Repeat-Associated Non-AUG (RAN) Translation and Other Molecular Mechanisms in Fragile X Tremor Ataxia Syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset inherited neurodegenerative disorder characterized by progressive intention tremor, gait ataxia and dementia associated with mild brain atrophy. The cause of FXTAS is a premutation expansion, of 55 to 200 CGG repeats localized within the 5′UTR of FMR1. These repeats are transcribed in the sense and antisense directions into mutants RNAs, which have increased expression in FXTAS. Furthermore, CGG sense and CCG antisense expanded repeats are translated into novel proteins despite their localization in putatively non-coding regions of the transcript. Here we focus on two proposed disease mechanisms for FXTAS: 1) RNA gain-of-function, whereby the mutant RNAs bind specific proteins and preclude their normal functions, and 2) repeat-associated non-AUG (RAN) translation, whereby translation through the CGG or CCG repeats leads to the production of toxic homopolypeptides, which in turn interfere with a variety of cellular functions. Here, we analyze the data generated to date on both of these potential molecular mechanisms and lay out a path forward for determining which factors drive FXTAS pathogenicity

Étude du taux d'hydroxyproline d'un muscle et de ses relations avec certaines caractéristiques de composition corporelle dans les races large white et de piétrain

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Sequestration of DROSHA and DGCR8 by Expanded CGG RNA Repeats Alters MicroRNA Processing in Fragile X-Associated Tremor/Ataxia Syndrome

SummaryFragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55–200 CGG repeats in the 5′ UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery

Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had \u3e 1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization, two iPSC lines from each subject were selected, differentiated into postmitotic neurons, and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs, iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover, repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.This work was supported by Genethon, the French Muscular Dystro-phy Association (AFM), and Spark Therapeutics. It was also sup-ported by the European Union’s Research and Innovation Programunder grant agreement number 667751 (to F.M.), the EuropeanResearch Council Consolidator Grant under grant agreement number617432 (to F.M.), and Marie Skłodowska-Curie Actions-IndividualFellowship (MSCA-IF) grant agreement number 797144 (to U.C.)S

Decreased DGCR8 expression and miRNA dysregulation in individuals with 22q11.2 deletion syndrome

Deletion of the 1.5-3 Mb region of chromosome 22 at locus 11.2 gives rise to the chromosome 22q11.2 deletion syndrome (22q11DS), also known as DiGeorge and Velocardiofacial Syndromes. It is the most common micro-deletion disorder in humans and one of the most common multiple malformation syndromes. The syndrome is characterized by a broad phenotype, whose characterization has expanded considerably within the last decade and includes many associated findings such as craniofacial anomalies (40%), conotruncal defects of the heart (CHD; 70-80%), hypocalcemia (20-60%), and a range of neurocognitive anomalies with high risk of schizophrenia, all with a broad phenotypic variability. These phenotypic features are believed to be the result of a change in the copy number or dosage of the genes located in the deleted region. Despite this relatively clear genetic etiology, very little is known about which genes modulate phenotypic variations in humans or if they are due to combinatorial effects of reduced dosage of multiple genes acting in concert. Here, we report on decreased expression levels of genes within the deletion region of chromosome 22, including DGCR8, in peripheral leukocytes derived from individuals with 22q11DS compared to healthy controls. Furthermore, we found dysregulated miRNA expression in individuals with 22q11DS, including miR-150, miR-194 and miR-185. We postulate this to be related to DGCR8 haploinsufficiency as DGCR8 regulates miRNA biogenesis. Importantly we demonstrate that the level of some miRNAs correlates with brain measures, CHD and thyroid abnormalities, suggesting that the dysregulated miRNAs may contribute to these phenotypes and/or represent relevant blood biomarkers of the disease in individuals with 22q11DS

rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.Peer reviewe

Cell Rep

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55-200 CGG repeats in the 5' UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.Peer reviewe

Modelling the interaction between turbidite deposition and salt-related deformation : a new experimental tectono-stratigraphic approach with application to deformation of allochthonous salt bodies

Le long d’une marge passive, la présence d'un niveau de sel mobile, ainsi que des apports sédimentaires détritiques importants peuvent déclencher une tectonique gravitaire déformant la couverture sédimentaire et le sel sous-jacent. Ces déformations sont contrôlées au niveau du glacis continental par la surcharge différentielle engendrée par le dépôt d'éventails turbiditiques (étalement gravitaire). Localement, lorsque la marge est mature, l'extrusion des évaporites forme des nappes de sel allochtone de quelques dizaines de kilomètres de longueur, pouvant être remobilisés et déformés par le dépôt de lobes/éventails turbiditiques. Deux styles structuraux se distinguent : Le premier (stepped counterregional) est dominé par la subsidence de la couverture sédimentaire et l’extrusion du sel en aval du système. Le second (roho) est dominé par des mouvements de translation horizontaux accommodés par de nombreuses structures extensives.Nous avons développé un nouveau type de dispositif expérimental permettant d’étudier les interactions entre sédimentation et tectonique salifère au sein des marges passives. Notre dispositif combine deux approches traditionnellement distinctes : l’approche stratigraphique, s’intéressant au transport et au dépôt sédimentaire, et l’approche tectonique, se focalisant sur les déformations salifères. Le bassin tectono-stratigraphique conçu au cours de cette thèse permet donc de simuler les processus de transport et de dépôt turbiditique ainsi que les déformations gravitaires induites par une surcharge sédimentaire différentielle sur un niveau mobile salifère à l’échelle régionale (étalement gravitaire) ou locale (remobilisation de nappe de sel allochtone).Salt-bearing passive margin with large clastic sediment supply are characterized by gravity-driven tectonics that deforms the sedimentary overburden and the underlying salt. These deformations are controlled along the continental rise by the differential loading generated by the deposition of turbidite fan (gravity spreading). Locally, along mature margin, extrusion of evaporites form allochthonous salt nappes (tens kilometres long) that are remobilized and deformed by the deposition of turbiditic lobes. Two end-members are recognized: the stepped counterregional structural style, which is dominated by subsidence of the sedimentary cover and distal salt extrusion. The roho structural style, which is dominated by lateral translation accommodated by several extensional structures.We designed a completely new kind of experimental apparatus for studying the interaction between sedimentation and salt tectonics along passive margin. Our device combines two approaches that are traditionally distinct: (1) the stratigraphic approach aiming to simulate sediment transport and deposition, and (2) the tectonic approach focusing on the deformation of the salt and its sedimentary cover. Thus, the tectono-stratigraphic basin built during this PhD work allows the modelling of turbiditic transport and deposition processes, as well as, salt-related deformations induced by differential loading of a mobile salt substratum at regional (gravity spreading) or local (allochthonous salt bodies remobilization)