Effects Of REM-sleep Deprivation On The Expression Of Endoplasmic Reticulum Stress Related Protein ATF4 In The Rat Brain

[Research Background and Objective]In modern society, people's sleeping time shows the tendency of declination, sleep deprivation has become a pattern of sleep disorder manifestation. Rapid Eye Movement (REM) -sleep is associated with cognitive psychological activity and the growth and development of brain, and plays a key role in memory consolidation, protein synthesis, and gene expression and so on. Sleep deprivation is similar to other stressors such as hunger and hypoxia, which can lead to disorders of normal physiological functions in the body. Clinical research shows that sleep deprivation can produce a series of symptoms including compromised immune function, loss of learning and memory abilities, and emotional disturbances. Currently very few studies in China and overseas are concerning about the association between sleep deprivation and endoplasmic reticulum stress, and in abroad it has been reported the levels of GRP78/BIP (Glucose-regulation Protein78/ bacterial intravenous protein) and the phosphorylation levels of eIF2α(Eukaryotic Initiation Factor 2) experienced significant changes after the deprivation of sleep in rats. Through the studies of our department in the past, we have noted that REM-sleep deprivation could enhance the expression of GRP94, the marked protein of endoplasmic reticulum stress. In order to probe into the pathophysiology mechanism of brain damage after REM-sleep deprivation, we choose REM-sleep deprivation as the subject of this trial. Endoplasmic reticulum, widely existing in eukaryotic cells, is the major site of intracellular protein synthesis and calcium storage. A number of factors, including the deficiency of cellular nutrition, and medicines which can affect endoplasmic reticulum (ER) calcium storage, oxidative stress, and viral infections, lead to the disequilibrium of endoplasmic reticulum functions, and thus endoplasmic reticulum stress (ERS) develops. A series of responses will be subsequently caused by ERS to reduce the damage of cellular functions and to protect the normal function of cells, including the following three specific aspects: 1. more chaperone proteins and protein folding-related enzymes could combine with the unfolding proteins to lessen damages and the unfolding protein could be enabled to refold appropriately; 2. temporal inhibition of protein synthesis decreases the load of endoplasmic reticulum; 3.the increase of enzymes related with protein degradation accelerates the degradation so as to further decrease the ER load. And the subsequent series of reactions caused by accumulation of protein is called Unfolded Protein Response (UPR). As a critical and essential protective measure of cells, however, ERS may induce apoptosis when the stress factors are too strong, and thus produce damages to tissues and organs in the body. Recently it has been found that ERS is closely related with the onset and development of many central nervous system diseases, including Alzheimer's disease, Parkinson's disease, cerebral infarction, and Huntnington disease.The objective of this study is to determine the correlation between sleep deprivation and ERS, which might be of theoretical importance for the research on the damage mechanism of sleep deprivation and appropriate intervention measures against it. As far as we know, there are three different pathways involved in the intracellular UPR signaling pathways, namely, Irel-dependent pathway, and another 2 pathways respectively with PERK (PKR-like Endoplasmic Reticulum Kinase) and ATF6 (Activating Transcription Factor 6) as their major conductive factor. We selected ATF4 (Activating Transcription Factor 4) as our study objective, which is involved in the pathway of eIF2αphosphorylation conducted by PERK. This pathway consists of the following parts:1. Suspension of Protein Synthesis in Primary Phase of ERSBecause BiP bound with PERK protein has to deal with a great number of unfolding proteins, the PERK proteins inside ER is exposed, which leads to their autophosphorylation and dimerization, subsequently the activated PERK proteins phosphorylating eIF2αlocated on lateral ER, the phosphorylated eIF2αcannot be accepted by the transition of GTP-GDP with eIF2β, ultimately the syntheses of most proteins are suspended at the initiation process.2. Emergency of Integrative Stress Reaction in Metaphase of ERSThe short-term suspension of protein syntheses may decrease the load and pressure which is brought by the need of nascent polypeptide chain for ER protein folding, but long-term protein synthesis inhibition can induce apoptosis. eIF2αphosphorylation can inhibit most of protein syntheses on one hand, but on the other it can facilitate the translation of ATF4 through its effect on the binding efficiency between ATF4 and ribosomal, the increase of ATF4 proteins level can enhance the transcription activation of a group of chaperone proteins, such as GADD34 (Growth Arrest and DNA Damage Inducible gene 34) and GADD153 (Growth Arrest and DNA Damage Inducible gene 153) etc. GADD34 dephosphorylates eIF2αso that syntheses of proteins can be recovered, and normal functions of cells are maintained; if the strength of stress is too much or the duration is too long for cells to be self-corrected, the apoptosis mechanism is induced by GADD153.ATF4,also called CREB2 (CAMP-response Element protein 2),is a member of ATF-CREB bZIP family. ATF4 mRNA is found in every kind of cells, the level of which is relatively higher in cranial bones, brain, thymus gland, and liver or other organs, but the ATF4 content in cells is quite low or even impossible to be measured. The present researchers account the phenomena for the structure of specific upstream 0pen reading frames (uORFs) in the 5′leader of ATF4 mRNA which contributes to the persistent inhibition status of ATF4 translation.When ERS occurs, eIF2αphosphorylation conducted by PERK can increase the binding efficiency of ATF4 mRNA and ribosome, and facilitate ATF4 translation. Higher content level of ATF4 can lead to the expression of a series of chaperone proteins, for instance, GADD34, GADD153 and so on, which are of great significance for ERS.We observed the expression of ATF4 protein after REM-sleep deprivation in PERK-eIF2αpathway, which proves that EMS occurs in the early phase of sleep deprivation and the translation of ATF4 is initiated, the increase of ATF4 proteins level can enhance the transcription activation of a group of chaperone proteins, such as GADD34 and GADD153, which plays an important role in many ways including recovery of protein syntheses, sustain of cellular functions and apoptosis control.[Methods] The rats are randomly divided into 3 groups, i.e. Sleep Deprivation(SD n=40), Tank Control (TC=10)and Cage Control(CC=10). And according to the duration of sleep deprivation, SD group was divided into 4 subgroups, 10 rats in each subgroup, respectively 6, 12, 24, 36 hours. Modified multiple plat—form method (MMPM) was applied to establish REM-sleep deprivation with different duration, and immunohistochemical techniques were applied to detect the distribution and contents of ATF4 in frontal lobe and hippocampus, while Western blot was used to detect the change of ATF 4 in frontal lobe and hippocampus specimen.[Results]a) There was no ATF detected in neither frontal lobe nor hippocampus in Cage Control (CC) group with immunohistochemical methods. The expression of ATF4 in hippocampus started from Tank control (TC) group, ATF4 expression in 6-hour-subgroup was higher than TC group (P<0.01)and peaked in 12-hour subgroup (compared with other groups and subgroups; P<0.01), it tended to decline since 24-hour and 72-hour subgroup, ATF4 expression in 72-hour subgroup was not significantly different from TC group (P>0.05). The expression in frontal lobe started from TC group, ATF4 expression in 6-hour-subgroup was higher than TC group (P<0.05)and peaked in 12-hour subgroup(compared with other groups and subgroups; P<0.01), it tended to decline since 24-hour and 72-hour subgroup, ATF4 expression in 72-hour subgroup was higher than TC group (P<0.05), 6-hour subgroup had no significant difference from72-hour subgroup (P>0.05).b) Western blot technique revealed no ATF4 detected in neither frontal lobe nor hippocampus in CC group. The expression of ATF4 in hippocampus started from TC group, ATF4 expression in 6-hour-subgroup was higher than TC group (P<0.05)and peaked in 12-hour subgroup (compared with other groups and subgroups; P<0.01), and began to decline since 24-hour and 72-hour subgroup, 6-hour subgroup had no significant difference from72-hour subgroup (P>0.05). The expression in frontal lobe started from TC group, and peaked in 12-hour subgroup (compared with other groups and subgroups; P<0.01), and began to decline after that, the expression of TC group is lower than any other group (P<0.01), 6-hour subgroup had no significant difference from72-hour subgroup (P>0.05).[Conclusions]a) ATF4 exists in normal rat brain tissue in the form of mRNA.b) REM-sleep deprivation can induce ER to initiate its protective function to maintain homeostasis—ERS.c) The timing and process of ERS in the frontal lobe and hippocampus induced by REM-sleep deprivation in almost the same.