Mechanisms of synapse development and homeostasis
Synapse development is a highly orchestrated process coordinated by intercellular communication between the pre- and postsynaptic compartments, and by neuronal activity itself. Understanding this coordination is the central objective of our research. Synaptogenesis requires three key processes: (1) recruiting the components at the proper site; (2) organizing those components to build synaptic structures, and (3) maturation and homeostasis of the synapse to optimize its activity. We address the molecular mechanisms underlying these processes using a comprehensive set of approaches including genetics, biochemistry, molecular biology, super resolution imaging and electrophysiology recordings in live animals and reconstituted systems.
Because of its many advantages, we choose to study these events in a powerful genetics system, Drosophila melanogaster, and to use the neuromuscular junction (NMJ) as a model for glutamatergic synapse development and function. The fact that individual NMJs can be reproducibly identified from animal to animal and are easily accessible for electrophysiological and optical analysis makes them uniquely suited for in vivo studies on synapse assembly, growth and plasticity. Furthermore, the fly NMJ is a glutamatergic synapse similar in composition and physiology to mammalian central synapses. The Drosophila NMJ can thus be used to analyze and model defects in the structural and physiological plasticity of glutamatergic synapses, which are associated with a variety of human pathologies from learning, memory deficits to autism.
Drosophila NMJ utilizes at least six ionotropic glutamate receptor (iGluR) subunits. They form (a) two distinct postsynaptic complexes (type-A and type-B) that co-exist within individual postsynaptic densities (PSDs) and enable NMJ functionality and plasticity and (b) a presynaptic complex that modulates basal neurotransmission. Until recently, our investigations were limited by the inability to reconstitute functional Drosophila iGluRs in heterologous systems. We have recently solved this problem and accomplished the first functional reconstitution of NMJ iGluRs in Xenopus oocytes. Furthermore, we have succeeded in expressing functional receptors in HEK293 cells, where the biophysical properties of these receptors can be further dissected using fast glutamate applications. The ability to examine the functional characteristics of iGluRs in heterologous systems opens up tremendous opportunities to study the modulation of iGluRs function and to identify the structural elements and the auxiliary subunits important for receptors assembly, surface delivery, synaptic recruitment and function.
The iGluR recruitment mechanism has been a long-standing question in our field. We identified Drosophila Neto as an obligatory subunit of the receptor complexes, essential for the synaptic recruitment of postsynaptic iGluRs. Neto belongs to a family of highly conserved proteins sharing an ancestral role in formation and modulation of glutamatergic synapses. Our studies indicate that trafficking of both iGluR subtypes on the muscle membrane, their synaptic recruitment and stabilization, and their function are tightly regulated by two Neto isoforms (alpha and beta), which modulate the iGluRs gating properties as well as their association with intracellular and extracellular synaptic components. More recently, we found that Neto-alpha also functions in the presynaptic compartment to modulate basal neurotransmission and to enable a presynaptic homeostatic response. Importantly, Neto has a modular structure and different Neto activities segregate to different domains of these proteins, allowing us to separate each of these functions and their requirements.
Modulation of BMP signaling
A recurrent theme in biology is that cells utilize a limited toolbox to establish their communication networks. BMPs modulate long-range signaling during patterning, but also ensure short-range communication at specialized cell-cell junctions, such as the NMJ. Through studies on extracellular modulators of BMP signaling, such as Short gastrulation (Sog) and Crossveinless-2 (Cv-2), we aim to elucidate mechanisms that shape cell-cell communication during early patterning and at NMJ synapses.
BMPs shape NMJ development via canonical and non-canonical signaling pathways. The canonical pathway activates transcriptional programs with distinct roles in the structural and functional development of the NMJ in response to accumulation of phosphorylated Smad (pMad) in motor neuron nuclei. A noncanonical, Mad-independent pathway, connects synaptic structures to microtubules to regulate synapse stability. Intriguingly, pMad also accumulates at synaptic locations but the biological relevance of this phenomenon remained a mystery for over a decade. We discovered that synaptic pMad is selectively lost at synapses with reduced levels of postsynaptic iGluRs. Moreover, the accumulation of synaptic pMad followed the activity of a particular subset of iGluRs, the type-A receptors, indicating that synaptic pMad functions as a local sensor for synapse activity.
Our data indicate that synaptic pMad marks a completely novel BMP pathway that is genetically distinct from all other known BMP signaling cascades. Super-resolution microscopy revealed that synaptic pMad accumulates at the active zones, the site of neurotransmitter release, and appears to associate with membrane-anchored presynaptic BMP/BMPR kinase complexes. Importantly, selective disruption of presynaptic pMad reduces the postsynaptic levels of type-A iGluRs, indicating that presynaptic pMad functions to stabilize active type-A iGluRs at postsynaptic locations.
How do postsynaptic glutamate receptors modulate presynaptic pMad and in turn are stabilized by it? Since synaptic pMad depends on active type-A iGluRs, we favor a model whereby Neto, the obligatory auxiliary subunit of the iGluR channels, connects active postsynaptic type-A iGluRs with presynaptic BMP/BMPR complexes. Such trans-synaptic complexes could offer a versatile means for relaying synapse activity status to the presynaptic neuron via fast conformational modifications. At the same time, these trans-synaptic complexes may facilitate interactions that stabilize the type-A iGluRs at PSD. This positive feedback could explain the Hebbian mode of type-A iGluRs incorporation at PSD and maturation of iGluR fields at larval NMJ. In addition, since BMPRs are limiting and shared among different BMP signaling modalities, the neurons may use this synaptic BMP pathway to monitor synapse activity then coordinate NMJ growth with synapse maturation and stabilization.