MUT-7 Provides Molecular Insight into the Werner Syndrome Exonuclease

Werner syndrome (WS) is a rare recessive genetic disease characterized by premature aging. Individuals with this disorder develop normally during childhood, but their physiological conditions exacerbate the aging process in late adolescence. WS is caused by mutation of the human WS gene (WRN), which encodes two main domains, a 3′-5′ exonuclease and a 3′-5′ helicase. Caenorhabditis elegans expresses human WRN orthologs as two different proteins: MUT-7, which has a 3′-5′ exonuclease domain, and C. elegans WRN-1 (CeWRN-1), which has only helicase domains. These unique proteins dynamically regulate olfactory memory in C. elegans, providing insight into the molecular roles of WRN domains in humans. In this review, we specifically focus on characterizing the function of MUT-7 in small interfering RNA (siRNA) synthesis in the cytoplasm and the roles of siRNA in directing nuclear CeWRN-1 loading onto a heterochromatin complex to induce negative feedback regulation. Further studies on the different contributions of the 3′-5′ exonuclease and helicase domains in the molecular mechanism will provide clues to the accelerated aging processes in WS

Endogenous Nuclear RNAi Mediates Behavioral Adaptation to Odor

SummaryMost eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that 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 whose downregulation 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.PaperFlic

Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides.

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

Expression of an expanded CGG-repeat RNA in a single pair of primary sensory neurons impairs olfactory adaptation in Caenorhabditis elegans.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X mental retardation 1 (FMR1) gene; current evidence supports a causal role of the expanded CGG repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons interfered with this plasticity without affecting either the innate odor-seeking response or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to diminish olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS

Expression of an expanded CGG-repeat RNA in a single pair of primary sensory neurons impairs olfactory adaptation in Caenorhabditis elegans.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X mental retardation 1 (FMR1) gene; current evidence supports a causal role of the expanded CGG repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons interfered with this plasticity without affecting either the innate odor-seeking response or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to diminish olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS

Severe denervation of FDB-2muscle in severe SMA mouse is restored by MO treatment.

<p>(A-C) The confocal images are Z-stack projection images at P9, stained with anti-synaptophysin antibody, anti-neurofilament antibody, and conjugated α-bungarotoxin protein from FDB-2 muscles of control, severe SMA, and MO-treated severe SMA mice. The images in parallel show individual channels of grey-scale images and merged images. (A) FDB-2 muscle from control mice (n = 3). NMJs are partially or fully innervated. (B) FDB-2 muscle from severe SMA mice (n = 3). Most NMJs are fully denervated (arrow). (C) FDB-2 muscle from severe SMA mice with MO treatment. Recovery is observed after MO treatment by SC injection at P0. (D) The endplate size of AT muscle in severe SMA mice, MO-treated SMA mice, and control mice at P9. (E) Quantification of fully innervated endplates (blue), partially denervated endplates (yellow), and fully denervated endplates (red) in FDB-2 muscles of control, severe SMA, and MO-treated severe SMA mice at P9. One hundred of individual muscles were counted in each mouse, and 3 pairs of control, severe SMA and MO-treated severe SMA mice were studied. All quantitative data are mean ± SEM, ***<i>P</i> < 0.001.</p

The full denervation of endplates is observed on the FDB-2 muscle of severe SMA mice at P9.

<p>The confocal images are Z-stack projection images, stained with anti-synaptophysin and anti-neurofilament antibodies and conjugated α-BTX protein, in control and Taiwanese severe SMA mice at P9. The images in parallel show individual channel grey-scale images and merge images. (A, B) FDB-2 muscle from control (n = 3) and severe SMA mice (n = 3). Most NMJs are fully denervated (arrow) or partially innervated in the FDB-2 muscle of severe SMA mice. (C, D) SPI muscles from control mice and severe SMA mice, (E, F) splenius capitis muscles from control mice and severe SMA mice, and (G, H) longissimus capitis muscles from control mice and severe SMA mice show little difference. (FDB, flexor digitorum brevis; SPI, serratus posterior inferior; α-BTX, α-bungarotoxin).</p

NMJ denervation in different pattern muscles of severe SMA mice at P9.

<p>(A) Quantification of fully innervated endplates in muscles of control (blue) and severe SMA (red) mice at end stage (P9). (B) Quantification of fully innervated endplates (blue), partially denervated endplates (yellow), and fully denervated endplates (red) in muscles of severe SMA mice at P9. One hundred NMJs of individual muscles were counted in each mouse, 3 pairs of severe SMA and control mice were studied. All quantitative data are mean ± SEM. (FDB, flexor digitorum brevis; AT, anterior tibialis; EDL, extensor digitorum longus; SPI, serratus posterior inferior)</p