Ⅰ Hippocampal LTP Triggers Proteasome-mediated SPAR Degradation Ⅱ Synapto-nuclear Messenger Protein Jacob Regulates Synaptic Transmission

Cognitive abilities, including learning and memory consolidation are determined by multifaceted regulation of interneuronal connectivity at the level of protein synthesis and degradation, as well as protein assembly and localization. Activity dependent synaptic plasticity, a term that refers to use-dependent changes in the efficacy or strength of synaptic transmission, plays a key role in mediating many different forms of learning and memory. The most extensively studied models of synaptic plasticity in the mammalian brain are the long-term potentiation (LTP) and long-term depression (LTD) which are generated at excitatory synapses in the hippocampus. LTP is an activity-dependent increase in synaptic efficacy that is regarded as one of the cellular mechanisms that underlie learning and memory.Many aspects of synaptic plasticity can be explained by the modification of two key processes in synaptic transmission:neurotransmitter release from the presynaptic neuron, and signal reception and transduction by the postsynaptic neuron. However, modulation of the synaptic transmission can be, at least in part, achieved by structural changes in neuronal connectivity, for example, in the structure and number of synapses but also by alternation of the balance of ionotropic or metabotropic receptors. This requires the remodulation of the postsynaptic density (PSD), with their uncounted number of proteins. Consequently, recent demonstrations that structural rearrangements accompanied synaptic plasticity have received much attention, which centered on the rearrangement of a specialized structure found at many synapses in the mammalian central nervous system:the dendritic spine.Recent research indicated that the ubiquitin-proteasome system (UPS)-mediated protein degradation is involved in the induction of long-term potentiation (LTP) of synaptic transmission in the rat hippocampus and so, in learning and memory. It is important to understand how synaptic transmission is regulated during memory formation and how activity-dependent changes of synapses are mediated at the molecular level and modulate neuronal network activity.However, it is not well understood how synaptic input specificity is achieved and whether there is any specific feedback between the nucleus and activated synapses that drive gene transcription. Thus, I studied two different aspects of activity dependent synaptic plasticity: i) the involvement of the PSD protein SPAR, which was suggested to play important roles in activity dependent remodulation of dendritic spines or PSD components and ii) the role of the putative synapto-nucelar messenger protein Jacob, for which it was speculated that it plays a role in the information transfer from activated synapses to the nucleus to modulated plasticity relevant gene expression.Thereby, the spine-associated Rap GTPase activating protein (SPAR) is a PSD protein that regulates spine morphogenesis and forms a complex with the scaffold protein PSD-95 and N-methyl-D-aspartate-type glutamate receptors. SPAR turnover depends upon the ubiquitin-proteasome pathway (UPP) since ubiquitinated SPAR accumulates in the presence of active Plk2 when proteasomes are inhibited. In the first part of the thesis, the dynamic property of spatial-temporal distribution of the PSD protein SPAR in hippocampal synaptic plasticity was characterized. The eGFP-tagged SPAR was over-expressed in acute hippocampal slices. Using confocal microscopy and electrophysiological tools to real-time monitor the tetanization induced changes of SPAR-eGFP fluorescence in apical dendrites of CA1 neurons, I found that LTP induction triggered a UPS-dependent decay of SPAR-eGFP fluorescence and this synaptic activity dependent degradation of SPAR was reduced upon inhibition of cyclin-dependent kinase 5 (CDK5) as well as by a protein synthesis inhibitor. At the same time, our results have revealed a protein synthesis-independent component of LTP-associated SPAR degradation. This second component required UPS and NMDA receptor activation but not CDK5. Thus, we conclude that the LTP triggers a down regulation of SPAR by two complementary mechanisms, one of which has previously been reported to mediate homeostatic plasticity.In the second part of the thesis, mechanisms of how Jacob modulates the synaptic transmission were addressed. Jacob is a novel PSD protein component identified as an interaction partner of the neuronal calcium sensor protein Caldendrin in rat brain. It was previously shown that after stimulation of (NMDA) receptors, Jacob translocates to the nucleus resulting in a rapid stripping of synaptic contacts and a drastically altered morphology of the dendritic tree. Using confocal microscopy and electrophysiological tools to real-time monitor the tetanization induced changes of eGFP-Jacob fluorescence in apical dendrites of CA1 neurons, I found that Jacob is translocating into the nucleus in response to induction of activity-synaptic plasticity during long-term potentiation (LTP), but not after induction of long-term depression (LTD). Immunocytochemical staining confirmed that Jacob had been accumulated in the nucleus after LTP induction but not LTD. Over-expression of Jacob constructs (△ Myr-S 180D-Jacob-GFP, △ Myr-S 180A-Jacob-GFP and △ NLS-Jacob-GFP) were tested by whole-cell voltage-clamp of mEPSCs in primary hippocampal cell culture. Our data has shown that mEPSC amplitude and frequency were increased in WT-Jacob-GFP transfected hippocampal neurons and in neurons over-expressing a phosphomimetic form of Jacob (△ Myr-S 180D-Jacob-GFP). Data indicate that synaptic transmission is controlled by the synaptic-nuclear messenger protein Jacob.Taken together, cognitive abilities, including memory consolidation are determined by complex regulatory mechanisms. Here these two proteins SPAR and Jacob are under our investigation using electrophysiological tools and imaging system.This research gives further insides how activity dependent synaptic-nuclear communication is achieved after induction of synaptic plasticity and a better understanding of the specific spatial-temporal dynamic of proteasome dependent degradation in various forms of hippocampal synaptic plasticity during memory formation..