Klf15 Is Critical for the Development and Differentiation of Drosophila Nephrocytes

Insect nephrocytes are highly endocytic scavenger cells that represent the only invertebrate model for the study of human kidney podocytes. Despite their importance, nephrocyte development is largely uncharacterised. This work tested whether the insect ortholog of mammalian Kidney Krüppel-Like Factor (Klf15), a transcription factor required for mammalian podocyte differentiation, was required for insect nephrocyte development. It was found that expression of Drosophila Klf15 (dKlf15, previously known as Bteb2) was restricted to the only two nephrocyte populations in Drosophila, the garland cells and pericardial nephrocytes. Loss of dKlf15 function led to attrition of both nephrocyte populations and sensitised larvae to the xenotoxin silver nitrate. Although pericardial nephrocytes in dKlf15 loss of function mutants were specified during embryogenesis, they failed to express the slit diaphragm gene sticks and stones and did not form slit diaphragms. Conditional silencing of dKlf15 in adults led to reduced surface expression of the endocytic receptor Amnionless and loss of in vivo scavenger function. Over-expression of dKlf15 increased nephrocyte numbers and rescued age-dependent decline in nephrocyte function. The data place dKlf15 upstream of sns and Amnionless in a nephrocyte-restricted differentiation pathway and suggest dKlf15 expression is both necessary and sufficient to sustain nephrocyte differentiation. These findings explain the physiological relevance of dKlf15 in Drosophila and imply that the role of KLF15 in human podocytes is evolutionarily conserve

Genetic and Chemical Modifiers of a CUG Toxicity Model in Drosophila

Non-coding CUG repeat expansions interfere with the activity of human Muscleblind-like (MBNL) proteins contributing to myotonic dystrophy 1 (DM1). To understand this toxic RNA gain-of-function mechanism we developed a Drosophila model expressing 60 pure and 480 interrupted CUG repeats in the context of a non-translatable RNA. These flies reproduced aspects of the DM1 pathology, most notably nuclear accumulation of CUG transcripts, muscle degeneration, splicing misregulation, and diminished Muscleblind function in vivo. Reduced Muscleblind activity was evident from the sensitivity of CUG-induced phenotypes to a decrease in muscleblind genetic dosage and rescue by MBNL1 expression, and further supported by the co-localization of Muscleblind and CUG repeat RNA in ribonuclear foci. Targeted expression of CUG repeats to the developing eye and brain mushroom bodies was toxic leading to rough eyes and semilethality, respectively. These phenotypes were utilized to identify genetic and chemical modifiers of the CUG-induced toxicity. 15 genetic modifiers of the rough eye phenotype were isolated. These genes identify putative cellular processes unknown to be altered by CUG repeat RNA, and they include mRNA export factor Aly, apoptosis inhibitor Thread, chromatin remodelling factor Nurf-38, and extracellular matrix structural component Viking. Ten chemical compounds suppressed the semilethal phenotype. These compounds significantly improved viability of CUG expressing flies and included non-steroidal anti-inflammatory agents (ketoprofen), muscarinic, cholinergic and histamine receptor inhibitors (orphenadrine), and drugs that can affect sodium and calcium metabolism such as clenbuterol and spironolactone. These findings provide new insights into the DM1 phenotype, and suggest novel candidates for DM1 treatments

"I know that you know that I know": neural substrates associated with social cognition deficits in DM1 patients

Myotonic dystrophy type-1 (DM1) is a genetic multi-systemic disorder involving several organs including the brain. Despite the heterogeneity of this condition, some patients with non-congenital DM1 can present with minimal cognitive impairment on formal testing but with severe difficulties in daily-living activities including social interactions. One explanation for this paradoxical mismatch can be found in patients' dysfunctional social cognition, which can be assessed in the framework of the Theory of Mind (ToM). We hypothesize here that specific disease driven abnormalities in DM1 brains may result in ToM impairments. We recruited 20 DM1 patients who underwent the "Reading the Mind in the Eyes" and the ToM-story tests. These patients, together with 18 healthy controls, also underwent resting-state functional MRI. A composite Theory of Mind score was computed for all recruited patients and correlated with their brain functional connectivity. This analysis provided the patients' "Theory of Mind-network", which was compared, for its topological properties, with that of healthy controls. We found that DM1 patients showed deficits in both tests assessing ToM. These deficits were associated with specific patterns of abnormal connectivity between the left inferior temporal and fronto-cerebellar nodes in DM1 brains. The results confirm the previous suggestions of ToM dysfunctions in patients with DM1 and support the hypothesis that difficulties in social interactions and personal relationships are a direct consequence of brain abnormalities, and not a reaction symptom. This is relevant not only for a better pathophysiological comprehension of DM1, but also for non-pharmacological interventions to improve clinical aspects and impact on patients' success in life

Elevated plasma levels of cardiac troponin-I predict left ventricular systolic dysfunction in patients with myotonic dystrophy type 1:A multicentre cohort follow-up study

Objective: High sensitivity plasma cardiac troponin-I (cTnI) is emerging as a strong predictor of cardiac events in a variety of settings. We have explored its utility in patients with myotonic dystrophy type 1 (DM1). Methods: 117 patients with DM1 were recruited from routine outpatient clinics across three health boards. A single measurement of cTnI was made using the ARCHITECT STAT Troponin I assay. Demographic, ECG, echocardiographic and other clinical data were obtained from electronic medical records. Follow up was for a mean of 23 months. Results: Fifty five females and 62 males (mean age 47.7 years) were included. Complete data were available for ECG in 107, echocardiography in 53. Muscle Impairment Rating Scale score was recorded for all patients. A highly significant excess (p = 0.0007) of DM1 patients presented with cTnI levels greater than the 99th centile of the range usually observed in the general population (9 patients; 7.6%). Three patients with elevated troponin were found to have left ventricular systolic dysfunction (LVSD), compared with four of those with normal range cTnI (33.3% versus 3.7%; p = 0.001). Sixty two patients had a cTnI level < 5ng/L, of whom only one had documented evidence of LVSD. Elevated cTnI was not predictive of severe conduction abnormalities on ECG, or presence of a cardiac device, nor did cTnI level correlate with muscle strength expressed by Muscle Impairment Rating Scale score. Conclusions: Plasma cTnI is highly elevated in some ambulatory patients with DM1 and shows promise as a tool to aid cardiac risk stratification, possibly by detecting myocardial involvement. Further studies with larger patient numbers are warranted to assess its utility in this setting

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders

Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis.

Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.We used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.Taken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell

Bird diversity and conservation in the lower delta of the Paraná River, Argentina

The Delta of the Paraná River, one of the most important wetlands in South America, harbors subtropical and temperate bird species. Although this region is key for biodiversity conservation, aspects such as species composition and conservation status, and their relationship with vegetation types are poorly known. Here we described bird richness and composition of this area, with emphasis on the relationship between vegetation type and the presence of key bird species. We compiled systematic studies conducted during the 2007-2020 period and performed new surveys to elaborate a checklist of bird species and assess completeness. We reviewed a total of 12 studies distributed along five landscape units and nine vegetation types. We recorded 245 species, though the species accumulation curve indicates that the number of species could be even higher for the area. Most species were recorded in fresh water marshes, watercourses and grasslands in Entre Ríos. We identified 14 (5.7%) threatened species (e.g., Spartonoica maluroides, Limnoctites rectirostris and Sporophila palustris), most of them registered in grasslands and freshwater marshes. To our best knowledge, the list of species is the most comprehensive one for the area, showing that 23.7% of all bird species known for Argentina. Our results suggest the importance of freshwater marshes, watercourses and grasslands as key vegetation types for birds.El delta del río Paraná, uno de los humedales más importantes de América del Sur, alberga especies de aves subtropicales y templadas. Si bien esta región es clave para la conservación de la biodiversidad, aspectos como la composición de especies presentes, el estado de conservación y su relación con los tipos de vegetación son poco conocidos. En este trabajo describimos la composición de aves del Bajo Delta del Río Paraná, relacionando la ubicación en el paisaje y el tipo de vegetación con la presencia de especies clave. Para elaborar una lista de especies de aves en el Bajo Delta y evaluar su integridad recopilamos estudios sistemáticos realizados durante 2007-2020 y realizamos nuevos muestreos. Revisamos un total de 12 estudios distribuidos a lo largo de cinco Unidades de paisaje y nueve tipos de vegetación. Registramos 245 especies, aunque la curva de acumulación de especies indica que el número de especies podría ser aún mayor para el área. El mayor número de especies se registraron en pajonales y juncales, cursos de agua y pastizales en Entre Ríos. Identificamos 14 (5.7%) especies en riesgo de extinción (por ejemplo, Spartonoica maluroides, Limnoctites rectirostris y Sporophila palustris), la mayoría de ellas registradas en pastizales y pajonales. La lista de especies que se presenta aquí es, a nuestro entender, la más completa para el área, mostrando que el 23.7% de todas las especies de aves conocidas para Argentina habitan en el Bajo Delta. Nuestros resultados sugieren la importancia de los pajonales y juncales, cursos de agua y pastizales como tipos de vegetación clave para las aves.EEA Delta del ParanáFil: Fracassi, Natalia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Delta del Paraná; ArgentinaFil: Sica, Yanina V. Universidad Nacional de San Martín. Instituto de Investigación e Ingeniería Ambiental; ArgentinaFil: Sica, Yanina V. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Magnano, Andrea Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Magnano, Andrea Laura. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Magnano, Andrea Laura. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Vaccaro, Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vaccaro, Anahí. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. IEGEBA; ArgentinaFil: Landó, Roberto. Arauco Argentina S.A.; ArgentinaFil: Artero, Diego. Arauco Argentina S.A.; ArgentinaFil: Gavier Pizarro, Gregorio Ignacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Gavier Pizarro, Gregorio Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bó, Roberto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. IEGEBA; ArgentinaFil: Somma, Daniel Jorge. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Delta del Paraná; ArgentinaFil: Quintana, Rubén D. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Quintana, Rubén D. Universidad Nacional de San Martín. Instituto de Investigación e Ingeniería Ambiental; ArgentinaFil: Rodriguez, María José. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia". División de Ornitología; ArgentinaFil: Cabanne, Gustavo Sebastián. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia". División de Ornitología; Argentin