Dehydroepiandrosterone alleviates hypoxia-induced learning and memory dysfunction by maintaining synaptic homeostasis

Abstract

Aims: Hypoxia causes plenty of pathologies in the central nervous system (CNS) including impairment of cognitive and memory function. Dehydroepiandrosterone (DHEA) has been proved to have therapeutic effects on CNS injuries by maintaining the homeostasis of synapses, yet its effect on hypoxia-induced CNS damage remains unknown.

Methods: In vivo and in vitro models were established. Concentrations of glutamate and γ GABA were tested by ELISA. Levels of synapse-associated proteins were measured by western blotting. Density of dendritic protrusions of hippocampal neurons was assessed by Golgi staining. Immunofluorescence was adopted to observe the morphology of primary neurons. The novel object recognition test (NORT) and shuttle box test were used to evaluate cognition.

Results: Dehydroepiandrosterone reversed abnormal elevation of glutamate levels, shortenings of neuronal processes, decreases in the density of dendritic protrusions, downregulation of synaptosome-associated protein (SNAP25), and impaired cognition caused by hypoxia. Hypoxia also resulted in notably downregulation of syntaxin 1A (Stx-1A). Overexpression of Stx-1A dramatically attenuated hypoxia-induced elevation of glutamate. Treatment with DHEA reversed the Stx-1A downregulation caused by hypoxic exposure.

Conclusion: Dehydroepiandrosterone may exert a protective effect on hypoxia-induced memory impairment by maintaining synaptic homeostasis. These findings offer a novel understanding of the therapeutic effect of DHEA on hypoxia-induced cognitive dysfunction.

Keywords: dehydroepiandrosterone; hippocampus; hypoxia; neuron; synaptic function.

FIGURE 1

Hypoxic exposure results in aberrant viability and neuronal dysfunction in HT22 cells. (A) Viability of HT22 cells was tested by CCK8 after hypoxic exposure for 24 and 48 h (n = 5, mean ± SEM). (B) Protein levels (HIF1α, PSD95, and SNAP25) were measured by western blotting after hypoxic exposure. (C) Gray intensity analysis of HIF1α, PSD95, and SNAP25 (n = 3, mean ± SEM). β‐actin was used as internal reference. (D) Glutamate concentrations were tested after hypoxic exposure (n = 3, mean ± SEM). (E) γ GABA concentrations were tested after hypoxic exposure (n = 3, mean ± SEM). ns, no significant; *p < 0.05, **p < 0.01, and ****p < 0.0001

FIGURE 2

DHEA reverses neuronal function impaired by hypoxic exposure. (A) Representative viability of HT22 cells was tested by CCK8 after treatment of DHEA in different concentrations (n = 5, mean ± SEM). (B) Representative cell viability of HT22 cells tested by CCK8 following hypoxic exposure with or without DHEA (n = 5, mean ± SEM). (C) Effects of DHEA on cell morphology in hypoxia‐exposed mice primary‐cultured hippocampal neurons (scale bar = 20 μm). (D) Neurite length of primary‐cultured hippocampal neurons after hypoxic exposure with or without treatment of DHEA (n = 5, mean ± SEM). (E) Protein level of HIF1α, PSD95, and SNAP25 in HT22 cells measured by western blotting after hypoxic exposure with or without DHEA. (F) Gray intensity analysis of HIF1α, PSD95, and SNAP25 (n = 3, mean ± SEM). (G) Glutamate concentration of HT22 cells tested after hypoxic exposure with or without DHEA (n = 3, mean ± SEM). (H) γ GABA concentration of HT22 cells tested after hypoxic exposure with or without DHEA (n = 3, mean ± SEM). ns, no significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

FIGURE 3

DHEA reverses mice memory function impairment caused by hypoxic exposure. (A) The experimental process proposal. (B) Body weight of mice after 2 weeks of hypoxic exposure (n = 10, mean ± SEM). (C) Representative locomotion tracking routes of mice exposed to hypoxia in NORT with or without DHEA. (D) Representative identification index of mice exposed to hypoxia for 2 weeks in NORT (n = 6, mean ± SEM). (E) Representative time of recognition mice used on new objects in the NORT test following 2 weeks of hypoxic exposure with or without DHEA (n = 6, mean ± SEM). (F) Representative active avoidance numbers of mice in shuttle box assay following 2 weeks of hypoxic exposure with or without DHEA (n = 6, mean ± SEM). (G) Representative mean reaction times of active avoidance of mice in shuttle box assay following 2 weeks of hypoxic exposure with or without DHEA (n = 6, mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

FIGURE 4

DHEA reverses impairment of dendritic plasticity and synaptic function in mice hippocampus induced by hypoxic exposure. (A) Left: Neurons in the hippocampus through Golgi staining (scale bar = 50 μm). DG = dentate gyrus. Right: Pyramidal neurons in CA1 through Golgi staining. (B) Basal and apical dendritic spines located in CA1 region of hippocampus (scale bar = 10 μm). (C, D) Statistical analysis of hypoxic exposure's influence on basal (C) and apical spines (D). Data are presented as mean ± SEM of per 10 μm spines in each group (32 neurons/16 mice for all groups). (E) Glutamate concentration of hippocampus tissues tested after hypoxic exposure with or without DHEA (n = 3, mean ± SEM). (F) γ GABA concentration of hippocampus tissues tested after hypoxic exposure with or without DHEA (n = 3, mean ± SEM). ns, no significant; *p < 0.05, **p < 0.01, and ****p < 0.0001

FIGURE 5

DHEA reverses hypoxia‐induced downregulation of Stx‐1A. (A) Protein level of HIF1α and Stx‐1A in mice hippocampus tissues measured by western blotting after hypoxic exposure with or without DHEA. (B) Gray intensity analysis of HIF1α and Stx‐1A (n = 3, mean ± SEM). (C) Protein level of Stx‐1A, Munc18‐1, and Rab 3a in HT22 cells measured by western blotting after hypoxic exposure with or without DHEA. (D–G) Gray intensity analysis of Stx‐1A, Munc18‐1, and Rab 3a (n = 3, mean ± SEM). β‐actin was used as internal reference. ns, no significant; *p < 0.05 and **p < 0.01

FIGURE 6

Stx‐1A is involved in hypoxia‐induced synaptic dysfunction. (A) Representative immunoblotting of Stx‐1A in HT22 cells under hypoxic exposure with or without overexpression of Stx‐1A. (B) Gray intensity analysis of Stx‐1A (n = 3, mean ± SEM). (C) Glutamate concentration of HT22 cells tested after hypoxic exposure with or without overexpression of Stx‐1A (n = 3, mean ± SEM). (D) Representative immunoblotting of Stx‐1A in HT22 cells with or without knockdown of Stx‐1A. (E) Gray intensity analysis of Stx‐1A (n = 3, mean ± SEM). (F) Glutamate concentration of HT22 cells tested after normoxia exposure with or without DHEA treatment and knockdown of Stx‐1A (n = 3, mean ± SEM). (G) Glutamate concentration of HT22 cells tested after hypoxic exposure with or without DHEA treatment and knockdown of Stx‐1A (n = 3, mean ± SEM). ns, no significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001