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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
Sigma-1 Receptor is a Potential Drug Target for Neuropathology Treatment
A.V. Bolshakova1 *, V.A. Zhemkov 12, A.N. Gainullina1, E.O. Kukanova1, S.A. Korban1,2, I.E. Bezprozvanny1,3
1 Laboratory of Molecular Neurodegeneration, Peter the Great St. Petersburg Polytechnic University, Russia;
2 Laboratory of Enzymology, National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, Gatchina, Russia;
3 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA. * Presenting e-mail: asyab@mail.ru
Sigma-1 receptor (S1R) is a trans-membrane chaperone protein at the endoplasmic reticulum (ER), which stabilizes the calcium signaling between ER and mitochondria. This receptor has a high affinity to bind wide range of chemical compounds of different structural classes with a variety of therapeutic and pharmacological properties [1]. Thereafter S1R has received the most interest in respect to pharmacology application. There are studies that SIR is involved in the formation of many neurological and psychiatric conditions [2-5]. It was proposed that S1R serves as a sensor of normal calcium homeostasis. Recent studies have demonstrated the role of calcium signaling in neuropathology, such as Alzheimer's (AD) and Huntington's diseases (HD). Changes in endoplasmic reticulum calcium homeostasis resulted in the violation of synaptic connections between neurons [6].
We aimed to study S1R role as a potential modulator of neuronal dysfunction, synaptic loss and eventually neurodegeneration on the cellular model of neurodegenerative diseases and using molecular and biochemical techniques. To understand the mechanism underlying the synaptic loss in HD we cultivated mixed cortico-striatal neurons in vitro. YAC128 mice model of HD was used in experiments [7]. Striatal spine loss in YAC128 medium spiny neurons (MSNs) on DIV21 was shown previously [8]. Using immunostaining with DARPP32 antibody of MSN we achieved that exposure of S1R agonist 3PPP leads to spine rescue in YAC128 MSNs. Knockdown of S1R resulted in decreasing of spine density in MSNs both in WT and YAC128 cell cultures.
For better understanding of the structure and functioning of S1R we cloned human SIGMAR1 gene into pMAL-p5x (New England Biolabs) bacterial expression vector which resulted in expression of MBP-S1R fusion protein. The same approach has been utilized by our group for structure determination of a number of difficult proteins [9-11]. In brief, E. coli cells (B834 strain, co-transformed with pRARE2 plasmid) were cultured until they reached OD(600)=1.0 when protein synthesis was induced by addition of IPTG. Cells were cultured at 25° C overnight, the following day cells were harvested and lysed by sonication Membrane fraction was sedimented by centrifugation and membrane proteins were extracted by DDM: Triton X-100 detergent mixture. MBP-S1R was purified by two chromatography steps: first on the amylose resin followed by size-exclusion chromatography. Protein purity was confirmed by SDS-PAGE and immunob-lotting against S1R. In order to confirm protein functionality we used pull-down assay with agarose-immobilized progesterone. Recombinant MBP-S1R can be pulled down by progesterone beads which indicate that the protein produced in bacteria is functionally active in terms of binding selective sigma ligands. We plan to use our expertise in crystallization of structurally difficult proteins for characterization of S1R receptor structure and function.
Thus, the obtained results indicate that SIR has a potential for therapeutic targeting for the development of pharmacological agents for the treatment of Huntington's disease and eventually other types of neuropathology. EphA receptors in trans.
Acknowledgements
This work was supported by the Russian Scientific Fund grant (14-25-00024). References
1. Su T.P. et al., The sigma-1 receptor chaperone as an inter-organelle signaling modulator. Trends Pharmacol Sci, 2010. 31(12): p. 557-66.
2. Meyer, D.A., et al., Neurosteroids enhance spontaneous glutamate release in hippocampal neurons. Possible role of metabotropic sigma1-like receptors. J Biol Chem, 2002. 277(32): p. 28725-32.
3. Yagasaki, Y., et al., Chronic antidepressants potentiate via sigma-1 receptors the brain-derived neurotrophic factor-induced signaling for glutamate release. J Biol Chem, 2006. 281(18): p. 12941-9.
4. Monnet, F.P. and T. Maurice, The sigma1 protein as a target for the non-genomic effects of neuro(active)steroids: molecular, physiological, and behavioral aspects. J Pharmacol Sci, 2006. 100(2): p. 93-118.
5. Martina, M., et al., The sigma-1 receptor modulates NMDA receptor synaptic transmission and plasticity via SK channels in rat hippocampus. J Physiol, 2007. 578(Pt 1): p. 143-57.
6. Bezprozvanny, I., Calcium signaling and neurodegenerative diseases. Trends Mol Med, 2009. 15(3): p. 89-100.
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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
7. Slow, E.J., et al., Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. Hum Mol Genet, 2003. 12(13): p. 1555-67.
8. Artamonov, D.N., et al., Characterization of Synaptic Dysfunction in an In Vitro Corticostriatal Model System of Huntington's Disease. Biologicheskie Membrany, 2013. 30(4): p. 276-288.
9. Kim, M.W., et al., Secondary structure of Huntingtin amino-terminal region. Structure, 2009. 17(9): p. 1205-12.
10. Kim, M., Beta conformation of polyglutamine track revealed by a crystal structure of Huntingtin N-terminal region with insertion of three histidine residues. Prion, 2013. 7(3): p. 221-8.
11. Zhemkov, V., et al., The 2.2-Angstrom resolution crystal structure of the carboxy-terminal region of ataxin-3. FEBS Open Bio, 2016. 6(3): p. 168-178.
hp1 and Endogenous Retroviruses in the Brain
Andrew Newman*
Charité Universitätsmedizin Berlin, Germany. * Presenting e-mail: andrew.newman@charite.de
Heterochromatin Protein 1 (HP1), a structural protein found in the nucleus, is highly conserved across plants and animals and has a mechanism of action that has remained enigmatic since it was first observed 20 years ago. Known for binding to the repressive histone mark H3K9me3 or H3K9me2 (tri- or di-methylated Lysine 9 of Histone 3 respectively), HP1 has naturally emerged to be an essential component in a host of epigenetic systems essential for survival. Briefly, HP1 proteins have been implicated in retrotransposon silencing and heterochromatin spreading, proviral HIV silencing, chromosome stability and mitosis, cell cycle exit, spermatogenesis, DNA methylation, transcription, and embryonic stem cell maintenance. However, whatever the essential interaction is between HP1, H3K9 and respective Lysine methyltransferases remains elusive due to conflicting evidence. Concomitant to this, a growing body of evidence now implicates HP1 proteins as important components of various transcriptionary regulatory systems important in tissue specification during development. Differential interactions have been observed between distinct HP1 proteins and specific members of Nucleosome remodelling histone deacetylase (NuRD) complexes, BRG1 associated factor (BAF) (also called SWI/SNF) complexes, Polycomb (PcG) complexes, as well interacting with Kruppel-associated box (KRAB) containing zinc finger family, including the neuronal specific REST/ CoREST complex.
Presented here are two novel findings: 1) Class II endogenous retrovirus (ERVs) are de-repressed in HP1ß and HP1y double knockouts, and 2) this is due to two independent mechanisms that are synergistic. Here we report the mechanisms responsible for Class II ERV repression, the effects of de-repression on behaviour and aging, and test occurrences of retrotransposition due to neuronal stimulation and DNA damage.
Greating of Adenoassotiated Viral Vector for Expressing of Neurotrophic Factor BDNF in Neuronal Cells
E.A. Epifanova *, E.V. Mitroshina, A.A. Babaev
Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia. * Presenting e-mail: epifa888@mail.ru
Brain-derived neurotrophic factor (BDNF) is important signaling molecule which takes part in regulation of neurogenesis, growth and survival of neurons in central nervous system. BDNF participates not only in neuronal differentiation and in formation of synaptic contacts during neurogenesis but also can correct the metabolism of mature neurons. According data from recent studies BDNF has strong neuroprotective properties, depresses cell apoptosis, stimulates growth and prevent neuronal death. In rehabilitation period after injuries, ischemic and neurodegenerative diseases it is important to stimulate endogenic reparation of functional neuronal nets. One of the approaches may be therapeutically rising of the BDNF level.
Recombinant adeno-associated virus is one of the most promising delivery vectors for gene therapy due to its nonpathogenic property, nonimmunogenecity to host and broad cell and tissue tropisms.
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