Synaptotagmin: Is 2 Better than 1?

The synaptotagmin protein family, characterized by two C-terminal calcium-binding motifs, is expressed throughout the brain (Grise et al., 2007). Synaptotagmin-1, the best characterized member, acts as a calcium sensor in the Ca²⁺-dependent release of neurotransmitter vesicles (Geppert et al., 1994) and is necessary for calcium-dependent exocytosis in invertebrates (Littleton et al., 1993). However, knock-out studies in mice demonstrated that synaptotagmin-1 was necessary for one type of Ca²⁺-dependent exocytosis (fast, synchronous release), but not for another type (asynchronous release) (Geppert et al., 1994). Of the synaptotagmin family, synaptotagmin-2 exhibits the highest degree of homology with synaptotagmin-1. Indeed, synaptotagmin-2 can rescue synaptotagmin-1 deficiency, and its expression overlaps partially but not completely with synaptotagmin-1 (Geppert et al., 1994; Nagy et al., 2006).

In a recent Journal of Neuroscience article, Pang et al. (2006) generated a synaptotagmin-2 knock-out mouse to examine the expression and function of synaptotagmin-2. Pang et al. (2006) characterized the expression pattern of the protein, the consequences of deletion on survival, and the electrophysiological properties of synaptotagmin-2-deficient neurons in the forebrain and neuromuscular junction (Table 1). The authors inserted LacZ in the exon-2 region of the synaptotagmin-2 gene, allowing them to simultaneously examine the expression of synaptotagmin-2 and the consequences of its deletion. The expression of other synaptic vesicle proteins was not altered except for a slight upregulation of synaptotagmin-1 in the spinal cord [Pang et al. (2006), their Fig. 1 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F1), Table 1 (http://www.jneurosci.org/cgi/content/full/26/52/13493/T1)].

The knock-out mice exhibited motor dysfunction and reduced body weight and growth and survived no longer than 24 d after birth, a survival length similar to that of synaptotagmin-1-deficient mice [Pang et al. (2006), their Fig. 2 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F2)]. Pang et al. (2006) suggest that expression of synaptotagmin-1 at the neuromuscular junction is initially sufficient to maintain synaptic transmission in the absence of synaptotagmin-2. However it would be interesting to know the time course of expression of the two proteins as the mice mature. For example, early expression of synaptotagmin-1 could initially compensate for the absent synaptotagmin-2 but then be switched off later in development.

Synaptotagmin-2 was expressed heavily in the brainstem and spinal cord but only weakly in forebrain. Expression was confined to only a few areas in the forebrain, including the striatum, hypothalamus, and reticular nucleus of the thalamus, whereas expression in the cerebellum, brainstem, and spinal cord was more robust [Pang et al. (2006), their Figs. 3 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F3), 4 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F4)]. Immunostaining confirmed that synaptotagmin-1 and -2 are coexpressed at neuromuscular junctions and that synaptotagmin-1 expression was upregulated in synaptotagmin-2-deficient endplates [Pang et al. (2006), their Fig. 5 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F5)]. The authors note that expression seemed to be confined to inhibitory neurons in the forebrain and cerebellum.

The authors examined the function of synaptotagmin-2 in forebrain neurons, as well as at the neuromuscular junction. In inhibitory neurons in the striatum, deletion of synaptotagmin-2 did not alter the amplitude or charge transfer of evoked IPSCs, but it did delay the time course of release, effectively slowing down the slow component of Ca²⁺-dependent release [Pang et al. (2006), their Fig. 6 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F6)]. This suggests that synaptotagmin-2 is not necessary for calcium-dependent release but can affect the slow component of release. Spontaneous release, as measured by miniature endplate potentials, was not affected.

At the neuromuscular junction, at which synaptotagmin-2 is highly expressed, deletion altered spontaneous release, evoked release, and high-frequency stimulation. The frequency of spontaneous release was increased, both in the presence of calcium and when calcium chelators were used [Pang et al. (2006), their Fig. 7 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F7)]. Thus, synaptotagmin-2 may have a function independent of Ca²⁺ sensing. Evoked synaptic responses and release probability were also decreased [Pang et al. (2006), their Figs. 8 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F8), 9 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F9)]. High-frequency stimuli led to an increase in failure rate [Pang et al. (2006), their Fig. 10 (http://www.jneurosci.org/cgi/content/full/26/52/13493/F10)]. This finding correlates well with the reduced evoked response and release probability. High-frequency stimuli also produced asynchronous vesicle release in synaptotagmin-2-deficient neuromuscular junctions and delayed release for several seconds after a stimulus train [Pang et al. (2006), their Fig. 11A,B (http://www.jneurosci.org/cgi/content/full/26/52/13493/F11)]. Stimulus trains (10–20 Hz) facilitated release in mutant neurons [Pang et al. (2006), their Fig. 11F,G (http://www.jneurosci.org/cgi/content/full/26/52/13493/F11)], which the authors suggest could indicate that a buildup of Ca²⁺ during the stimulus trains partially rescues the deficiency (Pang et al., 2006).

These experiments demonstrate that synaptotagmin-2 is not necessary for Ca²⁺-dependent release at forebrain and spinal cord neurons but that it can regulate synaptic transmission. It is interesting that the deletion of synaptotagmin-2 produces such disparate phenotypes in striatal and spinal neurons. The reasons for this disparity remain unclear but could involve other members of the synaptotagmin family. For example, synaptotag-min-6, -7, and -9 have similar Ca²⁺-sensing functions, suggesting that this family of molecules may work together to regulate synaptic transmission (Grise et al., 2007; Lynch and Martin, 2007; Monterrat et al., 2007).

Although previous studies suggested that synaptotagmin-2 has a similar function to synaptotagmin-1, Pang et al. (2006) demonstrate that synaptotag-min-2 plays additional, nonredundant roles in regulating synaptic transmission. Synaptotagmin-1 is known to act primarily as a Ca²⁺ sensor for fast synchronous release. Pang et al. (2006) show that, like synaptotagmin-1, synaptotagmin-2 plays an important role in Ca²⁺-triggered release. However, they also demonstrate that the deletion of synaptotagmin-2 does not entirely block release and also increases spontaneous release independently of calcium. Therefore, synaptotagmin-1 and -2 do not function in an identical manner, and the expression of one or the other at a specific synapse may be important in determining how those neurons fire.