A Tail-Anchored Myotonic Dystrophy Protein Kinase Isoform Induces Perinuclear Clustering of Mitochondria, Autophagy, and Apoptosis

Contains fulltext : 79678.pdf (publisher's version ) (Open Access)BACKGROUND: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. CONCLUSION/SIGNIFICANCE: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients

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The role of the melanocortin (MC) system in feeding behavior is well established. Food intake is potently suppressed by central infusion of the MC 3/4 receptor agonist α-melanocyte stimulating hormone (α-MSH), whereas the MC 3/4 receptor inverse-agonist Agouti Related Peptide (AGRP) has the opposite effect. MC receptors are widely expressed in both hypothalamic and extra-hypothalamic brain regions, including nuclei involved in food reward and motivation, such as the nucleus accumbens (NAc) and the ventral tegmental area. This suggests that MCs modulate motivational aspects of food intake. To test this hypothesis, rats were injected intracerebroventricularly with α-MSH or AGRP and their motivation for sucrose was tested under a progressive ratio schedule of reinforcement. Food motivated behavior was dose-dependently decreased by α-MSH. Conversely, AGRP increased responding for sucrose, an effect that was blocked by pretreatment with the dopamine receptor antagonist α-flupenthixol. In contrast to progressive ratio responding, free intake of sucrose remained unaltered upon α-MSH or AGRP infusion. In addition, we investigated whether the effects of α-MSH and AGRP on food motivation were mediated by the NAc shell. In situ hybridization of MC3 and MC4 receptor expression confirmed that the MC4 receptor was expressed throughout the NAc, and injection of α-MSH and AGRP into the NAc shell caused a decrease and an increase in motivation for sucrose, respectively. These data show that the motivation for palatable food is modulated by MC4 receptors in the NAc shell, and demonstrate cross-talk between the MC and dopamine system in the modulation of food motivation

Semen cryopreservation and usage rate for assisted reproductive technology in 898 men with cancer

An undesired side effect of cancer treatment is potential subfertility or infertility. Timely cryopreservation of semen is the best modality to ensure fertility. This retrospective data analysis established the usage rate of cryopreserved semen from cancer patients. Pubertal and post-pubertal patients who could become infertile as a result of cancer (treatment) were offered the option to cryopreserve semen prior to treatment. Of the 898 patients who cryopreserved their semen in our hospital, 96 (10.7%) used this for assisted reproductive technology. The live birth rates for intrauterine insemination, in-vitro fertilization, intracytoplasmic sperm injection and cryopreserved embryo transfer were 13%, 29%, 32% and 17%, respectively. Of all couples involved, 77% achieved parenthood, i.e. 60 of the 78 patients (with complete follow-up) fathered at least one child

Divergent Mitochondrial and Endoplasmic Reticulum Association of DMPK Splice Isoforms Depends on Unique Sequence Arrangements in Tail Anchors

Myotonic dystrophy protein kinase (DMPK) is a Ser/Thr-type protein kinase with unknown function, originally identified as the product of the gene that is mutated by triplet repeat expansion in patients with myotonic dystrophy type 1 (DM1). Alternative splicing of DMPK transcripts results in multiple protein isoforms carrying distinct C termini. Here, we demonstrate by expressing individual DMPKs in various cell types, including C(2)C(12) and DMPK(−/−) myoblast cells, that unique sequence arrangements in these tails control the specificity of anchoring into intracellular membranes. Mouse DMPK A and C were found to associate specifically with either the endoplasmic reticulum (ER) or the mitochondrial outer membrane, whereas the corresponding human DMPK A and C proteins both localized to mitochondria. Expression of mouse and human DMPK A—but not C—isoforms in mammalian cells caused clustering of ER or mitochondria. Membrane association of DMPK isoforms was resistant to alkaline conditions, and mutagenesis analysis showed that proper anchoring was differentially dependent on basic residues flanking putative transmembrane domains, demonstrating that DMPK tails form unique tail anchors. This work identifies DMPK as the first kinase in the class of tail-anchored proteins, with a possible role in organelle distribution and dynamics

Central melanocortins regulate the motivation for sucrose reward

The role of the melanocortin (MC) system in feeding behavior is well established. Food intake is potently suppressed by central infusion of the MC 3/4 receptor agonist α-melanocyte stimulating hormone (α-MSH), whereas the MC 3/4 receptor inverse-agonist Agouti Related Peptide (AGRP) has the opposite effect. MC receptors are widely expressed in both hypothalamic and extra-hypothalamic brain regions, including nuclei involved in food reward and motivation, such as the nucleus accumbens (NAc) and the ventral tegmental area. This suggests that MCs modulate motivational aspects of food intake. To test this hypothesis, rats were injected intracerebroventricularly with α-MSH or AGRP and their motivation for sucrose was tested under a progressive ratio schedule of reinforcement. Food motivated behavior was dose-dependently decreased by α-MSH. Conversely, AGRP increased responding for sucrose, an effect that was blocked by pretreatment with the dopamine receptor antagonist α-flupenthixol. In contrast to progressive ratio responding, free intake of sucrose remained unaltered upon α-MSH or AGRP infusion. In addition, we investigated whether the effects of α-MSH and AGRP on food motivation were mediated by the NAc shell. In situ hybridization of MC3 and MC4 receptor expression confirmed that the MC4 receptor was expressed throughout the NAc, and injection of α-MSH and AGRP into the NAc shell caused a decrease and an increase in motivation for sucrose, respectively. These data show that the motivation for palatable food is modulated by MC4 receptors in the NAc shell, and demonstrate cross-talk between the MC and dopamine system in the modulation of food motivation

An intact microtubular cytoskeleton enhances hDMPK A–induced perinuclear mitochondrial clustering.

<p><i>DMPK KO</i> myoblasts were transduced with YFP-hDMPK A or C-expressing adenoviruses in the presence of cytochalasin D (A) or nocodazole (B). F-actin was visualized by fluorescent phalloidin. The microtubular cytoskeleton was stained with an anti-tubulin antibody. Disruption of the actin cytoskeleton did not affect localization of mitochondria. Depolymerization of microtubules decreased mitochondrial clustering in YFP-hDMPK A-expressing cells, but mitochondria still appeared fragmented. The distribution of mitochondria in YFP-hDMPK C-transduced cells was unaffected by nocodazole treatment. Bars, 10 µm. (C) Quantification of mitochondrial clustering. The number of transduced cells that contain clustered mitochondria are expressed as percentage of the total amount of cells expressing hDMPK A at the MOM, with or without treatment of cytochalasin D or nocodazole (images shown in A and B; n = 3, ∼100 cells per experiment, P = 0.01). (D) Effect of nocodazole wash-out. Quantification of the percentage transduced cells with clustered mitochondria after a 12–16 hours treatment with nocodazole, followed by a 8 hours wash-out (n = 3, ∼30 cells per experiment, P<0.05).</p

Perinuclear clustering of mitochondria is induced by the C-terminal domain of hDMPK A.

<p>(A,B) C₂C₁₂ myoblasts transiently expressing YFP-hDMPK A or C fusion proteins or mock-transfected cells were stained with a cytochrome c oxidase antibody to visualize mitochondria. Typical examples of classes of mitochondrial morphology are shown in (A). Mitochondrial distribution was classified as fragmented, clustered or elongated. Frequencies of appearance are listed as percentage of the total number of cells expressing MOM-associated hDMPK A (n = 3, ∼50 cells analyzed per experiment). Around 40% of YFP-hDMPK A-expressing cells showed a cytosolic expression. These cells contained mitochondria with a typically elongated shape, but were disregarded in the analysis. (C) Schematic representation of YFP constructs used for transfection expression with protein domains, mutations and 3′ UTR indicated. (D) C₂C₁₂ myoblasts expressing YFP fusion proteins were stained with a cytochrome c oxidase antibody to visualize their ability to induce clustering of mitochondria. (E) Expression of constructs with altered 3′ UTRs demonstrated that the hDMPK A 3′ UTR was not involved in mitochondrial clustering. (F) Co-expression of YFP-hDMPK A and hDMPK C-HA in N2A cells resulted in mitochondrial clustering, whereas cells only expressing hDMPK C-HA exhibited normal, elongated mitochondria. Bars, 10 µm.</p

Expression of hDMPK A induces apoptosis.

<p>(A) The effect of apoptosis-inhibitor z-vad-fmk was tested on cell survival of DMPK KO myoblasts transduced with YFP-hDMPK A or C–expressing adenoviruses. YFP-positive cells were counted after 16, 24, and 48 hours. Z-vad-fmk greatly reduced cell death of YFP-hDMPK A–expressing cells, but had no effect when YFP-hDMPK C was expressed. Z-vad-fmk was applied immediately after transduction and maintained present for the remaining time of the experiment. Values at 16 hours were set at 100% (P<0.05, n = 3, >90 cells counted per experiment). (B) YFP-hDMPK A and C–expressing cells were stained for cytochrome c (cyt c). Cells expressing YFP-hDMPK A displayed a diffuse, cytosolic cytochrome c staining when mitochondria were clustered (upper panels). A clear mitochondrial staining was present when mitochondria appeared fragmented (middle panels). A discrete, mitochondrial cytochrome c staining was also observed in YFP-hDMPK C–expressing cells. Bars, 10 µm.</p

Human DMPK A expression affects mitochondrial function and cell viability.

<p>(A) C₂C₁₂ myoblasts were grown under different culture conditions and transfected with YFP-hDMPK A or C. The fraction of viable YFP positive cells was determined after 20 hours. When supplied with galactose and pyruvate, YFP-hDMPK A-expressing cells showed a significantly lower viability than YFP-hDMPK C-expressing cells (P<0.01, n = 3, >100 cells analyzed per experiment). (B) The MMP was determined in HeLa cells expressing YFP-hDMPK A or C. YFP-hDMPK A–expressing cells without clustered mitochondria showed a clear MMP signal (upper panels). No TMRM signal was found in YFP-hDMPK A–expressing cells with clustered mitochondria (middle panels, asterisks). YFP-hDMPK C–expressing cells demonstrated a clear mitochondrial signal (lower panels). Bars, 10 µm. (C) The MMP in YFP-hDMPK A–expressing cells was almost completely abolished and significantly lower than in YFP-hDMPK C and non-transfected (NT) cells (P<0.001, n = 3, >35 cells per experiment).</p