Multiplexed Metabolic Labeling of Glycoconjugates in Polarized Primary Cerebral Cortical Neurons

Choi, Ji Yu; Seo, Jung Soo; Park, Matthew; Kim, Mi-Hee; Kang, Kyung‐Tae; Choi, Insung S.

doi:10.1002/asia.201800996

Cited by 4 publications

(5 citation statements)

References 35 publications

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“…For example, glial cells, such as astrocytes, play supporting roles in the brain. Relevant reports showed that the in vitro neurotoxicity of unnatural monosaccharides to primary hippocampal neurons was mitigated when a hippocampal tissue was used 26,28 or a neuron/astrocyte coculture was employed. 27 In this respect, it could be explained that the coculture system of neuron and glia (2:1) did not show the neurotoxicity of CBD.…”

Section: Resultsmentioning

confidence: 99%

“…19 H 2 O 2 has been used as a model molecule to study oxidative stress-induced neural death and also to screen neuroprotective agents on the cultured neurons. 16,[20][21][22][23][24][25] As our on-going research program on neurochemistry and neurophysiology, [26][27][28][29][30][31] this study investigates the neuroprotective effect of CBD against H 2 O 2 in the hippocampal neuron culture.…”

Section: Introductionmentioning

confidence: 99%

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Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture

Jungnam

Choi

Seo

et al. 2021

Cannabis and Cannabinoid Research

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Introduction: Reports on the neurotoxic and neuroprotective effects of cannabidiol (CBD) have not been in complete accord, showing different and somewhat contradictory results depending upon the brain cell types and experimental conditions employed. This work systematically examines the neuroprotective capability of CBD against oxidative stress (i.e., hydrogen peroxide [H 2 O 2 ]) as well as its toxicity profile in the in vitro culture platform of primary hippocampal neurons. Materials and Methods: The low cell-density (100 neurons per mm 2) culture was used for analyzing the viability and morphology of neurons at a single-cell level with a confocal laser-scanning microscope (CLSM). Primary neurons were obtained from the hippocampal tissues of embryonic day-18 (E18) Sprague-Dawley rat pups and treated with CBD (0.1-100 lM) and/or H 2 O 2 (0.1-50 lM) at 1 DIV (days in vitro). Results: The lethal concentration 50 (LC 50) value (the concentration causing 50% cell death) of CBD was calculated to be 9.85 lM after 24 h of incubation, and that of H 2 O 2 was 2.46 lM under the same conditions. The neuroprotection ratio of CBD against H 2 O 2 ([H 2 O 2 ] = 10 lM) was 2.40 with 5 lM of CBD, increasing the cell viability to 57% from 24%. The CLSM analysis suggested that the cell-death mechanisms were different for CBD and H 2 O 2 , and CBD did not completely rescue the morphological alterations of primary hippocampal neurons caused by H 2 O 2 , such as neurite degeneration, at least in the in vitro neuron culture. Conclusion: Although CBD showed both neurotoxic and neuroprotective effects on hippocampal neurons in the in vitro setting, the use of low-concentrated (i.e., 5 lM) CBD, not causing toxic effects on the neurons, significantly rescued the neurons from the oxidative stress (H 2 O 2), confirming its neuroprotection capability.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture

Jungnam

Choi

Seo

et al. 2021

Cannabis and Cannabinoid Research

Self Cite

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“…In short, the sandwich coculture of neurons and astrocytes was carried out by placing the astrocytecultured plate parallel to the neuron-cultured plate with a narrow space in a face-to-face mode. [31,32] The sandwich platform makes it possible to analyze only neuron-cultured plate with the minimization of astrocyte contamination in the analysis. It also has been known that the supportive effects of astrocytes in the platform lead to the healthy growth of neurons: for example, most neurons die within 5 days in the neuron-alone culture, while neurons in the sandwich coculture with astrocytes survive healthily for more than 2 weeks.…”

Section: Resultsmentioning

confidence: 99%

“…[25][26][27][28] Our group also has reported that the neuron-astrocyte coculture in a sandwich platform abolishes the neurotoxicity of unnatural monosaccharides in metabolic labeling of the sialoglycans localized in neurons. [29][30][31] Therefore, it is equally crucial to investigate the neuronal responses to CBD under the support of astrocytes, for better understanding of the CBD actions to neurons in a more in-vivo-relevant manner. In this work, we studied the neuronal responses to CBD in the neuron-astrocyte coculture system, showing that astrocytes have the capability for rescuing neurons against CBD-induced damages, such as increase of intracellular calcium concentration ([Ca 2+ ] i ) and mitochondrial dysfunction.…”

Section: Introductionmentioning

confidence: 99%

Neutralization of Cannabidiol Neurotoxicity in Neuron‐Astrocyte Sandwich Coculture

2023

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Cannabidiol (CBD), a main nonpsychoactive phytocannabinoid in the Cannabis genus, has been in the limelight for its potential health benefits in various neurological diseases. However, the safety issue of CBD in the nervous system has not been settled fully, while CBD has been reported to have mild side effects including dizziness and somnolence. In this work, a platform of neuron‐astrocyte sandwich coculture to investigate the neurotoxicity of CBD, as well as the neuronal responses to CBD, in a more in vivo relevant mode is constructed. CBD (15 and 30 µm) causes the viability decrease, along with morphological damage, in the neuron‐alone culture, whereas its neurotoxic effects are significantly attenuated by the supports of astrocytes in the neuron‐astrocyte coculture. In addition, it is found that CBD‐induced increase of intracellular Ca2+ concentration and depolarization of mitochondrial membrane potential, via activation of transient receptor potential vanilloid 1, are noticeably ameliorated by coculturing neurons with astrocytes. This work provides crucial information in the development of CBD as therapeutics for neurological disorders, as well as in a fundamental understanding of how CBD works in the nervous system.

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“…Unfortunately, studies from the Choi group demonstrated that per- O -acetylated N -azidoacetylhexsosamine (Ac 4 HexNAz) including Ac 4 ManNAz and Ac 4 GalNAz were cytotoxic to cultured primary neurons. − Ac 4 ManNAz at concentrations above 50 μM suppressed neurite development and caused cell death in primary rat hippocampal neurons in a dose-dependent manner . Similar adverse effects were observed in rat cortical neurons with Ac 4 ManNAz as well as Ac 4 GalNAz . The mechanism of per- O -acetylated sugar-induced cytotoxicity in primary neurons remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Metabolic Glycan Labeling in Primary Neurons Enabled by Unnatural Sugars with No S-Glyco-Modification

Sun

Huang

et al. 2023

ACS Chem. Biol.

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It is of great interest to probe glycosylation in primary neuron cultures. However, per-O-acetylated clickable unnatural sugars, which have been routinely utilized in metabolic glycan labeling (MGL) for analyzing glycans, showed cytotoxicity to cultured primary neurons and thus led to the speculation that MGL was not compatible with primary neuron cell cultures. Here, we uncovered that neuron cytotoxicity of per-O-acetylated unnatural sugars was related to their reactions with protein cysteines via non-enzymatic S-glyco-modification. The modified proteins were enriched in biological functions related to microtubule cytoskeleton organization, positive regulation of axon extension, neuron projection development, and axonogenesis. We thus established MGL in cultured primary neurons without cytotoxicity using S-glyco-modification-free unnatural sugars including ManNAz, 1,3-Pr 2 ManNAz, and 1,6-Pr 2 ManNAz, which allowed for visualization of cell-surface sialylated glycans, probing the dynamics of sialylation, and large-scale identification of sialylated N-linked glycoproteins and the modification sites in primary neurons. Particularly, a total of 505 sialylated N-glycosylation sites distributed on 345 glycoproteins were identified by 1,6-Pr 2 ManNAz.

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Multiplexed Metabolic Labeling of Glycoconjugates in Polarized Primary Cerebral Cortical Neurons

Cited by 4 publications

References 35 publications

Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture

Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture

Neutralization of Cannabidiol Neurotoxicity in Neuron‐Astrocyte Sandwich Coculture

Metabolic Glycan Labeling in Primary Neurons Enabled by Unnatural Sugars with No S-Glyco-Modification

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