Familiar growth factors have diverse roles in neural network assembly.

The assembly of neuronal circuits during development depends on guidance of axonal growth cones by molecular cues deposited in their environment. While a number of families of axon guidance molecules have been identified and reviewed, important and diverse activities of traditional growth factors are emerging. Besides clear and well recognized roles in the regulation of cell division, differentiation and survival, new research shows later phase roles for a number of growth factors in promoting neuronal migration, axon guidance and synapse formation throughout the nervous system.

Growth Factors as Axon Guidance Molecules: Lessons From in vitro Studies.

Growth cones at the tips of extending axons navigate through developing organisms by probing extracellular cues, which guide them through intermediate steps and onto final synaptic target sites. Widespread focus on a few guidance cue families has historically overshadowed potentially crucial roles of less well-studied growth factors in axon guidance. In fact, recent evidence suggests that a variety of growth factors have the ability to guide axons, affecting the targeting and morphogenesis of growth cones in vitro. This review summarizes in vitro experiments identifying responses and signaling mechanisms underlying axon morphogenesis caused by underappreciated growth factors.

Cell adhesion and invasion mechanisms that guide developing axons.

Axon extension, guidance and tissue invasion share many similarities to normal cell migration and cancer cell metastasis. Proper cell and growth cone migration requires tightly regulated adhesion complex assembly and detachment from the extracellular matrix (ECM). In addition, many cell types actively remodel the ECM using matrix metalloproteases (MMPs) to control tissue invasion and cell dispersal. Targeting and activating MMPs is a tightly regulated process, that when dysregulated, can lead to cancer cell metastasis. Interestingly, new evidence suggests that growth cones express similar cellular and molecular machinery as migrating cells to clutch retrograde actin flow on ECM proteins and target matrix degradation, which may be used to facilitate axon pathfinding through the basal lamina and across tissues.

Density of α4ß2* nAChR on the surface of neurons is modulated by chronic antagonist exposure.

The expression of high-affinity α4ß2* nicotinic acetylcholine receptors (nAChR) increases following chronic exposure to nicotinic agonists. While, nAChR antagonists can also produce upregulation, these changes are often less pronounced than achieved with agonists. It is unknown if nAChR agonists and antagonists induce receptor upregulation by the same mechanisms. In this study, primary neuronal cultures prepared from cerebral cortex, hippocampus, diencephalon, and midbrain/hindbrain of C57BL/6J mouse embryos were treated chronically with nicotine (agonist), mecamylamine (noncompetitive antagonist) or dihydro-ß-erythroidine (competitive antagonist) or the combination of nicotine with each antagonist. The distribution of intracellular and surface [(125)I]epibatidine-binding sites were subsequently measured. Treatment with 1 µmol/L nicotine upregulated intracellular and cell surface [(125)I]epibatidine binding after 96 h. Chronic dihydro-ß-erythroidine (10 µmol/L) treatment also increased [(125)I]epibatidine binding on the cell surface; however, mecamylamine was ineffective in upregulating receptors by itself. The combination of 1 µmol/L nicotine plus 10 µmol/L mecamylamine elicited a significantly higher upregulation than that achieved by treatment with nicotine alone due to an increase of [(125)I]epibatidine binding on the cell surface. This synergistic effect of mecamylamine and nicotine was found in neuronal cultures from all four brain regions. Chronic treatment with nicotine concentrations as low as 10 nmol/L produced upregulation of [(125)I]epibatidine binding. However, the effect of mecamylamine was observed only after coincubation with nicotine concentrations equal to or greater than 100 nmol/L. Vesicular trafficking was required for both nicotine and nicotine plus mecamylamine-induced upregulation. Results presented here support the idea of multiple mechanisms for nAChR upregulation.

Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the ß(+)/α(-) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3ß2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-ß2D190, involving the C loop region of the ß2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-ß2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-ß2A127 and α3E173-ß2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation.

Chronic treatment with varenicline changes expression of four nAChR binding sites in mice.

INTRODUCTION: Chronic treatment with nicotine is known to increase the α4ß2-nAChR sites in brain, to decrease α6ß2-nAChR sites and to have minimal effect on α3ß4-and α7-nAChR populations. Varenicline is now used as a smoking cessation treatment, with and without continued smoking or nicotine replacement therapy. Varenicline, like nicotine, upregulates the α4ß2-nAChR sites; however, it is not known whether varenicline treatment changes expression of the other nAChR subtypes. METHODS: Using a mouse model, chronic treatments (10 days) with varenicline (0.12  mg/kg/h) and/or nicotine (1 mg/kg/hr), alone or in combination, were compared for plasma and brain levels of drugs, tolerance to subsequent acute nicotine and expression of four subtypes of nAChR using autoradiography. RESULTS: The upregulation of α4ß2-nAChR sites elicited by chronic varenicline was very similar to that elicited by chronic nicotine. Treatment with both drugs somewhat increased up-regulation, indicating that these doses were not quite at maximum effect. Similar down-regulation was seen for α6ß2-nAChR sites. Varenicline significantly increased both α3ß4-and α7-nAChR sites while nicotine had less effect on these sites. The drug combination was similar to varenicline alone for α3ß4-nAChR sites, while for α7 sites the drug combination was less effective than varenicline alone. Varenicline had small but significant effects on tolerance to acute nicotine. CONCLUSIONS: Effects of varenicline in vivo may not be limited to the α4ß2*-nAChR subtype. In addition, smoking cessation treatment with varenicline may not allow receptor numbers to be restored to baseline and may, in addition, change expression of other receptor subtypes.