Biomedical subjects

S M Strittmatter

Publications and source records attributed to S M Strittmatter.

At least 37 records · Page 2Linked to original sources

PubMed1998

The role of nitric oxide and NMDA receptors in the development of motor neuron dendrites.

Nitric oxide (NO) has been implicated in the establishment of precise synaptic connectivity throughout the neuroaxis in several species. To determine the contribution of NO to NMDA receptor-dependent dendritic growth in motor neurons, we administered the NMDA antagonist MK-801 to wild-type mice and neuronal nitric oxide synthase (nNOS) knock-out mice between postnatal days 7 and 14. Compared to saline-treated wild-type animals the number of dendritic bifurcations was significantly reduced in nNOS knock-out animals and MK-801-treated wild-type animals. There was no significant difference in dendritic bifurcation between MK-801-treated wild-type, MK-801-treated nNOS knock-out, and saline-treated nNOS knock-out animals, suggesting that nNOS knock-out and NMDA receptor block had similar effects. The path of the longest dendrite and the number of primary dendrites was the same in all treatment groups, indicating an effect specific to bifurcation. Sholl analysis revealed that differences in bifurcation numbers occurred between 160 and 320 micrometers from the cell body, the distance at which second, third, and fourth order dendrites are most prevalent. Dendrite order analyses confirmed a significant reduction in numbers, but not lengths, of third and fourth order dendrites in nNOS knock-out and drug-treatment groups. Finally, immunohistochemical examination of the developing spinal cord indicated that NMDA receptors and nNOS are colocalized within interneurons surrounding the motor neuron pool. These results support the view that at least part of NMDA receptor-dependent arborization of motor neuron dendrites is mediated by the local production of NO within the developing spinal cord.

Animals↗

PubMed1998

Neuropilin-1 extracellular domains mediate semaphorin D/III-induced growth cone collapse.

Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). Here, semD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus- (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, we show that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1, the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, mu) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein that in turn transduces a semD signal into axon repulsion.

Animals↗

PubMed1998

Semaphorins A and E act as antagonists of neuropilin-1 and agonists of neuropilin-2 receptors.

Neuropilin-1 (NP-1) has been identified as a necessary component of a semaphorin D (SemD) receptor that repulses dorsal root ganglion (DRG) axons during development. SemA and SemE are related to SemD and bind to NP-1, but do not repulse DRG axons. By expressing NP-1 in retinal neurons and NP-2 in DRG neurons, we demonstrate that neuropilins are sufficient to determine the functional specificity of semaphorin responsiveness. SemA and SemE block SemD binding to NP-1 and abolish SemD repulsion in axons expressing NP-1. SemA and SemE seem to have a newly discovered protein antagonist capacity at NP-1 receptors, whereas they act as agonists at receptors containing NP-2.

Animals↗

PubMed1998

GAP-43 augmentation of G protein-mediated signal transduction is regulated by both phosphorylation and palmitoylation.

The neuronal protein GAP-43 is concentrated at the growth cone membrane, where it is thought to amplify the signal transduction process. As a model for its neuronal effects, GAP-43 protein injection into Xenopus laevis oocytes strongly augments the calcium-sensitive chloride current evoked by the G protein-coupled receptor stimulation. We have now examined a series of GAP-43 mutants in this system and determined those regions of GAP-43 required for this increase in current flux. As expected, palmitoylation inhibits signal amplification in oocytes by blocking G protein activation. Unexpectedly, a second domain of GAP-43 (residues 35-50) containing a protein kinase C phosphorylation site at residue 41 is also necessary for augmentation of G protein-coupled signals in oocytes. This region is not required for activation of isolated Go but is necessary for GAP-43 binding to isolated calmodulin and to isolated protein kinase C. Substitution of Asp for Ser41 inactivates GAP-43 as a signal facilitator in oocytes. This mutation blocks GAP-43 binding to both protein kinase C and calmodulin. Thus, GAP-43 regulates an oocyte signaling cascade via coordinated, simultaneous G protein activation and interaction with either calmodulin or protein kinase C.

Amino Acid Substitution↗

PubMed1997

Neuronal and non-neuronal collapsin-1 binding sites in developing chick are distinct from other semaphorin binding sites.

The collapsin and semaphorin family of extracellular proteins contributes to axonal path finding by repulsing axons and collapsing growth cones. To explore the mechanism of collapsin-1 action, we expressed and purified a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4). This protein retains biological activity as a DRG growth cone collapsing agent and saturably binds to DRG neurons with low nanomolar affinity. Specific CAP-4 binding sites are present on DRG neurons, sympathetic neurons, and motoneurons, but not on retinal, cortical, or brainstem neurons. Outside the nervous system, high levels of CAP-4 binding sites are present in the mesenchyme surrounding major blood vessels and developing bone and in lung. These sites provide a substrate for the collapsin-1-dependent patterning of non-neuronal tissues perturbed in sema III (-/-) mice. The staining patterns for mouse semaphorin D/III and chick collapsin-1 fusion proteins are indistinguishable from one another but quite separate from that for semaphorin B and M-semaphorin F fusion proteins. These data imply that a family of high-affinity semaphorin binding sites similar in complexity to the semaphorin ligand family exists.

Animals↗

PubMed1997

Rac1 mediates collapsin-1-induced growth cone collapse.

Collapsin-1 or semaphorin III(D) inhibits axonal outgrowth by collapsing the lamellipodial and filopodial structures of the neuronal growth cones. Because growth cone collapse is associated with actin depolymerization, we considered whether small GTP-binding proteins of the rho subfamily might participate in collapsin-1 signal transduction. Recombinant rho, rac1, and cdc42 proteins were triturated into embryonic chick (DRG) neurons. Constitutively active rac1 increases the proportion of collapsed growth cones, and dominant negative rac1 inhibits collapsin-1-induced collapse of growth cones and collapsin-1 inhibition of neurite outgrowth. DRG neurons treated with dominant negative rac1 remain sensitive to myelin-induced growth cone collapse. Similar mutants of cdc42 do not alter growth cone structure, neurite elongation, or collapsin-1 sensitivity. Whereas the addition of activated rho has no effect, the inhibition of rho with Clostridium botulinum C3 transferase stimulates the outgrowth of DRG neurites. C3 transferase-treated growth cones exhibit little or no lamellipodial spreading and are minimally responsive to collapsin-1 and myelin. These data demonstrate a prominent role for rho and rac1 in modulating growth cone motility and indicate that rac1 may mediate collapsin-1 action.

ADP Ribose Transferases↗

PubMed1997

A novel action of collapsin: collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse.

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons.

Animals↗

PubMed1997

Brain CRMP forms heterotetramers similar to liver dihydropyrimidinase.

The cytoplasmic collapsin response mediator protein CRMP62 is involved in the signaling cascade initiated by collapsin-1, which collapses neuronal growth cones. To investigate the mechanism of CRMP action, we screened mouse and human fetal cDNA libraries by the yeast two-hybrid method with CRMP as bait. Clones encoding CRMP1 and CRMP4 were isolated, suggesting that the CRMPs form multimers. This finding was confirmed by expressing various rat CRMP cDNAs in the yeast two-hybrid system. Rat CRMP isoforms show differential association with one another. Heterooligomerization is preferred in both two-hybrid and in vitro binding assays. Purified bovine brain CRMP migrates as a tetramer during size exclusion chromatography. Examination of binding with truncated forms of CRMPs indicates that the avid association of CRMPs requires nearly intact proteins. Through the analysis of CRMP chimeras, CRMP amino acids 8-134 and 281-435 are found to be essential for CRMP oligomerization. The tetrameric structure of CRMP resembles that of liver dihydropyrimidinase (DHPase), a protein that shares sequence similarity with the CRMPs. Although purified brain CRMP does not hydrolyze several DHPase substrates, it is likely that a related activity accounts for CRMP participation in neuronal growth cone signaling.

Amidohydrolases↗

PubMed1996

A family of rat CRMP genes is differentially expressed in the nervous system.

Members of the collapsin/semaphorin family play an important role in creating the complex pattern of neuronal connectivity. Inhibition of growth cone motility by chick collapsin is mediated by the intraneuronal protein CRMP-62. We have now isolated four rat sequences that are highly related to chick CRMP-62. All four genes are expressed exclusively in the nervous system and primarily during development. Rat CRMP-2/TOAD-64 is most closely related to chick CRMP-62 and is the most widely expressed CRMP within the nervous system. Rat CRMP-1 and CRMP-4/rUlip are expressed during discrete periods of neuronal development and are not found in the adult nervous system. Rat CRMP-3 has a distinct distribution, being expressed transiently in developing spinal cord and selectively in the postnatal cerebellum. The differential expression of these genes suggests that CRMPs may transduce signals from different semaphorins and that semaphorins may regulate the plasticity of the adult nervous system.

Aging↗

PubMed1996

P2Y1 purinergic receptors in sensory neurons: contribution to touch-induced impulse generation.

Somatic sensation requires the conversion of physical stimuli into the depolarization of distal nerve endings. A single cRNA derived from sensory neurons renders Xenopus laevis oocytes mechanosensitive and is found to encode a P2Y1 purinergic receptor. P2Y1 mRNA is concentrated in large-fiber dorsal root ganglion neurons. In contrast, P2X3 mRNA is localized to small-fiber sensory neurons and produces less mechanosensitivity in oocytes. The frequency of touch-induced action potentials from frog sensory nerve fibers is increased by the presence of P2 receptor agonists at the peripheral nerve ending and is decreased by the presence of P2 antagonists. P2X-selective agents do not have these effects. The release of ATP into the extracellular space and the activation of peripheral P2Y1 receptors appear to participate in the generation of sensory action potentials by light touch.

Adenosine Triphosphate↗

PubMed1995

Collapsin-induced growth cone collapse mediated by an intracellular protein related to UNC-33.

Collapsin, a member of the newly recognized semaphorin family, contributes to axonal pathfinding during neural development by inhibiting growth cone extension. The mechanism of collapsin action is poorly understood. Here we use a Xenopus laevis oocyte expression system to identify molecules involved in collapsin signalling, because several experiments have raised the possibility that heterotrimeric GTP-binding proteins might participate in these events. A collapsin response mediator protein of relative molecular mass (M(r)) 62K (CRMP-62) required for collapsin-induced inward currents in X. laevis oocytes is isolated. CRMP-62 shares homology with UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. CRMP-62 is localized exclusively in the developing chick nervous system. Introduction of anti-CRMP-62 antibodies into dorsal root ganglion neurons blocks collapsin-induced growth cone collapse. CRMP-62 appears to be an intracellular component of a signalling cascade initiated by an unidentified transmembrane collapsin-binding protein.

Amino Acid Sequence↗

PubMed1995

An activated mutant of the alpha subunit of G(o) increases neurite outgrowth via protein kinase C.

The GTP-binding protein, G(o), is present at very high concentration in the neuronal growth cone membrane. The expression of activated mutants of the a subunit of G(o) increases neurite outgrowth. To determine the intracellular mechanism for this outgrowth, we have examined activated alpha o-dependent outgrowth in the presence of agents which modulate different signal transduction cascades. Activation of protein kinase C with phorbol esters or with diacylglycerol prevents the alpha o-dependent increase in neurite extension. Inhibition of protein kinase C with staurosporine, with H7, or with long-term, high dose phorbol ester treatment resulted in greater neurite elongation, and no further increase after activated alpha o transfection. The protein phosphatase inhibitor, okadaic acid, also blocked the effect of activated alpha o. In contrast, tyrosine kinase inhibitors and agents which alter cAMP levels did not alter activated alpha o-dependent neurite extension. We tested a number of compounds which alter intracellular calcium levels. TMB-8 and thapsigargin prevented an increase in outgrowth by activated alpha o, but diltiazem, Bay K8644 and dantrolene had no effect on activated alpha o-dependent outgrowth. These studies suggest that activated alpha o increases neurite outgrowth by inhibiting protein kinase C and by modulating intracellular calcium release.

Alkaloids↗

PubMed1995

Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43.

GAP-43 has been termed a "growth" or "plasticity" protein because it is expressed at high levels in neuronal growth cones during development and during axonal regeneration. By homologous recombination, we generated mice lacking GAP-43. The mice die in the early postnatal period. GAP-43-deficient retinal axons remain trapped in the chiasm for 6 days, unable to navigate past this midline decision point. Over the subsequent weeks of life, most GAP-43-deficient axons do enter the appropriate tracts, and the adult CNS is grossly normal. There is no evidence for interference with nerve growth rate, and cultured neurons extend neurites and growth cones in a fashion indistinguishable from controls. Thus, the GAP-43 protein is not essential for axonal outgrowth or growth cone formation per se, but is required at certain decision points, such as the optic chiasm. This is compatible with the hypothesis that GAP-43 serves to amplify pathfinding signals from the growth cone.

Animals↗

PubMed1994

An intrinsic guanine nucleotide exchange inhibitor in Gi2 alpha. Significance of G-protein self-suppression which antagonizes receptor signal.

The alpha subunit of Gi2 (Gi2 alpha) is a member of the heterotrimeric G protein family, which transduces receptor signals as a proto-oncogene product. We have found a novel self-suppressive region in Gi2 alpha near its C terminus. A polypeptide consisting of residues 338-352 of Gi2 alpha (Gi2 alpha-339-352) antagonizes receptor- and receptor peptide-stimulated Gi2 alpha activation, without affecting basal activity. Antagonism by Gi2 alpha-338-352 is attributable to an interaction with activated Gi2 alpha, which is not competitive with receptor polypeptides. Combined with the reports suggesting the presence of self-suppressive domains in a juxta-C-terminal portion of Gi2 alpha and G(o) alpha, this study supports the hypothesis that Gi2 alpha-338-352 constitutes an intrinsic guanine nucleotide exchange inhibitor, which in turn antagonizes receptor stimulation, suggesting that G proteins are activated by receptors through relaxation of a self-suppressive conformation.

Amino Acid Sequence↗

PubMed1994

An amino-terminal domain of the growth-associated protein GAP-43 mediates its effects on filopodial formation and cell spreading.

GAP-43 is a neuronal protein that is believed to be important to neuronal growth and nerve terminal plasticity. It is enriched on the inner surface of growth cone membranes, a localization that may depend upon palmitoylation of Cys3 and Cys4. It is a major substrate for protein kinase C, which phosphorylates Ser41. Isolated GAP-43 can bind to actin and to calmodulin, and can activate the heterotrimeric GTP-binding proteins, G(o) and Gi. A peptide consisting of the GAP-43 sequence 39-55 binds calmodulin, and an amino-terminal GAP-43 (1-10) peptide activates G(o), suggesting that these stretches may be functional domains of the intact protein. When expressed in non-neuronal cells, GAP-43 enhances filopodial extension and has effects upon cell spreading. We have examined the effects of various GAP-43 domains upon this assay, by expression of GAP-43, GAP-43 mutant proteins, and GAP-43-CAT fusion proteins in COS-7 cells. We find that the amino terminus (Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln) is an important contributor to these effects on cell shape. A GAP-43 protein mutant in Cys3 and Cys4 does not bind to the membrane, and is inactive. Mutants in Arg6 or Lys9 also are inactive, although they remain localized to particulate fractions; Arg7 mutants are active. A chimeric gene consisting of GAP-43 (1-10) fused to chloramphenicol acetyl transferase (CAT) also causes cell shape changes. As for GAP-43, the effects of this fusion protein are abolished by mutations of Cys3, Cys4, Arg6 or Lys9, but not by mutation of Arg7. Therefore, the cell surface activity of transfected GAP-43 depends upon its amino terminus, although other domains may regulate it in this regard. Since the amino-terminal domain includes the peptide stretch known to be capable of activating G(o) and Gi, we examined the effect of GAP-43 on a Gi-regulated second messenger system, the inhibition of cAMP production in A431 cells. A431 cells stably transfected with GAP-43 spread less well than do controls. In addition, they evidence decreased levels of forskolin-stimulated cAMP, consistent with chronic stimulation of Gi. Stimulation of adenylate cyclase by isoproterenol reverses the GAP-43-induced changes in cell shape. This suggests that G protein stimulation is involved in GAP-43 effects upon cell shape.

Amino Acid Sequence↗

PubMed1994

GAP-43 amino terminal peptides modulate growth cone morphology and neurite outgrowth.

The neuronal growth-associated protein GAP-43 is expressed maximally during development and regeneration, and is enriched at the cytosolic surface of the growth cone membrane. GAP-43 can activate the GTP-binding protein G(o) which is also a major component of the growth cone membrane. These findings have led to the hypothesis that GAP-43 might modulate neurite outgrowth by altering G-protein activity. Here we define the sequence requirements for GAP-43 amino terminal peptide stimulation of G(o), and test these peptides as potential modulators of neurite outgrowth. The first 10 amino acids of GAP-43, Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln, stimulate G(o). Substitutions at particular residues reveal that cys3, cys4, arg6, and lys9 are critical, but arg7 is not. Both the GAP-43(1-10) peptide and the G-protein-activating peptide mastoparan induce growth cone collapse and inhibit neurite extension from embryonic chick dorsal root ganglion and retinal neurons. This is likely to be mediated by G-proteins: pertussis toxin blocks the inhibition, and mutant peptides that do not activate G(o) do not alter outgrowth. In contrast to the case with embryonic chick dorsal root ganglion cells, neurite outgrowth from N1E-115 neuroblastoma cells is stimulated by GAP-43(1-10). This is probably also a G-protein-mediated event because it is blocked by pertussis toxin, because the sequence requirements match those for G(o) stimulation, and because mastoparan stimulates outgrowth from these cells. The longer GAP-43(1-25) peptide does not alter neurite outgrowth unless the cells are permeabilized, suggesting an intracellular site of action. These data identify a novel set of compounds that modulate neurite outgrowth, and also support the notion that GAP-43 can alter neurite extension by modulating pertussis toxin-sensitive G-protein activity in the growth cone.

Amino Acid Sequence↗

PubMed1994

Activated mutants of the alpha subunit of G(o) promote an increased number of neurites per cell.

The high concentration of the GTP-binding protein G(o) in the neuronal growth cone suggests that G(o) activation state may after neurite outgrowth. We find that activation of pertussis toxin-sensitive G-proteins by mastoparan increases neurite outgrowth from neuroblastoma cells. To examine G(o) activation specifically, point mutations homologous to activating, oncogenic mutations in alpha i2 and alpha s were introduced into the alpha subunit of G(o). The stability of the alpha o mutants to tryptic digestion confirms that they are activated. When expressed in PC12 or N1E-115 cells, activated alpha o doubles total neurites length per cell, primarily by increasing the number of neurites per cell. The growth cones of cells expressing activated alpha o are narrower than control growth cones. Expression of wildtype alpha o or the activated alpha subunits of other G-proteins did not affect total neurite length per cell. Thus, factors that lead to activation of G(o) can modulate neurite number per cell.

Amino Acid Sequence↗

PubMed1993

GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction.

Acetylcholine↗