Summary Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs) remains unclear. Here we show the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene who's down regulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation.

To navigate a complex and changing environment, an animal's sensory neurons must continually adapt to persistent cues while remaining responsive to novel stimuli. Long-term exposure to an inherently attractive odor causes Caenorhabditis elegans to ignore that odor, a process termed odor adaptation. Odor adaptation is likely to begin within the sensory neuron, because it requires factors that act within these cells at the time of odor exposure. The process by which an olfactory sensory neuron makes a decisive shift over time from a receptive state to a lasting unresponsive one remains obscure. In C. elegans, adaptation to odors sensed by the AWC pair of olfactory neurons requires the cGMP-dependent protein kinase EGL-4. Using a fully functional, GFP-tagged EGL-4, we show here that prolonged odor exposure sends EGL-4 into the nucleus of the stimulated AWC neuron. This odor-induced nuclear translocation correlates temporally with the stable dampening of chemotaxis that is indicative of long-term adaptation. Long-term adaptation requires cGMP binding residues as well as an active EGL-4 kinase. We show here that EGL-4 nuclear accumulation is both necessary and sufficient to induce longlasting odor adaptation. After it is in the AWC nucleus, EGL-4 decreases the animal's responsiveness to AWC-sensed odors by acting downstream of the primary sensory transduction. Thus, the EGL-4 protein kinase acts as a sensor that integrates odor signaling over time, and its nuclear translocation is an instructive switch that allows the animal to ignore persistent odors.neuron | plasticity | signaling | memory | integration S ensory neurons, the entry point for information about an animal's external environment, must both respond to relevant stimuli and dampen their response as a consequence of prolonged stimulation. This dampening or adaptation is thought to be a protective consequence of prolonged stimulation, because indeed, adaptation protects mammalian photoreceptors from stimulationinduced degeneration (1, 2). The cellular, molecular, and temporal controls that allow sensory neurons to switch from being responsive to refractory to a given stimulus are examined herein. The Caenorhabditis elegans AWC sensory neuronal pair can sense (3) and adapt (4) to inherently attractive odors, and although both sensory and interneurons within the olfactory circuit adapt to persistent stimulation (5, 6), this focus will be on the events that instruct long-lasting adaptation within the sensory neuron.The AWC neurons (3, 7) are postulated to express at least 20 different odor-responsive G protein-coupled receptors (8, 9), use cGMP as a second messenger (10-13), and hyperpolarize in response to odor (14). Odor adaptation begins within 10 min of odor exposure, and there is a mild decrease in the animal's attraction to the odor accompanied by an increase in phosphorylated MAP kinase in AWC (5, 15) that requires the G protein gamma, GPC-1 (16). After 30 min of exposure, attraction decreases further as the animal enters a phase of short-term adaptation...

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies.

Caenorhabditis elegans expresses human Werner syndrome protein (WRN) orthologs as two distinct proteins: MUT-7, with a 3'-5' exonuclease domain, and CeWRN-1, with helicase domains. How these domains cooperate remains unclear. Here, we demonstrate the different contributions of MUT-7 and CeWRN-1 to 22G small interfering RNA (siRNA) synthesis and the plasticity of neuronal signaling. MUT-7 acts specifically in the cytoplasm to promote siRNA biogenesis and in the nucleus to associate with CeWRN-1. The import of siRNA by the nuclear Argonaute NRDE-3 promotes the loading of the heterochromatin-binding protein HP1 homolog HPL-2 onto specific loci. This heterochromatin complex represses the gene expression of the guanylyl cyclase ODR-1 to direct olfactory plasticity in C. elegans. Our findings suggest that the exonuclease and helicase domains of human WRN may act in concert to promote RNA-dependent loading into a heterochromatin complex, and the failure of this entire process reduces plasticity in postmitotic neurons.