Image-based quantification of mitochondrial iron uptake via Mitoferrin-2.

Iron is a trace element that is critical for most living organisms and plays a key role in a wide variety of metabolic processes. In the mitochondrion, iron is involved in producing iron-sulfur clusters and synthesis of heme and kept within physiological ranges by concerted activity of multiple molecules. Mitochondrial iron uptake is mediated by the solute carrier transporters Mitoferrin-1 (SLC25A37) and Mitoferrin-2 (SLC25A28). While Mitoferrin-1 is mainly involved in erythropoiesis, the cellular function of the ubiquitously expressed Mitoferrin-2 remains less well defined. Furthermore, Mitoferrin-2 is associated with several human diseases, including cancer, cardiovascular and metabolic diseases, hence representing a potential therapeutic target. Here, we developed a robust approach to quantify mitochondrial iron uptake mediated by Mitoferrin-2 in living cells. We utilize HEK293 cells with inducible expression of Mitoferrin-2 and measure iron-induced quenching of rhodamine B[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA) fluorescence and validate this assay for medium-throughput screening. This assay may allow identification and characterization of Mitoferrin-2 modulators and could enable drug discovery for this target.

The iron chelator Deferasirox causes severe mitochondrial swelling without depolarization due to a specific effect on inner membrane permeability.

The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.

Targeting glycolysis in proliferative kidney diseases.

Glycolytic activity is increased in proliferating cells, leading to the concept that glycolysis could be a therapeutic target in cystic diseases and kidney cancer. Preclinical studies using the glucose analog 2-deoxy-d-glucose have shown promise; however, inhibiting glycolysis in humans is unlikely to be without risks. While proximal tubules are predominantly aerobic, later segments are more glycolytic. Understanding exactly where and why glycolysis is important in the physiology of the distal nephron is thus crucial in predicting potential adverse effects of glycolysis inhibitors. Live imaging techniques could play an important role in the process of characterizing cellular metabolism in the functioning kidney. The goal of this review is to briefly summarize recent findings on targeting glycolysis in proliferative kidney diseases and to highlight the necessity for future research focusing on glycolysis in the healthy kidney.

Effects of light and dark phase testing on the investigation of behavioural paradigms in mice: Relevance for behavioural neuroscience.

Different timing and light phases are critical factors in behavioural neuroscience, which can greatly affect the experimental outcomes of the performed tests. Despite the fact that time of testing is one of the most common factors that varies across behavioural laboratories, knowledge about the consequences of testing time on behavioural readouts is limited. Thus, in this study we systematically assessed the effect of this factor on the readout of a variety of elementary and recurrent behavioural paradigms in C57Bl/6 mice. Furthermore, we investigated potential neuronal correlates of this phenomenon by analysing how testing time influences the expression pattern of genes relevant for neuronal activation functions and the control of brain circadian rhythms. We show that animals tested in the light phase display reduced social approach behaviour and sensorimotor gating and increased locomotor activity, whereas anxiety-related behaviour and working memory are not affected. In addition, animals tested in the light phase also exhibit increased locomotor response to systemic amphetamine treatment, which is paralleled by alterations in the expression patterns of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the Nucleus Accumbens (NAc) and/or Midbrain (Mid). Lastly, we observed that neuronal activation, indexed by the gene expression levels of cFos, was increased in the NAc and Mid of animals tested during the light phase. Our data thus suggest that daily alterations in gene expression in mesolimbic brain structures might be involved in the different behavioural responses of mice tested in the light- versus the dark-phase. At the same time, our study adds further weight to the notion that the specific timing of testing can indeed strongly affect the readout of a given test. As comparison and reproducibility of findings is pivotal in science, experimental protocols should be clarified in detail to allow appropriate data comparison across different laboratories.