MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.National Institutes of Health (U.S.) (GM0077465)National Institutes of Health (U.S.) (DK080261

Activation of SK2 channels preserves ER Ca(2+) homeostasis and protects against ER stress-induced cell death

Alteration of endoplasmic reticulum (ER) Ca(2+) homeostasis leads to excessive cytosolic Ca(2+) accumulation and delayed neuronal cell death in acute and chronic neurodegenerative disorders. While our recent studies established a protective role for SK channels against excessive intracellular Ca(2+) accumulation, their functional role in the ER has not been elucidated yet. We show here that SK2 channels are present in ER membranes of neuronal HT-22 cells, and that positive pharmacological modulation of SK2 channels with CyPPA protects against cell death induced by the ER stressors brefeldin A and tunicamycin. Calcium imaging of HT-22 neurons revealed that elevated cytosolic Ca(2+) levels and decreased ER Ca(2+) load during sustained ER stress could be largely prevented by SK2 channel activation. Interestingly, SK2 channel activation reduced the amount of the unfolded protein response transcription factor ATF4, but further enhanced the induction of CHOP. Using siRNA approaches we confirmed a detrimental role for ATF4 in ER stress, whereas CHOP regulation was dispensable for both, brefeldin A toxicity and CyPPA-mediated protection. Cell death induced by blocking Ca(2+) influx into the ER with the SERCA inhibitor thapsigargin was not prevented by CyPPA. Blocking the K(+) efflux via K(+)/H(+) exchangers with quinine inhibited CyPPA-mediated neuroprotection, suggesting an essential role of proton uptake and K(+) release in the SK channel-mediated neuroprotection. Our data demonstrate that ER SK2 channel activation preserves ER Ca(2+) uptake and retention which determines cell survival in conditions where sustained ER stress contributes to progressive neuronal death.Cell Death and Differentiation advance online publication, 20 November 2015; doi:10.1038/cdd.2015.146.</p

The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation

Comparative Lipidomics in Clinical Isolates of Candida albicans Reveal Crosstalk between Mitochondria, Cell Wall Integrity and Azole Resistance

Prolonged usage of antifungal azoles which target enzymes involved in lipid biosynthesis invariably leads to the development of multi-drug resistance (MDR) in Candida albicans. We had earlier shown that membrane lipids and their fluidity are closely linked to the MDR phenomenon. In one of our recent studies involving comparative lipidomics between azole susceptible (AS) and azole resistant (AR) matched pair clinical isolates of C. albicans, we could not see consistent differences in the lipid profiles of AS and AR strains because they came from different patients and so in this study, we have used genetically related variant recovered from the same patient collected over a period of 2-years. During this time, the levels of fluconazole (FLC) resistance of the strain increased by over 200-fold. By comparing the lipid profiles of select isolates, we were able to observe gradual and statistically significant changes in several lipid classes, particularly in plasma membrane microdomain specific lipids such as mannosylinositolphosphorylceramides and ergosterol, and in a mitochondrial specific phosphoglyceride, phosphatidyl glycerol. Superimposed with these quantitative and qualitative changes in the lipid profiles, were simultaneous changes at the molecular lipid species levels which again coincided with the development of resistance to FLC. Reverse transcriptase-PCR of the key genes of the lipid metabolism validated lipidomic picture. Taken together, this study illustrates how the gradual corrective changes in Candida lipidome correspond to the development of FLC tolerance. Our study also shows a first instance of the mitochondrial membrane dysfunction and defective cell wall (CW) in clinical AR isolates of C. albicans, and provides evidence of a cross-talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance

Fibroblasts from patients with major depressive disorder show distinct transcriptional response to metabolic stressors

Major depressive disorder (MDD) is increasingly viewed as interplay of environmental stressors and genetic predisposition, and recent data suggest that the disease affects not only the brain, but the entire body. As a result, we aimed at determining whether patients with major depression have aberrant molecular responses to stress in peripheral tissues. We examined the effects of two metabolic stressors, galactose (GAL) or reduced lipids (RL), on the transcriptome and miRNome of human fibroblasts from 16 pairs of patients with MDD and matched healthy controls (CNTR). Our results demonstrate that both MDD and CNTR fibroblasts had a robust molecular response to GAL and RL challenges. Most importantly, a significant part (messenger RNAs (mRNAs): 26-33%; microRNAs (miRNAs): 81-90%) of the molecular response was only observed in MDD, but not in CNTR fibroblasts. The applied metabolic challenges uncovered mRNA and miRNA signatures, identifying responses to each stressor characteristic for the MDD fibroblasts. The distinct responses of MDD fibroblasts to GAL and RL revealed an aberrant engagement of molecular pathways, such as apoptosis, regulation of cell cycle, cell migration, metabolic control and energy production. In conclusion, the metabolic challenges evoked by GAL or RL in dermal fibroblasts exposed adaptive dysfunctions on mRNA and miRNA levels that are characteristic for MDD. This finding underscores the need to challenge biological systems to bring out disease-specific deficits, which otherwise might remain hidden under resting conditions

Modelling of the impact of universal added sugar reduction through food reformulation

© 2017 The Author(s). Food reformulation has been suggested to be one of the strategies to reduce population added sugar (AS) intake. This study aims to investigate the untested assumption that a reduction in AS through reformulation will result in a reduction in population intakes of AS and energy. Plausible dietary data from 4,140 respondents of an Australian national nutrition survey were used. Dietary modelling was performed at AS reductions of 10%, 15%, and 25% using four strategies: simple removal of AS or replacement with non-nutritive sweeteners (NNS), and replacement of AS with NNS and either: polyols, 50% fibres or 50% maltodextrin. Paired t-tests were conducted to compare the intake of energy, fat, and AS pre- and post-reformulation. The chosen reformulation strategies resulted in a projected reduction in AS and energy, with the greatest reduction found in 25% reformulation which was the highest level modelled. The overall projected mean (SD) reduction in energy and AS after 25% reformulation was 114 (92) kJ/day and 11.73 (7.52) g/day, p < 0.001. To conclude, product reformulation may be a potentially useful strategy for reducing AS intake. Although the magnitude of projected reduction was small at the individual level, the impact may be meaningful at a population level