Dezhi Liao

SynGAP: A synaptic RasGAP that associates with the PSD-95/SAP90 protein family

The PSD-95/SAP90 family of proteins has recently been implicated in the organization of synaptic structure. Here, we describe the isolation of a novel Ras-GTPase activating protein, SynGAP, that interacts with the PDZ domains of PSD-95 and SAP102 in vitro and in vivo. SynGAP is selectively expressed in brain and is highly enriched at excitatory synapses, where it is present in a large macromolecular complex with PSD-95 and the NMDA receptor. SynGAP stimulates the GTPase activity of Ras, suggesting that it negatively regulates Ras activity at excitatory synapses. Ras signaling at the postsynaptic membrane may be involved in the modulation of excitatory synaptic transmission by NMDA receptors and neurotrophins. These results indicate that SynGAP may play an important role in the modulation of synaptic plasticity.

Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration.

The microtubule-associated protein tau accumulates in Alzheimers and other fatal dementias, which manifest when forebrain neurons die. Recent advances in understanding these disorders indicate that brain dysfunction precedes neurodegeneration, but the role of tau is unclear. Here, we show that early tau-related deficits develop not from the loss of synapses or neurons, but rather as a result of synaptic abnormalities caused by the accumulation of hyperphosphorylated tau within intact dendritic spines, where it disrupts synaptic function by impairing glutamate receptor trafficking or synaptic anchoring. Mutagenesis of 14 disease-associated serine and threonine amino acid residues to create pseudohyperphosphorylated tau caused tau mislocalization while creation of phosphorylation-deficient tau blocked the mistargeting of tau to dendritic spines. Thus, tau phosphorylation plays a critical role in mediating tau mislocalization and subsequent synaptic impairment. These data establish that the locus of early synaptic malfunction caused by tau resides in dendritic spines.

Interaction of the N-Ethylmaleimide–Sensitive Factor with AMPA Receptors

Glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system (CNS) and play important roles in synaptic plasticity and neuronal development. Recently, protein-protein interactions with the C-terminal domain of glutamate receptor subunits have been shown to be involved in the modulation of receptor function and clustering at excitatory synapses. In this paper, we have found that the N-ethylmaleimide-sensitive factor (NSF), a protein involved in membrane fusion events, specifically interacts with the C terminus of the GluR2 and GluR4c subunits of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function.

Direct measurement of quantal changes underlying long-term potentiation in CA1 hippocampus

The modification responsible for the long-term synaptic potentiation (LTP) that follows a brief conditioning period is not known. To elucidate this change, we have resolved quantal levels of transmission before and after induction of LTP. We find an increase both in the number of quanta released and in quantal amplitude, consistent with combined pre- and postsynaptic modifications. On average, about 60% of LTP can be accounted for by presynaptic enhancement. The increase in either quantal amplitude or quantal content varies significantly among different experiments, but is inversely correlated with its initial value. These results may help to reconcile the different views concerning the site of LTP expression.

GRASP-1: A Neuronal RasGEF Associated with the AMPA Receptor/GRIP Complex

The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, we have identified several GRIP-associated proteins (GRASPs) that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduced the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 was rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity.

A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1.

We report the molecular cloning and characterization of 4.1N, a novel neuronal homolog of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). The 879 amino acid protein shares 70, 36, and 46% identity with 4.1R in the defined membrane-binding, spectrin-actin–binding, and C-terminal domains, respectively. 4.1N is expressed in almost all central and peripheral neurons of the body and is detected in embryonic neurons at the earliest stage of postmitotic differentiation. Like 4.1R, 4.1N has multiple splice forms as evidenced by PCR and Western analysis. Whereas the predominant 4.1N isoform identified in brain is ∼135 kDa, a smaller 100 kDa isoform is enriched in peripheral tissues. Immunohistochemical studies using a polyclonal 4.1N antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons. In certain neuronal locations, including the granule cell layers of the cerebellum and dentate gyrus, a distinct punctate-staining pattern was observed consistent with a synaptic localization. In primary hippocampal cultures, mouse 4.1N is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kDa (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker). By analogy with the roles of 4.1R in red blood cells, 4.1N may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin-actin–based cytoskeleton, integral membrane channels and receptors, and membrane-associated guanylate kinases.

Lipid binding regulates synaptic targeting of PICK1, AMPA receptor trafficking, and synaptic plasticity

The targeting and surface expression of membrane proteins are critical to their functions. In neurons, synaptic targeting and surface expression of AMPA-type glutamate receptors were found to be critical for synaptic plasticity such as long-term potentiation and long-term depression (LTD). PICK1 (protein interacting with C kinase 1) is a cytosolic protein that interacts with many membrane proteins, including AMPA receptors via its PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain. Its interactions with membrane proteins regulate their subcellular targeting and surface expression. However, the mechanism by which PICK1 regulates protein trafficking has not been fully elucidated. Here, we show that PICK1 directly binds to lipids, mainly phosphoinositides, via its BAR (Bin/amphiphysin/Rvs) domain. Lipid binding of the PICK1 BAR domain is positively regulated by its PDZ domain and negatively regulated by its C-terminal acidic domain. Mutation of critical residues of the PICK1 BAR domain eliminates its lipid-binding capability. Lipid binding of PICK1 controls the subcellular localization of the protein, because BAR domain mutant of PICK1 has diminished synaptic targeting compared with wild-type PICK1. In addition, the BAR domain mutant of PICK1 does not cluster AMPA receptors. Moreover, wild-type PICK1 enhances synaptic targeting of AMPA receptors, whereas the BAR domain mutant of PICK1 fails to do so. The BAR domain mutant of PICK1 loses its ability to regulate surface expression of the AMPA receptors and impairs expression of LTD in hippocampal neurons. Together, our findings indicate that the lipid binding of the PICK1 BAR domain is important for its synaptic targeting, AMPA receptor trafficking, and synaptic plasticity.

Rac1 Induces the Clustering of AMPA Receptors during Spinogenesis

Glutamatergic synapses switch from nonspiny synapses to become dendritic spines during early neuronal development. Here, we report that the lack of sufficient Rac1, a small RhoGTPase, contributes to the absence of spinogenesis in immature neurons. The overexpression of green fluorescence protein-tagged wild-type Rac1 initiated the formation of dendritic spines in cultured dissociated hippocampal neurons younger than 11 d in vitro, indicating that Rac1 is likely one of the missing pieces responsible for the lack of spines in immature neurons. The overexpression of wild-type Rac1 also induced the clustering of AMPA receptors (AMPARs) and increased the amplitude of miniature EPSCs (mEPSCs). The expression of constitutively active Rac1 induced the formation of unusually large synapses with large amounts of AMPAR clusters. Also, our live imaging experiments revealed that the contact of an axon induced the clustering of Rac1, and subsequent morphological changes led to spinogenesis. Additionally, the overexpression of wild-type Rac1 and constitutively active Rac1 increased the size of preexisting spines and the amplitude of mEPSCs in mature neurons (>21 d in vitro) within 24 h after transfection. Together, these results indicate that activation of Rac1 enhances excitatory synaptic transmission by recruiting AMPARs to synapses during spinogenesis, thus providing a mechanistic link between presynaptic and postsynaptic developmental changes. Furthermore, we show that Rac1 has two distinct roles at different stages of neuronal development. The activation of Rac1 initiates spinogenesis at an early stage and regulates the function and morphology of preexisting spines at a later stage.

Tau phosphorylation and tau mislocalization mediate soluble Aβ oligomer-induced AMPA glutamate receptor signaling deficits.

In our previous studies, phosphorylation‐dependent tau mislocalization to dendritic spines resulted in early cognitive and synaptic deficits. It is well known that amyloid beta (Aβ) oligomers cause synaptic dysfunction by inducing calcineurin‐dependent AMPA receptor (AMPAR) internalization. However, it is unknown whether Aβ‐induced synaptic deficits depend upon tau phosphorylation. It is also unknown whether changes in tau can cause calcineurin‐dependent loss of AMPARs in synapses. Here, we show that tau mislocalizes to dendritic spines in cultured hippocampal neurons from APPSwe Alzheimers disease (AD)‐transgenic mice and in cultured rat hippocampal neurons treated with soluble Aβ oligomers. Interestingly, Aβ treatment also impairs synaptic function by decreasing the amplitude of miniature excitatory postsynaptic currents (mEPSCs). The above tau mislocalization and Aβ‐induced synaptic impairment are both diminished by the expression of AP tau, indicating that these events require tau phosphorylation. The phosphatase activity of calcineurin is important for AMPAR internalization via dephosphorylation of GluA1 residue S845. The effects of Aβ oligomers on mEPSCs are blocked by the calcineurin inhibitor FK506. Aβ‐induced loss of AMPARs is diminished in neurons from knock‐in mice expressing S845A mutant GluA1 AMPA glutamate receptor subunits. This finding suggests that changes in phosphorylation state at S845 are involved in this pathogenic cascade. Furthermore, FK506 rescues deficits in surface AMPAR clustering on dendritic spines in neurons cultured from transgenic mice expressing P301L tau proteins. Together, our results support the role of tau and calcineurin as two intermediate signaling molecules between Aβ initiation and eventual synaptic dysfunction early in AD pathogenesis.

Characterization, Expression, and Distribution of GRIP Protein

lutamate receptors are the major receptors in excitatory synapses. They also play important roles in neuronal development, excitotoxicity, and synaptic plasticity. Glutamate receptors are highly enriched as clusters on the postsynaptic membrane. The mechanisms underlying the sorting, targeting, anchoring, and clustering of glutamate receptors have been unclear. Recently we have isolated a glutamate receptor interacting protein (GRIP) that directly binds to the GluR2 and three subunits of the AMPA receptors. GRIP may be involved in the mechanisms underlying the targeting and clustering of AMPA receptors. Here we characterize the expression and distribution of GRIP protein in brain (FIG.1). GRIP is expressed in many neurons in brain and is present in a somatodendritic staining pattern in cerebral cortex, hippocampus, and cerebellum (FIGS. 2, 3). Immunostaining of cultured hippocampal neurons shows that GRIP colocalizes with both AMPA receptors and GABAergic synapses, suggesting that GRIP might be involved in the function of both excitatory and inhibitory synapses (FIG. 4).