ISSN 16076729, Doklady Biochemistry and Biophysics, 2015, Vol. 461, pp. 76–79. © Pleiades Publishing, Ltd., 2015. Original Russian Text © Iu.F. Pastukhov, V.V. Simonova, M.A. Guzeev, D.A. Meshalkina, I.V. Guzhova, I.V. Ekimova, 2015, published in Doklady Akademii Nauk, 2015, Vol. 461, No. 2, pp. 228–231.
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
Chaperone Hsp70 Is Involved in the Molecular Mechanisms of Slow Wave Sleep Regulation Iu. F. Pastukhova, V. V. Simonovaa, M. A. Guzeeva, D. A. Meshalkinab, I. V. Guzhovab, and I. V. Ekimovaa Presented by Academician N. P. Veselkin September 2, 2014 Received September 11, 2014
DOI: 10.1134/S1607672915020040
In the last ten years, the hypotheses that chaper ones of the HSP70 (Heat Shock Protein 70 kDa) fam ily play an important role in the biosynthesis of pro teins and other macromolecules and in sleep recovery dominate in the literature [1–3]. These hypothesis are insufficiently substantiated due to the use of stress models, in which a large number of chaperones are expressed in different brain structures. In addition, these models use total rest/sleep deprivation; as a result, the role of chaperones in the slow wave sleep (SWS) and paradoxical sleep cannot be distinguished.
The lack of data and arguments in modern hypoth eses about the role of molecular chaperones in sleep regulation prompted us to create a model of a long term reduction in the expression of the Hsp70 chaper one in the main sleep “center” in the VLPO of the hypothalamus of Wistar rats based on RNA interfer ence technology. It was shown for the first time that, in the period of 6–15 days after the targeted transfection of the VLPO neurons with the lentivector pLKO.1 shRNAHsp70, the duration of SWS in the active phase of the day decreased by 32%, which was accom panied by a 60% decrease in the Hsp70 chaperone level in the sleep “center” in the VLPO. The results obtained in our model can be used in justifying the treatment of sleep disorders and nervous system dis eases, especially in the elderly age, which is character ized by a reduction in the level of chaperones in the brain and the SWS duration. The results of this study are of priority importance.
SWS accounts for approximately 80% of the total sleep time in humans, mammals, and birds [4]. Its important biological function is an increase in the rate of proteins synthesis required for the maintenance and recovery of the structure and function of nerve cells [2, 5, 6]. Earlier, we have shown for the first time that an increase in the levels of exogenous Hsp70 protein in the brain and the main sleep “center” in the ventrolat eral preoptic area (VLPO) of the hypothalamus is accompanied by a distinct increase in the natural slow sleep in rats and pigeons [1, 7, 8]. Hypotheses and our data do not provide an answer to the question as to how changes in the SWS depend on the level of HSP70 chaperones in the main sleep “center” in the VLPO. Since specific receptors for chaperones have not been found in the brain, it is impossible to solve this prob lem by the conventional methods.
The study was carried out in 11 male Wistar rats weighing 320–350 g at an ambient temperature of 21– 23°C and a photoperiod of 12 : 12. Manipulations with the animals were performed in accordance with the bioethical rules. The DSI4ET telemetry module was implanted subcutaneously under general anesthesia. Electroencephalogram, electrooculogram, elec tromyogram, and the body temperature of animals were continuously recorded for 48 h in conditions of free behavior of the animal using a Dataquest A.R.T. System (Data Sciences International, United States). To reduce the level of inducible Hsp70 chaperone in the VLPO neurons in the hypothalamus, we used the shRNAHsp70 lentiviral construction. Lentiviral par ticles were obtained according to the protocol described previously [9]. The solution containing these particles (concentration, 500–600 particles/µL) and the control solutions (saline and lentivector carry ing the green fluorescent protein gene GFP) were injected bilaterally (volume, 1.5 µL, 0.1 µL/min) into the VLPO of the hypothalamus of rats (Fig. 1) accord ing to the stereotactic atlas coordinates [10]. The
In this study, we investigated the characteristics of SWS and wakefulness using the method that allows a longterm targeted reduction in the level of a given chaperone in the sleep “center” in the VLPO using the RNA interference technology. a Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, pr. Morisa Toreza 44, St. Petersburg, 194223 Russia email:
[email protected] b Institute of Cytology, Russian Academy of Sciences, Tikhoretsii pr. 4, St. Petersburg, 194064 Russia
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Fig. 1. Histological control of the localization of the microinjection of the shRNAHsp70 lentiviral construc tion into the preoptic area of the hypothalamus of rats (light microscopy). The arrow indicates the track of the microinjection into the ventrolateral preoptic area (VLPO) of the hypothalamus. Frontal brain slices were immunohistochemically stained for the inducible Hsp70 protein. Scale, 50 µm.
pLKO.1 lentivirus transfection efficiency was deter mined by fluorescence immunohistochemistry and confocal microscopy, and changes in the level of the Hsp70 protein in the VLPO after transfection were estimated by immunoblotting. For identification and quantitative analysis of the temporal characteristics of sleep and wakefulness (the total time and the number and duration of episodes), we used the Sleep Pro pro
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gram developed in the laboratory for semiautomatic analysis and a program provided with the Dataguest device. The results were statistically processed using the parametric analysis of variance (ANOVA). Differences were regarded as statistically significant at p < 0.05. The results of the experiments confirmed the previ ous data [9] on the high degree of lentiviral transfec tion of the VLPO neurons and the absence of differ ences in the characteristics of the SWS and wakeful ness between the two control groups. It was demonstrated for the first time that in 2 weeks after the targeted transfection with the shRNAHsp70 lentivi ral construction, the content of the Hsp70 chaperone in the VLPO was reduced by 60% (Fig. 2). Significant changes in the characteristics of SWS and wakefulness were identified in the period of 6–15 days mainly in the active (dark) phase of the day. The total time of SWS, on average for 12 h, was reduced by 32% compared to the control (Fig. 3) solely due to the reduction in the number of its episodes (Fig. 4). The total time of wakefulness increased relative to the con trol (by 21%, p < 0.05) due to the increase in the dura tion of its episodes (by 65%, p < 0.05), where as the number of its episodes decreased (by 25%, p < 0.05). During the light (inactive) period of the day, the char acteristics of the SWS did not change, whereas the total time of wakefulness decreased (by 16%, p < 0.05) due to the reduction in the duration of its episodes (by 20%, p < 0.05). The average number of episodes per day and the total time of SWS reduced equally (by 10%, p < 0.05), the duration of episodes of wakefulness increased by 26% (p < 0.05), and the total time of wakefulness increased by 10% (p < 0.05). Thus, the inhibition of the synthesis of Hsp70 and the reduction in its level by 60% in VLPO neurons caused by the transfection with the lentivirus pLKO.1 shRNAHsp70 was accompanied by the inhibition of the mechanisms of initiation of SWS and by a reduc tion in its duration in the active phase of the day. This process apparently impairs the function of the inhibi Cortex
Hsp70 siRNA
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Tubulin Fig. 2. Changes in the content of the inducible Hsp70 protein in the ventrolateral preoptic area (PO) of the hypothalamus and motor cortex 15 days after the injection of the shRNAHsp70 lentivirus construction. Designations: C—injection of saline; GFP—injection of the lentivector carrying the green fluorescent protein gene; siRNAHsp70—injection of the lentivector car rying the hairpin Hsp70 RNA gene. DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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Fig. 3. Changes in the total time of SWS in rats during the dark phase of the day after the microinjection of the shRNAHsp70 lentiviral construction into the VLPO of the hypothalamus. The ordinate axis shows the mean val ues of the total time of SWS (expressed in percent of the recording time), represented as the arithmetic mean values for every 3 h and for 12 h of the dark phase of the day. The abscissa axis shows the time (h). LVC is the lentiviral con struction. Here and in Fig. 4, data are presented as M ± m, n = 6 (control) and n = 5 (LVC). * Differences from the control were significant at p < 0.05.
tory system of the VLPO and, according to the figura tive expression [11], “switches off the switching off” of the inhibitory effects of the neurons from the sleep “center” on the neurons from the wakefulness “cen ter” (in the hypothalamus, brain stem, and other areas of the brain). As a result, the duration of episodes and the total time of wakefulness in the active phase increase. It can be assumed that Hsp70 contained in the sleep “center” of the VLPO of the hypothalamus is involved in the molecular mechanisms of the “switch ing off” of wakefulness, which contribute to the imple mentation of the key function of the SWS—increase in the rate of protein synthesis in the brain. Earlier, we showed that, by increasing the content of exogenous Hsp70 in the brain and/or the VLPO, it is possible to increase the duration of natural SWS and to decrease the duration of wakefulness in rats and pigeons [7, 8]. These changes are implemented through the GABA(A) receptors in the VLPO and are accompanied by a decrease in several catabolic param eters [1, 7, 12]. The increase in the SWS duration and the decrease in the wakefulness duration were observed by us [9] in rats after the transfection of the VLPO neurons with the lentivector pLKO.1shRNA Hdj1, which led to a 80% reduction in the level of co chaperone Hdj1 (HSP40 family). It is known that Hdj1 closely interacts with Hsp70, regulating its bind ing to the target protein [13]. Therefore, the increase in the SWS duration depends on the increase in the level of Hsp70 [8] and/or the decrease in the level of its cochaperone Hdj1 [9] in the sleep “center.” These changes appar ently enhance the function of the inhibitory system in the VLPO on maintaining the wakefulness “centers”
Fig. 4. Changes in the number of the SWS episodes in rats during the dark phase of the day after the microinjection of the shRNAHsp70 lentiviral construction into the VLPO of the hypothalamus.
in the switchedoff state. Taken together, these data point to a close integration of the molecular systems HSP70 and HSP40 and their compensatory relation ships aimed at eliminating the imbalance between chaperones and normalizing the daily sleep–wake cycle. The data obtained in our model can be used in clinic practice in searching for the methods for mobi lizing the activity of chaperone systems [1] in order to justify the treatment of sleep disorders and nervous system diseases, especially in the elderly age, which is characterized by a longterm decline in the level of chap erones in the brain [14] and the SWS duration [15]. ACKNOWLEDGMENTS This work was supported by the program no. 7 of the Presidium of Russian Academy of Sciences “Mechanisms of Integration of Molecular Systems in the Implementation of Physiological Functions.” REFERENCES 1. Pastukhov, Yu.F., Khudik, K.A., and Ekimova, I.V., Ros. Fiziol. Zh. im. I.M. Sechenova, 2010, vol. 96, no. 7, pp. 708–725. 2. Mackiewicz, M., Naidoo, N., Zimmerman, J.E., et al., Ann. N. Y. Acad. Sci., 2008, vol. 1129, no. 1, pp. 335– 349. 3. Terao, A., Steininger, T.L., Hyder, K., et al., Neuro science, 2003, vol. 116, no. 1, pp. 187–200. 4. Koval’zon, V.M., Pastukhov, Yu.F., and Mukhame tov, L.M., in Mekhanizmy sna (Mechanisms of Sleep), Fiziologiya cheloveka i zhivotnykh (Human and Ani mal Physiology), Moscow: VINITI, 1986. 5. Nakanishi, H., Sun, Y., Nakamura, R.K., et al., J. Neu rosci., 1997, vol. 9, no. 2, pp. 271–279. 6. Siegel, J.M., Nature, 2005, vol. 437, no. 7063, pp. 1264–1271.
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CHAPERONE Hsp70 IS INVOLVED IN THE MOLECULAR MECHANISMS 7. Ekimova, I.V., Dokl. Biol. Sci., 2013, vol. 449, no. 6, pp. 89–92. 8. Pastukhov, Yu.F., Ekimova, I.V., Khudik, K.A., et al., Zh. Evol. Biokhim. Fiziol., 2008, vol. 44, no. 1, pp. 65– 71. 9. Pastukhov, Yu.F., Ekimova, I.V., Meshalkina, D.A., et al., Ros. Fiziol. Zh. im. I.M. Sechenova, 2012, vol. 98, no. 12, pp. 1530–1543. 10. Paxinos, G. and Watson, C., The Rat Brain in Stereo taxic Coordinates, San Diego: Academic, 1982.
12. 13. 14. 15.
Somnology: The Physiology and Neurochemistry of the Wake–Sleep Cycle), Moscow: BINOM, 2011. Ekimova, I.V. and Pastukhov, Yu.F., Zh. Evol. Biokhim. Fiziol., 2005, vol. 41, no. 4, pp. 356–363. Lazarev, V.F., Onokhin, K.V., Antimonova, O.I., et al., Biokhimiya, 2011, vol. 76, no. 5, pp. 724–730. Naidoo, N., Ferber, M., Master, M., et al., J. Neurosci., 2008, vol. 28, no. 26, pp. 6539–6548. Ohayon, M.M., Carskadon, M.A., Guilleminault, C., et al., Sleep, 2004, vol. 27, pp. 1255–1273.
11. Koval’zon, V.M., Osnovy somnologii: fiziologiya i neirokhimiya tsikla “bodrstvovanieson” (Principles of
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