Kate Prybylowski

The Synaptic Localization of NR2B-Containing NMDA Receptors Is Controlled by Interactions with PDZ Proteins and AP-2

The NMDA receptor (NMDAR) is a component of excitatory synapses and a key participant in synaptic plasticity. We investigated the role of two domains in the C terminus of the NR2B subunit--the PDZ binding domain and the clathrin adaptor protein (AP-2) binding motif--in the synaptic localization of NMDA receptors. NR2B subunits lacking functional PDZ binding are excluded from the synapse. Mutations in the AP-2 binding motif, YEKL, significantly increase the number of synaptic receptors and allow the synaptic localization of NR2B subunits lacking PDZ binding. Peptides corresponding to YEKL increase the synaptic response within minutes. In contrast, the NR2A subunit localizes to the synapse in the absence of PDZ binding and is not altered by mutations in its motif corresponding to YEKL of NR2B. This study identifies a dynamic regulation of synaptic NR2B-containing NMDARs through PDZ protein-mediated stabilization and AP-2-mediated internalization that is modulated by phosphorylation by Fyn kinase.

NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex

NMDA (N-methyl-D-aspartate) receptors (NMDARs) are targeted to dendrites and anchored at the post-synaptic density (PSD) through interactions with PDZ proteins. However, little is known about how these receptors are sorted from the endoplasmic reticulum and Golgi apparatus to the synapse. Here, we find that synapse-associated protein 102 (SAP102) interacts with the PDZ-binding domain of Sec8, a member of the exocyst complex. Our results show that interactions between SAP102 and Sec8 are involved in the delivery of NMDARs to the cell surface in heterologous cells and neurons. Furthermore, they suggest that an exocyst–SAP102–NMDAR complex is an important component of NMDAR trafficking.

PSD‐95 regulates NMDA receptors in developing cerebellar granule neurons of the rat

We transfected a green fluorescent protein‐tagged PSD‐95 (PSD‐95gfp) into cultured rat cerebellar granule cells (CGCs) to investigate the role of PSD‐95 in excitatory synapse maturation. Cells were grown in low potassium to favour functional synapse formation in vitro. Transfected cells displayed clear clusters of PSD‐95gfp, often at the extremities of the short dendritic trees. We recorded NMDA and AMPA miniature excitatory postsynaptic currents (NMDA‐ and AMPA‐mESPCs) in the presence of TTX and bicuculline. At days in vitro (DIV) 7–8 PSD‐95gfp‐transfected cells had NMDA‐mEPSCs with faster decay and smaller amplitudes than matching controls. In contrast, AMPA‐mEPSC frequencies and amplitudes were increased. Whole‐cell current density and ifenprodil sensitivity were reduced in PSD‐95gfp cells, indicating a reduction of NR2B subunits containing NMDA receptors. No changes were observed compared to control when cells were transfected with cDNA for PSD‐95gfp with palmitoylation site mutations that prevent targeting to the synapse. Overexpression of the NMDA receptor NR2A subunit, but not the NR2B subunit, prevented NMDA‐mEPSC amplitude reduction when cotransfected with PSD‐95gfp. PSD‐95gfp overexpression produced faster NMDA‐mEPSC decay when transfected alone or with either NR2 subunit. Surface staining of the epitope‐tagged NR2 subunits revealed that colocalization with PSD‐95gfp was higher for flag‐tagged NR2A subunit clusters than for flag‐tagged NR2B subunit clusters. These data suggest that PSD‐95 overexpression in CGCs favours synaptic maturation by allowing synaptic insertion of NR2A and depressing expression of NR2B subunits.

Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture

We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro.

Biochemical studies of the structure and function of the N-methyl-D-aspartate subtype of glutamate receptors.

TheN-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays a key role in synaptic transmission, synaptic plasticity, synaptogenesis, and excitotocity in the mammalian central nervous system. The NMDA receptor channel is formed from two gene products from two glutamate receptor subunit families, termed NR1 and NR2. Although the subunit composition of native NMDA receptors is incompletely understood, electrophysiological studies using recombinant receptors suggest that functional NMDA receptors consist of heteromers containing combinations of NR1, which is essential for channel activity, and NR2, which modulates the properties of the channels. The lack of agonists or antagonists selective for a given subunit of NMDA receptors has made it difficult to understand the subunit expression, subunit composition, and posttranslational modification mechanisms of native NMDA receptors. Therefore, most studies on NMDA receptors that examine regional expression and ontogeny have been focused at the level of the mRNAs encoding the different subunits using northern blotting, ribonuclease protection, andin situ hybridization techniques. However, the data from these studies do not provide clear information about the resultant subunit protein. To directly examine the protein product of the NMDA receptor subunit genes, the development of subunit-specific antibodies using peptides and fusion proteins has provided a good approach for localizing, quantifying, and characterizing the receptor subunits in tissues and transfected cell lines, and to study the subunit composition and the functional effects of posttranslational processing of the NMDA subunits, particularly the phosphorylation profiles of NMDA glutamate receptors.

The Role of the PDZ Protein GIPC in Regulating NMDA Receptor Trafficking

The NMDA receptor is an important component of excitatory synapses in the CNS. In addition to its synaptic localization, the NMDA receptor is also present at extrasynaptic sites where it may have functions distinct from those at the synapse. Little is known about how the number, composition, and localization of extrasynaptic receptors are regulated. We identified a novel NMDA receptor-interacting protein, GIPC (GAIP-interacting protein, C terminus), that associates with surface as well as internalized NMDA receptors when expressed in heterologous cells. In neurons, GIPC colocalizes with a population of NMDA receptors on the cell surface, and changes in GIPC expression alter the number of surface receptors. GIPC is mainly excluded from the synapse, and changes in GIPC expression do not change the total number of synaptic receptors. Our results suggest that GIPC may be preferentially associated with extrasynaptic NMDA receptors and may play a role in the organization and trafficking of this population of receptors.

Expression of splice variants of the NR1 subunit of the N-methyl-D-aspartate receptor in the normal and injured rat spinal cord.

Quantitative western blot analysis in laminectomy control spinal cords of adult rats was used to provide the first report of the normal expression patterns of the N1, C1, C2 and C2′ cassettes in the cervical, thoracic and lumbar regions of the spinal cord as a percent of total NR1 subunit protein. In all regions studied, the C1 and C2 cassettes were usually contained in less than 10% of total NR1 protein. In contrast, approximately 90% of total NR1 protein contained the C2′ cassette. A significant proportion of total NR1 protein (approximately 30%) also contained the N1 cassette. These data are consistent with expression of NR1000 (NR1–4a) and NR1100 (NR1–4b) as the dominant splice forms in the spinal cord. Splice variant expression was also studied following incomplete, contusive spinal cord injury (SCI) to the thoracic level 8 (T8) region. This injury did not change expression of the C1 or C2 cassette in any region of the spinal cord acutely at 24 h or chronically at 1 month. There was an increase in expression of the N1 cassette in the lumbar regions 1 month after injury (p < 0.05). These data indicate that SCI induces distal changes in NR1 splice variant expression, which may play a role in the adaptive response of neurons in the chronically injured spinal cord.

Increased Exon 5 Expression Alters Extrasynaptic NMDA Receptors in Cerebellar Neurons

Abstract: We investigated the ontogenic changes in expression of alternatively spliced forms of NR1 protein that contain the N1 cassette (exon 5) in comparison with the total population of NR1 within the rat cerebellum. The N1 cassette is strongly developmentally regulated in the cerebellum, with >80% of total NR1 protein in the adult rat containing the N1 cassette. In contrast, early in development, <20% of NR1 protein contained this cassette. Rat cortices from the same ages did not show an increase in the percent of NR1 protein expressing the N1 cassette, indicating that the developmental changes in the cerebellum are tissue‐specific. As the N1 cassette is known to determine NMDA receptor properties, including spermine sensitivity and decay kinetics of glutamate‐induced currents, changes in the characteristics of glutamate‐activated currents in granule cells from cerebellar slices were compared at postnatal day 7 versus 14. Glutamate responses exhibited fast deactivation kinetics and reduced potentiation by spermine at day 14 while maintaining sensitivity to an NR2B‐selective antagonist. These data are consistent with the possibility that N1 cassette expression may be a factor in the developmental changes in properties of extrasynaptic NMDA receptors in the cerebellum.