Analyzing PKA-dependent synaptic plasticity in the hippocampal mossy fiber synapse using novel optogenetic tools
Final Report Abstract
Synaptic plasticity is thought to be one of the main cellular processes that underlies learning and memory. It is indicated in several synapses, most notably the hippocampal mossy-fibers (MF), that a rise in presynaptic cAMP is a crucial component in elevating neurotransmitter release during MF Long Term Potentiation (LTP). So far, cAMP-dependent synaptic processes were obtained by using pharmacological agents like Forskolin in-order to elevate cAMP levels, but their effect is not specific nor reversible. In order to overcome these obstacles, we use bPAC; a Photoactivatable Adenylyl Cyclase from the marine bacterium Beggiatoa spec., which mediates light-dependent cAMP increase. The clear advantage is its ability to express its fast and reversible activation in a cell specific manner. In order to use this novel optogenetic tool, we search for a single florescent protein (FP) cAMP sensor that will allow tracking cAMP concentrations and dynamics in neurons following bPAC activation. In this work, we showed the implementation of dynamic cAMP measurements in PC12 cells and in neurons by screening Pink Flamindo, Flamindo2, and R-FlincA, 3 different single-FP cAMP indicators, and according to their performances, we continued to work with Pink Flamindo and Flamindo2. Next, we have co-expressed Pink Flamindo and bPAC in neurons and tracked cAMP concentrations and dynamics following bPAC activation. We activated bPAC in neurons by blue light, and tracked Pink Flamindo fluorescence before, during and following bPAC activation. We calibrated bPAC activation in Cortical cells followed by different durations of blue light Polychrome stimulations, or by different blue Laser intensities stimulations, accompanied by various cAMP analog concentrations. In order to quantify Pink Flamindo’s response to each bPAC stimulation or cAMP analog application, we measured 3 characteristics of the rising phase due to each manipulation: the change in FI, the time to peak FI, and the rising phase slope using original written Python scripts. In addition, an exact correlation between cAMP dynamics to synaptic potentiation is still missing. In order to surmount this challenge, we have tried to examine cAMP dynamics following an electrical High-Frequency stimulation (HFS) protocol. We have implemented a dual-imaging setup for recording cAMP in parallel to Ca2+ imaging; neurons co-transduced with Pink Flamindo and GCaMP6s sensors, respectively. Unfortunately, we found Pink Flamindo fluorescence is probably influenced by calcium- or voltage-dependency, introducing an artifact into our measurements, and we could not conclude yet for the exact correlation. In summary, we have developed an experimental system that allow us monitoring cAMP levels in a cell specific manner, and thus for a better understanding of cAMP dynamics in MF-terminals compared to Cortical cells. In parallel, we characterized novel Photoactivated Adenylyl Cyclases (PACs), either discovered in nature or generated by genetic engineering. The engineered membrane-bound PAC – PACmn – excels with negligible AC activity in dark or red light, as well as the recently discovered soluble biPAC. In addition we characterized light-sensitive phosphodiesterases for light-controlled cAMP degradation. These novel tools will eventually be transferred to synapses for the investigation of memory and learning.
Publications
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An engineered membrane-bound guanylyl cyclase with light-switchable activity. BMC Biology, 19(1).
Tian, Yuehui; Nagel, Georg & Gao, Shiqiang
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Effect of SARS-CoV-2 proteins on vascular permeability. eLife, 10.
Rauti, Rossana; Shahoha, Meishar; Leichtmann-Bardoogo, Yael; Nasser, Rami; Paz, Eyal; Tamir, Rina; Miller, Victoria; Babich, Tal; Shaked, Kfir; Ehrlich, Avner; Ioannidis, Konstantinos; Nahmias, Yaakov; Sharan, Roded; Ashery, Uri & Maoz, Ben Meir
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Hyperbaric oxygen therapy alleviates vascular dysfunction and amyloid burden in an Alzheimer’s disease mouse model and in elderly patients. Aging, 13(17), 20935-20961.
Shapira, Ronit; Gdalyahu, Amos; Gottfried, Irit; Sasson, Efrat; Hadanny, Amir; Efrati, Shai; Blinder, Pablo & Ashery, Uri
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Hypothalamic dopamine neurons motivate mating through persistent cAMP signalling. Nature, 597(7875), 245-249.
Zhang, Stephen X.; Lutas, Andrew; Yang, Shang; Diaz, Adriana; Fluhr, Hugo; Nagel, Georg; Gao, Shiqiang & Andermann, Mark L.
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miR-128 as a Regulator of Synaptic Properties in 5xFAD Mice Hippocampal Neurons. Journal of Molecular Neuroscience, 71(12), 2593-2607.
Shvarts-Serebro, Inna; Sheinin, Anton; Gottfried, Irit; Adler, Lior; Schottlender, Nofar; Ashery, Uri & Barak, Boaz
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PACmn for improved optogenetic control of intracellular cAMP. BMC Biology, 19(1).
Yang, Shang; Constantin, Oana M.; Sachidanandan, Divya; Hofmann, Hannes; Kunz, Tobias C.; Kozjak-Pavlovic, Vera; Oertner, Thomas G.; Nagel, Georg; Kittel, Robert J.; Gee, Christine E. & Gao, Shiqiang
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Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates. Biomolecules, 12(1), 88.
Tian, Yuehui; Yang, Shang; Nagel, Georg & Gao, Shiqiang
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Hippocampal cAMP-dependent synaptic potentiation, ERK-dependent immediate-early gene activation, and context-dependent fear conditioning in mice are linked through dependence on the guanine nucleotide exchange factor RapGEF2. Cold Spring Harbor Laboratory.
Jiang, Sunny Zhihong; Shahoha, Meishar; Tejeda, Hugo A.; Ashery, Uri & Eiden, Lee E.
