Neural Plast. 2016;2016:3289187. doi: 10.1155/2016/3289187
Dysfunction in ribosomal gene expression in the hypothalamus and hippocampus following chronic social defeat stress in male mice as revealed by RNA-Seq
Smagin DA, Kovalenko IL, Galyamina AG, Bragin AO, Orlov YL, Kudryavtseva NN
Modeling of Neuropathology Laboratory, Laboratory of Behavioral Neuroinformatics, Institute of Cytology and Genetics, Siberian Department of Russian Academy of Sciences, Novosibirsk, Russia
Chronic social defeat stress leads to the development of anxiety- and depression-like states in male mice and is accompanied by numerous molecular changes in brain. The influence of 21-day period of social stress on ribosomal gene expression in five brain regions was studied using the RNA-Seq database. Most Rps, Rpl, Mprs, and Mprl genes were upregulated in the hypothalamus and downregulated in the hippocampus, which may indicate ribosomal dysfunction following chronic social defeat stress. There were no differentially expressed ribosomal genes in the ventral tegmental area, midbrain raphe nuclei, or striatum. This approach may be used to identify a pharmacological treatment of ribosome biogenesis abnormalities in the brain of patients with “ribosomopathies.”
Two decades ago, we showed that chronic social defeat stress (CSDS) leads to the development of behavioral psychopathology, which is accompanied by anxiety- and depression-like states in male mice of C57BL/6J line [1, 2] similar to those in humans. Defeated mice developed a severe behavioral deficit: passive defense and immobile behavior, reduced ambulation in the open-field; increased immobility time in the Porsolt’s test. Clear-cut anxiety was revealed in the plus-maze test. These mice display disturbed social behavior, social indifference in any experimental situation and anhedonia – reduced sucrose consumption. A loss of weight, decreased plasma testosterone level and immune responsiveness, decreased stress reactivity have been revealed, too. This mixed anxiety/depression-like state in defeated mice was sensitive to antidepressants and anxiolytics. We also found numerous changes in neurotransmitter’ activity in brain of depressive mice [3, 4]. Our first data revealed the changes of expression of serotonergic genes in brain. These data suggested the existence of a new way of regulation – “from behavior to gene”. Later this suggestion was strongly confirmed . Moreover under CSDS, the adult brain undergoes numerous changes, including changes in DNA methylation, histone acetylation and chromatin remodeling as well as decrease in hippocampal neurogenesis [6-8].
We decided to do whole transcriptome analysis of some brain regions which are involved into regulation of depression. The collected samples from depressive mice and the controls were sequenced at JSC Genoanalytica (www.genoanalytica.ru, Moscow, Russia), where the mRNA was extracted using the Dynabeads mRNA Purification Kit (Ambion, USA). Choice of the brain regions selected for testing was based on their functions, localization of neurons of neurotransmitter systems and differential involvement in the mechanisms of a depression-like state in our experimental paradigm. These regions are as follows: the midbrain raphe nuclei, ventral tegmental area, striatum, hippocampus, and hypothalamus.
Figure 1. Chronic social defeat stress in male mice was induced by daily agonistic interactions with an aggressive partner [1, 2]. Animals were placed in cages bisected by a perforated transparent partition, which allowed the animals to see, hear and smell each other but prevented physical contact. Every day the partition was removed for 10 minutes to encourage agonistic interactions. The superiority of one of the mice was firmly established within two or three encounters with the same opponent. The winning mouse would attack, bite and chase the losing mouse, which would display only defensive behavior (sideways posture, upright posture, withdrawal, lying on the back or freezing). This procedure was performed for 21 days. Two groups of animals were analyzed in this experiment: 1) depressive mice: groups of chronically defeated mice; and 2) controls: the mice without any consecutive experience of agonistic interactions. Figure shows (from left to right) – experimental cage; defeater demonstrates defense posture during partner’ attacks; posture of depression, nose to the corner.
We assumed that the number of differentially expressed genes and direction of change (up or down) may be used as marker of more or less intensive involvement of any brain area into molecular mechanisms of depression-like state in mice. Analyzing the whole transcriptome using RNA-Seq in, we observed changes in the expression of numerous genes. This report was concentrated on the analysis of genes encoding ribosomal and mitochondrial ribosomal proteins (Rps, Rpl, Mrpl, Mrps), which are responsible for translation, transcription and cell proliferation and are involved in neural plasticity in healthy cells.
In the midbrain raphe nuclei, ventral tegmental area and striatum, the Rps, Rpl, Mrpl and Mrps genes did not change their expression under CSDS. In the hippocampus and hypothalamus, the major components of ribosomes — the small ribosomal subunit that reads the RNA (Rps) and the large subunit that connects amino acids to form a polypeptide chain (Rpl) — changed their expression under CSDS. In the hippocampus of depressive mice, the largest number of ribosomal genes (Rpl7, Rpl36a, Rpl39, Rps4x and Rps27a) was downregulated and only 2 genes (Rpl35 and Rpl18) were upregulated. We suggested that downregulation of the ribosomal genes may be associated with a decreased proliferation in the hippocampal dentate gyrus in defeated mice, as described by a authors using a similar experimental paradigm [7-8].
The hypothalamus is responsible for production of numerous hormones that are involved in the regulation of many physiological functions and psychoemotional states. Many diseases are connected with abnormal hypothalamic function, such as changed stress reactions, metabolism, loss or increase of appetite, changed emotional behavior, memory loss, sleep disorders, and affective and somatic states etc. Because decreased stress reactivity, weight loss, development of pronounced anxiety and depression-like state were observed in the mice after CSDS [1, 2], we suggest a significant involvement of the hypothalamus in these pathological processes. Support for this hypothesis comes from the observation that the largest number of all differentially expressed genes was observed in this brain region. In the hypothalamus the majority of differentially expressed genes were upregulated in depressive mice, and numerous ribosomal genes changed their expression under CSDS (14 Rps, 22 Rpl, 12 Mrps and Mrpl genes) (Figures 2).
Figure 2: Differentially expressed ribosomal RpS, Rpl, Mrpl and Mprs genes (from left to right) in the hypothalamus of depressive mice. The Cufflinks program was used to estimate the gene expression levels in FPKM (fragments per kilobase of transcript per million mapped reads) and then to detect the differentially expressed genes in the analyzed and control groups. The Rpsa, Rps2, Rps3, Rps5, Rps8, Rps9, Rps10, Rps14, Rps16, Rps19, Rps26, Rps6ka1genes were upregulated, whereas Rps6ka3 and Rps6ka6 were downregulated under CSDS in depressive mice. The Rpl37a, Rpl41, Rpl19, Rpl23a, Rpl37, Rpl8, Rpl10a, Rpl36, Rpl7a, Rpl12, Rpl35, Rpl34, Rplp0, Rpl6, Rpl28, Rpl18, Rplp2, Rpl13, Rpl18a, Rpl29, Rplp1 genes were upregulated, whereas the Rpl22l1 gene was downregulated. The Mrps18a, Mrps12, Mrpl54, Mrpl12, Mrpl38, Mrpl52, Mrpl28, Mrpl23, Mrpl34, Mrpl4 genes were upregulated, whereas Mrpl3 and Mrpl1were downregulated. The levels of the genes expression are presented in the control (blue columns) and depressive mice (dark blue columns). Statistical significance – P < 0.01 and q < 0.05, * P < 0.01; ** P < 0.001.
However, the question arises: does upregulation of numerous ribosomal and mitoribosomal genes present a feedback mechanism in response to hypothalamic activation under CSDS, or is a result of ribosomal gene dysfunction developing in depressive mice? Now we continue our study to answer on these questions.
In recent years a number of human diseases have been identified and categorized as “ribosomopathies” [9-10] caused by alterations in either the structure or function of ribosomal components, which are associated with distinct mutations in the ribosomal biogenesis pathway. These diseases include Diamond-Blackfan anemia, Shwachman-Diamond syndrome, dyskeratosis congenital etc. Interestingly, increased expression of the Rps19, Rps14, Rps10, Rps26 genes which are involved into Diamond-Blackfan anemia, was observed in depressive mice. Our observation concerning changes in the expression of ribosomal genes in the hippocampus and hypothalamus in mice indicates the involvement of translation, transcription and proliferation in ribosomes into pathophysiological processes of depression.
Additionally in the hypothalamus we found the development of possible mitochondrial protein dysfunctions: the mitochondrial ribosome genes Mrpl54, Mrpl12, Mrpl38, Mrpl52, Mrpl28, Mrpl23, Mrpl34, Mrpl4 Mrps18a, and Mrps12 were upregulated in depressive mice, whereas Mrpl1 and Mrpl3 were downregulated (Figure 2). Thus, we can assume a strong link between CSDS leading to the development of a depression-like state in mice and upregulation of mitochondrial ribosomal genes in the hypothalamus. This conclusion is indirectly confirmed by observations that patients with mitochondrial disorders can show primary psychiatric symptomatology, including mood disorder, cognitive impairment, psychosis and anxiety. As it is supposed, mitochondrial disorders may be caused by either acquired or inherited mutations in the mitochondrial DNA or in nuclear genes that code for mitochondrial components [11-12]. These disorders may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections or other environmental causes. The majority of mitochondrial disorders are associated with neurological abnormalities, including seizures and myoclonus, psychomotor retardation, dementia, ataxia, motor neuron disease, weakness, chronic fatigue etc. Depressive mice have been shown to demonstrate also motor retardation, immobility, helplessness in any situations. Undoubtedly it is difficult to find a direct association between the overexpression of ribosomal and mitochondrial ribosomal genes in the hypothalamus and the depression-like state in mice, which would help to understand causes and consequences of these processes. At this stage of research, it is impossible to elucidate the detailed sequence of neurochemical events, and molecular changes that occur due to restructuring brain regulation in depressive male mice. However, it is clear that, starting with a change in social behavior and psychoemotional state under CSDS, at certain stages this process launches a cascade of systemic changes at the whole brain, its regions, and specific neurons following changes in metabolism and reception of neurotransmitter systems. As a result, it leads to the changes in the expression of genes involved in the development of affective disorders. The changes observed in ribosomal and mitochondrial ribosomal genes expression may indicate ribosome dysfunction. Our model, which induces a mixed anxiety/depression-like state in male mice following CSDS [1, 2] may be used to identify a pharmacological treatment of ribosome biogenesis abnormalities in the brain.
Importance of the study:
It has been shown that chronic social stress induces changes in psychoemotional state which are accompanied by profound changes in hypothalamic cell functions involving the processes of translation, transcription and proliferation. We can suspect that depression in humans may be accompanied by ribosome and mitoribosome dysfunctions.
Our behavioral approach may be used for modeling of abnormalities in ribosome biogenesis in the brain, for the study of molecular mechanisms of these processes and for search of pharmacological treatment of ribosomopathies and mitochondrial disorders.
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Acknowledgements: This study was supported by Russian Science Foundation (No 14-15-00063) awarded to Natalia Kudryavtseva.
Prof. Natalia N. Kudryavtseva, Ph.D., Dr.Sci.
Modeling of Neuropathology Laboratory
Neurogenetics of Social Behavior Sector
Institute of Cytology and Genetics SD RAS
Ave. Ak. Lavrentjeva, 10,
Novosibirsk, 630090, Russia