Rare or neglicted diseases

Human erythrocytes release ATP by a novel pathway involving VDAC oligomerization independent of pannexin-1

Scientific Reports 2018, 8: 11834.

We previously demonstrated that the translocase protein TSPO2 together with the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT) were involved in a membrane transport complex in human red blood cells (RBCs). Because VDAC was proposed as a channel mediating ATP release in RBCs, we used TSPO ligands together with VDAC and ANT inhibitors to test this hypothesis. ATP release was activated by TSPO ligands, and blocked by inhibitors of VDAC and ANT, while it was insensitive to pannexin-1 blockers. TSPO ligand increased extracellular ATP (ATPe) concentration by 24–59% over the basal values, displaying an acute increase in [ATPe] to a maximal value, which remained constant thereafter. ATPe kinetics were compatible with VDAC mediating a fast but transient ATP efflux. ATP release was strongly inhibited by PKC and PKA inhibitors as well as by depleting intracellular cAMP or extracellular Ca2+, suggesting a mechanism involving protein kinases. TSPO ligands favoured VDAC polymerization yielding significantly higher densities of oligomeric bands than in unstimulated cells. Polymerization was partially inhibited by decreasing Ca2+ and cAMP contents. The present results show that TSPO ligands induce polymerization of VDAC, coupled to activation of ATP release by a supramolecular complex involving VDAC, TSPO2 and ANT.

TSPO ligands stimulate ZnPPIX transport and ROS accumulation leading to the inhibition of P. falciparum growth in human blood

Scientific Reports 2016, 6: 33516.

After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2–VDAC complex, which leads to an accumulation of ROS.

CMTX Disorder and CamKinase

Frontiers in Cellular Neuroscience, 2016, 10

Charcot-Marie-Tooth disease is a demyelinating peripheral neuropathy and is a heterogeneous inherited disorder (40 loci have been described so far) affecting peripheral nerves. Two forms, CMT1A and CMTX, account for 60 and 15% of patients, respectively, with clear familial transmission. CMTX is caused by mutations in the GBJ1 gene encoding the synthesis of connexin 32 (Cx32), which is a myelin protein related to PNS and CNS. Cx32 is a membrane protein located in gap junctions, which forms hexameric hemichannels called connexons. However, the mechanism by which anomalies in connexin 32 affect myelination and function of PNS remains is still unclear. Here, we share our opinion that CamKinase are involved in the pathophysiology of CMTX and likely involve genomic instability caused by mutations in Gjb1.

CMTX1 patients’ cells present genomic instability corrected by CamKII inhibitors.

Orphanet Journal of Rare Diseases, 2015, 10, 56

BACKGROUND: We previously described that fibroblasts from animal models of CMTX1 present genomic instability and poor connexon activity. In vivo, these transgenic mice present motor deficits. This phenotype could be significantly reverted by treatment with (CamKII) inhibitors. The objective of this study is to translate our findings to patients.
METHODS: We cultured fibroblasts from skin biopsies of CMTX1 patients and analyzed cells for genomic instabilty, connexon activity, and potential correction by CamKII inhibitors.
RESULTS: The phenotypic analysis of these cells confirmed strong similarities between the GJB1 transgenic mouse cell lines and CMTX1 patient fibroblast cell lines. Both present mitotic anomalies, centrosome overduplication, and connexon activity deficit. This phenotype is corrected by CamKII inhibitors.
CONCLUSIONS: Our data demonstrate that fibroblasts from CMTX1 patients present a phenotype similar to transgenic lines that can be corrected by CamKII inhibitors. This presents a track to develop therapeutic strategies for CMTX1 treatment.

A Yeast/Drosophila Screen to Identify New Compounds Overcoming Frataxin Deficiency

Oxidative Medicine and Cellular Longevity, 2015 , in press

Friedreich’s ataxia (FA) is a rare neurodegenerative disease which is very debilitating for the patients who progressively lose their autonomy. The lack of efficient therapeutic treatment of the disease strongly argues for urgent need to search for new active compounds that may stop the progression of the disease or prevent the appearance of the symptoms when the genetic defect is diagnosed early enough. In the present study, we used a yeast strain with a deletion of the frataxin homologue gene as a model of FA cells in a primary screen of two chemical libraries, a fraction of the French National Chemical Library (5500 compounds) and the Prestwick collection (880 compounds). We ran a secondary screen on Drosophila melanogaster flies expressing reduced levels of frataxin during larval development. Half of the compounds selected in yeast appeared to be active in flies in this developmental paradigm, and one of the two compounds with highest activities in this assay partially rescued the heart dilatation phenotype resulting from heart specific depletion of frataxin. The unique complementarity of these two frataxin-deficient models, unicellular and multicellular, appears to be very efficient to select new compounds with improved selectivity, bringing significant perspectives towards improvements in FA therapy.

CamKII inhibitors reduce mitotic instability, connexon anomalies and progression of the in vivo behavioral phenotype in transgenic animals expressing a mutated Gjb1 gene

Frontiers in Neuroscience, 2014, 8, 151

Mutation in the Gjb1 gene, coding for a connexin (Cx32), is associated with an inherited peripheral neuropathic disorder (X-linked Charcot-Marie-Tooth, CMTX). Our previous work reported that transgenic animals expressing a human Gjb1 transgene present polyploidy and abnormal over-duplication of the centrosome, suggesting a role for Gjb1 in mitotic stability. In this article, we propose mechanisms by which mutations in Gjb1 induce mitotic instability and discuss its potential relation with the CMTX phenotype. We showed that transgenic cells exhibit CamKII over-stimulation, a phenomenon that has been linked to mitotic instability (polyploidy, nuclear volume and centrosome over-duplication), that is reversed by CamKII inhibitors. We also demonstrate that connexon activity is partially restored in transgenic cells with CamKII inhibitors. Our model supports the role for Pim1, a kinase that has been associated with genomic instability in cancers, in genomic instability in Cx32 mutations. Regarding in vivo phenotype, we showed that degradation on the rotarod test in our transgenic mice is significantly lowered by treatment with a CamKII inhibitor (KN93). This effect was seen in two lines with different point mutations in GJB1, and stopping the treatment led to degradation of the phenotype.