Glutamate is the major excitatory neurotransmitter in the mammalian CNS and mediates fast synaptic transmission upon activation of glutamate-gated ion channels. In addition, glutamate modulates a ...variety of other synaptic responses and intracellular signaling by activating metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors. The mGluRs are also expressed in nonneuronal tissues and are implicated in a variety of normal biological functions as well as diseases. To study mGluR-activated calcium signaling in neurons, we generated mGluR5 transgenic animals using a Thy1 promoter to drive expression in the forebrain, and one founder unexpectedly developed melanoma. To directly investigate the role of mGluR5 in melanoma formation, we generated mGluR5 transgenic lines under a melanocyte-specific promoter, tyrosinase-related protein 1. A majority of the founders showed a severe phenotype with early onset. Hyperpigmentation of the pinnae and tail could be detected as early as 3–5 d after birth for most of the mGluR5 transgene-positive mice. There was 100% penetrance in the progeny from the tyrosinase-related protein 1-mGluR5 lines generated from founders that developed melanoma. Expression of mGluR5 was detected in melanoma samples by RT-PCR, immunoblotting, and immunohistochemistry. We evaluated the expression of several cancer-related proteins in tumor samples and observed a dramatic increase in the phosphorylation of ERK, implicating ERK as a downstream effector of mGluR5 signaling in tumors. Our findings show that mGluR5-mediated glutamatergic signaling can trigger melanoma in vivo. The aggressive growth and severe phenotype make these mouse lines unique and a potentially powerful tool for therapeutic studies.
PSD-95 is a scaffolding protein that regulates the synaptic localization of many receptors, channels, and signaling proteins. The NLGN gene family encodes single-pass transmembrane postsynaptic cell ...adhesion molecules that are important for synapse assembly and function. At excitatory synapses, NLGN1 mediates transsynaptic binding with neurexin, a presynaptic cell adhesion molecule, and also binds to PSD-95, although the relevance of the PSD-95 interaction is not clear. We now show that disruption of the NLGN1 and PSD-95 interaction decreases surface expression of NLGN1 in cultured neurons. Furthermore, PKA phosphorylates NLGN1 on S839, near the PDZ ligand, and dynamically regulates PSD-95 binding. A phosphomimetic mutation of NLGN1 S839 significantly reduced PSD-95 binding. Impaired NLGN1/PSD-95 binding diminished synaptic NLGN1 expression and NLGN1-mediated synaptic enhancement. Our results establish a phosphorylation-dependent molecular mechanism that regulates NLGN1 and PSD-95 binding and provides insights into excitatory synaptic development and function.
Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of ...neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Autism spectrum disorder (ASD) is more prevalent in males; however, the etiology for this sex bias is not well understood. Many mutations on X-linked cell adhesion molecule NLGN4X result in ASD or ...intellectual disability. NLGN4X is part of an X-Y pair, with NLGN4Y sharing ∼97% sequence homology. Using biochemistry, electrophysiology, and imaging, we show that NLGN4Y displays severe deficits in maturation, surface expression, and synaptogenesis regulated by one amino acid difference with NLGN4X. Furthermore, we identify a cluster of ASD-associated mutations surrounding the critical amino acid in NLGN4X, and these mutations phenocopy NLGN4Y. We show that NLGN4Y cannot compensate for the functional deficits observed in ASD-associated NLGN4X mutations. Altogether, our data reveal a potential pathogenic mechanism for male bias in NLGN4X-associated ASD.
•Despite sharing ∼97% amino acid identity, NLGN4X and NLGN4Y are differentially regulated•NLGN4Y cannot traffic to the surface due to one amino difference from NLGN4X•A cluster of autism-associated variants in NLGN4X surrounds the critical amino acid•NLGN4X autism-associated variants display a deficit in trafficking similar to NLGN4Y
NLGN4X and NLGN4Y are thought to have similar functions due to their ∼97% shared amino acid identity. Nguyen et al. demonstrate NLGN4Y has a deficit in trafficking due to one amino acid difference. The trafficking deficit phenotype is also found in a cluster of autism-associated variants in NLGN4X near the critical amino acid.
NMDA receptors (NMDARs) are ionotropic glutamate receptors that are crucial for neuronal development and higher cognitive processes. NMDAR dysfunction is involved in a variety of neurological and ...psychiatric diseases; however, the mechanistic link between the human pathology and NMDAR dysfunction is poorly understood. Rare missense variants within NMDAR subunits have been identified in numerous patients with mental or neurological disorders. We specifically focused on the GluN2B NMDAR subunit, which is highly expressed in the hippocampus and cortex throughout development. We analyzed several variants located in the GluN2B C terminus and found that three variants in patients with autism (S1415L) or schizophrenia (L1424F and S1452F) (S1413L, L1422F, and S1450F in rodents, respectively) displayed impaired binding to membrane-associated guanylate kinase (MAGUK) proteins. In addition, we observed a deficit in surface expression for GluN2B S1413L. Furthermore, there were fewer dendritic spines in GluN2B S1413L-expressing neurons. Importantly, synaptic NMDAR currents in neurons transfected with GluN2B S1413L in GluN2A/B-deficient mouse brain slices revealed only partial rescue of synaptic current amplitude. Functional properties of GluN2B S1413L in recombinant systems revealed no change in receptor properties, consistent with synaptic defects being the result of reduced trafficking and targeting of GluN2B S1413L to the synapse. Therefore, we find that GluN2B S1413L displays deficits in NMDAR trafficking, synaptic currents, and spine density, raising the possibility that this mutation may contribute to the phenotype in this autism patient. More broadly, our research demonstrates that the targeted study of certain residues in NMDARs based on rare variants identified in patients is a powerful approach to studying receptor function.
We have used a "bedside-to-bench" approach to investigate the functional regulation of NMDA receptors (NMDARs). Using information from deep sequencing of patients with neurological or psychiatric disorders, we investigated missense variants identified in the intracellular C-terminal domain of the GluN2B NMDAR subunit. We found several variants that displayed altered properties. In particular, one variant identified in a patient with autism, human GluN2B S1415L, displayed reduced surface expression and binding to PSD-95. Furthermore expression of GluN2B S1415L (S1413L in mouse) showed a deficit in rescue of synaptic NMDAR currents and fewer dendritic spines, consistent with other reports of spine abnormalities being associated with autism. More broadly, we demonstrate that using patient data is an effective approach to probing the structure/function relationship of NMDARs.
Rare variants in GRIN genes, which encode NMDAR subunits, are strongly associated with neurodevelopmental disorders. Among these, GRIN2A, which encodes the GluN2A subunit of NMDARs, is widely ...accepted as an epilepsy-causative gene. Here, we functionally characterize the de novo GluN2A-S1459G mutation identified in an epilepsy patient. We show that S1459 is a CaMKIIα phosphorylation site, and that endogenous phosphorylation is regulated during development and in response to synaptic activity in a dark rearing model. GluN2A-S1459 phosphorylation results in preferential binding of NMDARs to SNX27 and a corresponding decrease in PSD-95 binding, which consequently regulates NMDAR trafficking. Furthermore, the epilepsy-associated GluN2A-S1459G variant displays defects in interactions with both SNX27 and PSD-95, resulting in trafficking deficits, reduced spine density, and decreased excitatory synaptic transmission. These data demonstrate a role for CaMKIIα phosphorylation of GluN2A in receptor targeting and implicate NMDAR trafficking defects as a link to epilepsy.
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•The epilepsy-associated variant GluN2A-S1459G causes NMDAR trafficking defects•GluN2A-S1459G disrupts NMDAR interactions and leads to reduced mEPSC frequency•GluN2A is phosphorylated by CaMKII at S1459, which is regulated by neuronal activity•GluN2A-S1459 phosphorylation dictates binding to PDZ domain-containing proteins
Mota Vieira et al. identify CaMKII phosphorylation of the GluN2A subunit on S1459 as a mechanism regulating NMDAR trafficking. An epilepsy-associated rare variant at this same residue, GluN2A-S1459G, results in altered protein interactions, decreased NMDAR surface expression, and reduced synaptic function, providing potential insight into an epilepsy phenotype.
The RhoGEFs Kalirin-7 and Trio are regulators of synaptic plasticity, and their dysregulation is associated with a range of neurodevelopmental and neurodegenerative disorders. Although studies have ...implicated both Kalirin and Trio in certain diseases, such as tauopathies, they remarkably differ in their association with other disorders. Using unbiased proteomics, we identified interactomes of Kalirin-7 and Trio to ascertain distinct protein association networks associated with their respective function and revealed groups of proteins that preferentially interact with a particular RhoGEF. In comparison, we find Trio interacts with a range of axon guidance and presynaptic complexes, whereas Kalirin-7 associates with several synaptic adhesion molecules. Specifically, we show Kalirin-7 is an interactor of the cell adhesion molecule neuroligin-1 (NLGN1), and NLGN1-dependent synaptic function is mediated through Kalirin-7 in an interaction-dependent manner. Our data reveal not only the interactomes of two important disease-related proteins, but also provide an intracellular effector of NLGN1 function.
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•Kalirin-7 and Trio have discrete and coinciding protein-protein interaction networks•Kalirin-7 strongly associates with the synaptic adhesion molecule NLGN1•NLGN1 gain of function requires RhoGEF signaling•Binding of Kalirin-7 to NLGN1 is required to support NLGN1 function
Paskus et al. use quantitative proteomics to determine the synaptic interactomes of the disease-associated proteins Kalirin-7 and Trio, identifying Kalirin-7 as an interactor of NLGN1. Investigation of this interaction unveils Kalirin-7 as a primary intracellular effector of NLGN1 gain of function.
NR2C-containing N-methyl-d-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C ...exhibit a low conductance and reduced sensitivity to Mg2+, compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser1244), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser1244 on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser1244 does not influence this PDZ interaction. Furthermore, Ser1244 phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser1244 accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser1244 does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser1244 on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.
Neurolastin is a dynamin family GTPase that also contains a RING domain and exhibits both GTPase and E3 ligase activities. It is specifically expressed in the brain and is important for synaptic ...transmission, as neurolastin knockout animals have fewer dendritic spines and exhibit a reduction in functional synapses. Our initial study of neurolastin revealed that it is membrane-associated and partially co-localizes with endosomes. Using various biochemical and cell-culture approaches, we now show that neurolastin also localizes to mitochondria in HeLa cells, cultured neurons, and brain tissue. We found that the mitochondrial localization of neurolastin depends upon an N-terminal mitochondrial targeting sequence and that neurolastin is imported into the mitochondrial intermembrane space. Although neurolastin was only partially mitochondrially localized at steady state, it displayed increased translocation to mitochondria in response to neuronal stress and mitochondrial fragmentation. Interestingly, inactivation or deletion of neurolastin's RING domain also increased its mitochondrial localization. Using EM, we observed that neurolastin knockout animals have smaller but more numerous mitochondria in cerebellar Purkinje neurons, indicating that neurolastin regulates mitochondrial morphology. We conclude that the brain-specific dynamin GTPase neurolastin exhibits stress-responsive localization to mitochondria and is required for proper mitochondrial morphology.
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