The Mind Bending Quest for Cognitive Enhancers.

Adequate cognitive functioning is essential for daily activities. When there is an insult to the brain, cognitive abilities can suffer, which, in turn, produce substantial medical and functional impairment. Advances in neurobiology, circuit neuroscience, and clinical assessment technology are converging in a manner that holds promise for the development of new pharmacological agents for cognitive enhancement in neuropsychiatric disease.

Digital technologies for cognitive assessment to accelerate drug development in Alzheimer's disease.

For many neurological and psychiatric diseases, novel therapeutics have been elusive for decades. By focusing on attention interference in Alzheimer's disease (AD), we provide a future vision on how emerging mobile, computer, and device-based cognitive tools are converting classically noisy, subjective, data-poor clinical endpoints associated with neuropsychiatric disease assessment into a richer, scalable, and objective set of measurements. Incorporation of such endpoints into clinical drug trials holds promise for more quickly and efficiently developing new medicines.

Molecular determinants of NMDA receptor internalization.

Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites.

An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing.

Formation of mature excitatory synapses requires the assembly and delivery of NMDA receptors to the neuronal plasma membrane. A key step in the trafficking of NMDA receptors to synapses is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of an RXR-type ER retention/retrieval motif in the C-terminal tail of the NMDA receptor subunit NR1 that regulates receptor surface expression in heterologous cells and in neurons. In addition, we show that PKC phosphorylation and an alternatively spliced consensus type I PDZ-binding domain suppress ER retention. These results demonstrate a novel quality control function for alternatively spliced C-terminal domains of NR1 and implicate both phosphorylation and potential PDZ-mediated interactions in the trafficking of NMDA receptors through early stages of the secretory pathway.

Localization and stabilization of ionotropic glutamate receptors at synapses.

Appropriate targeting and clustering of ionotropic glutamate receptors (iGluRs) is critical for the formation and maintenance of excitatory synapses. Recent studies have demonstrated that the synaptic localization of iGluR subtypes is remarkably heterogeneous and subject to regulation over time scales ranging from minutes to months. These findings, together with the identification of key protein binding partners of iGluRs, have opened a window onto the complex cell biology of iGluR membrane trafficking. In this article, we review recent findings on the cellular and molecular mechanisms involved in localizing iGluRs at synapses and discuss their implications for synaptogenesis and synaptic plasticity.

Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting.

Both acute and chronic changes in AMPA receptor (AMPAR) localization are critical for synaptic formation, maturation, and plasticity. Here I report that AMPARs are differentially sorted between recycling and degradative pathways following endocytosis. AMPAR sorting occurs in early endosomes and is regulated by synaptic activity and activation of AMPA and NMDA receptors. AMPAR intemalization triggered by NMDAR activation is Ca2+-dependent, requires protein phosphatases, and is followed by rapid membrane reinsertion. Furthermore, NMDAR-mediated AMPAR trafficking is regulated by PKA and accompanied by dephosphorylation and rephosphorylation of GluR1 subunits at a PKA site. In contrast, activation of AMPARs without NMDAR activation targets AMPARs to late endosomes and lysosomes, independent of Ca2+, protein phosphatases, or PKA. These results demonstrate that activity regulates AMPAR endocytic sorting, providing a potential mechanistic link between rapid and chronic changes in synaptic strength.

Synapse structure: glutamate receptors connected by the shanks.

A family of proteins has been identified whose members, the Shanks, physically link two major receptor complexes at excitatory synapses - NMDA receptors and metabotropic glutamate receptors.

Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons.

Many excitatory synapses are thought to be postsynaptically 'silent', possessing functional NMDA but lacking functional AMPA glutamate receptors. The acquisition of AMPA receptors at silent synapses may be important in synaptic plasticity and neuronal development. Here we characterize a possible morphological correlate of silent synapses in cultured hippocampal neurons. Initially, most excitatory synapses contained NMDA receptors, but only a few contained detectable AMPA receptors. Synapses progressively acquired AMPA receptors as the cultures matured. AMPA receptor blockade increased the number, size and fluorescent intensity of AMPA receptor clusters and rapidly induced the appearance of AMPA receptors at 'silent' synapses. In contrast, NMDA receptor blockade increased the size, intensity and number of NMDA receptor clusters and decreased the number of AMPA receptor clusters, resulting in an increase in the proportion of 'silent' synapses. These results suggest that the number of silent synapses is regulated during development and by changes in synaptic activity.

Activity-dependent modulation of synaptic AMPA receptor accumulation.

Both theoretical and experimental work have suggested that central neurons compensate for changes in excitatory synaptic input in order to maintain a relatively constant output. We report here that inhibition of excitatory synaptic transmission in cultured spinal neurons leads to an increase in mEPSC amplitudes, accompanied by an equivalent increase in the accumulation of AMPA receptors at synapses. Conversely, increasing excitatory synaptic activity leads to a decrease in synaptic AMPA receptors and a decline in mEPSC amplitude. The time course of this synaptic remodeling is slow, similar to the metabolic half-life of neuronal AMPA receptors. Moreover, inhibiting excitatory synaptic transmission significantly prolongs the half-life of the AMPA receptor subunit GluR1, suggesting that synaptic activity modulates the size of the mEPSC by regulating the turnover of postsynaptic AMPA receptors.

Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors.

Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor-mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. We report here that the mutation of a specific calmodulin binding site in the CO region of the NR1 subunit of the NMDA receptor blocks CDI. Moreover, intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. Furthermore, this inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein alpha-actinin2 known to interact with the CO region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton.

Splice variant-specific interaction of the NMDA receptor subunit NR1 with neuronal intermediate filaments.

NMDA receptors are excitatory neurotransmitter receptors critical for synaptic plasticity and neuronal development in the mammalian brain. These receptors are found highly concentrated in the postsynaptic membrane of glutamatergic synapses. To investigate the molecular mechanisms underlying NMDA receptor localization, we used the yeast two-hybrid system to identify proteins expressed in the brain that interact with the NMDA receptor subunit NR1. Here we report that the 68 kDa neurofilament subunit NF-L directly interacts with the NR1 subunit. This interaction occurs between the cytoplasmic C-terminal domain of NR1 and the rod domain of NF-L. However, NR1 splice variants lacking the first C-terminal exon cassette (C1) failed to associate with NF-L. Immunogold electron microscopy revealed a preferential localization of NR1 at the ends of in vitro-assembled neurofilaments. Overexpression of C1 cassette-containing NR1 constructs in fibroblast cells disrupted the assembly of recombinant neurofilaments. In addition, NR1 and NF-L cofractionated in detergent-treated rat brain synaptic plasma membranes. Furthermore, NR1 and NF-L colocalize in the dendrites and growth cones of cultured hippocampal neurons. These results demonstrate the splice variant-specific association of NR1 with neurofilaments and suggest a possible mechanism for anchoring or localizing NMDA receptors in the neuronal plasma membrane.

The development of excitatory synapses in cultured spinal neurons.

Immunohistochemical studies of synapses in the CNS have demonstrated that glutamate receptors (GluRs) are concentrated at postsynaptic sites in vivo and in vitro (Baude et al., 1995). The mechanisms leading to receptor clustering at excitatory synapses are far less understood than those governing acetylcholine receptor accumulation at the neuromuscular junction () or glycine receptor aggregation at central inhibitory synapses (). Using cultured rat spinal cord neurons, we demonstrate that clustering of the AMPA receptor subunit GluR1 is among the earliest events in excitatory synapse formation in vitro, coincident with the onset of miniature EPSCs and in many cases preceding presynaptic vesicle accumulation. Postsynaptic receptor clustering is induced in a highly specific and reiterative pattern, independent of receptor activation, by contact with a subset of axons capable of inducing receptor clusters. The subunit composition of AMPA receptor clusters varied significantly between neurons but was invariant within a given neuron. The presence of either GluR2 or GluR3 was common to all receptor clusters. Neither high-affinity glutamate transporters nor NMDA receptors appeared to be concentrated with AMPA receptor subunits at these excitatory synapses.

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies.

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.

Synaptic targeting of glutamate receptors.

The proper targeting and clustering of neurotransmitter receptors at appropriate postsynaptic sites are principal requirements for the formation of functional synapses. Recently, new studies have begun to elucidate the mechanisms underlying the targeting and clustering of glutamate receptors at excitatory synapses in the brain. Members of the SAP90/PSD-95 family of proteins have emerged as potential regulators of glutamate-receptor membrane distribution. Further, targeting motifs within glutamate receptor subunits have been identified. These findings provide important clues in the effort to understand the molecular features of synaptic organization.

Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102.

Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl--aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.

Effect of dopaminergic drugs on the in vivo binding of [3H]WIN 35,428 to central dopamine transporters.

[11C]WIN 35,428 (also designated [11C]CFT) is now being used in several positron emission tomography (PET) centers to image dopamine (DA) transporter sites in the mammalian brain. Whether and to what extent in vivo WIN 35,428 binding is influenced by intra- and extrasynaptic dopamine levels are largely unknown. The purpose of the present study was to evaluate the effects of various drugs, known to affect DA levels and release, on the binding of [3H]WIN 35,428 to central DA transporters in the mouse brain. D-Amphetamine, which releases DA from neurons and blocks the DA transporter directly, inhibited striatal [3H]WIN 35,428 binding in dose-dependent manner. Similarly, alpha-methyl-DL-p-tyrosine, an inhibitor of tyrosine hydroxylase, blocked [3H]WIN 35,428 binding, possibly via competitive inhibition by the metabolite p-hydroxyamphetamine. Specific binding of [3H]WIN 35,428 was insensitive to changes in synaptic DA levels caused by pretreatment of the animals with high doses of D2 receptor agonists (apomorphine, bromocriptine), antagonists (haloperidol) or the inhibitor of dopaminergic neuron firing gamma-butyrolactone (GBL). High doses (> 50 mg/kg) of L-DOPA (in combination with benserazide), however, reduced [3H]WIN 35,428 binding significantly, yet for a relatively short time (approximately 2.5 h). Chronic treatment with L-deprenyl elicited no changes in in vivo [3H]WIN 35,428 accumulation in the striatum. Neurotoxic damage of DA neurons caused by administration of high doses of amphetamine was detected in the striatum by a significant reduction in [3H]WIN 35,428 binding 7 days after cessation of amphetamine treatment. Thus, [3H]WIN 35,428 binding was only affected by neurotoxic loss of neurons, by administration of uptake inhibitors, or by some treatments which significantly elevate DA levels. Compounds which inhibit DA release or deplete DA acutely do not increase [3H]WIN 35,428 binding, suggesting that normal or "resting" levels of DA are not sufficient to alter [3H]WIN 35,428 binding in vivo. These findings are important for our understanding of the function and regulation of the DA transporter, as well as the in vivo binding of the radioligand [3H/11 C]WIN 35,428. Moreover, they will be important for the interpretation of PET studies in which [11C]WIN 35,428 is used to assess the integrity of dopaminergic neurons.

Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit.

NMDA (N-methyl-D-aspartate) receptors are excitatory neurotransmitter receptors in the brain critical for synaptic plasticity and neuronal development. These receptors are Ca2+-permeable glutamate-gated ion channels whose physiological properties are regulated by intracellular Ca2+. We report here the purification of a 20 kDa protein identified as calmodulin that interacts with the NR1 subunit of the NMDA receptor. Calmodulin binding to the NR1 subunit is Ca2+ dependent and occurs with homomeric NR1 complexes, heteromeric NR1/NR2 subunit complexes, and NMDA receptors from brain. Furthermore, calmodulin binding to NR1 causes a 4-fold reduction in NMDA channel open probability. These results demonstrate that NMDA receptor function can be regulated by direct binding of calmodulin to the NR1 subunit, and suggest a possible mechanism for activity-dependent feedback inhibition and Ca2+-dependent inactivation of NMDA receptors.

Regulated subcellular distribution of the NR1 subunit of the NMDA receptor.

NMDA (N-methyl-D-aspartate) receptors are selectively localized at the postsynaptic membrane of excitatory synapses in the mammalian brain. The molecular mechanisms underlying this localization were investigated by expressing the NR1 subunit of the NMDA receptor in fibroblasts. NR1 splice variants containing the first COOH-terminal exon cassette (NR1A and NR1D) were located in discrete, receptor-rich domains associated with the plasma membrane. NR1 splice variants lacking this exon cassette (NR1C and NR1E) were distributed throughout the cell, with large amounts of NR1 protein present in the cell interior. Insertion of this exon cassette into the COOH-terminus of the GluR1 AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor was sufficient to cause GluR1 to be localized to discrete, receptor-rich domains. Furthermore, protein kinase C phosphorylation of specific serines within this exon disrupted the receptor-rich domains. These results demonstrate that amino acid sequences contained within the NR1 molecule serve to localize this receptor subunit to discrete membrane domains in a manner that is regulated by alternative splicing and protein phosphorylation.

NGF-stimulated retrograde transport of trkA in the mammalian nervous system.

The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo.