Super‐Resolution Infrared Imaging of Polymorphic Amyloid Aggregates Directly in Neurons

Loss of memory during Alzheimer's disease (AD), a fatal neurodegenerative disorder, is associated with neuronal loss and the aggregation of amyloid proteins into neurotoxic β‐sheet enriched structures. However, the mechanism of amyloid protein aggregation is still not well understood due to many challenges when studying the endogenous amyloid structures in neurons or in brain tissue. Available methods either require chemical processing of the sample or may affect the amyloid protein structure itself. Therefore, new approaches, which allow studying molecular structures directly in neurons, are urgently needed. A novel approach is tested, based on label‐free optical photothermal infrared super‐resolution microspectroscopy, to study AD‐related amyloid protein aggregation directly in the neuron at sub‐micrometer resolution. Using this approach, amyloid protein aggregates are detected at the subcellular level, along the neurites and strikingly, in dendritic spines, which has not been possible until now. Here, a polymorphic nature of amyloid structures that exist in AD transgenic neurons is reported. Based on the findings of this work, it is suggested that structural polymorphism of amyloid proteins that occur already in neurons may trigger different mechanisms of AD progression

Detection of pre-plaque amyloid aggregation using FTIR

Background: Alzheimer's disease (AD) is characterized by misfolding and aggregation of naturally occurring beta-amyloid peptides (Aβ). These aggregates are thought to be pathogenic to neurons, although the conformation of the pathogenic Aβ species remains unclear. Biochemical extraction methods and different microscopy techniques (TEM, confocal) can be used to identify pathogenic Aβ species in the brain, although such methods can alter protein conformation or are n ot designed to determine structural details of protein assemblies

Correlative optical photothermal infrared and X-ray fluorescence for chemical imaging of trace elements and relevant molecular structures directly in neurons

Alzheimer’s disease (AD) is the most common cause of dementia, costing about 1% of the global economy. Failures ofclinical trials targeting amyloid-βprotein (Aβ), a key trigger of AD, have been explained by drug inefficiency regardlessof the mechanisms of amyloid neurotoxicity, which are very difficult to address by available technologies. Here, wecombine two imaging modalities that stand at opposite ends of the electromagnetic spectrum, and therefore, can beused as complementary tools to assess structural and chemical information directly in a single neuron. Combininglabel-free super-resolution microspectroscopy for sub-cellular imaging based on novel optical photothermal infrared(O-PTIR) and synchrotron-based X-rayfluorescence (S-XRF) nano-imaging techniques, we capture elementaldistribution andfibrillary forms of amyloid-βproteins in the same neurons at an unprecedented resolution. Our resultsreveal that in primary AD-like neurons, iron clusters co-localize with elevated amyloidβ-sheet structures and oxidizedlipids. Overall, our O-PTIR/S-XRF results motivate using high-resolution multimodal microspectroscopic approaches tounderstand the role of molecular structures and trace elements within a single neuronal cell

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

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

Alzheimer’s disease (AD) accounts for about 70% of neurodegenerative diseases and is a cause of cognitive decline and death for one-third of seniors. AD is currently underdiagnosed, and it cannot be effectively prevented. Aggregation of amyloid-β (Aβ) proteins has been linked to the development of AD, and it has been established that, under pathological conditions, Aβ proteins undergo structural changes to form β-sheet structures that are considered neurotoxic. Numerous intensive in vitro studies have provided detailed information about amyloid polymorphs; however, little is known on how amyloid β-sheet-enriched aggregates can cause neurotoxicity in relevant settings. We used scattering-type scanning near-field optical microscopy (s-SNOM) to study amyloid structures at the nanoscale, in individual neurons. Specifically, we show that in well-validated systems, s-SNOM can detect amyloid β-sheet structures with nanometer spatial resolution in individual neurons. This is a proof-of-concept study to demonstrate that s-SNOM can be used to detect Aβ-sheet structures on cell surfaces at the nanoscale. Furthermore, this study is intended to raise neurobiologists’ awareness of the potential of s-SNOM as a tool for analyzing amyloid β-sheet structures at the nanoscale in neurons without the need for immunolabelin