| Popis: |
The molecular mechanisms involved in synaptic plasticity are thought to be the basis for the understanding of learning and memory. However, the complexity of the molecular interactions impedes a deep understanding of these mechanisms. Thus far, it has been well established that a common trigger of the synaptic plasticity mechanism is an increase in postsynaptic calcium concentration. Recently, the protein Copine-6 was found as a modulator of synaptic plasticity due to its ability to respond to calcium influx and subsequently to sequester components of the actin cytoskeleton to the postsynaptic membrane of excitatory synapses. Therefore, Copine-6 seems to be a good candidate involved in hippocampal long-term potentiation, learning and memory. Interestingly, Copine-6 has recently been related in different neurological disorders like intellectual disabilities, depression and epilepsy (Anazi et al., 2017; Han et al., 2018; Zhu et al., 2016). In the last years, our group generated a mouse line in which a calcium-binding mutant of Copine-6 was knocked-into the Cpne6 locus – called Cpne6D167N. Thereafter, we focused on the biochemical characterization of this mouse. We showed that the calcium-dependent enrichment of Copine-6 in membrane fractions of the mouse brain is abrogated in Cpne6D167N mice in the presence of calcium. Importantly, the calcium mutant Copine-6D167N is expressed at the same level as wild-type Copine-6. These data therefore shows that the exchange of Asp to Asn at position 167 of Copine-6 does not affect Copine-6 expression but suppresses its calcium-dependent binding to membranes. Furthermore, we also demonstrated that calcium binding to Copine-6 is crucial for its ability to act as a synaptic plasticity modulator. We found that expression of Copine-6D167N in the CA1 region of the hippocampus affects the relative proportion of spine types in vivo, as neurons of the hetero- and homozygous knock-in mice express a significantly higher proportion of thin spines at expense of mature spines, a phenotype that was not observed in Cpne6 knock-out (KO) mice. Differences in spine morphology were also observed in primary hippocampal neurons derived from homozygous Cpne6D167N mice, in which an increase in the number of "immature", filopodia-like, thin protrusions and a decrease in mushroom-like protrusions were found. These results suggest that either maturation of spines is delayed or that spines cannot be strengthened following Cpne6D167N mutation. Accordingly, we assessed synaptic strengthening of spines from wild-type, hetero- and homozygous Cpne6D167N neurons by inducing chemical long-term potentiation (cLTP). We found that while wild-type neurons responded with an increased number of mushroom spines and synapses after cLTP induction, phenotypes that have been correlated with synaptic strengthening (Papa et al., 1995; Hosokawa et al., 1995; Fortin et al., 2010) neurons from heterozygous and homozygous Cpne6D167N mice could not respond to the changes related to the cLTP induction paradigm. This suggests that both mutant genotypes failed to undergo synaptic strengthening. Interestingly, heterozygous Cpne6D167N neurons showed elevated numbers of filopodia-like spines after cLTP induction, possibly as a compensatory mechanism to establish synaptic connections. Finally, we also found in Cpne6D167N mice morphological simplifications of CA1 hippocampal pyramidal neurons when compared to wild-type. This result suggests that the binding of calcium to Copine-6 may indirectly affect neuronal morphology as a consequence of spine immaturity. In conclusion, the calcium-binding site point mutation of Copine-6 seems to have a more profound effect on spine structure plasticity than the complete absence of Copine-6. A similar phenomenon was observed when the phenotypes of mice deficient for CaMKII were compared with mice expressing a phosphorylation mutant of CaMKII (Giese, et al. 1998). Thus, the calcium binding site of Copine-6 seems to be a key element for its ability to act as a calcium sensor and as a further modulator of the synaptic plasticity mechanism. Finally, this work might help to deepen the molecular understanding of synaptic plasticity mechanisms and may also provide new avenues for the molecular understanding of related neurological disorders, revealing possible therapeutic targets. |
