Calcium Signaling and Contractility in Cardiac Myocyte of Wolframin Deficient Rats

Wolframin (Wfs1) is a membrane protein of the sarco/endoplasmic reticulum. Wfs1 mutations are responsible for the Wolfram syndrome, characterized by diabetic and neurological symptoms. Although Wfs1 is expressed in cardiac muscle, its role in this tissue is not clear. We have characterized the effect of invalidation of Wfs1 on calcium signaling-related processes in isolated ventricular myocytes of exon5-Wfs1 deficient rats (Wfs1-e5/-e5) before the onset of overt disease. Calcium transients and contraction were measured in field-stimulated isolated myocytes using confocal microscopy with calcium indicator fluo-3 AM and sarcomere length detection. Calcium currents and their calcium release-dependent inactivation were characterized in whole-cell patch-clamp experiments. At 4 months, Wfs1-e5/-e5 animals were euglycemic, and echocardiographic examination revealed fully compensated cardiac function. In field-stimulated isolated ventricular myocytes, both the amplitude and the duration of contraction of Wfs1-e5/-e5 animals were elevated relative to control Wfs1+/+ littermates. Increased contractility of myocytes resulted largely from prolonged cytosolic calcium transients. Neither the amplitude of calcium currents nor their voltage dependence of activation differed between the two groups. Calcium currents in Wfs1-e5/-e5 myocytes showed a larger extent of inactivation by short voltage prepulses applied to selectively induce calcium release-dependent inactivation of calcium current. Neither the calcium content of the sarcoplasmic reticulum, measured by application of 20 mmol/l caffeine, nor the expression of SERCA2, determined from Western blots, differed significantly in myocytes of Wfs1-e5/-e5 animals compared to control ones. These experiments point to increased duration of calcium release in ventricular myocytes of Wfs1-e5/-e5 animals. We speculate that the lack of functional wolframin might cause changes leading to upregulation of RyR2 channels resulting in prolongation of channel openings and/or a delay in termination of calcium release

Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome

International audienceDeficiency of the protein Wolfram syndrome 1 (WFS1) is associated with multiple neurological and psychiatric abnormalities similar to those observed in pathologies showing alterations in mitochondrial dynamics. The aim of this study was to examine the hypothesis that WFS1 deficiency affects neuronal function via mitochondrial abnormalities. We show that down-regulation of WFS1 in neurons leads to dramatic changes in mitochondrial dynamics (inhibited mitochondrial fusion, altered mitochondrial trafficking, and augmented mitophagy), delaying neuronal development. WFS1 deficiency induces endoplasmic reticulum (ER) stress, leading to inositol 1,4,5-trisphosphate receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn, alters mitochondrial dynamics. Importantly, ER stress, impaired Ca2+ homeostasis, altered mitochondrial dynamics, and delayed neuronal development are causatively related events because interventions at all these levels improved the downstream processes. Our data shed light on the mechanisms of neuronal abnormalities in Wolfram syndrome and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases

Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model

A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 μg/cm² exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n = 9-13). PLGA-PEO NPs were not toxic up to dose 3 μg/cm²; dose of 75 μg/cm² displays significant decrease in [³H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM > CON A, and no changes in PHA cultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) > monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised DNA bases in PLGA-PEO-treated cells. To conclude on immuno- and genotoxicity of PLGA-PEO NPs, more experiments with various particle size, charge and composition need to be done

WFS1 deficiency decreases mitochondrial membrane potential and cytosolic ATP level.

<p>(A) Primary cortical neurons were transfected with control or <i>Wfs1</i> siRNA using the N-TER nanoparticle siRNA transfection system and stained with JC-10, which emits light from 525 nm to 590 nm, depending on mitochondrial membrane potential. Values shown are corrected by subtracting the values obtained in the presence of FCCP incubation (5 μM). The red to green fluorescence ratio demonstrated a slight but significant decrease in the <i>Wfs1</i> siRNA group. (B) Neurons were transfected with plasmids expressing scrambled shRNA or <i>Wfs1</i> shRNA, firefly luciferase construct containing NRF2 binding site, and <i>Renilla</i> luciferase. Firefly luciferase signal normalized to <i>Renilla</i> signal demonstrates no change in NRF2 activity. NRF2 overexpression-induced reporter activity was used as a positive control. (C) Neurons transfected with the ATP sensor ATeam and treated with 2-deoxyglucose (12 mM)/oligomycin (2.5 μM) or glutamate (2 mM) (both used as positive controls) show a decrease in relative cytosolic ATP levels. (D) Neurons were transfected with the ATP sensor ATeam and scrambled or <i>Wfs1</i> shRNAs. WFS1-deficient neurons show a lower cytosolic ATP level as compared to control. **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 compared with respective control group. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002511#pbio.1002511.s001" target="_blank">S1 Data</a>.</p

Parameters of mitochondrial trafficking in neurons transfected with scrambled shRNA or <i>Wfs1</i> shRNA.

<p>Parameters of mitochondrial trafficking in neurons transfected with scrambled shRNA or <i>Wfs1</i> shRNA.</p

WFS1 deficiency induces mild ER stress in primary cortical neurons.

<p>(A) Neurons were transfected with plasmids expressing scrambled shRNA or <i>Wfs1</i> shRNA, firefly luciferase constructs containing ATF6 or ATF4 binding sites or a XBP-1 splicing reporter, and <i>Renilla</i> luciferase. Firefly luciferase signal normalized to <i>Renilla</i> signal demonstrates a moderate increase in ATF6 and ATF4 reporter activity. (B) Positive control experiments in which the above-mentioned reporter systems and <i>Renilla</i> luciferase were co-transfected with ATF4, ATF6, or IRE1. (C) The mitochondrial fusion rate is reduced by <i>Wfs1</i> shRNA and is restored by co-expressing wt HSPA5 (<i>p</i> = 0.001 for interaction, two-way ANOVA). (D) ATPase-deficient HSPA5 mutant (T37G) but not peptide binding-deficient mutant (P495L) restores the fusion rate reduced by <i>Wfs1</i> shRNA. (E) HSPA5 overexpression attenuates mitophagy activated by <i>Wfs1</i> silencing (<i>p</i> = 0.012 for interaction). (F–H) Activation of the primary ER stress pathways by overexpression of ATF6, ATF4, or IRE1 modulates neither fusion rate (F), mitochondrial length (G), nor mitophagy (H). (I–L) Silencing of <i>ATF6</i> or <i>ATF4</i> modulates neither fusion rate (I, J) nor mitophagy (K, L). *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared with respective control groups, or ^###<i>p</i> < 0.001 compared with the <i>Wfs1</i> shRNA-transfected control group and ^ns non-significant compared with the <i>Wfs1</i> shRNA-transfected control group. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002511#pbio.1002511.s001" target="_blank">S1 Data</a>.</p

WFS1 deficiency impairs mitochondrial dynamics.

<p>(A) Primary cortical neurons were transfected with the photoconvertible mitochondrially targeted construct mito-Kikume-Green and scrambled shRNA or <i>Wfs1</i> shRNA. Selected mitochondria were irradiated using a 405-nm laser line, thereby converting mito-Kikume-Green into mito-Kikume-Red. Fusion events between mito-Kikume-Green and photoactivated mito-Kikume-Red mitochondria are visible when mitochondria become yellow after mixing of the contents of the red and green mitochondria. (B–E) In primary cortical neurons, <i>Wfs1</i> shRNA significantly decreases fusion rate (B) and mitochondrial length (C). These parameters are restored by overexpression of wild-type (wt) WFS1 but not by P724L WFS1, a mutant found in Wolfram syndrome. Similar changes are observed in cortical neurons isolated from <i>Wfs1</i>^-/- mice. The lower fusion rate (D) and reduced mitochondrial length (E) in <i>Wfs</i>1^-/- neurons is restored by wt WFS1 overexpression, but <i>Wfs1</i> shRNA has no effect on these parameters. (F–H) Primary cortical neurons were transfected with mitochondrially targeted Keima (which changes its excitation spectrum under acidic conditions) and scrambled shRNA or <i>Wfs1</i> shRNA (F). The number of autolysosomes containing mitochondria increases in <i>Wfs1</i>-silenced neurons (G) and in neurons isolated from <i>Wfs1</i>^-/- mice (H). (I–K) Representative images of mitochondrial morphology and density in the axons of scrambled- and <i>Wfs1</i>-shRNA transfected neurons (I). The density of axonal mitochondria is reduced in <i>Wfs1</i>-silenced neurons (J) and in neurons isolated from <i>Wfs1</i>^-/- mice (K). This parameter is restored by wt WFS1 overexpression, but <i>Wfs1</i> shRNA has no effect. *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared with respective control groups. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002511#pbio.1002511.s001" target="_blank">S1 Data</a>.</p

WFS1 deficiency leads to impaired neuronal development.

<p>Primary cortical neurons were transfected with the neuronal marker pAAV-hSyn-DsRed1 and scrambled shRNA or <i>Wfs1</i> shRNA at DIV (day in vitro) 1, and neuronal morphology was assessed at different time points. (A) Cell morphology at different stages of development. (B) Morphological analysis demonstrating the retarded development of WFS1-deficient neurons. (C) Examples of reconstructed control and WFS1-deficient neurons at different DIV. (D–F) WFS1 deficiency retards growth of the longest axon (D) and growth of the axonal tree (E) and decreases the number of axon tips (F). (G) Survival of WFS1-deficient neurons is decreased when compared with control neurons (<i>n</i> = 61–62 individual dishes from 17 independent sister cultures at DIV 6–11). (H) Visualisation of synapses (red) using an antibody targeted against the post-synaptic marker PSD-95 in neurons transfected with GFP (green). The right panel shows a zoomed image. (I) WFS1-deficiency decreases synaptic density at late stages. *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared with respective scrambled shRNA-treated groups. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002511#pbio.1002511.s001" target="_blank">S1 Data</a>.</p