Choline transporter 1 maintains cholinergic function in choline acetyltransferase haploinsufficiency

Abstract

Choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine (ACh), is thought to be present in kinetic excess in cholinergic neurons. The rate-limiting factor in ACh production is the provision of choline to ChAT. Cholinergic neurons are relatively unique in their expression of the choline transporter 1 (CHT1), which exhibits high-affinity for choline and catalyzes its uptake from the extracellular space to the neuron. Multiple lines of evidence indicate that the activity of CHT1 is a key determinant of choline supply for ACh synthesis. We examined the interaction of ChAT and ChT activity using mice heterozygous for a null mutation in the Chat gene (Chat+/-). In these mice, brain ChAT activity was reduced by 40-50% relative to the wild type, but brain ACh levels as well as ACh content and depolarization-evoked ACh release in hippocampal slices were normal. However, the amount of choline taken up by CHT1 and ACh synthesized de novo from choline transported by CHT1 in hippocampal slices, as well as levels of CHT1 mRNA in the septum and CHT1 protein in several regions of the CNS, were 50-100% higher in Chat+/- than in Chat+/+ mice. Thus, haploinsufficiency of ChAT leads to an increased expression of CHT1. Increased ChT activity may compensate for the reduced ChAT activity in Chat+/- mice, contributing to the maintenance of apparently normal cholinergic function as reflected by normal performance of these mice in several behavioral assays.

Figure 1.

ChAT and AChE activity in Chat heterozygotes. a, ChAT activity is reduced in Chat+/- brain regions. In each of several regions of the brain (FCTX, frontal cortex; PCTX, parietal cortex; SPT, septum; STR, striatum; OB, olfactory bulb; HPC, hippocampus), ChAT activity (nmol/mg of protein/min) was reduced by almost half (FCTX, 41%; PCTX, 36%; HPC, 40%; SPT, 49%; STR, 42%; OB, 43%). Error bars represent SEM. Significant differences were observed between Chat+/+ mice and Chat+/- mice in all brain regions examined (FCTX, p < 0.0001; PCTX, p < 0.0005; HPC, p < 0.001; SPT, p < 0.001; STR, p < 0.005; OB, p < 0.0001). b, AChE activity in various brain regions of Chat+/+ and Chat+/- mice. No significant effect of genotype was found in any brain region.

Figure 2.

Behavioral analyses revealed no defects in Chat heterozygotes. a-d, Chat+/+ (closed circles) and Chat+/- (open squares) mice were tested in the activity chamber (a), the accelerating rotarod (b), either learning or relearning curves in the training phase of the water maze (c; arrow represents platform reversal to opposite quadrant), and recall in the probe trial of the water maze (d; time spent in each quadrant from the trial 4 hr after training on day 8 is shown). OPP, Opposite quadrant; AR, adjacent right quadrant; TARGET, correct quadrant; AL, adjacent left quadrant. Error bars represent SEM. ANOVA revealed no significant main effects of genotype in any of the behavioral tests, including the activity chamber (F_(1,25) = 0.03; p = 0.87), accelerating rotarod (F_(1,26) < 0.01; p = 0.99), learning and relearning in the training phase of the water maze (F_(1,32) < 0.05, p = 0.94 for learning; F_(1,32) = 0.27, p = 0.61 for relearning), or probe trial of the water maze (F_(1,32) = 0.41; p = 0.53) experiments shown here.

Figure 3.

Normal levels of ACh in Chat heterozygotes. a, Tissue ACh content in the striatum, hippocampus, and cortex of Chat+/+ and Chat+/- mice. Two separate methods were used to assay ACh content using HPLC as described in Materials and Methods. In both experiments, there were no significant differences between the two groups of mice in any of the brain regions assayed. STR, Striatum; FCTX, frontal cortex; HPC, hippocampus; CTX, cortex. b, c, Potassium-evoked ACh and choline release in Chat+/+ and Chat+/- mice. Hippocampal slices were incubated in each solution for 5 min, and ACh (b) and choline (c) levels in the medium were measured using HPLC as described in Materials and Methods. No significant differences in ACh or choline release were observed between the groups.

Figure 4.

Increased ChT activity, mRNA, and protein in Chat heterozygotes. a, [³H]Choline uptake was measured in the presence and absence of HC-3, an inhibitor of ChT activity, as described in Materials and Methods. The amount of ChT-transported choline was calculated by subtracting the amount of intracellular [³H]choline in the presence of HC-3 from the amount of [³H]choline in the absence of HC-3. The [³H]choline constituted a higher fraction of the HC-3-sensitive choline level in the Chat+/- mice compared with the wild-type mice (p < 0.05). ACh synthesized from [³H]choline was also measured in the presence and absence of HC-3. The amount of ACh synthesized from ChT-transported choline was calculated by subtracting the amount of ACh synthesized in the presence of HC-3 from the amount of ACh produced in the absence of HC-3. The newly made ACh constituted a higher fraction of the HC-3-sensitive ACh pool in the Chat+/- mice than it did in the wild-type littermates (p < 0.05). b, RT-PCR of CHT1 mRNA in Chat+/+ and Chat+/- mice. Septal RNA was used for RT-PCR of CHT1 and GAPDH. As expected, the PCR product from the amplification of CHT1 was present as an 840 bp band. The PCR product from the amplification of GAPDH was present as a 983 bp band. c, CHT1 product levels were quantified by Kodak Image Station Software and normalized to GAPDH product levels. The results are presented as a percentage of wild-type control ± range. d, CHT1 protein levels in Chat+/+ and Chat+/- mice. CHT1 protein levels in several CNS structures were measured by Western blot analysis. Chat+/- mice have an approximately twofold increase in CHT1 protein levels in each of the brain regions compared with Chat+/+ mice. e, CHT1 levels were quantified by Kodak Image Station Software and are presented as a percentage of wild-type control ± range. STR, Striatum; CTX, cerebral cortex; HPC, hippocampus; SC, spinal cord.