Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome

From MDPI via Jisc Publications RouterHistory: accepted 2021-11-17, pub-electronic 2021-11-18Publication status: PublishedFunder: Generalitat Valenciana; Grant(s): PROMETEU/2018/135Funder: European Commission; Grant(s): ERDF of Comunitat Valenciana 2014–2020Funder: ApoyoDravet; Grant(s): N/ADravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments

Mild Muscle Mitochondrial Fusion Distress Extends Drosophila Lifespan through an Early and Systemic Metabolome Reorganization

[EN] In a global aging population, it is important to understand the factors affecting systemic aging and lifespan. Mitohormesis, an adaptive response caused by different insults affecting the mitochondrial network, triggers a response from the nuclear genome inducing several pathways that promote longevity and metabolic health. Understanding the role of mitochondrial function during the aging process could help biomarker identification and the development of novel strategies for healthy aging. Herein, we interfered the muscle expression of the Drosophila genes Marf and Opa1, two genes that encode for proteins promoting mitochondrial fusion, orthologues of human MFN2 and OPA1. Silencing of Marf and Opa1 in muscle increases lifespan, improves locomotor capacities in the long term, and maintains muscular integrity. A metabolomic analysis revealed that muscle down-regulation of Marf and Opa1 promotes a non-autonomous systemic metabolome reorganization, mainly affecting metabolites involved in the energetic homeostasis: carbohydrates, lipids and aminoacids. Interestingly, the differences are consistently more evident in younger flies, implying that there may exist an anticipative adaptation mediating the protective changes at the older age. We demonstrate that mild mitochondrial muscle disturbance plays an important role in Drosophila fitness and reveals metabolic connections between tissues. This study opens new avenues to explore the link of mitochondrial dynamics and inter-organ communication, as well as their relationship with muscle-related pathologies, or in which muscle aging is a risk factor for their appearance. Our results suggest that early intervention in muscle may prevent sarcopenia and promote healthy aging.Work in the laboratory of M.I.G. was funded by PROMETEU/2018/135 from "Conselleria, de Sanitat de la Generalitat Valenciana". Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020).Tapia, A.; Palomino-Schätzlein, M.; Roca, M.; Lahoz, A.; Pineda-Lucena, A.; López Del Amo, V.; Galindo, MI. (2021). Mild Muscle Mitochondrial Fusion Distress Extends Drosophila Lifespan through an Early and Systemic Metabolome Reorganization. International Journal of Molecular Sciences. 22(22):1-20. https://doi.org/10.3390/ijms222212133120222

Missense mutations in the SH3TC2 protein causing Charcot-Marie-Tooth disease type 4C affect its localization in the plasma membrane and endocytic pathway

[EN] Mutations in SH3TC2 (KIAA1985) cause Charcot-Marie-Tooth disease (CMT) type 4C, a demyelinating inherited neuropathy characterized by early-onset and scoliosis. Here we demonstrate that the SH3TC2 protein is present in several components of the endocytic pathway including early endosomes, late endosomes and clathrin-coated vesicles close to the trans-Golgi network and in the plasma membrane. Myristoylation of SH3TC2 in glycine 2 is necessary but not sufficient for the proper location of the protein in the cell membranes. In addition to myristoylation, correct anchoring also needs the presence of SH3 and TPR domains. Mutations that cause a stop codon and produce premature truncations that remove most of the TPR domains are expressed as the wild-type protein. In contrast, missense mutations in or around the region of the first-TPR domain are absent from early endosomes, reduced in plasma membrane and late endosomes and are variably present in clathrin-coated vesicles. Our findings suggest that the endocytic and membrane trafficking pathway is involved in the pathogenesis of CMT4C disease. We postulate that missense mutations of SH3TC2 could impair communication between the Schwann cell and the axon causing an abnormal myelin formation.This work was supported by the Fondo de Investigacion Sanitaria [grant numbers PI08/90857, PI08/0889, CP08/00053] and the Spanish Ministry Science and Innovation [grant number SAF2006-01047]. V. L. is a recipient of JAE predoctoral fellowship from the Spanish Scientific Research Council (CSIC). M. I. G. has a `Ramon y Cajal' contract funded by the Ministry of Science and Innovation. C. E. has a `Miguel Servet' contract funded by the Fondo de Investigacion Sanitaria. Both CIBERER and CIBERNED are initiatives from the Instituto de Salud Carlos III. We are grateful to patients and their families for their kind collaboration. We thank B. Alarcón for his technical assistance and also anonymous reviewers for their invaluable insight and suggestionsLupo, V.; Galindo, MI.; Martínez-Rubio, D.; Sevilla, T.; Vílchez, JJ.; Palau, F.; Espinós-Armero, CÁ. (2009). Missense mutations in the SH3TC2 protein causing Charcot-Marie-Tooth disease type 4C affect its localization in the plasma membrane and endocytic pathway. Human Molecular Genetics. 18(23):4603-4614. https://doi.org/10.1093/hmg/ddp42746034614182

A Drosophila model of GDAP1 function reveals the involvement of insulin signalling in the mitochondria-dependent neuromuscular degeneration

[EN] Charcot-Marie-Tooth disease is a rare peripheral neuropathy for which there is no specific treatment. Some forms of Charcot-Marie-Tooth are due to mutations in the GDAP1 gene. A striking feature of mutations in GDAP1 is that they have a variable clinical manifestation, according to disease onset and progression, histology and mode of inheritance. Studies in cellular and animal models have revealed a role of GDAP1 in mitochondrial morphology and distribution, calcium homeostasis and oxidative stress. To get a better understanding of the disease mechanism we have generated models of over-expression and RNA interference of the Drosophila Gdapl gene. In order to get an overview about the changes that Gdapl mutations cause in our disease model, we have combined a comprehensive determination of the metabolic profile in the flies by nuclear magnetic resonance spectroscopy with gene expression analyses and biophysical tests. Our results revealed that both up- and down-regulation of Gdapl results in an early systemic inactivation of the insulin pathway before the onset of neuromuscular degeneration, followed by an accumulation of carbohydrates and an increase in the (3-oxidation of lipids. Our findings are in line with emerging reports of energy metabolism impairments linked to different types of neural pathologies caused by defective mitochondrial function, which is not surprising given the central role of mitochondria in the control of energy metabolism. The relationship of mitochondrial dynamics with metabolism during neurodegeneration opens new avenues to understand the cause of the disease, and for the discovery of new biomarkers and treatments.This work was supported by a project grant from the Association Francaise contre les Myopathies [AFM 18540 to M.I.G]; a collaborative grant from International Rare Diseases Research consortium (IRDiRC) and Institute de Salud Carlos III [IR11/TREAT-CMT to M.I.G. (partner 12) and F.V.P. (partner 8)]; funding from Institute de Salud Carlos III through Biomedical Network Research Center for Rare Diseases and the INGENIO 2010 program to F.V.P.; and a project grant from the Spanish Government (Secretaria de Estado de Investigacion, Desarollo e Innovacion, Ministerio de Economia y Competitividad) [SAF2014-53977-R to A.P.].Lopez Del Amo, V.; Palomino-Schätzlein, M.; Seco-Cervera, M.; Garcia-Gimenez, JL.; Pallardó-Calatayud, FV.; Pineda-Lucena, A.; Galindo-Orozco, MI. (2017). A Drosophila model of GDAP1 function reveals the involvement of insulin signalling in the mitochondria-dependent neuromuscular degeneration. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1863(3):801-809. https://doi.org/10.1016/j.bbadis.2017.01.003S8018091863

Peptides encoded by short ORFs control development and define a new eukaryotic gene family

Despite recent advances in developmental biology and in genomics, key questions remain regarding the organisation of cells into embryos. One possibility is that novel types of genes might await discovery and could provide some of the answers. Genome annotation depends strongly on comparison with previously known gene sequences, and so genes having previously uncharacterised structure and function can be missed. Here we present the characterisation of tarsal-less, a new such type of gene. Tarsal-less has two unusual features: first, it contains more than one coding unit, a structure more similar to some bacterial genes. Second, it codes for small peptides rather than proteins, and in fact these peptides represent the smallest gene products known to date. Functional analysis of this gene in the fruitfly Drosophila shows that it has important functions throughout development, including tissue morphogenesis and pattern formation. We identify genes similar to tarsal-less in other species, and thus define a tarsal-less-related gene family. We expect that a combination of bioinformatic and functional methods, such as the ones we use in this study, will identify and characterize more genes of this type. Potentially, thousands of such new genes may exist

Evolutionary History of the Smyd Gene Family in Metazoans: A Framework to Identify the Orthologs of Human Smyd Genes in Drosophila and Other Animal Species.

The Smyd gene family code for proteins containing a conserved core consisting of a SET domain interrupted by a MYND zinc finger. Smyd proteins are important in epigenetic control of development and carcinogenesis, through posttranslational modifications in histones and other proteins. Previous reports indicated that the Smyd family is quite variable in metazoans, so a rigorous phylogenetic reconstruction of this complex gene family is of central importance to understand its evolutionary history and functional diversification or conservation. We have performed a phylogenetic analysis of Smyd protein sequences, and our results show that the extant metazoan Smyd genes can be classified in three main classes, Smyd3 (which includes chordate-specific Smyd1 and Smyd2 genes), Smyd4 and Smyd5. In addition, there is an arthropod-specific class, SmydA. While the evolutionary history of the Smyd3 and Smyd5 classes is relatively simple, the Smyd4 class has suffered several events of gene loss, gene duplication and lineage-specific expansions in the animal phyla included in our analysis. A more specific study of the four Smyd4 genes in Drosophila melanogaster shows that they are not redundant, since their patterns of expression are different and knock-down of individual genes can have dramatic phenotypes despite the presence of the other family members

Generation and characterization of the Drosophila melanogaster paralytic gene knock-out as a model for Dravet syndrome

[EN] Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.This research has received funding from the ApoyoDravet patients association towards project running costs and stipends for Andrea Tapia. Work in the laboratory of MIG was funded by PROMETEU/2018/135 from "Conselleria, de Sanitat de la Generalitat Valenciana". Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020).Tapia, A.; Giachello, CN.; Palomino-Schätzlein, M.; Baines, RA.; Galindo, MI. (2021). Generation and characterization of the Drosophila melanogaster paralytic gene knock-out as a model for Dravet syndrome. Life. 11(11):1-18. https://doi.org/10.3390/life11111261118111

Transcriptional expression profile of <i>Drosophila</i> and mouse <i>Smyd4</i> class genes.

<p>We show a transcriptional study for <i>Drosophila melanogaster</i> genes <i>CG1868</i> (A-A”‘), <i>CG8378</i> (B-B”‘), <i>CG14122</i> (C-C”‘) and <i>CG7759</i> (D-D”‘) and <i>Mus musculus Smyd4</i> (E-G). For the <i>Drosophila</i> genes we show high throughput data from two consortia, the anatomical expression profile of FlyAtlas (A-D) and the temporal expression profile of modENCODE (A’-D’). For these genes we also determined the expression pattern by i<i>n situ</i> hybridization in embryos (anterior to the right, lateral view except where indicated). We show expression in extended germ band (A”-D”) and in stage 16 embryos (A”‘-D”‘). The ventral nerve cord in B”‘ is not visible as the embryo is slightly tilted. We also determined the expression of <i>Smyd4</i> in a 14.5E mouse embryo section. We show sections of the abdomen (E), dorsal trunk (F) and head (G). Abbreviations are used for the tissues where expression is detected as follows: GP, gut primordium; CP, cephalic primordium of the central nervous system; GT, gut; MS, mesoderm; CNS, central nervous system; SP, spinal cord; DRG, dorsal root ganglia; EN, encephalon.</p

Proposed evolutionary history of <i>Smyd</i> genes in metazoans.

<p>The schematic tree represents the accepted phylogeny for the phyla/subphyla represented in this work. From an original complement of <i>Smyd</i> genes, comprising Smyd3, Smyd4 and Smyd5, we indicate with white boxes the most likely events of gene gain (+) duplication (Dup.) and expansion (Exp.); and in black boxes the events of gene loss (-).</p

Sequence analysis of the confirmed and putative MYND domains.

<p>(A) For the protein sequences indicated on the left, the MYND Zn finger within the interrupted SET domain is compared to the PROSITE signature PS01360 and to the MYND hidden Markov model PFAM signature. For the PROSITE signature, the formula is indicated above with Zn-ligand residues in bold and highlighting the four Zn-ligand pairs. We have highlighted in grey all departures from the signature, including residues that do not match the consensus and stretches of the wrong length. For the PFAM model we indicate whether the sequence is a perfect match (+), a partial match of the right or left portions (+(r) and +(l) respectively), or not a match at all (-, highlighted grey). (B) Scheme of the cross-brace disposition of a MYND-type Zn finger, with the most common Zn-ligand residues depicted in black and Zn ions as empty circles; the numbers indicate the number of aminoacids between Zn-ligand residues according to the PS01360 formula.</p