Katherine Roche - US grants

The group I mGluRs (mGluR1 and mGluR5) are predominantly postsynaptic mGluRs that are coupled to phospholipase C, release of intracellular Ca2+, and activation of a variety of intracellular signaling molecules. PKC phosphorylation of mGluR5 has been known to affect Ca2+ signaling and receptor desensitization for some time, but rigorous studies on the direct phosphorylation of mGluR5 havent been conducted. Therefore, we are investigating the phosphorylation of mGluR5 and have identified several specific residues that are phosphorylated by protein kinase C. These PKC sites are located within the proximal one-third of the mGluR5 C-terminal domain. One phosphorylation site, Ser839, determines the regulation of intracellular calcium oscillations in response to mGluR5 activation. A second phosphorylation site that we have identified, Ser901, inhibits the binding of the protein calmodulin to mGluR5 and decreases mGluR5 surface expression. In parallel studies on the presynaptic receptor, mGluR7, we find that PKC phosphorylation of Ser862 inhibits calmodulin binding, and increases receptor surface expression. Therefore, we find a common mechanism to rapidly modify mGluR surface expression in response to increases in Ca2+, PKC activity, and changes in calmodulin binding;however, the changes in surface expression depend on receptor subtype and likely additional receptor subtype-specific binding proteins that are disrupted by calmodulin binding. These studies precisely defining the phosphorylation of mGluRs by PKC will allow us to study the functional consequences of glutamate receptor phosphorylation and the regulation of intracellular signaling and receptor trafficking.

The unique distribution of neurotransmitter receptors and their subtypes within a single cell and throughout the brain requires highly selective intracellular targeting mechanisms. My laboratory studies the regulation of glutamate receptor trafficking and localization using a combination of biochemical and molecular techniques. Glutamate receptors are the major excitatory neurotransmitter receptors in the mammalian brain and are a diverse family with many different subtypes. The ionotropic glutamate receptors include AMPA, NMDA, and kainate receptor subtypes, each of which are formed from a variety of subunits. The metabotropic glutamate receptors (mGluR1-8) are G protein-coupled receptors (GPCRs), which are assembled as homodimers. We focus on defining subunit-specific mechanisms that regulate the synaptic localization and functional regulation of glutamate receptors as well as synaptic scaffolding proteins. These mechanisms include posttranslational modifications such as phosphorylation and ubiquitination, as well as protein-protein interactions. A major focus of the lab is the study of the molecular mechanisms regulating the trafficking of NMDA receptors, which are multi-subunit complexes (GluN1; GluN2A-D; GluN3A-B). We have made significant progress in the detailed characterization of the synaptic expression of NMDARs and the role of GluN2A and GluN2B in receptor trafficking and synaptic expression. NMDA receptors are removed from synapses in an activity- and calcium-dependent manner, via casein kinase 2 (CK2) phosphorylation of the PDZ-ligand of the GluN2B subunit (S1480). We find that the NR2B subunit, and not NR2A, is specifically phosphorylated by CK2 and phosphorylation of NR2B increases in the second postnatal week and is important in the subunit switch (GluN2B to GluN2A), which takes place in many cortical regions during development and in response to activity. These data support unique contributions of the individual NMDA receptor subunits to NMDA receptor trafficking and localization. Our studies have shown that a single point mutation in the GluN2B C-terminus (E1479Q) totally blocks CK2 phosphorylation of S1480 and results in significant increases in synaptic GluN2B. We are currently generating a line of genetically-altered mice: a knock-in mouse expressing a point-mutated non-phosphorylatable GluN2B subunit (GluN2B E1479Q). This knock-in mouse will allow us to examine the precise regulation of GluN2B S1480 phosphorylation in neurons, in vivo, and without the requirement of exogenous protein overexpression. It is anticipated that these animals would show an impaired developmental GluN2 subunit switch (Sanz-Clemente et al, 2010). In addition, we are generating a knock-in mouse, GluN2B S1480E, which mimics phosphorylation of that key residue and constitutively blocks binding to PSD-95. Our experiments have shown us this mutation results in very low surface and synaptic expression of GluN2B receptors, thus we expect the mouse to have synaptic deficits as well. We are also exploring the role of tyrosine kinases and phosphatases on the regulation of synaptic NMDARs. GluN2B contains a classic tyrosine-based endocytic motif (-YEKL) that is a strong regulator of NMDAR surface expression. Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) target Y1472, which affects endocytosis and synaptic expression of receptors. In particular, STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs via direct binding to the C-terminal PDZ ligand (-ESDV). We have discovered that STEP61 binds to PSD-95 but not to other PSD-95 family members. In addition, PSD-95 expression triggers the degradation of STEP61 via ubiquitination and degradation by the proteasome. Surprisingly, we found that STEP61 is not enriched in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95-KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. An important consequence of STEP having low abundance at the PSD is that only extrasynaptic NMDAR expression and currents were increased upon STEP knock-down. Therefore, our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61. More recently, we have characterized the effect of STEP expression on AMPARs. We find that STEP regulates the synaptic expression of GluA2 and GluA3 subunits of AMPARs and we are currently exploring the molecular underpinnings of this effect in detail. Our data support a model in which STEP regulates the synaptic and extrasynaptic organization of AMPA and NMDA receptors. It is intriguing that removing STEP from neurons by knock-down or knock-out strategies results in increased extrasynaptic NMDA receptors, but increased synaptic AMPA receptors. And, the effects are subunit-specific. Over the last few years, we have focused on a new approach to studying structure/function of NMDARs. We are trying a bedside-to-bench approach to help guide us in testing receptor domains that are important for synaptic function. In particular, we used information from published papers and public databases that report variants identified by deep sequencing of patients with neurological or psychiatric disorders. We then began conducting experiments on missense variants identified in the intracellular C-terminal domain of the GluN2B NMDAR subunit. We found that one mutation in particular, identified in a patient with autism, reduced the surface expression of GluN2B as well as the binding to PSD-95. This variant, GluN2B S1415L (S1413L in mouse), showed a deficit in rescue of synaptic NMDAR currents and fewer dendritic spines. This phenotype is interesting because there are many examples in the literature of spine abnormalities being associated with autism. More broadly, this research shows that using patient data is an effective approach to probing the structure/function relationship of NMDARs. We have now generated a knock-in mouse of GluN2B S1413L and we are characterizing its phenotype. Our biochemical studies already reveal a region-specific effect, with hippocampus showing deficits in synaptic proteins. We are conducting behavioral analyses as well. Ubiquitination is a post-translational modification that dynamically regulates the synaptic expression of many proteins. Years ago, we performed a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, and identified two candidates. One of these, RNF112, is a brain-specific protein that we have characterized using a variety of approaches. We find that it is a functional GTPase, as well as an E3 ligase. We named it neurolastin (RNF112/Znf179) because it is most closely related to the dynamin superfamily GTPase, atlastin. We generated a knock-out line of mice and in our initial publication, we showed that neurolastin regulates endosome size and spine density in vivo. Neurolastin requires both an intact RING and GTPase domain to maintain spine density. Interestingly, mutations in the RING domain result in mistargeting of neurolastin from a primarily endosomal to primarily mitochondrial localization. We have now identified a mitochondrial targeting sequence on neurolastin. Furthermore, we have analyzed knock-out animals using EM and find mitochondrial defects.

Neuroligins (NLGNs) are brain-specific cell adhesion molecules. They are expressed on the postsynaptic membrane and bind to presynaptic neurexins (NRXNs) spanning the synaptic cleft. Interestingly, mutations in both NLGNs and NRXNs have been identified in Autism Spectrum Disorder (ASD) patients. This has led researchers to develop genetically engineered NLGN mouse models to study the etiology of ASDs. These studies have shown that NLGN dysfunction can shift the balance of inhibition and excitation in the brain. NLGN isoforms are highly conserved, yet display distinct synaptic localizations. However, the molecular mechanisms that regulate isoform-specific targeting and localization are not well understood. We focus on the role of protein-protein interactions and post-translational modifications in dictating NLGN trafficking and functional regulation. Over the last few years we have identified several different phosphorylation sites on the different neuroligin isoforms. We have been characterizing the kinases involved and the physiological relevance to synapse formation. ASDs are a group of neurodevelopmental disorders that have a high genetic predisposition and higher occurrence rates in males than females. A variety of point mutations have been identified in X-linked NLGN3 and 4X in patients with intellectual disability and symptoms characteristic of ASDs. Interestingly, all of the autism-associated point mutations in NLGN3 and NLGN4X reported thus far reside in their extracellular domains except for a single point mutation in the intracellular domain of NLGN4X at arginine (R) 704, which is modified to a cysteine (C). We discovered that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707 in human embryonic neurons. This autism mutation (R704C) eliminates T707 phosphorylation, which is critical for NLGN4X-mediated excitatory enhancement. Interestingly, unlike other NLGN ASD-associated mutations, R704C, did not disrupt the stability or surface expression of NLGN4X, yet still led to synaptic dysfunction. Mouse models of autism have uncovered a role for an imbalance of excitatory/inhibitory transmission, often resulting in direct increases in inhibitory transmission. Our results establish a potential causality between a genetic mutation, a key posttranslational modification, and robust synaptic changes and will provide insights in elucidating the pathophysiology of ASDs. In human there is also NLGN-4Y, which is located on the Y chromosome and is almost identical to NLGN-4X. In fact, NL-4X and NL-4Y have only eight amino acid differences in the extracellular domain and five in the intracellular domain. However, there are no studies on phosphorylation of NLGN-4Y. We have now compared the PKC phosphorylation of 4X vs 4Y using different kinases in conjunction with mass spectrometry, and find that NL-4X and NL-4Y are phosphorylated at different residues. Importantly, there is a difference in the levels of the phosphorylation of the PKC T707 site. These results suggest that NL-4X and NL-4Y are regulated in a fundamentally different manner, and we are following up on these findings. We believe through a better investigation of the sex-linked isoforms of NLGNs, we hope to understand the sex bias associated with ASDs. NLGN-1 and the scaffolding protein PSD-95 are both localized at excitatory synapses. In addition, NLGN-1 binds to PSD-95 through the PDZ ligand in the cytoplasmic tail. We have identified a protein kinase A (PKA) phosphorylation site on NLGN-1, near the PDZ ligand, in vitro and in heterologous cells. When we introduce a phospho-mimetic mutation at the NLGN-1 PKA site, the interaction between NLGN-1 and PSD-95 is decreased in vitro and in situ. Therefore, we find that phosphorylation regulates PSD-95 binding to NLGN-1, just as we have shown for NMDARs. We also have demonstrated that phosphorylation regulates NLGN-2, the neuroligin isoform that is located and specifically functions at inhibitory synapses. In particular, NLGN-2 is a substrate for PKA as detected using mass spectrometry. We have generated a phospho-specific antibody against this site, and demonstrate its specificity. With this great tool, we are now characterizing NLGN-2 phosphorylation and regulation in vivo. NLGN-2 phosphorylation is regulated by synaptic activity, and we are investigating the precise mechanisms by which this occurs. Finally, neuroligins undergo cleavage of their extracellular domain in response to synaptic activity. We have uncovered an isoform-specific regulation of this cleavage and are studying the mechanisms underlying its regulation. In particular, we are studying the proteases involved in neuroligin cleavage and the molecular determinants within the neuroligins.