Expression of expanded FMR1-CGG repeats alters mitochondrial miRNAs and modulates mitochondrial functions and cell death in cellular model of FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by an expansion of 55 to 200 CGG repeats located within 5′UTR of FMR1.These CGG repeats are transcribed into RNAs, which sequester several RNA binding proteins and alter the processing of miRNAs. CGG repeats are also translated into a toxic polyglycine-containing protein, FMRpolyG, that affects mitochondrial and nuclear functions reported in cell and animal models and patient studies. Nuclear-encoded small non-coding RNAs, including miRNAs, are transported to mitochondria; however, the role of mitochondrial miRNAs in FXTAS pathogenesis is not understood. Here, we analyzed mitochondrial miRNAs from HEK293 cells expressing expanded CGG repeats and their implication in the regulation of mitochondrial functions. The analysis of next generation sequencing (NGS) data of small RNAs from HEK293 cells expressing CGG premutation showed decreased level of cellular miRNAs and an altered pattern of association of miRNAs with mitochondria (mito-miRs). Among such mito-miRs, miR-320a was highly enriched in mitoplast and RNA immunoprecipitation of Ago2 (Argonaute-2) followed by Droplet digital PCR (ddPCR)suggested that miR-320a may form a complex with Ago2 and mitotranscripts. Finally, transfection of miR-320a mimic in cells expressing CGG permutation recovers mitochondrial functions and rescues cell death. Overall, this work reveals an altered translocation of miRNAs to mitochondria and the role of miR-320a in FXTAS pathology

PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD.

ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset

EMBO Mol Med

Mutations in amphiphysin-2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis-splicing of amphiphysin-2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin-2 orchestrates nuclear positioning and triad organization and how CNM-associated mutations lead to muscle dysfunction remains elusive. We find that N-WASP interacts with amphiphysin-2 in myofibers and that this interaction and N-WASP distribution are disrupted by amphiphysin-2 CNM mutations. We establish that N-WASP functions downstream of amphiphysin-2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b-dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N-WASP and amphiphysin-2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N-WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N-WASP in amphiphysin-2-dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology

Mercury release and speciation in chemical looping combustion of coal

In the in situ Gasification Chemical Looping Combustion of coal (iG-CLC), the fuel is gasified in situ in the fuel reactor and gasification products are converted to CO2 and H2O by reaction with the oxygen carrier. This work is the first study on mercury release in Chemical Looping Combustion of coal. The fraction of the mercury in coal vaporized in the fuel reactor depended mainly on the fuel reactor temperature and the coal type. In the fuel reactor, mercury was mainly emitted as Hg0 in the gas phase and the amount increased with the temperature. In the air reactor, mercury was mostly emitted as Hg2+. In a real CLC system, mercury emissions to the atmosphere will decrease compared to conventional combustion as only mercury released in the air reactor will reach the atmosphere. However, measures should be taken to reduce Hg0 in the CO2 stream before the purification and compression steps in order to avoid operational problems.The authors thank the Government of Aragón and La Caixa (2012-GA-LC-076 project) and the Spanish Ministry for Science and Innovation (ENE2010-19550 project) for the financial support. P. Gayán thanks CSIC for the financial support of the project 201180E102. The authors also thank to Alcoa Europe-Alúmina Española S.A. for providing the Fe-enriched sand fraction used in this work. G. Galo is acknowledged for his contribution to the experimental results.Peer reviewe

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

Transcriptional Mutagenesis Induced by 8-Oxoguanine in Mammalian Cells

Most of the somatic cells of adult metazoans, including mammals, do not undergo continuous cycles of replication. Instead, they are quiescent and devote most of their metabolic activity to gene expression. The mutagenic consequences of exposure to DNA–damaging agents are well documented, but less is known about the impact of DNA lesions on transcription. To investigate this impact, we developed a luciferase-based expression system. This system consists of two types of construct composed of a DNA template containing an 8-oxoguanine, paired either with a thymine or a cytosine, placed at defined positions along the transcribed strand of the reporter gene. Analyses of luciferase gene expression from the two types of construct showed that efficient but error-prone transcriptional bypass of 8-oxoguanine occurred in vivo, and that this lesion was not repaired by the transcription-coupled repair machinery in mammalian cells. The analysis of luciferase activity expressed from 8OG:T-containing constructs indicated that the magnitude of erroneous transcription events involving 8-oxoguanine depended on the sequence contexts surrounding the lesion. Additionally, sequencing of the transcript population expressed from these constructs showed that RNA polymerase II mostly inserted an adenine opposite to 8-oxoguanine. Analysis of luciferase expression from 8OG:C-containing constructs showed that the generated aberrant mRNAs led to the production of mutant proteins with the potential to induce a long-term phenotypical change. These findings reveal that erroneous transcription over DNA lesions may induce phenotypical changes with the potential to alter the fate of non-replicating cells

Identification and Characterization of Peripheral T-Cell Lymphoma-Associated SEREX Antigens

Peripheral T-cell lymphomas (PTCL) are generally less common and pursue a more aggressive clinical course than B-cell lymphomas, with the T-cell phenotype itself being a poor prognostic factor in adult non-Hodgkin lymphoma (NHL). With notable exceptions such as ALK+ anaplastic large cell lymphoma (ALCL, ALK+), the molecular abnormalities in PTCL remain poorly characterised. We had previously identified circulating antibodies to ALK in patients with ALCL, ALK+. Thus, as a strategy to identify potential antigens associated with the pathogenesis of PTCL, not otherwise specified (PTCL, NOS), we screened a testis cDNA library with sera from four PTCL, NOS patients using the SEREX (serological analysis of recombinant cDNA expression libraries) technique. We identified nine PTCL, NOS-associated antigens whose immunological reactivity was further investigated using sera from 52 B- and T-cell lymphoma patients and 17 normal controls. The centrosomal protein CEP250 was specifically recognised by patients sera and showed increased protein expression in cell lines derived from T-cell versus B-cell malignancies. TCEB3, BECN1, and two previously uncharacterised proteins, c14orf93 and ZBTB44, were preferentially recognised by patients' sera. Transcripts for all nine genes were identified in 39 cancer cell lines and the five genes encoding preferentially lymphoma-recognised antigens were widely expressed in normal tissues and mononuclear cell subsets. In summary, this study identifies novel molecules that are immunologically recognised in vivo by patients with PTCL, NOS. Future studies are needed to determine whether these tumor antigens play a role in the pathogenesis of PTCL

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

UVSSA and USP7, a new couple in transcription-coupled DNA repair

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes transcription-blocking lesions from our genome. Defects in this pathway are associated with two human disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite a similar cellular defect in the UV DNA damage response, patients with these syndromes exhibit strikingly distinct symptoms; CS patients display severe developmental, neurological, and premature aging features, whereas the phenotype of UVSS patients is mostly restricted to UV hypersensitivity. The exact molecular mechanism behind these clinical differences is still unknown; however, they might be explained by additional functions of CS proteins beyond TC-NER. A short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review. In addition, we will focus on two new players involved in TC-NER which were recently identified: UV-stimulated scaffold protein A (UVSSA) and ubiquitin-specific protease 7 (USP7). UVSSA has been found to be the causative gene for UVSS and, together with USP7, is implicated in regulating TC-NER activity. We will discuss the function of UVSSA and USP7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between UVSS and the more severe CS