scholarly journals Negative Regulation of Autophagy by UBA6-BIRC6–Mediated Ubiquitination of LC3

2019 ◽  
Author(s):  
Rui Jia ◽  
Juan S. Bonifacino
Keyword(s):  
Ubiquitin Ligase ◽  
Neurodegenerative Disorders ◽  
Hippocampal Neurons ◽  
Neuroblastoma Cells ◽  
Autophagic Flux ◽  
Protein Synthesis Inhibition ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Ubiquitin Ligase E3 ◽  
Autophagy Inhibition

AbstractAlthough the process of autophagy has been extensively studied, the mechanisms that regulate it remain insufficiently understood. The ability to manipulate autophagy is important not only for addressing fundamental biological questions, but also for its possible application to the treatment of various human diseases. To identify novel regulators of autophagy, we performed a whole-genome CRISPR/Cas9 knockout screen in H4 human neuroblastoma cells gene-edited to express the endogenous autophagy effector LC3B fused to a tandem of GFP and mCherry. Using this methodology, we identified the ubiquitin-activating (E1) enzyme UBA6 and the hybrid ubiquitin-conjugating (E2)/ubiquitin-ligase (E3) enzyme BIRC6 as important autophagy regulators. We found that these two enzymes cooperate to monoubiquitinate LC3B on lysine-51, targeting it for degradation by the proteasome. Knockout of UBA6 or BIRC6 increased the levels of LC3B as well as autophagic flux under conditions of nutrient deprivation or protein synthesis inhibition. Moreover, depletion of UBA6 or BIRC6 KO decreased the formation of aggresome-like induced structures in H4 cells, and aggregates of an α-synuclein mutant in the axon of rat hippocampal neurons. These findings demonstrate that UBA6 and BIRC6 negatively regulate autophagy by limiting the availability of LC3B, possibly to prevent the deleterious effects of excessive autophagy. Inhibition of UBA6 or BIRC6, on the other hand, could be used to enhance autophagic clearance of protein aggregates in neurodegenerative disorders.

scholarly journals Negative regulation of autophagy by UBA6-BIRC6–mediated ubiquitination of LC3

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rui Jia ◽  
Juan S Bonifacino
Keyword(s):  
Protein Synthesis ◽  
Ubiquitin Ligase ◽  
Neurodegenerative Disorders ◽  
Hippocampal Neurons ◽  
Negative Regulation ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Whole Genome ◽  
Protein Synthesis Inhibition ◽  
Ubiquitin Conjugating Enzyme

Although the process of autophagy has been extensively studied, the mechanisms that regulate it remain insufficiently understood. To identify novel autophagy regulators, we performed a whole-genome CRISPR/Cas9 knockout screen in H4 human neuroglioma cells expressing endogenous LC3B tagged with a tandem of GFP and mCherry. Using this methodology, we identified the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as autophagy regulators. We found that these enzymes cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation. Knockout of UBA6 or BIRC6 increased autophagic flux under conditions of nutrient deprivation or protein synthesis inhibition. Moreover, UBA6 or BIRC6 depletion decreased the formation of aggresome-like induced structures in H4 cells, and α-synuclein aggregates in rat hippocampal neurons. These findings demonstrate that UBA6 and BIRC6 negatively regulate autophagy by limiting the availability of LC3B. Inhibition of UBA6/BIRC6 could be used to enhance autophagic clearance of protein aggregates in neurodegenerative disorders.

scholarly journals 4-Hydroxychalcone Induces Cell Death via Oxidative Stress in MYCN-Amplified Human Neuroblastoma Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Amnah M. Alshangiti ◽  
Eszter Tuboly ◽  
Shane V. Hegarty ◽  
Cathal M. McCarthy ◽  
Aideen M. Sullivan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Cell Death ◽  
Cytotoxic Effect ◽  
Neuroblastoma Cells ◽  
Reactive Oxygen ◽  
Prognostic Indicator ◽  
Oxygen Species ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma

Neuroblastoma is an embryonal malignancy that arises from cells of sympathoadrenal lineage during the development of the nervous system. It is the most common pediatric extracranial solid tumor and is responsible for 15% of childhood deaths from cancer. Fifty percent of cases are diagnosed as high-risk metastatic disease with a low overall 5-year survival rate. More than half of patients experience disease recurrence that can be refractory to treatment. Amplification of the MYCN gene is an important prognostic indicator that is associated with rapid disease progression and a poor prognosis, highlighting the need for new therapeutic approaches. In recent years, there has been an increasing focus on identifying anticancer properties of naturally occurring chalcones, which are secondary metabolites with variable phenolic structures. Here, we report that 4-hydroxychalcone is a potent cytotoxin for MYCN-amplified IMR-32 and SK-N-BE (2) neuroblastoma cells, when compared to non-MYCN-amplified SH-SY5Y neuroblastoma cells and to the non-neuroblastoma human embryonic kidney cell line, HEK293t. Moreover, 4-hydroxychalcone treatment significantly decreased cellular levels of the antioxidant glutathione and increased cellular reactive oxygen species. In addition, 4-hydroxychalcone treatment led to impairments in mitochondrial respiratory function, compared to controls. In support of this, the cytotoxic effect of 4-hydroxychalcone was prevented by co-treatment with either the antioxidant N-acetyl-L-cysteine, a pharmacological inhibitor of oxidative stress-induced cell death (IM-54) or the mitochondrial reactive oxygen species scavenger, Mito-TEMPO. When combined with the anticancer drugs cisplatin or doxorubicin, 4-hydroxychalcone led to greater reductions in cell viability than was induced by either anti-cancer agent alone. In summary, this study identifies a cytotoxic effect of 4-hydroxychalcone in MYCN-amplified human neuroblastoma cells, which rationalizes its further study in the development of new therapies for pediatric neuroblastoma.

scholarly journals Cannabidiol induces autophagy via ERK1/2 activation in neural cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Talita A. M. Vrechi ◽  
Anderson H. F. F. Leão ◽  
Ingrid B. M. Morais ◽  
Vanessa C. Abílio ◽  
Antonio W. Zuardi ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Neurodegenerative Disorders ◽  
Molecular Mechanisms ◽  
Cannabis Sativa ◽  
Autophagic Flux ◽  
Neural Cells ◽  
Human Neuroblastoma ◽  
Trpv1 Receptor ◽  
The Central Nervous System ◽  
Catabolic Process

AbstractAutophagy is a lysosomal catabolic process essential to cell homeostasis and is related to the neuroprotection of the central nervous system. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid present in Cannabis sativa. Many therapeutic actions have been linked to this compound, including autophagy activation. However, the precise underlying molecular mechanisms remain unclear, and the downstream functional significance of these actions has yet to be determined. Here, we investigated CBD-evoked effects on autophagy in human neuroblastoma SH-SY5Y and murine astrocyte cell lines. We found that CBD-induced autophagy was substantially reduced in the presence of CB1, CB2 and TRPV1 receptor antagonists, AM 251, AM 630 and capsazepine, respectively. This result strongly indicates that the activation of these receptors mediates the autophagic flux. Additionally, we demonstrated that CBD activates autophagy through ERK1/2 activation and AKT suppression. Interestingly, CBD-mediated autophagy activation is dependent on the autophagy initiator ULK1, but mTORC1 independent. Thus, it is plausible that a non-canonical pathway is involved. Our findings collectively provide evidence that CBD stimulates autophagy signal transduction via crosstalk between the ERK1/2 and AKT kinases, which represent putative regulators of cell proliferation and survival. Furthermore, our study sheds light on potential therapeutic cannabinoid targets that could be developed for treating neurodegenerative disorders.

scholarly journals A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer’s disease

2021 ◽  
Author(s):  
Vega García-Escudero ◽  
Daniel Ruiz-Gabarre ◽  
Ricardo Gargini ◽  
Mar Pérez ◽  
Esther García ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Neuroblastoma Cells ◽  
Rna Seq ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Post Translational Modifications ◽  
Protein Levels ◽  
Tau Isoform ◽  
Human Specific

AbstractTauopathies, including Alzheimer’s disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer’s patients’ brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3β, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer’s disease and other tauopathies.

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