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1.
Analyst ; 149(3): 846-858, 2024 Jan 29.
Article in English | MEDLINE | ID: mdl-38167886

ABSTRACT

Lipid alterations in the brain are well-documented in disease and aging, but our understanding of their pathogenic implications remains incomplete. Recent technological advances in assessing lipid profiles have enabled us to intricately examine the spatiotemporal variations in lipid compositions within the complex brain characterized by diverse cell types and intricate neural networks. In this study, we coupled time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an amyotrophic lateral sclerosis (ALS) Drosophila model, for the first time, to elucidate changes in the lipid landscape and investigate their potential role in the disease process, serving as a methodological and analytical complement to our prior approach that utilized matrix-assisted laser desorption/ionization mass spectrometry. The expansion of G4C2 repeats in the C9orf72 gene is the most prevalent genetic factor in ALS. Our findings indicate that expressing these repeats in fly brains elevates the levels of fatty acids, diacylglycerols, and ceramides during the early stages (day 5) of disease progression, preceding motor dysfunction. Using RNAi-based genetic screening targeting lipid regulators, we found that reducing fatty acid transport protein 1 (FATP1) and Acyl-CoA-binding protein (ACBP) alleviates the retinal degeneration caused by G4C2 repeat expression and also markedly restores the G4C2-dependent alterations in lipid profiles. Significantly, the expression of FATP1 and ACBP is upregulated in G4C2-expressing flies, suggesting their contribution to lipid dysregulation. Collectively, our novel use of ToF-SIMS with the ALS Drosophila model, alongside methodological and analytical improvements, successfully identifies crucial lipids and related genetic factors in ALS pathogenesis.


Subject(s)
Amyotrophic Lateral Sclerosis , Animals , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Drosophila , Spectrometry, Mass, Secondary Ion , Lipids
2.
Mol Cells ; 45(11): 855-867, 2022 Nov 30.
Article in English | MEDLINE | ID: mdl-36172977

ABSTRACT

For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.


Subject(s)
Drosophila , alpha Karyopherins , Animals , Acetylation , Active Transport, Cell Nucleus , alpha Karyopherins/genetics , alpha Karyopherins/metabolism , Ataxin-3/genetics , Ataxin-3/metabolism , Calcium/metabolism , Drosophila/metabolism , Neurons/metabolism
3.
J Am Soc Mass Spectrom ; 32(10): 2536-2545, 2021 Oct 06.
Article in English | MEDLINE | ID: mdl-34448582

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a degenerative disease caused by motor neuron damage in the central nervous system, and it is difficult to diagnose early. Drosophila melanogaster is widely used to investigate disease mechanisms and discover biomarkers because it is easy to induce disease in Drosophila through genetic engineering. We performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to investigate changes in phospholipid distribution in the brain tissue of an ALS-induced Drosophila model. Fly brain tissues of several hundred micrometers or less were sampled using a fly collar to obtain reproducible tissue sections of similar sizes. MSI of brain tissues of Drosophila cultured for 1 or 10 days showed that the distribution of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI), was significantly different between the control group and the ALS group. In addition, the lipid profile according to phospholipids differed as the culture time increased from 1 to 10 days. These results suggest that disease indicators based on lipid metabolites can be discovered by performing MALDI-MSI on very small brain tissue samples from the Drosophila disease model to ultimately assess the phospholipid changes that occur in early-stage ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Molecular Imaging/methods , Phospholipids , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Brain/diagnostic imaging , Brain/metabolism , Brain Chemistry/physiology , Disease Models, Animal , Drosophila melanogaster , Phospholipids/analysis , Phospholipids/chemistry
4.
Hum Mol Genet ; 30(12): 1084-1100, 2021 06 09.
Article in English | MEDLINE | ID: mdl-33783499

ABSTRACT

RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP Staufen may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS-but not with mutated endogenous NLS-potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/-), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/- is protective. stau+/- also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Frontotemporal Dementia/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics , RNA-Binding Proteins/genetics , RNA/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Arginine/genetics , C9orf72 Protein/genetics , Cell Nucleus/genetics , Cytoplasm/genetics , Dipeptides/genetics , Disease Models, Animal , Drosophila melanogaster/genetics , Frontotemporal Dementia/pathology , Gene Knockdown Techniques , Humans , Neurons/metabolism , Neurons/pathology , Nuclear Localization Signals/genetics , RNA Processing, Post-Transcriptional/genetics
5.
Elife ; 92020 12 11.
Article in English | MEDLINE | ID: mdl-33305734

ABSTRACT

Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. Here, we identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS.


Subject(s)
Calcium/metabolism , Calpain/metabolism , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , alpha Karyopherins/metabolism , Active Transport, Cell Nucleus/physiology , Amyotrophic Lateral Sclerosis/metabolism , Animals , Drosophila melanogaster , Neurons/metabolism
6.
Front Cell Neurosci ; 14: 556461, 2020.
Article in English | MEDLINE | ID: mdl-33192307

ABSTRACT

Due to their enormous surface area compared to other cell types, neurons face unique challenges in properly handling supply and retrieval of the plasma membrane (PM)-a process termed PM turnover-in their distal areas. Because of the length and extensiveness of dendritic branches in neurons, the transport of materials needed for PM turnover from soma to distal dendrites will be inefficient and quite burdensome for somatic organelles. To meet local demands, PM turnover in dendrites most likely requires local cellular machinery, such as dendritic endocytic and secretory systems, dysregulation of which may result in dendritic pathology observed in various neurodegenerative diseases (NDs). Supporting this notion, a growing body of literature provides evidence to suggest the pathogenic contribution of dysregulated PM turnover to dendritic pathology in certain NDs. In this article, we present our perspective view that impaired dendritic endocytic and secretory systems may contribute to dendritic pathology by encumbering PM turnover in NDs.

7.
Mol Cells ; 43(9): 821-830, 2020 Sep 30.
Article in English | MEDLINE | ID: mdl-32975212

ABSTRACT

Altered dendritic morphology is frequently observed in various neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the cellular and molecular basis underlying these pathogenic dendritic abnormalities remains largely unclear. In this study, we investigated dendritic morphological defects caused by dipeptide repeat protein (DPR) toxicity associated with G4C2 expansion mutation of C9orf72 (the leading genetic cause of ALS and FTD) in Drosophila neurons and characterized the underlying pathogenic mechanisms. Among the five DPRs produced by repeat-associated non-ATG translation of G4C2 repeats, we found that arginine-rich DPRs (PR and GR) led to the most significant reduction in dendritic branches and plasma membrane (PM) supply in Class IV dendritic arborization (C4 da) neurons. Furthermore, expression of PR and GR reduced the number of Golgi outposts (GOPs) in dendrites. In Drosophila brains, expression of PR, but not GR, led to a significant reduction in the mRNA level of CrebA, a transcription factor regulating the formation of GOPs. Overexpressing CrebA in PR-expressing C4 da neurons mitigated PM supply defects and restored the number of GOPs, but the number of dendritic branches remained unchanged, suggesting that other molecules besides CrebA may be involved in dendritic branching. Taken together, our results provide valuable insight into the understanding of dendritic pathology associated with C9-ALS/FTD.


Subject(s)
Arginine/metabolism , C9orf72 Protein/metabolism , Drosophila Proteins/metabolism , Golgi Apparatus/metabolism , Neurons/metabolism , Animals , Dipeptides/metabolism , Drosophila
8.
Biointerphases ; 13(3): 03B414, 2018 03 30.
Article in English | MEDLINE | ID: mdl-29602282

ABSTRACT

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging elucidates molecular distributions in tissue sections, providing useful information about the metabolic pathways linked to diseases. However, delocalization of the analytes and inadequate tissue adherence during sample preparation are among some of the unfortunate phenomena associated with this technique due to their role in the reduction of the quality, reliability, and spatial resolution of the ToF-SIMS images. For these reasons, ToF-SIMS imaging requires a more rigorous sample preparation method in order to preserve the natural state of the tissues. The traditional thaw-mounting method is particularly vulnerable to altered distributions of the analytes due to thermal effects, as well as to tissue shrinkage. In the present study, the authors made comparisons of different tissue mounting methods, including the thaw-mounting method. The authors used conductive tape as the tissue-mounting material on the substrate because it does not require heat from the finger for the tissue section to adhere to the substrate and can reduce charge accumulation during data acquisition. With the conductive-tape sampling method, they were able to acquire reproducible tissue sections and high-quality images without redistribution of the molecules. Also, the authors were successful in preserving the natural states and chemical distributions of the different components of fat metabolites such as diacylglycerol and fatty acids by using the tape-supported sampling in microRNA-14 (miR-14) deleted Drosophila models. The method highlighted here shows an improvement in the accuracy of mass spectrometric imaging of tissue samples.


Subject(s)
Drosophila/chemistry , Histocytological Preparation Techniques/methods , Image Processing, Computer-Assisted/methods , Lipids/analysis , Spectrometry, Mass, Secondary Ion/methods , Animals , Drosophila/genetics , Gene Deletion , MicroRNAs/genetics , Reproducibility of Results
9.
Cell Rep ; 20(2): 356-369, 2017 07 11.
Article in English | MEDLINE | ID: mdl-28700938

ABSTRACT

Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. Here, we show that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, our results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.


Subject(s)
Dendrites/metabolism , Dendrites/pathology , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Neurons/metabolism , Neurons/pathology , Peptides/toxicity , Animals , CREB-Binding Protein/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chromatin Immunoprecipitation , Cyclic AMP Response Element-Binding Protein A/metabolism , Dendrites/drug effects , Drosophila Proteins/metabolism , Drosophila melanogaster , Neurons/drug effects
10.
Biochem Biophys Res Commun ; 463(1-2): 1-6, 2015.
Article in English | MEDLINE | ID: mdl-25979357

ABSTRACT

Parkin, an E3 ubuquitin ligase associated with Parkinson's disease (PD), has recently been implicated in mediating innate immunity. However, molecular details regarding parkin-mediated immune response remain to be elucidated. Here, we identified mitochondrial TSPO-VDAC complex to genetically interact with parkin in mediating responses against infection and wound in Drosophila. The loss-of-function mutation in parkin results in defective immune response against bacterial infection. Additionally, parkin mutant larvae showed hypersensitivity against wound regardless of bacterial infection. Interestingly, the combinatorial trans-heterozygotic mutations in parkin and TSPO, or parkin and VDAC showed similar lethal tendency with parkin homozygous mutants. Furthermore, knockdown of TSPO alone also resulted in defective responses to infection and wound analogously to parkin mutants. Taken together, we propose that parkin cooperates with TSPO-VDAC complex to mediate responses against infection and wound.


Subject(s)
Drosophila Proteins/immunology , Drosophila melanogaster/immunology , Ubiquitin-Protein Ligases/immunology , Animals , Animals, Genetically Modified , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Knockdown Techniques , Genes, Insect , Immunity, Innate/genetics , Infections/genetics , Infections/immunology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/immunology , Mutation , Receptors, GABA/immunology , Ubiquitin-Protein Ligases/genetics , Voltage-Dependent Anion Channels/immunology , Wounds and Injuries/genetics , Wounds and Injuries/immunology
11.
Cancer Res ; 65(7): 2804-14, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15805281

ABSTRACT

Fenretinide (N-4-hydroxyphenyl retinamide, 4HPR) is a synthetic anticancer retinoid that is a well-known apoptosis-inducing agent. Recently, we observed that the apoptosis induced by fenretinide could be effectively enhanced in hepatoma cells by a concomitant treatment with parthenolide, which is a known inhibitor of nuclear factor-kappaB (NF-kappaB). Furthermore, treatment with fenretinide triggered the activation of NF-kappaB during apoptosis, which could be substantially inhibited by parthenolide, suggesting that NF-kappaB activation during fenretinide-induced apoptosis has an antiapoptotic effect. This study investigated the molecular mechanism of this apoptotic potentiation by NF-kappaB inhibition. The genes involved in the enhanced fenretinide-induced apoptosis by parthenolide were identified using the differential display-PCR method and subsequent Northern blot or semiquantitative reverse transcriptase PCR analysis. This study identified 35 apoptosis-related genes including 12 unknown genes that were either up- or down-regulated by parthenolide. Interestingly, one up-regulated gene (HA1A2) was isolated and cloned from the liver cDNA, and was found to be identical to ANKRD1, which is also referred to as the CARP gene. Compared with controls treated with an empty vector or with antisense cDNA, the ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells. These results suggest that ANKRD1 and the other genes, whose expressions were substantially modulated by the parthenolide-mediated inhibition of NF-kappaB activation, play roles in the enhanced drug-induced apoptosis. In addition, this study suggests that those identified genes may be useful in anticancer strategies against hepatoma.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Carcinoma, Hepatocellular/drug therapy , Fenretinide/pharmacology , Liver Neoplasms/drug therapy , Sesquiterpenes/pharmacology , Amino Acid Sequence , Base Sequence , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Down-Regulation/drug effects , Drug Synergism , Fenretinide/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Molecular Sequence Data , Muscle Proteins , NF-kappa B/antagonists & inhibitors , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Polymerase Chain Reaction/methods , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Sesquiterpenes/administration & dosage , Up-Regulation/drug effects
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