic function in the normal healthy brain.