Neuroprotective properties of queen bee acid by autophagy induction

Autophagy is a conserved intracellular catabolic pathway that removes cytoplasmic components to contribute to neuronal homeostasis. Accumulating evidence has increasingly shown that the induction of autophagy improves neuronal health and extends longevity in several animal models. Therefore, there is a great interest in the identification of effective autophagy enhancers with potential nutraceutical or pharmaceutical properties to ameliorate age-related diseases, such as neurodegenerative disorders, and/or promote longevity. Queen bee acid (QBA, 10-hydroxy-2-decenoic acid) is the major fatty acid component of, and is found exclusively in, royal jelly, which has beneficial properties for human health. It is reported that QBA has antitumor, anti-inflammatory, and antibacterial activities and promotes neurogenesis and neuronal health; however, the mechanism by which QBA exerts these effects has not been fully elucidated. The present study investigated the role of the autophagic process in the protective effect of QBA. We found that QBA is a novel autophagy inducer that triggers autophagy in various neuronal cell lines and mouse and fly models. The beclin-1 (BECN1) and mTOR pathways participate in the regulation of QBA-induced autophagy. Moreover, our results showed that QBA stimulates sirtuin 1 (SIRT1), which promotes autophagy by the deacetylation of critical ATG proteins. Finally, QBA-mediated autophagy promotes neuroprotection in Parkinson’s disease in vitro and in a mouse model and extends the lifespan of Drosophila melanogaster. This study provides detailed evidences showing that autophagy induction plays a critical role in the beneficial health effects of QBA.This research was supported by a grant (IB18048) from Junta de Extremadura, Spain, and a grant (RTI2018-099259-A-I00) from Ministerio de Ciencia e Innovación, Spain. This work was also partially supported by “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. Part of the equipment employed in this work has been funded by Generalitat Valeciana and co-financed with ERDF funds (OP EDRF of Comunitat Valenciana 2014-2020). G.M-C is supported by University of Extremadura (ONCE Foundation). M.P-B is a recipient of a fellowship from the “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (University of Extremadura).” S.M.S.Y-D is supported by CIBERNED. E.U-C was supported by an FPU predoctoral fellowship FPU16/00684 from Ministerio de Educación, Cultura y Deporte. A.B. was supported by a postdoctoral fellowship (APOSTD2017/077). M.S.A. was supported by a predoctoral fellowship (ACIF/2018/071) both from the Conselleria d’Educació, Investigació, Cultura i Esport (Generalitat Valenciana). E.A-C was supported by a grant (IB18048) from Junta de Extremadura, Spain. S.C-C was supported by an FPU predoctoral fellowship FPU19/04435 from Ministerio de Educación, Cultura y Deporte. J.M.B-S. P was funded by the “Ramón y Cajal” program (RYC-2018-025099). J.M.F. received research support from the Instituto de Salud Carlos III, CIBERNED (CB06/05/004). M.N-S was funded by the “Ramon y Cajal” Program (RYC-2016-20883) Spain

Regulación transcripcional de muscleblind y nuevos mecanismos de patogénesis en un modelo de distrofia miotónica en Drosophila

La distrofia miotónica tipo I es una enfermedad genética con un patrón de herencia autosómico dominante. Se considera una enfermedad rara ya que afecta a 1 de cada 8000 personas, sin embargo, es la forma más común de distrofia en adultos. Es una enfermedad multisistémica que se caracteriza por la aparición de cataratas iridiscenctes, defectos en la conducción cardíaca, miotonía y atrofia muscular. Sin embargo, hasta la fecha no existen tratamientos efectivos. La enfermedad se produce por la expansión del triplete CTG en la región 3¿ no traducida del gen DMPK. Las expansiones forman unas horquillas de RNA capaces de secuestrar diversas proteínas de unión a RNA, entre ellas, el factor de splicing MBNL1, cuyo secuestro es el responsable de diversos fenotipos de la enfermedad. En el presente trabajo se estudió la regulación transcripcional de muscliblind, el ortólogo en Drosophila de MBNL1. De este modo, la compresión del funcionamiento del gen permite generar más opciones a la hora de desarrollar nuevos tratamientos. También se identificaron dos nuevos genes, TBPH y bsf, implicados en el mecanismo de patogénesis de la enfermedad. Por último, se identificó que la activación patológica de la apoptosis y de la autofagia en un modelo de la enfermedad en Drosophila eran los mecanismos responsables de la pérdida de masa muscular. De este modo, con los resultados obtenidos se abre la puerta a futuras investigaciones dirigidas a la búsqueda de tratamientos eficaces para la distrofia miotónica tipo I

miR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy

International audienceUnstable CTG expansions in the 3' UTR of the DMPK gene are responsible for myotonic dystrophy type 1 (DM1) condition. Muscle dysfunction is one of the main contributors to DM1 mortality and morbidity. Pathways by which mutant DMPK trigger muscle defects, however, are not fully understood. We previously reported that miR-7 was downregulated in a DM1 Drosophila model and in biopsies from patients. Here, using DM1 and normal muscle cells, we investigated whether miR-7 contributes to the muscle phenotype by studying the consequences of replenishing or blocking miR-7, respectively. Restoration of miR-7 with agomiR-7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth. Conversely, oligonucleotide-mediated blocking of miR-7 in normal myoblasts led to fusion and myotube growth defects. miR-7 was found to regulate autophagy and the ubiquitin-proteasome system in human muscle cells. Thus, low levels of miR-7 promoted both processes, and high levels of miR-7 repressed them. Furthermore, we uncovered that the mechanism by which miR-7 improves atrophy-related phenotypes is independent of MBNL1, thus suggesting that miR-7 acts downstream or in parallel to MBNL1. Collectively, these results highlight an unknown function for miR-7 in muscle dysfunction through autophagy- and atrophy-related pathways and support that restoration of miR-7 levels is a candidate therapeutic target for counteracting muscle dysfunction in DM1

Two enhancers control transcription of Drosophila muscleblind in the embryonic somatic musculature and in the central nervous system.

The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene

Msi2 enhances muscle dysfunction in a myotonic dystrophy type 1 mouse model

Background: Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the 3′ untranslated region of the DM1 protein kinase gene. Characteristic degenerative muscle symptoms include myotonia, atrophy, and weakness. We previously proposed an Musashi homolog 2 (MSI2)>miR-7>autophagy axis whereby MSI2 overexpression repressed miR-7 biogenesis that subsequently de-repressed muscle catabolism through excessive autophagy. Because the DM1 HSALR mouse model expressing expanded CUG repeats shows weak muscle-wasting phenotypes, we hypothesized that MSI2 overexpression was sufficient to promote muscle dysfunction in vivo. Methods: By means of recombinant AAV murine MSI2 was overexpressed in neonates HSALR mice skeletal muscle to induce DM1-like phenotypes. Results: Sustained overexpression of the murine MSI2 protein in HSALR neonates induced autophagic flux and expression of critical autophagy proteins, increased central nuclei and reduced myofibers area, and weakened muscle strength. Importantly, these changes were independent of MBNL1, MBNL2, and Celf1 protein levels, which remained unchanged upon Msi2 overexpression. Conclusions: Globally, molecular, histological, and functional data from these experiments in the HSALR mouse model confirms the pathological role of MSI2 expression levels as an atrophy-associated component that impacts the characteristic muscle dysfunction symptoms in DM1 patients

Genomic organization of the human MBNL1 gene.

<p>(A) Scale representation of 198 kb of the human MBNL1 locus. Green boxes correspond to exons and black lines to introns. Tested promoter regions are indicated as P1 and P2; a yellow circle denotes a predicted CpG island. (B) Schematic representation of H3K27Ac marks, typical of promoter regions, on seven human cell lines. (C) 5′-Ends of ESTs mapping to the MBNL1 locus support two potential transcription start sites for the gene. Data according to the UCSC Genome Browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093125#pone.0093125-Meyer1" target="_blank">[58]</a>. (E,F,H,I) Direct visualization of the eGFP reporter under the control of the ME enhancer. (D–F) Enhancer-less Hsa-P1 construct is a negative control. (G–I) Flies carrying the ME enhancer upstream of the human MBNL1 promoter (Hsa-P1) reproduce Muscleblind expression in the somatic musculature. (E,H) Lateral and (F,I) ventral views of late embryos. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up, unless otherwise stated.</p

ME reproduces <i>muscleblind</i> expression in the embryonic somatic musculature.

<p>(A) Localization of the putative <i>cis</i>-regulatory modules M1, M2, ML, H and M3, indicated as orange boxes, in the context of the <i>muscleblind</i> genomic locus. Fluorescence confocal images of lateral (B,E,H,) and ventral (C,F,I,) views of late <i>Drosophila</i> embryos. (D,G) Schematic representation of the reporter constructs used to transform the germline of <i>Drosophila</i>. In control <i>yw</i> flies (B,C) an anti-Mbl antibody detects robust expression in the somatic musculature and in the CNS (green). Direct visualization of the GFP reporter under the control of the ME enhancer in the pH-Stinger vector (E,F,H,I). Promoter-less ME constructs (D–F) do not activate GFP expression and serve as negative controls. Flies carrying the ME enhancer upstream of <i>Hsp70</i> (G–I) reproduce Muscleblind expression in the somatic musculature but not in the CNS. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up unless otherwise stated.</p

Sequence and melting temperature (Tm) of PCR primers used for cloning genomic DNA.

<p>Restriction sites are highlighted in bold.</p

NE reproduces <i>muscleblind</i> expression in the central nervous system.

<p>(A) Schematic representation of the reporter construct used to transform the <i>Drosophila</i> germline. Fluorescence confocal images of lateral (B–D) and ventral (E–J) views of late <i>Drosophila</i> embryos expressing construct (A) co-stained with anti-GFP (green) and anti-Mbl antibodies (red). NE drives expression in the CNS (arrows; C,I) overlapping Muscleblind expression (B,E,H; D,G,J shows the merge in yellow). No signal of the reporter was observed in tissues other than CNS. Endogenous Muscleblind expression in the muscles is in focus in (E,G,H,J). Micrographs were taken at 200× (B–G) and 400× magnification (H–J). Anterior is to the left and dorsal up unless otherwise stated.</p

Msi2 enhances muscle dysfunction in a myotonic dystrophy type 1 mouse model.

Background: Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the 3′ untranslated region of the DM1 protein kinase gene. Characteristic degenerative muscle symptoms include myotonia, atrophy, and weakness. We previously proposed an Musashi homolog 2 (MSI2)>miR7>autophagy axis whereby MSI2 overexpression repressed miR-7 biogenesis that subsequently de-repressed muscle catabolism through excessive autophagy. Because the DM1 HSALR mouse model expressing expanded CUG repeats shows weak muscle-wasting phenotypes, we hypothesized that MSI2 overexpression was sufficient to promote muscle dysfunction in vivo. Methods: By means of recombinant AAV murine MSI2 was overexpressed in neonates HSALR mice skeletal muscle to induce DM1-like phenotypes. Results: Sustained overexpression of the murine MSI2 protein in HSALR neonates induced autophagic flux and expression of critical autophagy proteins, increased central nuclei and reduced myofibers area, and weakened muscle strength. Importantly, these changes were independent of MBNL1, MBNL2, and Celf1 protein levels, which remained unchanged upon Msi2 overexpression. Conclusions: Globally, molecular, histological, and functional data from these experiments in the HSALR mouse model confirms the pathological role of MSI2 expression levels as an atrophy-associated component that impacts the characteristic muscle dysfunction symptoms in DM1 patients