Activity-dependent targeting of NMDA receptors (NMDARs) is a key feature of synapse formation and plasticity. Although mechanisms for rapid trafficking of glutamate receptors have been identified, the molecular events underlying chronic accumulation or loss of synaptic NMDARs have remained unclear. Here we demonstrate that activity controls NMDAR synaptic accumulation by regulating forward trafficking at the endoplasmic reticulum (ER). ER export is accelerated by the alternatively spliced C2' domain of the NR1 subunit and slowed by the C2 splice cassette. This mRNA splicing event at the C2/C2' site is activity dependent, with C2' variants predominating upon activity blockade and C2 variants abundant with increased activity. The switch to C2' accelerates NMDAR forward trafficking by enhancing recruitment of nascent NMDARs to ER exit sites via binding of a divaline motif within C2' to COPII coats. These results define a novel pathway underlying activity-dependent targeting of glutamate receptors, providing an unexpected mechanistic link between activity, mRNA splicing, and membrane trafficking during excitatory synapse modification.

Regulation of the abundance of NMDA receptors (NMDARs) at excitatory synapses is critical during changes in synaptic efficacy underlying learning and memory as well as during synapse formation throughout neural development. However, the molecular signals that govern NMDAR delivery, maintenance, and internalization remain unclear. In this study, we identify a conserved family of membraneproximal endocytic signals, two within the NMDAR type 1 (NR1) subunit and one within the NR2A and NR2B subunits, necessary and sufficient to drive the internalization of NMDARs. These endocytic motifs reside in the region of NMDAR subunits immediately after the fourth membrane segment, a region implicated in use-dependent rundown and NMDA channel inactivation. Although endocytosis driven by the distal C-terminal domain of NR2B is followed by rapid recycling, internalization mediated by membrane-proximal motifs selectively targets receptors to late endosomes and accelerates degradation. These results define a novel conserved signature of NMDARs regulating internalization and postendocytic trafficking.

Mutations in the PARK2 gene cause hereditary Parkinson disease (PD).The PARK2 gene product, termed parkin, is an E3 ubiquitin ligase that mediates the transfer of ubiquitin onto diverse substrate proteins. Despite progress in defining the molecular properties and substrates of parkin, little is known about its physiological function. Here, we show that parkin regulates the function and stability of excitatory glutamatergic synapses. Postsynaptic expression of parkin dampens excitatory synaptic transmission and causes a marked loss of excitatory synapses onto hippocampal neurons. Conversely, knockdown of endogenous parkin or expression of PD-linked parkin mutants profoundly enhances synaptic efficacy and triggers a proliferation of glutamatergic synapses. This proliferation is associated with increased vulnerability to synaptic excitotoxicity. Thus, parkin negatively regulates the number and strength of excitatory synapses. Increased excitatory drive produced by disruption of parkin may contribute to the pathophysiology of PD.excitotoxicity ͉ proteasome ͉ synapse ͉ glutamate P arkinson disease (PD) is the most common neurodegenerative movement disorder (1, 2). Although classic clinical symptoms arise as a result of the loss of dopaminergic neurons of the substantia nigra, widespread neurological abnormalities are present in animal models of PD and in human disease (3-6). Heightened responsiveness to the excitatory neurotransmitter glutamate and associated excitotoxicity has been implicated in the pathogenesis of PD (7-9). However, the molecular mechanisms linking PD risk factors to altered excitability and excitotoxic vulnerability remain unclear.Mutations responsible for rare hereditary forms of PD have been identified in several human genes (10, 11). Among these, PARK2 encodes a RING domain-containing E3 ubiquitin ligase that is widely expressed throughout the nervous system but whose cellular function is poorly understood (1,(12)(13)(14)(15)(16)(17). Among the reported substrates of parkin are several proteins implicated in synaptic transmission, including CDCrel-1 (15), glycosylated ␣-synuclein, synphilin, synaptotagmin XI (18), Eps15 (19), and protein interacting with C kinase 1 (20). Parkin associates with PDZ scaffold proteins in the postsynaptic density (PSD) (21) and protects postmitotic neurons from glutamate receptor-mediated excitotoxicity (16,22), suggesting a link between parkin and glutamatergic synapse function. Consistent with this notion, mice lacking parkin display both motor and cognitive behavioral deficits and altered excitability in the hippocampus and striatum (23, 24). We hypothesized that parkin may regulate the strength of excitatory synapses. Results Postsynaptic Parkin Dampens Excitatory Synaptic Transmission.To determine the effects of postsynaptic parkin on synaptic transmission, GFP-tagged parkin (parkin-WT) was expressed in cultured rat hippocampal neurons, and miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were recorded...