BIOLOGICAL OVERVIEW

Fas2 carries out multiple functions in the Drosophila nervous system. A member of the immunoglobulin superfamily, Fas2 has been implicated in the process of fasciculation, the adherence of growing axons to one another during the process of growth cone guidance. Fas2 has also been implicated in the process of presynaptic functional plasticity, the ability of synapses to undergo long-term changes in structure. Expressed both presynaptically and postsynaptically, Fas2 controls the number and stability of neuromuscular synapses. In this function, Fas2 is down regulated by mutations affecting cyclic AMP levels (See the learning pathway). In addition, decreased Fas2 is necessary and sufficient to cause synaptic sprouting. Finally, Fas2 and CREB (the cyclic AMP response element binding protein) act in parallel pathways to cause increases in strength of the neuromuscular synapse.

Fas2 functions in selective fasciculation, that is, adherence of growing axons to the MP1 fascicle. The MP1 fascicle, generated by the anterior extending growth cone of the pCC axon is easily identified because a specific monoclonal antibody will stain only very specific types of growth cones. These include aCC, pCC, MP1, dMP2 and vMP2. The pCC axon pioneers the MP1 fascicle. In the absence of Fas2, growth cones do not extend in the proper rostrocaudal direction and fail to fasciculate (Grenningloh, 1991).

Subsequent studies reveal an even more complex function for Fas2. In addition to selective fasciculation, Fas2 also functions in growth cone guidance. Three major motor nerves were analyzed: the intersegmental nerve (ISN) that innerates dorsal muscles; the segmental nerve a (SNa), innervating lateral muscles, and segmental nerve b (SNb), innervating ventral muscles. High specific defects in axonal guidance occur in over- or under-expression of Fas2. These do not appear to be fasciculation phenotypes because fascicles are not formed. They are instead guidance phenotypes in which axons wander from their normally specified courses. It would appear that guidance is not taking place here solely on the basis of self adhesion, but in the context of other competing attractive and repulsive cues. In this case, cell adhesion per se is not always the sole or the decisive factor (Lin, 1994 a and b).

Muscles 7 and 6 are each innervated by two motoneurons, RP3 and MN6/7b. This innervation occurs at stage 17. The two axons form about 18 synaptic boutons by the time of the initial larval hatch, and the number increases to approximately 180 boutons by the third instar larva. Although Fas2 is initially expressed by all embryonic motor axons and their growth cones during the period of axon outgrowth, the axonic Fas2 decreases upon innervation, and Fas2 is confined to the synaptic terminal. After synapse formation, Fas2 is localized both pre-synaptically and postsynaptically, where it controls synapse stabilization. In Fas2 mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and the resulting lethality is rescued by transgenes that drive Fas2 expression both pre- and postsynaptically; driving Fas2 expression on either side alone is insufficient.

Fas2 can also control synaptic growth: various Fas2 alleles lead to either an increase or decrease in sprouting depending upon the level of Fas2. A 50% decrease in Fas2 can lead to a 50% increase in the number of synaptic boutons (Schuster, 1996a).

Altering the level of neuronal activity or cAMP concentration leads to increased synaptic structure and function at the neuromuscular junction. ether à go-go and Shaker code for potassium channels; mutations in both can result in increased neuronal activity and increase in synaptic structure and branching. Mutation in K+ channels display greatly enhanced nerve activity as a result of reduction in K+ currents. K+ channel mutation is thought to increase motoneuron activity and synaptic transmission by increasing the cAMP second messenger signaling. Mutations in dunce, coding for c-AMP phosphodiesterase, result in increased cAMP levels, heightened neural activation, and also result in increased axonal branching (Schuster, 1996b and references).

Increase in synaptic growth in eag, Shaker and dunce mutants is accompanied by approximately 50% reduction in synaptic levels of Fas2. This decrease in Fas2 is both necessary and sufficient for presynaptic sprouting; Fas2 mutants that decrease Fas2 levels by 50% lead to sprouting similar to eag, Shaker and dunce mutants, while Fas2 transgenetic animals that maintain synaptic Fas 2 levels suppress sprouting in eag, Shaker and dunce mutants. However, Fas2 mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons. Thus these results show a requirement for the presynaptic downreguation of Fas2 in activity and cAMP-induced synaptic sprouting. It is speculated that activity or cAMP may trigger the down-regulation of synaptic Fas2 by actively removing it from the presynaptic terminal (Schuster, 1996b).

Since Fas2 mutants lead to an increase in the number of boutons without affecting synaptic strength, and increased cAMP in dnc mutants increases both synaptic structure and quantal content, there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB, the cyclic AMP response element-binding protein, mediates the transcriptional requirement of cAMP-dependent long-term synaptic change. Thus CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with FAS2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, dCREB2-b (an isoform of CREB), in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, dCREB2-a, increases synaptic strength by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of dCREB2-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release. Expression of dCREB2-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity, and cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, 1996).

PROTEIN STRUCTURE

Amino Acids

There is a phosphatidylinositol-linked form with 811 amino acids and a transmembrane form with 873 amino acids. They differ only in their C terminal amino acids (Grenningloh, 1991).

Structural Domains

FAS2 is a member of the immunoglobulin superfamily containing five Ig-like domains. It also contains two fibronectin-type III repeats (Grenningloh, 1991). The protein contains a signal sequence serving for protein secretion.

date revised: 28 MAY 97 

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