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Volume 16, Number 21, Issue of November 1, 1996 pp. 6695-6702
Copyright ©1996 Society for NeuroscienceA Murine Neural-Specific Homolog Corrects Cholinergic Defects in Caenorhabditis elegans unc-18 Mutants
Keiko Gengyo-Ando1, 2, Hitoshi Kitayama1, 3, Masahiro Mukaida2, and Yoji Ikawa1, 4 1 Laboratory of Molecular Oncology, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan, 2 Department of Forensic Medicine, National Defense Medical College, Tokorozawa, Saitama 359, Japan, 3 Department of Molecular Oncology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan, and 4 Department of Retroviral Regulation, Medical Research Division, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113, Japan
ABSTRACT
Caenorhabditis elegans UNC-18 protein, homologous to yeast Sec1p, is important in neurotransmitter release, because the unc-18 mutation leads to severe paralysis and presynaptic acetylcholine (ACh) accumulation. To examine the functional conservation in mammals, we tried to isolate unc-18 isoforms from mouse and human brain cDNA libraries and obtained two classes of isoforms
Key words: neurotransmitter release; unc-18; Caenorhabditis elegans; ACh; transgenic studyneural genes and ubiquitous genes. Neural genes were identical to Munc-18 (also known as n-Sec1 or rbSec1), identified in rat and bovine brains as a syntaxin-binding protein. According to ``Munc-18'' terminology, we call the neural genes Munc-18-1 and the ubiquitous genes Munc-18-3. These mammalian isoforms exhibit 58% (Munc-18-1) and 42-43% (Munc-18-3) amino acid sequence identity with UNC-18. Next, we constructed transgenic unc-18 mutants to test biological activity of mouse Munc-18-1 and Munc-18-3 under the control of C. elegans unc-18 promoter. Munc-18-1 compensates for severe locomotion disability and cholinergic defects, e.g., abnormal sensitivities to cholinesterase inhibitors and cholinergic receptor agonists in unc-18 mutants, but Munc-18-3 fails. These data suggest that Munc-18-1 and C. elegans unc-18 may play positive roles in ACh release and that the molecular mechanism of neuronal regulated secretion has been partially conserved from nematodes to mammals.
INTRODUCTION
Neurotransmitters stored in the synaptic vesicles are released into the synaptic gap by Ca2+-dependent exocytosis. According to the SNAP receptor (SNARE) hypothesis (Rothman, 1994
), the interaction of
esicular and
arget membrane proteins (referred to as v-SNARE and t-SNARE, respectively) leads to vesicle docking. Several neuronal components have been isolated from synaptosomes of vertebrates, and the molecular and biochemical properties of these components have been elucidated (Südhof and Jahn, 1991
The unc (
oordinated)-18 mutations of Caenorhabditis elegans cause several phenotypes implicating impairment of presynaptic functions, such as acetylcholine (ACh) accumulation and resistance to acetylcholinesterase inhibitors, as well as a paralytic phenotype (Hosono et al., 1987
In the present study, we cloned a brain-specific isoform (Munc-18/n-Sec1/rbSec1; Hata et al., 1993
MATERIALS AND METHODS
Molecular biology. Molecular biological procedures were performed as described previously, with minor modifications (Maniatis et al., 1989Cloning of C. briggsae unc-18. Southern blot analysis of a C. briggsae genomic DNA digested with EcoRI was performed using a C. briggsae cDNA fragment (+29 to +588) as a probe, which was amplified with RT-PCR; a 4 kb band was detected. Genomic DNA fragments of the proper size fraction were cloned into the EcoRI-digested
ZAP vector. The resulting phage clones were screened with the same probes under standard conditions. One of four positive clones was analyzed further. Because these clones did not contain the last exon, a cDNA clone of the 3
end of the message was isolated using the 3
cDNA isolation. Two degenerate oligonucleotides were designed to be located in highly conserved regions among the unc-18 of C. elegans and C. briggsae, and the sec1 family of S. cerevisiae. The forward oligo AG(A/G)(A/T)(G/C)CCA(A/G)CT(G/C)ATCATCATCGA(C/T)AG(A/G)GG(A/C)TA(C/T)GA, corresponding to amino acid residues 225-236, and the reverse oligo AG(A/G)TC(A/G)TA(A/G)CACAT(G/A)GC(T/C)TG, corresponding to residues 248-254 of UNC-18, were used in RT-PCR with poly(A+) RNA isolated from mouse adult brain. The fragments of the exact predicted size (89 bp) were subcloned and sequenced. Two clones (amp11 and amp15) were found to be ~70% homologous to unc-18 at the DNA level and were used as DNA probes to isolate cDNA clones. A mouse adult brain cDNA library in the
Human Munc-18-1 and Munc-18-3 cDNA clones were isolated from a human fetal brain cDNA library cloned in
RNA isolation and Northern blot analysis. Total cellular RNA was isolated from adult mouse tissues, and poly(A+) RNA was purified with oligo-dT cellulose columns (Life Technologies, Gaithersburg, MD) and separated on a 1% 0.01 M NaH2PO4 agarose gel. The glyoxylated RNA was transferred to Hybond-N+ (Amersham, Buckinghamshire, UK) for hybridization.
Nematodes. Conditions for growth and maintenance of C. elegans have been described by Brenner (1974)
Transgenic animals. For transgenic study, constructs carrying test cDNA (unc-18, mouse Munc-18-1, and Munc-18-3) under the control of unc-18 genomic flanking regions were generated. A 1 kb KpnI-XbaI genomic fragment containing the 3
The resulting DNA constructs, punc-18, pMunc-18-1, and pMunc-18-3 plasmids (20 µg/ml) were injected into unc-18(e81) by standard germline transformation techniques (Fire, 1986
Antibody staining. Immunohistochemistry was carried out using a whole-mount procedure (McIntire et al., 1992
RESULTS
Molecular identification of the unc-18 family
As a first step in isolating a mammalian unc-18 homolog, we tried to identify an evolutionally conserved region of the unc-18 gene from the nematode C. briggsae, which was expected to be functionally important. To determine the C. briggsae unc-18 sequence, we isolated genomic clones containing the C. briggsae unc-18-coding region. The predicted amino acid sequence of C. briggsae UNC-18 is highly conserved to that of C. elegans UNC-18 (572/590 identical; Fig. 1), and the intron-exon pattern of the C. briggsae unc-18 gene is also similar to that of C. elegans unc-18 gene (data not shown).
Fig. 1. The unc-18 families. A, Alignment of predicted amino acid sequences from the unc-18-related genes. An optimal alignment was produced with the Pile-Up program. Identical residues to the mouse Munc-18-1 are indicated by dots, and gaps caused by the alignment are indicated by dashes. The sequence of human Munc-18-1 is not shown in this alignment because it is identical to mouse Munc-18-1. B, Alternative C-terminal sequences of human Munc-18-1. Boxed sequences indicate amino acids identical in Munc-18-1A and Munc-18-1B. Munc-18-1B cDNA contains 126 bp of an additive sequence at position +1702 (indicated by arrow) of the cDNA sequence of Munc-18-1A and encodes a protein of 603 amino acids.
[View Larger Version of this Image (64K GIF file)]
Degenerate oligonucleotide primers based on the conserved amino acid regions among the UNC-18 of C. elegans and C. briggsae, and the yeast Sec1-related proteins Sec1p (Aalto et al., 1991
Distinct expression pattern of Munc-18-1 and Munc-18-3 mRNA in mouse tissues
The tissue distribution of Munc-18-1 and Munc-18-3 transcripts was investigated by Northern blot analysis. Poly(A+) RNA purified from various tissues was electrophoresed, blotted, and hybridized to 1.2 kb BamHI-BamHI coding fragments from a murine Munc-18-1 cDNA clone (2B-11) or a 0.7 kb EcoRI-PstI-coding fragment from a murine Munc-18-3 cDNA clone (5A-1).As expected, Munc-18-1 is expressed only in the brain as an ~4 kb transcript, whereas Munc-18-3 is expressed constitutively as a message of ~2.8 kb in all mouse tissues analyzed (Fig. 2).
Fig. 2. Northern blot analysis of mouse Munc-18 genes. Northern blots of poly(A+) RNA (2 µg) from seven different mouse tissues were hybridized with 32P-labeled probes derived from Munc-18-1, Munc-18-3, and actin. Whereas transcripts for Munc-18-1 are expressed exclusively in the brain, transcripts for Munc-18-3 are expressed in all tissues analyzed.
[View Larger Version of this Image (50K GIF file)]
Munc-18-1 rescues the cholinergic defects in unc-18 mutants
The fact that Munc-18-1 is a neural gene and has higher homology to unc-18 than does Munc-18-3 suggests that the Munc-18-1 protein might function in nervous system in a manner similar to UNC-18. Mutants defective in the unc-18 gene exhibit several characteristic phenotypes implicated in the impairment of presynaptic function, including severe locomotion disability, resistance to cholinesterase inhibitors, and hypersensitivity to cholinergic receptor agonists. To examine whether mouse Munc-18 genes can substitute for unc-18, we designed expression vectors containing the mouse genes under the control of promoter and poly(A) signal derived from the unc-18 genomic sequence (pMunc-18-1 and pMunc-18-3; Fig. 3A).
Fig. 3.
[View Larger Version of this Image (53K GIF file)]
For a positive control, an expression vector containing the unc-18 cDNA was also constructed (punc-18; Fig. 3A). unc-18(e81) strain was used as the recipient of the transgene, which has no detectable UNC-18 products. Obvious restoration of the coordinated phenotype was observed in animals (kEx2 and kEx3) transgenic for punc-18 as well as wild-type animals (kEx9) transgenic only for rol-6, indicating that the expression system was sufficient to examine the biological activities of mouse Munc-18 genes. Animals (kEx4 and kEx5) transgenic for the pMunc-18-1 construct showed a prominent restoration of locomotion compared to animals (kEx1) transgenic only for rol-6, whereas pMunc-18-3 induced no detectable rescue in two independent transformed lines (kEx6 and kEx7) (Table 1, Fig. 3B). We detected no mutation in the Munc-18-3 transgene and observed no obvious dominant effect in the wild-type worms transgenic for pMunc-18-3 (data not shown).
Table 1. Transgenic C. elegans strains and their properties
Plasmid Array Host genotype Motility Sensitivity for cholinergic reagents Trichlorfon (mM) Levamisole (mM) - - unc-18(+) wild-type 0.01 >0.30 - - unc-18(e81) unc 0.20 0.01 pRF4 kEx9 unc-18(+) wild-type 0.01 >0.30 pRF4 kEx1 unc-18(e81) unc 0.20 0.01 punc-18; pRF4 kEx2 unc-18(e81) wild-type 0.02 >0.30 punc-18; pRF4 kEx3 unc-18(e81) wild-type 0.02 >0.30 punc-18; pRF4 kIn2-1 unc-18(e81) wild-type 0.01 >0.30 punc-18; pRF4 kIn2-2 unc-18(e81) wild-type 0.01 >0.30 pMunc-18-1; pRF4 kEx4 unc-18(e81) wild-typea 0.01 >0.30 pMunc-18-1; pRF4 kEx5 unc-18(e81) wild-typea 0.01 >0.30 pMunc-18-1; pRF4 kIn4-1 unc-18(e81) wild-typea 0.01 >0.30 pMunc-18-1; pRF4 kIn4-2 unc-18(e81) wild-typea 0.01 >0.30 pMunc-18-3; pRF4 kEx6 unc-18(e81) unc 0.20 0.01 pMunc-18-3; pRF4 kEx7 unc-18(e81) unc 0.20 0.01 punc-18, pMunc-18-1, and pMunc-18-3 plasmids were injected into unc-18(e81) with the plasmid pRF4, which confers a dominant Roller phenotype. Extrachromosomal arrays that behave as unstable free duplications are named kEx, and stable arrays integrated into a chromosome are named kIn. The motility and the sensitivity for cholinergic reagents of unc-18 mutants are affected by these transgenes. a Wild-type means slightly slower motility than the motility of wild-type animals carrying rol-6. Three larvae were put onto NGM containing 10 different concentrations of reagents (0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, and 0.3 mM). The highest concentrations are shown in which animals were able to produce F2 progeny within 10 d in triplicate experiments. To investigate cholinergic functions in transformed C. elegans, we examined the sensitivity of these animals to a cholinesterase inhibitor and to cholinergic receptor agonists. The resistance of unc-18 animals to trichlorfon was fully reversed by the presence of the punc-18 transgene or by the presence of the pMunc-18-1 transgene (Table 1). In addition, pMunc-18-1 rescued the hypersensitivity to the ACh receptor agonists levamisole (Table 1) and carbachol (data not shown). As a control, we demonstrated that the presence of the rol-6 transgene by itself had no effect on the drug responses of transgenic animals. These results suggest that Munc-18-1 can correct presynaptic defects in cholinergic neurons of unc-18 mutants. In the case of pMunc-18-3 animals, however, there was no obvious change in sensitivity to cholinergic reagents (Table 1). To facilitate a comparison of the sensitivities of punc-18 and pMunc-18-1 animals to cholinergic reagents, we constructed chromosomal integrated lines of these plasmids in which non-Rol animals were not segregated. Figure 4 shows the growth rates of these animals on medium containing trichlorfon (Fig. 4A) or levamisole (Fig. 4B). The growth rates of wild-type and unc-18(e81) animals carrying only rol-6 are also shown for comparison.
Fig. 4. Sensitivities for cholinergic drugs in the transgenic nematodes. Animals were grown on NGM containing 40 µM trichlorfon (A) or 20 µM levamisole (B) at 20°C. Twenty to forty animal lengths were measured for each time point, and the mean values were plotted. Bars indicate SEM. The cholinergic defects in unc-18 mutants are partially suppressed by the Munc-18-1 transgene. Animals transgenic for punc-18 (kIn2-1) or pMunc-18-1 (kIn4-1) are more sensitive to levamisole than wild-type animals transgenic for rol-6. These effects may be attributable to copy number of transgenes.
[View Larger Version of this Image (15K GIF file)]
As expected, we found no detectable difference in sensitivity to trichlorfon between punc-18 and pMunc-18-1 animals. Moreover, pMunc-18-1 animals partially but significantly rescued the hypersensitive phenotype of levamisole.
Abnormal GABA immunoreactivities in unc-18 mutants
There are seven classes of motor neurons (DA, VA, DB, VB, DD, VD, and AS) innervating body wall muscles in the ventral nerve cord of C. elegans. Electrophysiological studies have shown that the DA, DB, and AS homologs of Ascaris are excitatory and that the DD and VD homologs are inhibitory (Walrond et al., 1985
Fig. 5. The higher GABA immunoreactivity in unc-18 mutants. Whole mounts of unc-18(e81) (B), wild-type N2 (C), and animals transgenic for Munc-18-1(kIn4-1) (D) and unc-47(e307) (E) were stained with an anti-GABA antiserum followed by a secondary biotin-conjugated goat anti-rabbit antiserum and ABC complex. The 12 immunoreactive neurons of the unc-18 animal at the left of B are schematically represented in A. The unc-18 mutants show abnormally high levels of GABA reactivity, similar to the unc-47 mutants. Scale bar, 50 µm.
[View Larger Version of this Image (29K GIF file)]
The GABAergic neurons in unc-18 (Fig. 5B) and unc-47 mutants (Fig. 5E) reacted similarly to anti-GABA antibody, and both reactions were more intense than those exhibited by wild-type N2 (Fig. 5C). These results suggest that mutations in the unc-18 gene cause some defect within GABAergic function. GABA immunoreactivity in transgenic nematodes for Munc-18-1 kIn4-1 (Fig. 5D) and kIn4-2 (data not shown) was slightly weaker than in recipient unc-18(e81).
DISCUSSION
To further our understanding of the precise role of the unc-18 family in vesicle traffic, we isolated and characterized the unc-18-related genes Munc-18-1 and Munc-18-3. Although the neuronal unc-18 isoform Munc-18-1 (n-Sec1/rbSec1) shows the highest homology to C. elegans unc-18 among mammalian homologs and exhibits specific binding to syntaxin (Hata et al., 1993It is interesting to clarify the relationship between the unc-18 family and the syntaxin family. In mammalian cells, six syntaxin isoforms with broad tissue distribution are characterized (Bennett et al., 1993
It has been shown that neurotransmitters common to vertebrates, such as ACh, GABA, serotonin, dopamine, and some neuropeptides, are involved in the functioning of the C. elegans nervous system. Immunocytochemical studies revealed that GABAergic neurons in unc-18 mutants contained abnormally high levels of GABA as well as those in unc-47 mutants (Fig. 5). Therefore, the unc-18 gene also appears to have some function in GABAergic neurons of C. elegans. A murine Munc-18-1 transgene could slightly reduce the abnormal GABA accumulation observed in the DDn motor neurons of unc-18 mutants. Further quantitative analyses of GABA accumulation and investigations of other GABA-related phenotype will facilitate an understanding of the roles of the unc-18 gene in GABAergic neurotransmission.
We found that human Munc-18-1 has an alternative form [referred to as Munc-18-1B (Fig. 1B), the same as in rat rbSec1B; Garcia et al., 1995
In mutants of the cha-1 and the closely linked unc-17 genes, which encode choline acetyltransferase and vesicular ACh transporter, respectively, homozygous null mutations lead to lethality in C. elegans, because cholinergic functions are fully blocked (Rand and Russell, 1984
Mutations in the C. elegans unc-18 gene lead to severe synaptic defects, suggesting that unc-18 may have positive functions in neuronal secretion. Binding of Munc-18-1(Munc-18/n-Sec1/rbSec1) to syntaxin, however, inhibits interaction of syntaxin with SNAREs (Pevsner et al., 1994b
GenBank accession numbers
The accession numbers for the sequences reported in this paper are D63504 (the C. briggsae genomic sequence), D63505 (the C. briggsae cDNA sequence), D45903 (the mouse Munc-18-1 cDNA sequence), D63851 (human Munc-18-1 cDNA sequence), D30798 (the mouse Munc-18-3 cDNA sequence), and D63506 (the human Munc-18-3 cDNA sequence).
FOOTNOTES
Received March 11, 1996; revised Aug. 8, 1996; accepted Aug. 13, 1996.
This study was supported by grants from the Science and Technology Agency of Japan, the Ministry of Education, Science, Sports, and Culture of Japan, and the Defense Agency of Japan. K.G.-A. acknowledges support by the Special Postdoctoral Researchers Program from the Science and Technology Agency of Japan. We thank Dr. R. Hosono (Kanazawa University) for kindly providing genomic unc-18 clone PE10 and unc-18 cDNA clone ASC, and for commenting on this manuscript. We also thank Dr. M. W. Schein (Rockville, MD) for his editing of this manuscript and Professor M. Noda (Kyoto University) for encouragement. Nematode strain unc-47 (e307) was provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources.
Correspondence should be addressed to Hitoshi Kitayama, Department of Molecular Oncology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606, Japan.
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