Dendritic spines are the major sites of excitatory synaptic input, and their morphological changes have been linked to learning and memory processes. Here, we report that growing microtubule plus ends decorated by the microtubule tip-tracking protein EB3 enter spines and can modulate spine morphology. We describe p140Cap/SNIP, a regulator of Src tyrosine kinase, as an EB3 interacting partner that is predominantly localized to spines and enriched in the postsynaptic density. Inhibition of microtubule dynamics, or knockdown of either EB3 or p140Cap, modulates spine shape via regulation of the actin cytoskeleton. Fluorescence recovery after photobleaching revealed that EB3-binding is required for p140Cap accumulation within spines. In addition, we found that p140Cap interacts with Src substrate and F-actin-binding protein cortactin. We propose that EB3-labeled growing microtubule ends regulate the localization of p140Cap, control cortactin function, and modulate actin dynamics within dendritic spines, thus linking dynamic microtubules to spine changes and synaptic plasticity.

The molecular mechanisms that determine the size and complexity of the neuronal dendritic tree are unclear. Here, we show that the phosphoinositide-3Ј kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway promotes the growth and branching of dendrites in cultured hippocampal neurons. Constitutively active mutants of Ras, PI3K, and Akt, or RNA interference (RNAi) knockdown of lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome Ten), induced growth and elaboration of dendrites that was blocked by mTOR inhibitor rapamycin and/or by overexpression of eIF-4E binding protein 1 (4E-BP1), which inhibits translation of 5Ј capped mRNAs. The effect of PI3K on dendrites was lost in more mature neurons (Ͼ14 d in vitro). Dendritic complexity was reduced by inhibition of PI3K and by RNAi knockdown of mTOR or p70 ribosomal S6 kinase (p70S6K, an effector of mTOR). A rapamycin-resistant mutant of mTOR "rescued" the morphogenetic effects of PI3K in the presence of rapamycin. By regulating global and/or local protein translation, and as a convergence point for multiple signaling pathways, mTOR could play a central role in the control of dendrite growth and branching during development and in response to activity.

Mammalian/mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that controls several important aspects of mammalian cell function. mTOR activity is modulated by various intra- and extracellular factors; in turn, mTOR changes rates of translation, transcription, protein degradation, cell signaling, metabolism, and cytoskeleton dynamics. mTOR has been repeatedly shown to participate in neuronal development and the proper functioning of mature neurons. Changes in mTOR activity are often observed in nervous system diseases, including genetic diseases (e.g., tuberous sclerosis complex, Pten-related syndromes, neurofibromatosis, and Fragile X syndrome), epilepsy, brain tumors, and neurodegenerative disorders (Alzheimer's disease, Parkinson's disease, and Huntington's disease). Neuroscientists only recently began deciphering the molecular processes that are downstream of mTOR that participate in proper function of the nervous system. As a result, we are gaining knowledge about the ways in which aberrant changes in mTOR activity lead to various nervous system diseases. In this review, we provide a comprehensive view of mTOR in the nervous system, with a special focus on the neuronal functions of mTOR (e.g., control of translation, transcription, and autophagy) that likely underlie the contribution of mTOR to nervous system diseases.

No abstract

Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubulebinding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin-3 (KIF1) and kinesin-4 (KIF21) subfamily that can also target dendrites. We found that microtubulebinding protein doublecortin-like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1-dependent dense-core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule-binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.

QZ1, 2-[2-chloro-6-hydroxy-3-oxo-5-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, and QZ2, 2-[6-hydroxy-3-oxo-4,5-bis-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, two fluorescein-based dyes derivatized with 8-aminoquinoline, have been prepared and their photophysical, thermodynamic, and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 have a large dynamic range, with 42-and 150-fold fluorescence enhancements upon Zn(II) coordination, respectively. These dyes have micromolar K d values for Zn(II) and are selective for Zn(II) over biologically relevant concentrations of the alkali and alkaline earth metals. The Zn(II) complexes also fluoresce brightly in the presence of excess Mn(II), Fe(II), Co(II), Cd(II), and Hg(II), offering improved specificity for Zn(II) over di(2-picolyl)amine-based Zn(II) sensors. Stopped-flow kinetic investigations indicate that QZ1 and QZ2 bind Zn(II) with k on values of (3-4) × 10 6 M -1 s -1 , compared to (6-8) × 10 5 M -1 s -1 for select ZP (Zinpyr) dyes, at 4.3 °C. Dissociation of Zn(II) from QZ1 and QZ2 occurs with k off values of 150 and 160 s -1 , over 5 orders of magnitude larger than those for ZP probes, achieving reversibility on the biological (millisecond) time scale. Laser scanning confocal and two-photon microscopy studies reveal that QZ2 is cell-permeable and Zn(II)-responsive in vivo. Because of its weaker affinity for Zn(II), QZ2 responds to higher concentrations of intracellular Zn (II) than members of the ZP family, illustrating that binding affinity is an important parameter for Zn(II) detection in vivo. (S5-S9); plots of k obs versus [Zn(II)] (S10-S14); Eyring plots (S15-S19); relative fluorescence emission of the Zn(II)-bound sensors (S20); and both confocal and two-photon microscopy images of biological samples (S21-S23); and experimental details for the representative kinetic analyses. This material is available free of charge via the Internet at

Matrix metalloproteinase-9 has recently emerged as an important molecule in control of extracellular proteolysis in the synaptic plasticity. However, no synaptic targets for its enzymatic activity had been identified before. In this report, we show that ␤-dystroglycan comprises such a neuronal activity-driven target for matrix metalloproteinase-9. This notion is based on the following observations. (i) Recombinant, autoactivating matrix metalloproteinase-9 produces limited proteolytic cleavage of ␤-dystroglycan. (ii) In neuronal cultures, ␤-dystroglycan proteolysis occurs in response to stimulation with either glutamate or bicuculline and is blocked by tissue inhibitor of metalloproteinases-1, a metalloproteinase inhibitor. (iii) ␤-Dystroglycan degradation is also observed in the hippocampus in vivo in response to seizures but not in the matrix metalloproteinase-9 knock-out mice. (iv) ␤-Dystroglycan cleavage correlates in time with increased matrix metalloproteinase-9 activity. (v) Finally, ␤-dystroglycan and matrix metalloproteinase-9 colocalize in postsynaptic elements in the hippocampus. In conclusion, our data identify the ␤-dystroglycan as a first matrix metalloproteinase-9 substrate digested in response to enhanced synaptic activity. This demonstration may help to understand the possible role of both proteins in neuronal functions, especially in synaptic plasticity, learning, and memory. Matrix metalloproteinases (MMPs)2 are a family of zinc-dependent endopeptidases acting outside the cells and therefore attributed with digesting extracellular matrix components. These enzymes are produced in a latent form, and after release to extracellular space, they are activated by cleavage off the propeptide (1, 2). MMPs are involved in a number of physiological and pathological conditions, including development, tissue remodeling, inflammation, and tumor metastasis (1-4). Specifically, multiple data show increased expression and activity of MMPs after brain injury and in certain diseases of the central nervous system (5). On the other hand, the physiological roles of MMPs in the adult brain have only recently been appreciated (4). In particular, MMP-9 (also known as gelatinase B) has been implicated in synaptic plasticity, learning, and memory (6, 7). Furthermore, a marked increase in MMP-9 mRNA protein and its enzymatic activity in the hippocampal dentate gyrus after kainate-evoked seizures has been shown (8). Kainate, a glutamate analog, produces excitotoxicity in the CA subfields of the hippocampus, sparing the granule neurons of the dentate gyrus that, however, undergo aberrant plastic changes (9).Despite data implicating MMP-9 in neuronal/synaptic plasticity, no synaptic targets for its enzymatic activity have as yet been identified in neurons. However, recent studies have suggested that this enzyme may digest the 43-kDa ␤-dystroglycan (␤-DG) to release a 30-kDa product from the full-length subunit. First, Yamada et al. (10) have shown that unidentified MMPs digest ␤-DG to reveal the 30-kDa product in the perip...