What are the molecular and cellular steps that govern presynaptic assembly and determine presynaptic function?

WHAT IS THE ROLE OF CELL ADHESION MOLECULES SUCH AS NEUREXIN IN PRESYNAPTIC ASSEMBLY?

Do synaptic cell adhesion molecules initiate presynaptic assembly through trans-synaptic interactions as originally proposed?

We have found that a recently-identified, conserved short isoform of neurexin, called gamma-neurexin, which does not contain any of the canonical extracellular binding domains, can nonetheless mediate presynaptic assembly and maturation. Our current studies are focused on elucidating the role of this short but seemingly redundant isoform in synapse development.

Moreover, we have found that the intracellular domain of neurexin alone is sufficient for presynaptic assembly. To understand the contribution of neurexin’s intracellular domain to presynaptic organization, we are undertaking proximity labeling approaches to identify neurexin’s intracellular binding partners.

SYNAPSE ASSEMBLY AT DISTINCT STAGES OF NEURONAL DEVELOPMENT

New synapses form behind the growth cone during axonal outgrowth. New synapses can also be added to pre-existing circuits after axon outgrowth is complete, as an organism grows. We have evidence that these two forms of synapse assembly are mediated by distinct molecular mechanisms. Projects in the lab are aimed at understanding this distinction, and the contribution of molecular motors, cell adhesion molecules and the cytoskeleton to these processes.

HOW IS THE ASSEMBLY OF PRESYNAPTIC SPECIALIZATIONS REGULATED?

We are performing forward genetic screens to look for mutants with defects in the assembly of active zone scaffolds, in order to understand what regulates this complex process.

MODELING HUMAN PATIENT CALCIUM CHANNEL MUTATIONS IN C. ELEGANS

Using C. elegans to understand how patient mutations in calcium channels affect their localization and function. A. Secondary structure of UNC-2/CaV2α. Adapted from Huang et al., 2019, eLife. B. A novel patient mutation in a highly homologous region confers developmental abnormalities. We have used Crispr/Cas9 to create worm models of this mutation. C. Worm locomotion is quantified using worm tracking software and analyzed to reveal behavioral defects. D. Reduced speed of locomotion in worms harboring the patient mutation mirrors mouse and human ataxia. We are using these models to delve deeper into potential defects in synapse structure and function resulting from patient mutations.

DEVELOPING A SINGLE-SYNAPSE OPTICAL READOUT FOR SYNAPSE FUNCTION

We are developing an optical physiology assay to look at synaptic function with single-synapse resolution. A-B. Using postsynaptically targeted GCaMP6 we can measure calcium transients opposite individual presynaptic release sites. C. We have developed an analysis pipeline to automatically detect regions of interest (ROIs) that correspond to individual synapses. D. Calcium transients at nearby ROIs are correlated but not identical to one another (and offset in time). E. ROIs form functional clusters.

THE WNT RECEPTOR FRIZZLED MEDIATES BOTH SYNAPSE ASSEMBLY AND SYNAPSE ELIMINATION THROUGH UNKNOWN PATHWAYS

Wnt, binding to the Frizzled (Fz) receptor, leads to Fz endocytosis and subsequent synapse elimination. To understand how this process works, we have undertaken forward genetic modifier screens and isolated suppressors of the Fz phenotype.