ABSTRACT
An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.
Subject(s)
Brain/metabolism , Neuromuscular Junction Diseases/etiology , Neuromuscular Junction/metabolism , Recognition, Psychology/physiology , Vesicular Acetylcholine Transport Proteins/deficiency , Acetylcholine/analysis , Acetylcholine/metabolism , Animals , Blotting, Northern , Blotting, Southern , Brain/pathology , Brain/physiopathology , Brain Chemistry , Chromatography, High Pressure Liquid , Female , Male , Membrane Potentials/physiology , Mice , Mice, Transgenic , Microdialysis , Motor Activity/physiology , Neuromuscular Junction/pathology , Neuromuscular Junction/physiopathology , Neuromuscular Junction Diseases/pathology , Neuromuscular Junction Diseases/physiopathology , Polymerase Chain Reaction , RNA, Messenger/analysis , Synaptic Transmission/physiology , Vesicular Acetylcholine Transport Proteins/geneticsABSTRACT
We isolated from a brain library a cDNA encoding an isoform of rat CED-6 that has not been previously described. This transcript results from alternative splicing of the ced-6 gene present on chromosome 9. We expressed this isoform as his-tagged protein in E. coli and used the purified protein to raise antibodies to investigate the expression of CED-6 in rat brain. Immunoblot analysis showed the presence of CED-6 as a doublet of approximately 34 and 33 kDa in cortex, hippocampus and cerebellum, indicating that the protein was present in different regions of the brain. Subcellular fractionation experiments showed that CED-6 immunoreactivity did not concentrate in GFAP-containing glial vesicles, whereas it showed a distribution similar to the synaptotagmin in synaptosomes-enriched fractions, suggesting that CED-6 is present in neurons. CED-6 immunoreactivity was also investigated using immunohistochemistry analysis and it was found in several brain regions, being particularly strong in the cell body of some groups of neurons such as Purkinje cell layer of cerebellum, and pyramidal cells of the hippocampal formation and also in epithelial cells from the choroid plexus. Importantly, CED-6 immunoreactivity colocalized with a neuronal marker but not with a glial marker. Considering that several PTB-containing proteins bind clathrin, we investigated whether rat CED-6 would also have this property. Yeast two-hybrid and GST pull-down analysis indicated that ratCED-6 interacts with clathrin and in cultured cells we detected colocalization between CED-6 and clathrin-coated vesicles. The present findings suggest that CED-6 may have a role in endocytic trafficking or signaling in neurons.
Subject(s)
Brain/metabolism , Clathrin/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Phosphoproteins/metabolism , Presynaptic Terminals/metabolism , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Base Sequence , Brain/anatomy & histology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Line, Transformed , Choroid Plexus/metabolism , Choroid Plexus/ultrastructure , Clathrin-Coated Vesicles , Endocytosis/physiology , Humans , Mice , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Neurons/cytology , Phosphoproteins/genetics , Phosphoproteins/isolation & purification , Presynaptic Terminals/ultrastructure , Protein Transport/physiology , Rabbits , Rats , Synaptic Transmission/physiology , Synaptosomes/metabolism , Synaptosomes/ultrastructureABSTRACT
The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site-directed mutagenesis to evaluate the contribution of the C-terminal tail of VAChT to the targeting of this transporter to synaptic-like microvesicles in cholinergic SN56 cells. We found that residues 481-490 contain the trafficking information necessary for VAChT localization and that within this region L485 and L486 are strictly necessary. Deletion and alanine-scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481-490, were still targeted to microvesicles. Moreover, we found that clathrin-mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles.
