Deferiprone targets aconitase: Implication for Friedreich's ataxia treatment

Background

Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly affected. As a mean to counteract disease progression, it has been suggested to chelate free mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of availability of deferiprone, a chelator preferentially targeting mitochondrial iron.

Method

Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a particular attention paid to iron-sensitive aconitase activity.

Results

We found that a direct consequence of chelating mitochondrial free iron in various cell systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity was shown to precede decreased cell proliferation.

Conclusion

We conclude that, if chelating excessive mitochondrial iron may be beneficial at some stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in order to avoid undesirable consequences.

     

The variability of the harlequin mouse phenotype resembles that of human mitochondrial-complex I-deficiency syndromes

Background

Despite the considerable progress made in understanding the molecular bases of mitochondrial diseases, no effective treatments have been developed to date. Faithful animal models would be extremely helpful for designing such treatments. We showed previously that the Harlequin mouse phenotype was due to a specific mitochondrial complex I deficiency resulting from the loss of the Apoptosis Inducing Factor (Aif) protein.

Methodology/Principal Findings

Here, we conducted a detailed evaluation of the Harlequin mouse phenotype, including the biochemical abnormalities in various tissues. We observed highly variable disease expression considering both severity and time course progression. In each tissue, abnormalities correlated with the residual amount of the respiratory chain complex I 20 kDa subunit, rather than with residual Aif protein. Antioxidant enzyme activities were normal except in skeletal muscle, where they were moderately elevated.

Conclusions/Significance

Thus, the Harlequin mouse phenotype appears to result from mitochondrial respiratory chain complex I deficiency. Its features resemble those of human complex I deficiency syndromes. The Harlequin mouse holds promise as a model for developing treatments for complex I deficiency syndromes.     

Interactions between oestrogen and 1α,25(OH)<Subscript>2</Subscript>-vitamin D<Subscript>3</Subscript> signalling and their roles in spermatogenesis and spermatozoa functions

Oestrogens and 1α,25(OH)₂-vitamin D₃ (1,25-D₃) are steroids that can provide effects by binding to their receptors localised in the cytoplasm and in the nucleus or the plasma membrane respectively inducing genomic and non-genomic effects. As confirmed notably by invalidation of the genes, coding for their receptors as tested with mice with in vivo and in vitro treatments, oestrogens and 1,25-D₃ are regulators of spermatogenesis. Moreover, some functions of ejaculated spermatozoa as viability, DNA integrity, motility, capacitation, acrosome reaction and fertilizing ability are targets for these hormones. The studies conducted on their mechanisms of action, even though not completely elicited, have allowed the demonstration of putative interactions between their signalling pathways that are worth examining more closely. The present review focuses on the elements regulated by oestrogens and 1,25-D₃ in the testis and spermatozoa as well as the interactions between the signalling pathways of both hormones.     

Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow.

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.     

Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis

In human cells, the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.     

Charybdotoxin-sensitive K(Ca) channel is not involved in glucose-induced electrical activity in pancreatic β-cells

Summary The effects of charybdotoxin (CTX) on single [Ca²⁺] _i -activated potassium channel (K _(Ca)) activity and whole-cell K⁺ currents were examined in rat and mouse pancreatic -cells in culture using the patch-clamp method. The effects of CTX on glucose-induced electrical activity from both cultured -cells and -cells in intact islets were compared. K_(Ca) activity was very infrequent at negative patch potentials (–70<V _m <0 mV), channel activity appearing at highly depolarizedV _m . K_(Ca) open probability at these depolarizedV _m values was insensitive to glucose (10 and 20mm) and the metabolic uncoupler 2,4 dinitrophenol (DNP). However, DNP blocked glucose-evoked action potential firing and reversed glucose-induced inhibition of the activity of K⁺ channels of smaller conductance.The venom fromLeiurus quinquestriatus hebreus (LQV) and highly purified CTX inhibited K_(Ca) channel activity when applied to the outer aspect of the excised membrane patch. CTX (5.8 and 18nm) inhibited channel activity by 50 and 100%, respectively. Whole-cell outward K⁺ currents exhibited an early transient component which was blocked by CTX, and a delayed component which was insensitive to the toxin. The individual spikes evoked by glucose, recorded in the perforated-patch modality, were not affected by CTX (20nm). Moreover, the frequency of slow oscillations in membrane potential, the frequency of action potentials and the rate of repolarization of the action potentials recorded from pancreatic islet -cells in the presence of glucose were not affected by CTX.We conclude that the K_(Ca) does not participate in the steady-state glucose-induced electrical activity in rodent pancreatic islets.