Morio Ueyama

Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31

Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. We report that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. We revealed that motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.

Identification and characterization of a novel Drosophila 3'-phosphoadenosine 5'-phosphosulfate transporter.

Sulfation of macromolecules requires the translocation of a high energy form of nucleotide sulfate, i.e. 3′-phosphoadenosine 5′-phosphosulfate (PAPS), from the cytosol into the Golgi apparatus. In this study, we identified a novel Drosophila PAPS transporter gene dPAPST2 by conducting data base searches and screening the PAPS transport activity among the putative nucleotide sugar transporter genes in Drosophila. The amino acid sequence of dPAPST2 showed 50.5 and 21.5% homology to the human PAPST2 and SLALOM, respectively. The heterologous expression of dPAPST2 in yeast revealed that the dPAPST2 protein is a PAPS transporter with an apparent Km value of 2.3 μm. The RNA interference of dPAPST2 in cell line and flies showed that the dPAPST2 gene is essential for the sulfation of cellular proteins and the viability of the fly. In RNA interference flies, an analysis of the genetic interaction between dPAPST2 and genes that contribute to glycosaminoglycan synthesis suggested that dPAPST2 is involved in the glycosaminoglycan synthesis and the subsequent signaling. The dPAPST2 and sll genes showed a similar ubiquitous distribution. These results indicate that dPAPST2 may be involved in Hedgehog and Decapentaplegic signaling by controlling the sulfation of heparan sulfate.

Increased Apoptosis of Myoblasts in Drosophila Model for the Walker-Warburg Syndrome

Walker-Warburg syndrome, a progressive muscular dystrophy, is a severe disease with various kinds of symptoms such as muscle weakness and occasional seizures. The genes of protein O-mannosyltransferases 1 and 2 (POMT1 and POMT2), fukutin, and fukutin-related protein are responsible for this syndrome. In our previous study, we cloned Drosophila orthologs of human POMT1 and POMT2 and identified their activity. However, the mechanism of onset of this syndrome is not well understood. Furthermore, little is known about the behavioral properties of the Drosophila POMT1 and POMT2 mutants, which are called rotated abdomen (rt) and twisted (tw), respectively. First, we performed various kinds of behavioral tests and described in detail the muscle structures by using these mutants. The mutant flies exhibited abnormalities in heavy exercises such as climbing or flight but not in light movements such as locomotion. Defective motor function in mutants appeared immediately after eclosion and was exaggerated with aging. Along with motor function, muscle ultrastructure in the tw mutant was altered, as seen in human patients. We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively. Flies expressing RNAi had reduced lifespans. These findings clearly demonstrate that Drosophila POMT mutants are models for human muscular dystrophy. We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity. In this paper, we propose a novel mechanism for the development of muscular dystrophy: POMT mutation causes high myoblast density and position derangement, which result in apoptosis, muscle disorganization, and muscle cell defects.

Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS

In humans, mutations in the fused in sarcoma (FUS) gene have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Cabeza (Caz) is the Drosophila ortholog of human FUS. Previously, we established Drosophila models of ALS harboring Caz-knockdown. These flies develop locomotive deficits and anatomical defects in motoneurons (MNs) at neuromuscular junctions; these phenotypes indicate that loss of physiological FUS functions in the nucleus can cause MN degeneration similar to that seen in FUS-related ALS. Here, we aimed to explore molecules that affect these ALS-like phenotypes of our Drosophila models with eye-specific and neuron-specific Caz-knockdown. We examined several previously reported ALS-related genes and found genetic links between Caz and ter94, the Drosophila ortholog of human Valosin-containing protein (VCP). Genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown strongly enhanced the rough-eye phenotype and the MN-degeneration phenotype caused by Caz-knockdown. Conversely, the overexpression of wild-type ter94 in the background of Caz-knockdown remarkably suppressed those phenotypes. Our data demonstrated that expression levels of Drosophila VCP ortholog dramatically modified the phenotypes caused by Caz-knockdown in either direction, exacerbation or remission. Our results indicate that therapeutic agents that up-regulate the function of human VCP could modify the pathogenic processes that lead to the degeneration of MNs in ALS.

Wolbachia-Mediated Male Killing Is Associated with Defective Chromatin Remodeling

Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon.

P62 Plays a Protective Role in the Autophagic Degradation of Polyglutamine Protein Oligomers in Polyglutamine Disease Model Flies

Background: Oligomers of pathogenic proteins are implicated in the pathomechanisms of neurodegenerative diseases. Results: Depletion of p62 delays the degradation of polyglutamine protein oligomers via autophagy and exacerbates neurodegeneration in polyglutamine disease model flies. Conclusion: p62 plays a protective role via autophagic degradation of polyglutamine protein oligomers. Significance: p62 should be a therapeutic target for the polyglutamine diseases. Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases because it can degrade even large oligomers. Although p62/sequestosome 1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. In this study, to elucidate the role of p62 in such pathogenic conditions in vivo, we used Drosophila models of neurodegenerative diseases. We found that p62 predominantly co-localizes with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function resulted in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. We also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model as well as in ALS model flies. We therefore conclude that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases and possibly for other neurodegenerative diseases.

Functional Analysis of Proteoglycan Galactosyltransferase II RNA Interference Mutant Flies

Heparan sulfate proteoglycan plays an important role in developmental processes by modulating the distribution and stability of the morphogens Wingless, Hedgehog, and Decapentaplegic. Heparan and chondroitin sulfates share a common linkage tetrasaccharide structure, GlcAβ1,3Galβ1,3Galβ1,4Xylβ-O-Ser. In the present study, we identified Drosophila proteoglycan galactosyltransferase II (dβ3GalTII), determined its substrate specificity, and performed its functional analysis by using RNA interference (RNAi) mutant flies. The enzyme transferred a galactose to Galβ1,4Xyl-pMph, confirming that it is the Drosophila ortholog of human proteoglycan galactosyltransferase II. Real-time PCR analyses revealed that dβ3GalTII is expressed in various tissues and throughout development. The dβ3GalTII RNAi mutant flies showed decreased amounts of heparan sulfate proteoglycans. A genetic interaction of dβ3GalTII with Drosophila β1,4-galactoslyltransferase 7 (dβ4GalT7) or with six genes that encode enzymes contributing to the synthesis of glycosaminoglycans indicated that dβ3GalTII is involved in heparan sulfate synthesis for wing and eye development. Moreover, dβ3GalTII knock-down caused a decrease in extracellular Wingless in the wing imaginal disc of the third instar larvae. These results demonstrated that dβ3GalTII contributes to heparan sulfate proteoglycan synthesis in vitro and in vivo and also modulates Wingless distribution.

Parkinson’s disease-linked DNAJC13 mutation aggravates alpha-synuclein-induced neurotoxicity through perturbation of endosomal trafficking

Mutations in DNAJC13 gene have been linked to familial form of Parkinsons disease (PD) with Lewy pathology. DNAJC13 is an endosome-related protein and believed to regulate endosomal membrane trafficking. However, the mechanistic link between DNAJC13 mutation and α-synuclein (αSYN) pathology toward neurodegeneration remains poorly understood. In this study, we showed that PD-linked N855S-mutant DNAJC13 caused αSYN accumulation in the endosomal compartment, presumably due to defective cargo trafficking from the early endosome to the late and/or recycling endosome. In vivo experiments using human αSYN transgenic flies showed that mutant DNAJC13 not only increased the amount of insoluble αSYN in fly head but also induced dopaminergic neurodegeneration, rough eye phenotype and age-dependent locomotor impairment. Together, these findings suggest that DNAJC13 mutation perturbs multi-directional endosomal trafficking, resulting in the aberrant endosomal retention of αSYN, which might predispose to the neurodegenerative process that leads to PD.

NPM-hMLF1 fusion protein suppresses defects of a Drosophila FTLD model expressing the human FUS gene

Fused in sarcoma (FUS) was identified as a component of typical inclusions in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). In FTLD, both nuclear and cytoplasmic inclusions with wild-type FUS exist, while cytoplasmic inclusions with a mutant-form of FUS occur in many ALS cases. These observations imply that FUS plays a role across these two diseases. In this study, we examined the effect of several proteins including molecular chaperons on the aberrant eye morphology phenotype induced by overexpression of wild-type human FUS (hFUS) in Drosophila eye imaginal discs. By screening, we found that the co-expression of nucleophosmin–human myeloid leukemia factor 1 (NPM-hMLF1) fusion protein could suppress the aberrant eye morphology phenotype induced by hFUS. The driving of hFUS expression at 28 °C down-regulated levels of hFUS and endogenous cabeza, a Drosophila homolog of hFUS. The down-regulation was mediated by proteasome dependent degradation. Co-expression of NPM-hMLF1 suppressed this down-regulation. In addition, co-expression of NPM-hMLF1 partially rescued pharate adult lethal phenotype induced by hFUS in motor neurons. These findings with a Drosophila model that mimics FTLD provide clues for the development of novel FTLD therapies.

Repeat Expansion Disease Models

Repeat expansion disorders are a group of inherited neuromuscular diseases, which are caused by expansion mutations of repeat sequences in the disease-causing genes. Repeat expansion disorders include a class of diseases caused by repeat expansions in the coding region of the genes, producing mutant proteins with amino acid repeats, mostly the polyglutamine (polyQ) diseases, and another class of diseases caused by repeat expansions in the noncoding regions, producing aberrant RNA with expanded repeats, which are called noncoding repeat expansion diseases. A variety of Drosophila disease models have been established for both types of diseases, and they have made significant contributions toward elucidating the molecular mechanisms of and developing therapies for these neuromuscular diseases.