scholarly journals Nanoscopic investigation of C9orf72 poly-GA oligomers on nuclear membrane disruption by a photoinducible platform

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Hung-Ming Chien ◽  
Ruei-Yu He ◽  
Chi-Chang Lee ◽  
Yung-An Huang ◽  
I-Ju Hung ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Cortical Neurons ◽  
Dna Binding Protein ◽  
Nucleocytoplasmic Transport ◽  
Time Dependent ◽  
Membrane Disruption ◽  
Dependent Manner ◽  
Alanine Dipeptide ◽  
Lateral Sclerosis ◽  
In Cells

AbstractGlycine-alanine dipeptide repeats (GA DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). While GA DPRs aggregates have been suggested as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers has not been explored due to its unstable nature. In this study, we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows monitoring the oligomerization process of GA DPRs in cells. By applying advanced microscopies, we examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provided direct evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane. Moreover, we found GA DPRs hamper nucleocytoplasmic transport in cells and cause cytosolic retention of TAR DNA-binding protein 43 in cortical neurons. Our results highlight the toxicity of GA DPRs oligomers, which is a key step toward elucidating the pathological roles of C9orf72 DPRs.

scholarly journals Nanoscopic investigation of C9orf72 poly-GA oligomers on nuclear membrane disruption by photoinducible platform

2021 ◽  
Author(s):  
Hung-Ming Chien ◽  
Ruei-Yu He ◽  
Chi-Chang Lee ◽  
Yung-An Huang ◽  
I-Ju Hung ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Cortical Neurons ◽  
Ex Vivo ◽  
Membrane Integrity ◽  
Super Resolution ◽  
Dependent Manner ◽  
Nucleocytoplasmic Shuttling ◽  
Als Patients ◽  
Lateral Sclerosis

Abstract Dipeptide repeats (DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). Within these DPRs, the most abundant glycine-alanine (GA) DPRs form insoluble inclusions in C9orf72-ALS patients. While GA DPRs aggregates have been considered as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers generated during the amyloidogenesis has not yet been explored due to its unstable and fast equilibrium nature. In this study, we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows to monitor the oligomerization process of GA DPRs in cells in nanoscale. By combining lifetime-based and super-resolution fluorescence microscopies with biophysical tools, we thoroughly examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provide direct ex vivo and in vitro evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane integrity. In addition, we found GA DPRs sabotage Ran protein gradient, hamper nucleocytoplasmic shuttling in neurons, and cause the mislocalization of TAR DNA-binding protein 43 in primary cortical neurons. Our results highlight the nanoscopic properties and toxicity of GA DPRs oligomers in living cells, which is a key step toward elucidating the pathological roles of C9orf72 DPRs in disease.

scholarly journals The Corepressor mSin3A Regulates Phosphorylation-Induced Activation, Intranuclear Location, and Stability of AML1

2004 ◽  
Vol 24 (3) ◽  
pp. 1033-1043 ◽  
Author(s):  
Yoichi Imai ◽  
Mineo Kurokawa ◽  
Yuko Yamaguchi ◽  
Koji Izutsu ◽  
Eriko Nitta ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Modification ◽  
Transcriptional Activity ◽  
Regulatory Mechanism ◽  
Dna Binding Protein ◽  
Time Dependent ◽  
Myeloid Differentiation ◽  
Dependent Manner ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

ABSTRACT The AML1 (RUNX1) gene, one of the most frequent targets of translocations associated with human leukemias, encodes a DNA-binding protein that plays pivotal roles in myeloid differentiation through transcriptional regulation of various genes. Previously, we reported that AML1 is phosphorylated on two serine residues with dependence on activation of extracellular signal-regulated kinase, which positively regulates the transcriptional activity of AML1. Here, we demonstrate that the interaction between AML1 and the corepressor mSin3A is regulated by phosphorylation of AML1 and that release of AML1 from mSin3A induced by phosphorylation activates its transcriptional activity. Furthermore, phosphorylation of AML1 regulates its intranuclear location and disrupts colocalization of AML1 with mSin3A in the nuclear matrix. PEBP2β/CBFβ, a heterodimeric partner of AML1, was shown to play a role in protecting AML1 from proteasome-mediated degradation. We show that mSin3A also protects AML1 from proteasome-mediated degradation and that phosphorylation-induced release of AML1 from mSin3A results in degradation of AML1 in a time-dependent manner. This study provides a novel regulatory mechanism for the function of transcription factors mediated by protein modification and interaction with cofactors.

scholarly journals Glycine-alanine dipeptide repeats spread rapidly in a repeat length- and age-dependent manner in the fly brain

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Javier Morón-Oset ◽  
Tessa Supèr ◽  
Jacqueline Esser ◽  
Adrian M. Isaacs ◽  
Sebastian Grönke ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Genetic Screens ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions ◽  
Alanine Dipeptide ◽  
The Brain ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

AbstractHexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the expansions are translated by repeat-associated non-AUG translation into five dipeptide repeat proteins (DPRs). Of these, the polyGR, polyPR and, to a lesser extent, polyGA DPRs are neurotoxic, with polyGA the most abundantly detected DPR in patient tissue. Trans-cellular transmission of protein aggregates has recently emerged as a major driver of toxicity in various neurodegenerative diseases. In vitro evidence suggests that the C9 DPRs can spread. However, whether this phenomenon occurs under more complex in vivo conditions remains unexplored. Here, we used the adult fly brain to investigate whether the C9 DPRs can spread in vivo upon expression in a subset of neurons. We found that only polyGA can progressively spread throughout the brain, which accumulates in the shape of aggregate-like puncta inside recipient cells. Interestingly, GA transmission occurred as early as 3 days after expression induction. By comparing the spread of 36, 100 and 200 polyGA repeats, we found that polyGA spread is enhanced upon expression of longer GA DPRs. Transmission of polyGA is greater in older flies, indicating that age-associated factors exacerbate the spread. These data highlight a unique propensity of polyGA to spread throughout the brain, which could contribute to the greater abundance of polyGA in patient tissue. In addition, we present a model of early GA transmission that is suitable for genetic screens to identify mechanisms of spread and its consequences in vivo.

scholarly journals Abnormal microtubule dynamics disrupt nucleocytoplasmic transport in tau-mediated frontotemporal dementia

2018 ◽  
Author(s):  
F. Paonessa ◽  
L. Evans ◽  
R. Solanki ◽  
D. Larrieu ◽  
S. Wray ◽  
...  
Keyword(s):  
Frontotemporal Dementia ◽  
Nuclear Membrane ◽  
Cortical Neurons ◽  
Neuronal Cell ◽  
Nucleocytoplasmic Transport ◽  
Microtubule Dynamics ◽  
Early Event ◽  
Pathogenic Mechanisms ◽  
Neuronal Cell Bodies ◽  
High Incidence

SUMMARYThe neuronal microtubule-associated protein tau (MAPT) is central to the pathogenesis of many dementias, including Alzheimer’s disease. Autosomal dominant mutations in MAPT cause inherited frontotemporal dementia (FTD), but the underlying pathogenic mechanisms are unclear. Using human stem cell models of FTD due to MAPT mutations, we find that tau becomes hyperphosphorylated and mislocalises to neuronal cell bodies and dendrites in cortical neurons, recapitulating a key early event in FTD. Mislocalised tau in the cell body leads to abnormal microtubule dynamics in FTD-MAPT neurons that grossly deform the nuclear membrane, resulting in defective nucleocytoplasmic transport. Neurons in the post-mortem human FTD-MAPT cortex have a high incidence of nuclear deformation, indicating that tau-mediated nuclear membrane dysfunction is an important pathogenic process in FTD. Defects in nucleocytoplasmic transport in FTD point to important commonalities in the pathogenic mechanisms of both tau-mediated dementias and ALS-FTD due to TDP-43 and C9orf72 mutations.

scholarly journals Loss of C9orf72 perturbs the Ran-GTPase gradient and generates compositionally diverse cytoplasmic Importin β-1 granules in motor and cortical neurons in vivo

2021 ◽  
Author(s):  
Philip McGoldrick ◽  
Agnes Lau ◽  
Zhipeng You ◽  
Thomas M Durcan ◽  
Janice Robertson
Keyword(s):  
Cortical Neurons ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Ran Gtpase ◽  
Repeat Expansions ◽  
Neuronal Subtype ◽  
Link Loss ◽  
Lateral Sclerosis

Repeat expansions in C9orf72 cause Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) eliciting toxic effects through generation of RNA foci, dipeptide repeat proteins and/or loss of C9orf72 protein. Defects in nucleocytoplasmic transport (NCT) have been implicated as a pathogenic mechanism underlying repeat expansion toxicity. Here, we show that loss of C9orf72 causes neuronal specific phenotypes, disrupting the Ran-GTPase gradient both in vitro and in vivo. We describe compositionally different types of cytoplasmic Importin β-1 granules that exhibit neuronal subtype-specific properties in vivo. We show that the abundance of Importin β-1 granules is increased in the context of C9orf72 deficiency, disrupting interactions with nuclear pore complex proteins. These granules appear to bud from the nuclear envelope and are co-immunoreactive for G3BP1 and K63-ubiquitin. These findings link loss of C9orf72 protein to gain-of-function mechanisms and defects in NCT.

Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger

2004 ◽  
Vol 287 (3) ◽  
pp. C797-C806 ◽  
Author(s):  
Olga Chernysh ◽  
Madalina Condrescu ◽  
John P. Reeves
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Time Dependent ◽  
Activation Process ◽  
Dependent Manner ◽  
Wild Type ◽  
Sodium Calcium Exchanger ◽  
Experimental Conditions ◽  
Activated State ◽  
In Cells

Allosteric regulation by cytosolic Ca2+ of Na+/Ca2+ exchange activity in the Ca2+ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca2+ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, Δ(241–680), that operates constitutively; i.e., its activity does not require allosteric Ca2+ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca2+ concentration ([Ca2+]i) compared with nontransfected cells. During Ca2+ entry through store-operated Ca2+ channels, Ca2+ efflux by the wild-type exchanger became evident only after [Ca2+]i approached 100–200 nM. A subsequent decline in [Ca2+]i was observed, suggesting that the activation process was time dependent. In contrast, Ca2+ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca2+]i, the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca2+]i had returned to resting levels. We conclude that Ca2+ efflux activity by the wild-type exchanger is allosterically activated by Ca2+, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca2+]i to resting levels.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

scholarly journals Synthesis and Evaluation of the Antitumor Activity of Novel 1-(4-Substituted phenyl)-2-ethyl Imidazole Apoptosis Inducers In Vitro

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4293
Author(s):  
Zhen-Wang Li ◽  
Chun-Yan Zhong ◽  
Xiao-Ran Wang ◽  
Shi-Nian Li ◽  
Chun-Yuan Pan ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Cells ◽  
Antiproliferative Activity ◽  
Antitumor Agent ◽  
Positive Control ◽  
Selectivity Index ◽  
Time Dependent ◽  
Potential Candidate ◽  
Dependent Manner

Novel imidazole derivatives were designed, prepared, and evaluated in vitro for antitumor activity. The majority of the tested derivatives showed improved antiproliferative activity compared to the positive control drugs 5-FU and MTX. Among them, compound 4f exhibited outstanding antiproliferative activity against three cancer cell lines and was considerably more potent than both 5-FU and MTX. In particular, the selectivity index indicated that the tolerance of normal L-02 cells to 4f was 23–46-fold higher than that of tumor cells. This selectivity was significantly higher than that exhibited by the positive control drugs. Furthermore, compound 4f induced cell apoptosis by increasing the protein expression levels of Bax and decreasing those of Bcl-2 in a time-dependent manner. Therefore, 4f could be a potential candidate for the development of a novel antitumor agent.

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