Implicaciones en el desarrollo y la longevidad del gen gale-1 en Caenorhabditis elegans: Un modelo animal de galactosemia tipo III

Tesis Doctoral presentada por la licenciada Ana María Brokate-Llanos en la Universidad Pablo de Olavide de Sevilla,El objetivo inicial de este trabajo ha sido caracterizar el gen gale-1 y entender su relación con el proceso de longevidad a través de mutantes de pérdida o reducción de función en Caenorhabditis elegans. En la caracterización del mutante de reducción de función, gale-1(pv18), encontramos una gran variedad de fenotipos: la termotolerancia a 30ºC, la letalidad embrionaria a 25ºC, la longevidad en el estadio L4 a 25 y 20ºC, puesta de huevos y progenie tardía a 20ºC, retraso en el crecimiento, vulva protuberante y malformación de gónadas a cualquier temperatura y condición de crecimiento, sin embargo, el mutante de pérdida de función, gale-1(tm3267), resultó ser letal. El hecho de que mutantes de reducción de función del gen homólogo a gale-1 en humanos (GALE) genere galactosemia tipo III, una enfermedad poco estudiada, ha hecho que el objetivo se haya ampliado para entender esta enfermedad en un modelo biológico animal. En resumen, podemos decir que la falta total de función es letal como se ha propuesto en humanos y que una reducción de función debido a la mutación pv18 que es mas similar a los alelos encontrados en humanos, es viable aunque presenta numerosos problemas, sensibilidad a galactosa y problemas de desarrollo, atribuibles a fallos en glicosilación, los cuales concuerdan con fenotipos descritos en humanos. Además, la longevidad de gale-1(pv18) no se debe a una supervivencia a la infección, ni una reducción del proceso de N-glicosilación y si puede estar relacionada con una restricción dietaría. También se han encontrado interacciones con rutas relacionadas con la longevidad, la ruta de la Unfolded Protein Response y la Ruta de la Insulina, que puede ser los factores intrínsecos de gale-1 para conferir un incremento de longevidad.Departamento de Biología Molecular e Ingeniería Bioquímica para optar al grado de Doctor.Peer Reviewe

Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date. After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International).-- et al.Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo-or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16:GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance.This work was supported by a grant from the Instituto de Salud Carlos III (ISCIII) (Exp. PI12/01554). R.J. and D.B. were supported by a Natural Sciences and Engineering Research Council (NSERC) Canada grant. D.B. holds a Canada Research Chair. J.C. is a Miguel Servet Researcher (ISCIII). E.C. was supported with a CTP-AIRE fellowship from AGAUR (Generalitat de Catalunya) and a TRANSBIO SUDOE collaborative project.Peer Reviewe

Defects in female fertility due to mutations in cohesins

Meiosis is a fundamental process in the improvement of genetic diversity, thus allowing the generation of new allelic combinations.This process consists of two consecutive rounds of nuclear divisions, known as meiosis I (MI) and meiosis II (MII), which ensure the correct distribution of chromosomes from diploid parental cells to haploid gametes (León-Periñán & Fernández-Álvarez., 2020). Throughout this process, cohesion is required to hold sister chromatids together until their separation in anaphase. Cohesion is created by subunit complexes of multiple proteins called cohesins; these proteins are essential for chromosome structure and segregation during meiosis. To date, several studies link defects in cohesins to female infertility. The model organism Schizosaccharomyces pombe is used to study the molecular mechanisms related to cohesins. During meiotic prophase, chromosome oscillations driven by cytoskeletal forces aim to move chromosomes to promote recognition and pairing between homologues and defects in these events lead to negative impacts on chromosome segregation (Hopkins et al., 2014). To observe these events, the functions of some proteins or factors involved in the behavior of cohesins such as the Pds5, Rec8 or Rec7 proteins have been studied. Different studies agree that the Pds5 protein determines the binding of cohesin to chromosomes, controls the binding capacity of sister chromatids and participates in gene expression (Misulovin et al., 2018). In order to observe the impact of this protein, S. pombe strains with a deletion in the Pds5 gene are studied by fluorescence microscopy using the DeltaVision microscope. As results, altered chromosome oscillations and segregations have been obtained in Pds5 mutants with respect to controls without this type of mutation, which could be extrapolated to a nefarious meiotic process in this context. In short, our results position Pds5 as a marker that can anticipate small fertility failures in women

Transgenerational inheritance of environmental information in Caenorhabditis elegans

Motivation: In 1906 Luther Burbank, an American botanist, said “Heridity is only the sum of all past environment” (Burbank, 1906). This idea has always been present in spite of being left behind after sustaining that the germline was isolated of the rest of the organism and, therefore, of the environment. With the rediscovery of epigenetics as a regulatory system which controls gene expression without affecting the composition of genes themselves, this idea has regained importance. Not only a great number of cases of environmental changes that influenced the epigenetics in a great variety of species began to be observed; but also the effects of these changes could last for three or more generations. This phenomenon was known as transgenerational epigenetic inheritance (Heard and Martienssen, 2014). This project aims to study the phenomenon of transgenerational epigenetic inheritance in the Caenorhabditis elegans model animal. The idea is to set the parameters where we can identify the phenomenon and reveal the molecular mechanism involved in the process.Methods: We set two different experiments, resistance to pathogen and behavioral respond to the pathogen. For the resistance experiment, plates with Escherichia coli (OP50, main source of nematode's food) and plates with Pseudomonas aeruginosa (PA14, a pathogen) were used to grow mothers and then the percentage of descendants that reach adulthood were scored. For the behavioral experiment, plates with OP50 and plates with both bacteria (PA14 and OP50) were used to grow mothers. Then their eggs were placed on plates containing OP50 and PA14. We counted the percent of worms that went to OP50 and to PA14.Results: It has been observed that individuals whose mothers have been exposed to Pseudomonas aeruginosa are more resistant to it than those whose mothers have never been in contact with the pathogen. On the other hand, it has been observed that this inheritance not only affects the defense mechanisms of the organism, but also influences its behavior. At this time we are studying the genetic pathways involved in this process by using mutants affected in epigenetic and in other candidate pathways.Conclusions: Our results suggest that there is not only an inheritance to stress resistance but also an acquired behavioral inheritance. Genes involved in epigenetic seem to be involved in the process

The study of the effect of mannose in GALE Caenorhabditis elegans mutants using as food source Escherichia Coli mutants defective in mannose metabolism pathway

Type III galactosemia is a rare disease characterized by mutations in the GALE gene that encodes the enzyme UDP-galactose 4-epimerase. The deficit of this enzyme gives rise to various physical and mental problems in humans (Walter et al. 1999). Several studies in gale-1 mutants of C. elegans worms have shown a  positive effect of mannose (Brokate-Llanos et al. 2014), on the longevity and development of these mutants. These worms have as their main diet E. coli (OP50), so that the positive effects of mannose can be influenced by the transformation these bacteria can produce through metabolic transformation of mannose. Trying to eliminate this possible influence of OP50 mannose metabolism on these experiments, UV radiation inactivated  E.coli  has been used as worm food in the presence of different concentrations of mannose. This approach presents, among others, the problem that by inactivating bacteria by UV radiation we are eliminating their replication, but not their complete metabolism,demanding other experimental approaches as using E. coli strains unable to metabolize mannose to feed the worms. Therefore, the objective of this work is to study the effects of mannose in C. elegans, fed with OP50 mutants defective in different steps of mannose metabolism to assess the direct effects of mannose on C. elegans GALE mutants. - Construction of OP50 isogenic strains defective in different steps of mannose metabolism by transformation with mutant alleles obtained from E. coli (K12) mutant strains. OP50 was transformed by homologous recombination with fragments containing deletions in ΔmanA (gene for the enzyme of the sugars metabolism mannose derived pathway), ΔmanB (gene for the enzyme of the lipopolysaccharide synthesis mannose derived pathway) and ΔmanX (gene for one of the mannose transporters within the cell). These deleted genes were replaced by a kanamycin resistance gene. - Preparation of eggs of C. elegans and incubation in plates with different concentrations of mannose (0%, 1%, 2%, 3%).The E. coli used are the different mutant strains obtained and a control (wild type OP50). OP50 mutants for the genes indicated above were correctly obtained. They were tested with the GALE worms, and we observed that ΔmanA and ΔmanX show a great improvement in GALE mutants growth and development with respect to the wt OP50 control. Nevertheless, we did not observe these changes with ΔmanB. It seems that LPS pathway is probably important for the mannose asimilation

Optimization of a new protocol that allows speeding up the process of screening genes and candidate drugs for Spinal Muscular Atrophy in C. elegans.

Spinal Muscular Atrophy (SMA) is a rare genetic disease that affects 1 in 8,000 people. It is caused by a recessive mutation in the SMN1 gene, which produces the SMN protein and is highly conserved in invertebrates [1]. In humans, the SMN2 gene is also found, which differs by 5 nucleotides from SMN1, which means that only 10% of the translated proteins are complete [2]. For the study of SMA, a strain of C. elegans, developed by our group, is used, which presents the smn1 gene fused to the mCherry fluorescence marker, in such a way that it allows measuring the expression of SMN in response to different candidates (drugs and RNAi ) through fluorescence. These candidates have been chosen by using the ASACO bioinfomatic tool, which makes it possible to generate an expression profile opposite to SMN1. However, normally, to measure fluorescence, a confocal microscope is used, which slows down this procedure considerably and makes it difficult to carry out large screenings of candidates. For this reason, in this work a new method has been sought to measure fluorescence in a faster way and that, like the confocal method, gives reliable results. For this, tests have been carried out with two pieces of equipment: the Apotome microscope and a fluorimeter, and candidates were used, both drugs and RNAi, which had been confirmed by confocal microscopy as capable of increasing SMN levels. In this way, tests were carried out with both teams to see which of the two provides results of fluorescence levels more similar to the confocal one. The results show the fluorimeter as the most appropriate equipment for this task, since, in addition to presenting very similar results to the confocal microscope, it is a faster method that allows for large scrutinies of candidates. In the future, using the ASACO tool, new candidate genes and drugs will be selected and fluorescence levels will be measured using this new method

A Functional Pipeline of Genome-Wide Association Data Leads to Midostaurin as a Repurposed Drug for Alzheimer’s Disease

Genome-wide association studies (GWAS) constitute a powerful tool to identify the different biochemical pathways associated with disease. This knowledge can be used to prioritize drugs targeting these routes, paving the road to clinical application. Here, we describe DAGGER (Drug Repositioning by Analysis of GWAS and Gene Expression in R), a straightforward pipeline to find currently approved drugs with repurposing potential. As a proof of concept, we analyzed a meta-GWAS of 1.6 × 107 single-nucleotide polymorphisms performed on Alzheimer’s disease (AD). Our pipeline uses the Genotype-Tissue Expression (GTEx) and Drug Gene Interaction (DGI) databases for a rational prioritization of 22 druggable targets. Next, we performed a two-stage in vivo functional assay. We used a C. elegans humanized model over-expressing the Aβ1-42 peptide. We assayed the five top-scoring candidate drugs, finding midostaurin, a multitarget protein kinase inhibitor, to be a protective drug. Next, 3xTg AD transgenic mice were used for a final evaluation of midostaurin’s effect. Behavioral testing after three weeks of 20 mg/kg intraperitoneal treatment revealed a significant improvement in behavior, including locomotion, anxiety-like behavior, and new-place recognition. Altogether, we consider that our pipeline might be a useful tool for drug repurposing in complex diseases.This work was mainly financed by Programa Operativo FEDER funds from the European Union through grant UMA20-FEDERJA-133. We thank Fundacion SantÁngela for co-funding with grant 83/23.04.2021. P.G.-G. is supported by the CIBERNED employment plan CNV-304-PRF-866. CIBERNED is integrated into Instituto de Salud Carlos III. I.d.R is supported by a national grant from the Instituto de Salud Carlos III FI20/00215. A.R. is supported by national grants PI13/02434, PI16/01861, PI17/01474, PI19/01240, and PI19/01301. A.M.B.-L. and M.J.M. were funded by grant PID2020-120463RB-I00 funded by the Spanish Ministerio de Ciencia e Innovación. A.C.-Z. holds a postdoctoral research contract from Secretaría General de Universidades, Investigación y Tecnología–Junta de Andalucía (POSTDOC21_00365). B.P.S (IFI21/00024) holds an “iPFIS” predoctoral contract from the National System of Health, EU-ERDF-ISCIII. M.d.C.M.-P. holds predoctoral grants from the Spanish Ministry of Science, Innovation and Universities (FPU17/00276). P.R. (CP19/00068) holds a “Miguel Servet” research contract from the National System of Health, ISCIII co-funded by the European Social Fund, “Investing in your future,” Gobierno de España. This research was funded by Delegación del Gobierno para el Plan Nacional sobre Drogas, Ministerio de Salud, Gobierno de España (PND2020/048). Ethovision XT software v17 (Noldus, Wageningen, The Netherlands) funded by Plan Propio, Universidad de Málaga

Escherichia coli carbon source metabolism affects longevity of its predator Caenorhabditis elegans

Nutrition is probably the most determinant factor affecting aging. Microorganisms of the intestinal flora lay in the interface between available nutrients and nutrients that are finally absorbed by multicellular organisms. They participate in the processing and transformation of these nutrients in a symbiotic or commensalistic relationship. In addition, they can also be pathogens. Alive Escherichia coli OP50 are usually used to culture the bacteriovorus nematode Caenorhabditis elegans. Here, we report a beneficial effect of low concentration of saccharides on the longevity of C. elegans. This effect is only observed when the bacterium can metabolize the sugar, suggesting that physiological changes in the bacterium feeding on the saccharides are the cause of this beneficial effect.Peer Reviewe

Sugar and longevity in gale-1 mutant

Resumen del trabajo presentado al 4th Spanish Worm Meeting, celebrado en Carmona (Sevilla) del 14 al 15 de marzo de 2013.A reduction of food intake without malnutrition increases longevity in almost any of the model animals where have been tested. In order to understand this process our group has isolated a collection of mutants that are long lived even in normal diet. One of those mutants is gale-1(pv18) which encoded to a UDP-4-galactosa-epimerase. This mutant, in addition to be long lived, show morphological organ defects, probably due to an alteration in the concentration of UDP-sugars. In humans, mutation in the homologue gene to gale-1 generate a rare disease known as galactosemia type III, the characterization of this mutant may help to better understand this disease in humans.Peer Reviewe

Developmental defects in a Caenorhabditis elegans model for type III galactosemia

Type III galactosemia is a metabolic disorder caused by reduced activity of UDP-galactose-4-epimerase, which participates in galactose metabolism and the generation of various UDP-sugar species. We characterized gale-1 in Caenorhabditis elegans and found that a complete loss-of-function mutation is lethal, as has been hypothesized for humans, whereas a nonlethal partial loss-offunction allele causes a variety of developmental abnormalities, likely resulting from the impairment of the glycosylation process. We also observed that gale-1 mutants are hypersensitive to galactose as well as to infections. Interestingly, we found interactions between gale-1 and the unfolded protein response.This work was supported by the Junta de Andalucía (P07-CVI-02697) and the Spanish Ministry of Science and Innovation (BFU2006-07391/BMC) and Ministry of Economy and Competitiveness (BFU2013-46923-P). A.M.B.-L. was supported by a Plan Propio de Investigación fellowship from the Universidad Pablo de Olavide. J.M.M. was supported by the Formación del personal Universitario program of the Spanish Ministry of Science and Innovation.Peer Reviewe