Emerging Functional Connections Between Metabolism and Epigenetic Remodeling in Neural Differentiation.

Stem cells possess extraordinary capacities for self-renewal and differentiation, making them highly valuable in regenerative medicine. Among these, neural stem cells (NSCs) play a fundamental role in neural development and repair processes. NSC characteristics and fate are intricately regulated by the microenvironment and intracellular signaling. Interestingly, metabolism plays a pivotal role in orchestrating the epigenome dynamics during neural differentiation, facilitating the transition from undifferentiated NSC to specialized neuronal and glial cell types. This intricate interplay between metabolism and the epigenome is essential for precisely regulating gene expression patterns and ensuring proper neural development. This review highlights the mechanisms behind metabolic regulation of NSC fate and their connections with epigenetic regulation to shape transcriptional programs of stemness and neural differentiation. A comprehensive understanding of these molecular gears appears fundamental for translational applications in regenerative medicine and personalized therapies for neurological conditions.

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes.

T-cell activation induces a metabolic switch generating energy for proliferation, survival, and functions. We used noninvasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NAD(P)H co-enzyme during T lymphocyte activation. This provides a readout of the OXPHOS and glycolysis rates at a single-cell level. Analyzes were performed in the CD4+ leukemic T cell line Jurkat, and in human CD4+ primary T cells. Cells were activated on glass surfaces coated with activating antibodies mimicking immune synapse formation. Comparing the fraction of bound NAD(P)H between resting and activated T cells, we show that T-cell activation induces a rapid switch toward glycolysis. This occurs after 10 min and remains stable for one hour. Three-dimensional analyzes revealed that the intracellular distribution of fraction of bound NAD(P)H increases at the immune synapse in activated cells. Finally, we show that fraction of bound NAD(P)H tends to negatively correlate with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study highlights that 2P-FLIM measurement of fraction of bound NAD(P)H is well suited to follow a fast metabolic switch in three dimensions, in single T lymphocytes with subcellular resolution.

FLUTE: A Python GUI for interactive phasor analysis of FLIM data.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The fit-free phasor approach to FLIM data is increasingly being used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available in Python, thus limiting the widespread use of phasor analysis in biomedical research. Here, we present Fluorescence Lifetime Ultimate Explorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of the analysis of FLIM data acquired in the time domain, such as calibrating the FLIM data, performing interactive exploration of the phasor plot, displaying phasor plots and FLIM images with different lifetime contrasts simultaneously, and calculating the distance from known molecular species. After applying desired filters and thresholds, the final edited datasets can be exported for further user-specific analysis. FLUTE has been tested using several FLIM datasets including autofluorescence of zebrafish embryos and in vitro cells. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets, and accelerates FLIM analysis for non-specialized labs.

Coordinated metabolic transitions and gene expression by NAD+ during adipogenesis.

Adipocytes are the main cell type in adipose tissue, which is a critical regulator of metabolism, highly specialized in storing energy as fat. Adipocytes differentiate from multipotent mesenchymal stromal cells (hMSCs) through adipogenesis, a tightly controlled differentiation process involving close interplay between metabolic transitions and sequential programs of gene expression. However, the specific gears driving this interplay remain largely obscure. Additionally, the metabolite nicotinamide adenine dinucleotide (NAD+) is becoming increasingly recognized as a regulator of lipid metabolism, and a promising therapeutic target for dyslipidemia and obesity. Here, we explored how NAD+ bioavailability controls adipogenic differentiation from hMSC. We found a previously unappreciated repressive role for NAD+ on adipocyte commitment, while a functional NAD+-dependent deacetylase SIRT1 appeared crucial for terminal differentiation of pre-adipocytes. Repressing NAD+ biosynthesis during adipogenesis promoted the adipogenic transcriptional program, while two-photon microscopy and extracellular flux analyses suggest that SIRT1 activity mostly relies on the metabolic switch. Interestingly, SIRT1 controls subcellular compartmentalization of redox metabolism during adipogenesis.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA-induced metabolic stress in reconstructed human skin.

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.