Oligomeric Forms of Insulin Amyloid Aggregation Disrupt Outgrowth and Complexity of Neuron-Like PC12 Cells

Abstract

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.

Preconditioning of Mesenchymal Stem Cells by Sevoflurane to Improve Their Therapeutic Potential

“…Neuron-like PC12 cells have been widely studied as a model for neuronal cells [19]–[21]. In the current study, a 24 h H/SD resulted in an obvious apoptosis in the neuron-like PC12 cells.…”

Structural, morphological, and functional diversity of amyloid oligomers

“…On the other hand, early stage oligomers derived from the folded proteins such as insulin [100,101], SOD-1 [102], and acylphosphatase [103,104] largely preserved the native fold of these proteins. Formation of these early oligomers resembles the process of the surfactant micelle formation, although due to the required conformational changes in protein molecules, the oligomer formation process is much slower [105].…”

Insulin aggregation tracked by its intrinsic TRES

“…Several techniques have been employed to study fibril formation including circular dichroism (CD), 14,15 transmission electron microscopy (TEM), 16 atomic force microscopy (AFM), 17 X-ray diffraction 18 and others. [19][20][21] CD spectroscopy studies showed that insulin adopts -helical conformations that can convert to appreciably -pleated structures as a function of time.…”