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First published online 27 February 2007
doi: 10.1242/jcs.03401
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Research Article |
1 Department of Genetics, Eötvös Loránd University, Budapest, H-1117, Hungary
2 Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, H-1117, Hungary
* Author for correspondence (e-mail: vellai{at}falco.elte.hu)
Accepted 10 January 2007
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Neurodegeneration, Ion channel subunits, Autophagy, C. elegans, Dopamine neurons, TOR signaling
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Introduction |
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; Yuan et al., 2003; Rubinsztein et al., 2005). The majority of these pathologies are induced by dominant mutations that cause late-onset loss of the affected nerve cells. Accumulating data suggest that autophagy – a process in which eukaryotic cells self-digest parts of their contents during development and to survive nutrient deprivation (Klionsky and Emr, 2000; Noda et al., 2002; Cuervo, 2004) – is implicated in various pathological states of the nervous system (Rubinsztein et al., 2005). Autophagy involves the formation of subcellular membrane structures termed autophagic vacuoles to sequester cargo for breakdown by acidic lysosomal hydrolases. In some cases, autophagic degradation of cytoplasmic constituents occurs as part of programmed cell death (Muller et al., 2004).
A strong association between the autophagic process and neuronal function results from studies on Lurcher mice bearing a gain-of-function mutation in the orphan delta 2 glutamate receptor (GluR
2) (Zuo et al., 1997). This mutation causes neuronal degeneration in vivo. Characterization of a protein complex bound to GluR2 identified the autophagy protein Beclin1 (Liang et al., 1999) as a component of the complex (Yue et al., 2002). This finding raises the possibility that autophagy contributes to Lurcher pathology. Indeed, degrading Lurcher purkinje cells display intense autophagic activity (Doughty et al., 2000). Neuronal degeneration following traumatic brain injury in mice has also been shown to induce upregulation of Beclin1 (Erlich et al., 2006). Furthermore, accumulation of vacuole-like structures and lysosomes in neurons were reported in patients with a variety of neurodegenerative pathologies (Rubinsztein et al., 2005; Cataldo et al., 1994; Webb et al., 2003; Qin et al., 2003).
In Caenorhabditis elegans, gain-of-function mutant alleles of genes encoding specific ion channel subunits result in necrotic-like swelling and death of certain neurons. For example, gain-of-function mutations in deg-3, which encodes the 
-subunit of the neuronal nicotinic acetylcholine receptor, trigger degeneration of several sensory neurons and PVC interneurons (Treinin and Chalfie, 1995). Gain-of-function alleles of two degenerin ion channel genes, deg-1 and mec-4, cause the degenerative state of the IL1, ASH and PVC neurons, and six touch receptor neurons, respectively, rendering mutants incapable of mechanosensation (Chalfie and Wolinsky, 1990; Driscoll and Chalfie, 1991). In these genetic models, the identity of the dying neurons (Fig. 1A,B) and the time in development at which these cells die are essentially invariant among individuals (Chalfie and Sulston, 1981).
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). The function of certain proteases is thus likely to contribute to necrotic cell death. The execution of degenerative processes in dying neurons is also influenced by acidic intracellular conditions because the vacuolar H+-ATPase, a membrane pump that acidifies the cell interior, has been found to contribute to toxic ion channel-induced necrosis (Syntichaki et al., 2005).
Ultrastructural analysis of dying neurons in mec-4(gf) and deg-1(gf) mutants has shown extensive degradation of cytoplasmic contents as well as formation of cytoplasmic vacuoles and membranous whorls during mid to late stages of cell death (Hall et al., 1997). Structural changes involving membrane rearrangements are also characteristic features in a wide range of cells with autophagic activity (Meléndez et al., 2003). Furthermore, lysosome function, which is essential for autophagy, is crucial for neuronal degeneration in C. elegans (Artal-Sanz et al., 2006). Here we investigate the role of three C. elegans autophagy genes, bec-1, unc-51 and lgg-1 (Meléndez et al., 2003; Kovács et al., 2004; Takács-Vellai et al., 2005), in necrotic-like cell death induced by mutations affecting ion channel subunits or by the neurotoxin 6-hydroxidopamine (6-OHDA). Our results demonstrate that inactivation of these genes influences neuronal degeneration in this organism.
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Results |
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; Kovács et al., 2004). To evaluate whether these components, or at least some of them, are required for structural membrane changes including vacuolization that precede the death of toxic ion-channel-poisoned neurons, we first performed a genetic analysis of degenerin mutants with reduced or no BEC-1 activity. BEC-1 is similar to the yeast and mammalian autophagy proteins Atg6/Vps30 and Beclin1, and essential for development (Meléndez et al., 2003; Takács-Vellai et al., 2005). Two mutations in bec-1, ok691 and ok700, result in a highly penetrant (92.7% and 85.1%, respectively) embryonic arrest phenotype (Takács-Vellai et al., 2005). Homozygous bec-1 mutants that pass embryonic development die at different larval stages or at young adulthood. These animals often fail to emerge from the larval cuticle, are fragile and, if they pass the larval stages, become sterile. We found that in double mutant animals bearing the gain-of-function mutation u231 in mec-4, bec-1 mutant alleles suppress vacuolarization and swelling of touch receptor neurons (Fig. 2A). mec-4(u231);bec-1(ok691) and mec-4(u231);bec-1(ok700) double mutant animals had fewer vacuolated neurons than mec-4(u231) single mutants. Note that vacuolization is an unambiguous morphological sign of cell death in this neurodegeneration paradigm (Fig. 1D) (Hall et al., 1997; Syntichaki et al., 2002; Syntichaki et al., 2005). We rescued the lethality of bec-1(–) mutants by a non-integrated and thus unstable array containing wild-type copies of bec-1 (this array actually consists of copies of a translational fusion functional BEC-1::GFP reporter: swEx520[pbec-1::BEC-1::GFP + rol-6(su1006)], see Materials and Methods). According to our data, vacuolization of touch receptor neurons caused by the mec-4 alleles u231 (Fig. 2A) and e1611 (data not shown) was significantly suppressed in bec-1(–);Ex[bec-1(+)] genetic background. Together, these data suggest a general requirement for BEC-1 in neuronal degeneration.
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We also assayed cell death at the young adult stage in mec-4(u231) mutant hermaphrodites deficient for BEC-1 by scoring the number of intact touch receptor neurons that express a pmec-4::GFP reporter (Syntichaki et al., 2005). This reporter is expressed consistently in the touch receptor neurons from the late L3 larval stage until the postreproductive period. GFP driven solely by the mec-4 promoter (pmec-4::GFP) was expressed in 5.6 touch receptor neurons on average (n=493) in an otherwise wild-type background (Fig. 1B and Fig. 2B). By contrast, pmec-4::GFP accumulated only in 1.92 touch receptor neurons on average (n=635) in mec-4(u231) mutants (Fig. 2B). We found an increased number of pmec-4::GFP-positive neurons at the young adult stage in homozygous mec-4(u231); bec-1(–) mutants, compared with mec-4(u231) single mutant animals (Fig. 2B). The expression intensity of both this transcriptional fusion pmec-4::GFP reporter and a translational fusion pmec-4::MEC-4::GFP reporter in wild-type animals was comparable with that observed in animals with reduced or eliminated BEC-1 activity (Fig. 2C). This implies that suppression of necrotic death by deregulation of bec-1 was not merely a consequence of a reduction in the quantity of toxic MEC-4. It is also important to note that mec-4::GFP expression was not significantly affected in bec-1(–) single mutant animals (see the legend of Fig. 2), suggesting that BEC-1 is not required for the development of touch receptor neurons.
We also checked the effects of BEC-1 deficiency on the survival of neurons that express toxic DEG-1 or DEG-3 proteins. By counting the number of vacuolated cells at the L1 larval stage, we found that inactivation and reduced activity of BEC-1 markedly suppress neuronal degeneration in both deg-1(u506) and deg-3(u662) mutants (Fig. 2D,E). Vacuolization of dying neurons differs from the formation of typical autophagic vacuoles as the latter are only 1 µm in diameter in C. elegans (Kovács et al., 2004). Similar but less effective reactions were obtained by RNA-interference-mediated depletion of BEC-1 in mutants with toxic ion channel subunit variants (Fig. 2A, data not shown). In summary, our data demonstrate that downregulation of BEC-1 is protective against necrotic cell death.
Mutational inactivation of unc-51/atg1 suppresses ion-channel-dependent neuronal degeneration
We next assessed the function of another C. elegans autophagy gene, unc-51, in neuronal cell loss. unc-51 encodes a serine/threonine kinase similar to the yeast Atg1 protein (Meléndez et al., 2003; Ogura et al., 1994). Two loss-of-function mutations in unc-51, e369 and e1189, result in viable nematodes with uncoordinated movement (Unc). We found that homozygous mec-4(u231);unc-51(–) double mutant animals contain fewer vacuolated and more pmec-4::GFP-positive cells than mec-4(u231) single mutants (Fig. 2A,B). In addition, vacuolated cell corpses were detectable for a slightly longer period in these double mutants than in mec-4(u231) single mutant animals [as corpses were eliminated in 6-8 hours in mec-4(u231) and in 8-10 hours in mec-4(u231);unc-51(e369) background]. Mutations in unc-51 also diminished vacuolization of specific neurons in deg-1(u506) and deg-3(u662) backgrounds (Fig. 2D,E). Thus, we conclude that UNC-51 contributes to neurotoxicity caused by hyperactive MEC-4, DEG-1 or DEG-3.
LGG-1/Atg8 deficiency influences ion-channel-dependent neurotoxicity
The third autophagy gene we tested in relation to neuronal degeneration was lgg-1. LGG-1 is the worm counterpart of the yeast ubiquitin-like protein Atg8, which constitutes a major component of the preautophagosomal complex (Meléndez et al., 2003; Hara et al., 2000). Because LGG-1 has not yet been characterized by mutations, we used RNAi to deplete its function. lgg-1 RNAi treatment completely abolished the expression of a lgg-1::gfp reporter. Our results showed a weaker, but still significant reduction in the number of dying neurons in mec-4, deg-1 and deg-3 gain-of-function mutant animals treated with lgg-1 double-stranded RNA, compared with animals fed with bacteria expressing the empty vector (see statistics for lgg-1 data in the legend of Fig. 2A,B,D,E). This weaker effect of lgg-1 RNAi on cell survival was probably due to the fact that RNAi is often ineffective for genes expressed in mature neurons. In spite of the weaker reaction, the statistically significant difference (for statistics see the legend of Fig. 2) shows that LGG-1 also influences genetically induced necrotic cell death. In summary, inactivation of bec-1, unc-51 and lgg-1 suppresses neuronal degeneration in C. elegans.
BEC-1 and UNC-51 deficiency restores mechanosensitivity in mutants with toxic ion channel variants
The data showing that BEC-1 contributes to ion channel-dependent neuronal degeneration prompted us to investigate whether inactivation of bec-1 rescues the mechanosensation-defective phenotype of degenerin mutants. We performed a behavioral assay in which nematodes moving backwards were touched gently with an eyelash at the tail position and scored the percentage of those capable of moving away from the stimulus. While touch sensitivity of randomly chosen bec-1 mosaic adults was comparable with the wild type, BEC-1 deficiency significantly enhanced the ability of mec-4(u231) and deg-1(u506) mutants to respond to touch stimulus (Fig. 3). This indicates that the surviving touch receptors may transmit some touch function.
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Neuronal expression of the C. elegans autophagy genes
Because inactivation of bec-1, unc-51 and lgg-1 protects neurons from ion channel-dependent toxicity, we assumed that these autophagy genes are expressed in the nervous system throughout development. Indeed, UNC-51 was reported to appear in neurons (Ogura et al., 1997). Using a translational fusion GFP reporter driven by bec-1 or lgg-1 regulatory sequences, we found that both BEC-1::GFP and LGG-1::GFP are expressed in the cell bodies and processes of neurons, including touch receptor neurons, ventral cord neurons and the nerve ring in the head (Fig. 4A-D). A similar pattern was obtained for the TOR (for target of rapamycin) kinase (Fig. 4E) that is predicted to downregulate the autophagic machinery (Long et al., 2002).
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) at the early stages of degenerative processes. GFP::LGG-1 often accumulated in a punctate pattern in neurons showing early signs of vacuolization (Fig. 4F-H). These GFP-positive foci of LGG-1 have been suggested to label preautophagosomal and autophagic structures (Meléndez et al., 2003). Thus, we conclude that autophagy might indeed occur in neurons undergoing necrosis.
Inactivation of unc-51 or bec-1 suppresses neurotoxin-induced cell death
Ion channel-induced neurotoxicity depends on the vacuolar H+-ATPase (Syntichaki et al., 2005). Whether autophagy genes interact with the necrosis process itself or alternatively may affect the activity of ion channels or the vacuolar H+-ATPase needs clarification. To address this issue we examined the role of UNC-51 and BEC-1 in another necrotic cell death paradigm that is independent of toxic channels. In C. elegans, like in mammals, selective degeneration of dopamine neurons can be triggered through exposure to the neurotoxin 6-hydroxidopamine (Nass et al., 2002). To visualize the three pairs of head dopamine neurons (two pairs of CEP neurons and one pair of ADE neurons) and necrotic processes, we examined hermaphrodites carrying a dopamine transporter (DAT) reporter, pdat-1::GFP (Nass et al., 2002). Treatment with 6-OHDA often caused a complete loss of pdat-1::GFP expression in the CEP and ADE neurons in the wild-type background (Fig. 5A,B,E). As previously shown by Nass and colleagues (Nass et al., 2002), the loss of GFP signal is unlikely to be a consequence of the transcriptional repression of the pdat-1::GFP transgene. Disruption of GFP expression in the head dopamine neurons by 6-OHDA was significantly suppressed in animals defective for UNC-51 or BEC-1 (Fig. 5C-E). Treatment with 6-OHDA caused a similar rate of lethality in wild-type and autophagy mutant genetic backgrounds (data not shown). This suggests that deregulation of unc-51 and bec-1 is unlikely to block the uptake of the toxin. Together, our data imply that these autophagy genes contribute to 6-OHDA-induced toxicity of dopamine neurons.
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TOR signaling prevents, starvation promotes neuronal cell loss
Autophagy genes are known to be downregulated by a nutrient-sensing system that involves the TOR-kinase-mediated pathway (Klionsky and Emr, 2000). In C. elegans, TOR controls life span, reproductive growth and metabolism (Long et al., 2002; Vellai et al., 2003; Jia et al., 2004). We depleted TOR by RNAi and monitored whether this treatment affects neurotoxicity. We found a marked increase in the number of vacuolated (dying) touch receptor neurons in mec-4(u231) mutants with reduced CeTOR activity, compared with those treated with the empty vector (Fig. 6). This suggests that TOR signaling affects ion channel-induced neuronal degeneration.
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). These worms were then transferred into favorable conditions and the survival rate of the six touch receptor neurons at the early adult stage was determined. mec-4(u231) mutant animals subjected to starvation contained significantly fewer pmec-4::GFP-positive neurons than those grown under well-fed conditions throughout development (Fig. 6). As a control, neither hyperactivation nor downregulation of the RTK-Ras signaling pathway (Fig. 6), which is also implicated in cellular response to nutritional stress, had a significant impact on neuronal loss. Together, environmental insults such as food limitation can make neurons susceptible to fatal necrotic injury. However, it must be noted that TOR has many downstream effectors, thus it cannot be excluded that its effect on neuronal degeneration is independent of autophagy.
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Discussion |
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Although autophagy genes seem to be involved in necrotic cell death, it remains an open question whether autophagy itself is also implicated. Various pathological states of the nervous system such as Alzheimer's, Parkinson's and Huntington's diseases have been suggested to involve autophagy (Klionsky and Emr, 2000; Cuervo, 2004). Alzheimer's disease is defined by neuritic plaques consisting of extracellularly aggregated 
-amyloid or by intraneuronal fibrillary tangles containing aggregated forms of the microtubule-associated protein tau (Yankner, 1996). In patients with Alzheimer's disease, neurons progressively accumulate lysosomes as they become metabolically compromised. In addition, autophagic vacuoles often contain -amyloid, suggesting that they may be involved in -amyloidogenesis. In Parkinson's disease, degeneration of specific dopaminergic neurons results from the accumulation of filamentous cytoplasmic inclusions termed Lewy bodies that consist of mainly -synuclein. Degradation of normal -synuclein has been shown to occur via chaperone-mediated autophagy (Cuervo et al., 2004). Furthermore, forced expression of -synuclein in neuronal cell lines causes a marked induction of autophagy (Stefanis et al., 2001). Huntington's disease is caused by mutations of CAG trinucleotide repeat expansion, resulting in long polyglutamine tracts in the mutant proteins. Inhibition of the autophagic-lysosomal pathway suspends the clearance of mutant huntingtin proteins (Yamada et al., 2002). Moreover, these proteins are often localized into lysosomes. Consistent with these findings, lysosomal function that is required for autophagy has been shown to be crucial for neuronal degeneration in C. elegans (Artal-Sanz et al., 2006). Together, these data imply that conditions overactivating the autophagic process contribute to neuronal cell loss. However, it has also been demonstrated that loss of autophagy in the central nervous system causes neurodegeneration in mice (Komatsu et al., 2006; Yokoyama et al., 2006). Therefore, both overactivation and deregulation of autophagy seem to induce neuronal degeneration, suggesting that, depending on the actual cellular environment, autophagy may have a dual role in cell survival, acting both as a contributor and a protector to cell demise (Takács-Vellai et al., 2006). On the one hand, the basal activation of autophagy may protect neurons by providing energy for cell functioning during starvation (self-digestion) and facilitating removal of damaged organelles and misfolded proteins. Autophagy is thus essential for cell survival. On the other hand, prolonged or vigorous activation of autophagy may lead to an effective elimination of the affected cell.
So far autophagic vacuoles have not been demonstrated in the neurodegenerative process in C. elegans (Hall et al., 1997). Indeed, a growing number of membrane-mediated functions, such as endocytosis and vesicular trafficking, that are seemingly unrelated to the autophagic process itself appear to involve the product of various autophagy genes (Klionsky and Emr, 2000). It might be possible that autophagy proteins have a role in neuronal degeneration independently of autophagic vacuole formation. The involvement of autophagy in neuronal cell loss and other neuronal functions are important areas for future investigations.
Recently BEC-1 was shown to act both in autophagy and apoptosis (Meléndez et al., 2003; Takács-Vellai et al., 2005). Our study reveals a novel role of autophagy genes in a distinct degenerative process, necrosis. Therefore, the fascinating possibility might emerge that three cell death processes might act in a concerted manner in the cell death machinery that is induced by genetic or environmental factors. Furthermore, identifying autophagy genes as mediators of necrotic-like neuronal cell loss in C. elegans will be essential for understanding how genetic lesions affecting ion channel subunits as well as adverse environmental conditions can lead to human neurodegenerative pathologies (Driscoll and Gerstbrein, 2003; Yuan et al., 2003; Rubinsztein et al., 2005).
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Materials and Methods |
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). Wild-type worms were C. elegans var. Bristol (N2). Other strains were: FR758 swEx520[pbec-1::BEC-1::GFP + rol-6(su1006)], FR853 bec-1(ok691)IV; swEx520[pbec-1::BEC-1::GFP + rol-6(su1006)], FR854 bec-1(ok700)IV; swEx520[pbec-1::BEC-1::GFP + rol-6(su1006)], CB369 unc-51(e369)V, CB1189 unc-51(e1189)V, [pmec-4::MEC-4::GFP + rol-6(su1006)], [pmec-4::gfp + rol-6(su1006)], TU1747 deg-3(u662)V, mec-4(u231)X, CB1611 mec-4(e1611)X, TU1366 deg-1(u506)X, MT4866 let-60(n2021)IV, MT2124 let-60(n1046gf)IV, BY200 [pdat-1::gfp + rol-6(su1006)], Ex[plgg-1::GFP::LGG-1 + rol-6(su1006)], BU070 Ex[plgg-1::LGG-1::GFP + rol-6(su1006)], FR297 swEx226[CeTor::gfp + rol-6(su1006)].
RNA interference and plasmid construction
Specific cDNA fragments were amplified by RT-PCR, and cloned into the vector pPD129.36 (kindly provided by A. Fire, Stanford University, Stanford, CA). Sequences of the forward and reverse primers used were as follows: lgg-1, 5'-CAT GCC ATG GCA TGT GGG CTT ACA AGG AGG AGA AC-3' and 5'-CAT GCC ATG GCA TGT TCC CTT CTT TTC GAC CTC TCC-3'; let-363/CeTor, 5'-CAT GCC ATG GCA TGA ACA ATT GGC AAA TTT CGT G-3' and 5'-CAT GCC ATG GCA TGT GCA CGT AAC GAT GGA GAA C-3'. RNAi experiments were carried out as described (Kamath et al., 2001). To generate a translational fusion BEC-1::GFP reporter, a 9 kb genomic fragment was amplified with the following forward and reverse primers: 5'-GCT ACTCCT GCA GGC ATA GCG CGT AAT TAC TAT TGC GTT CTC G-3' and 5'-CGG GAT CCC GAA TAG GCG ATC TGA GAG CAT CG-3'. The PCR product was digested with SbfI and BamHI, and cloned into pPD95.75. This construct contains 5.6 kb upstream sequences of the ATG translation initiation site and the full-length bec-1 coding region fused with gfp reporter gene. Transgenic worms were generated by injecting 50 µg/ml DNA construct with 30 µg/ml co-transformation marker pRF4 [rol-6(su1006)] into wildtype hermaphrodites. To examine the expression of lgg-1, we also generated a lgg-1::gfp reporter. A 3746 bp-long genomic fragment was amplified by the following forward and reverse primers: 5'-AAC TGC AGA ACC AAT GCA TTG GCC GAG GGA AAA GAC GAA GAG-3' and 5'-TCC CCC GGG GGA CGA CCT CTC CTC CAT ACA CAC-3'. The PCR product was cloned into pPD95.75 (kindly provided by A. Fire) by PstI and SmaI. Germ-line transformation was performed according to standard protocols. This strain, BU070 Ex[plgg-1::LGG-1::GFP], showed an expression profile consistent with that previously reported (Meléndez et al., 2003), using a functional GFP::LGG-1 reporter.
Cell death assays
Nematodes were treated and assayed as described (Syntichaki et al., 2002). Mutations in bec-1 arrest development at different developmental stages (Takács-Vellai et al., 2005). Thus, homozygous mec-4(d);bec-1(–) mutant animals were selected among the progeny of hermaphrodites with mec-4(d/d);bec-1(–/+) genotype. In addition, lethality caused by bec-1 mutant alleles was rescued by transforming a non-integrated transgene, swEx520[pbec-1::BEC-1::GFP + rol-6(su1006)], that carries the wild-type copy of bec-1. The number of vacuolated cells was scored at specific developmental stages in animals of bec-1(–); Ex[bec-1(+)] mosaic genotype. The percentage of intact (pmec-4::GFP-positive) touch receptor neurons at the young adulthood in mec-4(u231) mutants was also determined. Starvation conditions were as follows. Newly hatched mec-4(u231) mutant L1 larvae were deprived of food for 24 hours, and then transferred onto plates containing bacteria where they developed further. GFP-positive cells were scored at young adulthood. Treatment of nematodes with 6-OHDA was performed as described (Nass et al., 2002). Briefly, nematodes were exposed in liquid suspension to 50 mM 6-OHDA in 10 mM ascorbic acid solution for 1 hour, and then transferred to plates lacking the toxin. pdat-1::GFP expression was analyzed the following day at 25°C. The percentage of nematodes with intact head dopamine neurons was determined. Control animals were treated with 10 mM ascorbic acid.
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Acknowledgments |
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