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Abstract
Cholinergic neurotransmission supports motor, autonomic, and cognitive function and is compromised in myasthenias, cardiovascular diseases, and neurodegenerative disorders. Presynaptic uptake of choline via the sodium-dependent, hemicholinium-3-sensitive choline transporter (CHT) is believed to sustain acetylcholine (ACh) synthesis and release. Analysis of this hypothesis in vivo is limited in mammals because of the toxicity of CHT antagonists and the early postnatal lethality of CHT-/- mice (Ferguson et al., 2004). In Caenorhabditis elegans, in which cholinergic signaling supports motor activity and mutant alleles impacting ACh secretion and response can be propagated, we investigated the contribution of CHT (CHO-1) to facets of cholinergic neurobiology. Using the cho-1 promoter to drive expression of a translational, green fluorescent protein-CHO-1 fusion (CHO-1:GFP) in wild-type and kinesin (unc-104) mutant backgrounds, we establish in the living nematode that the transporter localizes to cholinergic synapses, and likely traffics on synaptic vesicles. Using embryonic primary cultures, we demonstrate that CHO-1 mediates hemicholinium-3-sensitive, high-affinity choline uptake that can be enhanced with depolarization in a Ca(2+)-dependent manner supporting ACh synthesis. Although homozygous cho-1 null mutants are viable, they possess 40% less ACh than wild-type animals and display stress-dependent defects in motor activity. In a choline-free liquid environment, cho-1 mutants demonstrate premature paralysis relative to wild-type animals. Our findings establish a requirement for presynaptic choline transport activity in vivo in a model amenable to a genetic dissection of CHO-1 regulation.
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Figures
Figure 1.
CHO-1:GFP localization in vivo and…
Figure 1.
CHO-1:GFP localization in vivo and its UNC-104-dependent trafficking to cholinergic synapses. A ,…
Figure 1. CHO-1:GFP localization in vivo and its UNC-104-dependent trafficking to cholinergic synapses. A, A CHO-1:GFP fusion protein localizes to punctate regions of cholinergic nerve processes and colabels with VAMP:mRFP1. Top panel, Transgenic C. elegans expressing a CHO-1:GFP fusion protein under the control of the endogenous cho-1 promoter in a wild-type background (BY503). The arrows indicate en passant synapses of a cholinergic sublateral neuron. Bottom panels, Transgenic animals coexpressing CHO-1:GFP (BY503) and VAMP:mRFP1 (BY508) in a cholinergic sublateral neuron. Scale bars, 5 μm. B, CHO-1:GFP traffics on synaptic vesicles. Shown are head neurons expressing the CHO-1:GFP fusion protein in the wild-type (BY503) and unc-104 (vtIs16; unc-104(e1265)) mutant backgrounds. The arrows indicate normal synaptic localization in the wild-type background and the loss of synaptic localization in the unc-104 mutant background. The asterisks indicate where the fusion protein is trapped in the cell bodies of these neurons.
Figure 2.
Characterization of high-affinity choline uptake…
Figure 2.
Characterization of high-affinity choline uptake in C. elegans primary cultures. A , Primary…
Figure 2. Characterization of high-affinity choline uptake in C. elegans primary cultures. A, Primary cultures prepared from animals expressing the CHO-1:GFP fusion protein (BY503). The arrows indicate the ends of neurite projections contacting adjacent cells. DIC, Differential interference contrast. Scale bars, 10 μm. B, Saturation kinetics for primary cultures derived from BY503 (filled triangles) and WT (filled squares) animals. Overexpression of CHO-1:GFP in wild-type cells increased the Vmax of [3H]choline transport (BY503, 3.73 ± 0.180 pmol · mg protein−1 · min−1; WT, 1.69 ± 0.049 pmol · mg protein−1 · min−1) with no change in apparent Km (BY503, 1.083 ± 0.22 μm choline; WT, 1.014 ± 0.12 μm choline) relative to nonexpressing controls. [3H]Choline uptake from cho-1 knock-out cultures was defined as nonspecific uptake. Data are from two independent experiments performed in triplicate and are plotted as mean ± SEM. C–F, [3H]Choline uptake monitored from primary cultures prepared from WT and cho-1 mutant animals. C, Saturation kinetics of [3H]choline uptake in WT cultures. Nonspecific binding was defined by 1 μm HC-3 (mean ± SEM; n = 3 in triplicate). D, Normal [3H]choline uptake is Na+ and Cl− dependent. Uptake measured from WT (black) and cho-1 knock-out (gray) cultures in the presence of Na+ and Cl− (standard), the absence of Na+ (+NMDG), and the absence of Cl− (+Na-gluconate). Nonspecific binding was defined by uptake at 4°C (mean ± SEM; n = 3 in triplicate). E, High-affinity [3H]choline uptake is lost in cho-1 knock-out cultures. [3H]Choline uptake was measured in WT (black) and cho-1 mutant (gray) cultures in the presence and absence of 1 μm HC-3. Data are expressed as percent WT control (mean ± SEM; n = 3 in triplicate), and nonspecific binding was determined by uptake performed at 4°C. Statistical analysis was performed using a Student’s t test; asterisks indicate statistical significance as described in text. F, High-affinity [3H]choline uptake is inhibited by HC-3. [3H]Choline uptake was measured in WT (filled squares) and cho-1 knock-out (filled triangles) cultures in the presence of increasing concentrations of HC-3. Data are plotted as mean ± SEM of two experiments performed in triplicate, and nonspecific uptake is defined by uptake at 4°C.
Figure 3.
High-affinity choline uptake is enhanced…
Figure 3.
High-affinity choline uptake is enhanced with activity in C. elegans primary cultures. A …
Figure 3. High-affinity choline uptake is enhanced with activity in C. elegans primary cultures. A, [3H]Choline uptake is enhanced with depolarization and requires active calcium channels. Uptake was measured from WT cultures under normal conditions (Control), and under depolarizing conditions (+30 mm KCl). This activity-dependent enhancement of [3H]choline uptake was abolished when voltage-gated calcium channels were blocked with 100 μm CdCl2. Data shown are from a representative experiment performed in triplicate, plotting mean + SEM as percent control (n = 3). Nonspecific uptake was defined by uptake in the presence of 2 μm HC-3. B, Activity-dependent enhancement of [3H]choline uptake is persistent. WT cultures were briefly depolarized with 30 mm KCl, and then assayed immediately for [3H]choline uptake (Initial) or placed into standard uptake buffer for 5, 10, or 30 min before measuring uptake [shown as 5, 10, and 30′ post dep(olarization)]. Data are shown as percentage control (control treated identically in parallel with “Initial” cells but without depolarization) and are mean ± SEM of a representative experiment performed in triplicate (n = 2). Uptake at 4°C was subtracted from these data sets as nonspecific uptake. Asterisks designate statistical significance as described in text.
Figure 4.
Effects of a loss of…
Figure 4.
Effects of a loss of high-affinity choline transport on ACh synthesis in vitro …
Figure 4. Effects of a loss of high-affinity choline transport on ACh synthesis in vitro. Results of a combined pulse-labeling/neurochemistry approach reveal that CHO-1 is the only source of high-affinity transport supporting ACh synthesis in primary cultures. A, Total ACh content of WT (black) and cho-1 knock-out (KO) (gray) cultures at steady state as assayed by HPLC. B, Total [3H]choline uptake in WT (black) and cho-1 mutant (gray) cultures over 10, 20, and 40 min periods assayed. Total uptake (mean ± SEM) were as follows: WT, 0.955 ± 0.009 pmol at 10 min, 2.288 ± 0.013 pmol at 20 min, and 3.440 ± 0.002 at 40 min; cho-1 knock-out, 0.053 ± 0.003 pmol at 10 min, 0.208 ± 0.030 pmol at 20 min, and 0.284 ± 0.017 pmol at 40 min. ANOVA comparison of means, p < 0.0001. Uptake was performed with 100 nm [3H]choline, and nonspecific uptake is defined by uptake at 4°C. C, Incorporation of [3H]choline into ACh. WT (black) and cho-1 knock-out (gray) cultures were incubated with 100 nm [3H]choline for 10, 20, or 40 min, and then processed for neurochemistry. Peak fractions for ACh were collected and analyzed by liquid scintillation spectrometry to analyze the conversion of [3H]choline into [3H]ACh. For all panels, shown are mean ± SEM, n = 3 in triplicate; asterisks indicate statistical significance as described in the text.
Figure 5.
The low-affinity system supports ACh…
Figure 5.
The low-affinity system supports ACh synthesis only at high substrate concentrations. Results of…
Figure 5. The low-affinity system supports ACh synthesis only at high substrate concentrations. Results of a similar pulse-labeling approach as described in Figure 3, except performed with increasing concentrations of [14C]choline. A, Total ACh content in WT (black) and cho-1 mutant (gray) cultures as detected by HPLC. ANOVA comparison of the means: p = 0.0001 (WT vs cho-1 mutant); p > 0.05 (cho-1−/− cells comparing the four choline concentrations). B, Total [14C]choline uptake in WT (black) and cho-1 mutant (gray) cultures, expressed as percentage of WT control. Uptake was performed in parallel for 10 min at 0.1, 10, 50, and 100 μm [14C]choline, and nonspecific binding was defined by uptake at 4°C. C, Incorporation of [14C]choline into ACh. Conversion of [14C]choline into [14C]ACh was determined as described above (Fig. 3) for WT (black) and cho-1 mutant (gray) cultures. Data are shown as the percentage of total ACh that is [14C]ACh. For all panels, shown are mean ± SEM, n = 3 in triplicate; asterisks indicate statistical significance as described in the text.
Figure 6.
Phospholipase D contribution to ACh…
Figure 6.
Phospholipase D contribution to ACh synthesis, ChAT activity in the absence of CHO-1,…
Figure 6. Phospholipase D contribution to ACh synthesis, ChAT activity in the absence of CHO-1, and high-affinity uptake in ChAT knock-out (KO) cultures. A, [3H]Choline uptake in WT (black), cho-1 mutant (dark gray), and cho-1;pld-1 double-mutant (light gray) cultures. Data are plotted as percentage of WT control. *p < 0.0001; ns, no significant difference (p > 0.05); t test. B, Total ACh concentrations in WT (black), cho-1 mutant (dark gray), and cho-1;pld-1 double-mutant (light gray) cells as determined by HPLC analysis. *p < 0.01; ns, no significant difference (p > 0.05); t test. C, ChAT activity in WT (black) and cho-1 mutant (gray) cultures. Data are expressed as percentage of WT control. For A–C, n = 2 in triplicate. D, Total [3H]choline uptake in WT (black), cho-1 mutant (dark gray), and ChAT mutant (cha-1(cn101); light gray) cultures. Data are expressed as percentage of WT control (n = 3 in triplicate). For all panels, shown are mean ± SEM; uptake was performed using 0.1 μm [3H]choline.
Figure 7.
Effects of loss of cho-1 …
Figure 7.
Effects of loss of cho-1 activity on total choline and ACh stores. A …
Figure 7. Effects of loss of cho-1 activity on total choline and ACh stores. A, Total choline extracted from adult worms and detected by HPLC. No significant difference in choline levels was detected between WT animals, cho-1 mutants, and cho-1 mutants expressing a CHO-1:GFP fusion protein in the cholinergic nervous system (BY506) (ANOVA, p > 0.05). Results are plotted as mean ± SEM from samples performed in triplicate (n = 4). B, Total ACh in adult worms as detected by HPLC. cho-1 mutants animals had 36% less total ACh relative to WT animals (ANOVA test, p < 0.01; mean ± SEM from samples performed in triplicate; n = 4). Normal ACh levels were restored in BY506 animals (ANOVA test, WT vs BY506, p > 0.05; mean ± SEM). C, cho-1 mutants are visibly impaired in locomotory activity. Adult cho-1 mutant animals placed in a choline-free liquid medium demonstrated a 16% reduction in thrashing behavior relative to WT animals, as defined by the number of body bends per minute (WT, n = 32; cho-1 mutant, n = 36). This thrashing defect was rescued in BY506 animals (n = 32). D, The percentage difference in total ACh between WT and cho-1 mutant animals does not increase when animals are subjected to sustained physical stress. Shown is the total ACh extracted from adult worms and detected by HPLC after allowing animals to thrash for 2 h in a choline-free liquid environment (mean ± SEM; n = 3 in triplicate). KO, Knock-out. Asterisks indicate statistical significance as described in the text.
Figure 8.
Characterization of cho-1 mutant motor…
Figure 8.
Characterization of cho-1 mutant motor activity. A , B , Group analysis demonstrates…
Figure 8. Characterization of cho-1 mutant motor activity. A, B, Group analysis demonstrates an early fatigue phenotype of cho-1 mutants that is influenced by exogenous choline. Shown are representative analyses of 15 worms per genotype (WT, red; cho-1 mutants, blue) in a standard liquid thrashing assay, monitored visually. Plotted are the percentage of total animals immobile per genotype at each minute of a 120 min assay. A, WT and cho-1 mutant animals analyzed after growth on thick lawns of NA22 bacteria. B, WT and cho-1 mutant animals analyzed after growth on sparse lawns of HB101 bacteria. This graph is representative of an extreme example of this phenotype. C, Representative graphs of single-worm automated assays showing animal movement in a choline-free liquid medium over the course of 2 h. Shown are movement traces for WT (top panel) and cho-1 knock-out (bottom panel) animals. Plotted are FFTs of movement frequencies (in hertz) per 6 s window, for a total of 30 min per segment. Shown are 2 h assays broken down into 30 min segments. Periods of inactivity (or fatigue) are shown as a flatline at 0 Hz; the red line is provided as a visual guide to this drop in activity. D, Graph summarizing the amount of time spent immobile (or fatigued) by WT (black) and cho-1 mutant (gray) animals. The data analyzed correspond to the 30 min windows depicted in A but are the combined data of multiple recordings (n = 17 for WT; n = 23 for cho-1 mutants). cho-1 mutant animals displayed a significant increase in fatigue throughout the 2 h assay, with a marked increase in inactivity during the first 30 min relative to WT animals. Error bars indicate SEM; asterisks indicate statitsical significance as described in the text. All figures (8) See this image and copyright information in PMC
References
Apparsundaram S, Ferguson SM, Blakely RD (2001). Molecular cloning and characterization of a murine hemicholinium-3-sensitive choline transporter. Biochem Soc Trans 29:711–716. - PubMed
Aquilonius SM, Schuberth J, Sundwall A (1970). Studies on choline in cerebrospinal fluid. Acta Pharmacol Toxicol (Copenh) 28:35. - PubMed
Barker LA, Mittag TW (1975). Comparative studies of substrates and inhibitors of choline transport and choline acetyltransferase. J Pharmacol Exp Ther 192:86–94. - PubMed
Barker LA, Dowdall MJ, Mittag TW (1975). Comparative studies on synaptosomes: high-affinity uptake and acetylation of N-[Me-3H]choline and N-[Me-3H]N-hydroxyethylpyrrolidinium. Brain Res 86:343–348. - PubMed
Bauerfeind R, Regnier-Vigouroux A, Flatmark T, Huttner WB (1993). Selective storage of acetylcholine, but not catecholamines, in neuroendocrine synaptic-like microvesicles of early endosomal origin. Neuron 11:105–121. - PubMed
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