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Chimera RNA interference knockdown of ?-synuclein in human cortical astrocytes results in mitotic catastrophe.

ABSTRACT: Elevated levels of ?-synuclein (?-syn) expression have been noted in the progression of glioblastomas, and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases. ?-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes. Internalized ?-syn results in increased cellular proliferation, brain derived neurotrophic factor release and astroprotection. However, the function of endogenous ?-syn in primary astrocytes, and the relationship to these two opposing disease states are unknown. ?-Syn is expressed by astrocytes in the human cortex, and to gain a better understanding of the role of endogenous ?-syn, primary human cortical astrocytes were treated with chimera RNA interference (RNAi) targeting ?-syn after release from cell synchronization. Quantitative polymerase chain reaction analysis demonstrated an increase in endogenous ?-syn expression 48 hours after release from cell synchronization, while RNAi reduced ?-syn expression to control levels. Immunocytochemistry of Ki67 and 5-bromodeoxyuridine showed chimera RNAi ?-syn knockdown reduced cellular proliferation at 24 and 48 hours after release from cell synchronization. To further investigate the consequence of ?-syn knockdown on the astrocytic cell cycle, phosphorylated histone H3 pSer10 (pHH3) and phosphorylated cyclin dependent kinase-2 pTyr15 (pCDK2) levels were observed via western blot analysis. The results revealed an elevated expression of pHH3, but not pCDK2, indicating ?-syn knockdown leads to disruption of the cell cycle and chromosomal compaction after 48 hours. Subsequently, flow cytometry with propidium iodide determined that increases in apoptosis coincided with ?-syn knockdown. Therefore, ?-syn exerts its effect to allow normal astrocytic progression through the cell cycle, as evidenced by decreased proliferation marker expression, increased pHH3, and mitotic catastrophe after knockdown. In this study, we demonstrated that the knockdown of ?-syn within primary human cortical astrocytes using chimera RNAi leads to cell cycle disruption and apoptosis, indicating an essential role for ?-syn in regulating normal cell division in astrocytes. Therefore, disruption to ?-syn function would influence astrocytic proliferation, and could be an important contributor to neurological diseases.

SUBMITTER: Le T

PROVIDER: S-EPMC7513975 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe.

Le Timmy T Winham Cynthia L CL Andromidas Fotis F Silver Adam C AC Jellison Evan R ER Levesque Aime A AA Koob Andrew O AO

Neural regeneration research 20201001 10

Elevated levels of γ-synuclein (γ-syn) expression have been noted in the progression of glioblastomas, and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases. γ-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes. Internalized γ-syn results in increased cellular proliferation, brain derived neurotrophic factor release and astroprotection. However, the function of endogenous γ-syn in primary astrocy ...[more]

PMID: 32246638

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Project description:Background and purposeStem-like tumor cells are regarded as highly resistant to ionizing radiation (IR). Previous studies have focused on apoptosis early after irradiation, and the apoptosis resistance observed has been attributed to reduced DNA damage or enhanced DNA repair compared to non-stem tumor cells. Here, early and late radioresponse of patient-derived stem-like glioma cells (SLGCs) and differentiated cells directly derived from them were examined for cell death mode and the influence of stem cell-specific growth factors.Materials and methodsPrimary SLGCs were propagated in serum-free medium with the stem-cell mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Differentiation was induced by serum-containing medium without EGF and FGF. Radiation sensitivity was evaluated by assessing proliferation, clonogenic survival, apoptosis, and mitotic catastrophe. DNA damage-associated γH2AX as well as p53 and p21 expression were determined by Western blots.ResultsSLGCs failed to apoptose in the first 4 days after irradiation even at high single doses up to 10 Gy, but we observed substantial cell death later than 4 days postirradiation in 3 of 6 SLGC lines treated with 5 or 10 Gy. This delayed cell death was observed in 3 of the 4 SLGC lines with nonfunctional p53, was associated with mitotic catastrophe and occurred via apoptosis. The early apoptosis resistance of the SLGCs was associated with lower γH2AX compared to differentiated cells, but we found that the stem-cell culture cytokines EGF plus FGF-2 strongly reduce γH2AX levels. Nonetheless, in two p53-deficient SLGC lines examined γIR-induced apoptosis even correlated with EGF/FGF-induced proliferation and mitotic catastrophe. In a line containing CD133-positive and -negative stem-like cells, the CD133-positive cells proliferated faster and underwent more γIR-induced mitotic catastrophe.ConclusionsOur results suggest the importance of delayed apoptosis, associated mitotic catastrophe, and cellular proliferation for γIR-induced death of p53-deficient SLGCs. This may have therapeutic implications. We further show that the stem-cell culture cytokines EGF plus FGF-2 activate DNA repair and thus confound in vitro comparisons of DNA damage repair between stem-like and more differentiated tumor cells.

Project description:There is serious concern about arsenic in the natural environment, which exhibits neurotoxicity and increases the risk of neurodevelopmental disorders. Adverse effects of arsenic have been demonstrated in neurons, but it is not fully understood how arsenic affects other cell types in the brain. In the current study, we examined whether sodium arsenite (NaAsO2) affects the cell cycle, viability, and apoptosis of in vitro-cultured astrocytes isolated from the cerebral cortex of mice. Cultured astrocytes from transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were subjected to live imaging analysis to assess the effects of NaAsO2 (0, 1, 2, and 4 μM) on the cell cycle and number of cells. Fucci was designed to express monomeric Kusabira Orange2 (mKO2) fused with the ubiquitylation domain of hCdt1, a marker of G1 phase, and monomeric Azami Green (mAG) fused with the ubiquitylation domain of hGem, a marker of S, G2, and M phases. NaAsO2 concentration-dependently decreased the peak levels of the mAG/mKO2 emission ratio when the ratio had reached a peak in astrocytes without NaAsO2 exposure, which was due to attenuating the increase in the mAG-expressing cell number. In contrast, the mAG/mKO2 emission ratio and number of mAG-expressing cells were concentration-dependently increased by NaAsO2 before their peak levels, indicating unscheduled S phase entry. We further examined the fate of cells forced to enter S phase by NaAsO2. We found that most of these cells died up to the end of live imaging. In addition, quantification of the copy number of the glial fibrillary acidic protein gene expressed specifically in astrocytes revealed a concentration-dependent decrease caused by NaAsO2. However, NaAsO2 did not increase the amount of nucleosomes generated from DNA fragmentation and failed to alter the gene expression of molecules relevant to unscheduled S phase entry-coupled apoptosis (p21, p53, E2F1, E2F4, and Gm36566). These findings suggest that NaAsO2 adversely affects the cell cycle and viability of astrocytes by inducing unscheduled S phase entry coupled with cell death that may be caused by mechanisms other than apoptosis.

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Chimera RNA interference knockdown of ?-synuclein in human cortical astrocytes results in mitotic catastrophe.

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Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe.

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