SUMMARYWe expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNAi, in C. elegans neurons. This expression increased the response of neurons to dsRNA delivered by feeding. Mutations in the lin-15b and lin-35 genes further enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased non-neuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(−) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation.

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG͞ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.prohibitin-domain proteins ͉ TRP channels ͉ DEG/ENaC channels ͉ slit diaphragm ͉ mechanosensation T he prohibitin homology (PHB)-domain proteins constitute a family of Ϸ1,800 proteins (SMART database; http:͞͞smart. embl-heidelberg.de) (1) all of which share an Ϸ150-aa domain similar to that in the mitochondrial protein prohibitin (2). More than 340 of these proteins, many of which have an N-terminal adjacent hydrophobic region that places them on the inner leaflet of the lipid bilayer, have been identified in animals. These membrane-associated proteins regulate osmotic homeostasis, mechanosensation, and cell signaling (3-5). Several of the animal PHB-domain proteins including flotillin, Podocin, prohibitin, stomatin, UNC-1, UNC-24, and the UNC-24-like mammalian protein SLP-1 are found in cholesterol-rich membrane fractions derived from the plasma membrane (reviewed in ref.2).In this article, we investigate the function of these proteins using two members of the family, MEC-2 from Caenorhabditis elegans and Podocin from mouse. MEC-2 (6) and Podocin (7) have a single, central hydrophobic domain that embeds these proteins in the inner leaflet of the plasma membrane with their N-and C-terminal tails facing the cytoplasm (Fig. 1a). Although the two proteins contain different N and C termini, they have hydrophobic regions that are 35% identical and 75% similar and PHB-domains that are 50% identical and 80% similar (Fig. 1b). The PHB domain is critical for the action of both proteins (8, 9).

Axonal degeneration is a key event in the pathogenesis of neurodegenerative conditions. We show here that mec-4d triggered axonal degeneration of Caenorhabditis elegans neurons and mammalian axons share mechanistical similarities, as both are rescued by inhibition of calcium increase, mitochondrial dysfunction, and NMNAT overexpression. We then explore whether reactive oxygen species (ROS) participate in axonal degeneration and neuronal demise. C. elegans dauers have enhanced anti-ROS systems, and dauer mec-4d worms are completely protected from axonal degeneration and neuronal loss. Mechanistically, downregulation of the Insulin/IGF-1-like signaling (IIS) pathway protects neurons from degenerating in a DAF-16/FOXO–dependent manner and is related to superoxide dismutase and catalase-increased expression. Caloric restriction and systemic antioxidant treatment, which decrease oxidative damage, protect C. elegans axons from mec-4d-mediated degeneration and delay Wallerian degeneration in mice. In summary, we show that the IIS pathway is essential in maintaining neuronal homeostasis under pro-degenerative stimuli and identify ROS as a key intermediate of neuronal degeneration in vivo. Since axonal degeneration represents an early pathological event in neurodegeneration, our work identifies potential targets for therapeutic intervention in several conditions characterized by axonal loss and functional impairment.

The dynamic response of organisms exposed to environmental pathogens determines their survival or demise, and the outcome of this interaction depends on the host’s susceptibility and pathogen-dependent virulence factors. The transmission of acquired information about the nature of a pathogen to progeny may ensure effective defensive strategies for the progeny’s survival in adverse environments. Environmental RNA interference (RNAi) is a systemic and heritable mechanism and has recently been linked to antibacterial and antifungal defenses in both plants and animals. Here, we report that the second generation of Caenorhabditis elegans living on pathogenic bacteria can avoid bacterial infection by entering diapause in an RNAi pathway-dependent mechanism. Furthermore, we demonstrate that the information encoding this survival strategy is transgenerationally transmitted to the progeny via the maternal germ line.