E12 - Naturally occurring betanin modulates oligomer formation of Alzheimer's disease amyloid-β protein

Introduction: : Alzheimer’s disease (AD) is credited as the seventh leading cause of death in the United States. This chronic neurodegenerative disorder is distinguished by increases in aggregation of amyloid-β (Aβ) into senile plaques and their deposition in the brain. Among the few available therapeutics, cholinesterase inhibitors and N-methyl D-aspartate (NMDA) aim to treat symptoms. In contrast, monoclonal antibodies target underlying biochemical origins of AD but are expensive.

The development of one or more effective, naturally occurring therapeutics will maximize accessibility of treatment to AD patients worldwide, whose numbers are forecast to more than double by the year 2050. Among phytochemicals of intrigue, multiple polyphenols have been demonstrated to affect Aβ aggregation by reducing the formation of large, mature assemblies; limiting the prevalence of oligomers - small soluble, intermediary aggregates established as the most neurotoxic conformation of Aβ; or even destabilizing pre-formed Aβ aggregates.

Betanin is a beetroot pigment belonging to the betalain sub-class of polyphenol analogs, a group that often exhibits strong antioxidative and anti-inflammatory properties, which offer promise in ameliorating certain diseases. To date, some inhibitory properties of betanin in Aβ oligomerization and fibril formation have been documented. In this study, anti-aggregation phenomena by betanin during Aβ oligomer preparation are highlighted by 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) spectroscopy, atomic force microscopy (AFM), and western blot. The results conclusively reveal susceptibility of Aβ oligomerization to inhibition by betanin, motivating additional mechanistic study.

Materials and

Methods: : Aβ(1-42) peptide is solubilized in dimethyl sulfoxide (DMSO) at 0.5mM. To initiate oligomerization (45min, 25°C), Aβ is diluted to 10μM in 12mM phosphate buffer (pH 7.4) containing 1μM NaCl alone (control) or in the presence of 50μM or 500μM betanin or dextrin - a stabilizing vehicle in which betanin is commercially available.

Oligomers are loaded onto black-wall, clear-bottom microplates for bis-ANS or freshly cleaved mica disks for AFM. Quadruplicate wells of oligomer samples are diluted to 2μM in phosphate buffer (pH 7.4) with 4μM bis-ANS, before evaluating fluorescence intensity (excitation=390nm; emission=450-600nm). Profiles are baseline-subtracted, integrated (IAUC), and normalized to the control.

AFM images are curated via a TT-2 AFM equipped with AFMWORKSHOP™ Control and Gwyddion™ 64-bit software, with 4-5 images recorded of each disk. Average oligomer height is determined by multiplying heights by density probability.

For western blotting, end products of Aβ oligomerization are halted from further aggregation by dilution (3:1) in 0.4% Tween-20. Oligomers are mixed (1:1) with 2X Laemmli buffer and loaded onto a 4-20% Tris-glycine gel, adjacent to a standard. Following electrophoresis and separation, samples are transferred to a 0.2μm nitrocellulose membrane and blocked overnight in 5%(v/v) nonfat milk with phosphate buffer containing Tween-20. Detection by 6E10 primary antibody and treatment with HRP-conjugated, anti-mouse secondary antibody are followed by chemiluminescence reading with Invitrogen™ iBright1500. Densitometric analyses are performed in ImageJ.

Statistical analyses are achieved with GraphPad Prism™ 9.2.0 software, including ordinary ANOVA with Dunnett’s multiple comparisons tests. Significance is defined by p< 0.05.

Results, Conclusions, and Discussions:: Baseline-subtracted bis-ANS spectra (Figure 1A) demonstrate a blue shift in emission maxima, characteristic of bound bis-ANS fluorophore. Comparison of integrated areas under the curve (IAUC) (Figure 1B) reveals significantly elevated bis-ANS activity with Aβ oligomers formed in the presence of 500μM betanin (p < 0.01) or 500μM dextrin (p < 0.05), relative to control (dashed line).

AFM analysis reveals an average height of 2.31nm among oligomers formed in the absence of betanin or dextrin (control) (Figure 2F). For Aβ oligomers formed in the presence of 500μM betanin (Figure 2C), the average height is 1.38nm, marking a 40% decrease from the control (p < 0.05). In contrast, oligomers formed in the presence of dextrin (Figure 2D-E) exhibit no statistically significant difference in height, relative to the control.

SDS-PAGE with western blotting establishes a baseline smear of Aβ oligomers, in which the mass of aggregates ranges from 25 to 250kDa (Figure 3A). The presence of 500μM betanin most significantly reduces the proportion of large oligomers (100-250kDa) by over 60% (p < 0.05) (Figure 3B).

Overall, these characterizations of Aβ oligomers, which include hydrophobic residue exposure demonstrated by bis-ANS; size captured by AFM; and extent of aggregation illustrated by western blot, comprise three critical evaluations of Aβ oligomerization in the presence of betanin. They demonstrate betanin as an Aβ oligomerization inhibitor. As a naturally occurring biomaterial, betanin is hereby implicated for therapeutic potential in vivo, where it may similarly modulate the formation of highly toxic oligomeric morphologies of Aβ. Toward this end, an area for future experimentation is the impact of Aβ oligomers formed in the presence or absence of betanin upon the health and survival of neuronal primary cell lines, which could be measured via assays like neurite outgrowth, live/dead, or caspase-3 and-7 apoptosis.

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