Energy Metabolism and Intracellular pH Alteration in Neural Spheroids Carrying Down Syndrome

Abstract

Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects.

Trisomy 21-driven metabolite alterations are linked to cellular injuries in Down syndrome

“…For example, mitochondria ATP metabolism was altered in trisomic hiPSCs [ 41 ]. Energy metabolism and intracellular pH were altered in neural spheroids derived from DS hiPSCs [ 43 ]. However, despite previous reports observing altered metabolisms in DS patients [ 44 – 48 ], comprehensive investigations into the influence of exCh21 on these metabolic aberrations or the roles of exCh21-driven differential metabolites have remained elusive.…”

FLIM for Evaluation of Difference in Metabolic Status between Native and Differentiated from iPSCs Dermal Papilla Cells

“…For 3D models from primary DP cells and differentiated DP cell lines (iDP-DYP0730, iDP-iPSDP and iDP-hES), BCECF (cat #B1170) (Invitrogen, Waltham, Massachusetts, USA) staining with a ratiometric pH dye was used. In preliminary experiments, calibrations were performed with SypHer-2 and BCECF according to the previously described method [ 20 ]. Following the data, calibration curves were constructed.…”

“…The switching of metabolic pathways is directly related to changes in intracellular pH. More alkaline pH values are known to promote glycolysis in cells [ 20 ]. Fluorescence microscopy combined with genetically encoded sensors allows the analysis of pH changes with a minimal invasive impact on samples.…”

Rigor and reproducibility in human brain organoid research: Where we are and where we need to go

“…This method can indicate the mitochondrial nicotinamide adenine dinucleotide (NAD+/NADH) redox potential ( Blacker and Duchen, 2016 ), where higher redox ratio suggests prevalence of oxidative phosphorylation (OXPHOS) ( Meleshina et al., 2017 ), as well as oxidized glutathione ( Jiang et al., 2017 , 2019 ), which can reflect reactive oxygen species (ROS) production in a cell. In turn, FLIM measures the contribution of exogenous and endogenous fluorophores over time ( Meleshina et al., 2016 ) and has been employed to estimate the relative amounts of free or enzyme-bound NAD(P)H in brain spheroids ( Kashirina et al., 2021 ; Stringari et al., 2012 ).…”

“…Specific glucose and lactate assays offer the capability to measure the respective metabolite concentration in both tissue and culture medium ( Cho et al., 2021 ). Simultaneously, genetically encoded pH sensors, designed to selectively target distinct cellular compartments ( Martynov et al., 2018 ), assess tissue pH ( Kashirina et al., 2021 ). Collectively, these assays provide information on tissue acidity, indirectly indicating anaerobic conditions and cellular stress.…”