An official website of the United States government
Abstract Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.
more »
« less
The CHD Protein, Kismet, is Important for the Recycling of Synaptic Vesicles during Endocytosis
Abstract Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, theDrosophilahomolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound todap160andendophilin Btranscription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition,kismetmutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored atkismetmutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.
more »
« less
- Award ID(s):
- 1856439
- PAR ID:
- 10153807
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Ewer, John (Ed.)Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis inDrosophila, as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, suggesting that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, indicating that the Inc C-terminus has the properties of a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and support the notion that Inc-Cul3 complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Inc-Cul3 complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated withCul3mutations.more » « less
-
Hart, Anne C (Ed.)Modulation of neurotransmission is key for organismal responses to varying physiological contexts such as during infection, injury, or other stresses, as well as in learning and memory and for sensory adaptation. Roles for cell autonomous neuromodulatory mechanisms in these processes have been well described. The importance of cell non-autonomous pathways for inter-tissue signaling, such as gut-to-brain or glia-to-neuron, has emerged more recently, but the cellular mechanisms mediating such regulation remain comparatively unexplored. Glycoproteins and their G protein-coupled receptors (GPCRs) are well-established orchestrators of multi-tissue signaling events that govern diverse physiological processes through both cell-autonomous and cell non-autonomous regulation. Here, we show that follicle stimulating hormone receptor, FSHR-1, the soleCaenorhabditis elegansortholog of mammalian glycoprotein hormone GPCRs, is important for cell non-autonomous modulation of synaptic transmission. Inhibition offshr-1expression reduces muscle contraction and leads to synaptic vesicle accumulation in cholinergic motor neurons. The neuromuscular and locomotor defects infshr-1loss-of-function mutants are associated with an underlying accumulation of synaptic vesicles, build-up of the synaptic vesicle priming factor UNC-10/RIM, and decreased synaptic vesicle release from cholinergic motor neurons. Restoration of FSHR-1 to the intestine is sufficient to restore neuromuscular activity and synaptic vesicle localization tofshr-1-deficient animals. Intestine-specific knockdown of FSHR-1 reduces neuromuscular function, indicating FSHR-1 is both necessary and sufficient in the intestine for its neuromuscular effects. Re-expression of FSHR-1 in other sites of endogenous expression, including glial cells and neurons, also restored some neuromuscular deficits, indicating potential cross-tissue regulation from these tissues as well. Genetic interaction studies provide evidence that downstream effectorsgsa-1/GαS,acy-1/adenylyl cyclase andsphk-1/sphingosine kinase and glycoprotein hormone subunit orthologs, GPLA-1/GPA2 and GPLB-1/GPB5, are important for intestinal FSHR-1 modulation of the NMJ. Together, our results demonstrate that FSHR-1 modulation directs inter-tissue signaling systems, which promote synaptic vesicle release at neuromuscular synapses.more » « less
-
Gupton, Stephanie (Ed.)Following exocytosis at active zones, synaptic vesicle membranes and membrane-bound proteins must be recycled. The endocytic machinery that drives this recycling accumulates in the periactive zone (PAZ), a region of the synapse adjacent to active zones, but the organization of this machinery within the PAZ, and how PAZ composition relates to active zone release properties, remains unknown. The PAZ is also enriched for cell adhesion proteins, but their function at these sites is poorly understood. Here, using Airyscan and stimulated emission depletion imaging of Drosophila synapses, we develop a quantitative framework describing the organization and ultrastructure of the PAZ. Different endocytic proteins localize to distinct regions of the PAZ, suggesting that subdomains are specialized for distinct biochemical activities, stages of membrane remodeling, or synaptic functions. We find that the accumulation and distribution of endocytic but not adhesion PAZ proteins correlate with the abundance of the scaffolding protein Bruchpilot at active zones—a structural correlate of release probability. These data suggest that endocytic and exocytic activities are spatially correlated. Taken together, our results identify novel relationships between the exocytic and endocytic apparatus at the synapse and provide a new conceptual framework to quantify synaptic architecture.more » « less
-
Abstract Neurons are highly polarized, specialized cells that must overcome immense challenges to ensure the health and survival of the organism in which they reside. They can spread over meters and persist for decades yet communicate at sub-millisecond and millimeter scales. Thus, neurons require extreme levels of spatial-temporal control. Neurons employ molecular motors to transport coding and noncoding RNAs to distal synapses. Intracellular trafficking of RNAs enables neurons to locally regulate protein synthesis and synaptic activity. The way in which RNAs get loaded onto molecular motors and transported to their target locations, particularly following synaptic plasticity, is explored below.more » « less
