Live-Cell imaging and quantification of PolyQ aggregates by stimulated raman scattering of selective deuterium labeling

Abstract

Polyglutamine (polyQ) diseases are a group of neurodegenerative disorders, involving the deposition of aggregationprone proteins with long polyQ expansions. However, the cytotoxic roles of these aggregates remain controversial, largely due to a lack of proper tools for quant. and nonperturbative interrogations. Common methods including in vitro biochem., spectroscopic assays, and live-cell fluorescence imaging all suffer from certain limitations. Here, we propose coupling stimulated Raman scattering microscopy with deuterium-labeled glutamine for live-cell imaging, quantification, and spectral anal. of polyQ aggregates with subcellular resoln. First, through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) exon1 proteins for Huntington's disease, we achieved sensitive and specific stimulated Raman scattering (SRS) imaging of carbon-deuterium bonds (C-D) from aggregates without GFP labeling, which is commonly employed in fluorescence microscopy. We revealed that these aggregates became 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantifications, which indicate a surprising dependence of protein compns. on aggregation sizes. Our further calcns., for the first time, reported the abs. concns. for sequestered mHtt and non-mHtt proteins within the same aggregates. Third, we adopted hyperspectral SRS for Raman spectroscopic studies of aggregate structures. By inducing a cellular heat shock response, a potential therapeutic approach for inhibiting aggregate formation, we found an aggregation intermediate state. Vibrational line shapes of the polyQ aggregates suggested that they experience a hyper-hydrated environment during the intermediate state[KM1] . Our method should fill the gap and serve as a suitable tool to study native polyQ aggregates. It may unveil new features of polyQ aggregates and pave the way for comprehensive in vivo investigations.

Additional details