LETM1-Mediated K+ and Na+ Homeostasis Regulates Mitochondrial Ca2+ Efflux

HIGHLIGHTS \u2022 Monovalent cation homeostasis is dysregulated upon LETM1 depletion \u2022 K+/H+ exchange activity is decreased in LETM1 knockdown cells \u2022 LETM1 depletion results in K+ accumulation in the mitochondrial matrix \u2022 LETM1 knockdown does not affect expression of major mitochondrial Ca2+ transport modulators \u2022 LETM1-regulated mitochondrial Ca2+ fluxes are dependent on Na+ Ca2+ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. It is therefore tightly regulated. It has been recently proposed that LETM1\u2014an IMM protein with a crucial role in mitochondrial K+/H+ exchange and volume homeostasis\u2014also acts as a Ca2+/H+ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K+/H+ and Na+/H+ exchange. Decreased exchange activity resulted in matrix K+ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na+/Ca2+ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca2+ Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca2+ fluxes may reflect those on Na+/H+ exchange activity

Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation

The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca2+ uptake capacity. This defect is neither a consequence of global alteration in cellular Ca2+ homeostasis nor of the reduced driving force for Ca2+ internalization within mitochondria, since cytosolic Ca2+ transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca2+ uptake speed in Afg3l2−/− cells, indicating the presence of functional Ca2+ uptake machinery. Our results show that the defective Ca2+ handling in Afg3l2−/− cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca2+ elevations, hampering the proper Ca2+ diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2−/− MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca2+ buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca2+ homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutation

Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD

Amyotrophic lateral sclerosis (ALS) manifests pathological changes in motor neurons and various other cell types. Compared to motor neurons, the contribution of the other cell types to the ALS phenotypes is understudied. G4C2 repeat expansion in C9ORF72 is the most common genetic cause of ALS along with frontotemporal dementia (C9-ALS/FTD), with increasing evidence supporting repeat-encoded poly(GR) in disease pathogenesis. Here, we show in Drosophila muscle that poly(GR) enters mitochondria and interacts with components of the Mitochondrial Contact Site and Cristae Organizing System (MICOS), altering MICOS dynamics and intra-subunit interactions. This impairs mitochondrial inner membrane structure, ion homeostasis, mitochondrial metabolism, and muscle integrity. Similar mitochondrial defects are observed in patient fibroblasts. Genetic manipulation of MICOS components or pharmacological restoration of ion homeostasis with nigericin effectively rescue the mitochondrial pathology and disease phenotypes in both systems. These results implicate MICOS-regulated ion homeostasis in C9-ALS pathogenesis and suggest potential new therapeutic strategies

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca2+ handling

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (A\u3b2) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca2+ handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4R\u394C20), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8-10\u202fnm) and the long- (40-50\u202fnm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca2+ homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca2+ content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4R\u394C20 tau, indicating that Ca2+ mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies

Image-guided temporal bone dissection course

Introduction: Temporal bone anatomy is complex and demands a profound anatomical knowledge. Association between surgery and imaging helps in the process of learning three-dimensional (3D) anatomy and surgical techniques. High definition temporal bone imaging can play an important role in dissection training. Objective: To describe a computed tomography (CT) image-guided temporal bone dissection course for surgical training in otolaryngology and to verify the satisfaction level of the students with the course. Methods: Descriptive research. The course took place at a research laboratory, with three experienced temporal bone surgeons. The participants were 12 otolaryngology residents. The laboratory has 7 modern workstations with microscope and monitors linked with a computerized video system. Cadaveric temporal bones were donated to the university. Imaging acquisition of the cadaveric temporal bones used in the course was performed in a multislice CT scanner. The CT images of cadaveric temporal bones were available with real-time access on the laboratory monitor's screens during dissections. Results: A total of 13 temporal bones were included for dissection. Students had the opportunity to view on the same screen, simultaneously, both the dissection video and the respective CT images of their temporal bone anatomical specimens. This allowed correlating surgical and imaging aspects of temporal bone anatomy. At the end of the course, participants answered a satisfaction survey. Conclusion: Considering imaging methods are routinely used during most otologic surgeries, detailed knowledge of CT imaging should be explored in conjunction with the temporal bone anatomical dissection

Emilin-1 controls arterial blood pressure by regulating contractility of vascular smooth muscle cells

Emilin-1 is a protein of the elastic extracellular matrix (ECM) expressed in interstitial connective tissue and in the cardiovascular system. Emilin1 null mice display hypotrophic remodeling of the wall of conductance arteries and increased blood pressure. The protein regulates the bioavailability of TGF-b by inhibiting proteolysis of the proTGF-b precursor to LAP/TGF-b, a complex from which the growth factor can be subsequently released for receptor binding. In the absence of Emilin-1, the amount of active TGF-b is increased. As Emilin-1 is expressed in blood vessels starting from early stages of embryonic development to adulthood, a key question concerning the function of the protein is whether the Emilin1-/- phenotype is the result of a developmental defect or the function of the protein is required for the regulation of blood pressure and arterial structure also in the adult. The conditional gene targeting procedure chosen to inactivate the Emilin1 gene in smooth muscle cells (SMCs) of adult mice included the use of floxed Emilin1 and CreERT2 (a tamoxifen inducible Cre recombinase) under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. Tamoxifen administration induced activity of Cre specifically in vascular and visceral SMCs, as revealed by X-gal staining of tissues from animals with the Rosa26R mutation. When Emilin1flox/flox mice carrying the Smmhc-CreERT2 transgene were given tamoxifen for 7 days, Emilin-1 disappeared completely in 10-12 days from start of treatment. In the same time, blood pressure increased of about 20 mmHg, a level that was stably maintained thereafter. The myogenic response of second branch meseteric arteries, evaluated using a pressure myograph, was found to be increased in Emilin1-/- mice. Additional experiments with aorta and mesenteric artery SMC cultures from control and mutant mice showed that lack of Emilin-1  enhanced phosphorylation of myosin light chain 20 when cells were stimulated with the a1-adrenergic receptor agonists phenylephrine or with angiotensin II. Moreover, basal cytosolic Ca2+ levels and calcium transients induced by stimulation with phenylephrine and angiotensin II were increased in SMCs from Emilin1-/- mutants. The data suggest that Emilin-1 expression is continuously required for regulation of blood pressure and that the increase of TGF-b activity induced by diminished Emilin-1  stimulates, likely through alteration of intracellular calcium homeostasis, contractility of vascular SMC to mechanical and chemical stimuli with ensuing hypertension

Monitoring calcium handling by the plant endoplasmic reticulum with a low‐Ca 2+ ‐affinity targeted aequorin reporter

Precise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca2+-affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non-cleavable N-terminal ER signal peptide (fl2). The correct targeting of fl2-AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well-established in animal cells – consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling – was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+]ER) were recorded in response to different environmental stresses, displaying stimulus-specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set-up of an ER-targeted aequorin chimera extends and complements the currently available toolkit of organelle-targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system

A Novel Function for Fragile X Mental Retardation Protein in Translational Activation

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism