Transcriptomic analysis of human astrocytes in vitro reveals hypoxia-induced mitochondrial dysfunction, modulation of metabolism, and dysregulation of the immune response

Hypoxia is a feature of neurodegenerative diseases, and can both directly and indirectly impact on neuronal function through modulation of glial function. Astrocytes play a key role in regulating homeostasis within the central nervous system, and mediate hypoxia-induced changes in response to reduced oxygen availability. The current study performed a detailed characterization of hypoxia-induced changes in the transcriptomic profile of astrocytes in vitro. Human astrocytes were cultured under normoxic (5% CO2, 95% air) or hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 h, and the gene expression profile assessed by microarray analysis. In response to hypoxia 4904 genes were significantly differentially expressed (1306 upregulated and 3598 downregulated, FC ≥ 2 and p ≤ 0.05). Analysis of the significant differentially expressed transcripts identified an increase in immune response pathways, and dysregulation of signalling pathways, including HIF-1 (p = 0.002), and metabolism, including glycolysis (p = 0.006). To assess whether the hypoxia-induced metabolic gene changes observed affected metabolism at a functional level, both the glycolytic and mitochondrial flux were measured using an XF bioanalyser. In support of the transcriptomic data, under physiological conditions hypoxia significantly reduced mitochondrial respiratory flux (p = 0.0001) but increased basal glycolytic flux (p = 0.0313). However, when metabolically stressed, hypoxia reduced mitochondrial spare respiratory capacity (p = 0.0485) and both glycolytic capacity (p = 0.0001) and glycolytic reserve (p < 0.0001). In summary, the current findings detail hypoxia-induced changes in the astrocyte transcriptome in vitro, identifying potential targets for modifying the astrocyte response to reduced oxygen availability in pathological conditions associated with ischaemia/hypoxia, including manipulation of mitochondrial function, metabolism, and the immune response

Geometry-preserving expansion microscopy microplates enable high-fidelity nanoscale distortion mapping

Expansion microscopy (ExM) is a versatile super-resolution microscopy pipeline, leveraging nanoscale biomolecular crosslinking and osmotically driven swelling of hydrogels. Currently, ExM is a laborious and skill-intensive technique, involving manual handling of the hydrogels that can compromise the integrity of the gels and capacity to track gel isotropy, hence diminishing reproducibility. We have developed a 3D-printable microplate system to contain the entire ExM workflow within each well, enabling in situ image acquisition and eliminating the need for direct handling of the hydrogels. The preservation of the gel geometry and orientation of the microplate wells enables convenient tracking of gel expansion, pre- and post-ExM image acquisition, and distortion mapping of every cell or region of interest. We demonstrate the utility of this approach with both single-color and multiplexed ExM of cultured HeLa cells and dissected pupal Drosophila melanogaster wing tissue to reveal distortion-prone structures ranging from sub-cellular organelles to micron-scale tissue regions

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human HeLa cell line. When combined with the super-resolution technique expansion microscopy (ExM) involving swellable acrylamide hydrogels, fluorescent esters reveal nanoscale features including cytoplasmic membrane-bound compartments and nucleolar densities. We observe differential labelling patterns linked to the biochemical properties of the conjugated dye. Alterations in staining density and compartment specificity were seen depending on the timepoint of application in the ExM protocol. Additional complexity in labelling patterns was detected arising from inter-ester interactions. Our findings raise a number of considerations for the use of fluorescent esters. We demonstrate esters as a useful addition to the repertoire of stains of the cellular proteome, whether applied either on their own to visualise overall cellular morphology, or as counterstains providing ultrastructural context alongside specific target markers like antibodies

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human HeLa cell line. When combined with the super-resolution technique expansion microscopy (ExM) involving swellable acrylamide hydrogels, fluorescent esters reveal nanoscale features including cytoplasmic membrane-bound compartments and nucleolar densities. We observe differential labelling patterns linked to the biochemical properties of the conjugated dye. Alterations in staining density and compartment specificity were seen depending on the timepoint of application in the ExM protocol. Additional complexity in labelling patterns was detected arising from inter-ester interactions. Our findings raise a number of considerations for the use of fluorescent esters. We demonstrate esters as a useful addition to the repertoire of stains of the cellular proteome, whether applied either on their own to visualise overall cellular morphology, or as counterstains providing ultrastructural context alongside specific target markers like antibodies