CaMKII is one of the most studied synaptic proteins, but many critical issues regarding its role in synaptic function remain unresolved. Using a CRISPR-based system to delete CaMKII and replace it with mutated forms in single neurons, we have rigorously addressed its various synaptic roles. In brief, basal AMPAR and NMDAR synaptic transmission both require CaMKIIα, but not CaMKIIβ, indicating that, even in the adult, synaptic transmission is determined by the ongoing action of CaMKIIα. While AMPAR transmission requires kinase activity, NMDAR transmission does not, implying a scaffolding role for the CaMKII protein instead. LTP is abolished in the absence of CaMKIIα and/or CaMKIIβ and with an autophosphorylation impaired CaMKIIα (T286A). With the exception of NMDAR synaptic currents, all aspects of CaMKIIα signaling examined require binding to the NMDAR, emphasizing the essential role of this receptor as a master synaptic signaling hub.

Phosphorylation regulates surface and synaptic expression of NMDA receptors (NMDARs). Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) are known to target the NMDA receptor subunit GluN2B on tyrosine 1472, which is a critical residue that mediates NMDAR endocytosis. 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. However, nothing is known about a potential functional interaction between STEP and PSD-95. We now report that STEP 61 binds to PSD-95 but not to other PSD-95 family members. We find that PSD-95 expression destabilizes STEP 61 via ubiquitination and degradation by the proteasome. Using subcellular fractionation, we detect low amounts of STEP 61 in the PSD fraction. However, STEP 61 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 STEP 61 from the PSD. Importantly, only extrasynaptic NMDAR expression and currents were increased upon STEP knockdown, as is consistent with low STEP 61 localization in the PSD. 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 STEP 61 .PSD-95 | NMDA receptor | STEP | ubiquitination N MDA receptors (NMDARs) are ionotropic glutamate receptors that are expressed throughout the nervous system and play crucial roles in neuronal development, synaptic plasticity, and learning and memory (1-4). Functional NMDARs are heterotetrameric complexes that are composed of homologous subunits (GluN1, GluN2A-D, and GluN3A-B). Two GluN1 subunits typically combine with two GluN2 subunits, which modulate channel activity and receptor properties (5-8). GluN2A and GluN2B are the predominant GluN2 subunits in hippocampus and cortex, and the subunit composition varies during neuronal development (9)(10)(11)(12). Therefore the precise regulation of NMDAR subunit expression, composition, trafficking, and localization is critical for proper neuronal function. NMDAR activity is dynamically regulated by protein phosphorylation, e.g. NMDAR currents are potentiated by tyrosine kinases and suppressed by tyrosine phosphatases (13). In fact, GluN2B is the most prominent tyrosine phosphorylated protein within postsynaptic densities (PSDs) (14), and phosphorylation is increased during long-term potentiation (LTP) in CA1 hippocampus (15). Moreover, tyrosine phosphorylation of GluN2B has been shown to increase in several pathological conditions, including ischemia and seizures (16)(17)(18)(19).Striatal-enriched protein tyrosine phosphatase (STEP, also known as "PTPN5") is a brain-specific protein phosphatase that is expressed in the striatum, hippocampus, and cortex (20, 21). The STEP family of protein tyrosine phosphatases includes both membrane-associated [str...

SUMMARY One of the most powerful ways to test the function of a protein is to characterize the consequences of its deletion. In the past, this has involved inactivation of the gene by homologous recombination either in the germline or later through conditional deletion. RNA interference (RNAi) provides an alternative way to knock down proteins, but both of these approaches have their limitations. Recently, the CRISPR/Cas9 system has suggested another way to selectively inactivate genes. We have now tested this system in postmitotic neurons by targeting two well-characterized synaptic proteins, the obligatory GluN1 subunit of the NMDA receptor and the GluA2 subunit of the AMPA receptor. Expression of CRISPR/Cas9 in hippocampal slice cultures completely eliminated NMDA receptor and GluA2 function. CRISPR/Cas9 thus provides a powerful tool to study the function of synaptic proteins.

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific protein phosphatase that regulates a variety of synaptic proteins, including NMDA receptors (NAMDRs). To better understand STEP's effect on other receptors, we used mass spectrometry to identify the STEP 61 interactome. We identified a number of known interactors, but also ones including the GluA2 subunit of AMPA receptors (AMPARs). We show that STEP 61 binds to the C termini of GluA2 and GluA3 as well as endogenous AMPARs in hippocampus. The synaptic expression of GluA2 and GluA3 is increased in STEP-KO mouse brain, and STEP knockdown in hippocampal slices increases AMPAR-mediated synaptic currents. Interestingly, STEP 61 overexpression reduces the synaptic expression and synaptic currents of both AMPARs and NMDARs. Furthermore, STEP 61 regulation of synaptic AMPARs is mediated by lysosomal degradation. Thus, we report a comprehensive list of STEP 61 binding partners, including AMPARs, and reveal a central role for STEP 61 in differentially organizing synaptic AMPARs and NMDARs.

The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPA receptors containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca2+-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPA receptors. Electrophysiological, biochemical and quantitative electron microscopy studies revealed that sucrose training (7 days) induced a stable (>24 hr) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 hr) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7-day protocol of daily ingestion of a 3% solution of saccharin, a non-caloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multi-step GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.