Analysis of Caenorhabditis elegans acetylcholine synthesis mutants reveals a temperature-sensitive requirement for cholinergic neuromuscular function

Abstract

In Caenorhabditis elegans, the cha-1 gene encodes choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine. We have analyzed a large number of cha-1 hypomorphic mutants, most of which are missense alleles. Some homozygous cha-1 mutants have approximately normal ChAT immunoreactivity; many other alleles lead to consistent reductions in synaptic immunostaining, although the residual protein appears to be stable. Regardless of protein levels, neuromuscular function of almost all mutants is temperature-sensitive, i.e., neuromuscular function is worse at 25° than at 14°. We show that the temperature effects are not related to acetylcholine release, but specifically to alterations in acetylcholine synthesis. This is not a temperature-dependent developmental phenotype, because animals raised at 20° to young adulthood and then shifted for 2 h to either 14° or 25° had swimming and pharyngeal pumping rates similar to animals grown and assayed at either 14° or 25°, respectively. We also show that the temperature-sensitive phenotypes are not limited to missense alleles; rather, they are a property of most or all severe cha-1 hypomorphs. We suggest that our data are consistent with a model of ChAT protein physically, but not covalently, associated with synaptic vesicles; and there is a temperature-dependent equilibrium between vesicle-associated and cytoplasmic (i.e., soluble) ChAT. Presumably, in severe cha-1 hypomorphs, increasing the temperature would promote dissociation of some of the mutant ChAT protein from synaptic vesicles, thus removing the site of acetylcholine synthesis (ChAT) from the site of vesicular acetylcholine transport. This, in turn, would decrease the rate and extent of vesicle-filling, thus increasing the severity of the behavioral deficits.

Keywords: Caenorhabditis elegans; ChAT; cholinergic gene locus; synaptic vesicle; temperature-sensitive mutants.

Figure 1

cha-1 exon structure and cha-1 mutations. Shown are the splicing pattern and exon structure of the C. elegans cha-1 transcript (Alfonso et al. 1994b). Protein-coding regions are blue, and the 5ʹ- and 3ʹ-untranslated regions are gray. Also shown are the locations of missense mutations and their predicted amino acid substitutions (see Table 1) as well as the sites of the previously described Tc1 transposon insertions and m324 deletion (Alfonso et al. 1994b). The mutations shown in red are homozygous lethal alleles.

Figure 2

Synaptic ChAT immunostaining at different temperatures. ChAT immunoreactivity shows similar patterns in wild-type and cha-1 mutants with the predominantly synaptic antibodies (mAb 1402 and 1414) at 15° (A, C, E) and 25° (B, D, F). (Duerr et al. 2008). Staining in wild type (A, B), cha-1(p503) (C, D), and cha-1(md39) (E, F). Overall intensity appears somewhat lower after 1 day at 25° in all strains; immunoreactivity in neuronal processes, such as the anterior sublateral nerve cords (arrows) is similar in wild type and cha-1(p503) and lower in cha-1(md39). Anterior is left; dorsal is up; NR, nerve ring; DNC, dorsal nerve cord; VNC, ventral nerve cord. Scale bar is 30 μm.

Figure 3

Somatic ChAT and synaptic VAChT immunostaining at different temperatures. ChAT immunoreactivity shows similar patterns in wild type and cha-1 mutants with the predominantly somatic antibodies (Mab 1401, 1415, 1432). Color panels show partial colocalization of anti-VAChT (green) and anti-ChAT (red) in cholinergic neurons; black and white panels show ChAT alone for clarity. Young adults were kept 1 day at 15° (A, B, E, F, I, J) or 25° (C, D, G, H, K, L) (Duerr et al. 2008). Staining in wild type (A, B, C, D), cha-1(cn101) (E, F, G, H), and cha-1(y226) (I, J, K, L). Somatic staining is similar in all strains. Arrows point to characteristic ChAT immunoreactivity in processes in the isthmus of the pharynx, clearly seen in wild type. This staining was fainter in the cha-1 mutants at 25°; arrows indicate where the isthmus could be seen in the original images. Arrowheads point to neuronal cell somas in the ventral ganglia that show punctate ChAT immunoreactivity. Anterior is left; dorsal up; NR, nerve ring; DNC, dorsal nerve cord; VNC, ventral nerve cord. Scale bar is 20 µm.

Figure 4

Behavioral analysis of cha-1 and other synaptic mutants. Synchronous cultures of all strains were grown at 20° and shifted to the desired assay temperature for 2 h before assay. Pharyngeal pumping (A, C, E, G) and swimming behavior (B, D, F, H) of cha-1 mutants demonstrate the behavioral effects of temperature. Pharyngeal pumping (I) and swimming behavior (J) of other synaptic mutants demonstrate increased rates at higher temperature. Pumping data represent the mean ± SD of 50 animals of each strain measured for 1 min each. Body bends were measured for 3 min for 10 animals of each strain, presented as the mean body bends/min ± SD. Statistical analysis utilized the Mann–Whitney U-test; for each behavioral test of each cha-1 strain (A–H), the 25° set of values was compared with either the 14° or the 20° data set (whichever had the higher mean value). For I and J, the 25° set of values was compared to the 14° data set. *P < 0.05; **P < 0.0005; ***P < 0.00005.

Figure 5

Reversibility of temperature effects. (A) Synchronized cultures of wild type (N2) and the indicated cha-1 mutants were grown at 20° until they were young adults. They were then transferred to 25° for 1 h, followed by alternating incubations at 14° and 25° for 1 h each. Before each transfer, pharyngeal pumping rate was measured for 10 worms for 1 min each. Two different experimental protocols were employed: (1) we started with 100–200 synchronized young adult worms on a plate, and at each time point, we picked 10 worms to assay, and those worms were then discarded (solid lines); and (2) we picked 10 synchronized young adults to separate plates at the start of the experiment and followed the same 10 worms through each time point (dashed lines). Each data point represents the mean (±SD) of 10 worms assayed for 1 min each. Note: one of the 10 md39 homozygotes died during the first 25° incubation, and another one died during the second 25° incubation. (B) Controls. Synchronized cultures of wild-type animals were grown at either 14° or 25° until they were young adults, and pharyngeal pumping rate was measured. Each data point represents the mean (±SD) of 50 worms assayed for 1 min each.

Figure 6

Phylogenetic Conservation of C. elegans ChAT Mutation Sites. (A) Sites of C. elegans cha-1 Missense Alleles: 145 different species were divided into four groups: the first group contained 56 other species from the genus Caenorhabditis; the second group (Other Nematodes) contained 39 representative species; the third group (non-nematode Invertebrates) included 32 species; and the fourth group (Chordates and Vertebrates) included 18 species (Supplementary File S2). Except for the group of Caenorhabditis species, the second, third, and fourth groups included representative species from diverse clades. The ChAT protein sequences within each species group were aligned with each other and with the C. elegans ChAT sequence. For each of the 12 sites corresponding to a cha-1 missense mutation (Table 1), we scored the number of species in each group containing the same amino acid at that site. If at least 85% (an arbitrary cutoff) of the species in a group matched the C. elegans amino acid, that amino acid was considered to be conserved. In the upper matrix , each cell represents a missense site evaluated in one of the four groups of species. The percent of species matching the C. elegans amino acid is shown, along with the fractional raw data count. Note that in some of the cells, the denominator of the fraction is less than the total number of species in the group—this is because some of the species have small assembly gaps within the ChAT genomic sequence. (B) Control: Conservation of Every 50th Amino Acid: A control set of 12 amino acids was chosen—every 50th position in the C. elegans ChAT sequence from #50 to #600. This set of amino acids was compared to the sets of aligned ChAT sequences already described, and the resulting matches are presented as above. For A and B, percentages >85% are shown in magenta. ^a The species in each group are listed in Supplementary File S2. ^b md88 and p1154 are independently isolated cha-1 mutations associated with exactly the same C>T transition mutation.

Figure 7

A thermal dissociation model. According to this model, in wild-type C. elegans cholinergic neurons, some of the ChAT protein is physically, but not covalently, associated with synaptic vesicles. The vesicle-associated ChAT would therefore produce ACh in the immediate neighborhood of the vesicular ACh transporter (A); this would provide an efficient mechanism for rapid filling of synaptic vesicles. Since the ChAT-vesicle association is not covalent, it seems likely that the soluble and vesicle-associated forms of ChAT would be in equilibrium, and presumably, the extent of physical association would depend on the temperature. Thus, as ambient temperature is increased from 14° to 25°, less of the ChAT protein would remain associated with vesicles (B). However, under normal conditions, wild-type C. elegans have at least a 20-fold excess of ACh production capacity (Rand and Russell 1984), so that if even half of the vesicle-associated ChAT were to diffuse away from the vesicle, there would be almost no measurable decrease in the rate of vesicle-filling. However, severe hypomorphic (loss-of-function) cha-1 mutants have only a few percent of wild-type ACh production capacity remaining (C) (Rand and Russell 1984); this may be due to a decrease in ChAT protein at synapses, or to altered catalytic properties of the remaining enzyme (or both), but in any event, even at 14°, the ability to synthesize ACh and load it into vesicles is greatly reduced, which severely impairs overall cholinergic function. In such mutants, increasing the temperature would promote dissociation of some ChAT protein from synaptic vesicles (D), thus reducing even further the amount of ACh synthesized in the immediate vicinity of the transport sites. This, in turn, would decrease the rate and extent of vesicle-filling, thereby increasing the severity of the cha-1 behavioral deficits.