Infrared microspectroscopy: a multiple-screening platform for investigating single-cell biochemical perturbations upon prion infection.
Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP(Sc)) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrP(C), into nascent PrP(Sc). The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level.
Laboratory of Prion Biology, Neurobiology Sector, Scuola Internazionale Superiore di Studi Avanzati (SISSA), via Bonomea 265,I-34136 Trieste, Italy.
Alessandro
A
Didonna
Lisa
L
Vaccari
Alpan
A
Bek
Giuseppe
G
Legname
D000818:Animals; D051379:Mice; D015591:Microspectrophotometry / Q000379:methods; D018620:PrPSc Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D017096:Prion Diseases / Q000378:metabolism / Q000473:pathology*; D059010:Single-Cell Analysis / Q000379:methods; D013055:Spectrophotometry, Infrared / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D018620:PrPSc Proteins