Positive Effect of YB-1 and Mesenchymal Stromal Cells on Primary Hippocampal Culture under Conditions of ACE2 Receptor Blockade

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Although the current COVID-19 incidence situation is not an emergency, more new strains of SARS-CoV-2 coronavirus continue to emerge worldwide, some of which are more virulent than the original virus. Studies have shown that patients with Alzheimer's disease (AD) had a high risk of severe COVID-19, but the molecular and cellular mechanism of this predisposition is not fully elucidated. In this study, we developed a cellular model of the initial stage of COVID-19 on primary hippocampal culture of 5xFAD mice, a familial AD model, using a specific ACE2 receptor inhibitor, MLN-4760. This model is based on the experimentally proven decrease in ACE2 receptor activity observed in COVID-19 patients due to internalization of the receptor inside the cell after binding to coronavirus. Using immunochemical staining with specific antibodies to detect neurons (marker MAP2) and astroglia (marker GFAP), it was found that 24 h after the addition of MLN-4760 (0.2 nmol per 1 mL of medium) to the culture medium, there was a decrease in the density of astrocytes and neurons, a change in their morphology with a sharp reduction in the length and density of neurites, which led to the death of the cell culture. The transgenic culture was more sensitive to the effect of the inhibitor compared to the control hippocampal culture of native mice. In the second part of the study the possibilities of preventing the destructive effect of MLN-4760 on the hippocampal culture condition were studied. It was shown that administration of YB-1, an endogenous polyfunctional stress protein, promoted restoration of cell culture structure and resulted in stimulation of neurite growth and astroglia activation. Introduction of multipotent mesenchymal stromal cells (MMSCs) after ACE2 blockade was also accompanied by improved culture survival, restoration of cell morphology, and increased density of astrocytes and neurons. The obtained results indicate that YB-1 and cell therapy using MMSCs are promising options for the development of new effective methods to prevent the pathological effect of the virus on brain tissue, which is an important link in the treatment of infection caused by SARS-CoV-2 virus.

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Sobre autores

D. Zhdanova

Institute of Cell Biophysics, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Autor responsável pela correspondência
Email: ddzhdanova@mail.ru
Rússia, Pushchino, 142290

A. Chaplygina

Institute of Cell Biophysics, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Email: ddzhdanova@mail.ru
Rússia, Pushchino, 142290

N. Bobkova

Institute of Cell Biophysics, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Email: ddzhdanova@mail.ru
Rússia, Pushchino, 142290

R. Poltavtseva

National Medical Research Center for Obstetrics, Gynecology, and Perinatology named after Academician V.I. Kulakov, Ministry of Healthcare of the Russian Federation

Email: ddzhdanova@mail.ru
Rússia, Moscow, 117997

G. Sukhikh

National Medical Research Center for Obstetrics, Gynecology, and Perinatology named after Academician V.I. Kulakov, Ministry of Healthcare of the Russian Federation

Email: ddzhdanova@mail.ru
Rússia, Moscow, 117997

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2. Fig. 1. Effect of ACE2 inhibitor on the area of immunopositive staining of astrocyte marker (GFAP - green) and neuronal marker (MAP2 - red) in primary cell cultures of hippocampus of nontransgenic (nTg) and transgenic (Tg) mice. a - Native primary culture of hippocampus of nTg mouse (nTg). b - Effect of MLN-4760 on native primary culture of hippocampus of nTg mouse (nTg MLN). c - Native primary culture of hippocampus of Tg mouse 5xFAD (Tg). d - Effect of MLN-4760 on primary culture of hippocampus of Tg mouse 5xFAD (Tg MLN). e - Neuron and astrocyte density (in %). * p < 0.05, one-factor ANOVA followed by Dunn's posttests. Scale bar is 250 μm.

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3. Fig. 2. Structural changes of astrocytes (GFAP - green) and neurons (MAP2 - red) in primary nTg hippocampal cell cultures after MLN-4760 action. nTg - Native primary hippocampal cell culture. MLN - Effect of MLN-4760 on primary hippocampal cultures. Scale bar - 125 μm.

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4. Fig. 3. Positive effect of YB-1 on neuronal and astrocyte density in cultures of Tg and nTg mice exposed to ACE2 blocker. Immunopositivity to an astrocyte marker (GFAP in green) and a neuronal marker (MAP2 in red). a - Effect of YB-1 on nTg culture exposed to ACE2 blocker (nTg). b - Effect of YB-1 on Tg culture exposed to ACE2 blocker (Tg). c - Neuronal and astrocyte density (in %) in nTg culture in the absence of MLN-4760 (nTg), under the influence of MLN-4760 (MLN), after the effect of YB-1 (MLN + YB-1). d - Neuronal and astrocyte density (in %) in 5xFAD Tg culture in the absence of MLN-4760 (nTg), under the influence of MLN-4760 (MLN), after the effect of YB-1 (MLN + YB-1). * p < 0.05, one-factor ANOVA followed by Dunn's posttests. Scale bar is 250 μm.

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5. Fig. 4. Effect of YB-1 administration on neurons (MAP2 - red) and astrocytes (GFAP - green) in primary nTg neuroglial cultures of hippocampus exposed to MLN-4760. Scale bar is 125 μm.

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6. Fig. 5. Effect of MMSCs on neurons (MAP2 - red) and astrocytes (GFAP - green) in nTg hippocampal cell culture exposed to MLN-4760. Scale bar is 125 μm.

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7. Fig. 6. Effect of MMSCs (MMSC nuclei - blue) on immunopositivity of astrocytes (marker GFAP - green) and neurons (marker MAP2 - red) in primary hippocampal cultures of nTg and Tg mice. a - MMSCs were injected into nTg culture after ACE2 inhibition. b - MMSCs were injected into Tg culture 5xFAD after ACE2 inhibition. c - Density of neurons and astrocytes (in %) in nTg culture in the absence of MLN-4760 (nTg), under the influence of MLN-4760 (MLN), after the action of MMSCs (MLN + MMSCs). d - Neuronal and astrocyte density (in %) in 5xFAD Tg culture in the absence of MLN-4760 (Tg), under the influence of MLN-4760 (MLN), after the action of MMSCs (MLN + MMSCs).** p < 0.001, one-factor ANOVA followed by Bonferroni posttests. * p < 0.05, one-factor ANOVA followed by Dunn's posttests. Scale bar was 250 μm.

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