Houhui Xia

Interaction of Nitric Oxide Synthase with the Postsynaptic Density Protein PSD-95 and α1-Syntrophin Mediated by PDZ Domains

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy

Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

Role of AMPA Receptor Cycling in Synaptic Transmission and Plasticity

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

Signaling from synapse to nucleus: the logic behind the mechanisms

Signaling from synapse to nucleus is vital for activity-dependent control of neuronal gene expression and represents a sophisticated form of neural computation. The nature of specific signal initiators, nuclear translocators and effectors has become increasingly clear, and supports the idea that the nucleus is able to make sense of a surprising amount of fast synaptic information through intricate biochemical mechanisms. Information transfer to the nucleus can be conveyed by physical translocation of messengers at various stages within the multiple signal transduction cascades that are set in motion by a Ca(2+) rise near the surface membrane. The key role of synapse-to-nucleus signaling in circadian rhythms, long-term memory, and neuronal survival sheds light on the logical underpinning of these signaling mechanisms.

Regulation of Synaptic Strength by Protein Phosphatase 1

We investigated the role of postsynaptic protein phosphatase 1 (PP1) in regulating synaptic strength by loading CA1 pyramidal cells either with peptides that disrupt PP1 binding to synaptic targeting proteins or with active PP1. The peptides blocked synaptically evoked LTD but had no effect on basal synaptic currents mediated by either AMPA or NMDA receptors. They did, however, cause an increase in synaptic strength following the induction of LTD. Similarly, PP1 had no effect on basal synaptic strength but enhanced LTD. In cultured neurons, synaptic activation of NMDA receptors increased the proportion of PP1 localized to synapses. These results suggest that PP1 does not significantly regulate basal synaptic strength. Appropriate NMDA receptor activation, however, allows PP1 to gain access to synaptic substrates and be recruited to synapses where its activity is necessary for sustaining LTD.

Regulation of neuronal nitric oxide synthase through alternative transcripts.

Nitric oxide (NO) participates in diverse physiological processes ranging from neurotransmission to muscle relaxation. Neuronal-derived NO can be either beneficial or detrimental depending on the cellular context. Neuronal NO synthase (nNOS) must therefore be tightly regulated. One level of regulation involves synthesis of numerous nNOS mRNA transcripts. At least six distinct molecular species of nNOS mRNA are expressed in a tissue and developmentally-regulated manner. Alternative splicing allows the creation of nNOS proteins differing in both enzymatic characteristics and structural features. As one example, we find that there are nNOS mRNAs lacking exon 2. One isoform, nNOS beta, retains full enzymatic activity but lacks a major protein-protein interaction domain (PDZ domain) responsible for targeting nNOS to synaptic membranes. This alternative splicing produces a mislocalized but fully active protein which may be relevant to certain pathologies. As evidence of this, we find that many human brain tumors express an alternatively spliced form of nNOS that co-migrates with nNOS beta, and lacks exon 2. Finally, we also find a 2.5-kb testis-specific nNOS mRNA corresponding to the C-terminal reductase domain of nNOS whose function is unclear.

Differential roles for NSF and GRIP/ABP in AMPA receptor cycling

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) stability and movement at synapses are important factors controlling synaptic strength. Here, we study the roles of proteins [N-ethylmaleimide-sensitive fusion protein (NSF), glutamate receptor AMPAR binding protein (ABP)-interacting protein (GRIP)/(ABP), and protein interacting with C-kinase-1 (PICK1) that interact with the GluR2 subunit in the control of the surface expression and cycling of AMPARs. Epitope-tagged GluR2 formed functional receptors that exhibited targeting to synaptic sites. Constructs in which binding to NSF, PDZ proteins (GRIP/ABP and PICK1), or GRIP/ABP alone was eliminated each exhibited normal surface targeting and constitutive cycling. The lack of NSF binding, however, resulted in receptors that were endocytosed to a greater extent than wild-type receptors in response to application of AMPA or N-methyl-d-aspartate (NMDA). Conversely, the behavior of the GluR2 mutants incapable of binding to GRIP/ABP suggests that these PDZ proteins play a role in the stabilization of an intracellular pool of AMPARs that have been internalized on stimulation, thus inhibiting their recycling to the synaptic membrane. These results provide further evidence for distinct functional roles of GluR2-interacting proteins in AMPAR trafficking.

An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits.

The molecular mechanisms that control the surface expression of NMDA receptors (NMDARs) and AMPA receptors (AMPARs) are unknown. To determine the role of the intracellular C-terminal tails of glutamate receptor subunits in the synaptic targeting of AMPARs and NMDARs, we fused the tails of the AMPAR subunits, GluR1 and GluR2, and the NMDAR subunit, NR1, to the human T lymphocyte membrane protein CD8 and expressed these constructs in HEK293 cells and cultured hippocampal neurons. The GluR1 and GluR2 fusion proteins exhibited robust surface expression in the plasma membrane of neurons at synapses as did CD8 alone. In contrast, the NR1 fusion protein was retained intracellularly in both HEK293 cells and neurons because of the presence of an ER retention signal in the C1 cassette. This ER retention signal was overridden either by the addition of a PDZ domain-binding motif or by mimicking phosphorylation at a site adjacent to the retention signal. These results provide further evidence that the intracellular trafficking of AMPAR and NMDAR subunits are regulated independently at least in part because of differences in the protein-protein interactions of their intracellular C-terminal tails.

Nitric oxide synthase and cyclic GMP-dependent protein kinase concentrated at the neuromuscular endplate

Nitric oxide mediates diverse functions in development and physiology of vertebrate skeletal muscle. Neuronal type nitric oxide synthase-mu is enriched in fast-twitch fibers and binds to syntrophin, a component of the sarcolemmal dystrophin glycoprotein complex. Here, we show that cyclic GMP-dependent protein kinase type I, a primary effector for nitric oxide, occurs selectively at the neuromuscular junction, in mice and rats, and both neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I remain at skeletal muscle endplates at least two weeks following muscle denervation. Expression of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I are up-regulated following fusion of cultured primary myotubes. Interestingly, the highest levels of neuronal type nitric oxide synthase-mu in muscle are found complexed with dystrophin at the sarcolemma of intrafusal fibers in muscle spindles. Localization of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I at the neuromuscular junction suggests functions for nitric oxide and cyclic GMP in the regulation of synaptic actions of intra- and extrafusal muscle fibers.

Inactivation of CREB mediated gene transcription by HDAC8 bound protein phosphatase

CREB activation via phosphorylation at serine 133 and resulting CREB mediated gene expression is a critical event which can have a significant effect on many cellular processes, including cell survival and plasticity. CREB can be activated by many kinases, for example, it can be phosphorylated by PKA, MAPK, and CaMKIV. The various signaling pathways leading to CREB activation have been extensively studied. On the other hand, CREB is inactivated by PP1 through dephosphorylation at S133 and not much attention has been paid to this aspect of the signaling pathway. It was shown recently that PP1 can be targeted to CREB, for efficient dephosphorylation, through PP1 binding protein HDAC1. In this study, we found that another class-I HDAC family protein, HDAC8, localized in the nucleus of HEK293 cells and also bound to both CREB and PP1. Expression of recombinant HDAC8 results in decreased CREB activation and CREB mediated gene transcription in response to forskolin application. Our study thus elucidated that more than one class-I HDAC family members can regulate the duration of CREB mediated gene transcription.