The Role Of Jacob In Synapto-nuclear Communication Of Activity-dependent Synaptic Plasticity

Part I The role of Jacob protein overexpression in the synapto-nuclear communicationBackground:It is generally believed that synapse-to-nucleus communication plays an important role in long-term memory formation. However, the control of activity-dependent gene expression by synaptic signals is still far from being understood. In recent years several potential synapto-nuclear protein messengers have been characterized that are thought to be involved in regulation of gene expression after induction of activity-dependent related synaptic plasticity, and that have the activity-dependent nuclear import. However, there is a surprising paucity of demonstrations visualizing the nuclear import of such proteins in response to activity-dependent synaptic plasticity. Only recently it was found that the transcription factor cyclic AMP response element binding protein2(CREB2) transits to the nucleus during long-term depression (LTD), but not during long-term potentiation (LTP) of synaptic transmission in hippocampal primary neurons. Jacob is another messenger that couples NMDA-receptor-activity to nuclear gene expression. We therefore aimed to study whether Jacob accumulates in the nucleus in physiological relevant models of activity-dependent synaptic plasticity.Methodology/Principal Findings:We have analyzed the dynamics of Jacob’s nuclear import following induction of NMDA-receptor dependent LTP or LTD at Schaffer collateral-CAl synapses in hippocampal slices. Using time-lapse imaging of neurons expressing a Jacob-Green-Fluorescent-Protein we found that Jacob rapidly translocates from dendrites to the nucleus already during the tetanization period of LTP, but not after induction of LTD. Immunocytochemical stainings confirmed the nuclear accumulation of endogenous Jacob in comparison to apical dendrites after induction of LTP but not LTD. Moreover, we analyzed the resulting of LTP in hippocampal slices expressing Wild-type Jacob (full length) or Delta-NLS Jacob (no nuclear translocation signal). Using Semliki Forest Virus (SFV) based transduction of hippocampal CA1neurons we observed that only Delta-NLS Jacob overexpression enhances fEPSP-slope potentiation, and the effect of overexpressed Delta-NLS Jacob on LTP is mediated through NR2B receptors, and the overexpression of wild type Jacob does not alter LTP. In addition, we analyzed the data of western blot for the nuclear protein in CA1neurons from wistar rat hippocampal slices after induction electrical LTP. Western blot showed that Jacob protein was phosphorylated in nucleus during LTP maintainance but not immediately occurred after LTP induced.Conclusions/Significance:Taken together, our findings suggest that the two major forms of NMDA-receptor dependent synaptic plasticity, LTP and LTD, elicit a nuclear transition of Jacob differently and NR2B-receptor activation is required during LTP induction. And the finding that overexpression of wild type Jacob does not alter LTP, might point towards its efficiency to interfere with plasticity related processes at endogenous Jacob protein levels sufficiently. These experiments point towards that Jacob is involved in mechanisms underlying the formation of activity-dependent synaptic plasticity in hippocampus. Part II Functional analysis of Jacob knockout or Caldendrin knockout in activity-dependent synaptic plasticityBackground:NMDA (N-methyl-D-aspartate) receptors and calcium can play multiple and divergent effects in neurons, therefore impacting on synaptic plasticity, neuronal degeneration, cell death and so on. Caldendrin is a neuronal Ca2+sensor and a postsynaptic density component with high similarity to calmodulin. And Jacob is a recently identified caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Caldendrin binds to Jacob’s nuclear localization signal in a Ca2+-dependent manner. Strictly depending upon activation of NMDA-receptors, Jacob is recruited to neuronal nuclei.Methodology/Principal Findings:Our previous work showed that Jacob translocates into the nucleus after1) NMDA-receptor activation and2) induction of synaptic plasticity by electrical stimulation of Schaffer-collateral CA1synapses, but the question remained, whether this described activity-dependent translocation is a requirement for the potentiation of synaptic transmission. To answer the question, acute hippocampal slices from a knock out mice which do not express Jacob or Caldendrin were analyzed and tested for synaptic plasticity as well as basal hippocampal synaptic transmission. Long-term potentiation (LTP) and long-term depression (LTD) are two major forms of NMDA-receptor dependent synaptic plasticity. Using CRE+Flox Jacob mice strain (Jacob knockout) and Caldendrin knockout mice strain (CaBP1KO) we found that Jacob knockout and Caldendrin knockout have similarly impact on long-term potentiation (LTP) but differently impact on long-term depression (LTD). Jacob knockout mice are characterized by a deficit of the synaptic transmission efficiency in comparison to wild type mice.This indicated that Jacob is involved in activity-dependent synaptic potentiation. Interestingly, even if a nuclear Jacob translocation was not detectable after induction of LTD, the Jacob knockout experiments indicated an involvement of Jacob in synaptic depression. However, presynaptic vesicle release is not affected, as suggested by the non-altered paired pulse facilitation. Immunocytochemical stainings confirmed the nuclear phospholated CREB (pCREB) increased in CA1neurons of wild-type hippocampal slices and decreased in CA1neurons of Jacob knockout hippocampal slices after induction of L-LTP. In comparison to wild type mice, Caldendrin knockout mice are also characterized in LTP by a deficit of the synaptic transmission efficiency and in LTD on the contrary. This indicated that Caldendrin is involved in activity-dependent synaptic potentiation and depression.Conclusions/Significance:These experiments might point towards that no Caldendrin binding to Jacob’s nuclear localization signal even if NMDA-receptor was activated, LTP still does not keep maintainance. It gives a deeper insight about the functional role of Caldendrin-Jacob binding complexes in the expression of synaptic plasticity in hippocampus.