This is the first report of synaptic vesicle recycling in an animal model that mimics the condition of nonmanifesting carriers of the ΔE-torsinA mutation (knock-in mice). FM dyes allowed us to distinguish general features of synaptic vesicle exocytosis and endocytosis from the features of processes that depend on the properties of endogenous neurotransmitters, for example, transport from the extracellular space to the cytosol, transport from the cytosol to the synaptic vesicle, and activation of postsynaptic receptors. Capitalizing on the usefulness of FM dyes, we have evaluated the presynaptic effects of ΔE-torsinA. Our results demonstrate (summarized in Fig. 5 B) that a single copy of ΔE-torsinA causes an increase in exocytosis without an accompanying change in TRP size. We also found that a single copy of ΔE-torsinA accelerates exocytosis following cellular exposure to high activity. These enhancements of synaptic vesicle exocytosis in heterozygous neurons suggest that wild-type torsinA inhibits synaptic transmission through a presynaptic mechanism, and that ΔE-torsinA relieves this brake, especially after high activity due to strong stimulation. It is of note that homozygous neurons exhibited not only an increase in exocytosis, but also a reduction in TRP size that was not detectable in heterozygous neurons. This raises the possibility that torsinA has more than one site of action in synaptic vesicle recycling.
Synaptic vesicle recycling in ΔE-torsinA knock-in mice
Prior to this study, Warner and coworkers first identified defective synaptic vesicle recycling in cell models of DYT1 dystonia, triggering this study to explore further the presynaptic abnormalities. However, the results from the previous study were different from ours. For example, it was claimed that the overexpression of ΔE-torsinA in neuroblastoma cells increased the uptake of FM dye (FM1-43), suggesting that vesicular endocytosis was enhanced (Granata et al.,2008). This contradiction from our results seems to have arisen from the complexity of synaptic vesicle recycling, in addition to the differences in cellular preparations (neuroblastoma vs. hippocampal neurons) and in expression levels of torsinA (overexpression vs. endogenous level). FM dyes can be loaded into not only recycling synaptic vesicles, but also intracellular compartments that do not allow immediate rerelease, and this leads to residual FM staining even after extensive stimulation (Granata et al.,2008). Given that this residual fraction does not represent the recycling vesicles, it should be subtracted from the FM signal, as in our analysis of ΔFM. However, collectively, our results and those from Warner and coworkers indicated the presence of abnormal presynaptic features in models of DYT1 dystonia.
The main, novel features of our findings are common to both heterozygous and homozygous ΔE-torsinA neurons. Specifically: (1) exocytosis in response to a single round of electrical stimulation involves a higher percentage of TRP of vesicles (Fig. 3 A) and (2) this release is accelerated when the neurons are stimulated prior to destaining (i.e., high activity staining method; Figs. 3 B and 5 B). These enhancements in exocytosis demonstrate that ΔE-torsinA induces fundamental changes in synaptic-vesicle recycling. These enhancements are expected to correlate with increased neurotransmitter release during high-frequency firing of action potentials, especially in heterozygous neurons as they released higher amount of FM dye (ΔFM1 round; Fig. 2 B). Thus, these enhancements are expected to have a strong impact on the efficiency of synaptic transmission.
An intriguing finding in the knock-in mice was that prior stimulation of the neurons (high activity staining method) accelerated FM destaining (Fig. 3 Bc, d). The fact that this phenotype was not evident with the basal activity staining method (Fig. 3 Ba, b) indicates that high activity can serve as a key trigger for the neuronal phenotype. The mechanism that leads to accelerated activity is expected to influence that controlling the intracellular Ca2+ concentration, as there was no difference in the sensitivity to ionomycin (Fig. 4). One possible explanation for this acceleration is that synaptic plasticity was induced by high activity staining method. A similar acceleration of FM destaining in nerve terminals was noted during long-term potentiation of synaptic transmission in the hippocampus of wild-type rodents (Zakharenko et al.,2001). Interestingly, the acceleration was observed only in forms of long-term potentiation whose induction relied on voltage-gated Ca2+ channels (L-type) (Zakharenko et al.,2001). The possibility that ΔE-torsinA might influence synaptic plasticity is consistent with the observation that the basal ganglia of transgenic mice expressing this protein exhibit enhanced long-term potentiation (Martella et al.,2009) and modified voltage-gated Ca2+ channel (N-type) activity (Pisani et al.,2006; Sciamanna et al.,2011). Another possible explanation for the apparent acceleration in exocytosis is that the two staining methods led to labeling of different pools of vesicles; this would be independent of synaptic plasticity, and consistent with the notion that spontaneous and evoked release in hippocampal neurons involves separate vesicle pools (Chung et al.,2010). However, it should be noted that the concept of such different vesicle pools remains controversial, because contradictory results have also been reported (Hua et al.,2010).
Mice homozygous for ΔE-torsinA die within 2–3 days after birth, whereas heterozygous mice are indistinguishable from wild-type mice (Goodchild et al.,2005). In this study, the synaptic phenotypes of the cultured homozygous neurons did not differ drastically from those of their heterozygous counterparts. These findings are consistent with the notion that the lethality in homozygous knock-in mice is associated with the reported abnormalities in the nuclear membranes of these neurons (Yokoi et al.,2011). However, it is also possible that the lethality is due to more severe functional abnormalities in other areas of the central nervous system, such as the spinal cord.
Implications for DYT1 dystonia
The ΔE-torsinA knock-in mice used in this study do not show overt motor symptoms, but do show abnormalities in the circuitry of and metabolic activity in the brain. As such, the phenotype resembles that in human, nonmanifesting, mutation carriers of DYT1 dystonia (Ulug et al.,2011). This is in sharp contrast to the phenotype of ΔE-torsinA transgenic mice that demonstrate motor symptoms (e.g., Grundmann et al.,2007; Shashidharan et al.,2005). Therefore, our results have implications for the pathophysiology of nonmanifesting DYT1 dystonia, with the caveat that the hippocampus may not be the main site affected in manifesting DYT1 dystonia (see the last paragraph).
First, the presence of synaptic phenotypes indicates that synaptic abnormalities are prevalent among carriers of the mutation, and this may underlie some of the endophenotypes (i.e., subclinical markers) for nonmanifesting human carriers. These include a number of deficits, for example, in electrophysiological responses (Edwards et al.,2003b), temporal processing of sensory stimuli (Fiorio et al.,2007), motor sequence learning (Carbon et al.,2011; Ghilardi et al.,2003), and sensorimotor cortical activity (Carbon et al.,2010), as well as an increase in susceptibility to recurrent major depression (Heiman et al.,2004).
Second, our results demonstrate that strong neuronal activity can alter the synaptic phenotype, and therefore may serve as a “second hit” in ΔE-torsinA neurons. The low penetrance and phenotypic variability of DYT1 dystonia imply that genetic polymorphisms (Kock et al.,2006; Risch et al.,2007) or environmental factors (Edwards et al.,2003a; Gioltzoglou et al.,2006) play important roles in the pathogenesis of DYT1 dystonia, as is the case for other CNS disorders such as schizophrenia (Ayhan et al.,2009). Our finding of activity-induced change may reflect one cellular mechanism whereby environmental insults contribute to the emergence of symptoms. The details of this mechanism are unclear at present, and it remains to be determined which other cellular responses (e.g., inflammation and ischemia) can serve as environmental insults and modifiers of neuronal phenotypes. Our experimental system represents a useful tool for dissecting the gene-environment interactions that influence dystonia.
Traditionally, the striatum (mostly the putamen) has been considered the primary site of dysfunction in dystonia. More recently, dystonia was hypothesized to be a network disorder that involves at least the cerebellum, thalamus, striatum, and cerebral cortex (Neychev et al.,2011). How torsinA or other dystonia-causing proteins alter this circuit remains unknown. We acknowledge that the hippocampus is unlikely to be the primary trigger of the pathogenic process. Nevertheless, there are several reasons to study synaptic physiology in this particular region of the brain. First, the hippocampus is a well-characterized experimental system that is widely used to explore synaptic physiology (e.g., Ruiz et al.,2010; Terashima et al.,2008; Zhou et al.,2000) and the effects of defective proteins (e.g., Deak et al.,2004; Feng et al.,2010). Second, the fact that hippocampal neurons express torsinA at high levels (Allen_Mouse_Brain_Atlas,2009; Augood et al.,1999; Walker et al.,2001) and upregulate its expression further as a consequence of ischemia (Zhao et al.,2008) suggests that torsinA plays a functional role in the context of neuronal stress in this region. Third, hippocampal cells also express other proteins, such as ε-sarcoglycan, associated with other forms of dystonia (Ritz et al.,2011). Fourth, the hippocampus is one of the highly activated sites after the systemic administration of L-type Ca2+ channel activators triggered dystonia in rodents (Jinnah et al.,2003). Thus, expanding studies of dystonia into this well-established mammalian neuronal system represents a step toward better understanding the roles of torsinA in mammalian synapses. However, it will fall to future studies to address whether this is a universal phenomenon or restricted to specific neuronal subtypes.