Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, we studied the attractive responses of Xenopus spinal axons to netrin-1, which are mediated by DCC. We show that attraction is converted to repulsion by expression of UNC5 proteins in these cells, that this repulsion requires DCC function, that the UNC5 cytoplasmic domain is sufficient to effect the conversion, and that repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. We provide evidence that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion.

Activity-induced synaptic modification is essential for the development and plasticity of the nervous system. Repetitive correlated activation of pre- and postsynaptic neurons can induce persistent enhancement or decrement of synaptic efficacy, commonly referred to as long-term potentiation or depression (LTP or LTD). An important unresolved issue is whether and to what extent LTP and LTD are restricted to the activated synapses. Here we show that, in the CA1 region of the hippocampus, reduction of postsynaptic calcium influx by partial blockade of NMDA (N-methyl-D-aspartate) receptors results in a conversion of LTP to LTD and a loss of input specificity normally associated with LTP, with LTD appearing at heterosynaptic inputs. The induction of LTD at homo- and heterosynaptic sites requires functional ryanodine receptors and inositol triphosphate (InsP3) receptors, respectively. Functional blockade or genetic deletion of type 1 InsP3 receptors led to a conversion of LTD to LTP and elimination of heterosynaptic LTD, whereas blocking ryanodine receptors eliminated only homosynaptic LTD. Thus, postsynaptic Ca2+, deriving from Ca2+ influx and differential release of Ca2+ from internal stores through ryanodine and InsP3 receptors, regulates both the polarity and input specificity of activity-induced synaptic modification.

Signalling by intracellular second messengers such as cyclic nucleotides and Ca2+ is known to regulate attractive and repulsive guidance of axons by extracellular factors. However, the mechanism of interaction among these second messengers in determining the polarity of the guidance response is largely unknown. Here, we report that the ratio of cyclic AMP to cyclic GMP activities sets the polarity of netrin-1-induced axon guidance: high ratios favour attraction, whereas low ratios favour repulsion. Whole-cell recordings of Ca2+ currents at Xenopus spinal neuron growth cones indicate that cyclic nucleotide signalling directly modulates the activity of L-type Ca2+ channels (LCCs) in axonal growth cones. Furthermore, cGMP signalling activated by an arachidonate 12-lipoxygenase metabolite suppresses LCC activity triggered by netrin-1, and is required for growth-cone repulsion mediated by the DCC-UNC5 receptor complex. By linking cAMP and cGMP signalling and modulation of Ca2+ channel activity in growth cones, these findings delineate an early membrane-associated event responsible for signal transduction during bi-directional axon guidance.

Pathfinding by growing axons in the developing nervous system is guided by diffusible or bound factors that attract or repel the axonal growth cone. The cytoplasmic signalling mechanisms that trigger the responses of the growth cone to guidance factors are mostly unknown. Previous studies have shown that the level and temporal patterns of cytoplasmic Ca2+ can regulate the rate of growth-cone extension in vitro and in vivo. Here we report that Ca2+ also mediates the turning behaviour of the growth cones of cultured Xenopus neurons that are induced by an extracellular gradient of netrin-1, an established diffusible guidance factor in vivo. The netrin-1-induced turning response depends on Ca2+ influx through plasma membrane Ca2+ channels, as well as Ca2+-induced Ca2+ release from cytoplasmic stores. Reduction of Ca2+ signals by blocking either of these two Ca2+ sources converted the netrin-1-induced response from attraction to repulsion. Activation of Ca2+-induced Ca2+ release from internal stores with a gradient of ryanodine in the absence of netrin-1 was sufficient to trigger either attractive or repulsive responses, depending on the ryanodine concentration used. These results support the model that cytoplasmic Ca2+ signals mediate growth-cone guidance by netrin-1, and different patterns of Ca2+ elevation trigger attractive and repulsive turning responses.

The axon guidance cue netrin is importantly involved in neuronal development. DCC (deleted in colorectal cancer) is a functional receptor for netrin and mediates axon outgrowth and the steering response. Here we show that different regions of the intracellular domain of DCC directly interacted with the tyrosine kinases Src and focal adhesion kinase (FAK). Netrin activated both FAK and Src and stimulated tyrosine phosphorylation of DCC. Inhibition of Src family kinases reduced DCC tyrosine phosphorylation and blocked both axon attraction and outgrowth of neurons in response to netrin. Mutation of the tyrosine phosphorylation residue in DCC abolished its function of mediating netrin-induced axon attraction. On the basis of our observations, we suggest a model in which DCC functions as a kinase-coupled receptor, and FAK and Src act immediately downstream of DCC in netrin signaling.During embryonic development, neurons are guided to specific targets by extracellular cues in their environment. Axon growth cones sense various chemoattractive and chemorepulsive signals and translate these signals, via intracellular signal transduction pathways, into cellular movements that ultimately steer them to their correct targets. Several axon guidance molecules have been discovered and characterized, including a soluble family of proteins called netrins 1-3 . Netrins can stimulate axon growth in addition to eliciting both attractive and repulsive responses 4 .Correspondence should be addressed to K.-L.G. (kunliang@umich.edu). Competing Interests Statement:The authors declare that they have no competing financial interests.Note: Supplementary information is available on the Nature Neuroscience website. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2008 May 6. Published in final edited form as:Nat Neurosci. 2004 November ; 7(11): 1213-1221. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptGenetic studies in Caenorhabditis elegans indicate that UNC-40 and UNC-5 are functional receptors for UNC-6, a netrin homolog 5-7 . The mammalian homolog of UNC-40 is DCC, originally identified as a tumor suppressor gene 8 . DCC mediates both the axon growth and the chemoattractive function of netrin 6,7,9 . In addition, DCC mediates growth cone repulsion when in a complex with the UNC-5 receptor 5,10-13 . The UNC-40 and UNC-5 receptor complex is required for the dorsoventral repulsion of both neurons and gonads in C. elegans. The biological functions of netrin and its receptors in axon growth and growth cone guidance have been well studied; the intracellular signal transduction pathways downstream of the netrin receptors, however, are only partially understood.Tyrosine phosphorylation may be involved downstream of guidance receptors, as phosphorylation of both DCC and UNC-5 has been observed 14 . Furthermore, coexpression of Src, a tyrosine kinase, increases UNC-5 tyrosine phosphorylation, indicating a Results Netrin stimulates tyrosine phosphorylation of DCC and FAKIt has b...

TitleFemtosecond soft x-ray spectroscopy of solvated transition metal complexes: Deciphering the interplay of electronic and structural dynamics RECEIVED DATE:*Authors to whom correspondence should be addressed. E-mail: nhuse@lbl.gov, tkkim@pusan.ac.kr. 2ABSTRACT: We present the first implementation of femtosecond soft X-ray spectroscopy as an ultrafast direct probe of the excited-state valence orbitals in solution-phase molecules. This method is applied to photo-induced spin-crossover of [Fe(tren(py) 3 )] 2+, where the ultrafast spin-state conversion of the metal ion-initiated by metal-to-ligand charge transfer excitation-is directly measured using the intrinsic spin-state selectivity of the soft X-ray L-edge transitions. Our results provide important experimental data concerning the mechanism of ultrafast spin-state conversion and subsequent electronic and structural dynamics, highlighting the potential of this technique to study ultrafast phenomena in solution phase.

Cyclic nucleotide-gated channels (CNGCs) transduce external signals required for sensory processes, e.g., photoreception, olfaction, and taste. Nerve growth cone guidance by diffusible attractive and repulsive molecules is regulated by differential growth cone Ca2+ signaling. However, the Ca2+-conducting ion channels that transduce guidance molecule signals are largely unknown. We show that rod-type CNGC-like channels function in the repulsion of cultured Xenopus spinal neuron growth cones by Sema3A, which triggers the production of the cGMP that activates the Xenopus CNGA1 (xCNGA1) subunit-containing channels in interneurons. Downregulation of xCNGA1 or overexpression of a mutant xCNGA1 incapable of binding cGMP abolished CNG currents and converted growth cone repulsion to attraction in response to Sema3A. We also show that Ca2+ entry through xCNGCs is required to mediate the repulsive Sema3A signal. These studies extend our knowledge of the function of CNGCs by demonstrating their requirement for signal transduction in growth cone guidance.

Aberrant ion channel activity plays a causative role in several human disorders. Inappropriately regulated channel activity also appears to be the basis for neurodegeneration induced by dominant mutations of Caenorhabditis elegans mec-4 (mec-4(d)), a member of the degenerin gene family postulated to encode a subunit of a mechanosensory channel. The degenerin gene family has been defined by two C. elegans genes, mec-4 and deg-1, which can mutate to gain-of-function alleles that induce degeneration of specific groups of neurons. A related mammalian gene, rat alpha-rENaC, induces an amiloride-sensitive Na+ current when introduced to Xenopus oocytes, strongly suggesting that degenerin genes encode ion channel proteins. Deduced amino-acid sequences of the degenerins include two predicted membrane-spanning domains. Here we show that conserved amino acids within the second membrane-spanning domain (MSDII) are critical for MEC-4 activity and that specific substitutions within MSDII, whether encoded in cis or in trans to a mec-4(d) mutation, block or delay the onset of degeneration. Remarkably, MSDII from two other family members, C. elegans deg-1 and rat alpha-rENaC, can functionally substitute for MEC-4 MSDII in chimaeric proteins. Our results support a structural model for a mechanosensory channels in which multiple MEC-4 subunits are oriented such that MSDII lines the channel pore, and a neurodegeneration model in which aberrant ion flow through this channel is a key event.