Publications (19) View all
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Article: Synaptic circuit abnormalities of motor-frontal layer 2/3 pyramidal neurons in an RNA interference model of methyl-CpG-binding protein 2 deficiency.
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ABSTRACT: Rett syndrome, an autism spectrum disorder with prominent motor and cognitive features, results from mutations in the gene for methyl-CpG-binding protein 2 (MeCP2). Here, to identify cortical circuit abnormalities that are specifically associated with MeCP2 deficiency, we used glutamate uncaging and laser scanning photostimulation to survey intracortical networks in mouse brain slices containing motor-frontal cortex. We used in utero transfection of short hairpin RNA constructs to knock down MeCP2 expression in a sparsely distributed subset of layer (L) 2/3 pyramidal neurons in wild-type mice, and compared input maps recorded from transfected-untransfected pairs of neighboring neurons. The effect of MeCP2 deficiency on local excitatory input pathways was severe, with an average reduction in excitatory synaptic input from middle cortical layers (L3/5A) of >30% compared with MeCP2-replete controls. MeCP2 deficiency primarily affected the strength, rather than the topography, of excitatory intracortical pathways. Inhibitory synaptic inputs and intrinsic eletrophysiological properties were unaffected in the MeCP2-knockdown neurons. These studies indicate that MeCP2 deficiency in individual postsynaptic cortical pyramidal neurons is sufficient to induce a pathological synaptic defect in excitatory intracortical circuits.Journal of Neuroscience 10/2009; 29(40):12440-8. · 7.11 Impact Factor -
Article: Subcellular dynamics of type II PKA in neurons.
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ABSTRACT: Protein kinase A (PKA) plays multiple roles in neurons. The localization and specificity of PKA are largely controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA in neurons and the roles of specific AKAPs are poorly understood. We imaged the distribution of type II PKA in hippocampal and cortical layer 2/3 pyramidal neurons in vitro and in vivo. PKA was concentrated in dendritic shafts compared to the soma, axons, and dendritic spines. This spatial distribution was imposed by the microtubule-binding protein MAP2, indicating that MAP2 is the dominant AKAP in neurons. Following cAMP elevation, catalytic subunits dissociated from the MAP2-tethered regulatory subunits and rapidly became enriched in nearby spines. The spatial gradient of type II PKA between dendritic shafts and spines was critical for the regulation of synaptic strength and long-term potentiation. Therefore, the localization and activity-dependent translocation of type II PKA are important determinants of PKA function.Neuron 06/2009; 62(3):363-74. · 14.74 Impact Factor -
Article: Amyloid-beta alters trafficking of internalized acetylcholinesterase and dextran.
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ABSTRACT: Amyloid-beta (Aβ), the main peptide constituent of senile plaques, is a suspected pathogenic mediator in Alzheimer's Disease (AD). Plaques also contain acetylcholinesterase (AChE), which may promote Aβ toxicity. We previously found that Aβ increased AChE levels in neuron-like N1E.115 neuroblastoma cells by reducing AChE degradation and surface shedding. Here we show that Aβ also alters the intracellular fate of surface AChE. When surface AChE was tagged with FITC-conjugated Fasciculin II (FasII), fluorescence gradually accumulated in intracellular particles. In the presence of extracellular Aβ this accumulation increased and shifted from the juxtanuclear zone to more peripheral cytoplasm. The cytoplasmic FasII-positive structures were positive for Lysosomal-Associated Membrane Protein 1, identifying them as late endosomes and early lysosomes. Thus, surface AChE trafficked into the lysosomal compartment, but further transport was impaired. Aβ also affected the transport or disposition of fluorescent dextran, an index of pinocytosis, and caused a 60% increase in intracellular accumulation similar to the lysosomotropic effects of chloroquine. On the other hand, Aβ caused no apparent changes in clathrin- and caveolae-mediated endocytosis. Overall it appears that selective alteration of endocytic mechanisms and an accumulation of organelles containing improperly processed substrates might contribute to the neuronal damage associated with age and disease-related accumulation of neurotoxic Aβ in the human brain.International Journal of Physiology, Pathophysiology and Pharmacology 01/2009; 1(1):15-24. -
Article: Calcineurin selectively docks with the dynamin Ixb splice variant to regulate activity-dependent bulk endocytosis.
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ABSTRACT: Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.Journal of Biological Chemistry 07/2011; 286(35):30295-303. · 4.77 Impact Factor -
Article: Glia.
Nature 11/2010; 468(7321):213. · 36.28 Impact Factor
