Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditis elegans

Abstract

The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20°and 14°resulted in locomotion rates that were 3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14°and 20°showed a 10-and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14°and switched to 25°showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.KEYWORDS Caenorhabditis elegans; KIF1A; axonal transport; Cdk5; liprin; SAD-A; dynein N EURONS have a unique requirement to transport synaptic vesicles (SVs) long distances along microtubule tracks in axons to establish synapses for the regulated secretion of neurotransmitters. Microtubules have an intrinsic plus-and minus-end polarity. Axonal microtubules are oriented with their plus-ends pointing toward the synaptic region (Burton and Paige 1981;Heidemann et al. 1981;Baas and Lin 2011). The plus-end-directed kinesin motor KIF1A carries SVs outward to the stable SV clusters at synapses (Hall and Hedgecock 1991;Okada et al. 1995;Pack-Chung et al. 2007). However, for reasons not immediately clear, the transport is bidirectional, alternating between minus-end-directed retrograde transport and plus-end-directed anterograde transport, with plus-end transport ultimately dominating to achieve SV accumulation at synapses . Dynein mediates the minus-end-directed transport of SVs (Koushika et al. 2004;. Neurons also use KIF1A to transport neuropeptide-carrying dense core vesicles (DCVs) from the cell soma to the synaptic region (Jacob and Kaplan 2003;Pack-Chung et al. 2007), again in a bidirectional manner in concert with dynein (Barkus et al. 2008;Goodwin et al. 2012).Although null alleles of KIF1A are lethal, forward genetic studies in Drosophila and Caenorhabditis elegans have generated many useful reduction-of-function alleles for investigating the consequences of impaired SV and DCV transport and/or for studying the...

UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons

“…We conclude that STRD-1 (STRADa) and SAD-1 are part of the same system to regulate the trafficking of lysosomes in axons and dendrites. The data further show that neither protein can function without the other in Discovery of the CSS System 125 regulating dendritic lysosome trafficking, but SAD-1 remains partially functional in the absence of STRD-1 for axonal lysosome trafficking.KIF1A (UNC-104) is not the motor that carries lysosomes to the axonal synaptic region in unc-16 mutantsIn the accompanying article in this issue, we found that the synapse assembly proteins SYD-1, SYD-2, and SAD-1 promote the plus-end-directed trafficking of synaptic vesicles by the kinesin KIF1A (UNC-104), preventing their accumulation in dendrites and cell somas in C. elegans cholinergic motor neurons (Edwards et al 2015). However, UNC-104 does not contribute to the accumulation of lysosomes in unc-16 mutant axons because, when we compared unc-16 single mutants to unc-16; unc-104 doubles, the cell soma and axonal levels of lysosomes in motor neurons in the unc-16; unc-104 double were not significantly lower than the unc-16 single mutant ( Figure S6).…”

“…The data revealed that UNC-16's organelle transport regulatory function is unexpectedly mediated by synapse assembly proteins acting as part of a newly defined organelle transport regulatory system: the CSS system. In an accompanying article in this issue, we show that several CSS system proteins also regulate synaptic vesicle transport, further underscoring the counterintuitive dual roles of some synapse assembly proteins in regulating active transport and synapse assembly/stability (Edwards et al 2015). …”

“…However, neurons outside of the cortex, including peripheral motor neurons and many other types of central neurons, do not require the SAD kinases or LKB1 for axon formation/identity (Lilley et al 2013). In the current study, we show that sad-1 mutants have wildtype locomotion rates, a result that seems inconsistent with a motor neuron axon/dendrite identity defect.In the accompanying article in this issue, we provide evidence that the accumulation of synaptic vesicles in sad-1, Discovery of the CSS System 135 syd-1, and syd-2 motor neuron dendrites results from a defect in the regulation of synaptic vesicle transport, as opposed to an axon/dendrite identity defect (Edwards et al 2015). Indeed, the regulation of synaptic vesicle and/or dense core vesicle transport appears to be another common theme that ties together several of CSS system proteins.…”

“…In the accompanying article in this issue, we found that the synapse assembly proteins SYD-1, SYD-2, and SAD-1 promote the plus-end-directed trafficking of synaptic vesicles by the kinesin KIF1A (UNC-104), preventing their accumulation in dendrites and cell somas in C. elegans cholinergic motor neurons (Edwards et al 2015). However, UNC-104 does not contribute to the accumulation of lysosomes in unc-16 mutant axons because, when we compared unc-16 single mutants to unc-16; unc-104 doubles, the cell soma and axonal levels of lysosomes in motor neurons in the unc-16; unc-104 double were not significantly lower than the unc-16 single mutant ( Figure S6).…”

“…High-pressure freezing electron microscopy followed by tomographic reconstructions of active zones show that SYD-2 has roles in tethering/clustering synaptic vesicles near the active zone (Stigloher et al 2011;) and in determining the size of the dense projection to which the synaptic vesicles are tethered . In syd-2 mutants, the rate at which synaptic vesicles dissociate from mature synapses is increased (Wu et al 2013), and synaptic vesicles or clusters of vesicles accumulate in distal regions of the axon that are normally devoid of synapses and synaptic vesicles (Li et al 2014;Edwards et al 2015).The phenotype of accumulation of synaptic vesicles in the distal asynaptic region of motor neurons, which could be considered diagnostic of reduced synaptic vesicle clustering activity at synapses, is also shared by syd-1 and sad-1 mutants (Li et al 2014;Edwards et al 2015), and prior studies also demonstrated defects in synaptic vesicle clustering at other synapses in C. elegans sad-1 mutants (Crump et al 2001;Patel et al 2006). Other studies have proposed that C. elegans sad-1 and syd-1 mutants also have defects in axon identity/fate, referred to as a "polarity defect," based largely on the accumulation of synaptic vesicles in dendrites (Hallam et al 2002;Kim et al 2008.…”