Caenorhabditis elegans PAQR-2 and IGLR-2 Protect against Glucose Toxicity by Modulating Membrane Lipid Composition.

In spite of the worldwide impact of diabetes on human health, the mechanisms behind glucose toxicity remain elusive. Here we show that C. elegans mutants lacking paqr-2, the worm homolog of the adiponectin receptors AdipoR1/2, or its newly identified functional partner iglr-2, are glucose intolerant and die in the presence of as little as 20 mM glucose. Using FRAP (Fluorescence Recovery After Photobleaching) on living worms, we found that cultivation in the presence of glucose causes a decrease in membrane fluidity in paqr-2 and iglr-2 mutants and that genetic suppressors of this sensitivity act to restore membrane fluidity by promoting fatty acid desaturation. The essential roles of paqr-2 and iglr-2 in the presence of glucose are completely independent from daf-2 and daf-16, the C. elegans homologs of the insulin receptor and its downstream target FoxO, respectively. Using bimolecular fluorescence complementation, we also show that PAQR-2 and IGLR-2 interact on plasma membranes and thus may act together as a fluidity sensor that controls membrane lipid composition.

Correction: Caenorhabditis elegans PAQR-2 and IGLR-2 Protect against Glucose Toxicity by Modulating Membrane Lipid Composition.

[This corrects the article DOI: 10.1371/journal.pgen.1005982.].

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway.

Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction.

The mevalonate pathway in C. elegans.

The mevalonate pathway in human is responsible for the synthesis of cholesterol and other important biomolecules such as coenzyme Q, dolichols and isoprenoids. These molecules are required in the cell for functions ranging from signaling to membrane integrity, protein prenylation and glycosylation, and energy homeostasis. The pathway consists of a main trunk followed by sub-branches that synthesize the different biomolecules. The majority of our knowledge about the mevalonate pathway is currently focused on the cholesterol synthesis branch, which is the target of the cholesterol-lowering statins; less is known about the function and regulation of the non-cholesterol-related branches. To study them, we need a biological system where it is possible to specifically modulate these metabolic branches individually or in groups. The nematode Caenorhabditis elegans (C. elegans) is a promising model to study these non-cholesterol branches since its mevalonate pathway seems very well conserved with that in human except that it has no cholesterol synthesis branch. The simple genetic makeup and tractability of C. elegans makes it relatively easy to identify and manipulate key genetic components of the mevalonate pathway, and to evaluate the consequences of tampering with their activity. This general experimental approach should lead to new insights into the physiological roles of the non-cholesterol part of the mevalonate pathway. This review will focus on the current knowledge related to the mevalonate pathway in C. elegans and its possible applications as a model organism to study the non-cholesterol functions of this pathway.

The C. elegans M3 neuron guides the growth cone of its sister cell M2 via the Krüppel-like zinc finger protein MNM-2.

The invariant cell-cell interactions occurring during C. elegans development offer unique opportunities to discover how growing axons may receive guidance cues from neighboring cells. The mnm-2 mutant was isolated because of its defects in the axon trajectory of the bilateral M2 pharyngeal neurons in C. elegans. We found that mnm-2 enhances the effects of many growth cone guidance mutations on these axons, suggesting that it performs a novel function during axon guidance. We cloned mnm-2 and found that it encodes a protein with three C2H2 zinc finger domains related to the Krüppel-like Factor protein family. mnm-2 is expressed only transiently in the M2 neuron, but exhibits a sustained expression in its sister cell, the M3 neuron. Strikingly, the expression of mnm-2 is not sustained in the M3 cell of the mnm-2 mutant, indicating that this gene positively regulates itself in that cell. Electropharyngeograms also indicate that the M3 cell is functionally impaired in the mnm-2 mutant. We used an M3-specific promoter to show that the M2 axon defect can be rescued by expression of mnm-2 in its sister cell M3. The same promoter was used to express the pro-apoptotic gene egl-1 to kill the M3 cell, which resulted in an M2 axon guidance defect similar to that found in the mnm-2 mutant. Our results suggest an M2 axon guidance model in which the M3 cell provides an important signal to the growth cone of its sister M2 and that this signal and the proper differentiation of M3 both depend on mnm-2 expression. This mechanism of axon guidance regulation allows fine-tuning of trajectories between sister cells.

Developmental genetics of the C. elegans pharyngeal neurons NSML and NSMR.

BACKGROUND: We are interested in understanding how the twenty neurons of the C. elegans pharynx develop in an intricate yet reproducible way within the narrow confines of the embryonic pharyngeal primordium. To complement an earlier study of the pharyngeal M2 motorneurons, we have now examined the effect of almost forty mutations on the morphology of a bilateral pair of pharyngeal neurosecretory-motor neurons, the NSMs. RESULTS: A careful description of the NSM morphology led to the discovery of a third, hitherto unreported process originating from the NSM cell body and that is likely to play a proprioceptive function. We found that the three NSM processes are differently sensitive to mutations. The major dorsal branch was most sensitive to mutations that affect growth cone guidance and function (e.g. unc-6, unc-34, unc-73), while the major sub-ventral branch was more sensitive to mutations that affect components of the extracellular matrix (e.g. sdn-1). Of the tested mutations, only unc-101, which affects an adaptin, caused the loss of the newly described thin minor process. The major processes developed synaptic branches post-embryonically, and these exhibited activity-dependent plasticity. CONCLUSION: By studying the effects of nearly forty different mutations we have learned that the different NSM processes require different genes for their proper guidance and use both growth cone dependent and growth cone independent mechanisms for establishing their proper trajectories. The two major NSM processes develop in a growth cone dependent manner, although the sub-ventral process relies more on substrate adhesion. The minor process also uses growth cones but uniquely develops using a mechanism that depends on the clathrin adaptor molecule UNC-101. Together with the guidance of the M2 neuron, this is the second case of a pharyngeal neuron establishing one of its processes using an unexpected mechanism.

The twisted pharynx phenotype in C. elegans.

BACKGROUND: The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development. Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype but no careful studies have been made to directly address this phenomenon. In this study, the twisting mutants dig-1, mig-4, mnm-4 and unc-61 are examined in detail and the nature of the twist is investigated. RESULTS: We find that the twisting phenotype worsens throughout larval development, that in most mutants the pharynx retains its twist when dissected away from the worm body, and that double mutants between mnm-4 and mutants with thickened pharyngeal domains (pha-2 and sma-1) have less twisting in these regions. We also describe the ultrastructure of pharyngeal tendinous organs that connect the pharyngeal basal lamina to that of the body wall, and show that these are pulled into a spiral orientation by twisted pharynges. Within twisted pharynges, actin filaments also show twisting and are longer than in controls. In a mini screen of adhesionmolecule mutants, we also identified one more twisting pharynx mutant, sax-7. CONCLUSION: Defects in pharyngeal cytoskeleton length or its anchor points to the extracellular matrix are proposed as the actual source of the twisting force. The twisted pharynx is a useful and easy-to-score phenotype for genes required in extracellular adhesion or organ attachment, and perhaps forgenes required for cytoskeleton regulation.

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells.

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations.

MicroRNA Regulation of nAChR Expression and Nicotine-Dependent Behavior in C. elegans.

Chronic exposure to nicotine upregulates nicotinic acetylcholine receptors (nAChRs), and such upregulation is critical for the development of nicotine dependence in humans and animal models. However, how nicotine upregulates nAChRs is not well understood. Here, we identify a key role for microRNA in regulating nicotine-dependent behavior by modulating nAChR expression in C. elegans. We show that the nAChR gene acr-19 and alg-1, a key Argonaute-family member in the microRNA machinery, are specifically required for nicotine withdrawal response following chronic nicotine treatment. Chronic exposure to nicotine downregulates alg-1, leading to upregulation of acr-19. This effect is mediated by the microRNA miR-238 that recognizes the 3' UTR of acr-19 transcript. Our results unveil a previously unrecognized role for microRNA in nicotine signaling, providing insights into how chronic nicotine administration leads to upregulation of nAChR and ultimately nicotine dependence.

Immunization with recombinant adenovirus synthesizing the secretory form of Japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis.

Replication-defective recombinant adenoviruses (RAds) were constructed that synthesized the pre-membrane and envelope (E) proteins of Japanese encephalitis virus (JEV). Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa replicated poorly in HEK 293A cells and synthesized lower amounts of E protein. Oral immunization of mice with RAds generated low titers of anti-JEV antibodies that had little JEV neutralizing activity. Intra-muscular (IM) immunization of mice with either RAd generated high titers of anti-JEV antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing antibodies. Titers were significantly higher in case of RAdEs immunization. Splenocytes from mice immunized IM with RAds secreted large amounts of interferon-gamma and moderate amounts of interleukin-5 in the presence of JEV and showed cytotoxic activity against JEV-infected cells. Naïve mice immunized IM with RAdEs showed complete protection against a lethal dose of JEV given intra-cerebrally. In order to study the effect of the pre-existing adenovirus 5 (Ad5) immunity on the outcome of the RAdEs immunization, mice were exposed to Ad5 through IM or intra-nasal (IN) routes before immunization with RAdEs. Mice exposed to Ad5 through the IN route, when immunized with RAdEs given IM, or those exposed to Ad5 through the IM route, when immunized with RAdEs given IN, were completely protected against lethal JEV challenge.

A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans.

We previously showed that inhibition of the mevalonate pathway in C. elegans causes inhibition of protein prenylation, developmental arrest and lethality. We also showed that constitutive activation of the mitochondrial unfolded protein response, UPR(mt), is an effective way for C. elegans to become resistant to the negative effects of mevalonate pathway inhibition. This was an important finding since statins, a drug class prescribed to lower cholesterol levels in patients, act by inhibiting the mevalonate pathway, and it is therefore possible that some of their undesirable side effects could be alleviated by activating the UPR(mt). Here, we screened a chemical library and identified 4 compounds that specifically activated the UPR(mt). One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Methacycline hydrochloride and ethidium bromide, a known UPR(mt) activator, were also tested in mice: only ethidium bromide significantly activate the UPR(mt) in skeletal muscles.

A Mutation in Caenorhabditis elegans NDUF-7 Activates the Mitochondrial Stress Response and Prolongs Lifespan via ROS and CED-4.

The mevalonate pathway is responsible for the synthesis of cholesterol, coenzyme Q, and prenyl groups essential for small GTPase modification and function, and for the production of dolichols important for protein glycosylation. Statins, i.e., cholesterol-lowering drugs that inhibit the rate-limiting enzyme in the mevalonate pathway, HMG-CoA reductase, are lethal to Caenorhabditis elegans even though this animal lacks the branch of the mevalonate pathway that leads to cholesterol synthesis. To better understand the effects of statins that are not related to cholesterol, we have adopted the strategy of isolating statin-resistant C. elegans mutants. Previously, we showed that such mutants often have gain-of-function mutations in ATFS-1, a protein that activates the mitochondrial unfolded protein response. Here, we describe the isolation of a statin-resistant mutant allele of the NDUF-7 protein, which is a component of complex I in the mitochondrial electron transport chain. The novel nduf-7(et19) mutant also exhibits constitutive and ATFS-1-dependent activation of the mitochondrial unfolded protein response (UPR(mt)) and prolonged life span, both of which are mediated through production of ROS. Additionally, lifespan extension, but not activation, of the mitochondrial unfolded protein response was dependent on the pro-apoptotic gene ced-4. We conclude that the nduf-7(et19) mutant allele causes an increase in reactive oxygen species that activate ATFS-1, hence UPR(mt)-mediated statin resistance, and extends life span via CED-4.

Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies.

In order to evaluate the possibility of developing an oral vaccine against Japanese encephalitis virus (JEV), mice were fed with recombinant JEV envelope (E) protein synthesized in Escherichia coli. The protein was administered orally to mice with or without an immunostimulatory cytosine-phosphate-guanosine (CpG) motif containing synthetic oligodeoxynucleotide (ODN) as an adjuvant. The immunized mice made high-titered anti-E and anti-JEV antibodies. Mice immunized with JEV E protein along with the ODN adjuvant produced higher antibody titers and these were predominantly IgG2a type. These antibodies, however, failed to neutralize JEV activity in vitro, and the immunization did not protect the mice against lethal JEV challenge. Splenocytes from the immunized mice secreted large amounts of interferon (IFN)-gamma and showed proliferation in the presence of JEV E protein. Our results indicate that JEV E protein delivered orally to mice together with ODN generated both humoral and cellular immune responses to JEV, and these were of the Th1 type.

Loss of HMG-CoA reductase in C. elegans causes defects in protein prenylation and muscle mitochondria.

HMG-CoA reductase is the rate-limiting enzyme in the mevalonate pathway and the target of cholesterol-lowering statins. We characterized the C. elegans hmgr-1(tm4368) mutant, which lacks HMG-CoA reductase, and show that its phenotypes recapitulate that of statin treatment, though in a more severe form. Specifically, the hmgr-1(tm4368) mutant has defects in growth, reproduction and protein prenylation, is rescued by exogenous mevalonate, exhibits constitutive activation of the UPRer and requires less mevalonate to be healthy when the UPRmt is activated by a constitutively active form of ATFS-1. We also show that different amounts of mevalonate are required for different physiological processes, with reproduction requiring the highest levels. Finally, we provide evidence that the mevalonate pathway is required for the activation of the UPRmt.

When a TRP goes bad: transient receptor potential channels in addiction.

Drug addiction is a psychiatric disease state, wherein a drug is impulsively and compulsively self-administered despite negative consequences. This repeated administration results in permanent changes to nervous system physiology and architecture. The molecular pathways affected by addictive drugs are complex and inter-dependent on each other. Recently, various new proteins and protein families have been discovered to play a role in drug abuse. Emerging players in this phenomenon include TRP (Transient Receptor Potential) family channels, which are primarily known to function in sensory systems. Several TRP family channels identified in both vertebrates and invertebrates are involved in psychostimulant-induced plasticity, suggesting their involvement in drug dependence. This review summarizes various observations, both from studies in humans and other organisms, which support a role for these channels in the development of drug-related behaviors.

Immunogenicity in mice of a cationic microparticle-adsorbed plasmid DNA encoding Japanese encephalitis virus envelope protein.

Previously, we described a plasmid pMEa, synthesizing the anchored form of Japanese encephalitis virus (JEV) envelope protein that generated virus-neutralizing antibodies in mice upon intra-muscular injection. The immunized mice showed significant protection against lethal JEV challenge. In order to improve the efficacy of plasmid DNA immunization against JEV, methods need to be employed that would enhance neutralizing antibody titers. Plasmid DNA adsorbed to cationic microparticles has recently been shown to significantly improve its immunogenicity. In the present study, we have adsorbed the plasmid pMEa on cationic microparticles and have compared its immunogenicity with the naked plasmid DNA. As seen in ELISA, the microparticle-adsorbed DNA induced higher titers of anti-JEV antibodies when compared to those induced by the naked DNA. No difference, however, was seen in JEV neutralization titers. The microparticle-adsorbed DNA induced a mixed Th1-Th2 kind of immune responses as opposed to Th1 type of immune responses elicited by the naked DNA.

pha-2 encodes the C. elegans ortholog of the homeodomain protein HEX and is required for the formation of the pharyngeal isthmus.

The pha-2 mutant was isolated in 1993 by Leon Avery in a screen for worms with visible defects in pharyngeal feeding behavior. In pha-2 mutant worms, the pharyngeal isthmus is abnormally thick and short and, in contrast to wild-type worms, harbors several cell nuclei. We show here that pha-2 encodes a homeodomain protein and is homologous to the vertebrate homeobox gene, Hex (also known as Prh). Consistent with a function in pharyngeal development, the pha-2 gene is expressed in the pharyngeal primordium of Caenorhabditis elegans embryos, particularly in pm5 cells that form the bulk of the isthmus. We show that in the pha-2 mutant there is a failure of the pm5 cells to elongate anteriorly while keeping their nuclei within the nascent posterior bulb to form the isthmus during the 3-fold embryonic stage. We also present evidence that pha-2 regulates itself positively in pm5 cells, that it is a downstream target of the forkhead gene pha-4, and that it may also act in the isthmus as an inhibitor of the ceh-22 gene, an Nkx2.5 homolog. Finally, we have begun characterizing the regulation of the pha-2 gene and find that intronic sequences are essential for the complete pha-2 expression profile. The present report is the first to examine the expression and function of an invertebrate Hex homolog, that is, the C. elegans pha-2 gene.