Alain Burette

The Rac1-GEF Tiam1 Couples the NMDA Receptor to the Activity-Dependent Development of Dendritic Arbors and Spines

NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. We report that the Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with RNAi and dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines.

NGL family PSD-95–interacting adhesion molecules regulate excitatory synapse formation

Synaptic cell adhesion molecules (CAMs) regulate synapse formation through their trans-synaptic and heterophilic adhesion. Here we show that postsynaptic netrin-G ligand (NGL) CAMs associate with netrin-G CAMs in an isoform-specific manner and, through their cytosolic tail, with the abundant postsynaptic scaffold postsynaptic density–95 (PSD-95). Overexpression of NGL-2 in cultured rat neurons increased the number of PSD-95–positive dendritic protrusions. NGL-2 located on heterologous cells or beads induced functional presynaptic differentiation in contacting neurites. Direct aggregation of NGL-2 on the surface membrane of dendrites induced the clustering of excitatory postsynaptic proteins. Competitive inhibition by soluble NGL-2 reduced the number of excitatory synapses. NGL-2 knockdown reduced excitatory, but not inhibitory, synapse numbers and currents. These results suggest that NGL regulates the formation of excitatory synapses.

Regulation of Dendritic Spine Morphogenesis by Insulin Receptor Substrate 53, a Downstream Effector of Rac1 and Cdc42 Small GTPases

The small GTPases Rac1 and Cdc42 are key regulators of the morphogenesis of actin-rich dendritic spines in neurons. However, little is known about how activated Rac1/Cdc42 regulates dendritic spines. Insulin receptor substrate 53 (IRSp53), which is highly expressed in the postsynaptic density (PSD), is known to link activated Rac1/Cdc42 to downstream effectors for actin regulation in non-neural cells. Here, we report that IRSp53 interacts with two specific members of the PSD-95 family, PSD-95 and chapsyn-110/PSD-93, in brain. An IRSp53 mutant lacking the C-terminal PSD-95-binding motif shows significant loss of synaptic localization in cultured neurons. Overexpression of IRSp53 in cultured neurons increases the density of dendritic spines but does not affect their length or width. Conversely, short-interfering RNA-mediated knock-down of IRSp53 reduces the density, length, and width of spines. In addition, the density and size of spines are decreased by a dominant-negative IRSp53 with a point mutation in the Src homology 3 (SH3) domain and a dominant-negative proline-rich region of WAVE2 (Wiskott-Aldrich syndrome protein family Verprolin-homologous protein), a downstream effector of IRSp53 that binds to the SH3 domain of IRSp53. These results suggest that PSD-95 interaction is an important determinant of synaptic IRSp53 localization and that the SH3 domain of IRSp53 links activated Rac1/Cdc42 to downstream effectors for the regulation of spine morphogenesis.

A Critical Role for Myosin IIB in Dendritic Spine Morphology and Synaptic Function

Dendritic spines show rapid motility and plastic morphology, which may mediate information storage in the brain. It is presently believed that polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Here, we show that myosin IIB, a molecular motor that binds and contracts actin filaments, is essential for normal spine morphology and dynamics and represents a distinct biophysical pathway to control spine size and shape. Myosin IIB is enriched in the postsynaptic density (PSD) of neurons. Pharmacologic or genetic inhibition of myosin IIB alters protrusive motility of spines, destabilizes their classical mushroom-head morphology, and impairs excitatory synaptic transmission. Thus, the structure and function of spines is regulated by an actin-based motor in addition to the polymerization state of actin.

Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections

Presynaptic proteins are readily identified by light microscopic immunocytochemistry, but immunodetection of postsynaptic proteins in brain sections proves difficult. We performed immunofluorescent double labeling for the NR1 subunit of the N‐methyl‐D‐aspartate receptor (NMDAR) and the vesicular glutamate transporter 1 (VGLUT1). In material fixed with 4% paraformaldehyde, NMDAR staining in somatosensory cortex was restricted to the section surface, whereas presynaptic staining extended deeper into the tissue. Staining for postsynaptic proteins was enhanced in weakly fixed material and in tissue treated with pepsin, as previously reported, but tissue quality was impaired. Staining was also markedly enhanced, and without impairment of tissue quality, by treatment during perfusion with a mixture of inhibitors of proteases and the ubiquitin/proteosome system. We performed quantitative analysis of confocal images to study how immunostaining varies with depth into the tissue. Virtually all puncta immunopositive for VGLUT1 colocalized with synaptophysin puncta; these presynaptic puncta were most numerous 1–2 μm beneath the section surface. In contrast, puncta immunopositive for the NR1 subunit were most numerous at the surface, as were puncta immunopositive for the NR2 subunit, SynGAP, and CaMKII. Punctate staining for all postsynaptic proteins, but not presynaptic markers, was substantially enhanced in material pretreated with antiproteolytic agents. The large majority of NR1‐positive puncta at the surface associated with VGLUT1 in this material are likely to represent synaptic contacts. Approximately eighty‐five percent of VGLUT1‐positive puncta in layers II–III of SI are associated with NR1‐positive puncta, and ∼80% are associated with NR2, SynGAP, and CaMKII. This approach may permit systematic analysis of the chemistry of glutamatergic synapses with light microscopic immunocytochemistry. J. Comp. Neurol. 492:495–509, 2005.

SAP97 concentrates at the postsynaptic density in cerebral cortex

SAP97, a PDZ‐containing protein, is reported to concentrate in axon terminals, where its function remains unknown. Using highly specific new antibodies, we show that SAP97 in rat cerebral cortex is associated with heteromeric AMPA receptors via a selective biochemical interaction between SAP97 and the GluR1 subunit. Using light and electron microscopic immunocytochemistry, we demonstrate cellular and synaptic colocalization of SAP97 and GluR1, and show that SAP97 concentrates at synapses that contain GluR1 but not necessarily GluR2 or GluR3. Using quantitative postembedding immunogold electron microscopy, we find that SAP97 is at highest concentration within the postsynaptic density of asymmetric synapses. These data suggest that SAP97 may help to anchor GluR1‐containing AMPA receptors at the synapse. As a multifunctional scaffolding protein, SAP97 may organize components of AMPA‐related intracellular signalling pathways, including those associated with calcium‐permeable homomeric GluR1 channels.

Maternal Loss of Ube3a Produces an Excitatory/Inhibitory Imbalance through Neuron Type-Specific Synaptic Defects

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of the maternally inherited allele of UBE3A. AS model mice, which carry a maternal Ube3a null mutation (Ube3a(m-/p+)), recapitulate major features of AS in humans, including enhanced seizure susceptibility. Excitatory neurotransmission onto neocortical pyramidal neurons is diminished in Ube3a(m-/p+) mice, seemingly at odds with enhanced seizure susceptibility. We show here that inhibitory drive onto neocortical pyramidal neurons is more severely decreased in Ube3a(m-/p+) mice. This inhibitory deficit follows the loss of excitatory inputs and appears to arise from defective presynaptic vesicle cycling in multiple interneuron populations. In contrast, excitatory and inhibitory synaptic inputs onto inhibitory interneurons are largely normal. Our results indicate that there are neuron type-specific synaptic deficits in Ube3a(m-/p+) mice despite the presence of Ube3a in all neurons. These deficits result in excitatory/inhibitory imbalance at cellular and circuit levels and may contribute to seizure susceptibility in AS.

Assembly of a β2‐adrenergic receptor—GluR1 signalling complex for localized cAMP signalling

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the β2‐adrenergic receptor (β2AR), the trimeric Gs protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA‐type glutamate receptor subunit GluR1, which is linked to the β2AR through stargazin and PSD‐95 and their homologues. Only GluR1 associated with the β2AR is phosphorylated by PKA on β2AR stimulation. Peptides that interfere with the β2AR–GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic β2AR–cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.

Distribution of soluble guanylyl cyclase in the rat brain

The diffusible messenger nitric oxide (NO) acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprising α and β subunits. We used immunohistochemistry to study the distribution of both sGC subunits in the brain of adult rats. α and β subunits gave similar widespread staining throughout the CNS, which was strongest in neostriatum, olfactory tubercle, and supraoptic nucleus. Double‐labeling experiments showed striking cellular colocalization in most brain regions, suggesting that the two subunits may be organized into enzymatically active α/β heteromers. Mismatches were observed in cerebellar cortex: Purkinje cells and Bergmann glia were positive for both subunits, whereas granule cells and interneurons in the molecular layer were strongly immunopositive for β but only weakly stained for the α subunit. By using multiple labeling, we compared the localization of sGC with neuronal nitric oxide synthase (NOS‐I, the NO‐producing enzyme in neurons). In forebrain, the distribution of sGC and NOS‐I was complementary, with only occasional colocalization. In contrast, cellular colocalization was common in midbrain and cerebellum. These data support a widespread role for the NO/sGC/cGMP pathway in the CNS and suggest that, in addition to its role as paracrine messenger, NO may also be an intracellular autocrine agent. J. Comp. Neurol. 472:437–448, 2004.

Isoform-specific distribution of the plasma membrane Ca2+ ATPase in the rat brain

Regulation of cytoplasmic calcium is crucial both for proper neuronal function and cell survival. The concentration of Ca2+ in cytoplasm of a neuron at rest is 10,000 times lower than in the extracellular space, pointing to the importance of the transporters that extrude intracellular Ca2+. The family of plasma membrane calcium‐dependent ATPases (PMCAs) represent a major component of the Ca2+ regulatory system. However, little information is available on the regional and cellular distribution of these calcium pumps. We used immunohistochemistry to investigate the distribution of each of the four PMCA isoforms (PMCA1–4) in the rat brain. Each isoform exhibited a remarkably precise and distinct pattern of distribution. In many cases, PMCA isoforms in a single brain structure were differentially expressed within different classes of neurons, and within different subcellular compartments. These data show that each isoform is independently organized and suggest that PMCAs may play a more complex role in calcium homeostasis than generally recognized. J. Comp. Neurol. 467:464–476, 2003.