The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cells

Dietary overload of toxic, free metabolic intermediates leads to disrupted insulin signalling and fatty liver disease. However, it was recently reported that this pathway might not be universal: depletion of histone deacetylase (HDAC) enhances insulin sensitivity alongside hepatic lipid accumulation in mice, but the mechanistic role of microscopic lipid structure in this effect remains unclear. Here we study the effect of Entinostat, a synthetic HDAC inhibitor undergoing clinical trials, on hepatic lipid metabolism in the paradigmatic HepaRG liver cell line. Specifically, we statistically quantify lipid droplet morphology at single cell level utilizing label-free microscopy, coherent anti-Stokes Raman scattering, supported by gene expression. We observe Entinostat efficiently rerouting carbohydrates and free-fatty acids into lipid droplets, upregulating lipid coat protein gene Plin4, and relocating droplets nearer to the nucleus. Our results demonstrate the power of Entinostat to promote lipid synthesis and storage, allowing reduced systemic sugar levels and sequestration of toxic metabolites within protected protein-coated droplets, suggesting a potential therapeutic strategy for diseases such as diabetes and metabolic syndrome

Unraveling amyloid formation paths of Parkinson\u27s disease protein alpha-synuclein triggered by anionic vesicles

Amyloid formation of the synaptic brain protein alpha-synuclein (alpha S) is related to degeneration of dopaminergic neurons in Parkinson\u27s disease patients. aS is thought to function in vesicle transport and fusion and it binds strongly to negatively charged vesicles in vitro. Here we combined circular dichroism, fluorescence and imaging methods in vitro to characterize the interaction of alpha S with negatively charged vesicles of DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1\u27-rac-glycerol), sodium salt) and the consequences of such interactions on alpha S amyloid formation. We found that lipid head-group chemistry modulates alpha S interactions and also affects amyloid fiber formation. During the course of the experiments, we made the unexpected discovery that pre-formed alpha S oligomers, typically present in a small amount in the alpha S starting material, acted as templates for linear growth of anomalous amyloid fibers in the presence of vesicles. At the same time, the remaining alpha S monomers were restricted from vesicle-mediated nucleation of amyloid fibers. Although not a dominant process in bulk experiments, this hidden alpha S aggregation pathway may be of importance in vivo

Nonlinear nearfield microscopy

Higher-order nonlinearity of light-matter interactions, such as second and third harmonic generation (SHG & THG) and Coherent anti-Stokes Raman Scattering (CARS) can be used for improving spatial resolution in microscopy as a consequence of the confinement of the nonlinear polarization to the high-intensity region of the focal volume. However, the resolution is limited to similar to 300 nm, not sufficient to resolve macromolecules or nanostructures of interest in the bio-, life- and nano-sciences. In the strive to push the resolution beyond the diffraction limit, allowing for nanoscale imaging, we have equipped a nonlinear optical microscope with a scanning-probe setup operated in tapping-mode feedback. A tapered, gold-coated, open-aperture tip with an aperture diameter of similar to 150 nm is scanned over the sample, probing the nonlinear nearfield generated by free-beam excitation. First nonlinear coherent Raman nearfield images of biological macromolecules and metallic nanostructures are shown. Limitations and future challenges with nonlinear nearfield microscopy are discussed

Nonlinear nearfield microscopy

Higher-order nonlinearity of light-matter interactions, such as second and third harmonic generation (SHG & THG) and Coherent anti-Stokes Raman Scattering (CARS) can be used for improving spatial resolution in microscopy as a consequence of the confinement of the nonlinear polarization to the high-intensity region of the focal volume. However, the resolution is limited to similar to 300 nm, not sufficient to resolve macromolecules or nanostructures of interest in the bio-, life- and nano-sciences. In the strive to push the resolution beyond the diffraction limit, allowing for nanoscale imaging, we have equipped a nonlinear optical microscope with a scanning-probe setup operated in tapping-mode feedback. A tapered, gold-coated, open-aperture tip with an aperture diameter of similar to 150 nm is scanned over the sample, probing the nonlinear nearfield generated by free-beam excitation. First nonlinear coherent Raman nearfield images of biological macromolecules and metallic nanostructures are shown. Limitations and future challenges with nonlinear nearfield microscopy are discussed

The processes of α-synuclein amyloid protein complexes involved in the pathogenesis of Parkinson’s disease

Objective. To analyze interactions between α-synuclein (αS) protein and lipids using biophysical methods. Material and methods. Recombinant α-synuclein synthesized in prokaryotic cells was used. To characterize the interaction of αS with negatively charged vesicles of DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), sodium salt) and the consequences of such interactions on αS amyloid formation, combined circular dichroism, fluorescence and imaging methods in vitro were applied. Results and conclusion. Lipid head-group chemistry modulates αS interactions and also affects amyloid fiber formation. Pre-formed αS oligomers, typically present in a small amount in the αS starting material, acted as templates for linear growth of anomalous amyloid fibers in the presence of vesicles. At the same time, the remaining αS monomers were restricted from vesicle-mediated nucleation of amyloid fibers. Although not a dominant process in bulk experiments, this hidden αS aggregation pathway may be of importance in vivo

Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy

Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r2 = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation

CARS microscopy of Alzheimer\u27s diseased brain tissue

Alzheimera\u27s disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field

Imaging lipids in live microalgae

Microalgae are capable of producing lipids from CO2 and sunlight and as such the primary producers of n-3 fatty acids. Intense research is underway to understand the conditions under which optimal lipid accumulation occurs, not only for neutraceutical applications, but also for biodiesel production. To aid this research we propose the application of a powerful microscopic technique that allows monitoring of lipids with chemical specificity at intra-cellular level in living cells: Coherent anti-Stokes Raman spectroscopy (CARS). CARS is a non-linear microscopy technique that can be used to probe C-H bonds especially abundant in lipids by a process involving four-wave mixing: two or three coherent beams of different near-infra red (NIR) wavelengths are tuned to induce a resonant vibration in the C-H bonds, and generate a blue-shifted CARS signal. The NIR light used to probe the sample allows good penetration which in turn makes optical sectioning possible. By taking many optical sections of the sample, a 3D image can be constructed, from which the volume of lipids in the cell can be calculated, allowing quantitative studies of lipid accumulation in single microalgae. In our study, we used Phaeodactylum tricornutum grown under normal, light-starved and nitrogen-starved conditions. CARS microscopy detected statistically significant differences in lipid droplet number and their volumes when comparing growth conditions at single cell level. The whole population was then subjected to traditional lipid extraction and chromatographic separation of fatty acids. Average lipid volumes, as calculated from CARS microscopy, correlated well with traditional chemical analysis. As a conclusion, CARS could be applied to the in vivo study of culture condition effect on lipid accumulation in microalgae

Imaging lipids in live microalgae

Microalgae are the primary producers of n-3 fatty acids in the aquatic food web, capable of producing lipids from CO2 and sunlight. Intense research is underway to understand the conditions under which optimal lipid accumulation occurs. To aid this research we propose the application of a powerful microscopic technique that allows monitoring of lipids with chemical specificity at intra-cellular level in living cells. While fluorescence microscopy with an appropriate dye can be highly specific, cells must be fixed and permeabilized, excluding live-cell studies; other techniques, such as phase contrast microscopy can be applied to living cells, but they lack chemical specificity. Coherent anti-Stokes Raman spectroscopy (CARS) is a non-linear microscopy technique that probes C-H bonds especially abundant in lipids by a process involving four-wave mixing: two or three coherent beams of different near-infra red (NIR) wavelengths are tuned to induce a resonant vibration in the C-H bonds, and generate a blue-shifted CARS signal in the visible region. Multi-photon autofluorescence from e.g. pigments in the sample can be detected simultaneously. The NIR light used to illuminate the sample allows good penetration which in turn makes optical sectioning possible. By taking many optical sections of the sample a 3D image can be constructed, from which the volume of lipids in the cell can be calculated, allowing quantitative studies of lipid accumulation in single microalgae. We herein present a proof-of-concept in the comparison of Phaeodactylum tricornutum grown under normal light-starved and nitrogen-starved conditions