LARGE, an intellectual disability-associated protein, regulates AMPA-type glutamate receptor trafficking and memory.

Mutations in the human LARGE gene result in severe intellectual disability and muscular dystrophy. How LARGE mutation leads to intellectual disability, however, is unclear. In our proteomic study, LARGE was found to be a component of the AMPA-type glutamate receptor (AMPA-R) protein complex, a main player for learning and memory in the brain. Here, our functional study of LARGE showed that LARGE at the Golgi apparatus (Golgi) negatively controlled AMPA-R trafficking from the Golgi to the plasma membrane, leading to down-regulated surface and synaptic AMPA-R targeting. In LARGE knockdown mice, long-term potentiation (LTP) was occluded by synaptic AMPA-R overloading, resulting in impaired contextual fear memory. These findings indicate that the fine-tuning of AMPA-R trafficking by LARGE at the Golgi is critical for hippocampus-dependent memory in the brain. Our study thus provides insights into the pathophysiology underlying cognitive deficits in brain disorders associated with intellectual disability.

Neuronal calcium channel α1 subunit interacts with AMPA receptor, increasing its cell surface localisation.

Voltage-activated Ca2+ channels (Cav) play critical roles in excitable cells including neurons. Unlike the well-defined roles of Cav2 for pre-synaptic neurotransmission, the post-synaptic function of Cav2 is unclear. Based on our previous study demonstrating the postsynaptic association of the Cav2 with the AMPA receptor (AMPA-R), in this study we sought to further analyse the Cav2-AMPA-R association. We used a step-by-step dissociation of partially purified native Cav2-AMPA-R complexes and co-immunoprecipitation of the Cav2-AMPA-R complexes expressed in HEK293T cells to demonstrate that the main subunit of Cav, α1, formed a complex with the AMPA-R without the auxiliary subunits ß, α2δ, γ2/3. The α1 subunit increased the cell-surface localisation of the AMPA-R, which could be a post-synaptic function of the Cav2.

Proteogenomics of the human hippocampus: The road ahead.

The hippocampus is one of the most essential components of the human brain and plays an important role in learning and memory. The hippocampus has drawn great attention from scientists and clinicians due to its clinical importance in diseases such as Alzheimer's disease (AD), non-AD dementia, and epilepsy. Understanding the function of the hippocampus and related disease mechanisms requires comprehensive knowledge of the orchestration of the genome, epigenome, transcriptome, proteome, and post-translational modifications (PTMs) of proteins. The past decade has seen remarkable advances in the high-throughput sequencing techniques that are collectively called next generation sequencing (NGS). NGS enables the precise analysis of gene expression profiles in cells and tissues, allowing powerful and more feasible integration of expression data from the gene level to the protein level, even allowing "-omic" level assessment of PTMs. In addition, improved bioinformatics algorithms coupled with NGS technology are finally opening a new era for scientists to discover previously unidentified and elusive proteins. In the present review, we will focus mainly on the proteomics of the human hippocampus with an emphasis on the integrated analysis of genomics, epigenomics, transcriptomics, and proteomics. Finally, we will discuss our perspectives on the potential and future of proteomics in the field of hippocampal biology. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Spatially-resolved exploration of the mouse brain glycome by tissue glyco-capture (TGC) and nano-LC/MS.

Tissue glyco-capture (TGC), a highly sensitive MS-compatible method for extraction of glycans from tissue, was combined with structure-specific nano-LC/MS for sensitive and detailed profiling of the mouse brain glycome. Hundreds of glycan structures were directly detected by accurate mass MS and structurally elucidated by MS/MS, revealing the presence of novel glycan motifs such as antennary fucosylation, sulfation, and glucuronidation that are potentially associated with cellular signaling and adhesion. Microgram-level sensitivity enabled glycomic analysis of specific regions of the brain, as demonstrated on not only brain sections (with a one-dimensional spatial resolution of 20 µm) but also isolated brain structures (e.g., the hippocampus). Reproducibility was extraordinarily high (R > 0.98) for both method and instrumental replicates. The pairing of TGC with structure-specific nano-LC/MS was found to be an exceptionally powerful platform for qualitative and quantitative exploration of the brain glycome.

Proteomic analysis of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor complexes.

The AMPA receptor (AMPA-R) is a major excitatory neurotransmitter receptor in the brain. Identifying and characterizing the neuronal proteins interacting with AMPA-Rs have provided important information about the molecular mechanisms underlying synaptic transmission and plasticity. In this study, to identify more AMPA-R interactors in vivo, we performed proteomic analyses of AMPA-R complexes from the brain. AMPA-R complexes were isolated from the brain through various combinations of biochemical techniques for solubilization, enrichment, and immunoprecipitation. Mass spectrometry analyses of these isolated complexes identified several novel components of the AMPA-R complexes as well as some previously identified components. The identification of these novel components helps to further define the complex mechanisms involved in the regulation of AMPA receptor function and synaptic plasticity.

AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade.

AMPA receptors (AMPA-R) are major mediators of synaptic transmission and plasticity in the developing and adult central nervous system. Activity-dependent structural plasticity mediated by dynamic changes in the morphology of spines and dendrites is also essential for the formation and tuning of neuronal circuits. RhoA and Rac1 are known to play important roles in the regulation of spine and dendrite development in response to neuronal activity. These Rho GTPases are activated by guanine nucleotide exchange factors (GEFs). In this study, we identified GEF-H1/Lfc as a component of the AMPA-R complex in the brain. GEF-H1 is enriched in the postsynaptic density and is colocalized with GluR1 at spines. GEF-H1 activity negatively regulates spine density and length through a RhoA signaling cascade. In addition, AMPA-R-dependent changes in spine development are eliminated by down-regulation of GEF-H1. Altogether, these results strongly suggest that GEF-H1 is an important mediator of AMPA-R activity-dependent structural plasticity in neurons.

Cerebellar 5HT-2A receptor mediates stress-induced onset of dystonia.

Stress is a key risk factor for dystonia, a debilitating motor disorder characterized by cocontractions of muscles leading to abnormal body posture. While the serotonin (5HT) system is known to control emotional responses to stress, its role in dystonia remains unclear. Here, we reveal that 5HT neurons in the dorsal raphe nuclei (DRN) send projections to the fastigial deep cerebellar nuclei (fDCN) and that photostimulation of 5HT-fDCN induces dystonia in wild-type mice. Moreover, we report that photoinhibition of 5HT-fDCN reduces dystonia in a1A tot/tot mice, a genetic model of stress-induced dystonia, and administration of a 5HT-2A receptor inverse agonist (MDL100907; 0.1 to 1 mg/kg) or shRNA-mediated knockdown of the ht2ar gene in fDCN can notably reduce the onset of dystonia in a1A tot/tot mice. These results support the serotonin theory of dystonia and suggest strategies for alleviating symptoms in human patients by blocking 5HT-2A receptors.

Synaptic incorporation of AMPA receptors during LTP is controlled by a PKC phosphorylation site on GluR1.

Incorporation of GluR1-containing AMPA receptors into synapses is essential to several forms of neural plasticity, including long-term potentiation (LTP). Numerous signaling pathways that trigger this process have been identified, but the direct modifications of GluR1 that control its incorporation into synapses are unclear. Here, we show that phosphorylation of GluR1 by PKC at a highly conserved serine 818 residue is increased during LTP and critical for LTP expression. GluR1 is phosphorylated by PKC at this site in vitro and in vivo. In addition, acute phosphorylation at GluR1 S818 by PKC, as well as a phosphomimetic mutation, promotes GluR1 synaptic incorporation. Conversely, preventing GluR1 S818 phosphorylation reduces LTP and blocks PKC-driven synaptic incorporation of GluR1. We conclude that the phosphorylation of GluR1 S818 by PKC is a critical event in the plasticity-driven synaptic incorporation of AMPA receptors.

AMPA receptor trafficking and synaptic plasticity require SQSTM1/p62.

SQSTM1/p62 is a multidomain/scaffold for the atypical protein kinase Cs (aPKC). Phosphorylation of AMPA receptors by PKC has been shown to regulate their insertion in the postsynaptic membrane. Here, we directly tested whether p62 could interact with AMPA receptor subunits and influence their trafficking and phosphorylation. GluR1 receptor intracellular loop L2-3 and the ZZ-type zinc finger domain of p62 are essential for the interaction between these two proteins. In this context, both p62 and aPKC-mediated phosphorylation were necessary for surface delivery of the receptor. Our findings reveal that p62 is the first protein identified that interacts with a region of the GluR receptor other than the C-terminal tail. Furthermore, mice deficient in p62 displayed impaired hippocampal CA1 long-term potentiation (LTP), along with diminished surface expression of GluR1 and phosphorylation of S818. Lastly, we identify a conserved sequence (ISExSL) shared by all p62 interacting-aPKC substrates. These findings support a model where p62 interaction and aPKC phosphorylation act together to mediate AMPA receptor trafficking and long-term synaptic plasticity in the hippocampus.

Stress-induced changes in social dominance are scaled by AMPA-type glutamate receptor phosphorylation in the medial prefrontal cortex.

The establishment and maintenance of social dominance are critical for social stability and the survival and health of individual animals. Stress lead to depression and a decrease in the social status of depressed persons is a risk factor for suicide. Therefore, we explored the mechanistic and behavioral links among stress, depression, and social dominance and found that mice subjected to chronic restraint stress (CRS), an animal model of stress-induced depression, showed decreased social dominance as measured by a dominance tube test. Importantly, this submissive behavior was occluded by the antidepressant, fluoxetine, a selective serotonin reuptake inhibitor. It is known that social dominance is controlled by synaptic efficacy in the medial prefrontal cortex (mPFC) and that AMPA-type glutamate receptor (AMPA-R) is a key molecule for synaptic efficacy. We found that the phosphorylation on AMPA-R was bidirectionally changed by CRS and fluoxetine in the mPFC of mice with CRS. Moreover, we found a strong correlation between social dominance and AMPA-R phosphorylation that regulates synaptic efficacy by modulating the synaptic targeting of AMPA-R. Our correlational analysis of the behavior and biochemistry of the CRS model suggests that AMPA-R phosphorylation in the mPFC may serve as a biomarker of social dominance related to stress.

The Possible Role of Neurobeachin in Extinction of Contextual Fear Memory.

Established fear memory becomes vulnerable to disruption after memory retrieval and extinction; this labile state is critical for inhibiting the return of fear memory. However, the labile state has a very narrow time window after retrieval, and underlying molecular mechanisms are not well known. To that end, we isolated the hippocampus immediately after fear memory retrieval and performed proteomics. We identified Neurobeachin (NBEA), an autism-related regulator of synaptic protein trafficking, to be upregulated after contextual fear memory retrieval. NBEA protein expression was rapid and transient after fear memory retrieval at the synapse. Nbea mRNA was enriched at the synapses, and the rapid induction of NBEA expression was blocked by inhibition of the mammalian target of rapamycin (mTOR)-dependent signaling pathway. Mice with cornu ammonis 1 (CA1)-specific Nbea shRNA knockdown showed normal fear acquisition and contextual fear memory but impaired extinction, suggesting an important role of Nbea in fear memory extinction processes. Consistently, Nbea heterozygotes showed normal fear acquisition and fear memory recall but showed impairment in extinction. Our data suggest that NBEA is necessary either for induction of memory lability or for the physiological process of memory extinction.

SALM4 suppresses excitatory synapse development by cis-inhibiting trans-synaptic SALM3-LAR adhesion.

Synaptic adhesion molecules regulate various aspects of synapse development, function and plasticity. These functions mainly involve trans-synaptic interactions and positive regulations, whereas cis-interactions and negative regulation are less understood. Here we report that SALM4, a member of the SALM/Lrfn family of synaptic adhesion molecules, suppresses excitatory synapse development through cis inhibition of SALM3, another SALM family protein with synaptogenic activity. Salm4-mutant (Salm4(-/-)) mice show increased excitatory synapse numbers in the hippocampus. SALM4 cis-interacts with SALM3, inhibits trans-synaptic SALM3 interaction with presynaptic LAR family receptor tyrosine phosphatases and suppresses SALM3-dependent presynaptic differentiation. Importantly, deletion of Salm3 in Salm4(-/-) mice (Salm3(-/-); Salm4(-/-)) normalizes the increased excitatory synapse number. These results suggest that SALM4 negatively regulates excitatory synapses via cis inhibition of the trans-synaptic SALM3-LAR adhesion.

Long-term regulation of voltage-gated Ca(2+) channels by gabapentin.

Gabapentin (GBP) is a gamma-aminobutyric acid analog effective in the treatment of seizures. A high-affinity interaction between GBP and the alpha(2)delta subunit of the voltage-gated Ca(2+) channels has been documented. In this report, we examined the effects of the chronic treatment with GBP on neuronal recombinant P/Q-type Ca(2+) channels expressed in Xenopus oocytes. GBP did not affect significantly the amplitude or the voltage dependence of the currents. Exposure to the drug did, however, slow down the kinetics of inactivation in a dose-dependent fashion. In addition, biochemical analysis showed that the integrity of Ca(2+) channel complex is not apparently affected by GBP binding, suggesting that chronic treatment with the drug might cause the channel kinetic modification through subtle conformational changes of the protein complex.

Reactive oxygen species (ROS) modulate AMPA receptor phosphorylation and cell-surface localization in concert with pain-related behavior.

Sensitization of dorsal horn neurons (DHNs) in the spinal cord is dependent on pain-related synaptic plasticity and causes persistent pain. The DHN sensitization is mediated by a signal transduction pathway initiated by the activation of N-methyl-d-aspartate receptors (NMDA-Rs). Recent studies have shown that elevated levels of reactive oxygen species (ROS) and phosphorylation-dependent trafficking of GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) are a part of the signaling pathway for DHN sensitization. However, the relationship between ROS and AMPA-R phosphorylation and trafficking is not known. Thus, this study investigated the effects of ROS scavengers on the phosphorylation and cell-surface localization of GluA1 and GluA2. Intrathecal NMDA- and intradermal capsaicin-induced hyperalgesic mice were used for this study since both pain models share the NMDA-R activation-dependent DHN sensitization in the spinal cord. Our behavioral, biochemical, and immunohistochemical analyses demonstrated that: 1) NMDA-R activation in vivo increased the phosphorylation of AMPA-Rs at GluA1 (S818, S831, and S845) and GluA2 (S880) subunits; 2) NMDA-R activation in vivo increased cell-surface localization of GluA1 but decreased that of GluA2; and 3) reduction of ROS levels by ROS scavengers PBN (N-tert-butyl-α-phenylnitrone) or TEMPOL (4-hydroxy-2, 2, 6, 6-tetramethylpiperidin-1-oxyl) reversed these changes in AMPA-Rs, as well as pain-related behavior. Given that AMPA-R trafficking to the cell surface and synapse is regulated by NMDA-R activation-dependent phosphorylation of GluA1 and GluA2, our study suggests that the ROS-dependent changes in the phosphorylation and cell-surface localization of AMPA-Rs are necessary for DHN sensitization and thus, pain-related behavior. We further suggest that ROS reduction will ameliorate these molecular changes and pain.

Emotion enhances learning via norepinephrine regulation of AMPA-receptor trafficking.

Emotion enhances our ability to form vivid memories of even trivial events. Norepinephrine (NE), a neuromodulator released during emotional arousal, plays a central role in the emotional regulation of memory. However, the underlying molecular mechanism remains elusive. Toward this aim, we have examined the role of NE in contextual memory formation and in the synaptic delivery of GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors during long-term potentiation (LTP), a candidate synaptic mechanism for learning. We found that NE, as well as emotional stress, induces phosphorylation of GluR1 at sites critical for its synaptic delivery. Phosphorylation at these sites is necessary and sufficient to lower the threshold for GluR1 synaptic incorporation during LTP. In behavioral experiments, NE can lower the threshold for memory formation in wild-type mice but not in mice carrying mutations in the GluR1 phosphorylation sites. Our results indicate that NE-driven phosphorylation of GluR1 facilitates the synaptic delivery of GluR1-containing AMPARs, lowering the threshold for LTP, thereby providing a molecular mechanism for how emotion enhances learning and memory.

Synapse-specific regulation of AMPA receptor function by PSD-95.

PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of "silent" synapses i.e., synapses containing N-methyl-d-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDAR-mediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapse-specific role for PSD-95 in controlling synaptic function that is independent of spine morphology.

A functional AMPA receptor-calcium channel complex in the postsynaptic membrane.

Ca(2+) channels play critical roles in the regulation of synaptic activity. In contrast to the well established function of voltage-activated Ca(2+) channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the Ca(2+) channels in the postsynaptic membrane. In this study, we demonstrated the functional association of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal Ca(2+) channels. A series of biochemical studies showed the specific association of Ca(v)2.1 (alpha(1A)-class) and Ca(v)2.2 (alpha(1B)-class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and Ca(2+) imaging analyses of recombinant Ca(v)2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of Ca(2+) ([Ca(2+)](i)) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of Ca(2+) channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity.

The voltage-dependent calcium channel beta subunit contains two stable interacting domains.

Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.

Phenotypic heterogeneity in the stargazin allelic series.

The stargazer mutant mouse is characterized by its ataxic gait, head tossing, and absence seizures. The mutation was identified in the gamma 2 subunit gene of the high voltage-dependent calcium channel, Cacng2. Subsequently, two allelic variants of stargazer have arisen, waggler and stargazer 3J. In this study, we have compared these new alleles to the original stargazer allele. All three mutations affect the Cacng2 mRNA levels as they all arise from disruptions within the introns of this gene. Our results show that the mutations cause reduced Cacng2 mRNA and protein levels. Stargazer and waggler mice have the least amount of mRNA and undetectable protein, whereas stargazer 3J appears to be the mildest allele, both in terms of the phenotype and protein expression. Electroencephalographic (EEG) analysis confirmed that stargazer has frequent spike-wave discharges (SWDs); the average duration of each discharge burst is 5 seconds and recurs every minute. The waggler allele causes a greater variation in SWD activity depending on the individual mouse, and the stargazer 3J mouse has no SWDs. The preliminary characterization of this heterogeneous allelic series provides a basis to explore more biochemical and physiological parameters relating to the role of the Cacng2 product in calcium channel activity, AMPA receptor localization, and cerebellar disturbances.

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit.

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.