Amyloid Structural Changes Studied by Infrared Microspectroscopy in Bigenic Cellular Models of Alzheimer’s Disease

Abstract

Alzheimer’s disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of β-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of β-sheet structures in different monogenic and bigenic cellular models of Alzheimer’s disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-β, α-synuclein) and (amyloid-β, Tau) neuron-like cells display changes in β-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer’s disease.

Nano-Infrared Imaging of Primary Neurons

“…We demonstrate that by using s-SNOM, we can select exosomes deposited on the surface (via a specific band that corresponds to lipids). Analyzing s-SNOM spectra, we did not detect β-sheet structures in these isolated exosomes, but because the presence of Aβ has been previously shown in exosomes [44], it could indicate that the exosomes carried β-sheet free Aβ. However, more experiments are needed to investigate why β-sheet-enriched structures were not detected in exosomes isolated from APP/PS1 transgenic neurons by a sequential centrifugation protocol and determine if modifications of the protocol are needed to preserve exosomes that may carry aggregated Aβ.…”

“…However, the molecular mechanisms behind neuronal damage induced by Aβ aggregation in neurons are not well understood. In our previous work [44], we demonstrated that µ-FTIR and O-PTIR can be used to study molecular structures in primary neurons that produce aggregation-prone amyloid proteins such as Aβ. Here, we demonstrate that s-SNOM can simultaneously characterize the morphology and the complex optical (reflectivity and absorption) responses of the sample surface with a spatial resolution of~50 nm.…”

“…Further, using s-SNOM, we examined protein structures in exosomes. It has been shown that exosomes isolated from APP/PS1 primary neurons contain Aβ [44]; however, the conformational states of Aβ cannot be assessed using WB or immunochemistry due to the chemical processing in these methods, which may alter Aβ conformations. Therefore, to address conformational states of Aβ, nanospectroscopic approaches are needed.…”

Diffraction-limited mid-infrared microspectroscopy to reveal a micron-thick interfacial water layer signature

“…Infrared microspectroscopy down to the diffraction limit has been widely applied to study biological samples in the past 40 years. It allowed exploring individual cells for revealing the damage mechanisms and modification of protein structure, 2,3 diagnosing diseases, [4][5][6][7][8] studying microbiology and bacteria, [9][10][11][12][13] biochemical and bio-physical modification processes, [14][15][16][17][18][19] and variation of chemical composition. [20][21][22][23] In all that, working at the diffraction limit was crucial to downscale at the size of the individual biological object.…”