243
Neuroscience Letters, 128 (1991) 243-244 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100366L NSL07886
Additional genes which result in an elevation of acetylcholine levels by mutations in Caenorhabditis elegans Ryuji H o s o n o a n d Y a s u k o K a m i y a Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa (Japan) (Received 21 February 1991; Revised version received 10 April 1991; Accepted 12 April 1991)
Key words: Trichlorfon; C. elegans; Acetylcholine; unc-11; unc-63; unc-64 Four mutant genes (unc-17, unc-18, unc-41 and unc-13) have been identified that result in abnormal accumulation of acetylcholine (ACh) [3, 4]. We have now identified 3 more such genes (unc-63, unc-ll and unc-64). In addition to the abnormal accumulation of ACh, mutants in these 7 genes possess common phenotypes in locomotion, resistance to inhibitors of acetylcholinesterase (ACHE) and in post-embryonic development. These resuits suggest that the 7 genes are involved in some related functions.
Mutations affecting neurotransmitter kinetics should contribute much to our understanding of signal transduction in the nervous system. We isolated a temperature-sensitive cha-1 allele which decreases the activity of choline acetyltransferase (CHAT) as well as acetylcholine (ACh) levels when the culture temperature is raised [3, 4, 10]. Accompanying these phenotypes, the mutant becomes resistant to acetylcholinesterase (ACHE) inhibitors. Based on extensive recombination analyses, cha-1 is not a single gene but is believed to be composed of a complex with the closely linked unc-17 gene [10]. The unc-17 mutants are also resistant to AChE inhibitors as seen in cha-1, although their ChAT activities are normal. However, ACh levels in unc-17 mutants are abnormally high [4]. We investigated the number of genes present in C. elegans which elevate ACh levels by mutations. Therefore, we extensively screened genes resistant to trichlorfon, an organophosphate which is neurotoxic by virtue of its AChE-inhibitory effect. We identified 7 trichlorfon resistant genes (unc-3, unc-lO, cha-1, unc-17, unc-18, unc-41 and unc-13) and found that 4 of them had abnormally higher ACh levels than the wild type [5]. Recently, J. Rand (Wisconsin University) informed us of resistants to aldicarb, another AChE inhibitor. In the present study, we undertook testing trichlorfon resistance and the ACh levels of these mutants, and found that mutations in 3 genes affected ACh levels.
Correspondence: R. Hosono, Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920, Japan.
J. Rand (personal communication) has found that some mutations of unc-1, unc-32, unc-36, unc-65, unc-63, unc-64 and unc-ll are resistant to aldicarb. We found that of these, alleles of unc-63, unc-64 and unc-11 were also resistant to trichlorfon. We have previously identified 4 trichlorfon-resistant genes, unc-17, unc-18, unc-13 and unc-41 whose mutations cause an abnormal accumulation of ACh [3]. The ACh levels of the 3 recently identified mutations (Fig. 1) were all significantly higher than the wild type. Although the number of strains assayed was limited, ACh levels of the following alleles were all abnormally high: unc-17 (e245, cn355), unc-18 (e81, cn347, md118, md120, md183, md193, b403), unc-13 (e1019, cn490), unc-41 (cn252, e268), unc-64 (e246), unc-11 (e47) and unc-63 (e384). The extent of resistance and locomotion defectiveness was also compared (Table I). The extent of the trichlorfon resistance was diverse, that is, the unc-11 mutant was slightly, whereas the unc-63 and the unc-64 mutants were intermediately resistant. The locomotion defectiveness also varied. Movement is greatly damaged in the unc-64 mutant, although not so harmfully as in the unc-18 and unc-13 mutants. The unc-ll and unc-63 mutants can move forwards smoothly but are defective in backward movement as are the unc-17 and unc-41 mutants. We have already shown that the 4 mutants (unc-17, unc-18, unc-41 and unc-13) are accompanied by retardation of post-embryonic development and small body size in adulthood. The 3 mutants (unc-11, unc-63 and unc-64) are also abnormal in post-embryonic development (data not shown). Therefore, the 7 mutants presented here
244 Acetytcholine 0.2
0.4
nrnotes/rng 0.6
0.8
J
i
protein 1,0
1.2
i
1.4
1.6
J
w i l d type
is n o t observed as in wild type even when A C h hydrolysis is inhibited [4]. Therefore, the gene p r o d u c t might
un c - 17(e 2 4 5 )
{--q
unc- 18 (on J 4 7 )
c o n t r i b u t e to the flow o f A C h after the synthesis, includ-
unc- 4 1 ( ¢ n 2 5 2 )
F
unc-13(elOIg)
I
3q Y~
unc- 6 5 ( e 3 8 4 ) uric- 6 4 ( e 2 4 6 ) unc-II (e47) i
i
i
i~
I
i
Fig. 1. ACh levels of trichlorfon-resistant mutants. ACh was measured according to the slightly modified method of McCaman and Stezler [8l (Hosono et al., submitted). ACh was extracted from an asynchronous population of C. elegans. Standard deviations are indicated by vertical bars. share c o m m o n phenotypes with regard to A C h levels, p h a r m a c o l o g i c a l response, l o c o m o t i o n , a n d development, indicating that these m u t a n t s are defective in overlapped functions. The structural gene for choline acetyltransferase [9], acetylcholinesterase [2, 6], a n d potential receptors for A C h [7] have been m a p p e d at different sites of the 7 genes. There are two possible e x p l a n a t i o n s for the elevation o f A C h levels in the 7 gene m u t a n t s . Firstly, A C h might n o t be n o r m a l l y released a n d therefore accumulates at the presynaptic terminals. This is because the m u t a n t p h e n o t y p e s are similar to those o f cha1 which are defective in t r a n s m i t t e r release o n a c c o u n t TABLE I PHENOTYPES OF TRICHLORFON-RESISTANT MUTANTS ACCOMPANYING AN ELEVATION OF ACETYLCHOLINE To determine sensitivityto trichlorfon, 3 animals of the 4th larval stage were each placed onto NGM [I] containing 11 different concentrations of trichlorfon (0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.0 mM) and grown at 20°C. The highest concentration of trichlorton is indicated, in which animals were able to produce F2 progeny within 10 days. The ability to move was tested by touch stimulation with toothpicks at either the head or the tail regions. + + + and indicate normal and indiscernible movement, respectively. The extent of the defectivenessof locomotion between + + + and - was divided into 3 steps (_, +, + +). Resistancy to trichlorfon (mM)
forward
backward
wild type
0.02
+++
+++
unc-17 (e245) unc-18 (cn347) unc-41 (cn252) unc-13 (elO19) unc-63 (e384) uric-64 (e246) unc-11 (e47)
1.00
++
-
0.20 0.20 0.20 0.20 0.20 0.04
++ -++ + ++
+ -+ + +
Genotype
o f insufficiency of the t r a n s m i t t e r supply. F u r t h e r m o r e , an elevation o f A C h levels in the case o f u n c - 1 7 m u t a n t
Locomotion
ing steps such as axonal transport, storage or its release. Secondly, the p r i m a r y defects o f the nervous system in these genes m a y result in A C h a c c u m u l a t i o n . The u n c - 1 3 m u t a n t is a b n o r m a l in the gap j u n c t i o n between intern e u r o n s a n d m o t o r n e u r o n s (I. M a r u y a m a , personal c o m m u n i c a t i o n ) , a n d the u n c - 1 8 m u t a n t is a b n o r m a l in the d e v e l o p m e n t of the dorsal nerve cord (R. H o s o n o et al., submitted). In s u m m a r y , we add here 3 genes in a d d i t i o n to those 4 previously identified that show a n a b n o r m a l accumulation of A C h by m u t a t i o n s . D a t a from the present a n d previous studies show that m u t a t i o n s in these 7 genes share overlapped phenotypes. Supported by G r a n t - i n - A i d for Scientific Research o n Priority Areas (Molecular basis o f neural connection), Ministry of E d u c a t i o n , Science a n d C u l t u r e a n d by funds for Medical T r e a t m e n t of the Elderly, School o f Medicine K a n a z a w a University, 1990. 1 Brenner, S., The genetics of Caenorhabditis elegans, Genetics, 77 (1974) 71-94. 2 Culotti, J.G., von Ehrenstein, G., Culotti, M.R. and Russell, R.L., A second class of acetylcholinesterase-deficientmutants of the nematode Caenorhabditis elegans, Genetics, 97 (1981) 281 305. 3 Hosono, R., Kuno, S. and Midsukami, M., Temperature-sensitive mutation causing reversible paralysis in Caenorhabditis elegans, J. Exp. Zool., 235 (1985)409-421. 4 Hosono, R., Sassa, T. and Kuno, S., Mutations affectingacetylcholine levelsin the nematode Caenorhabditis elegans, J. Neurochem., 49 (1987) 1820-1823. 5 Hosono, R., Sassa, T. and Kuno, S., Spontaneous mutations of trichlorfon resistance in the nematode Caenorhabditis elegans, Zool. Sci., 6 (1989) 697-708. 6 Johnson, C.D., Rand, J.B., Herman, R.K., Stern, B.D. and Russell, R.L., The acetylcholinesterasegenes of C. elegans: identification of a third gene (ace-3) and mosaic mapping of a synthetic lethal phenotype, Neuron, 1 (1988) 165-173. 7 Lewis, J.A., Elmer, J.S., Skimming, J., McLafferty, S., Fleming, J. and McGee, T., Cholinergic receptor mutants of the nematode Caenorhabditis elegans, J. Neurosci., 7 (1987) 3059-3071. 8 McCaman, R.E. and Stetzler, J., Radiochemical assay of ACh: modification for sub-picomole measurements, J. Neurochem., 28 (1977) 669-671. 9 Rand, J.B. and Russell, R.L., Choline acetyltransferase-deficient mutants of the nematode Caenorhabditis elegans, Genetics, 106 (1984) 227-248. I0 Sassa, T., Hosono, R. and Kuno, S., Choline acetyltransferase from a temperature-sensitive mutant of C. elegans, Neurochem. Int., l 1 (1987) 323 329.
Neuroscience Letters, 128 (1991) 243-244 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100366L NSL07886
Additional genes which result in an elevation of acetylcholine levels by mutations in Caenorhabditis elegans Ryuji H o s o n o a n d Y a s u k o K a m i y a Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa (Japan) (Received 21 February 1991; Revised version received 10 April 1991; Accepted 12 April 1991)
Key words: Trichlorfon; C. elegans; Acetylcholine; unc-11; unc-63; unc-64 Four mutant genes (unc-17, unc-18, unc-41 and unc-13) have been identified that result in abnormal accumulation of acetylcholine (ACh) [3, 4]. We have now identified 3 more such genes (unc-63, unc-ll and unc-64). In addition to the abnormal accumulation of ACh, mutants in these 7 genes possess common phenotypes in locomotion, resistance to inhibitors of acetylcholinesterase (ACHE) and in post-embryonic development. These resuits suggest that the 7 genes are involved in some related functions.
Mutations affecting neurotransmitter kinetics should contribute much to our understanding of signal transduction in the nervous system. We isolated a temperature-sensitive cha-1 allele which decreases the activity of choline acetyltransferase (CHAT) as well as acetylcholine (ACh) levels when the culture temperature is raised [3, 4, 10]. Accompanying these phenotypes, the mutant becomes resistant to acetylcholinesterase (ACHE) inhibitors. Based on extensive recombination analyses, cha-1 is not a single gene but is believed to be composed of a complex with the closely linked unc-17 gene [10]. The unc-17 mutants are also resistant to AChE inhibitors as seen in cha-1, although their ChAT activities are normal. However, ACh levels in unc-17 mutants are abnormally high [4]. We investigated the number of genes present in C. elegans which elevate ACh levels by mutations. Therefore, we extensively screened genes resistant to trichlorfon, an organophosphate which is neurotoxic by virtue of its AChE-inhibitory effect. We identified 7 trichlorfon resistant genes (unc-3, unc-lO, cha-1, unc-17, unc-18, unc-41 and unc-13) and found that 4 of them had abnormally higher ACh levels than the wild type [5]. Recently, J. Rand (Wisconsin University) informed us of resistants to aldicarb, another AChE inhibitor. In the present study, we undertook testing trichlorfon resistance and the ACh levels of these mutants, and found that mutations in 3 genes affected ACh levels.
Correspondence: R. Hosono, Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920, Japan.
J. Rand (personal communication) has found that some mutations of unc-1, unc-32, unc-36, unc-65, unc-63, unc-64 and unc-ll are resistant to aldicarb. We found that of these, alleles of unc-63, unc-64 and unc-11 were also resistant to trichlorfon. We have previously identified 4 trichlorfon-resistant genes, unc-17, unc-18, unc-13 and unc-41 whose mutations cause an abnormal accumulation of ACh [3]. The ACh levels of the 3 recently identified mutations (Fig. 1) were all significantly higher than the wild type. Although the number of strains assayed was limited, ACh levels of the following alleles were all abnormally high: unc-17 (e245, cn355), unc-18 (e81, cn347, md118, md120, md183, md193, b403), unc-13 (e1019, cn490), unc-41 (cn252, e268), unc-64 (e246), unc-11 (e47) and unc-63 (e384). The extent of resistance and locomotion defectiveness was also compared (Table I). The extent of the trichlorfon resistance was diverse, that is, the unc-11 mutant was slightly, whereas the unc-63 and the unc-64 mutants were intermediately resistant. The locomotion defectiveness also varied. Movement is greatly damaged in the unc-64 mutant, although not so harmfully as in the unc-18 and unc-13 mutants. The unc-ll and unc-63 mutants can move forwards smoothly but are defective in backward movement as are the unc-17 and unc-41 mutants. We have already shown that the 4 mutants (unc-17, unc-18, unc-41 and unc-13) are accompanied by retardation of post-embryonic development and small body size in adulthood. The 3 mutants (unc-11, unc-63 and unc-64) are also abnormal in post-embryonic development (data not shown). Therefore, the 7 mutants presented here
244 Acetytcholine 0.2
0.4
nrnotes/rng 0.6
0.8
J
i
protein 1,0
1.2
i
1.4
1.6
J
w i l d type
is n o t observed as in wild type even when A C h hydrolysis is inhibited [4]. Therefore, the gene p r o d u c t might
un c - 17(e 2 4 5 )
{--q
unc- 18 (on J 4 7 )
c o n t r i b u t e to the flow o f A C h after the synthesis, includ-
unc- 4 1 ( ¢ n 2 5 2 )
F
unc-13(elOIg)
I
3q Y~
unc- 6 5 ( e 3 8 4 ) uric- 6 4 ( e 2 4 6 ) unc-II (e47) i
i
i
i~
I
i
Fig. 1. ACh levels of trichlorfon-resistant mutants. ACh was measured according to the slightly modified method of McCaman and Stezler [8l (Hosono et al., submitted). ACh was extracted from an asynchronous population of C. elegans. Standard deviations are indicated by vertical bars. share c o m m o n phenotypes with regard to A C h levels, p h a r m a c o l o g i c a l response, l o c o m o t i o n , a n d development, indicating that these m u t a n t s are defective in overlapped functions. The structural gene for choline acetyltransferase [9], acetylcholinesterase [2, 6], a n d potential receptors for A C h [7] have been m a p p e d at different sites of the 7 genes. There are two possible e x p l a n a t i o n s for the elevation o f A C h levels in the 7 gene m u t a n t s . Firstly, A C h might n o t be n o r m a l l y released a n d therefore accumulates at the presynaptic terminals. This is because the m u t a n t p h e n o t y p e s are similar to those o f cha1 which are defective in t r a n s m i t t e r release o n a c c o u n t TABLE I PHENOTYPES OF TRICHLORFON-RESISTANT MUTANTS ACCOMPANYING AN ELEVATION OF ACETYLCHOLINE To determine sensitivityto trichlorfon, 3 animals of the 4th larval stage were each placed onto NGM [I] containing 11 different concentrations of trichlorfon (0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.0 mM) and grown at 20°C. The highest concentration of trichlorton is indicated, in which animals were able to produce F2 progeny within 10 days. The ability to move was tested by touch stimulation with toothpicks at either the head or the tail regions. + + + and indicate normal and indiscernible movement, respectively. The extent of the defectivenessof locomotion between + + + and - was divided into 3 steps (_, +, + +). Resistancy to trichlorfon (mM)
forward
backward
wild type
0.02
+++
+++
unc-17 (e245) unc-18 (cn347) unc-41 (cn252) unc-13 (elO19) unc-63 (e384) uric-64 (e246) unc-11 (e47)
1.00
++
-
0.20 0.20 0.20 0.20 0.20 0.04
++ -++ + ++
+ -+ + +
Genotype
o f insufficiency of the t r a n s m i t t e r supply. F u r t h e r m o r e , an elevation o f A C h levels in the case o f u n c - 1 7 m u t a n t
Locomotion
ing steps such as axonal transport, storage or its release. Secondly, the p r i m a r y defects o f the nervous system in these genes m a y result in A C h a c c u m u l a t i o n . The u n c - 1 3 m u t a n t is a b n o r m a l in the gap j u n c t i o n between intern e u r o n s a n d m o t o r n e u r o n s (I. M a r u y a m a , personal c o m m u n i c a t i o n ) , a n d the u n c - 1 8 m u t a n t is a b n o r m a l in the d e v e l o p m e n t of the dorsal nerve cord (R. H o s o n o et al., submitted). In s u m m a r y , we add here 3 genes in a d d i t i o n to those 4 previously identified that show a n a b n o r m a l accumulation of A C h by m u t a t i o n s . D a t a from the present a n d previous studies show that m u t a t i o n s in these 7 genes share overlapped phenotypes. Supported by G r a n t - i n - A i d for Scientific Research o n Priority Areas (Molecular basis o f neural connection), Ministry of E d u c a t i o n , Science a n d C u l t u r e a n d by funds for Medical T r e a t m e n t of the Elderly, School o f Medicine K a n a z a w a University, 1990. 1 Brenner, S., The genetics of Caenorhabditis elegans, Genetics, 77 (1974) 71-94. 2 Culotti, J.G., von Ehrenstein, G., Culotti, M.R. and Russell, R.L., A second class of acetylcholinesterase-deficientmutants of the nematode Caenorhabditis elegans, Genetics, 97 (1981) 281 305. 3 Hosono, R., Kuno, S. and Midsukami, M., Temperature-sensitive mutation causing reversible paralysis in Caenorhabditis elegans, J. Exp. Zool., 235 (1985)409-421. 4 Hosono, R., Sassa, T. and Kuno, S., Mutations affectingacetylcholine levelsin the nematode Caenorhabditis elegans, J. Neurochem., 49 (1987) 1820-1823. 5 Hosono, R., Sassa, T. and Kuno, S., Spontaneous mutations of trichlorfon resistance in the nematode Caenorhabditis elegans, Zool. Sci., 6 (1989) 697-708. 6 Johnson, C.D., Rand, J.B., Herman, R.K., Stern, B.D. and Russell, R.L., The acetylcholinesterasegenes of C. elegans: identification of a third gene (ace-3) and mosaic mapping of a synthetic lethal phenotype, Neuron, 1 (1988) 165-173. 7 Lewis, J.A., Elmer, J.S., Skimming, J., McLafferty, S., Fleming, J. and McGee, T., Cholinergic receptor mutants of the nematode Caenorhabditis elegans, J. Neurosci., 7 (1987) 3059-3071. 8 McCaman, R.E. and Stetzler, J., Radiochemical assay of ACh: modification for sub-picomole measurements, J. Neurochem., 28 (1977) 669-671. 9 Rand, J.B. and Russell, R.L., Choline acetyltransferase-deficient mutants of the nematode Caenorhabditis elegans, Genetics, 106 (1984) 227-248. I0 Sassa, T., Hosono, R. and Kuno, S., Choline acetyltransferase from a temperature-sensitive mutant of C. elegans, Neurochem. Int., l 1 (1987) 323 329.
