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Triose phosphate isomerase (TPI) deficiency glycolytic enzymopathy is a progressive neurodegenerative condition that remains poorly understood. The disease is caused exclusively by specific missense mutations affecting the TPI protein and clinically features hemolytic anemia, adult-onset neurological impairment, degeneration, and reduced longevity. TPI has a well-characterized role in glycolysis, catalyzing the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P); however, little is known mechanistically about the pathogenesis associated with specific recessive mutations that cause progressive neurodegeneration. Here, we describe key aspects of TPI pathogenesis identified using the [TPI.sup.sugarkill] mutation, a Drosophila model of human TPI deficiency. Specifically, we demonstrate that the mutant protein is expressed, capable of forming a homodimer, and is functional. However, the mutant protein is degraded by the 20S proteasome core leading to loss-of-function pathogenesis.

Heritable mutations, known as inborn errors of metabolism, cause numerous devastating human diseases, typically as a result of a deficiency in essential metabolic products or the accumulation of toxic intermediates. We have isolated a missense mutation in the Drosophila sugarkill (sgk) gene that causes phenotypes analogous to symptoms of triosephosphate isomerase (TPI) deficiency, a human familial disease, characterized by anaerobic metabolic dysfunction resulting from pathological missense mutations affecting the encoded TPI protein. In Drosophila, the sgk gene encodes the glycolytic enzyme TPI. Our analysis of sgk mutants revealed TPI impairment associated with reduced longevity, progressive locomotor deficiency, and neural degeneration. Biochemical studies demonstrate that mutation of this glycolytic enzyme gene does not result in a bioenergetic deficit, suggesting an alternate cause of enzymopathy associated with TPI impairment.

Triosephosphate isomerase (TPI) is a glycolytic enzyme that converts dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3- phosphate (GAP). Glycolytic enzyme dysfunction leads to metabolic diseases collectively known as glycolytic enzymopathies. Of these enzymopathies, TPI deficiency is unique in the severity of neurological symptoms. The Drosophila sugarkill mutant closely models TPI deficiency and encodes a protein prematurely degraded by the proteasome. This led us to question whether enzyme catalytic activity was crucial to the pathogenesis of TPI sugarkill neurological phenotypes. To study TPI deficiency in vivo we developed a genomic engineering system for the TPI locus that enables the efficient generation of novel TPI genetic variants. Using this system we demonstrate that TPI sugarkill can be genetically complemented by TPI encoding a catalytically inactive enzyme. Furthermore, our results demonstrate a non-metabolic function for TPI, the loss of which contributes significantly to the neurological dysfunction in this animal model. [ABSTRACT FROM AUTHOR]

Numerous mitochondrial DNA mutations cause mitochondrial encephalomyopathy: a collection of related diseases for which there exists no effective treatment. Mitochondrial encephalomyopathies are complex multisystem diseases that exhibit a relentless progression of severity, making them both difficult to treat and study. The pathogenic and compensatory metabolic changes that are associated with chronic mitochondrial dysfunction are not well understood. The Drosophila ATP61 mutant models human mitochondrial encephalomyopathy and allows the study of metabolic changes and compensation that occur throughout the lifetime of an affected animal. ATP61animals have a nearly complete loss of ATP synthase activity and an acute bioenergetic deficit when they are asymptomatic, but surprisingly we discovered no chronic bioenergetic deficit in these animals during their symptomatic period. Our data demonstrate dynamic metabolic compensatory mechanisms that sustain normal energy availability and activity despite chronic mitochondrial complex V dysfunction resulting from an endogenous mutation in the mitochondrial DNA. ATP61animals compensate for their loss of oxidative phosphorylation through increases in glycolytic flux, ketogenesis and Kreb's cycle activity early during pathogenesis. However, succinate dehydrogenase activity is reduced and mitochondrial supercomplex formation is severely disrupted contributing to the pathogenesis seen in ATP61 animals. These studies demonstrate the dynamic nature of metabolic compensatory mechanisms and emphasize the need for time course studies in tractable animal systems to elucidate disease pathogenesis and novel therapeutic avenues. [ABSTRACT FROM AUTHOR]

Mitochondrial encephalomyopathies are common and devastating multisystem genetic disorders characterized by neuromuscular dysfunction and tissue degeneration. Point mutations in the human mitochondrial ATP6 gene are known to cause several related mitochondrial disorders: NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh's syndrome), and FBSN (familial bilateral striatal necrosis). We identified a pathogenic mutation in the Drosophila mitochondrial ATP6 gene that causes progressive, adult-onset neuromuscular dysfunction and myodegeneration. Our results demonstrate ultrastructural defects in the mitochondrial innermembrane, neural dysfunction, and a marked reduction in mitochondrial ATP synthase activity associated with this mutation. This Drosophila mutant recapitulates key features of the human neuromuscular disorders enabling detailed in vivo studies of these enigmatic diseases. [ABSTRACT FROM AUTHOR]

Examines the impact of Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) on axon guidance receptor. Role of Dscam in neural development; Correlation between Dscam and messenger RNA; Relevance of receptor molecules on extracellular ligands.

Mitochondrial encephalomyopathies (MEs) result from mutations in mitochondrial genes critical to oxidative phosphorylation. Severe and untreatable ME results from mutations affecting each endogenous mitochondrial encoded gene, including all 13 established protein coding genes. Effective techniques to manipulate mitochondrial genome are limited and targeted mitochondrial protein expression is currently unavailable. Here we report the development of a mitochondrial-targeted RNA expression ( mtTRES ) vector capable of protein expression within mitochondria ( mtTRES Pro ). We demonstrate that mtTRES Pro expressed RNAs are targeted to mitochondria and are capable of being translated using EGFP encoded constructs in vivo . We additionally test mtTRES Pro constructs encoding wild type ATP6 for their ability to rescue an established ATP6 1 Drosophila model of ME. Genetic rescue is examined including tests with co-expression of mitochondrial targeted translational inhibitors TLI-NCL::ATP6 RNAs that function to reduce expression of the endogenous mutant protein. The data demonstrate allotopic RNA expression of mitochondrial targeted wild type ATP6 coding RNAs are sufficient to partially rescue a severe and established animal model of ME but only when combined with a method to inhibit mutant protein expression, which likely competes for incorporation into complex V. [ABSTRACT FROM AUTHOR]

Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase-mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain-mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting. [ABSTRACT FROM AUTHOR]

Endogenous mitochondrial genes encode critical oxidative phosphorylation components and their mutation results in a set of disorders known collectively as mitochondrial encephalomyopathies. There is intensive interest in modulating mitochondrial function as organelle dysfunction has been associated with numerous disease states. Proteins encoded by the mitochondrial genome cannot be genetically manipulated by current techniques. Here we report the development of a mitochondrial-targeted RNA expression system (mtTRES) utilizing distinct non-coding leader sequences (NCLs) and enabling in vivo expression of small mitochondrial-targeted RNAs. mtTRES expressing small chimeric antisense RNAs was used as translational inhibitors (TLIs) to target endogenous mitochondrial protein expression in vivo. By utilizing chimeric antisense RNA we successfully modulate expression of two mitochondrially-encoded proteins, ATP6 and COXII, and demonstrate the utility of this system in vivo and in human cells. This technique has important and obvious research and clinical implications. [ABSTRACT FROM AUTHOR]

Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase-mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain-mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting., (© 2015 Dong et al.)

Reactive oxygen species (ROS) play essential roles in cell signaling, survival, and homeostasis. Aberrant ROS lead to disease and contribute to the aging process. Numerous enzymes and vigilant antioxidant pathways are required to regulate ROS for normal cellular health. Mitochondria are a major source of ROS, and mechanisms to prevent elevated ROS during oxidative phosphorylation require super oxide dismutase (SOD) activity. SOD2, also known as MnSOD, is targeted to mitochondria and is instrumental in regulating ROS by conversion of superoxides to hydrogen peroxide, which is further broken down into H(2)O and oxygen. Here, we describe the identification of a novel mutation within the mitochondrial SOD2 enzyme in Drosophila that results in adults with an extremely shortened life span, sensitivity to hyperoxia, and neuropathology. Additional studies demonstrate that this novel mutant, SOD2(bewildered), exhibits abnormal brain morphology, suggesting a critical role for this protein in neurodevelopment. We investigated the basis of this neurodevelopmental defect and discovered an increase in aberrant axonal that could underlie the aberrant neurodevelopment and brain morphology defects. This novel allele, SOD2(bewildered), provides a unique opportunity to study the effects of increased mitochondrial ROS on neural development, axonal targeting, and neural cell degeneration in vivo.

The fruit fly, Drosophila melanogaster, is a powerful model genetic organism that has been used since the turn of the previous century in the study of complex biological problems. In the last decade, numerous researchers have focused their attention on understanding neurodegenerative diseases by utilizing this model system. Numerous Drosophila mutants have been isolated that profoundly affect neural viability and integrity of the nervous system with age. Additionally, many transgenic strains have been developed as models of human disease conditions. We review the existing Drosophila neurodegenerative mutants and transgenic disease models, and discuss the role of the fruit fly in therapeutic development for neurodegenerative diseases.

Single-strand conformational polymorphism analysis has been used successfully to identify single nucleotide changes within sequences based on the fact that multidetection enhancement gels will separate molecules based on their conformation rather than their size. We have expanded the utility of this technique to analyze easily the alternative splicing of pre-mRNAs containing multiple mutually exclusive exons of the same size. We have used this technique to study the Caenorhabditis elegans let-2 gene containing two alternative exons and the Drosophilia melanogaster Dscam gene, which contains 12 mutually exclusive exons. The ease and the quantitative nature of this technique should be very useful.

RNA interference (RNAi) is a useful tool for degrading targeted messenger RNAs (mRNAs) and thus "knocking down" the abundance of the encoded protein. We have been using RNAi in cultured Drosophila cells to evaluate the effect of "knocking down" numerous mRNA processing factors on the alternative splicing of specific pre-mRNAs. This relatively simple technique has allowed us to identify a number of splicing factors that impact the alternative splicing of particular alternatively spliced exons. This approach can be extended to examine the splicing of nearly any gene.

The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.