The Dunce cAMP phosphodiesterase PDE-4 negatively regulates G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the Caenorhabditis elegans synaptic signaling network.

Forward genetic screens for mutations that rescue the paralysis of ric-8 (Synembryn) reduction-of-function mutations frequently reveal mutations that cause hyperactivation of one or more components of the G alpha(s) pathway. Here, we report that one of these mutations strongly reduces the function of the Dunce cAMP phosphodiesterase PDE-4 by disrupting a conserved active site residue. Loss of function and neural overexpression of PDE-4 have profound and opposite effects on locomotion rate, but drug-response assays suggest that loss of PDE-4 function does not affect steady-state acetylcholine release or reception. Our genetic analysis suggests that PDE-4 regulates both G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the neurons controlling locomotion rate. By immunostaining, PDE-4 is strongly expressed throughout the nervous system, where it localizes to small regions at the outside boundaries of synaptic vesicle clusters as well as intersynaptic regions. The synaptic subregions containing PDE-4 are distinct from those containing active zones, as indicated by costaining with an antibody against the long form of UNC-13. This highly focal subsynaptic localization suggests that PDE-4 may exert its effects by spatially regulating intrasynaptic cAMP pools.

Presynaptic UNC-31 (CAPS) is required to activate the G alpha(s) pathway of the Caenorhabditis elegans synaptic signaling network.

C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal G alpha(s) pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal G alpha(s) are different parts of the same pathway and that the UNC-31/G alpha(s) pathway is functionally distinct from the presynaptic G alpha(q) pathway with which it interacts. UNC-31 acts upstream of G alpha(s) because mutations that activate the G alpha(s) pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (+) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the G alpha(s) pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal G alpha(s) pathway near synaptic active zones.

Mutations that rescue the paralysis of Caenorhabditis elegans ric-8 (synembryn) mutants activate the G alpha(s) pathway and define a third major branch of the synaptic signaling network.

To identify hypothesized missing components of the synaptic G alpha(o)-G alpha(q) signaling network, which tightly regulates neurotransmitter release, we undertook two large forward genetic screens in the model organism C. elegans and focused first on mutations that strongly rescue the paralysis of ric-8(md303) reduction-of-function mutants, previously shown to be defective in G alpha(q) pathway activation. Through high-resolution mapping followed by sequence analysis, we show that these mutations affect four genes. Two activate the G alpha(q) pathway through gain-of-function mutations in G alpha(q); however, all of the remaining mutations activate components of the G alpha(s) pathway, including G alpha(s), adenylyl cyclase, and protein kinase A. Pharmacological assays suggest that the G alpha(s) pathway-activating mutations increase steady-state neurotransmitter release, and the strongly impaired neurotransmitter release of ric-8(md303) mutants is rescued to greater than wild-type levels by the strongest G alpha(s) pathway activating mutations. Using transgene induction studies, we show that activating the G alpha(s) pathway in adult animals rapidly induces hyperactive locomotion and rapidly rescues the paralysis of the ric-8 mutant. Using cell-specific promoters we show that neuronal, but not muscle, G alpha(s) pathway activation is sufficient to rescue ric-8(md303)'s paralysis. Our results appear to link RIC-8 (synembryn) and a third major G alpha pathway, the G alpha(s) pathway, with the previously discovered G alpha(o) and G alpha(q) pathways of the synaptic signaling network.

Convergent, RIC-8-dependent Galpha signaling pathways in the Caenorhabditis elegans synaptic signaling network.

We used gain-of-function and null synaptic signaling network mutants to investigate the relationship of the G alpha(q) and G alpha(s) pathways to synaptic vesicle priming and to each other. Genetic epistasis studies using G alpha(q) gain-of-function and null mutations, along with a mutation that blocks synaptic vesicle priming and the synaptic vesicle priming stimulator phorbol ester, suggest that the G alpha(q) pathway generates the core, obligatory signals for synaptic vesicle priming. In contrast, the G alpha(s) pathway is not required for the core priming function, because steady-state levels of neurotransmitter release are not significantly altered in animals lacking a neuronal G alpha(s) pathway, even though these animals are strongly paralyzed as a result of functional (nondevelopmental) defects. However, our genetic analysis indicates that these two functionally distinct pathways converge and that they do so downstream of DAG production. Further linking the two pathways, our epistasis analysis of a ric-8 null mutant suggests that RIC-8 (a receptor-independent G alpha guanine nucleotide exchange factor) is required to maintain both the G alpha(q) vesicle priming pathway and the neuronal G alpha(s) pathway in a functional state. We propose that the neuronal G alpha(s) pathway transduces critical positional information onto the core G alpha(q) pathway to stabilize the priming of selected synapses that are optimal for locomotion.