Modulation of TGF-beta signaling

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Bone morphogenetic proteins (BMPs) are potent secreted signaling factors that function at long- and short-range and elicit critical cellular responses during development and homeostasis. Long-range signaling is key to the formation and function of morphogen gradients. Such gradients control the cell fate and tissue allocation and influence the patterning of early embryos as well as later developmental processes. Short-range signaling sculpts cellular junctions and has been implicated in the growth, development and homeostasis of synaptic junctions, such as Drosophila neuromuscular junction (NMJ). The fly NMJ is a glutamatergic synapse similar in composition and physiology to mammalian central synapses. The fact that individual NMJs can be reproducibly identified and are easily accessible for electrophysiological and optical analysis makes this genetic model system uniquely suited for in vivo studies on synapse assembly, growth and plasticity. In flies, BMPs shape NMJ development via multiple signaling pathways. A 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. In recent work we discovered that synaptic pMad is selectively lost at synapses with reduced levels of postsynaptic ionotropic glutamate receptors (iGluRs). Moreover, mutants that lack a particular receptor subtype, GluRIIA, show complete loss of synaptic pMad signals but normal Mad-positive signals in the motor neuron nuclei. The expression of BMP target genes remains unaffected in GluRIIA mutant animals, indicating a specific impairment in the pMad production/ maintenance at synaptic terminals. More importantly, the accumulation of synaptic pMad followed the activity and not the net levels of GluRIIA-containing (type-A) iGluRs. Thus, synaptic pMad appears to function as a local sensor for NMJ synapse activity. Our data indicate that synaptic pMad marks a completely novel BMP pathway that is genetically distinguishable from all other known BMP signaling cascades. Unlike the BMP retrograde signaling pathway, this novel pathway does not require the BMP7 ortholog, Glass bottom boat (Gbb), but depends on presynaptic BMP receptors (Wit, Put and Sax) and postsynaptic type-A iGluRs. Also, nuclear and synaptic pMad are independently regulated: Genetic manipulations of type-A iGluRs induce proportional changes in synaptic pMad but have no effect on nuclear pMad. Conversely, excess Mad-GFP in motor neurons induces strong accumulation of nuclear pMad but has no effect on the synaptic pMad. Super-resolution microscopy revealed that synaptic pMad accumulates at the active zones as presynaptic discs, parallel with the iGluR fields and Brp-positive rings, which mark the sites of neurotransmitter release. The size and shape of the pMad-positive domains suggest that pMad associates with membrane-anchored complexes at the active zone. Since BMP signals are generally short lived, these domains likely represent pMad that, upon phosphorylation, remains associated with the BMP/BMPR kinase complexes at presynaptic sites. Interestingly, selective disruption of presynaptic pMad reduces the postsynaptic levels of type-A iGluRs, indicating that synaptic pMad functions to stabilize active type-A iGluRs at synaptic 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, an obligatory 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 GluRIIA 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. To learn more about the structural features of Mad that may influence its association with the BMPRs, we have collected most of existing Drosophila Mad alleles and compared them side-by side for their ability to sustain the two Smad-dependent signaling modalities: the canonical BMP signaling, marked by pMad accumulation in motor neuron nuclei, and the synaptic BMP signaling, marked by pMad accumulation at synaptic terminals. Within this comprehensive collection, we found that strong Mad alleles generally disrupt both synaptic and nuclear pMad accumulation, whereas moderate Mad alleles have a wider range of phenotypes and can differentially impact different BMP signaling modalities. In particular, Mad8 showed drastically reduced synaptic pMad levels but only moderately diminished nuclear pMad signals. The postsynaptic composition and electrophysiological properties of Mad8 NMJs were likewise altered. Using biochemical assays and structural modeling, we examined how point mutations such as S359L, present in Mad8, could influence the Mad-Tkv interface. Our study identified a new molecular determinant for this Mad-Tkv interaction, the highly class conserved H2 helix. Several genetic variants identified in human patients map to H2, underscoring the relevance of this motif for normal development and function. To examine how motor neurons (MNs) respond to different BMP signaling modalities or compensate for various synaptic perturbations, we are currently using droplet microfluidics (10x Chromium) methodology to compare MNs transcriptomes in control and various mutants. For this work we are collaborating with Stephen Coon (Molecular Genomics Core, NICHD). We have already developed a set of protocols to generate and analyze single cell RNAseq datasets for larval ventral nerve cords (VNCs), the fly equivalent of the spinal cord. We use un-supervised algorithms, reiterative processes and known specific transcripts to cluster these cells and identify different cell types. Based on sequences from more than 30,000 single cells (from multiple experimental sets), we are in the process of assembling an atlas of the larval VNC; it includes several glia subtypes, neuroblast and newborn neurons, motor neurons and interneurons. Next, we will examine how VNCs, in particular MNs, respond to synaptic perturbations or disruptions of various BMP signaling modalities.