Chemogenetic emulation of intraneuronal oxidative stress affects synaptic plasticity

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Overproduction of reactive oxygen species (ROS) and oxidative cell damage are commonly associated with most brain pathologies [1, 2]. Dysregulation of redox homeostasis in the aging brain is thought to be responsible for impaired synaptic transmission and plasticity, leading to reduced neuronal computational capacity and learning and memory deficits. Studying the contribution of oxidative stress to the development of diseases, such as age-related dementia and Alzheimer’s disease, is complex due to the lack of methods for modeling isolated oxidative damage in individual cell types [3]. We introduce a chemogenetic approach utilizing D-amino acid oxidase (DAAO) from yeast to produce hydrogen peroxide intraneuronally, which is one of the most stable ROS [4]. H2O2 generation was evaluated in primary cultured neurons and acute mouse brain slices through the utilization of a genetically encoded fluorescent biosensor, HyPer7, to validate the methodology [5]. The changes in the fluorescence signal of HyPer7 after treating neurons that expressed DAAO with D-Norvaline (D-Nva), a substrate for DAAO, confirmed the targeted production of H2O2 through chemogenetics. Using electrophysiological recordings in acute brain slices, we demonstrated that intraneuronal oxidative stress induced by chemogenetics did not affect basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals. However, it diminished long-term potentiation (LTP) at the single-cell level.

Astrocytes have the ability to metabolize d-amino acids, rendering the proposed approach ineffective in vivo experiments. Consequently, in vivo testing of the tool was necessary for validation. To achieve this, an optical setup for exciting and detecting the HyPer7 signal was developed and implanted into the mouse brain via optical fibers. By using this approach, we were able to demonstrate the generation of H2O2 in DAAO-expressing neurons in vivo, upon intraperitoneal administration of D-amino acids. The results demonstrate that using a DAAO-based chemogenetic tool, along with electrophysiological recordings, clarifies numerous unanswered queries regarding the part of ROS-dependent signaling in typical brain activities and the impact of oxidative stress on the development of cognitive aging and preliminary neurodegenerative stages. The suggested method is valuable for detecting initial indicators of neuronal oxidative stress. Additionally, it can be used for evaluating probable antioxidants that can effectively combat neuronal oxidative harm.

全文:

Overproduction of reactive oxygen species (ROS) and oxidative cell damage are commonly associated with most brain pathologies [1, 2]. Dysregulation of redox homeostasis in the aging brain is thought to be responsible for impaired synaptic transmission and plasticity, leading to reduced neuronal computational capacity and learning and memory deficits. Studying the contribution of oxidative stress to the development of diseases, such as age-related dementia and Alzheimer’s disease, is complex due to the lack of methods for modeling isolated oxidative damage in individual cell types [3]. We introduce a chemogenetic approach utilizing D-amino acid oxidase (DAAO) from yeast to produce hydrogen peroxide intraneuronally, which is one of the most stable ROS [4]. H2O2 generation was evaluated in primary cultured neurons and acute mouse brain slices through the utilization of a genetically encoded fluorescent biosensor, HyPer7, to validate the methodology [5]. The changes in the fluorescence signal of HyPer7 after treating neurons that expressed DAAO with D-Norvaline (D-Nva), a substrate for DAAO, confirmed the targeted production of H2O2 through chemogenetics. Using electrophysiological recordings in acute brain slices, we demonstrated that intraneuronal oxidative stress induced by chemogenetics did not affect basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals. However, it diminished long-term potentiation (LTP) at the single-cell level.

Astrocytes have the ability to metabolize d-amino acids, rendering the proposed approach ineffective in vivo experiments. Consequently, in vivo testing of the tool was necessary for validation. To achieve this, an optical setup for exciting and detecting the HyPer7 signal was developed and implanted into the mouse brain via optical fibers. By using this approach, we were able to demonstrate the generation of H2O2 in DAAO-expressing neurons in vivo, upon intraperitoneal administration of D-amino acids. The results demonstrate that using a DAAO-based chemogenetic tool, along with electrophysiological recordings, clarifies numerous unanswered queries regarding the part of ROS-dependent signaling in typical brain activities and the impact of oxidative stress on the development of cognitive aging and preliminary neurodegenerative stages. The suggested method is valuable for detecting initial indicators of neuronal oxidative stress. Additionally, it can be used for evaluating probable antioxidants that can effectively combat neuronal oxidative harm.

ADDITIONAL INFORMATION

Funding sources. Electrophysiological recordings were funded by the RSF grant No. 22-15-00293; in vivo validation of the DAAO-based optogenetic tool was funded by the RSF grant No. 23-75-30023; fiber probes development was funded by RSF grant No. 22-22-00590.

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.

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作者简介

D. Maltsev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Institute of Fundamental Neurology, Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency

编辑信件的主要联系方式.
Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow

А. Kalinichenko

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Lomonosov Moscow State University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow

D. Jappy

Institute of Fundamental Neurology, Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency; Kazan Federal University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Kazan

M. Solotenkov

Lomonosov Moscow State University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow

G. Solius

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow

L. Mukhametshina

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Lomonosov Moscow State University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow

E. Elesina

Lomonosov Moscow State University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow

R. Sokolov

Pirogov Russian National Research Medical University; Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Nizhny Novgorod

A. Tsopina

Lomonosov Moscow State University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow

I. Fedotov

Lomonosov Moscow State University; Russian Quantum Center “Skolkovo”

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow

A. Moshchenko

Institute of Fundamental Neurology, Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow

A. Fedotov

Lomonosov Moscow State University; Russian Quantum Center “Skolkovo”

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow

V. Shaydurov

Institute of Fundamental Neurology, Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency; Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow

A. Rozov

Institute of Fundamental Neurology, Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow

O. Podgorny

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Institute of Fundamental Neurology, Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency; Pirogov Russian National Research Medical University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow; Moscow

V. Belousov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Institute of Fundamental Neurology, Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency; Pirogov Russian National Research Medical University

Email: maltsev.d@fccps.ru
俄罗斯联邦, Moscow; Moscow; Moscow

参考

  1. Sies H, Berndt C, Jones DP. Oxidative Stress. Annual Review of Biochemistry. 2017;86:715–748. doi: 10.1146/annurev-biochem-061516-045037
  2. Cobley JN, Fiorello ML, Bailey DM. 13 reasons why the brain is susceptible to oxidative stress. Redox Biology. 2018;15:490–503. doi: 10.1016/j.redox.2018.01.008
  3. Kumar A, Yegla B, Foster TC. Redox Signaling in Neurotransmission and Cognition During Aging. Antioxidants & Redox Signaling. 2018;28(18):1724–1745. doi: 10.1089/ars.2017.7111
  4. Pollegioni L, Langkau B, Tischer W, et al. Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis. Journal of Biological Chemistry. 1993;268(19):13850–13857. doi: 10.1016/S0021-9258(19)85181-5
  5. Pak VV, Ezeriņa D, Lyublinskaya OG, et al. Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function. Cell Metabolism. 2020;31(3):642–653.e6. doi: 10.1016/j.cmet.2020.02.003

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