mTORC1 and PKB/Akt control the muscle response to denervation by regulating autophagy and HDAC4

Loss of innervation of skeletal muscle is a determinant event in several muscle diseases. Although several effectors have been identified, the pathways controlling the integrated muscle response to denervation remain largely unknown. Here, we demonstrate that PKB/Akt and mTORC1 play important roles in regulating muscle homeostasis and maintaining neuromuscular endplates after nerve injury. To allow dynamic changes in autophagy, mTORC1 activation must be tightly balanced following denervation. Acutely activating or inhibiting mTORC1 impairs autophagy regulation and alters homeostasis in denervated muscle. Importantly, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogates denervation-induced synaptic remodeling and causes neuromuscular endplate degeneration. We establish that PKB/Akt activation promotes the nuclear import of HDAC4 and is thereby required for epigenetic changes and synaptic gene up-regulation upon denervation. Hence, our study unveils yet-unknown functions of PKB/Akt-mTORC1 signaling in the muscle response to nerve injury, with important implications for neuromuscular integrity in various pathological conditions

A Randomized, Double Blind, Placebo-Controlled Trial of Pioglitazone in Combination with Riluzole in Amyotrophic Lateral Sclerosis

BACKGROUND: Pioglitazone, an oral anti-diabetic that stimulates the PPAR-gamma transcription factor, increased survival of mice with amyotrophic lateral sclerosis (ALS). METHODS/PRINCIPAL FINDINGS: We performed a phase II, double blind, multicentre, placebo controlled trial of pioglitazone in ALS patients under riluzole. 219 patients were randomly assigned to receive 45 mg/day of pioglitazone or placebo (one: one allocation ratio). The primary endpoint was survival. Secondary endpoints included incidence of non-invasive ventilation and tracheotomy, and slopes of ALS-FRS, slow vital capacity, and quality of life as assessed using EUROQoL EQ-5D. The study was conducted under a two-stage group sequential test, allowing to stop for futility or superiority after interim analysis. Shortly after interim analysis, 30 patients under pioglitazone and 24 patients under placebo had died. The trial was stopped for futility; the hazard ratio for primary endpoint was 1.21 (95% CI: 0.71-2.07, p = 0.48). Secondary endpoints were not modified by pioglitazone treatment. Pioglitazone was well tolerated. CONCLUSION/SIGNIFICANCE: Pioglitazone has no beneficial effects on the survival of ALS patients as add-on therapy to riluzole. TRIAL REGISTRATION: Clinicaltrials.gov NCT00690118

Genetic correlation between amyotrophic lateral sclerosis and schizophrenia

A. Palotie on työryhmän Schizophrenia Working Grp Psychiat jäsen.We have previously shown higher-than-expected rates of schizophrenia in relatives of patients with amyotrophic lateral sclerosis (ALS), suggesting an aetiological relationship between the diseases. Here, we investigate the genetic relationship between ALS and schizophrenia using genome-wide association study data from over 100,000 unique individuals. Using linkage disequilibrium score regression, we estimate the genetic correlation between ALS and schizophrenia to be 14.3% (7.05-21.6; P = 1 x 10(-4)) with schizophrenia polygenic risk scores explaining up to 0.12% of the variance in ALS (P = 8.4 x 10(-7)). A modest increase in comorbidity of ALS and schizophrenia is expected given these findings (odds ratio 1.08-1.26) but this would require very large studies to observe epidemiologically. We identify five potential novel ALS-associated loci using conditional false discovery rate analysis. It is likely that shared neurobiological mechanisms between these two disorders will engender novel hypotheses in future preclinical and clinical studies.Peer reviewe

Analyse der Funktion von MORF-Proteinen im mitochondrialen RNA-Editing in Arabidopsis thaliana

Durch den Prozess des RNA-Editing werden in mitochondrialen Transkripten der Modellpflanze Arabidopsis thaliana 400-600 Cytidine zu Uridinen umgewandelt. Möglich wird diese Veränderung der RNA-Sequenz durch einen Proteinkomplex, der sich aus verschiedenen Editingfaktoren zusammensetzt. Zwei Hauptinteraktionspartner stellen dabei die PPR-Proteine (PPR: Pentatricopeptide Repeat) und die MORF-Proteine (MORF: Multiple Organellar RNA Editing Factor) dar. Es war bereits bekannt, dass das PPR-Protein MEF13 am Editing der betreffenden Cytidine in den Transkripten ccb452-50, cox3-314, nad2-59, nad4-158, nad5-1665, nad5-1916 und nad7-213 beteiligt ist. Durch die Analyse der EMS-Linie mef13-1 konnte bewiesen werden, dass dieser Editingfaktor ebenfalls an der Modifikation der Editingstelle ccb452-415 teilhat. Im Vergleich zum Wildtyp Columbia wies die Mutante mef13-1 ein verlangsamtes Wachstum auf. Dieser Makrophänotyp konnte durch die Transformation von mef13-1 mit dem Transgen MEF13 aufgehoben werden. Alle genannten Editingstellen weisen ebenfalls verminderte Editingeffizienzen in den Mutationslinien morf3/rip3 und morf8/rip1 auf. Mittels einer Yeast Three-Hybrid-Analyse konnte belegt werden, dass die zusätzliche Expression des Proteins MORF8 einen stabilisierenden Effekt auf die Interaktion von MORF3 und MEF13 ausübt. Diese Stabilisierung konnte nicht durch die Expression des Editingfaktors MORF1 beobachtet werden. Dies weist auf eine funktionelle Einheit des PPR-Proteins MEF13 mit einem MORF3-MORF8-Heteromer hin. Alle MORF-Proteine beinhalten einen ca. 100 Aminosäuren umfassenden konservierten Bereich, der als MORF-Box bezeichnet wird. Dieses Areal kann wiederum in vier Sequenzmotive unterteilt werden, welche innerhalb dieser Proteinfamilie, bezüglich ihrer Position und Aminosäureausstattung, leicht variieren. Jedes dieser konservierten Motive stellt ein potentielles Interaktionsareal für die Bildung von Homo- und Heteromeren mit weiteren Editingfaktoren dar. Mittels einer Yeast Two-Hybrid-Analyse wurde untersucht, ob verschiedene Deletionskonstrukte des trans-Faktors MORF1 zur Bildung von Homo- respektive Heteromeren fähig waren. Hierbei konnte belegt werden, dass die Interaktion zwischen MORF1 und MORF3 hauptsächlich über die konservierten Motive 2 und 3 vermittelt wird. Diese beiden Motive befinden sich in der C-terminalen Hälfte der MORF-Box. In der EMS-Linie morf1-1 wurde untersucht, ob chimäre Konstrukte aus MORF1 und MORF3 in der Lage waren, Editing an den betroffenen Cytidinen wieder herzustellen. Zu diesem Zweck wurden die Chimären als Transgene in morf1-1 exprimiert. Die cDNA verschiedener Transkripte wurde auf die Effizienz des C-zu-U-Editing hin überprüft. Die Positionen der Verbindungsstellen zwischen den MORF1- bzw. MORF3-basierten Sequenzen der chimäre Konstrukte wurden so gewählt, dass jeweils mindestens eines der konservierten Motive, zwischen den schlussendlich exprimierten Proteinen, ausgetauscht wurde. Mit wenigen Ausnahmen konnte gezeigt werden, dass Chimären deren N-Terminus MORF3-basiert war, nicht in der Lage sind, den Editingdefekt in morf1-1 auszugleichen. Es wurden ebenfalls Chimären hergestellt, deren Termini in umgekehrter Orientierung angeordnet waren. Folglich basierten die N-terminalen Sequenzen auf MORF1 und der C-Terminus war MORF3-kodiert. Diese chimären Konstrukte waren größtenteils in der Lage, den Editingdefekt in morf1-1 auszugleichen. Ein Vergleich mit bekannten Editingfaktoren belegte, dass die Wiederherstellung einer geeignet hohen Editingeffizienz an Stellen auftrat, deren cis-Elemente von PPR-Proteinen der E-Unterklasse erkannt werden. Demgegenüber waren Vertreter dieser Chimären nicht in der Lage Editing an Stellen zu erzeugen, die von PPR-Proteinen der DYW-Untergruppe erkannt werden

The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana.

RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C) to uridine (U) mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35) to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209), for cytochrome b (cob-286) and for subunit 4 of complex I (nad4-1373), respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins

The <i>rpl16</i>-209 editing site is located in a highly conserved environment.

<p><b>(A)</b> Comparison of nucleotide identities in the <i>cis</i>-recognition sequence around the <i>rpl16</i>-209 editing site with the homologous editing sites in other plant species reveals the high degree of conservation. This is presumably imposed by the functional constraints on the conservation of the amino acids surrounding the <i>rpl16</i>-209 editing site. Some of the plants compared here encode a genomic T at this position to maintain the amino acid identity. <b>(B)</b> The absence of RNA editing event <i>rpl16</i>-209 results in <i>Arabidopsis</i> in the incorporation of the genomically encoded threonine rather than the isoleucine specified by the edited codon number 70. The amino acid isoleucine is conserved in even distant plant species. Nucleotides and amino acids derived by RNA editing are given in bold letters and are underlined. Nucleotides and amino acids differing from the consensus are shown in inverse shading.</p

MEF35 interacts with the MORF1, MORF2 and MORF8 proteins.

<p>Yeast 2-hybrid (Y2H) assays reveal interactions with the mitochondrially located MORF1, the plastid MORF2 and the dual targeted MORF8. In a mutant of MORF1, the MEF35 target sites are not affected [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.ref022" target="_blank">22</a>], some slight effects are seen upon knock-down in other assays [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.ref028" target="_blank">28</a>]. In a respective MORF8 (also termed RIP1) mutant, editing at two of the three MEF35 target sites is reduced, suggesting that the interactions with MORF8 maybe functionally relevant. Controls include cotransfection of the pGBKT7-MEF35 construct with the pGADT7 vector without any MORF insert (empty) as well as the positive interaction control of murine p53 (pGBKT7-53) with the SV40 large T-antigen in pGADT7 (T) and no connection of human lamin C (pGBKT7-Lam) with the SV40 large T-antigen (T). The fusion protein pGBKT7-53 does not interact with the protein product of the empty pGADT7 vector (empty).</p

The MEF35 protein is required for RNA editing at the <i>rpl16</i>-209, <i>nad4</i>-1373 and <i>cob</i>-286 editing sites in mitochondria of <i>Arabidopsis thaliana</i>.

<p><b>(A)</b> Schematic structure of the MEF35 PPR-protein encoded by locus At4g14050. Locations of the T-DNA insertions in the two mutants and their respective left borders (LB) are indicated. The predicted types of PPR elements, P, L or S, as well as the E and DYW motifs are labelled. The region marked E+ is also considered part of the E domain. <b>(B)</b> Analysis of editing in <i>mef35-1</i> and <i>mef35-2</i> mutant plants. Comparison of the cDNA sequence analysis of three RNA editing sites (boxed) in the mitochondrial <i>rpl16</i>, <i>nad4</i> and <i>cob</i> mRNAs between wild type <i>Arabidopsis thaliana</i> (wt) and the <i>mef35-1</i> and <i>mef35-2</i> mutant plants shows that both mutants have lost the ability of C to U editing at these sites. Three other editing sites in the respective same mRNAs are shown as controls, these sites are correctly edited in wild type and both mutant plants. In the cDNA strands analysed, the detected T nucleotide (red trace) corresponds to the edited U, the observed C (blue trace) is derived from an unedited C. The right hand analyses (<i>mef35-2+MEF35</i>) show that the Col <i>MEF35</i> gene sequence restores the ability for RNA editing in transgenic plants of mutant line <i>mef35-2</i>. (C) The plants of mutant line <i>mef35-1</i> show developmental and adult phenotypes indistinguishable from the wild type. Seedlings of mutant line <i>mef35-2</i> initially develop a little slower in comparison to the wild type Col plantlets, but look similar at the flowering stage (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.s001" target="_blank">S1 Fig</a>). Transgenic complemented plants of <i>mef35-2</i> also display the retarded growth indicating that this phenotype is not related to the <i>mef35-2</i> T-DNA insertion. Four weeks old plants grown side by side are shown.</p