Late onset of Synaptotagmin 2a expression at synapses relevant to social behavior.

As they form, synapses go through various stages of maturation and refinement. These steps are linked to significant changes in synaptic function, potentially resulting in emergence and maturation of behavioral outputs. Synaptotagmins are calcium-sensing proteins of the synaptic vesicle exocytosis machinery, and changes in Synaptotagmin proteins at synapses have significant effects on vesicle release and synaptic function. Here, we examined the distribution of the synaptic vesicle protein Synaptotagmin 2a (Syt2a) during development of the zebrafish nervous system. Syt2a is widely distributed throughout the midbrain and hindbrain early during larval development but very weakly expressed in the forebrain. Later in development, Syt2a expression levels in the forebrain increase, particularly in regions associated with social behavior, and most intriguingly, around the time social behavior becomes apparent. We provide evidence that Syt2a localizes to synapses onto neurons implicated in social behavior in the ventral forebrain and show that Syt2a is colocalized with tyrosine hydroxylase, a biosynthetic enzyme in the dopamine pathway. Our results suggest a developmentally important role for Syt2a in maturing synapses in the forebrain, coinciding with the emergence of social behavior.

Calmodulin suppresses synaptotagmin-2 transcription in cortical neurons.

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca(2+) binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca(2+) binding to an unidentified secondary Ca(2+) sensor. To test whether CaM could be the enigmatic Ca(2+) sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca(2+) binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca(2+)/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.

Anti-sense RNA inhibits the expression of synaptotagmin II in RBL-2H3 and enhances the exocytosis of lysosomes in RBL-2H3.

The expression of synaptotagmin II (Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8% and 10% of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.