Disruption of protein kinase expression in cortical neurons due to the deletion of transcription factor Satb1 leads to neuronal network hyperexcitation and determines hypoxia sensitivity
- Authors: Celis Suesсun J.C.1, Gavrish M.S.1, Turovsky E.A.1,2, Varlamova E.G.2
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Affiliations:
- Institute of Neurosciences, National Research Lobachevsky State University of Nizhny Novgorod
- Institute of Cell Biophysics of the Russian Academy of Sciences “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”
- Issue: Vol 18, No 4 (2023)
- Pages: 456-459
- Section: Conference proceedings
- URL: https://genescells.ru/2313-1829/article/view/623236
- DOI: https://doi.org/10.17816/gc623236
- ID: 623236
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Abstract
Neuronal transcription factors regulate the expression of receptors and intracellular signaling molecules involved in excitatory neurotransmission. The transcription factor Satb1 received significant attention for its role in regulating the growth and development of various types of brain neurons in both embryonic and postnatal periods. The depletion of the transcription factor Satb1 can be considered a fundamental mechanism for triggering hyperexcitation in the neural network, given the alterations in the activity and expression levels of proteins, such as glutamate receptors, protein kinases, and cell viability regulators. Although numerous studies were conducted on the role of Satb1 in neurogenesis, none showed any changes in intracellular calcium signaling or protein expression levels following Satb1 deletion in neurons.
Mice with complete (Satb1-null) and partial (Satb1-deficient) deletion of the Satb1 transcription factor were used in this study. Neuroglial cultures were obtained from the cerebral cortex of neonatal mice and were cultivated in-vitro for ten days. The cells were then loaded with a calcium-sensitive Fura-2 fluorescent probe and the dynamics of cytosolic Ca2+ ([Ca2+]i) were recorded using a fluorescent microscope. Spontaneous calcium activity was observed in the cell cultures, and epileptiform activity was modeled using conventional methods, i.e. the medium was depleted of magnesium (magnesium-free model) and 10 µM bicuculline was added (bicuculline model). The control group comprised cerebral cortex cells obtained from normal mice. To analyze expression patterns of genes encoding kinases, total RNA was extracted from cell cultures, and real-time PCR analysis was conducted.
Complete and incomplete deletion of the transcription factor Satb1 had distinct effects on protein kinase expression and genes that regulate cell viability. Satb1-deficient neurons exhibited increased expression of phosphoinositide-3-kinase, protein kinase C, protein kinase B, mitogen-activated protein kinase, as well as Bcl-2, Creb, and Nf-kB genes. The deletion of Satb1 in cortical neurons led to increased expression of only phosphoinositide-3-kinase, while the expression of all other studied protein kinases decreased in the presence of pro-inflammatory factors Nf-kB, Caspase-3, and Tnfα.
At the neurotransmission level, the complete deletion of Satb1 led to heightened spontaneous Ca2+ neuron activity alongside amplified Ca2+ oscillation frequencies and amplitudes when modeling epileptiform activity. Since hyperexcitation of the network is a symptom observed in neuronal networks during hypoxia, and the response of neurons to hypoxia is dependent on the activity of the kinases being studied, experiments were conducted to simulate hypoxia using neurons obtained from mice who lack the Satb1 transcription factor. Concurrently with recording [Ca2+]i, we measured pO2 using a somatic oximeter. The drop in pO2 signified the start of Ca2+ responses in neurons. Initially appearing in 8–10% of cells, hypoxia-triggered the first Ca2+ responses in WT neurons when pO2 dropped to 40 mm Hg. The remaining cells within the microscope field of view did not react. Satb1-null neurons responded to hypoxia at 60 mm Hg, exhibiting high-amplitude Ca2+ oscillations in over 15% of cells. Furthermore, Satb1-deficient neurons exhibited high-frequency Ca2+ oscillations and an increase in [Ca2+]i baseline to a new stationary level when pO2 decreased to 75–80 mm Hg. With over 20% of neurons responding, this suggests that Satb1-null neurons are more sensitive to hypoxia.
Keywords
Full Text
Neuronal transcription factors regulate the expression of receptors and intracellular signaling molecules involved in excitatory neurotransmission. The transcription factor Satb1 received significant attention for its role in regulating the growth and development of various types of brain neurons in both embryonic and postnatal periods. The depletion of the transcription factor Satb1 can be considered a fundamental mechanism for triggering hyperexcitation in the neural network, given the alterations in the activity and expression levels of proteins, such as glutamate receptors, protein kinases, and cell viability regulators. Although numerous studies were conducted on the role of Satb1 in neurogenesis, none showed any changes in intracellular calcium signaling or protein expression levels following Satb1 deletion in neurons.
Mice with complete (Satb1-null) and partial (Satb1-deficient) deletion of the Satb1 transcription factor were used in this study. Neuroglial cultures were obtained from the cerebral cortex of neonatal mice and were cultivated in-vitro for ten days. The cells were then loaded with a calcium-sensitive Fura-2 fluorescent probe and the dynamics of cytosolic Ca2+ ([Ca2+]i) were recorded using a fluorescent microscope. Spontaneous calcium activity was observed in the cell cultures, and epileptiform activity was modeled using conventional methods, i.e. the medium was depleted of magnesium (magnesium-free model) and 10 µM bicuculline was added (bicuculline model). The control group comprised cerebral cortex cells obtained from normal mice. To analyze expression patterns of genes encoding kinases, total RNA was extracted from cell cultures, and real-time PCR analysis was conducted.
Complete and incomplete deletion of the transcription factor Satb1 had distinct effects on protein kinase expression and genes that regulate cell viability. Satb1-deficient neurons exhibited increased expression of phosphoinositide-3-kinase, protein kinase C, protein kinase B, mitogen-activated protein kinase, as well as Bcl-2, Creb, and Nf-kB genes. The deletion of Satb1 in cortical neurons led to increased expression of only phosphoinositide-3-kinase, while the expression of all other studied protein kinases decreased in the presence of pro-inflammatory factors Nf-kB, Caspase-3, and Tnfα.
At the neurotransmission level, the complete deletion of Satb1 led to heightened spontaneous Ca2+ neuron activity alongside amplified Ca2+ oscillation frequencies and amplitudes when modeling epileptiform activity. Since hyperexcitation of the network is a symptom observed in neuronal networks during hypoxia, and the response of neurons to hypoxia is dependent on the activity of the kinases being studied, experiments were conducted to simulate hypoxia using neurons obtained from mice who lack the Satb1 transcription factor. Concurrently with recording [Ca2+]i, we measured pO2 using a somatic oximeter. The drop in pO2 signified the start of Ca2+ responses in neurons. Initially appearing in 8–10% of cells, hypoxia-triggered the first Ca2+ responses in WT neurons when pO2 dropped to 40 mm Hg. The remaining cells within the microscope field of view did not react. Satb1-null neurons responded to hypoxia at 60 mm Hg, exhibiting high-amplitude Ca2+ oscillations in over 15% of cells. Furthermore, Satb1-deficient neurons exhibited high-frequency Ca2+ oscillations and an increase in [Ca2+]i baseline to a new stationary level when pO2 decreased to 75–80 mm Hg. With over 20% of neurons responding, this suggests that Satb1-null neurons are more sensitive to hypoxia.
ADDITIONAL INFORMATION
Funding sources. This work was supported by the Russian Science Foundation grant No. 22-24-00712.
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.
About the authors
J. C. Celis Suesсun
Institute of Neurosciences, National Research Lobachevsky State University of Nizhny Novgorod
Author for correspondence.
Email: juancamilo1297@gmail.com
Russian Federation, Nizhny Novgorod
M. S. Gavrish
Institute of Neurosciences, National Research Lobachevsky State University of Nizhny Novgorod
Email: juancamilo1297@gmail.com
Russian Federation, Nizhny Novgorod
E. A. Turovsky
Institute of Neurosciences, National Research Lobachevsky State University of Nizhny Novgorod; Institute of Cell Biophysics of the Russian Academy of Sciences “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”
Email: juancamilo1297@gmail.com
Russian Federation, Nizhny Novgorod; Pushchino
E. G. Varlamova
Institute of Cell Biophysics of the Russian Academy of Sciences “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”
Email: juancamilo1297@gmail.com
Russian Federation, Pushchino
References
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