Axonal transport has been intensively examined as a good model for studying the mechanism of organelle transport in cells, but it is still unclear how different types of membrane organelles are transported through the nerve axon. To elucidate the function of this mechanism, we have cloned KIF1A, a novel neuron-specific kinesin superfamily motor that was discovered to be a monomeric, globular molecule and that had the fastest reported anterograde motor activity (1.2 microns/s). To identify its cargo, membranous organelles were isolated from the axon. KIF1A was associated with organelles that contained synaptic vesicle proteins such as synaptotagmin, synaptophysin, and Rab3A. However, this organelle did not contain SV2, another synaptic vesicle protein, nor did it contain presynaptic membrane proteins, such as syntaxin 1A or SNAP-25, or other known anterograde motor proteins, such as kinesin and KIF3. Thus, we suggest that the membrane proteins are sorted into different classes of transport organelles in the cell body and are transported by their specific motor proteins through the axon.

Alzheimer amyloid beta-peptide (Abeta) is a physiological peptide constantly anabolized and catabolized under normal conditions. We investigated the mechanism of catabolism by tracing multiple-radiolabeled synthetic peptide injected into rat hippocampus. The Abeta1-42 peptide underwent full degradation through limited proteolysis conducted by neutral endopeptidase (NEP) similar or identical to neprilysin as biochemically analyzed. Consistently, NEP inhibitor infusion resulted in both biochemical and pathological deposition of endogenous Abeta42 in brain. This NEP-catalyzed proteolysis therefore limits the rate of Abeta42 catabolism, up-regulation of which could reduce the risk of developing Alzheimer's disease by preventing Abeta accumulation.

␣-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate the majority of excitatory synaptic transmission in the brain. Recent studies have shown that activation of PKA regulates the membrane trafficking of the AMPA receptor Glu receptor 1 (GluR1) subunit, but the role of direct phosphorylation of GluR1 in regulating receptor redistribution is not clear. Here we show that phosphorylation of the GluR1 subunit on serine 845 by PKA is required for PKA-induced increases in AMPA receptor cell-surface expression because it promotes receptor insertion and decreases receptor endocytosis. Furthermore, dephosphorylation of GluR1 serine 845 triggers NMDA-induced AMPA receptor internalization. These findings strongly suggest that dynamic changes in direct phosphorylation of GluR1 by PKA are crucial in the modulation of AMPA receptor trafficking and synaptic plasticity.glutamate receptors ͉ long-term potentiation ͉ synaptic plasticity A MPA (␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, which are responsible for the majority of excitatory synaptic transmissions in the mammalian central nervous system, undergo constant trafficking between the plasma membrane and cytosolic compartments. Alterations of these dynamic processes will redistribute AMPA receptors in neurons and might underlie changes in synaptic strength, such as long-term potentiation and long-term depression (1-5).AMPA receptor trafficking is regulated by a variety of mechanisms (1-5). Activation of glutamatergic receptors, including NMDA receptors, AMPA receptors, and metabotropic Glu receptors (mGluR), triggers rapid AMPA receptor internalization (4), whereas selective activation of synaptic NMDA receptors facilitates AMPA receptor surface insertion (6, 23). Interactions of AMPA receptors with multiple proteins such as PICK1, NSF, and stargazin play a key role in AMPA receptor dynamics and its synaptic accumulation (4, 5).The intracellular signaling pathways leading to AMPA receptor relocation are being actively examined and are still controversial. The involvement of different kinases and phosphatases in protein redistribution suggests that protein phosphorylation is a major cellular mechanism in regulating intracellular trafficking (7). AMPA receptors are members of a substrate of protein kinases that includes PKA, PKC, and Ca 2ϩ /calmodulin-dependent PK II (1). Phosphorylation of Glu receptor 1) (GluR1) subunits is closely correlated with AMPA receptor redistribution and expression of both long-term potentiation and long-term depression, indicating that PKA might regulate AMPA receptor trafficking via direct phosphorylation of GluR1 subunits (1,8,9). However, the specific role of GluR1 phosphorylation has not been clearly defined. Here we report that PKA phosphorylation of GluR1 at serine 845 (GluR1S845) increases AMPA receptor cell-surface expression as the result of a combination of increased receptor insertion and decreased internalization and that a dynamic dephosphorylation of this site is critical in NMDA-dependen...

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

Postsynaptic AMPA receptor (AMPAR) trafficking mediates some forms of synaptic plasticity that are modulated by NMDA receptor (NMDAR) activation and N-ethylmaleimide sensitive factor (NSF). We report that NSF is physiologically S-nitrosylated by endogenous, neuronally derived nitric oxide (NO). S-nitrosylation of NSF augments its binding to the AMPAR GluR2 subunit. Surface insertion of GluR2 in response to activation of synaptic NMDARs requires endogenous NO, acting selectively upon the binding of NSF to GluR2. Thus, AMPAR recycling elicited by NMDA neurotransmission is mediated by a cascade involving NMDA activation of neuronal NO synthase to form NO, leading to S-nitrosylation of NSF which is thereby activated, enabling it to bind to GluR2 and promote the receptor's surface expression.

␣-Amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate excitatory synaptic transmission and are dynamically regulated during synaptic plasticity in the CNS. The membrane trafficking of AMPA receptors to synapses is critical for the regulation of the efficacy of excitatory synaptic transmission. Direct imaging of AMPA receptors in various cell compartments is important to dissecting the regulation of distinct steps in receptor membrane trafficking. In this study, we have developed an approach for the imaging of receptor trafficking with subunits tagged with a 13-aa ␣-bungarotoxin (BTX)-binding site (BBS). The small polypeptide neurotoxin BTX has been used for decades to study the nicotinic acetylcholine receptor. Similar high-affinity ligands are rarely available for most receptors. Engineering the BBS tag into receptor subunits allowed the high-affinity binding of fluorescent, radioactive, and biotinylated BTX to the tagged receptor subunits. By using this approach, the total receptor expression, surface expression, internalization, and insertion of receptors into the plasma membrane could be visualized and quantified in fixed or live cells including cultured neurons. The BBS tag is a flexible approach for labeling membrane proteins and studying their dynamic trafficking.GFP ͉ glutamate receptor ͉ live imaging ͉ synapses ͉ tag I onotropic glutamate receptors mediate rapid excitatory transmission in the CNS and play critical role in synaptic transmission and plasticity, in neuronal development, and in several neurological and psychiatric disorders (1-4). The ionotropic glutamate receptors include the ␣-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) subtypes (1, 2). AMPA receptors mediate the majority of excitatory synaptic transmission in the CNS and are heterotetramers of four different subunits (GluR1-GluR4) (1, 2). The GluR1-GluR4 subunits have a large N-terminal extracellular domain, three transmembrane domains, a reentrant membrane loop that forms the central ion channel, and an intracellular C-terminal domain (1, 2). The regulation of AMPA-receptor membrane trafficking to synapses is critical for the dynamic modulation of excitatory synaptic transmission and is required for several forms of synaptic plasticity in the brain (3-5).Recent studies on AMPA-receptor membrane trafficking have used common cell biological techniques. GFP fusion proteins of AMPA-receptor subunits have been used to observe trafficking and targeting of AMPA receptors (6-8). In addition, labeling of live cells with antibodies against extracellular epitopes or tags has been used to visualize the surface expression and internalization of receptors (9-12). Moreover, surface biotinylation techniques have been used to biochemically measure the relative levels of surface and internalized receptors (13,14). Although these approaches have been used successfully to study AMPA-receptor trafficking and are widely used in many systems, there are several drawbacks to these techniq...

The matrix metalloproteinase (MMP) family, comprising more than 20 isoforms, modulates the extracellular milieu by degrading extracellular matrix (ECM) proteins. Because MMP is one of the few groups of proteinases capable of hydrolysing insoluble fibrillar proteins, they are likely to play crucial roles in regulating both normal and pathophysiological processes in the brain. However, little is yet known about their possible neuronal functions due presumably to their unusual redundancy and to the absence of a complete catalogue of isoforms. As an initial step in understanding the MMP system in the brain, we analysed an expression spectrum of MMP in rat brain using RT-PCR and discovered a novel brain-specific MMP, MT5-MMP. MT5-MMP was the predominant species among the nongelatinase-type isoforms in brain. MT5-MMP, present in all brain tissues examined, was most strongly expressed in cerebellum and was localized in the membranous structures of expressing neurons, as assessed biochemically and immunohistochemically. In cerebellum, its expression was regulated developmentally and was closely associated with dendritic tree formation of Purkinje cells, suggesting that MT5-MMP may contribute to neuronal development. Furthermore, its stable postdevelopmental expression and colocalization with senile plaques in Alzheimer brain indicates possible roles in neuronal remodeling naturally occurring in adulthood and in regulating pathophysiological processes associated with advanced age.

Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain. We previously demonstrated that neprilysin is the major Abeta-degrading enzyme in vivo. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating Abeta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on Abeta metabolism. The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wild-type neprilysin, but not the mutant, led to significant decreases in both the Abeta40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expression also resulted in reducing cell-associated Abeta, which could be more neurotoxic than extracellular Abeta. These results indicate that the manipulation of neprilysin activity in neurons, the major source of Abeta in the brain, would be a relevant strategy for controlling the Abeta levels and thus the Abeta-associated pathology in brain tissues.