Michael L. Nonet

Loss of the Putative RNA-Directed RNA Polymerase RRF-3 Makes C. elegans Hypersensitive to RNAi

RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.

A post-docking role for active zone protein Rim

Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.

SYNAPTIC TRANSMISSION DEFICITS IN CAENORHABDITIS ELEGANS SYNAPTOBREVIN MUTANTS

Synaptobrevins are vesicle-associated proteins implicated in neurotransmitter release by both biochemical studies and perturbation experiments that use botulinum toxins. To test these models in vivo, we have isolated and characterized the first synaptobrevin mutants in metazoans and show that neurotransmission is severely disrupted in mutant animals. Mutants lackingsnb-1 die just after completing embryogenesis. The dying animals retain some capability for movement, although they are extremely uncoordinated and incapable of feeding. We also have isolated and characterized several hypomorphic snb-1 mutants. Although fully viable, these mutants exhibit a variety of behavioral abnormalities that are consistent with a general defect in the efficacy of synaptic transmission. The viable mutants are resistant to the acetylcholinesterase inhibitor aldicarb, indicating that cholinergic transmission is impaired. Extracellular recordings from pharyngeal muscle also demonstrate severe defects in synaptic transmission in the mutants. The molecular lesions in the hypomorphic alleles reside on the hydrophobic face of a proposed amphipathic–helical region implicated biochemically in interacting with the t-SNAREs syntaxin and SNAP-25. Finally, we demonstrate that double mutants lacking both the v-SNAREs synaptotagmin and snb-1 are phenotypically similar tosnb-1 mutants and less severe than syntaxin mutants. Our work demonstrates that synaptobrevin is essential for viability and is required for functional synaptic transmission. However, our analysis also suggests that transmitter release is not completely eliminated by removal of either one or both v-SNAREs.

rpm-1, a conserved neuronal gene that regulates targeting and synaptogenesis in C. elegans.

Little is known of mechanisms regulating presynaptic differentiation. We identified rpm-1 in a screen for mutants with defects in patterning of a presynaptic marker at certain interneuronal synapses. The predicted RPM-1 protein contains zinc binding, RCC1, and other conserved motifs. In rpm-1 mutants, mechanosensory neurons fail to accumulate tagged vesicles, retract synaptic branches, and ectopically extend axons. Some motor neurons branch and overgrow; others show altered synaptic organization. Expression of RPM-1 in the presynaptic mechanosensory neurons is sufficient to rescue phenotypes in these cells. Certain rpm-1 phenotypes are temperature sensitive, revealing that RPM-1 function can be bypassed by maintaining mutants at the permissive temperature at stages commensurate with synapse formation in wild-type animals. These results indicate that RPM-1 functions cell autonomously during synaptogenesis to regulate neuronal morphology.

Defects in synaptic vesicle docking in unc-18 mutants

Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing unc-18 mutants of the nematode Caenorhabditis elegans. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked vesicles at the active zone in unc-18 mutants, suggesting that UNC-18 functions, directly or indirectly, as a facilitator of vesicle docking.

Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions

Synaptic specializations are difficult to visualize at the light microscope level in living preparations. To circumvent this problem, synaptic vesicle proteins were fused to green fluorescent protein (GFP) and expressed in C. elegans neurons. C. elegans synaptobrevin-GFP and synaptogyrin-GFP fusion proteins were observed to target to synaptic sites. This localization allowed the visualization of synaptic specializations in living animals with light microscopy. Restricted expression of synaptobrevin-GFP fusions in subsets of neurons enables the visualization of individual presynaptic varicosities. The cell biology underlying the sorting of synaptic vesicle proteins, trafficking of vesicles to terminals, and the development of presynaptic specializations is now more amenable to forward genetic analysis using these synaptic markers.

SLO-1 Potassium Channels Control Quantal Content of Neurotransmitter Release at the C. elegans Neuromuscular Junction

Six mutants of SLO-1, a large-conductance, Ca(2+)-activated K(+) channel of C. elegans, were obtained in a genetic screen for regulators of neurotransmitter release. Mutants were isolated by their ability to suppress lethargy of an unc-64 syntaxin mutant that restricts neurotransmitter release. We measured evoked postsynaptic currents at the neuromuscular junction in both wild-type and mutants and observed that the removal of SLO-1 greatly increased quantal content primarily by increasing duration of release. The selective isolation of slo-1 as the only ion channel mutant derived from a whole genomic screen to detect regulators of neurotransmitter release suggests that SLO-1 plays an important, if not unique, role in regulating neurotransmitter release.

The Sodium-Activated Potassium Channel Is Encoded by a Member of the Slo Gene Family

Na(+)-activated potassium channels (K(Na)) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. We now report that K(Na) is encoded by the rSlo2 gene (also called Slack), the mammalian ortholog of slo-2 in C. elegans. rSlo2, heterologously expressed, shares many properties of native K(Na) including activation by intracellular Na(+), high conductance, and prominent subconductance states. In addition to activation by Na(+), we report that rSLO-2 channels are cooperatively activated by intracellular Cl(-), similar to C. elegans SLO-2 channels. Since intracellular Na(+) and Cl(-) both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis revealed that the SLO-2 current is a major component of the delayed rectifier. We demonstrate in C. elegans that slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily.

SYD-2 Liprin-alpha organizes presynaptic active zone formation through ELKS.

A central event in synapse development is formation of the presynaptic active zone in response to positional cues. Three active zone proteins, RIM, ELKS (also known as ERC or CAST) and Liprin-α, bind each other and are implicated in linking active zone formation to synaptic vesicle release. Loss of function in Caenorhabditis eleganssyd-2 Liprin-α alters the size of presynaptic specializations and disrupts synaptic vesicle accumulation. Here we report that a missense mutation in the coiled-coil domain of SYD-2 causes a gain of function. In HSN synapses, the syd-2(gf) mutation promotes synapse formation in the absence of syd-1, which is essential for HSN synapse formation. syd-2(gf) also partially suppresses the synaptogenesis defects in syg-1 and syg-2 mutants. The activity of syd-2(gf) requires elks-1, an ELKS homolog; but not unc-10, a RIM homolog. The mutant SYD-2 shows increased association with ELKS. These results establish a functional dependency for assembly of the presynaptic active zone in which SYD-2 plays a key role.

aex-3 Encodes a Novel Regulator of Presynaptic Activity in C. elegans

C. elegans aex-3 mutations cause pleiotropic behavioral defects that are suggestive of reduced synaptic transmission. aex-3 mutations also show strong genetic interactions with mutations in unc-31 and unc-64, two other genes implicated in synaptic transmission. Physiological and pharmacological studies indicate that aex-3 defects are presynaptic. In aex-3 mutants, the synaptic vesicle-associated RAB-3 protein aberrantly accumulates in neuronal cell bodies and is reduced in synapse-rich axons. This localization defect is specific to RAB-3, since other synaptic proteins are localized normally in aex-3 mutants. aex-3 encodes a 1409 amino acid protein with strong homology to DENN, a human protein of unknown function. In C. elegans, aex-3 is expressed in all or nearly all neurons. These results suggest that AEX-3 is a novel regulator of presynaptic activity that interacts with RAB-3 to regulate synaptic vesicle release.