Satellite signaling at synapses

Abstract

Neural function requires effective communication between neurons and their targets at synapses. Thus, proper formation, growth and plasticity of synapses are critical to behavior. A retrograde (muscle to neuron) BMP signal is required to promote synaptic growth, homeostasis and stability at Drosophila neuromuscular junctions (NMJs).^1-4 We recently demonstrated that this signal constitutes an instructive signal that sculpts synaptic growth in a graded manner and uncovered a presynaptic endocytic mechanism that modulates BMP signaling levels. In the absence of this regulation, excessive BMP signaling results in overgrown NMJs with a proliferation of ectopic boutons.⁵

Regulation of NMJ Growth

Analysis of Drosophila mutants with altered NMJ morphology has uncovered a broad variety of cellular processes and molecular signaling pathways that regulate synaptic growth. Cell adhesion, cytoskeletal regulation, endocytosis, neuronal activity, protein degradation and RNA localization along with BMP, Wnt and MAP Kinase and heparan sulfate proteoglycan signaling pathways all play important roles in determining NMJ size and structure.^1,4,6-19 Recent studies identifying regulators of these regulators underscore the high level of molecular control over synaptic growth and highlight the importance to the organism of getting it right.^5,7,15,20,21

Mutations in these pathways result in diverse morphological perturbations of the NMJ—overgrowth, undergrowth, abnormal branching, large boutons, small boutons, mislocalization and disorganization of synaptic components within boutons, and more. A distinctive phenotype that has been the subject of recent attention is the formation of “supernumerary” or “satellite” boutons—small ectopic boutons that emerge from the main nerve terminal or that bud excessively from primary boutons. Many endocytic mutants exhibit satellite bouton formation.^9,11,13 Our recent study of nervous wreck(nwk) identifies retrograde BMP growth signaling as an endocytic target and demonstrates that disrupted regulation of this pathway results in satellite bouton formation.⁵

We previously demonstrated that loss of Nwk, an SH3 domain-containing adaptor protein, results in NMJ overgrowth characterized by formation of satellite boutons through a mechanism that involved actin assembly.⁸ Strong support for this mechanistic model comes from recent work demonstrating the cooperative action of Nwk and the small GTPase Cdc42 in Wasp-dependent actin polymerization.¹⁵ Endocytosis requires actin polymerization at multiple steps and many components of the endocytic machinery are also actin regulators, so we investigated the possibility that endocytosis is impaired in nwk. Consistent with this idea, a conserved domain in Nwk was recently identified as an F-BAR domain, which promotes membrane invagination.²² Nwk interacts functionally and physically with key components of the endocytic machinery, including dynamin and Dap160/Intersectin.^5,15 Since nwk does not affect synaptic vesicle endocytosis,⁸ we focused on receptor-mediated endocytosis. Because Nwk is a negative regulator of synaptic growth, if nwk mutations do perturb the endocytosis of a signaling receptor, this signal must promote growth. The BMP ligand Glass bottom boat (Gbb) initiates a crucial positive growth-signaling cascade at NMJs.^1,4,23 We found that NMJ overgrowth in nwk depends on wild-type levels of BMP signaling and that excessive BMP signaling alone could cause formation of numerous satellite boutons. This phenotype is exacerbated by loss of Nwk and suppressed by Nwk overexpression. To confirm that these interactions are indeed mediated by endocytosis, we investigated BMP interactions with other endocytic proteins and found that Dap160 displays analogous interactions with the BMP pathway. Our finding that Nwk binds Thickveins, a type I BMP receptor at presynaptic terminals, establishes a direct physical link between Nwk and BMP signaling. Finally, we found that synaptic levels of pMAD, the downstream effector of BMP signaling, are elevated in nwk and other endocytic mutants and reduced by Nwk overexpression in the presence of ectopic BMP signaling. In fact, we observe a general correlation between presynaptic pMAD levels and synaptic growth, most strikingly in the form of satellite boutons, indicating that the BMP signal is not simply permissive but an instructive regulator of synapse size and complexity.

These results support a model in which Nwk links BMP receptors with the endocytic machinery to attenuate BMP signaling via regulation of presynaptic receptor trafficking. The precise nature of this regulation remains unclear. Our studies do not reveal gross differences in ectopic GFP-tagged receptor levels indicating that Nwk likely does not regulate their degradation. Nwk localizes to a Rab11-positive recycling-endosome compartment, which raises the possibility that Nwk modulates neuronal responsiveness to the BMP ligand by regulating the trafficking of BMP receptors to and from the membrane (Fig. 1).¹⁵ Consistent with this model of BMP receptor regulation, the Drosophila hereditary spastic paraplegia protein Spichthyin localizes to recycling endosomes and attenuates BMP signaling at NMJs.²¹

Figure 1.

Model of Nervous Wreck function. Nwk binds Dap160, dynamin and Thickveins to negatively regulate the BMP signaling pathway. Because Nwk specifically localizes to Rab11-positive endocytic compartments, we hypothesize that Nwk regulates receptor recycling through an RE-dependent pathway. Shunting Thickveins away from the more rapid RE-independent recycling route would have the effect of reducing plasma membrane receptor concentrations and, thus, neuronal responsiveness to the retrograde BMP signal. Early endosome (EE); Recycling endosome (RE); Wishful Thinking Type II BMP receptor (Wit).

In addition to excessive BMP signaling, perturbations in other regulatory mechanisms may generate satellite boutons. In fact, ectopic FasII/Amyloid Precursor Protein (Appl) signaling leads to satellite bouton formation, as does loss of the glycogen synthase kinase 3β Shaggy, a negatively regulated target of presynaptic Wnt signaling.^24-26 Satellite boutons also occur in response to integrin mislocalization and loss of multiple components of the actin-regulatory Scar complex.^16,27-29 It will be of interest to determine whether any of these pathways intersect with presynaptic endocytic mechanisms to affect satellite bouton formation or whether they operate via distinct processes.

Functional Consequences of Aberrant Synaptic Morphology

Mutations resulting in satellite boutons also have pronounced effects on synaptic transmission. Mutations in nwk and dap160 lead to decreases in baseline neurotransmitter release.^8,13 Similarly, presynaptic vesicle release, or quantal content as estimated by the ratio of excitatory junctional potential (EJP) amplitude to miniature EJP amplitude (quantal size), is decreased by presynaptic overexpression of APPL or pre- and postsynaptic overexpression of FasII.²⁴ Decreased quantal content, however, is not responsible for satellite bouton formation as there are many mutants with decreased quantal content that do not form satellite boutons. In addition, blockage of synaptic transmission by expression of tetanus toxin in motor neurons does not result in satellite bouton formation.⁹ These findings suggest that decreased vesicle release may be a functional consequence of satellite bouton formation. However, it is also possible that perturbations in bouton formation and defects in synaptic vesicle release are two separate consequences of mutations in key regulatory proteins that play important but distinct roles in both mechanisms.

An appealing idea is that satellite boutons are exactly what they look like—physically and functionally immature boutons. EM and light microscopic analysis of APPL-induced satellite boutons revealed no obvious abnormalities.²⁴ On the other hand, three-dimensional reconstructions of satellite boutons in the endocytic mutant synaptojanin (synj) revealed a higher density of active zones and presynaptic dense bodies.⁹ However, as more active zones and dense bodies would be expected to result in a greater probability of neurotransmitter release, these changes may reflect a compensatory response to decreased neurotransmission in synj mutants. Focal recordings of satellite boutons will be necessary to characterize the function of satellite boutons in detail, although compensatory mechanisms that maintain synaptic function could complicate the analysis.

Genetic identification and investigation of the genes required for the morphological development of Drosophila NMJs is revealing the complex molecular regulation that underlies synaptic growth. Highlighting the importance of these insights, mutations in human Nwk family member WRP/MEGAP have been linked to 3p deletion syndrome, a devastating developmental disorder characterized by severe motor and cognitive impairment.³⁰ Future studies at the Drosophila NMJ should continue to shed light on how the elaboration of synaptic structure is regulated and how this affects synaptic function.