Abstract

Synaptic Plasticity Via Receptor Tyrosine Kinase and G Protein-Coupled Receptor Crosstalk

Kim JH, Lao-Peregrin C, Xiang GQ, Srivastava I, Fall A, Gerhard D, Kohtala P, Song MS, Garcia-Marcos M, Levitz J, Lee FS
ACNP 62nd Annual Meeting. 2023.

Background: Cognition and mood regulation are higher order functions of the brain that arise from the proper regulation of neuronal circuits and synaptic activity. Dysfunctional synaptic receptors binding neurotransmitters, such as glutamate, and neurotrophic growth factors, such as brain-derived neurotrophic factor (BDNF), have been associated with aberrant synaptic plasticity and neural circuits underlying psychopathology. Abnormal plasticity mediated by the metabotropic glutamate receptor 5 (mGluR5) is driven by accentuated mGluR5-mediated long-term depression (LTD), correlated with decreased dendritic growth and increased synapse loss. This leads to disruptions in learning, planning and execution of goal-directed activities, and mood regulation. Similarly ubiquitous as mGluR5, tropomyosin receptor kinase B (TrkB), the predominant CNS neurotrophin receptor that binds BDNF, promotes long-term potentiation (LTP), increasing dendritic growth and promoting synapse stabilization. Normalizing aberrant BDNF-TrkB signaling is believed to be critical for treatment strategies for neuropsychiatric diseases. Intriguingly, growing evidence has implicated functional interactions between the glutamate system and the neurotrophin system. While evidence exists that receptor tyrosine kinases (RTKs) can be indirectly regulated by GPCRs, it is unknown whether RTKs can potentiate or attenuate GPCR signaling. Given mGluR5 and TrkB’s essential roles in synaptic plasticity, as well as their shared associations with neuropsychiatric disorders, we hypothesized that TrkB engages mGluR5 to augment a distinctive profile of intracellular signaling to mediate BDNF-induced potentiation in the hippocampus.

Methods: Using acute slice electrophysiology and primary neuronal cultures, we probe the functional and structural plasticity induced by BDNF. We primarily focus on a chemically induced form of LTP, known as BDNF-LTP, in which we perfuse recombinant BDNF protein onto an acute hippocampal slice and measure field excitatory postsynaptic potentials (fEPSP) within the CA3-CA1 synapse. We used specific pharmacological agents to inhibit and enhance activity of mGluR5 on acute hippocampal slices and primary neuronal cultures to assess BDNF-LTP and BDNF-induced spine density changes. We also injected AAV5-CamKII-mCherry-Cre into the dorsal CA1 of the hippocampus in mGluR5 FL/FL mice to mediate a region-specific genetic mGluR5 knockout to establish the necessity of TrkB/mGluR5 crosstalk to achieve LTP. We then performed a series of mutagenesis signaling studies of the two receptors in HEK 293 cells to pinpoint the mechanism of this signaling crosstalk. Finally, we use a series of G protein manipulations in acute slice electrophysiology, primary neurons, and HEK 293 cells to pinpoint the mechanism of the TrkB/mGluR5 crosstalk in modulating intracellular calcium signaling and hippocampal functional and structural synaptic plasticity.

Results: Through pharmacological (n = 10 mice/group, p < 0.001) and genetic manipulations of mGluR5 (n = 6-8/group, p < 0.01), we found that BDNF-TrkB-induced potentiation is dependent on mGluR5. This dependence of BDNF-TrkB-induced potentiation in acute hippocampal slices correlated with structural plasticity changes in spine density in primary neuronal cultures (n = 40-60 neurons/group, p < 0.001). We then show that BDNF-induced neuronal calcium response is diminished with pharmacological inhibition of mGluR5 (n = 6 neurons/group, p < 0.001) and enhanced with positive allosteric modulation of mGluR5 (n = 6 neurons/group, p. < 0.05). Similarly in HEK 293 cells, activation of TrkB by BDNF enhanced the constitutive mGluR5 activity in HEK 293 cells (n = 10-18/group, p < 0.001). When TrkB and mGluR5 are co-expressed, BDNF leads to a mode-switch to a sustained, oscillatory calcium signaling, characteristic of mGluR5-mediated calcium signaling. Positive allosteric modulation of mGluR5 also led to an enhancement in BDNF-induced oscillatory calcium signaling (n = 6/group, p < 0.001) as well as enhanced MAPK activation (n = 7 preps/group). Finally, through pharmacological and genetic manipulations in HEK 293 cells, primary neurons, and acute hippocampal slice electrophysiology, we show that this TrkB-mGluR5 crosstalk is mediated by synergy between the G-beta-gamma subunit released by TrkB and the Gq-GTP released by mGluR5. This BDNF-mediated crosstalk between these two ubiquitous synaptic proteins highlights physiologically relevant RTK/GPCR crosstalk that has been previously uncharacterized.

Conclusions: We have identified a novel mode of RTK/GPCR crosstalk, with a specific focus on understanding BDNF-induced TrkB/mGluR5 crosstalk in the hippocampus. Our findings support a new synergistic model, in which activation of both an RTK and a GPCR drives non-linear signal summation to shape the long-term consequences of receptor activation. Both mGluR5 and TrkB-mediated signaling have been shown to have broad clinical relevance. Enhanced mGluR-induced LTD as well insufficient BDNF-TrkB-induced LTP have been both shown to be underlying negative mood and cognitive processes seen across the lifespan, such as in depression, autism spectrum disorders, and Alzheimer’s disease. Our study defines this important interplay between these two systems, which is crucial to understanding mechanisms of how pharmacological agents can be designed to work in concert with one another to produce maximal therapeutic benefits.