Dystonia-associated protein torsinA is not detectable at the nerve terminals of central neurons.

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.

Imaging single synaptic vesicles undergoing repeated fusion events: kissing, running, and kissing again.

At synapses of the mammalian central nervous system, release of neurotransmitter occurs at rates transiently as high as 100 Hz, putting extreme demands on nerve terminals with only tens of functional vesicles at their disposal. Thus, the presynaptic vesicle cycle is particularly critical to maintain neurotransmission. To understand vesicle cycling at the most fundamental level, we studied single vesicles undergoing exo/endocytosis and tracked the fate of newly retrieved vesicles. This was accomplished by minimally stimulating boutons in the presence of the membrane-fluorescent styryl dye FM1-43, then selecting for terminals that contained only one dye-filled vesicle. We then observed the kinetics of dye release during single action potential stimulation. We found that most vesicles lost only a portion of their total dye during a single fusion event, but were able to fuse again soon thereafter. We interpret this as direct evidence of "kiss-and-run" followed by rapid reuse. Other interpretations such as "partial loading" and "endosomal splitting" were largely excluded on the basis of multiple lines of evidence. Our data placed an upper bound of <1.4 s on the lifetime of the kiss-and-run fusion event, based on the assumption that aqueous departitioning is rate limiting. The repeated use of individual vesicles held over a range of stimulus frequencies up to 30 Hz and was associated with neurotransmitter release. A small percentage of fusion events did release a whole vesicle's worth of dye in one action potential, consistent with a classical picture of exocytosis as fusion followed by complete collapse or at least very slow retrieval.