PKC Signaling Is Impaired in BDV-Infected Neurons

<p>Western blots of neuronal extracts from NI and BDV-infected cultures stimulated with glycine using antibodies specific for: (A) Diphosphorylated ERK 1/2 (pERK) and total ERK 1/2; (B) phospho-synapsin I (pSynapsin I; Site 3, a site specific for CaMK II), and total synapsin I; (C) phospho-synapsin I (Site 1, a site specific for PKA and CamK I) and total synapsin I; (D) phospho-MARCKS (pMARCKS; PKC site at Ser152/156) and β-tubulin III. Results are representative of four independent experiments. Single asterisk (*) indicates <i>p</i> < 0.05; double asterisks (**) indicate <i>p</i> < 0.01 by unpaired <i>t</i> test.</p

Spontaneous and Evoked SV Recycling Are Not Affected by BDV Infection

<div><p>(A) Cumulative probability distributions of fluorescence ratios (F1/F2) for individual synapses for spontaneous SV recycling. No difference is observed between control (NI) and BDV-infected neurons (<i>p</i> = 0.112, KS test).</p><p>(B) Cumulative probability distributions of fluorescence ratios (F1/F2) for individual synapses for 70 mM K⁺ evoked and spontaneous SV recycling. In both NI and BDV-infected neurons, the level of SV recycling is significantly increased in neurons stimulated with 70 mM K⁺ (<i>p</i> < 0.001, KS test). The overview of the experimental stimulation protocol is depicted above each graph.</p><p>(C) Pairwise comparisons of mean presynaptic activities in independent experiments involving at least 300 synapses (small symbols). Bigger squares correspond to mean values of all independent experiments. n.s., not significant using paired <i>t</i> test.</p></div

PMA, but Not Forskolin-Induced Potentiation of Vesicular Recycling Is Impaired in BDV-Infected Neurons

<p>Cumulative probability distributions of fluorescence ratios (F1/F2) for individual synapses for the analysis of SV recycling in spontaneous conditions and after (A) PMA treatment or (B) forskolin treatment. The overview of the experimental stimulation protocol is depicted above each graph. The level of SV recycling is significantly lower in BDV-infected neurons compared to NI neurons after stimulation with PMA (<i>p</i> < 0.001 using KS test). Pairwise comparisons of mean presynaptic activities in independent experiments involved at least 180 synapses (small symbols). Bigger squares correspond to mean values of all independent experiments. Similar results were found in four independent experiments. Also shown are results for NI neurons treated with the PKC inhibitor Bis or the PKA inhibitor H89. Hash mark (#) indicates <i>p</i> < 0.05. n.s., not significant, by paired <i>t</i> test.</p

Synapse Integrity Is Preserved in Neurons Persistently Infected with BDV

<div><p>(A) Western blots of neuronal extracts. Comparative analysis of the expression levels of a panel of neuronal proteins from non-infected (NI) and BDV-infected cultures. Results are representative of three independent experiments.</p><p>(B) Outline of the experimental protocol to measure vesicular cycling at synapses. In a first step, recycling vesicles are labeled with a mouse monoclonal antibody directed against the luminal domain of synaptotagmin 1 (Syt-ecto). After washes, neurons are fixed and permeabilized and incubated with a rabbit antibody directed against the cytoplasmic domain of synaptotagmin 1 (Total-Syt).</p><p>(C) Examples of Syt-ecto and total-Syt labeling. Bottom panels show neurons treated with botulinum B toxin (Bot B). Bar: 20 μm.</p><p>(D) Quantification of total-Syt fluorescence staining (<i>n</i> = 381 and 386 synapses for BDV-infected and NI neurons, respectively).</p></div

Analysis of PKC Signaling in NI and BDV-Infected Neurons

<p>Western blot analyses of (A) MARCKS and (B) Munc18 phosphorylation by PKC. β-Tubulin III and total Munc18 were used to normalize expression. Results are representative of three to five independent experiments. Double asterisks (**) indicate <i>p</i> < 0.01. n.s., not significant, using unpaired <i>t</i> test.</p

Analysis of PKC Activation

<div><p>(A) Cytosol-to-membrane recruitment of PKC. Subcellular distribution of PKC isoforms in NI and BDV-infected neurons after treatment with PMA. The detergent-soluble and particulate fractions, corresponding to cytosolic and membrane fractions, were subjected to Western blots for the detection of PKCα, PKCγ, PKCɛ, and PKCδ. Note the shift in the subcellular distribution of PKC isoforms after treatment with PMA. Results are representative of three independent experiments.</p><p>(B) In vitro measurements of basal and cofactor-stimulated PKC activities. The cofactor-stimulated specific activity represents kinase activity in the presence of Ca²⁺ and lipid activators. Results are expressed as percentage of increase over the mean of unstimulated controls.</p></div

Rotenone potentiates RLR signalling.

<p>HEK 293T cells were treated with 1 μM rotenone alone or in combination with 0.1 μg/ml or 1 μg/ml of the intracellularly delivered RLR ligand poly(I:C)/Lyovec (poly(I:C)-LV) for 16h. <b>(A)</b> IFNβ mRNA levels and <b>(B)</b> CXCL10 mRNA levels were quantified by qRT-PCR and normalized to the level of β actin mRNA. Shown are the mean ± standard error of the mean (sem) of three independent experiments. *, p ≤ 0.05</p

Rotenone and Sendai virus infection trigger oligomerization of the TM domain of the unrelated outer mitochondrial membrane protein OMP25.

<p><b>(A)</b> Schema of Flag_PERK-KD_OMP25-TM, a N-terminally Flag-tagged fusion protein between the kinase domain of PERK and the TM domain of OMP25, an outer mitochondrial membrane protein. The construct encoding this protein was introduced into Flp-In T-Rex-293 cells by recombination <b>(B)</b> Intracellular localization of Flag_PERK-KD_OMP25-TM following doxycycline induction in Flp-In T-Rex-293 cells. Cells were stained for the Flag tag and the mitochondrial specific dye MitoTracker. Images were acquired sequentially on a confocal microscope. The merge panel shows an overlap of Flag and Mitotracker signals. <b>(C)</b> Immunoblot of Flag_PERK-KD fused to the TM domain of MAVS (MAVS-TM) or to the TM domain of OMP25 (OMP25-TM). Cells were treated with 10 μM rotenone (Rot) for 90 min, or infected with 100 HA/ml Sendai virus for 16h (SeV). The percentage of PERK-KD phosphorylation is indicated under each sample.</p

Increased MAVS-TM domain oligomerization in cells with elevated reactive oxygen species.

<p><b>(A)</b> Schema of Flag_PERK-KD_MAVS-TM, a N-terminally Flag-tagged fusion protein between the kinase domain of PERK and the TM domain of MAVS. The construct encoding this protein was introduced into Flp-In T-Rex-293 cells by recombination <b>(B)</b> Intracellular localization of Flag_PERK-KD_MAVS-TM following doxycycline induction in Flp-In T-Rex-293 cells. Cells were stained for the Flag tag and the mitochondrial specific dye MitoTracker. Images were acquired sequentially on a confocal microscope. The merge panel shows an overlap of Flag and Mitotracker signals. <b>(C)</b> Immunoblot of Flag_PERK-KD_MAVS-TM immunopurified from Flp-In T-Rex-293 cells. Cells were either left untreated (non-induced) or exposed to doxycycline to induce the expression of Flag_PERK-KD_MAVS-TM. In addition, cells were treated for 90min with the indicated doses of rotenone. The position of hypophosphorylated (0) and phosphorylated (P) Flag_PERK-KD_MAVS-TM is indicated in the top panel. The bottom panel is an immunoblot of the same samples treated in vitro with λ-Phosphatase. <b>(D)</b> Immunoblot of Flag_PERK-KD_MAVS-TM immunopurified from Flp-In T-Rex-293 cells treated with 10 μM rotenone or 40 μg/ml antimycin A for the indicated time. <b>(E)</b> Immunoblot of Fv2E-PERK, immunopurified from cells treated with 10 μM rotenone (Rot) or 40 μg/ml antimycin A (Ant A), or with the Fv2E-PERK dimerizing ligand AP20187 (AP) at 10 nM for 90 min. <b>(F)</b> Immunoblot of Flag_PERK-KD_MAVS-TM immunopurified from Flp-In T-Rex-293 cells exposed for 2 hours to the indicated concentrations of hydrogen peroxide. The percentage of PERK-KD phosphorylation is indicated under each sample.</p

Proposed model for mROS effects on MAVS signalling.

<p>MAVS is predominantly monomeric on the outer mitochondrial membrane of cells with low levels of mROS (top panel). Increased mROS production promotes lipid-dependent oligomerization of MAVS TM domain (lower panel). Upon viral infection, RLRs bind to viral RNA and expose their CARD domain, which is now available to interact with the CARD domain of MAVS. RLR induced MAVS CARD domains homo-oligomerization is required for MAVS signalling. Lipid-dependent TM domain oligomerization could prime MAVS for RLR-induced MAVS CARD domains homooligomerization, therefore leading to increased MAVS signalling.</p