Hippo, Drosophila MST, is a novel modifier of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS.

Mutations in the Fused in Sarcoma (FUS) gene have been identified in familial ALS in human. Drosophila contains a single ortholog of human FUS called Cabeza (Caz). We previously established Drosophila models of ALS targeted to Caz, which developed the locomotive dysfunction and caused anatomical defects in presynaptic terminals of motoneurons. Accumulating evidence suggests that ALS and cancer share defects in many cellular processes. The Hippo pathway was originally discovered in Drosophila and plays a role as a tumor suppressor in mammals. We aimed to determine whether Hippo pathway genes modify the ALS phenotype using Caz knockdown flies. We found a genetic link between Caz and Hippo (hpo), the Drosophila ortholog of human Mammalian sterile 20-like kinase (MST) 1 and 2. Loss-of-function mutations of hpo rescued Caz knockdown-induced eye- and neuron-specific defects. The decreased Caz levels in nuclei induced by Caz knockdown were also rescued by loss of function mutations of hpo. Moreover, hpo mRNA level was dramatically increased in Caz knockdown larvae, indicating that Caz negatively regulated hpo. Our results demonstrate that hpo, Drosophila MST, is a novel modifier of Drosophila FUS. Therapeutic targets that inhibit the function of MST could modify the pathogenic processes of ALS.

Loss-of-function mutation in Hippo suppressed enlargement of lysosomes and neurodegeneration caused by dFIG4 knockdown.

Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuropathy, and more than 80 CMT-causing genes have been identified to date. CMT4J is caused by a loss-of-function mutation in the Factor-Induced-Gene 4 (FIG4) gene, the product of which plays important roles in endosome-lysosome homeostasis. We hypothesized that Mammalian sterile 20-like kinase (MST) 1 and 2, tumor-suppressor genes, are candidate modifiers of CMT4J. We therefore examined the interaction between dFIG4 and Hippo (hpo), Drosophila counterparts of FIG4 and MSTs, respectively, using the Drosophila CMT4J model with the knockdown of dFIG4. The loss-of-function allele of hpo improved the rough eye morphology, locomotive dysfunction accompanied by structural defects in the presynaptic terminals of motoneurons, and the enlargement of lysosomes caused by the knockdown of dFIG4. Therefore, we identified hpo as a modifier of phenotypes induced by the knockdown of dFIG4. These results in Drosophila may provide an insight into the pathogenesis of CMT4J and contribute toward the development of disease-modifying therapy for CMT. We also identified the regulation of endosome-lysosome homeostasis as a novel probable function of Hippo/MST.

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.

Genetic screening of the genes interacting with Drosophila FIG4 identified a novel link between CMT-causing gene and long noncoding RNAs.

Neuron-specific knockdown of the dFIG4 gene, a Drosophila homologue of human FIG4 and one of the causative genes for Charcot-Marie-Tooth disease (CMT), reduces the locomotive abilities of adult flies, as well as causing defects at neuromuscular junctions, such as reduced synaptic branch length in presynaptic terminals of the motor neurons in third instar larvae. Eye imaginal disc-specific knockdown of dFIG4 induces abnormal morphology of the adult compound eye, the rough eye phenotype. In this study, we carried out modifier screening of the dFIG4 knockdown-induced rough eye phenotype using a set of chromosomal deficiency lines on the second chromosome. By genetic screening, we detected 9 and 15 chromosomal regions whose deletions either suppressed or enhanced the rough eye phenotype induced by the dFIG4 knockdown. By further genetic screening with mutants of individual genes in one of these chromosomal regions, we identified the gene CR18854 that suppressed the rough eye phenotype and the loss-of-cone cell phenotype. The CR18854 gene encodes a long non-coding RNA (lncRNA) consisting of 2566 bases. Mutation and knockdown of CR18854 patially suppressed the enlarged lysosome phenotype induced by Fat body-specific knockdown of dFIG4. Further characterization of CR18854, and a few other lncRNAs in relation to dFIG4 in neuron, using neuron-specific dFIG4 knockdown flies indicated a genetic link between the dFIG4 gene and lncRNAs including CR18854 and hsrω. We also obtained data indicating genetic interaction between CR18854 and Cabeza, a Drosophila homologue of human FUS, which is one of the causing genes for amyotrophic lateral sclerosis (ALS). These results suggest that lncRNAs such as CR18854 and hsrω are involved in a common pathway in CMT and ALS pathogenesis.

Overexpression of ter94, Drosophila VCP, improves motor neuron degeneration induced by knockdown of TBPH, Drosophila TDP-43.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterized by the motor neuron degeneration that eventually leads to complete paralysis and death within 2-5 years after disease onset. One of the major pathological hallmark of ALS is abnormal accumulation of inclusions containing TAR DNA-binding protein-43 (TDP-43). TDP-43 is normally found in the nucleus, but in ALS, it localizes in the cytoplasm as inclusions as well as in the nucleus. Loss of nuclear TDP-43 functions likely contributes to neurodegeneration. TBPH is the Drosophila ortholog of human TDP-43. In the present study, we confirmed that Drosophila models harboring TBPH knockdown develop locomotive deficits and degeneration of motoneurons (MNs) due to loss of its nuclear functions, recapitulating the human ALS phenotypes. We previously suggested that ter94, the Drosophila ortholog of human Valosin-containing protein (VCP), is a modulator of degeneration in MNs induced by knockdown of Caz, the Drosophila ortholog of human FUS. In this study, to determine the effects of VCP on TDP-43-assosiated ALS pathogenic processes, we examined genetic interactions between TBPH and ter94. Overexpression of ter94 suppressed the compound eye degeneration caused by TBPH knockdown and suppressed the morbid phenotypes caused by neuron-specific TBPH knockdown, such as locomotive dysfunction and degeneration of MN terminals. Further immunocytochemical analyses revealed that the suppression is caused by restoring the cytoplasmically mislocalized TBPH back to the nucleus. In consistent with these observations, a loss-of-function mutation of ter94 enhanced the compound eye degeneration caused by TBPH knockdown, and partially enhanced the locomotive dysfunction caused by TBPH knockdown. Our data demonstrated that expression levels of ter94 influenced the phenotypes caused by TBPH knockdown, and indicate that reagents that up-regulate the function of human VCP could modify MN degeneration in ALS caused by TDP-43 mislocalization.

An Adult Case of Anti-Myelin Oligodendrocyte Glycoprotein (MOG) Antibody-associated Multiphasic Acute Disseminated Encephalomyelitis at 33-year Intervals.

Acute disseminated encephalomyelitis (ADEM) followed by optic neuritis (ON) has been reported as a distinct phenotype associated with anti-myelin oligodendrocyte protein (MOG) antibody. We herein report the case of a 37-year-old woman who was diagnosed with ADEM at 4 years old of age and who subsequently developed ON followed by recurrent ADEM 33 years after the initial onset. A serum analysis showed anti-MOG antibody positivity. This phenotype has only previously been reported in pediatric cases. Neurologists thus need to be aware that the phenotype may occur in adult patients, in whom it may be assumed to be atypical multiple sclerosis.

[Head drop syndrome in a patient with immune-mediated necrotizing myopathy with anti-signal recognition particle antibody: a case report].

We report an 87-year-old female patient who presented a dropped head and progressive weakness in proximal muscles over five months. The value of serum creatine kinase was 2,708 IU/l and the antibody against signal recognition particle (SRP) was detected by means of immunoprecipitation. The computed tomography of skeletal muscles revealed atrophy and fatty degeneration preferentially in the neck extensors and paraspinal muscles. The biopsied specimen of the deltoid muscle showed necrotic fibers scattered in fascicles with marked myophagia. The mononuclear cells in necrotic fibers were positive against CD68, leading to the diagnosis of immune-mediated necrotizing myopathy. We hypothesize that a group of patients with necrotizing myopathy can present a preferential involvement in neck extensors resulting in dropped head syndrome.