KIBRA-PKCγ signaling pathway modulates memory performance in mice and humans

Human memory is a polygenic cognitive trait that is fundamental to individual competence. Genome-wide association studies (GWAS) have identified KIBRA as a novel gene associated with human memory performance. KIBRA interacts with AMPA receptors (AMPARs) and proteins essential for synaptic plasticity. The deletion of Kibra in mice impairs synaptic plasticity and learning and memory. However, the molecular basis through which KIBRA regulates dynamic AMPAR trafficking underlying synaptic plasticity is still unknown. Here we report that KIBRA interacts with the neuronal specific kinase PKCγ to modulate AMPAR trafficking upon learning, and KIBRA-PKCγ signaling pathway also associates with human memory performance. We find PKCγ is an essential kinase that phosphorylates AMPARs upon learning, and the loss of KIBRA in mouse brain impedes PKCγ-AMPAR interaction. Activation of PKCγ enables KIBRA to recruit phosphorylated AMPARs to the synapse to promote LTP and learning. We further performed transcriptomic and genetic analyses in human postmortem brain samples, and behavioral and fMRI evaluations in living human subjects, to demonstrate the genetic interactions between KIBRA and PRKCG on memory performance and memory associated physiological engagement of the hippocampal memory system. Overall, our results support that the KIBRA-PKCγ signaling pathway is crucial for modulating memory performance in mice and humans.

Molecular Characterization of AMPA-Receptor-Containing Vesicles

Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPA-containing vesicles (ACVs) from whole mouse brains via immunoisolation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography–tandem mass spectrometry (LC–MS/MS). We identified several proteins on ACVs that were previously found to play a role in AMPAR trafficking, including synaptobrevin-2, Rabs, the SM protein Munc18-1, the calcium-sensor synaptotagmin-1, as well as several new candidates, including synaptophysin and synaptogyrin on ACV membranes. Additionally, we identified two populations of ACVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ACVs, and larger transferrin- receptor-, GluA1-, GluA2-, and GluA3-containing ACVs. The small-diameter population of ACVs may represent a fusion-capable population of vesicles due to the presence of synaptobrevin-2. Because the fusion of ACVs may be a requisite of LTP, this population could represent trafficking vesicles related to LTP.

A Multisubcellular Compartment Model of AMPA Receptor Trafficking for Neuromodulation of Hebbian Synaptic Plasticity

Neuromodulation can profoundly impact the gain and polarity of postsynaptic changes in Hebbian synaptic plasticity. An emerging pattern observed in multiple central synapses is a pull–push type of control in which activation of receptors coupled to the G-protein Gs promote long-term potentiation (LTP) at the expense of long-term depression (LTD), whereas receptors coupled to Gq promote LTD at the expense of LTP. Notably, coactivation of both Gs- and Gq-coupled receptors enhances the gain of both LTP and LTD. To account for these observations, we propose a simple kinetic model in which AMPA receptors (AMPARs) are trafficked between multiple subcompartments in and around the postsynaptic spine. In the model AMPARs in the postsynaptic density compartment (PSD) are the primary contributors to synaptic conductance. During LTP induction, AMPARs are trafficked to the PSD primarily from a relatively small perisynaptic (peri-PSD) compartment. Gs-coupled receptors promote LTP by replenishing peri-PSD through increased AMPAR exocytosis from a pool of endocytic AMPAR. During LTD induction AMPARs are trafficked in the reverse direction, from the PSD to the peri-PSD compartment, and Gq-coupled receptors promote LTD by clearing the peri-PSD compartment through increased AMPAR endocytosis. We claim that the model not only captures essential features of the pull–push neuromodulation of synaptic plasticity, but it is also consistent with other actions of neuromodulators observed in slice experiments and is compatible with the current understanding of AMPAR trafficking.

Molecular characterization of AMPA receptor trafficking vesicles

Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPAR trafficking vesicles (ATVs) from whole mouse brains via immunoprecipitation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified several proteins on ATVs that were previously found to play a role in AMPAR trafficking, including SNARES (including synaptobrevin 2), Rabs, the SM protein Munc18-1, a calcium-sensor (synaptotagmin-1), as well as several new markers, including synaptophysin and synaptogyrin on ATV membranes. Additionally, we identified two populations of ATVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ATVs and larger transferrin-receptor-, GluA1-, GluA2-, GluA3-containing ATVs. The smaller population of ATVs likely represents a trafficking vesicle whose fusion is essential for LTP. These findings reveal the important role of AMPAR sorting into fusion-competent trafficking vesicles that are implicated in synaptic strength modification and reveal candidates of putative effectors and regulators of AMPAR trafficking.

RAB39B-mediated trafficking of the GluA2-AMPAR subunit controls dendritic spine maturation and intellectual disability-related behaviour

Mutations in the RAB39B gene cause X-linked intellectual disability (XLID), comorbid with autism spectrum disorders or early Parkinson’s disease. One of the functions of the neuronal small GTPase RAB39B is to drive GluA2/GluA3 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) maturation and trafficking, determining AMPAR subunit composition at glutamatergic postsynaptic neuronal terminals. Taking advantage of the Rab39b knockout murine model, we show that a lack of RAB39B affects neuronal dendritic spine refinement, prompting a more Ca2+-permeable and excitable synaptic network, which correlates with an immature spine arrangement and behavioural and cognitive alterations in adult mice. The persistence of immature circuits is triggered by increased hypermobility of the spine, which is restored by the Ca2+-permeable AMPAR antagonist NASPM. Together, these data confirm that RAB39B controls AMPAR trafficking, which in turn plays a pivotal role in neuronal dendritic spine remodelling and that targeting Ca2+-permeable AMPARs may highlight future pharmaceutical interventions for RAB39B-associated disease conditions.

VER/VEGF receptors regulate AMPA receptor surface levels and glutamatergic behavior

Several intracellular trafficking pathways contribute to the regulation of AMPA receptor (AMPAR) levels at synapses and the control of synaptic strength. While much has been learned about these intracellular trafficking pathways, a major challenge is to understand how extracellular factors, such as growth factors, neuropeptides and hormones, impinge on specific AMPAR trafficking pathways to alter synaptic function and behavior. Here, we identify the secreted ligand PVF-1 and its cognate VEGF receptor homologs, VER-1 and VER-4, as regulators of glutamate signaling in C. elegans. Loss of function mutations in ver-1, ver-4, or pvf-1, result in decreased cell surface levels of the AMPAR GLR-1 and defects in glutamatergic behavior. Rescue experiments indicate that PVF-1 is expressed and released from muscle, whereas the VERs function in GLR-1-expressing neurons to regulate surface levels of GLR-1 and glutamatergic behavior. Additionally, ver-4 is unable to rescue glutamatergic behavior in the absence of pvf-1, suggesting that VER function requires endogenous PVF-1. Inducible expression of a pvf-1 rescuing transgene suggests that PVF-1 can function in the mature nervous system to regulate GLR-1 signaling. Genetic double mutant analysis suggests that the VERs act together with the VPS-35/retromer recycling complex to promote cell surface levels of GLR-1. Our data support a genetic model whereby PVF-1/VER signaling acts with retromer to promote recycling and cell surface levels of GLR-1 to control behavior.

Subunit-dependent and independent rules of AMPA receptor trafficking during long-term depression in hippocampal neurons

ABSTRACTLong-term potentiation (LTP) and depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity-induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how, certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Here, we show that phosphorylation of the membrane-proximal region (MPR), which only occurs in GluA1 AMPAR subunits, mediates the subunit-dependent endosomal transport of AMPARs during LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit-independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD.

GRIP1 regulates synaptic plasticity and learning and memory

Hebbian plasticity is a key mechanism for higher brain functions, such as learning and memory. This form of synaptic plasticity primarily involves the regulation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) abundance and properties, whereby AMPARs are inserted into synapses during long-term potentiation (LTP) or removed during long-term depression (LTD). The molecular mechanisms underlying AMPAR trafficking remain elusive, however. Here we show that glutamate receptor interacting protein 1 (GRIP1), an AMPAR-binding protein shown to regulate the trafficking and synaptic targeting of AMPARs, is required for LTP and learning and memory. GRIP1 is recruited into synapses during LTP, and deletion of Grip1 in neurons blocks synaptic AMPAR accumulation induced by glycine-mediated depolarization. In addition, Grip1 knockout mice exhibit impaired hippocampal LTP, as well as deficits in learning and memory. Mechanistically, we find that phosphorylation of serine-880 of the GluA2 AMPAR subunit (GluA2-S880) is decreased while phosphorylation of tyrosine-876 on GluA2 (GluA2-Y876) is elevated during chemically induced LTP. This enhances the strength of the GRIP1–AMPAR association and, subsequently, the insertion of AMPARs into the postsynaptic membrane. Together, these results demonstrate an essential role of GRIP1 in regulating AMPAR trafficking during synaptic plasticity and learning and memory.

Mechanism underlying hippocampal long-term potentiation and depression based on competition between endocytosis and exocytosis of AMPA receptors

N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) and long-term depression (LTD) of signal transmission form neural circuits and thus are thought to underlie learning and memory. These mechanisms are mediated by AMPA receptor (AMPAR) trafficking in postsynaptic neurons. However, the regulatory mechanism of bidirectional plasticity at excitatory synapses remains unclear. We present a network model of AMPAR trafficking for adult hippocampal pyramidal neurons, which reproduces both LTP and LTD. We show that the induction of both LTP and LTD is regulated by the competition between exocytosis and endocytosis of AMPARs, which are mediated by the calcium-sensors synaptotagmin 1/7 (Syt1/7) and protein interacting with C-kinase 1 (PICK1), respectively. Our result indicates that recycling endosomes containing AMPAR are always ready for Syt1/7-dependent exocytosis of AMPAR at peri-synaptic/synaptic membranes. This is because molecular motor myosin Vb constitutively transports the recycling endosome toward the membrane in a Ca2+-independent manner.