Reduction of depression-like behavior in rat model induced by ShRNA targeting norepinephrine transporter in locus coeruleus

Xiangdong Du,Nian Yuan,Siyun Zou,Guangya Zhang,Lian Yuan,Yan Fan,Thomas R Kosten,Xiang Yang Zhang,Xueli Zhao,Zhe Li,Xiaoli Lv,Ming Yin,Wei Deng

doi:10.1038/s41398-020-0808-8

Abstract

Depression may be associated with reduced monoamine neurotransmission, particularly serotonin and norepinephrine (NE). Reuptake of NE by the norepinephrine transporter (NET) is the primary mechanism by which many of the antidepressants are high-affinity substrates for NET. This study aimed to examine the effect of lentivirus-mediated shRNA targeting NET in locus coeruleus (LC) on depression-like behaviors of rats. We randomly assigned 60 male Wistar rats to 6 experimental groups: (1) Control group: without chronic unpredictable mild stress (CUMS) and without NET-shRNA treatment; (2) shRNA group: without CUMS + NET-shRNA; (3) CUMS group: 3-week CUMS without NET-shRNA; (4) CUMS + nonsense shRNA group; (5) CUMS + amygdala (Amy)-shRNA group; (6) CUMS+ locus coeruleus (LC)-shRNA group. First, recombinant lentiviral vector expressing shRNA (ShRNA-629, ShRNA-330, ShRNA-1222, ShRNA-1146 or ShRNA- negative control) against NET were produced, and their efficiency in knocking down of NET in PC12 cells were assessed by Q-PCR and western blot analysis. Second, shRNA was injected into the rat LC bilaterally to investigate whether it could prevent the depressive-like behavior induced by 3-week CUMS. Third, we tested the depressive-like behavior of the rats in the forced swimming test, the open field test, the sucrose preference test, as well as the body weight gain at the end of the seventh week. Finally, the protein expressions of NET was measured by western blot and the NE levels were measured by high performance liquid chromatography. Q-PCR and western blot showed that the ShRNA-1146 had the best interference efficiency targeting on NET in PC12 cells (p < 0.01). Compared to the depression model group, the immobility time in the forced swimming test was significantly reduced (p < 0.01), but the sucrose preference and the total scores in the open field test were significantly increased (all p < 0.01) in the group treated with shRNA in LC. Furthermore, compared with the depression model group, NET levels were significantly decreased (p < 0.01), but NE levels were significantly increased in the group treated with shRNA in LC (p < 0.05). Our findings suggest that Lentivirus-mediated shRNA targeting NET in LC downregulated NET both in vitro and in vivo, resulting in a significant decrease in depressive-like behavior of rats.

Highlights

Major depressive disorder (MDD) is a common, serious, life-threatening mental illness, whose incidence is high, affecting 120 million people around the world[1]
Rats were randomly assigned into 6 groups, with 10 rats per group: (1) Control group: without chronic unpredictable mild stress (CUMS) and without norepinephrine transporter (NET)-shRNA treatment; (2) shRNA group: rats were injected with NET-shRNA into the locus coeruleus (LC) bilaterally without CUMS; (3) CUMS group: rats were treated with CUMS for 3 weeks without shRNA treatment; (4) CUMS + nonsense shRNA group: rats were injected with nonsense shRNA into the LC bilaterally after 3 weeks of CUMS; (5) CUMS + Amy-shRNA group: rats which were injected with NET-shRNA into the amygdala (Amy) bilaterally after 3 weeks of CUMS; (6) CUMS + LCshRNA group, rats were injected with NET-shRNA into the LC bilaterally after 3 weeks of CUMS
We assessed the efficiency of the NET-shRNA in knocking down of NET in PC12 cells using Q-PCR and western Blot

Summary

Introduction

Major depressive disorder (MDD) is a common, serious, life-threatening mental illness, whose incidence is high, affecting 120 million people around the world[1]. Many preclinical and clinical studies have shown that NE involves extensive physiological activities such as learning, memory, awakening and sleep, all of which are important for mental illness[4] These activities are important for depression and stress-induced autonomic regulation[5,6]. The central noradrenergic pathways originate from the locus coeruleus (LC), and is primarily projected to the frontal cortex, as well as to the limbic system, including its main components such as the amygdala, hippocampus and hypothalamus, all of which are associated with mood and cognition[14] These NE brain targets and associated functions are modified in depressed patients, which include appetite, pleasure, sexual satisfaction, aggression and response to pain[15,16]

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