A Drosophila model of neuronal ceroid lipofuscinosis CLN4 reveals a hypermorphic gain of function mechanism

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of CLN4 by expressing CLN4 mutant human CSPα (hCSPα) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

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ADrosophilamodel of neuronal ceroid lipofuscinosisCLN4reveals a hypermorphic gain of function mechanism

10.1101/579771 ◽

2019 ◽

Author(s):

Elliot Imler ◽

Jin Sang Pyon ◽

Selina Kindelay ◽

Yong-quan Zhang ◽

Sreeganga S. Chandra ◽

...

Keyword(s):

Membrane Trafficking ◽

Gene Dosage ◽

Neuronal Ceroid Lipofuscinoses ◽

Dependent Manner ◽

Membrane Structures ◽

Gain Of Function ◽

Function Mechanism ◽

Ubiquitinated Proteins ◽

Activity Dependent ◽

Dose Dependent

AbstractThe autosomal dominant neuronal ceroid lipofuscinoses (NCL)CLN4is caused by mutations in the synaptic vesicle (SV) protein CSPα, which is a critical co-chaperone of Hsc70 protecting synapses from activity-dependent degeneration. We developed the first animal models ofCLN4by expressing eitherCLN4mutant human CSPα (hCSPα) orDrosophilaCSP (dCSP) in fly neurons. Similar to patients,CLN4mutations induced excessive oligomerization of mutant hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, mostCLN4mutant hCSPα abnormally accumulated in axons and somata, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructurally, abnormal multi-laminar membrane structures were frequently observed in axons and somata next to degenerative abnormalities. The lethality, oligomerization and prelysosomal accumulation induced byCLN4mutations was attenuated by reducing wild type (WT) dCSP levels and enhanced by increasing WT dCSP or hCSPα levels, which indicates that bothCLN4alleles resemble dominant hypermorphic gain of function mutations. Furthermore, reducing the gene dosage of Hsc70 also attenuatedCLN4phenotypes. Taken together, we suggest thatCLN4alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

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Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner

Bioscience Reports ◽

10.1042/bsr20080143 ◽

2009 ◽

Vol 29 (3) ◽

pp. 173-181 ◽

Cited By ~ 12

Author(s):

Kristina Oresic ◽

Britta Mueller ◽

Domenico Tortorella

Keyword(s):

Quality Control ◽

Protein Extraction ◽

Neuronal Ceroid Lipofuscinosis ◽

Point Mutations ◽

Lysosomal Storage Diseases ◽

Neuronal Ceroid Lipofuscinoses ◽

Dependent Manner ◽

Lysosomal Storage ◽

Er Quality Control ◽

Recessive Mutations

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6G123D and Cln6M241T) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6G123D and Cln6M241T polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.

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Hepatitis C Virus Induces the Localization of Lipid Rafts to Autophagosomes for Its RNA Replication

Journal of Virology ◽

10.1128/jvi.00541-17 ◽

2017 ◽

Vol 91 (20) ◽

Cited By ~ 22

Author(s):

Ja Yeon Kim ◽

Linya Wang ◽

Jiyoung Lee ◽

Jing-hsiung James Ou

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Rafts ◽

Membrane Trafficking ◽

Rna Replication ◽

Host Cells ◽

Hcv Rna ◽

Dependent Manner ◽

Human Pathogens ◽

Membrane Structures

ABSTRACT Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication. IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this study, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells.

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Codon-optimized TDP-43-mediated neurodegeneration in a Drosophila model for ALS/FTLD

10.1101/696963 ◽

2019 ◽

Author(s):

Tanzeen Yusuff ◽

Shreyasi Chatterjee ◽

Ya-Chu Chang ◽

Tzu-Kang Sang ◽

George R. Jackson

Keyword(s):

The Novel ◽

Genetic Screens ◽

Gain Of Function ◽

Function Mechanism ◽

Promoter Fusion ◽

Drosophila Model ◽

Weight Protein ◽

Photoreceptor Neurons ◽

And Function

ABSTRACTTransactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains of affected patients remains unclear. In a novel Drosophila melanogaster model, we report gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on our observations, we propose that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets.

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C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons

Human Molecular Genetics ◽

10.1093/hmg/ddab089 ◽

2021 ◽

Author(s):

Eun Seon Kim ◽

Chang Geon Chung ◽

Jeong Hyang Park ◽

Byung Su Ko ◽

Sung Soon Park ◽

...

Keyword(s):

Retinal Degeneration ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Accumulation ◽

Heterozygous Mutation ◽

Pathological Feature ◽

Dependent Manner ◽

Cellular Processes ◽

Drosophila Model ◽

Post Transcriptional Regulation

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.

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Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

Eukaryotic Cell ◽

10.1128/ec.00049-07 ◽

2007 ◽

Vol 6 (6) ◽

pp. 931-939 ◽

Cited By ~ 96

Author(s):

Fang Li ◽

Michael J. Svarovsky ◽

Amy J. Karlsson ◽

Joel P. Wagner ◽

Karen Marchillo ◽

...

Keyword(s):

Candida Albicans ◽

Cell Adhesion ◽

Biofilm Formation ◽

Fungal Infections ◽

Gene Dosage ◽

Venous Catheter ◽

Flow Chamber ◽

Dependent Manner

ABSTRACT Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.

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The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0500 ◽

2004 ◽

Vol 15 (2) ◽

pp. 481-496 ◽

Cited By ~ 34

Author(s):

Josefa Andrade ◽

Hu Zhao ◽

Brian Titus ◽

Sandra Timm Pearce ◽

Margarida Barroso

Keyword(s):

Endoplasmic Reticulum ◽

Conformational Changes ◽

Membrane Trafficking ◽

Secretory Pathway ◽

Network Formation ◽

Binding Protein ◽

Dependent Manner ◽

Microtubule Depolymerization ◽

Independent Manner ◽

Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200405018 ◽

2004 ◽

Vol 167 (1) ◽

pp. 75-85 ◽

Cited By ~ 79

Author(s):

Brenton L. Scott ◽

Jeffrey S. Van Komen ◽

Hassan Irshad ◽

Song Liu ◽

Kirilee A. Wilson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Fusion ◽

Membrane Trafficking ◽

Snare Complex ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bud Neck ◽

Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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Modelling of Neuronal Ceroid Lipofuscinosis Type 2 in Dictyostelium discoideum Suggests That Cytopathological Outcomes Result from Altered TOR Signalling

Cells ◽

10.3390/cells8050469 ◽

2019 ◽

Vol 8 (5) ◽

pp. 469 ◽

Cited By ~ 2

Author(s):

Paige K. Smith ◽

Melodi G. Sen ◽

Paul R. Fisher ◽

Sarah J. Annesley

Keyword(s):

Dictyostelium Discoideum ◽

Neuronal Ceroid Lipofuscinosis ◽

Clinical Manifestations ◽

Batten Disease ◽

Neuronal Ceroid Lipofuscinoses ◽

Antisense Inhibition ◽

Phenotypic Characterisation ◽

Cellular Slime ◽

Multiple Cell

The neuronal ceroid lipofuscinoses comprise a group of neurodegenerative disorders with similar clinical manifestations whose precise mechanisms of disease are presently unknown. We created multiple cell lines each with different levels of reduction of expression of the gene coding for the type 2 variant of the disease, Tripeptidyl peptidase (Tpp1), in the cellular slime mould Dictyostelium discoideum. Knocking down Tpp1 in Dictyostelium resulted in the accumulation of autofluorescent material, a characteristic trait of Batten disease. Phenotypic characterisation of the mutants revealed phenotypic deficiencies in growth and development, whilst endocytic uptake of nutrients was enhanced. Furthermore, the severity of the phenotypes correlated with the expression levels of Tpp1. We propose that the phenotypic defects are due to altered Target of Rapamycin (TOR) signalling. We show that treatment of wild type Dictyostelium cells with rapamycin (a specific TOR complex inhibitor) or antisense inhibition of expression of Rheb (Ras homologue enriched in the brain) (an upstream TOR complex activator) phenocopied the Tpp1 mutants. We also show that overexpression of Rheb rescued the defects caused by antisense inhibition of Tpp1. These results suggest that the TOR signalling pathway is responsible for the cytopathological outcomes in the Dictyostelium Tpp1 model of Batten disease.

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