title: Dystonia-associated protein torsinA is not detectable at the nerve terminals of central neurons.
abstract: Presynaptic functions of the mammalian central neurons are regulated by a network of protein interactions. Synaptic vesicle recycling in and neurotransmitter release from the presynaptic nerve terminals are altered when a glutamate-deleting mutation is present in the torsinA protein (ΔE-torsinA). This mutation is linked with a hereditary form of the movement disorder dystonia known as DYT1 dystonia. Although torsinA expression is prevalent throughout the central nervous system, its subcellular localization - in particular with respect to presynaptic nerve terminals - remains unclear. This information would be useful in narrowing down possible models for how wild-type torsinA affects presynaptic function, as well as the nature of the presynaptic dysfunction that arises in the context of ΔE-torsinA mutation. Here we report on an analysis of the presynaptic localization of torsinA in cultured neurons obtained from a knock-in mouse model of DYT1 dystonia. Primary cultures of neurons were established from heterozygous and homozygous ΔE-torsinA knock-in mice, as well as from their wild-type littermates. Neurons were obtained from the striatum, cerebral cortex and hippocampus of these mice, and were subjected to immunocytochemistry. This analysis revealed the expression of both proteins in the somata and dendrites. However, neither the nerve terminals nor axonal shafts were immunoreactive. These results were confirmed by fluorogram-based quantitation. Our findings indicate that neither the wild-type nor the ΔE-torsinA mutant protein is present at substantial levels in the presynaptic structures of cultured neurons. Thus, the effects of torsinA, in wild-type and mutant forms, appear to influence presynaptic function indirectly, without residing in presynaptic structures.
authors.affiliation: Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, Iowa City, IA, USA.
authors.forename: J-Y
authors.initials: JY
authors.lastname: Koh
authors.forename: S
authors.initials: S
authors.lastname: Iwabuchi
authors.forename: N C
authors.initials: NC
authors.lastname: Harata
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D019256:Cadmium Chloride / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018832:Molecular Chaperones / Q000378:metabolism*; D009411:Nerve Endings / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D020867:omega-Agatoxin IVA / Q000494:pharmacology; D020866:omega-Conotoxin GVIA / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; C491548:Dyt1 protein, mouse; D018832:Molecular Chaperones; D020867:omega-Agatoxin IVA; D020866:omega-Conotoxin GVIA; D019256:Cadmium Chloride; D011188:Potassium
keywords: DIC; FBS; GAD65; HEPES; LED; MEM; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; NF; PBS; Syp; VGLUT1; central neurons; confocal and widefield fluorescence microscopy; deletion of a single GAG codon in Tor1A gene; differential interference contrast; dystonia; fetal bovine serum; glutamic acid decarboxylase 65kDa; immunocytochemistry; light-emitting diode; mEPSCs; mIPSCs; miniature excitatory postsynaptic currents; miniature inhibitory postsynaptic currents; minimum essential media; nerve terminals; neurofilament; phosphate-buffered saline; synaptophysin; torsinA; vesicular glutamate transporter 1; ΔE