Effect of conversion cocktail on astrocyte and neuronal status in the primary hippocampal culture of 5xFAD mice with angiotensin-converting enzyme 2 inhibition

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Abstract

Neurodegenerative diseases are intricate pathological conditions characterized by the progressive degeneration and death of neurons in the nervous system. Consequently, researchers are increasingly focusing on strategies that utilize combinations of bioactive chemical compounds to convert other, more stable cell types into functional neurons. Chemical conversion has shown promise, particularly in models consisting solely of astrocytes; however, more realistic experimental systems include various cell types whose interactions may influence the response to chemical conversion. In this study, we investigated the impact of a multicomponent chemical cocktail on cells in mixed astro-neuronal cultures derived from the hippocampus of transgenic mice from the 5xFAD line, a genetic model of Alzheimer's disease (AD). Additionally, we recreated a model that simulates the reduction in ACE2 receptor activity observed in COVID-19 patients, which occurs due to internalization of the receptor after it binds to the coronavirus in order to study the consequences of chemical conversion upon disruption of this enzyme activity in the brain. Our findings indicate that the increase in neuronal density and the emergence of new neurons following exposure to the conversion cocktail in complex multicomponent cell systems become apparent only at later time points in cultures derived from non-transgenic animals, as well as in cultures from the 5xFAD mouse line. This may be attributed to the natural rise in astroglial levels during culture degradation. Notably, ACE2 inhibition significantly impacts the morphology of individual astrocytes and neurons. When we assessed the effects of the chemical cocktail, we observed that its efficacy was influenced by both the transgenic status of the culture and the timing of the conversion cocktail administration in relation to ACE2 inhibition. Cultures derived from transgenic animals exhibited higher susceptibility to both the ACE2 inhibitor and the chemical conversion agents.

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A. V. Chaplygina

Institute of Cell Biophysics of the Russian Academy of Sciences

Author for correspondence.
Email: shadowhao@yandex.ru
Russian Federation, Pushchino

D. Y. Zhdanova

Institute of Cell Biophysics of the Russian Academy of Sciences

Email: shadowhao@yandex.ru
Russian Federation, Pushchino

R. A. Poltavtseva

Research Center for Obstetrics, Gynecology and Perinatology named after аcademician V.I.Kulakov Ministry of Health of the Russian Federation

Email: shadowhao@yandex.ru
Russian Federation, Moscow

N. V. Bobkova

Institute of Cell Biophysics of the Russian Academy of Sciences

Email: shadowhao@yandex.ru
Russian Federation, Pushchino

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2. Fig. 1. Effect of chemical conversion cocktail on aged primary cell cultures of the hippocampus of non-transgenic and transgenic 5xFAD mice. Immunopositivity to the astrocyte marker (GFAP – green) and the neuronal marker (MAP2 – red). Scale bar – 250 µm. (a) – Native aged primary cell culture from the hippocampus of non-transgenic animals (nTg). (b) – Effect of conversion cocktail on aged primary cell culture of the hippocampus of non-transgenic animals (nTg conversion). (c) – Native aged primary cell culture of the hippocampus of transgenic animals (Tg). (d) – Effect of conversion cocktail on aged primary cell culture of the hippocampus of transgenic animals (Tg conversion). (e) – Neuronal and astrocytic densities (%) in primary hippocampal cell cultures of non-transgenic animals before and after chemical conversion. (f) – Neuronal and astrocytic densities (%) in primary hippocampal cell cultures of transgenic animals before and after chemical conversion. * – p ≤ 0.001, Mann–Whitney U-test, ** – p ≤ 0.001, t-test. →

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3. Fig. 2. Effect of chemical conversion cocktail on primary hippocampal cell cultures of non-transgenic animals upon ACE2 inhibition. Immunopositivity to the astrocyte marker (GFAP – green) and neuronal marker (MAP2 – red). Scale bar – 250 µm for panels a–d, scale bar 125 µm for panels a1–d1. (a–a1) – Native primary hippocampal cell culture of non-transgenic mice (nTg). (b–b1) – ACE2 inhibition with MLN-4760. (c–c1) – ​​Administration of conversion cocktail after ACE2 inhibition (Treatment). (d–d1) – Administration of conversion cocktail before ACE2 inhibition (Priming). (e) – Neuronal and astrocytic densities (%) in primary hippocampal cell cultures from non-transgenic mice. * – p < 0.05, one-way ANOVA, Dunn’s post-hoc test. (f) – Area of ​​astrocytes (%) exhibiting double immunopositivity to GFAP/MAP2 markers. * – p < 0.001, one-way ANOVA, Bonferroni’s post-hoc test. (j) – Schematic diagram of the experiment with group assignment. →

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4. Fig. 3. Effect of chemical conversion cocktail on primary hippocampal cell cultures of transgenic 5xFAD mice upon ACE2 inhibition. Immunopositivity to astrocyte marker (GFAP – green) and neuronal marker (MAP2 – red). Scale bar – 250 µm for panels a–d, scale bar – 125 µm for panels a1–d1. (a–a1) – Native primary hippocampal cell culture of transgenic mice (Tg). (b–b1) – ACE2 inhibition with MLN-4760. (c–c1) – ​​Administration of conversion cocktail after ACE2 inhibition (Treatment). (d–d1) – Administration of conversion cocktail before ACE2 inhibitor (Priming). (e) – Neuronal and astrocytic densities (%) in primary hippocampal cell cultures of transgenic mice. * – p < 0.05, one-way ANOVA, Dunn’s post-hoc test. (f) – Astrocyte area (%) exhibiting double immunopositivity to GFAP/MAP2 markers. * – p < 0.001, one-way ANOVA, Bonferroni’s post-hoc test.

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