Abstract

Mutations or multiplications in α-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type α-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating α-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of α-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C . elegans (Tg worms) that overexpress human α-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed α-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in α-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7 ) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing α-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. α-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of α-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2 , an AP-2 subunit, caused an accumulation of phosphorylated α-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and α-synuclein-induced neurotoxicity in synucleinopathy.

INTRODUCTION

α-Synuclein is the major component of Lewy bodies (LB) that are pathognomonic intraneuronal inclusions in a subset of neurodegenerative diseases including Parkinson disease (PD) and dementia with Lewy bodies (DLB), collectively referred to as synucleinopathies ( 1 , 2 ). A subset of patients inherit PD or DLB as an autosomal dominant trait, and missense mutations (A53T, A30P and E46K) ( 3–5 ) and multiplications (duplication and triplication) ( 6–8 ) of α-synuclein gene have been identified in these families. It has also been shown that α-synuclein deposited in affected cells of synucleinopathy brains is phosphorylated at residue Ser129 ( 9 , 10 ). In vitro studies suggested that α-synuclein forms filaments similar to those deposited in synucleinopathy brains, and pathogenic mutations of α-synuclein accelerated the fibril formation ( 11–14 ). Collectively, these findings implicate deposition of α-synuclein in the pathogenesis of synucleinopathies.

α-Synuclein is an abundant neuronal protein that is normally localized to the presynaptic termini ( 15 ), and a portion of α-synuclein has been found to be loosely associated with synaptic vesicles ( 16–18 ). Although the normal function of α-synuclein remains elusive, studies in α-synuclein knockout mice suggest that α-synuclein may play a role in the regulation of neurotransmitter release ( 19 ) and recycling and/or maintenance of synaptic vesicles ( 20 ). α-Synuclein has also been identified as a negative regulator of phospholipase D2 (PLD2), which is essential to membrane vesicle formation ( 21 ). However, it is not clear whether these putative functions of α-synuclein, especially those related to the regulation of synaptic vesicles, are involved in neuronal dysfunction and degeneration in synucleinopathy brains.

To elucidate the pathogenic mechanism of α-synuclein in neurodegeneration, a variety of transgenic (Tg) model animals that overexpress α-synuclein using heterologous organisms (i.e. mouse ( 22–26 ), Drosophila ( 27 , 28 ), C . elegans ( 29–32 ) and yeast ( 33 )) have been developed, and abnormal behavioral or pathological phenotypes have been documented in a subset of these models. We previously reported that Tg C . elegans that overexpress human α-synuclein in dopamine neurons exhibit a partial defect in food sensing, a function inherent in C . elegans dopamine neurons, in a manner dependent on pathogenic mutations ( 32 ). Together with other reports on aberrant phenotypes in α-synuclein Tg worms ( 29–31 ), these findings support the view that the neurotoxic property of α-synuclein can be recapitulated in invertebrate neurons. C . elegans offers a number of unique advantages for modeling neurodegeneration: (i) genetic manipulations are easily achieved and a number of mutants are already available, (ii) the anatomy and connection of all neurons (302 in total) have been thoroughly mapped and (iii) a clear link has been established between behaviors and functions of a subset of neurons. For these reasons, C . elegans has recently been adopted as models of a variety of human neurodegenerative disorders, e.g. polyglutamine disease or tauopathy ( 34–38 ). Another advantage of C . elegans is the wide applicability of RNA interference (RNAi), by feeding worms with bacteria expressing double-stranded RNAs (feeding RNAi). In fact, preliminary studies on a systematic RNAi screen of modifier genes have been reported in C . elegans models overexpressing tau in neurons ( 39 ) or fluorescent protein-tagged α-synuclein in body wall muscles ( 40 , 41 ).

Here we report a genetic screen for modifier genes of α-synuclein neurotoxicity by a systematic RNAi using Tg C . elegans overexpressing human α-synuclein in a pan-neuronal fashion. To overcome the major obstacle that C . elegans neurons display high resistance to RNAi, we crossed the Tg worms with eri-1(mg366) the latter being isolated as a neuronal RNAi-sensitive mutant and recently applied in RNAi screen for modifier genes of synaptic function ( 42 ). By this approach, we identified 10 genes that resulted in behavioral and/or growth disabilities selectively in α-synuclein Tg worms upon RNAi treatment. Interestingly, four among the 10 genes whose knockdown-enhanced α-synuclein-induced dysfunction were related to endocytosis, suggesting an involvement of endocytic pathway in α-synuclein neurotoxicity.

RESULTS

Generation and characterization of transgenic C. elegans overexpressing human α-synuclein in a pan-neuronal manner

To establish a C . elegans model of synucleinopathy, we generated transgenic (Tg) C . elegans that overexpress human α-synuclein in the entire nervous system under the control of an unc-51 promoter. DNA constructs encoding wild-type (synWT), or A53T or A30P pathogenic mutant (synA53T and synA30P) α-synuclein fused to unc-51 promoter sequence (P unc-51 ::α-synuclein, Fig.  1 A) were microinjected, together with P unc-51 ::EGFP as an expression marker, to generate extrachromosome strains. The transgenes were then integrated into chromosomes by UV irradiation. We also generated a Tg line expressing EGFP (EGFP-Tg) as a control strain. Expression of α-synuclein protein was detected by the western-blot analysis (Fig.  1 B), and that of EGFP was confirmed by fluorescence microscopy (Fig.  1 C). The level of α-synuclein protein expression was comparable in synWT and synA53T worms, and was slightly lower in synA30P worms, whereas no expression of α-synuclein was detected in N2 or EGFP-Tg worms. Immunohistochemical analyses showed that the expression of α-synuclein was abundant throughout the nervous system, which was particularly accentuated in the nerve ring, a cluster of neurites and synapses located rostral to the pharynx (Fig.  1 D). The latter finding suggested that a majority of α-synuclein expressed in C . elegans were transported into axons and synapses as in mammals, despite the lack of endogenous α-synuclein. We then assessed the phenotypic abnormalities caused by pan-neuronal overexpression of α-synuclein in the Tg lines. Neither motor deterioration, as determined by the thrashing rate in the liquid, nor retardation in growth (both in 3-day-old young-adult worms) was apparent, suggesting that the α-synuclein toxicity was at a subthreshold level, or C . elegans neurons were not sensitive enough to α-synuclein neurotoxicity.

Figure 1.

Characterization of Tg worms pan-neuronally overexpressing human α-synuclein. ( A ) Schematic representation of the transgene. cDNAs encoding wild-type (synWT) or FPD-mutant (synA53T, synA30P) human α-synuclein or EGFP were inserted downstream of the unc-51 promoter, which drives pan-neuronal expression. ( B ) Immunoblot analysis of the whole-lysates of Tg worms with anti-α-synuclein (LB509) or anti-α-tubulin (DM1A) antibodies. α-tubulin is a loading control. Expression levels of α-synuclein were comparable between synWT and synA53T worms, lower in synA30P worms, and not detected in N2 or EGFP worms. ( C ) EGFP fluorescence detected in the C . elegans nervous system. ( D ) Immunohistochemical analysis of α-synuclein Tg worms. Formalin-fixed, paraffin-embedded section was immunostained with anti-α-synuclein antibody LB509. α-Synuclein was abundantly expressed in the whole nervous system, particularly in the ‘nerve ring’ (arrows), which consists of bundle of neurites with accumulated synapses.

Figure 1.

Characterization of Tg worms pan-neuronally overexpressing human α-synuclein. ( A ) Schematic representation of the transgene. cDNAs encoding wild-type (synWT) or FPD-mutant (synA53T, synA30P) human α-synuclein or EGFP were inserted downstream of the unc-51 promoter, which drives pan-neuronal expression. ( B ) Immunoblot analysis of the whole-lysates of Tg worms with anti-α-synuclein (LB509) or anti-α-tubulin (DM1A) antibodies. α-tubulin is a loading control. Expression levels of α-synuclein were comparable between synWT and synA53T worms, lower in synA30P worms, and not detected in N2 or EGFP worms. ( C ) EGFP fluorescence detected in the C . elegans nervous system. ( D ) Immunohistochemical analysis of α-synuclein Tg worms. Formalin-fixed, paraffin-embedded section was immunostained with anti-α-synuclein antibody LB509. α-Synuclein was abundantly expressed in the whole nervous system, particularly in the ‘nerve ring’ (arrows), which consists of bundle of neurites with accumulated synapses.

A systematic RNAi screen for suppressors/enhancers of α-synuclein-induced neurotoxicity in transgenic C. elegans

We then searched for genes that modify the manifestations of α-synuclein-induced neurotoxicity by a systematic RNAi screen. To maximize the RNAi effects, we crossed the Tg worms with eri-1(mg366) , the latter being reported as a mutant sensitized to RNAi in neurons ( 42 ). First, we examined the effect of eri-1 on the RNAi efficacy of a subset of genes that are known to be expressed in neurons and essential to locomotion (Table  1 ). Worms crossed with eri-1(mg366) frequently showed an Unc phenotype (uncoordinated movement) when these genes were knocked down, whereas worms of wild-type background showed little phenotypic changes, suggesting that the mutation in eri-1 gene enhanced RNAi. The RNAi efficacy in eri-1;lin-15B double mutant, which has also been used in a neuronal RNAi screen ( 43 ), was comparable to that in eri-1 single mutant, while eri-1;lin-15B double mutant frequently showed sterility (data not shown). Therefore, we decided to use eri-1 mutant for the RNAi screen. It is noteworthy that the expression levels of α-synuclein were decreased by ∼50% upon crossing with eri-1(mg366) , presumably due to transgene silencing, an effect that has been reported in a subset of RNAi-hypersensitive mutants ( 44 , and Supplementary Material, Fig. S1A and B ). However, all Tg lines crossed with eri-1 mutant exhibited detectable levels of α-synuclein expression by western blotting ( Supplementary Material, Fig. S1C ).

Table 1.

RNAi effects upon eri-1 mutant worms

  % worms with Unc phenotype
 
RNAi gene synA53T  synA53T; eri-1 
egl-30 20 
unc-13 20 
unc-104 10 40 
rbf-1 40 
egl-8 30 
snb-1 15 50 
unc-22a 90 95 
  % worms with Unc phenotype
 
RNAi gene synA53T  synA53T; eri-1 
egl-30 20 
unc-13 20 
unc-104 10 40 
rbf-1 40 
egl-8 30 
snb-1 15 50 
unc-22a 90 95 

a Non-neuronal gene used as a positive control.

We carried out a systematic feeding RNAi screen on 1673 genes using α-synuclein; eri-1 lines. A large proportion of the 1673 genes (listed in Supplementary Material, Table S1 and functional classification in Fig.  2 A) were selected from 2072 genes that had been tested in a previous report, in which genes involved in synaptic function of C . elegans were screened ( 43 ). Upon initial screen, we used the following three strains: synWT; eri-1 , synA53T; eri-1 and eri-1 (non-Tg), and RNAi clones that led to different phenotypes, e.g. uncoordinated movement (Unc) or growth retardation (Gro), between the α-synuclein-Tg lines (synWT-Tg and synA53T-Tg) and eri-1 , were selected. Various abnormal phenotypes, e.g. Unc, Gro, Lva (larval arrest) or Emb (embryonic lethal), were observed by RNAi knockdown of 278 genes (at least in one of the lines), which accounted for ∼17% of the total genes tested. This result supports the validity of the RNAi screen method, since the percentage of the genes exhibiting RNAi phenotypes was ∼10% in previous reports on feeding RNAi screen in C . elegans ( 45 , 46 ). Among the 278 genes, we isolated 66 genes that caused different phenotypes between α-synuclein Tg lines and eri-1 as positive clones in the first screening (listed in Supplementary Material, Table S2 ). We subsequently performed a second screen, in which the following five strains were examined: synWT; eri-1 , synA53T; eri-1 , eri-1 , synA30P; eri-1 and EGFP; eri-1 , and the RNAi clones that showed phenotypic changes between α-synuclein-Tg worms (synWT-Tg, synA53T-Tg and synA30P-Tg) and controls ( eri-1 and EGFP-Tg) were selected. The same experiment was repeated three times in order to test the reproducibility of the results (Fig.  2 B).

Figure 2.

Overview of the RNAi screen. ( A ) Classification of 1673 genes selected in this screen. ( B ) Flowchart of the first and second screens.

Figure 2.

Overview of the RNAi screen. ( A ) Classification of 1673 genes selected in this screen. ( B ) Flowchart of the first and second screens.

As a result, we identified 10 genes that, upon knockdown, gave rise to abnormal phenotypes (e.g. Unc and Gro) selectively in α-synuclein Tg worms (Table  2 ). In addition, we isolated cst-1 , whose knockdown caused abnormal phenotypes selectively in control lines ( eri-1 and EGFP-Tg). These results suggested genetic interactions between the respective 11 genes and α-synuclein overexpressed in neurons. All genes have orthologs in human genome, and at least five genes ( apa-2, aps-2, eps-8, cdk-4 and cst-1 ) have been documented to be expressed in neurons of C . elegans ( 43 , 47–49 , and WormBase. http://www.wormbase.org/ ). Interestingly, among the 10 genes that enhanced α-synuclein toxicity, four genes ( apa-2, aps-2, eps-8 and rab-7 ) were known to be involved in the endocytic machinery. The result that defects in endocytic machinery exacerbated neurotoxicity of α-synuclein supports the notion that overexpressed α-synuclein may potentially inhibit the endocytic pathway, and that α-synuclein toxicity is manifested when endocytosis is partially affected.

Table 2.

List of RNAi genes that caused different phenotypes between α-synuclein-Tg worms and control (non-Tg and EGFP-Tg) worms

Gene Annotation RNAi phenotype, strength Human ortholog? Express in neurons? 
apa-2a AP-2 α complex synTg→Unc, ++ Yes Yes 
aps-2a AP-2 σ2 complex synTg→Unc, + Yes Yes 
eps-8a EGF receptor kinase substrate synTg→Gro, ++ Yes Yes 
cdk-4 Cyclin dependent kinase synTg→Unc, ++ Yes Yes 
ubc-9 Ubiquitin conjugating enzyme (E2) synTg→Gro, Unc, ++ Yes – 
cdk-9 Cyclin dependent kinase synTg→Gro, ++ Yes – 
csnk-1 Casein kinase synTg→Gro, Unc, ++ Yes – 
rab-7a GTPase synTg→Unc, + Yes – 
lit-1 Nemo-like MAPK-related kinase synTg→Gro, ++ Yes – 
pat-10 Troponin C synTg→Gro, ++ Yes – 
cst-1 Ste20-like kinase MST controls→Gro,Unc,++ Yes Yes 
Gene Annotation RNAi phenotype, strength Human ortholog? Express in neurons? 
apa-2a AP-2 α complex synTg→Unc, ++ Yes Yes 
aps-2a AP-2 σ2 complex synTg→Unc, + Yes Yes 
eps-8a EGF receptor kinase substrate synTg→Gro, ++ Yes Yes 
cdk-4 Cyclin dependent kinase synTg→Unc, ++ Yes Yes 
ubc-9 Ubiquitin conjugating enzyme (E2) synTg→Gro, Unc, ++ Yes – 
cdk-9 Cyclin dependent kinase synTg→Gro, ++ Yes – 
csnk-1 Casein kinase synTg→Gro, Unc, ++ Yes – 
rab-7a GTPase synTg→Unc, + Yes – 
lit-1 Nemo-like MAPK-related kinase synTg→Gro, ++ Yes – 
pat-10 Troponin C synTg→Gro, ++ Yes – 
cst-1 Ste20-like kinase MST controls→Gro,Unc,++ Yes Yes 

Unc, uncoordinated movement; Gro, slow growth.

a Endocytosis-related gene.

RNAi knockdown or genetic deletion of AP-2 complex causes motor/developmental disabilities selectively in α-synuclein Tg worms

Among the four endocytosis-related genes identified in this screen, gene products of apa-2 and aps-2 represent subunits of the adaptor protein complex AP-2, an important component involved in endocytosis. AP-2 recruits clathrin and cargo receptors to the endocytic pits, which in turn are progressively invaginated and internalized into the cytosol by forming vesicles. AP-2 is a heterotetramer composed of two large subunits, α and β2, and two small subunits, μ2 and σ2 ( 50 ), of which α- and σ2-subunits are encoded by apa-2 and aps-2 , respectively. RNAi knockdown of either apa-2 or aps-2 caused growth retardation (Gro) and uncoordinated movement (Unc) exclusively in α-synuclein Tg worms (Fig.  3 A and Supplementary Material, Movies ). The immunoblot analysis validated marked reduction of apa-2 expression by apa-2 RNAi, while the α-synuclein level was not changed (Fig.  3 B). To quantitatively examine the effects of RNAi of these two genes on different α-synuclein Tg lines (i.e. synWT-Tg, synA53T-Tg and synA30P-Tg) and control lines ( eri-1 and EGFP-Tg), we counted the number of worms that display Unc phenotype upon knockdown of apa-2 or aps-2 (Fig.  3 C and D). All α-synuclein Tg lines showed significantly higher percentage of Unc compared to non-Tg or EGFP-Tg lines upon knockdown of apa-2 (Fig.  3 C). RNAi of aps-2 also resulted in higher Unc ratio selectively in α-synuclein Tg lines (Fig.  3 D). Notably, synA53T-Tg was most severely affected by RNAi of either of the genes, and toxicity of synA30P may also be higher than that of synWT, considering the relatively lower expression level of α-synuclein in synA30P-Tg worm (Fig.  1 B and Supplementary Material, Fig. S1 C). Thus, the pathogenic mutations of α-synuclein gene possibly aggravated the phenotypes of α-synuclein Tg worms manifested by the inhibition of AP-2/endocytic machinery.

Figure 3.

The phenotypes induced by RNAi of the subunits of AP-2 complex. ( A ) The appearances of the worms treated with apa-2 RNAi. While most of eri-1 (non-Tg) worms grew up and moved normally (left), synA53T; eri-1 worms presented severe growth retardation and uncoordinated movement (right). Scale bar = 500 µm. ( B ) Immunoblot analysis for the validation of the RNAi knockdown of apa-2 level. eri-1 or synA53T; eri-1 were fed with bacteria expressing mock or apa-2 dsRNA. Total lysates of these worms were separated by SDS–PAGE and detected with anti-APA-2, anti-α-synuclein and anti-α-tubulin antibodies. ( C and D ) Percentage of the worms presenting uncoordinated movement (Unc) upon knockdown of apa-2 (AP-2 α-subunit) (C) and aps-2 (AP-2 σ2-subunit) (D). A total of 100–200 worms per each line were examined. Mean ± SE, n = 3, * P < 0.01 compared to both eri-1 and EGFP; eri-1 .

Figure 3.

The phenotypes induced by RNAi of the subunits of AP-2 complex. ( A ) The appearances of the worms treated with apa-2 RNAi. While most of eri-1 (non-Tg) worms grew up and moved normally (left), synA53T; eri-1 worms presented severe growth retardation and uncoordinated movement (right). Scale bar = 500 µm. ( B ) Immunoblot analysis for the validation of the RNAi knockdown of apa-2 level. eri-1 or synA53T; eri-1 were fed with bacteria expressing mock or apa-2 dsRNA. Total lysates of these worms were separated by SDS–PAGE and detected with anti-APA-2, anti-α-synuclein and anti-α-tubulin antibodies. ( C and D ) Percentage of the worms presenting uncoordinated movement (Unc) upon knockdown of apa-2 (AP-2 α-subunit) (C) and aps-2 (AP-2 σ2-subunit) (D). A total of 100–200 worms per each line were examined. Mean ± SE, n = 3, * P < 0.01 compared to both eri-1 and EGFP; eri-1 .

To confirm the results of RNAi experiments, we then conducted cross experiments using aps-2(tm2912) , in which the σ2 subunit of AP-2 is mutated: aps-2(tm2912) harbors a deletion of 194 bp in the coding region of aps-2 gene and displays Gro/Unc phenotypes in a temperature-dependent manner, almost normally growing at 15°C but partially arresting at 20°C. We crossed the α-synuclein Tg worms with aps-2(tm2912) and examined the changes in the degrees of growth arrest at 20°C. The growths of aps-2 crossed with synWT, synA53T or synA30P were all severely arrested, whereas aps-2 crossed with EGFP-Tg or non-crossed aps-2 showed much milder manifestations of arrest (Table  3 ). In addition, motor activities of aps-2 ; α-synuclein-Tg worms also were severely impaired, while those of aps-2 ; EGFP-Tg or aps-2 (non-crossed) were almost normal ( Supplementary Material, Fig. S2 ). These results further supported the notion that neurotoxicity of α-synuclein is manifested when AP-2 function is potentially inhibited.

Table 3.

The number of the progeny a at every growth stage of each Tg line before/after crossing with aps-2(tm2912)

 Adult L3–L4 arrest L1–L2 arrest Embryonic arrest Total Adult/total (%) 
EGFP 981 981 100 
synWT 1134 1134 100 
synA53T 116 116 100 
synA30P 835 835 100 
aps-2 342 19 103 19 483 70.8 
EGFP; aps-2 190 38 28 11 267 71.2 
synWT; aps-2 38 23 131 17 209 18.2 
synA53T; aps-2 10 24 99 137 7.3 
synA30P; aps-2 15 115 59 194 7.7 
 Adult L3–L4 arrest L1–L2 arrest Embryonic arrest Total Adult/total (%) 
EGFP 981 981 100 
synWT 1134 1134 100 
synA53T 116 116 100 
synA30P 835 835 100 
aps-2 342 19 103 19 483 70.8 
EGFP; aps-2 190 38 28 11 267 71.2 
synWT; aps-2 38 23 131 17 209 18.2 
synA53T; aps-2 10 24 99 137 7.3 
synA30P; aps-2 15 115 59 194 7.7 

a Ten individual L4 stage worms grown at a permissive temperature of 15°C were transferred to the new 35 mm plate, and cultured at 20°C for 8–10 days. The parent worms were transferred every other day to new fresh plates. The numbers of the progeny at every growth stage were counted under visual inspection.

RNAi knockdown of AP-2 ( apa-2 ) or AP180 ( unc-11 ) exacerbates impairment in touch sensitivity caused by overexpression of α-synuclein

To further examine the involvement of endocytosis in α-synuclein-induced neuronal toxicity, we tested the RNAi effects of a set of endocytosis-related genes other than AP-2. Because endocytosis of synaptic vesicles is considered to be particularly important in neurons, we selected a subset of genes that are involved in synaptic vesicle endocytosis, and examined their RNAi effects in Tg lines Is(P mec-7 ::synWT) and Is(P mec-7 ::synA53T) that overexpress WT or A53T mutant α-synuclein specifically in six touch-receptor neurons under the control of mec-7 promoter (Fig.  4 A). Expression of α-synuclein was confirmed by immunohistochemistry (Fig.  4 B). These α-synuclein Tg lines showed moderate impairments in touch response, a function of touch-receptor neurons to reverse the direction of movement against gentle touch stimulus. Is(P mec-7 ::synWT) and Is(P mec-7 ::synA53T) were initially crossed with eri-1(mg366) to sensitize RNAi, and then young-adult worms of these lines, together with eri-1(mg366) , were treated by RNAi of endocytosis-related genes for 3 days. The touch sensitivities in young adults (3-day-old) of the next generation were then examined. RNAi knockdown of apa-2 caused additional decremental effects on touch response in both synWT-Tg and synA53T-Tg lines (Fig.  4 C). Furthermore, knockdown of unc-11 , an ortholog of mammalian AP180 involved in synaptic vesicle endocytosis, aggravated touch response in synA53T-Tg line (Fig.  4 C). In contrast, eri-1 mutant showed normal touch response by RNAi of apa-2 or unc-11 . These results lent further support to the view that impaired endocytosis exacerbates α-synuclein-induced neurotoxicity.

Figure 4.

Touch sensitivities of Tg worms pretreated with RNAi for synaptic vesicle endocytosis-related genes. ( A ) Schematic representation of the transgene. ( B ) Tg worm overexpressing synA53T in touch neurons (Is(P mec-7 ::synA53T)) was immunostained with anti-α-synuclein antibody LB509. Arrowheads indicate the cell bodies of PLM touch neurons localized near the tail. Scale bar = 10 µm. ( C ) Is(P mec-7 ::synWT); eri-1 , Is(P mec-7 ::synA53T); eri-1 and non-Tg ( eri-1 ) pretreated with RNAi of indicated genes were subjected to the touch assay in a blind manner. Data represent mean ± SE n = 8, * P < 0.05 compared to mock RNAi of the same line.

Figure 4.

Touch sensitivities of Tg worms pretreated with RNAi for synaptic vesicle endocytosis-related genes. ( A ) Schematic representation of the transgene. ( B ) Tg worm overexpressing synA53T in touch neurons (Is(P mec-7 ::synA53T)) was immunostained with anti-α-synuclein antibody LB509. Arrowheads indicate the cell bodies of PLM touch neurons localized near the tail. Scale bar = 10 µm. ( C ) Is(P mec-7 ::synWT); eri-1 , Is(P mec-7 ::synA53T); eri-1 and non-Tg ( eri-1 ) pretreated with RNAi of indicated genes were subjected to the touch assay in a blind manner. Data represent mean ± SE n = 8, * P < 0.05 compared to mock RNAi of the same line.

α-synuclein Tg worms display decreased neurotransmitter release similarly to endocytosis-defective mutants

It has been shown that the release of neurotransmitters at presynaptic termini is strongly inhibited in synaptic vesicle endocytosis-defective mutants of C . elegans ( 51 , 52 ). This prompted us to examine the presynaptic neurotransmitter release in α-synuclein Tg worms. The release of acetylcholine (Ach) at the neuromuscular junction can be assessed using aldicarb, an Ach esterase (AchE) inhibitor, as well as levamisole, an agonist of Ach receptors. Both drugs render normal cholinergic synapses in a hyperactive state, leading to muscle hypercontraction and paralysis ( 51 ). Therefore, worms defective in Ach release are resistant to aldicarb but still sensitive to levamisole, while worms defective in Ach receptors or muscle function are resistant to both aldicarb and levamisole. A set of Tg lines (synWT, synA53T, synA30P and EGFP), non-Tg (N2 strain) as well as an endocytosis-defective mutant unc-11 (as a positive control) were tested for sensitivities to aldicarb and levamisole. As a result, all α-synuclein Tg lines (synWT, synA53T and synA30P) were significantly more resistant to aldicarb, synA53T-Tg being the most resistant, compared to the sensitivity of N2 or EGFP-Tg line (Fig.  5 A). In contrast, sensitivities to levamisole were not significantly different between α-synuclein Tg lines and the control N2 or EGFP-Tg line (Fig.  5 B), indicating an intact postsynaptic function. Taken together with the results of RNAi experiments, it was strongly suggested that overexpression of α-synuclein caused a presynaptic defect in Ach neurotransmission through defects in synaptic vesicle endocytosis or recycling.

Figure 5.

α-Synuclein Tg worms showed impaired neurotransmission. ( A ) Worms were incubated with an acetylcholine esterase inhibitor aldicarb, and the percentages of the paralyzed worms were assessed over time. Mean ± SE for three trials, *: P < 0.05 compared to N2. ( B ) Worms were incubated with an acetylcholine receptor agonist levamisole, and the paralyzed worms were assessed. Mean ± SE for three trials.

Figure 5.

α-Synuclein Tg worms showed impaired neurotransmission. ( A ) Worms were incubated with an acetylcholine esterase inhibitor aldicarb, and the percentages of the paralyzed worms were assessed over time. Mean ± SE for three trials, *: P < 0.05 compared to N2. ( B ) Worms were incubated with an acetylcholine receptor agonist levamisole, and the paralyzed worms were assessed. Mean ± SE for three trials.

Accumulation of Ser129-phosphorylated α-synuclein in neuronal cell bodies by apa-2 knockdown

We finally examined the immunohistochemical changes in α-synuclein Tg worms treated by apa-2 RNAi. Neither neuronal death revealed by TUNEL staining nor abnormality in nerve processes visualized by EGFP coexpression was detected (data not shown). However, immunohistochemical analyses revealed an alteration in the subcellular localization of α-synuclein phosphorylated at serine 129, whose accumulation is characteristic of synucleinopathy lesions ( 9 , 10 ), upon knockdown of apa-2 . In Tg lines overexpressing α-synuclein and treated by mock RNAi, a fraction of α-synuclein was phosphorylated and detected at the nerve ring comprised by axons and synapses (Fig.  6 A). In contrast, RNAi knockdown of apa-2 elicited a relocation of phosphorylated α-synuclein, leading to its accumulation in the cell bodies (Fig.  6 B). In addition, neuronal cell bodies located in the tail region also became immunoreactive to phosphorylated α-synuclein upon apa-2 knockdown (Fig.  6 C and D). Compared to the altered immunoreactivity for phosphorylated α-synuclein, the staining pattern of total α-synuclein, as detected by anti-α-synuclein antibody LB509, was apparently unchanged (Fig.  6 E and F). The ratio of neuronal cell bodies immunopositive for phosphorylated α-synuclein at the head (Fig.  6 G) and tail (Fig.  6 H) regions were higher upon apa-2 RNAi compared to those upon mock RNAi. Thus, suppression of AP-2 recapitulates abnormal accumulation of phosphorylated α-synuclein species in neuronal cell bodies in a manner reminiscent of synucleinopathy lesions.

Figure 6.

Immunohistochemical analyses of α-synuclein Tg worms treated with apa-2 RNAi. ( A and B ) Head region of α-synuclein (A53T) Tg worms treated with RNAi of mock (A) or apa-2 (B) immunostained with an antibody that specifically detect α-synuclein phosphorylated at Ser129. Arrowheads indicate the localization of phosphorylated α-synuclein at the nerve ring, and arrows show the accumulation of phosphorylated α-synuclein in neuronal cell bodies, which are detected upon treatment with apa-2 RNAi (B). ( C and D ) Tail region of α-synuclein (A53T) Tg worms treated with RNAi of mock (C) or apa-2 (D) immunostained with anti-phospho-α-syn antibody. Immunopositive neuronal cell bodies (arrows) were observed upon apa-2 RNAi. ( E and F ) α-Synuclein (A53T) Tg worms treated with RNAi for an empty vector (mock) (E) or apa-2 (F) immunostained with an anti-α-synuclein antibody LB509. Arrowheads indicate the nerve ring positively stained for α-synuclein. Scale bars in (A)–(F) is 10 µm. ( G and H ) Percentage of the worms in which phosphorylated α-synuclein (p-Syn) were detected in the neuronal cell bodies of the head (G) and tail (H) regions upon RNAi of mock or apa-2 . Thirty worms per each line were examined.

Figure 6.

Immunohistochemical analyses of α-synuclein Tg worms treated with apa-2 RNAi. ( A and B ) Head region of α-synuclein (A53T) Tg worms treated with RNAi of mock (A) or apa-2 (B) immunostained with an antibody that specifically detect α-synuclein phosphorylated at Ser129. Arrowheads indicate the localization of phosphorylated α-synuclein at the nerve ring, and arrows show the accumulation of phosphorylated α-synuclein in neuronal cell bodies, which are detected upon treatment with apa-2 RNAi (B). ( C and D ) Tail region of α-synuclein (A53T) Tg worms treated with RNAi of mock (C) or apa-2 (D) immunostained with anti-phospho-α-syn antibody. Immunopositive neuronal cell bodies (arrows) were observed upon apa-2 RNAi. ( E and F ) α-Synuclein (A53T) Tg worms treated with RNAi for an empty vector (mock) (E) or apa-2 (F) immunostained with an anti-α-synuclein antibody LB509. Arrowheads indicate the nerve ring positively stained for α-synuclein. Scale bars in (A)–(F) is 10 µm. ( G and H ) Percentage of the worms in which phosphorylated α-synuclein (p-Syn) were detected in the neuronal cell bodies of the head (G) and tail (H) regions upon RNAi of mock or apa-2 . Thirty worms per each line were examined.

DISCUSSION

In this study, we carried out an RNAi screen using lines of Tg C . elegans that pan-neuronally overexpress human WT or pathogenic mutant α-synuclein, and identified 11 genes that exert modifying effects on the phenotypes of α-synuclein Tg worms. These genes may be more closely related to the α-synuclein-induced neurotoxicity compared to those reported in the previous C . elegans -based RNAi screens ( 40 , 41 ), because the latter studies were designed to search for modulators of formation of α-synuclein inclusions. Another methodological advantage of the present RNAi study is the adoption of eri-1 mutant to enhance RNAi efficacy in C . elegans neurons. Although eri-1 mutation induces transgene silencing to some extent, the protein level of α-synuclein was high enough to elicit toxic effects, and the RNAi efficacy was maintained after cross with eri-1 .

We conducted an RNAi screen on 1673 genes related to nervous system or synaptic functions and identified eleven genes that specifically modified the phenotypes of α-synuclein Tg but not of wild-type worms. Among these were four genes involved in endocytosis, which included two subunits of the AP-2 complex. Furthermore, we confirmed that genetic deletion of an AP-2 subunit, aps-2 , dramatically aggravates the phenotypes of α-synuclein Tg worms as observed in RNAi experiments, supporting the genetic link between endocytosis/AP-2 function and α-synuclein neurotoxicity. These findings raise the possibility that accumulation of α-synuclein in neurons perturbs the endocytic pathway (Fig.  7 ). We further investigated the relationship between α-synuclein and endocytosis, and found that knockdown of AP-2 or AP180 ( unc-11 ), both being important for the synaptic vesicle endocytosis, exacerbated the touch response in Tg worms overexpressing α-synuclein in touch neurons (Fig.  4 ), and that acetylcholine release in α-synuclein Tg worms was impaired as in the synaptic vesicle endocytosis-defective mutant (Fig.  5 ). These findings collectively support the view that defective endocytosis of synaptic vesicles is involved in the toxicity of neuronally expressed α-synuclein. Recently, a similar observation that defects in synaptic vesicle endocytosis enhanced the neuronal dysfunction was reported in a C . elegans model of polyglutamine disease ( 53 ).

Figure 7.

Hypothetical model for the toxic effect of overexpressed α-synuclein in neurons. α-Synuclein may inhibit endocytosis at the presynaptic terminals or cell bodies, and the dysfunction becomes apparent when endocytic machineries are defective.

Figure 7.

Hypothetical model for the toxic effect of overexpressed α-synuclein in neurons. α-Synuclein may inhibit endocytosis at the presynaptic terminals or cell bodies, and the dysfunction becomes apparent when endocytic machineries are defective.

Genetic interactions between α-synuclein and the membrane trafficking pathway have been reported in previous studies using model organisms, e.g. yeast or C . elegans , in which overexpression of genes involved in ER-Golgi trafficking ( 54 ) or related vesicular trafficking ( 40 ) rescued α-synuclein toxicity. In addition, a genetic study using mice has demonstrated that α-synuclein ameliorated inhibition of SNARE complex assembly, the latter being important in exocytosis ( 55 ). These results are not apparently consistent with our present data suggesting that endocytosis is involved in α-synuclein toxicity. However, it is conceivable that these cellular trafficking pathways are closely linked to each other under related mechanisms. In fact, the endocytic mutants of C . elegans demonstrated the altered localization of synaptobrevin, a molecule involved in exocytosis and neurotransmission, and showed resistance to aldicarb ( 52 , 56 , 57 ), suggesting interactions between exocytosis and endocytosis. Nonetheless, the fact that all the positively selected genes from our RNAi screen of 1673 genes, that originally included multiple genes involved in exocytosis (e.g. unc-18, rab-3, snt-1 and aex-3 ) (Fig.  2 A), were endocytic genes underscores the specific genetic interaction between α-synuclein and endocytosis.

In fact, previous studies have implicated α-synuclein in the endocytic pathway. Analyses of α-synuclein knockout mice suggested that α-synuclein is required for the formation and/or maintenance of a reserve pool of presynaptic vesicles ( 20 ). In addition, treatment of primary neurons with α-synuclein antisense oligonucleotides led to a reduction in the distal pool of synaptic vesicles ( 58 ). This regulation of synaptic vesicles is possibly mediated by direct binding of α-synuclein to the vesicles at nerve termini, since in vitro studies have shown an interaction of N-terminal repeat domain of α-synuclein to membrane lipids ( 16–18 ). It has also been shown that α-synuclein inhibits activity of PLD2 in vitro ( 21 ), and that α-synuclein binds to PLD isozymes and inhibits its activation in cultured cells ( 59 ). PLD2 is a membrane-bound enzyme located in plasma and endosomal membranes, and hydrolyzes phosphatidylcholine into lysophosphatidylcholine and phosphatidic acid in response to external stimuli. PLD2-derived phosphatidic acid then recruits AP-2 and triggers the budding of vesicles from donor membranes ( 60 ). Thus, it is also possible that α-synuclein inhibits endocytosis by negatively regulating PLD2 activity. The finding that A53T α-synuclein inhibited PLD2 more potently than WT α-synuclein ( 61 ) agrees with our observation that synA53T-Tg worms presented the most severe phenotype upon knockdown of components of the AP-2 complex. Thus, there remains the possibility that the defects in synaptic vesicle endocytosis caused by overexpression of α-synuclein are caused by upregulation of the normal function of α-synuclein, although it is also conceivable that these phenotypes were caused by abnormal toxic functions of accumulated α-synuclein conferred by altered conformation.

We have also shown that α-synuclein, especially its phosphorylated form, was accumulated in neuronal cell bodies upon knockdown of apa-2 (Fig.  6 ). This is interesting because accumulation and aggregation of phosphorylated α-synuclein in neuronal somata is one of the hallmark lesions of human synucleinopathies that we intended to model. Although the precise mechanism that led to this altered subcellular localization of α-synuclein is unclear, previous reports have suggested that α-synuclein is transported to the presynaptic region via binding to the membrane vesicles ( 17 ), and that phosphorylation of α-synuclein disrupts its binding to membranes ( 62 ). Moreover, a subset of endocytosis-defective mutants such as unc-11 , unc-26 or unc-57 have been shown to cause impaired transport of synaptic vesicles ( 52 , 56 , 57 ). Taken together, it is possible that phosphorylated form of α-synuclein failed to be attached to vesicles and transported to synaptic termini, resulting in its accumulation in cell bodies. Further studies will clarify whether the accumulation of phosphorylated α-synuclein in neuronal cell bodies is the cause or result of the aberrant phenotypes of α-synuclein Tg worms.

One important caveat in this RNAi screen study is that we cannot completely exclude the possibility that the RNAi effects of identified genes are additional, and not functionally synergistic, to the α-synuclein-induced toxicity. In fact, we could not find a physical binding of α-synuclein to human AP-2α or its C . elegans ortholog APA-2 by a co-immunoprecipitation assay ( Supplementary Material, Fig. S3 ). Nonetheless, the following lines of evidence strongly support the synergistic relationship: (i) 11 genes that modulate α-synuclein neurotoxicity were specifically detected out of 1673 genes, (ii) RNAi phenotypes in the control lines (EGFP-Tg or non-Tg) were not apparent or at minimal levels (Fig.  3 C and D and 4 C) and (iii) cross with the aps-2 mutant resulted in severe growth/motor disabilities of the α-synuclein-Tg lines, whereas no phenotypic aggravation was observed in the EGFP-Tg line. In addition to these genetic data, we also showed the accumulation of phosphorylated α-synuclein in neuronal cell bodies upon apa-2 knockdown. These data collectively support the existence of a pathogenic link between the endocytosis and α-synuclein.

In addition to AP-2, a subset of genes that likely modulate the α-synuclein toxicity were identified. Among these were protein kinases such as cdk-4 , cdk-9 , csnk-1 , lit-1 or cst-1 , suggesting the involvement of phosphorylation-mediated signaling pathways in α-synuclein toxicity. A muscle-specific gene pat-10 , an ortholog of troponin C, also was identified as a modifier of α-synuclein toxicity. Although the effect of knockdown of pat-10 is considered to be indirect to that of α-synuclein expressed in neurons, it may have contributed to the impaired neuromuscular transmission in α-synuclein Tg worms. In fact, the sensitivities to aldicarb were significantly decreased in these worms (Fig.  5 A).

It is still unclear which types of neurons are most vulnerable to the failure of AP-2 complex/endocytic machinery when combined with α-synuclein overexpression. The results of aldicarb/levamisole assays suggest the failure of cholinergic transmission upon overexpression of α-synuclein, although the phenotypic changes we observed in the present study should be regarded largely as pan-neuronal effects. In fact, neuronal dysfunction and α-synuclein accumulation are supposed to occur in multiple neuronal systems in dementia with LB.

Recent reports have shown transgenic C . elegans lines overexpressing human α-synuclein in dopaminergic neurons that exhibit signs of neurodegeneration as suggested by reduced fluorescence of coexpressed GFP, which are rescued by modifications of candidate genes ( 30 , 54 ), although these are not readily applicable to a large-scale screen to explore genetic or pharmacological modifiers of α-synuclein-induced toxicity. In our present experimental model, α-synuclein toxicity can be easily evaluated by simple observation of the worms treated with RNAi of the components of AP-2 complex, or by that of aps-2 ;α-synuclein-Tg lines. Thus, the present C . elegans model will be useful in high-throughput analyses, e.g. screen of chemical compounds or genes that inhibit toxicity of α-synuclein, as well as for the elucidation of the pathomechanisms of human synucleinopathies.

MATERIALS AND METHODS

Plasmid construction

Full-length cDNA encoding human α-synuclein was kindly provided by Dr T. Nakajo. pU51P plasmid containing unc-51 promoter sequence was kindly provided by Dr H. Kuroyanagi. To create the P unc-51 ::α-synuclein construct, α-synuclein sequence was PCR-amplified and inserted into the Nhe I/Kpn I site of the pU51P vector. The A53T and A30P mutant α-synuclein cDNAs were generated by the overlapping PCR-mutagenesis strategy. To create the P mec-7 ::α-synuclein construct, EGFP sequence in the P mec-7 ::EGFP vector ( 63 ) was Not I/Bgl II digested and replaced with Not I/Bgl II fragment containing human α-synuclein (wild-type or A53T, A30P-mutant) sequence. All constructs were sequenced using Thermo Sequenase™ (Amersham Biosciences) on an automated sequencer (Li-Cor, Lincolin, NE, USA).

C . elegans protocols

Nematodes were handled by standard methods ( 64 ). N2 Bristol is the wild-type strain. eri-1(mg366) , unc-11(e47) strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota, St Paul, MN, USA). aps-2(tm2912) was isolated as previously described ( 65 ). Worms were grown on the nematode growth medium (NGM) agar plates inoculated with Escherichia coli ( E . coli ) strain OP50 at 20°C unless otherwise noted. For the generation of Tg worms pan-neuronally expressing α-synuclein, plasmid DNAs encoding P unc-51 ::α-synuclein were mixed with a marker plasmid P unc-51 ::EGFP (enhanced green fluorescent protein), and the mixtures (each 200 µg/ml) were co-injected into the gonads of young adult hermaphrodite N2 worms. For the generation of Tg worms expressing α-synuclein in touch neurons, mixture of plasmid DNAs encoding P mec-7 ::α-synuclein and a marker pRF4 ( rol-6 ) (each 200 µg/ml) were co-injected as above. Transgenic progeny carrying α-synuclein as extrachromosomal arrays (Ex strains) was selected on the basis of the expression of markers. Transgenes were integrated into chromosomes by ultraviolet irradiation as described previously ( 66 ), and each isolated animal was out-crossed for four times. Worms carrying both α-synuclein transgene and eri-1 mutation were generated by mating each α-synuclein Tg line with eri-1(mg366) , and the genotyping of eri-1 (allele structure described in 42) was carried out by PCR using following set of primers: 5′-GATAAAACTTCGGAACATATGGGGC-3′ and 5′-ACTGATGGGTAAGGAATCGAAGACG-3′. Worms carrying both α-synuclein transgene and aps-2 mutation were generated by mating α-synuclein Tg worm with aps-2(tm2912) and genotyping was performed by nested PCR using following two set of primers: external primers; 5′-GCGGATTTCAGCTTTCCGCA-3′ and 5′-CTTAGAATACCTCCGGCAAC-3′, internal primers; 5′-CCGCAAGGTCTTCGCTAAGT-3′ and 5′-GAATACCTCCGGCAACATTG-3′.

RNAi screen

E . coli feeding RNAi library was obtained from GeneService Ltd. (Cambridge, UK). Feeding RNAi screen was performed essentially as described ( 67 ). Each RNAi clone stocked at −80°C was streaked on the LB agar plate containing 25 µg/ml carbenicillin (Sigma), and the colony was cultured in LB medium containing 200 µg/ml ampicillin (Wako) at 37°C for 12 h. Each cultured E . coli was then seeded onto 35 mm NGM agar plate containing 1 m m isopropyl β-D-thiogalactoside (Wako) and 25 μg/ml carbenicillin (Sigma), and these RNAi plates were incubated overnight at 30°C to induce dsRNA expression. Worms were synchronized by isolation of eggs via the hypochlorite method ( 68 ). Eggs were incubated in M9 buffer (22 m m KH 2 PO 4 /22 m m Na 2 HPO 4 /85 m m NaCl/1 m m MgSO 4 ) at 20°C for 16 h, and the worms arrested at the L1 larval stage were plated on NGM plates and incubated another 48 h at 20°C to generate L4 larvae. Three individuals of L4 larva were transferred to each plate seeded with RNAi E . coli and incubated at 20°C for 3 days. The phenotypes of the next generation (i.e. young-adult stage when normally developed) were assessed by visual observation. The identities of library clones selected from this RNAi screening were confirmed by sequencing the plasmid DNA isolated from the library E . coli . These isolated plasmids were re-transformed into the E . coli HT115 and the reproducibility of the RNAi effects was confirmed. Mock RNAi was performed by feeding worms with E . coli HT115 that were transformed with an empty vector L4440, which was originally used in the feeding RNAi library. The number of worms presenting Unc phenotype was recorded by counting those failed to respond to harsh touch stimuli.

Immunohistochemistry

Worms were pelleted by a brief centrifugation and then fixed overnight at 4°C with 4% paraformaldehyde, dehydrated in graded ethanol, embedded in paraffin and sectioned at 3 μm thick. Sections were first blocked with 10% calf serum in phosphate buffered saline, pH 7.4, and sequentially reacted with primary and secondary antibodies (biotin-conjugated horse anti-mouse IgG, Vector Laboratories). Biotin-labeled antibodies were reacted with VECTASTAIN ABC elite kit (Vector laboratories) to form avidin–biotin complex conjugated to horseradish peroxidase and visualized with diaminobenzidine. α-Synuclein was detected with a mouse monoclonal antibody LB509 ( 2 ) and α-synuclein phosphorylated at serine 129 was detected with a mouse monoclonal antibody PSyn#64 ( 10 ).

Immunoblotting

Worms were washed three times with M9 buffer, and then worm pellets were temporarily frozen at –80°C. Extracts were prepared by resuspending frozen pellets in SDS sample buffer and sonicating. The sonicates were centrifuged at 10 000× g for 5 min and the supernatants were collected. Forty micrograms of the proteins per each sample were separated by SDS–PAGE and immunoblotting as previously described ( 9 ). APA-2 protein was detected with an anti-APA-2 polyclonal antibody against the C-terminal region of APA-2 (gift of Dr B. D. Grant, Rutgers University). α-Synuclein was detected with antibody LB509 ( 2 ), and α-tubulin was detected with antibody DM1A (Sigma).

Fluorescence microscopy

Worms were anesthetized by placing in a drop of 50 m m sodium azide in M9 buffer, which was put on the solidified pads of 5% agarose laid on the slides. After addition of a coverslip, worms were examined with FLUOVIEW FV300 confocal microscope (Olympus, Tokyo, Japan).

Touch assay

Synchronized young-adult worms were transferred to fresh NGM plates, and 5 h later, the anterior and the posterior part of the body were gently touched with an eyelash. Touch sensitivity was scored as a number of times of proper response among five touches. The average score among 20 individuals was defined as a result of each trial. Plates were coded so that the experimenter was blinded to the genotype of the animal.

Aldicarb and levamisole assays

Acute sensitivities to aldicarb and levamisole were determined by placing 30 young-adult worms on 60 mm NGM plates containing 1 m m aldicarb (ChemService, West Chester, PA, USA) or 0.5 m m levamisole (Sigma). The time course of the onset of paralysis following exposure to these drugs was assayed by prodding worms every 15 min over a 2 h period. Worms that failed to respond at all to harsh touch were classified as paralyzed.

Statistics

All data presented are expressed as the mean ± SE. The results were statistically evaluated for significance by using one-way analysis of variance (ANOVA) with Fisher’s PLSD post hoc test. Differences were considered significant when P < 0.01 unless otherwise noted.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

FUNDING

This work was supported by grants-in-aid from the Ministry of Education, Science, Culture and Sports for the 21st Century Center of Excellence Program.

ACKNOWLEDGEMENTS

The authors thank Caenorhabditis Genetics Center, funded by the National Institutes of Health National Center for Research Resources, for providing eri-1(mg366) and unc-11(e47) mutants, Dr Barth D. Grant (Rutgers University) for providing anti-APA-2 antibody. We also thank lab members of Departments of Health Chemistry and Physiological Chemistry, Graduate School of Pharmaceutical Sciences in Univ. Tokyo, for kind instructions on C . elegans feeding RNAi screen, G. Ito, Y. Hanno, S. Takatori and S. Kamikawaji for helpful suggestions and discussions.

Conflict of Interest statement . The Authors have no conflicting interests to declare.

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