Effects of amyloid-β-mimicking peptide hydrogel matrix on neuronal progenitor cell phenotype

Abstract

Self-assembling peptide-based hydrogels have become a highly attractive scaffold for three-dimensional (3D) in vitro disease modeling as they provide a way to create tunable matrices that can resemble the extracellular matrix (ECM) of various microenvironments. Alzheimer's disease (AD) is an exceptionally complex neurodegenerative condition; however, our understanding has advanced due to the transition from two-dimensional (2D) to 3D in vitro modeling. Nonetheless, there is a current gap in knowledge regarding the role of amyloid structures, and previously developed models found long-term difficulty in creating an appropriate model involving the ECM and amyloid aggregates. In this report, we propose a multi-component self-assembling peptide-based hydrogel scaffold to mimic the amyloid-beta (β) containing microenvironment. Characterization of the amyloid-β-mimicking hydrogel (Col-HAMA-FF) reveals the formation of β-sheet structures as a result of the self-assembling properties of phenylalanine (Phe, F) through π-π stacking of the residues, thus mimicking the amyloid-β protein nanostructures. We investigated the effect of the amyloid-β-mimicking microenvironment on healthy neuronal progenitor cells (NPCs) compared to a natural-mimicking matrix (Col-HAMA). Our results demonstrated higher levels of neuroinflammation and apoptosis markers when NPCs were cultured in the amyloid-like matrix compared to a natural brain matrix. Here, we provided insights into the impact of amyloid-like structures on NPC phenotypes and behaviors. This foundational work, before progressing to more complex plaque models, provides a promising scaffold for future investigations on AD mechanisms and drug testing. STATEMENT OF SIGNIFICANCE: In this study, we engineered two multi-component hydrogels: one to mimic the natural extracellular matrix (ECM) of the brain and one to resemble an amyloid-like microenvironment using a self-assembling peptide hydrogel. The self-assembling peptide mimics β-amyloid fibrils seen in amyloid-β protein aggregates. We report on the culture of neuronal progenitor cells within the amyloid-mimicking ECM scaffold to study the impact through marker expressions related to inflammation and DNA damage. This foundational work, before progressing to more complex plaque models, offers a promising scaffold for future investigations on AD mechanisms and drug testing.

Keywords: Alzheimer's disease; Amyloid fibrils; Hydrogel; Neuronal cells; Peptide self-assembly.

Figure 1.. Scheme of the overall project.

Scheme showing the construction of a “healthy” (left) and an “amyloid” (right) state hydrogel matrix and the expression of certain neuronal markers when cultured in each one. Scale bar 50 μm.

Figure 2.. Rheological characterization of Col-HAMA and Col-HAMA-FF hydrogels.

A. Scheme showing components of hydrogel matrix. B. Frequency sweep at 1% strain. C. amplitude sweep. D. temperature sweep from 10–50°C. E. step strain sweeps showing the storage (purple) and loss (blue) modulus of Col-HAMA and the storage (green) and loss (orange) modulus of Col-HAMA-FF hydrogels in repeating cycles from 1 to 200%. F. Viscosity of Col-HAMA (purple) and Col-HAMA-FF hydrogel.

Figure 3.. ThT and β-sheet characterization in Col-HAMA and Col-HAMA-FF hydrogels.

A. Scheme showing when ThT binds to amyloid aggregates, it gives rise to fluorescent signals. B. ThT tests on our hydrogel components alone and its various mixtures show the formation of aggregates in the Col-HAMA-FF hydrogels through fluorescent intensity (FI). C. FT-IR studies showing the formation of β-sheet functional groups.

Figure 4.. In vitro cytotoxicity effects.

A. Live/dead studies of HDF cells with both hydrogel matrices Scale bars: 200 μm. B. Quantification of live/dead cells. C. Presto Blue study to show cell proliferation after indirect contact with gels. D. Live/dead studies of NPCs cultured in various matrices. (Scale bars: 100 μm; n = 3) (Two-way Anova, Tukey’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.005, ****p<0.001).

Figure 5.. Pax-6 and Nestin staining of NPCs cultured in healthy versus amyloid hydrogel matrices.

A. Microscopy images. B. Pax-6 quantification (control, CH, n=5, CHFF, n=6). C. Nestin quantification (n=6). Scale bar = 50 μm. (One-way Anova, Tukey’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.005, ****p<0.001).

Figure 6.. β-tubulin and TNF-α staining of NPCs cultured in both hydrogel states.

A. Microscopy images. B. β-tubulin quantification (n=5). C. TNF-α quantification (n=5). Scale bar = 50 μm.; (One-way Anova, Tukey’s post hoc test p < 0.05, p < 0.01, p < 0.005, **** p<0.001).

Figure 7.. Ki-67 and PARP staining of NPCs cultured in both hydrogel states.

A. Microscopy images. B. Ki-67 quantification (control, CHFF n=5, CH, n=6). C. PARP quantification (control, CH, n=6, CHFF, n=5). Scale bar = 50 μm. (One-way Anova, Tukey’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.005, ****p<0.001).