(718f) Conjugated Polyelectrolytes for Biosensing and Theranostic Applications in Protein Misfolding Diseases

A major pathological hallmark of Alzheimer’s disease is the deposition of amyloid plaques composed of the amyloid-β (Aβ) peptide, which results from the abnormal misfolding and aggregation of the peptides into small oligomers that subsequently grow into large fibrils. (1) The oligomers, which are transient and heterogeneous in nature, are known to be more neurotoxic than the mature fibrils. (2) Aβ fibrils also play a key role in neurodegeneration through impairment of axonal transport or by inducing the aggregation of tau protein and seeding the formation of neurofibrillary tangles. (3) Additionally, amyloid aggregates are also involved in the rapid and predictable spatiotemporal disease progression through cell-to-cell transmission. (4) Because of the central roles amyloid aggregates play in Alzheimer’s disease pathogenesis, their selective detection, degradation, and clearance is an attractive therapeutic approach.

Methods

In this work, we studied novel super luminescent conjugated polyelectrolytes as in vitro and ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE₁^2− and cationic OPE₂⁴⁺, as in vitro sensors of amyloid aggregates and ex vivo stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer’s disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). Additionally, as the OPEs are also photosensitizers upon binding to molecular targets, we evaluated the potential of OPE₁^2− as a selective photo-oxidizer for Aβ fibrils and compared its activity to the well-known but non-specific photosensitizer methylene blue (MB). Oxidation of both Aβ monomers and fibrils with light exposure in the presence of OPE₁^2− or MB was characterized. Oxidized amino acids on Aβ fibrils were identified and quantified, and the effect of fibril oxidation on fibril morphology, secondary structures, cell toxicity, and fibril seeding potency were evaluated.

Results and Discussion

In vitro sensing: We found that OPEs selectively bind to and detect β-sheet rich amyloid fibrils in vitro (5, 6, 7). The small and negatively charged OPE₁^2− detects fibrils made of two model amyloid proteins, insulin and lysozyme (5,6), and two disease-relevant proteins, Aβ and α-synuclein (7). More importantly, OPE₁^2− is also capable of selectively detecting the more toxic, pre-fibrillar aggregates of Aβ42 and α-synuclein (7). OPE’s superior sensor performance compared to that of the commonly used thioflavin T dye (ThT) could be attributed to its high sensitivity to fluorescence quenching wherein the conjugated sensor is quenched in an aqueous solvent and binding to amyloid aggregates reverses quenching and leads to fluorescence recovery or turn-on. The ability of OPE₁^2− to detect a wider set of protein aggregate conformations stems from the combination of different modes that leads to fluorescence turn-on of the OPEs, including hydrophobic unquenching, backbone planarization, and OPE complexation upon binding to amyloid aggregates.

Ex vivo sensing: We found that the anionic OPE₁^2− selectively stained tau NFTs in mouse and human tissue at 5 µM. (8) The cationic OPE₂⁴⁺ at 5 µM showed some non-specific binding in all brain sections, but at a lower concentration of 0.5 µM, this problem was resolved and the sensor displayed selectivity for staining tau NFTs. OPE₁^2− staining co-localized with the binding of the phosphorylated tau antibody AT180 and ThT in mouse and human sections, confirming that the OPE-stained tau NFTs. Furthermore, quantitative analysis showed that OPE₁^2− is better at staining NFTs than AT180. In the rat AD model, OPE₂⁴⁺ displayed selectivity for Aβ plaques at 5 µM with minimal background fluorescence in the Fisher-344 wildtype sections. OPE₂⁴⁺ co-localized with Aβ specific antibody 4G8 on the same plaques but stained the core, while the antibody stained the more diffuse morphology. We also evaluated the OPEs’ toxicity toward N2a cells using the MTT assay. Neither OPE caused a significant loss of cell viability at concentrations up to 10 µM. In comparison, ThT at a tissue staining concentration of 1.56 mM caused a complete loss of viability of the N2a cells. Our results thus show that OPEs are effective ex vivo markers for the selective staining of tau NFTs and Aβ amyloid plaques at concentrations orders of magnitude lower than ThT.

Selective and controllable photosensitizing activity of OPEs: We showed that MB non-selectively oxidizes both Aβ monomers and fibrils, while OPE₁^2− only oxidizes Aβ fibrils. (9) Three amino acids on the fibril are oxidized by OPE photosensitization, His13, His14 and Met35, which proceeds through binding induced generation of ¹O₂. Photooxidation causes fibrils to disassemble into shorter, but non-toxic oxidized fibrils. The oxidized fibrils also retain their ability to seed further Aβ40 aggregation, albeit fibrils of a lower β-sheet content were produced. Overall, we demonstrated the ability of OPE₁^2− to photo-sensitize the oxidation of Aβ fibrils controllably and selectively. The selective nature of OPE’s photosensitizing activity overcomes the major drawback of off-target oxidation from using conventional photosensitizers such as MB in photodynamic therapy.

Conclusions

OPEs displayed selective sensing of amyloid aggregates both in vitro and ex vivo. OPE₁^2− detected PHFs in fluorimetry assays and strong staining of NFTs in mouse and human brain tissue sections, while OPE₂⁴⁺ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. In addition, concomitant with fluorescence turn-on, OPE also photosensitizes singlet oxygen under illumination through the generation of a triplet state. We found that, while MB photo-oxidized both monomeric and fibrillar conformers of Aβ40, OPE oxidized only Aβ40 fibrils, targeting two histidine residues on the fibril surface and a methionine residue located in the fibril core. Oxidized fibrils were shorter and more dispersed but retained the characteristic β-sheet rich fibrillar structure and the ability to seed further fibril growth. Importantly, the oxidized fibrils displayed low toxicity. Combined with its selective fluorescence sensing capabilities, our results support the further development of OPEs as potential theranostics for the simultaneous detection and clearance of amyloid aggregates in protein misfolding diseases such as Alzheimer’s and Parkinson’s diseases.

References

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