in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in <em>Drosophila melanogaster</em>

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

University of Wisconsin-Madison

Traumatic brain injury (TBI) is a substantial health issue worldwide, yet the mechanisms responsible for its complex spectrum of pathologies remains largely unknown. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. The model allows us to take advantage of the wealth of experimental tools available in flies. Closed head TBI was inflicted with a mechanical device that subjects flies to rapid acceleration and deceleration. Similar to humans with TBI, flies with TBI exhibited temporary incapacitation, ataxia, activation of the innate immune response, neurodegeneration, and death. Our data indicate that TBI results in death shortly after a primary injury only if the injury exceeds a certain threshold and that age and genetic background, but not sex, substantially affect this threshold. Furthermore, this threshold also appears to be dependent on the same cellular and molecular mechanisms that control normal longevity. This study demonstrates the potential of flies for providing key insights into human TBI that may ultimately provide unique opportunities for therapeutic intervention.

A Drosophila model of closed head traumatic brain injury.

Genetic variability affects the response to numerous xenobiotics but its role in the clinically-observed irregular responses to general anesthetics remains uncertain. To investigate the pharmacogenetics of volatile general anesthetics (VGAs), we developed a Serial Anesthesia Array apparatus to expose multiple Drosophila melanogaster samples to VGAs and behavioral assays to determine pharmacokinetic and pharmacodynamic properties of VGAs. We studied the VGAs isoflurane and sevoflurane in four wild type strains from the Drosophila Genetic Reference Panel, two commonly used laboratory strains (Canton S and w  ), and a mutant in Complex I of the mitochondrial electron transport chain (ND23  ). In all seven strains, isoflurane was more potent than sevoflurane, as predicted by their relative lipid solubilities, and emergence from isoflurane was slower than from sevoflurane, reproducing cardinal pharmacokinetic and pharmacodynamic properties in mammals. In addition, ND23  flies were more sensitive to both agents, as observed in worms, mice, and humans carrying Complex I mutations. Moreover, we found substantial variability among the fly strains both in absolute and in relative pharmacokinetic and pharmacodynamic profiles of isoflurane and sevoflurane. These data indicate that naturally occurring genetic variations measurably influence cardinal pharmacologic properties of VGAs and that flies can be used to identify relevant genetic variations.

Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster.

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions  are sparse. Here we describe the characterization of a mutation in , a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the  mutant phenotype.  mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of  A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of  mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant , including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an  demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease.

Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a  Model of Leigh Syndrome.

A screen for neuroprotective genes in  led to the identification of a mutation that causes extreme, progressive loss of adult brain neuropil in conjunction with massive brain overgrowth. We mapped the mutation to the  () locus, which encodes a tripartite motif-NCL-1, HT2A, and LIN-41 (TRIM-NHL) RNA-binding protein with established roles limiting stem cell proliferation in developing brain and ovary. However, a neuroprotective role for  in the adult  brain has not been described previously. The new allele,  ( ), carries a mutation in the coiled-coil domain of the TRIM motif, and is temperature-sensitive. We demonstrate that mRNA and protein levels of neural stem cell genes are increased in heads of adult  mutants and that the over-proliferation phenotype initiates prior to adult eclosion. We also report that disruption of an uncharacterized gene coding for a presumptive prolyl-4-hydroxylase strongly enhances the over-proliferation and neurodegeneration phenotypes. Together, our results reveal an unexpected role for  that could be relevant to human cancer and neurodegenerative diseases.

Article	Year
A Drosophila model of closed head traumatic brain injury. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24127584	2013
Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster. Scientific reports\| PubMed ID: 29402974	2018
Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Model of Leigh Syndrome. Genetics\| PubMed ID: 29496745	2018
A Novel Mutation in Brain Tumor Causes Both Neural Over-Proliferation and Neurodegeneration in Adult . G3 (Bethesda, Md.)\| PubMed ID: 30126833	2018

Carin Loewen

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