Copper Dependent Modulation of α-Synuclein Phosphorylation in Differentiated SHSY5Y Neuroblastoma Cells

Greco, Marco; Spinelli, Chiara; Riccardis, Lidia De; Buccolieri, Alessandro; Giulio, Simona Di; Musarò, Debora; Pagano, Claudia; Manno, D.; Maffia, Michele

doi:10.3390/ijms22042038

Cited by 10 publications

(7 citation statements)

References 67 publications

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“…For this reason, EVs are implicated in diseases strictly linked to oxidative stress, for instance neurodegenerative disorders ( Fowler and Hill, 2019 ; Meldolesi, 2021 ; Qi et al, 2021 ). Human neuroblastoma SH-SY5Y cell line, widely used as a model to investigate neurodegeneration and oxidative stress ( Martínez et al, 2020 ; Greco et al, 2021 ; Chen et al, 2022 ), has been chosen to perform a comparative morphological and quantitative analysis of membrane budding and isolated microvesicles-like vesicles (MVs), in physiological conditions (control) or under oxidative stress (H2O2-induced), by electron microscopy (EM) and atomic force microscopy (AFM). The main goal of this work was to define a new method to study EVs morphological features and their alterations under stress conditions, evaluating the efficacy of the system compared to conventional methods.…”

Section: Introductionmentioning

confidence: 99%

High-resolution atomic force microscopy as a tool for topographical mapping of surface budding

Sbarigia

Tacconi²,

Mura³

et al. 2022

Front. Cell Dev. Biol.

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Extracellular vesicles (EVs) are membranous nanoparticles secreted by almost all cell types. Reflecting the physiopathological state of the parental cell, EVs circulate in all body fluids, reaching distant cell targets and delivering different bioactive cargoes. As biological carriers, EVs influence their microenvironment altering cellular responses, being considered promising biomarkers for both physiological and pathological conditions. EVs are heterogeneous in terms of size and composition, depending on cell type and exposure to stimuli, and different methods have been developed to characterize their morphological, biophysical, and biochemical features. Among them, electron microscopy (EM) is the main technique used, however, the lack of standardized protocols makes it difficult to characterize EVs with a good reproducibility, thus using multiple approaches may represent a way to obtain more precise information. Furthermore, the relationship between architecture and function, not only in a molecular, but also in a cellular level, is gaining growing emphasis, characterizing morphometric parameters may represent a distinct, but effective approach to study the physiopathological state of the cell. Atomic force microscopy (AFM), may represent a promising method to study in detail EVs dynamics throughout the cell surface and its variations related to the physiological state, overcoming the limits of EM, and providing more reliable information. In this study, human neuroblastoma SH-SY5Y cell line, a cellular model to investigate neurodegeneration and oxidative stress, has been used to perform a comparative morphological and quantitative analysis of membrane budding and isolated large vesicles-enriched (microvesicles-like vesicles; MVs) fraction from control or oxidative stressed cells. Our main goal was to build up a methodology to characterize EVs morphology and spatial distribution over the cell surface in different physiological conditions, and to evaluate the efficacy of AFM against conventional EM. Interestingly, both microscopy techniques were effective for this analysis, but AFM allowed to reveal a differential profiling of plasma membrane budding between the physiological and the stress condition, indicating a potential relationship between mechanical characteristics and functional role. The results obtained may provide interesting perspectives for the use of AFM to study EVs, validating a morphometric approach to understand the pathophysiological state of the cell related to EVs trafficking.

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Section: Introductionmentioning

confidence: 99%

High-resolution atomic force microscopy as a tool for topographical mapping of surface budding

Sbarigia

Tacconi²,

Mura³

et al. 2022

Front. Cell Dev. Biol.

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“…It is known that cells exposed to low concentrations of Cu can attenuate its cytotoxic effect by binding it to different ligands . In addition, some pieces of evidence showed that exposing SH-SY5Y cells to 50 μM Cu does not increase ROS in a significant manner with respect to control . Considering this, we hypothesize that we do not observe Chol synthesis changes after 50 μM Cu exposure because it is too low, making cells able to buffer this low Cu overload.…”

Section: Resultsmentioning

confidence: 79%

“… 66 In addition, some pieces of evidence showed that exposing SH-SY5Y cells to 50 μM Cu does not increase ROS in a significant manner with respect to control. 67 Considering this, we hypothesize that we do not observe Chol synthesis changes after 50 μM Cu exposure because it is too low, making cells able to buffer this low Cu overload. On the other hand, after 400 μM, Cu is too elevated.…”

Section: Resultsmentioning

confidence: 86%

Effect of Sublethal Copper Overload on Cholesterol De Novo Synthesis in Undifferentiated Neuronal Cells

et al. 2022

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Although copper (Cu) is an essential trace metal for cells, it can induce harmful effects as it participates in the Fenton reaction. Involuntary exposure to Cu overload is much more common than expected and has been linked with neurodegeneration, particularly with Alzheimer’s disease (AD) evidenced by a positive correlation between free Cu in plasma and the severity of the disease. It has been suggested that Cu imbalance alters cholesterol (Chol) homeostasis and that high membrane Chol promotes the amyloidogenic processing of the amyloid precursor protein (APP) secreting the β-amyloid (Aβ) peptide. Despite the wide knowledge on the effects of Cu in mature brain metabolism, the consequence of its overload on immature neurons remains unknown. Therefore, we used an undifferentiated human neuroblastoma cell line (SH-SY5Y) to analyze the effect of sublethal concentrations of Cu on 1— de novo Chol synthesis and membrane distribution; 2—APP levels in cells and its distribution in membrane rafts; 3—the levels of Aβ in the culture medium. Our results demonstrated that Cu increases reactive oxygen species (ROS) and favors Chol de novo synthesis in both ROS-dependent and independent manners. Also, at least part of these effects was due to the activation of 3-hydroxy-3-methyl glutaryl CoA reductase (HMGCR). In addition, Cu increases the Chol/PL ratio in the cellular membranes, specifically Chol content in membrane rafts. We found no changes in total APP cell levels; however, its presence in membrane rafts increases with the consequent increase of Aβ in the culture medium. We conclude that Cu overload favors Chol de novo synthesis in both ROS-dependent and independent manners, being at least in part, responsible for the high Chol levels found in the cell membrane and membrane rafts. These may promote the redistribution of APP into the rafts, favoring the amyloidogenic processing of this protein and increasing the levels of Aβ.

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“…PLK2 mainly phosphorylates soluble α-Syn ( 151 ); Inhibition of PLK2 triggers autophagic elimination of α-Syn ( 152 ). Oxidative stress might play a key role in PLK2 phosphorylation of α-Syn, and antioxidant NAC could completely block iron-induced up-regulation of PLK2, CK2 and p-Ser129 α-Syn ( 140 ); However, as PLK2 induces elevation of α-Syn in copper-treated SHSY5Y neuroblastoma cells, both PP2A level and oxidative status remains unchanged ( 153 ). Glutamate-mediated excitotoxicity is often considered as the mechanism of cell death in PD ( 154 ); Group II metabotropic glutamate receptors (mGLU2/3) are highly expressed in the preterminal region of subthalamic synapses, and activation of them could inhibit glutamate release from the presynaptic membrane ( 155 , 156 ).…”

Section: Role Of Plk2 In Diseasesmentioning

confidence: 99%

Polo-Like Kinase 2: From Principle to Practice

Zhang

2022

Front. Oncol.

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Polo-like kinase (PLK) 2 is an evolutionarily conserved serine/threonine kinase that shares the n-terminal kinase catalytic domain and the C-terminal Polo Box Domain (PBD) with other members of the PLKs family. In the last two decades, mounting studies have focused on this and tried to clarify its role in many aspects. PLK2 is essential for mitotic centriole replication and meiotic chromatin pairing, synapsis, and crossing-over in the cell cycle; Loss of PLK2 function results in cell cycle disorders and developmental retardation. PLK2 is also involved in regulating cell differentiation and maintaining neural homeostasis. In the process of various stimuli-induced stress, including oxidative and endoplasmic reticulum, PLK2 may promote survival or apoptosis depending on the intensity of stimulation and the degree of cell damage. However, the role of PLK2 in immunity to viral infection has been studied far less than that of other family members. Because PLK2 is extensively and deeply involved in normal physiological functions and pathophysiological mechanisms of cells, its role in diseases is increasingly being paid attention to. The effect of PLK2 in inhibiting hematological tumors and fibrotic diseases, as well as participating in neurodegenerative diseases, has been gradually recognized. However, the research results in solid organ tumors show contradictory results. In addition, preliminary studies using PLK2 as a disease predictor and therapeutic target have yielded some exciting and promising results. More research will help people better understand PLK2 from principle to practice.

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Copper Dependent Modulation of α-Synuclein Phosphorylation in Differentiated SHSY5Y Neuroblastoma Cells

Cited by 10 publications

References 67 publications

High-resolution atomic force microscopy as a tool for topographical mapping of surface budding

High-resolution atomic force microscopy as a tool for topographical mapping of surface budding

Effect of Sublethal Copper Overload on Cholesterol De Novo Synthesis in Undifferentiated Neuronal Cells

Polo-Like Kinase 2: From Principle to Practice

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