Role of RIPK1 kinase in neuronal-glial network adaptation under hypoxic conditions
- 作者: Loginova M.M.1,2, Yarkov R.S.1, Vedunova M.V.1, Mitroshina E.V.1
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隶属关系:
- National Research Lobachevsky State University of Nizhny Novgorod
- Privolzhsky Research Medical University
- 期: 卷 18, 编号 4 (2023)
- 页面: 508-511
- 栏目: Conference proceedings
- URL: https://journals.rcsi.science/2313-1829/article/view/256257
- DOI: https://doi.org/10.17816/gc623467
- ID: 256257
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详细
Cerebral hypoxia is a condition characterized by a reduced oxygen supply to tissues that plays a crucial role in the pathogenesis of numerous neurodegenerative diseases. In hypoxia, intracellular signaling cascades are activated, ultimately leading to various forms of nerve cell death. The initiation of necroptosis under hypoxic conditions is governed by PIPK1 kinase, and its inhibition could potentially offer neuroprotection against hypoxic damage [1–3]. Studies investigating the effects of blocking RIPK1 kinase on the activity of neuronal-glial networks are currently lacking. Consequently, RIPK1 kinase constitutes a promising objective for further exploration. Thus, the aim of this work is to examine the part played by RIPK1 kinase in the adjustment of neuronal-glial networks amid hypoxia.
The study focused on primary cultures of nerve cells in the hippocampus of mouse embryos belonging to the C57Bl/6 line. Hypoxia was induced in vitro on the 14th day of culturing the nerve cells. The RIPK1 kinase inhibitor was administered 20 minutes prior to, during, and after the hypoxia intervention. After 7 days of the stress induction, the calcium and bioelectrical activity of the neuron-glial networks were evaluated. Calcium activity was assessed via the Oregon Green 488 BAPTA-1, AM (Thermo Fisher Scientific, USA) using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss, Germany). The experiments assessed total percentage of oscillating cells in culture, the frequency, and duration of calcium events. The analysis of bioelectrical activity was conducted using the MEA 60 multi-electrode arrays from Multichannel Systems. The registered signal from the arrays was processed through the MEAMAN algorithms in MATLAB (Certificate of State Registration of Computer Program No. 2012611190). The average number of small network packages and spikes was estimated.
Under physiological conditions, spontaneous calcium activity is observed by the 21st day of neuron-glial network development. The percentage of cells exhibiting calcium events is 60.64 ± 3.68%, the frequency of calcium oscillations is 1.52±0.22 osc/min, and the duration is 9.63±0.75 s. During hypoxia modeling, the percentage of cells exhibiting calcium events decreased to 34.77±4.08%, and the frequency of calcium oscillations decreased to 0.64±0.08 osc/min. The inhibition of RIPK1 kinase maintains the percentage of cells exhibiting calcium events at the level of intact cultures, which is 60.38±3.4%.
By the 21st day of culturing primary nerve cell cultures under physiological conditions, spontaneous bioelectrical activity forms. This is indicated by parameters such as the average number of small network clusters and spikes. Hypoxia modeling has a negative impact on the development of spontaneous bioelectrical activity. In the “Intact” group, the average number of small network packs was 36.12±4.27 packs per 10 minutes, while in the cell culture with hypoxia, it was 15.87±3.03 packs per 10 minutes. Additionally, the “Intact” group had an average of 90.22±12.32 spikes, while the cell culture with hypoxia had only 11.58±4.7 spikes. However, blocking RIPK1 kinase during hypoxia preserved the average number of small network packs (23,49±2,14 packs/10 min).
Thus, inhibiting RIPK1 kinase under hypoxic conditions preserves the proportion of cells that exhibit spontaneous calcium events and partially preserves spontaneous bioelectrical activity.
全文:
Cerebral hypoxia is a condition characterized by a reduced oxygen supply to tissues that plays a crucial role in the pathogenesis of numerous neurodegenerative diseases. In hypoxia, intracellular signaling cascades are activated, ultimately leading to various forms of nerve cell death. The initiation of necroptosis under hypoxic conditions is governed by PIPK1 kinase, and its inhibition could potentially offer neuroprotection against hypoxic damage [1–3]. Studies investigating the effects of blocking RIPK1 kinase on the activity of neuronal-glial networks are currently lacking. Consequently, RIPK1 kinase constitutes a promising objective for further exploration. Thus, the aim of this work is to examine the part played by RIPK1 kinase in the adjustment of neuronal-glial networks amid hypoxia.
The study focused on primary cultures of nerve cells in the hippocampus of mouse embryos belonging to the C57Bl/6 line. Hypoxia was induced in vitro on the 14th day of culturing the nerve cells. The RIPK1 kinase inhibitor was administered 20 minutes prior to, during, and after the hypoxia intervention. After 7 days of the stress induction, the calcium and bioelectrical activity of the neuron-glial networks were evaluated. Calcium activity was assessed via the Oregon Green 488 BAPTA-1, AM (Thermo Fisher Scientific, USA) using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss, Germany). The experiments assessed total percentage of oscillating cells in culture, the frequency, and duration of calcium events. The analysis of bioelectrical activity was conducted using the MEA 60 multi-electrode arrays from Multichannel Systems. The registered signal from the arrays was processed through the MEAMAN algorithms in MATLAB (Certificate of State Registration of Computer Program No. 2012611190). The average number of small network packages and spikes was estimated.
Under physiological conditions, spontaneous calcium activity is observed by the 21st day of neuron-glial network development. The percentage of cells exhibiting calcium events is 60.64 ± 3.68%, the frequency of calcium oscillations is 1.52±0.22 osc/min, and the duration is 9.63±0.75 s. During hypoxia modeling, the percentage of cells exhibiting calcium events decreased to 34.77±4.08%, and the frequency of calcium oscillations decreased to 0.64±0.08 osc/min. The inhibition of RIPK1 kinase maintains the percentage of cells exhibiting calcium events at the level of intact cultures, which is 60.38±3.4%.
By the 21st day of culturing primary nerve cell cultures under physiological conditions, spontaneous bioelectrical activity forms. This is indicated by parameters such as the average number of small network clusters and spikes. Hypoxia modeling has a negative impact on the development of spontaneous bioelectrical activity. In the “Intact” group, the average number of small network packs was 36.12±4.27 packs per 10 minutes, while in the cell culture with hypoxia, it was 15.87±3.03 packs per 10 minutes. Additionally, the “Intact” group had an average of 90.22±12.32 spikes, while the cell culture with hypoxia had only 11.58±4.7 spikes. However, blocking RIPK1 kinase during hypoxia preserved the average number of small network packs (23,49±2,14 packs/10 min).
Thus, inhibiting RIPK1 kinase under hypoxic conditions preserves the proportion of cells that exhibit spontaneous calcium events and partially preserves spontaneous bioelectrical activity.
ADDITIONAL INFORMATION
Funding sources. This study was supported by the Federal Academic Leadership Program “Priority 2030” of the Ministry of Science and Higher Education of the Russian Federation.
Authors' contribution. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, and final approval of the version to be published and agree to be accountable for all aspects of the work.
Competing interests. The authors declare that they have no competing interests.
作者简介
M. Loginova
National Research Lobachevsky State University of Nizhny Novgorod; Privolzhsky Research Medical University
编辑信件的主要联系方式.
Email: pandaagron@ya.ru
俄罗斯联邦, Nizhny Novgorod; Nizhny Novgorod
R. Yarkov
National Research Lobachevsky State University of Nizhny Novgorod
Email: pandaagron@ya.ru
俄罗斯联邦, Nizhny Novgorod
M. Vedunova
National Research Lobachevsky State University of Nizhny Novgorod
Email: pandaagron@ya.ru
俄罗斯联邦, Nizhny Novgorod
E. Mitroshina
National Research Lobachevsky State University of Nizhny Novgorod
Email: pandaagron@ya.ru
俄罗斯联邦, Nizhny Novgorod
参考
- Newton K, Dugger DL, Maltzman A, et al. RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury. Cell Death & Differentiation. 2016;23(9):1565–1576. doi: 10.1038/cdd.2016.46
- Cruz SA, Qin Z, Stewart AFR, Chen HH. Dabrafenib, an inhibitor of RIP3 kinase-dependent necroptosis, reduces ischemic brain injury. Neural Regeneration Research. 2018;13(2):252–256. doi: 10.4103/1673-5374.226394
- Zhang YY, Liu WN, Li YQ, et al. Ligustroflavone reduces necroptosis in rat brain after ischemic stroke through targeting RIPK1/RIPK3/MLKL pathway. Naunyn-Schmiedeberg’s Archives of Pharmacology. 2019;392(9):1085–1095. doi: 10.1007/s00210-019-01656-9
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