Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolinbinding motif, a coiled-coil structure, a calmodulinbinding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234 -243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca 2؉ . In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.Striatin is an intracellular protein mostly present in neurons of mammalian basal ganglia and cranial and spinal motor nuclei (1, 2). Electron microscopy showed that it is present in the somato-dendritic compartment of neurons, especially in dendritic spines (1). Brain fractionation shows that striatin is both cytosolic and associated with membranes. This multimodular protein possesses, from the N to the C terminus, four domains mediating protein-protein interactions: a caveolinbinding domain (aa 1 55-63) (3), a putative coiled-coil structure (aa 70 -116), a Ca 2ϩ -calmodulin (CaM)-binding domain (aa 149 -166) (4),and a WD repeat domain (aa 419 -780). The WD repeat family is composed of homologous, structurally related, but functionally diverse proteins able to organize multiple simultaneous or consecutive protein-protein interactions (5). The richness of striatin in domains mediating protein-protein interactions suggests that striatin is both a signaling protein and a multimodular platform protein. A study aimed at elucidating the function of striatin revealed two sets of data demonstrating its central role both in embryonic neurons and in adult brain (6). On the one hand, we showed that the expression of striatin is essential for the maintenance and growth of dendrites in rat embryonic motoneurons in culture. On the other hand, we showed that striatin is involved in the control of motor function in adult rats. Albeit a quantitatively minor protein, striatin thus appears to play major cellular and physiological roles.We have previously reported that the sequence of SG2NA, a 713 aa, supposedly nuclear protein discovered by Muro et al. (7), is 80% similar to and 66% identical...

Abstract.A rat brain synaptosomal protein of 110,000

The axon initial segment (AIS) plays a key role in maintaining the molecular and functional polarity of the neuron. The relationship between the AIS architecture and the microtubules (MTs) supporting axonal transport is unknown. Here we provide evidence that the MT plus-end-binding (EB) proteins EB1 and EB3 have a role in the AIS in addition to their MT plus-end tracking protein behavior in other neuronal compartments. In mature neurons, EB3 is concentrated and stabilized in the AIS. We identified a direct interaction between EB3/EB1 and the AIS scaffold protein ankyrin G (ankG). In addition, EB3 and EB1 participate in AIS maintenance, and AIS disassembly through ankG knockdown leads to cell-wide up-regulation of EB3 and EB1 comets. Thus, EB3 and EB1 coordinate a molecular and functional interplay between ankG and the AIS MTs that supports the central role of ankG in the maintenance of neuronal polarity. N eurons are highly polarized cells that rely on microtubules (MTs) for maintenance of their architecture and long-range polarized trafficking (1). MTs supporting axonal transport travel through the axon initial segment (AIS), a compartment that is essential for the generation of action potentials (2) and the maintenance of neuronal polarity (3). Generation of action potentials depends on the concentration of voltage-gated sodium (Nav) and potassium channels at the AIS, which are tethered at the plasma membrane via their interaction with ankyrin G (ankG (4-7). ankG, in turn, is linked to the actin cytoskeleton via βIV-spectrin and organizes AIS formation by recruiting membrane proteins and βIV-spectrin to the nascent AIS (3).The AIS maintains neuronal polarity by forming a diffusion barrier for membrane constituents at the cell surface (4,8,9) and also by dampening intracellular diffusion and vesicular transport through the AIS (10). Both phenomena depend on ankG, because depletion of ankG results in the disappearance of AIS and the acquisition of dendritic identity by the proximal axon (11,12). However, the molecular role of ankG in the intracellular AIS organization is still unknown. The dependence of the AIS intracellular filter on ankG (10) and the disorganization of MT bundles in the AIS of Purkinje cells from ankG-deficient mice (12) suggest an unknown link between ankG and MTs in the AIS.The end-binding (EB) proteins family, composed of three members (EB1-3), has been described as plus-end-tracking proteins (+TIPs) that coordinate a network of dynamic proteins on the growing MT plus-ends (13). In neurons, EB1 has been implicated in axonal transport (14, 15), whereas EB3 has been characterized as a molecular link between MTs and the actin cytoskeleton (16, 17). We hypothesized that EB proteins could have a role in the AIS via interaction with the ankG/βIV-spectrin scaffold. We first found that EB3 is accumulated and stabilized in the AIS of mature neurons. Both EB3 and EB1 bind to ankG and participate in the maintenance of the AIS scaffold. Reciprocally, altering neuronal polarity through ankG knockdown in...

The glia-derived, neurotrophic protein S100␤ has been implicated in development and maintenance of the nervous system. However, S100␤ has also been postulated to play a role in mechanisms of neuropathology, because of its specific localization and selective overexpression in Alzheimer's disease. To begin to address the question of whether S100␤ can induce potentially toxic signaling pathways, we examined the effects of the protein on nitric oxide synthase (NOS) activity in cultures of rat cortical astrocytes. S100␤ treatment of astrocytes induced a time-and dose-dependent increase in accumulation of the NO metabolite, nitrite, in the conditioned medium. The S100␤-stimulated nitrite production was blocked by cycloheximide and by the NOS inhibitor N-nitro-L-arginine methylester, but not by the inactive D-isomer of the inhibitor. Direct measurement of NOS enzymatic activity in cell extracts and analysis of NOS mRNA levels showed that the NOS activated by S100␤ addition is the calcium-independent, inducible isoform. Furthermore, the specificity of the effects of S100␤ on activation of NOS was demonstrated by the inability of S100␣ and calmodulin to induce an increase in nitrite levels. Our data indicate that S100␤ can induce a potent activation of inducible NOS in astrocytes, an observation that might have relevance to the role of S100␤ in neuropathology.The normal development and maintenance of the brain involves the temporal and spatial coordination and proper functioning of a number of intracellular and cell-cell signaling events, and the contribution of glial cells to these signaling processes is becoming more widely appreciated. The classical concept of the role of glia in brain function is rapidly changing with newer evidence of the crucial nature of these cells in controlling neurotransmitter levels, maintaining calcium homeostasis, and synthesizing and releasing neurotrophic and growth factors (for review, see Ref. 1). One such glia-derived factor is S100␤, a protein that promotes neuritic outgrowth of specific neuronal populations (e.g. cortical (2, 3), dorsal root ganglia (4), serotonergic (5, 6), and motoneurons (7)) and enhances survival of neurons during development (7,8) and after insult (9). S100␤ is also a glial mitogen, inducing phosphoinositide hydrolysis, increases in intracellular calcium, and protooncogene expression (10, 11). These trophic functions require nanomolar concentrations of a disulfide-linked S100␤ dimer (see Ref. 12). Thus, S100␤ may be beneficial during development of the nervous system, and increased S100␤ expression and secretion following acute glial activation in response to central nervous system injury may be one mechanism the brain uses in attempts to repair injured neurons.However, S100␤ may also reach concentrations that are deleterious, e.g. in neurodegenerative diseases like Alzheimer's disease and Down syndrome where chronic glial activation occurs (13). It has been found that S100␤ levels in severely affected brain regions of Alzheimer's disease patients are severalfol...

Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity between Drosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulinbinding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.

No abstract

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolinbinding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein^protein association domains, among which a putative caveolin-binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970^19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca 2 -dependent signalling proteins [Bartoli et al. (1999)

The axon initial segment (AIS) is a highly specialized neuronal compartment that plays a key role in neuronal development and excitability. It concentrates multiple membrane proteins such as ion channels and cell adhesion molecules (CAMs) that are recruited to the AIS by the scaffold protein ankyrin G (ankG). The crucial function of ankG in the anchoring of AIS membrane components is well established, but a reciprocal role of membrane partners in ankG targeting and stabilization remained elusive. In rat cultured hippocampal neurons and cortical organotypic slices, we found that shRNA-mediated knockdown of ankG membrane partners (voltage-gated sodium channels (Nav) or neurofascin-186) led to a decrease of ankG concentration and perturbed the AIS formation and maintenance. These effects were rescued by expressing a recombinant AIS-targeted Nav or by a minimal construct containing the ankyrin-binding domain of Nav1.2 and a membrane anchor (mABD). Moreover, overexpressing mABD in mature neurons led to ankG mislocalization. Altogether, these results demonstrate that a tight and precocious association of ankG with its membrane partners is a key step for the establishment and maintenance of the AIS.