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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
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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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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain Aims
The main goal of this research is to create adenoassociated viral vector for expressing of neurotrophic factor BDNF in neuronal cells. For this study we created the adenoassotiated viral vector with fragment of BDNF gene and EGFP and infected transgenic cell line HEK293T and primary neuronal culture to estimate the viral expression.
Methods
We used standard cloning techniques to create adenoassotiated viral vector with fragment of BDNF gene. We extracted mouse total RNA, made PCR with reverse transcription and amplify the cloning fragment of BDNF gene (900 base). Then we cloned the BDNF fragment into AAV-Syn-kid2 plasmid containing strong human synapsin promoter and Woodchuck hepatitis posttranscriptional regulatory element which enhances the synapsin promoter.
Afterwards we contransfected with this plasmid and helper plasmids the transgenic HEK293T cell culture and collected the virus. We had two different AAV packaging system - DJ and pDP5. Next we cleaned the virus using benzonase treatment and additionally cleaned and concentrated the virus on Amicon Ultra columns to achieve necessary purity of virus so far as the purity of viral sample is important for primary neuronal cultures. The last step was the infection of primary neuronal culture and transgenic HEK293T cell culture and estimating the viral expression using confocal microscopy. Primary neuronal culture was received from E18 mouse.
Results
Adenoasstiated virus containing fragment of BDNF gene has strong expression in HEK293T cell culture and primary neuronal culture. The expression of adenoassotiated virus is more efficient in case of using DJ AAV packaging system. Strong expression appeared on 3-4 day in case of HEK293T cell culture infection and on 5-7 day in case of primary neuronal culture infection. The expression was proved by confocal microscopy, immunocytochemistry and PCR methods.
Conclusions
Thereby it was created the adenoassotiated viral vector containing the fragment of BDNF gene and it was tested on primary neurons culture.
Acknowledgements
The research was supported by the Federal Target Program "Research and development in priority areas of the development of the scientific and technological complex of Russia for 2014-2020" of the Ministry of Education and Science of Russia, contract 14.581.21.0016 (Project ID RFMEFI58115X0016).
Effect of Epileptiform Activity on Hippocampal Astrocytes
A. Lebedeva1 *, O. Tyurikova1,2, A. Plata1, V. Tovpyga1, P. Denisov1, S. Makovkin1,I. Ivlev1, A. Semyanov1
1 Institute of Neuroscience, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia;
2 Department of Clinical and Experimental Epilepsy, University College London, London, UK. * Presenting e-mail: lebedeva@neuro.nnov.ru
Over decades, epilepsy has been considered a neurogenerative disease mainly manifested by abnormal neuronal firing. However, some recent findings suggest that electrically passive astrocytes strongly contribute to neural function in health and disease, owing to the release of a variety of signalling molecules (such as glutamate, ATP, D-serine) that target receptors in both neuronal and non-neuronal cells. Astrocytes also participate in neuronal signalling trough high-affinity uptake of neurotransmitters and spatial buffering of extracellular K+. Glutamate transporters deal with up to 80% of the released glutamate, as well as exchanging also Na+ H+ and K+. However, during strong neuronal activity, extracellular K+ concentration can significantly increase from 2.5 mM to 10-12 mM. In pathological condition, such as epileptiform activity, this concentration can even rise up to 30mM [Verkhratsky, Butt, 2007]. According to the kinetics of glutamate transporter, increase of extracellular K+ could interfere with local glutamate uptake. How synaptic activity is affected by epilepsy remains poorly understood. We investigated astrocytic activity in rat slices from control animals and lithium-pilocarpine epilepsy model animals. Ca2+ dynamics was monitored in astrocytes loaded with sulforhodamine 101 (200nM), a specific astrocytic marker, and Oregon Green BAPTA AM (7.95 |oM), a Ca2+ sensor. Transporter and K+ currents in astrocytes were measured with patch pipette in response to electric stimulation of Schaffer collaterals in baseline conditions and subsequently after pharmacological blockade of the excitatory amino acid transporters (EAATs) with TBOA (50 |oM). The present study demonstrates changes in astrocytes associated with epileptogenesis.
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