Neuronal Activity-Dependent Transcriptional Regulation of the Synaptic Cell Adhesion Molecules Lrrtm1 and Lrrtm2

The large processing capacity of our brain is the result of properly formed synaptic contacts and their maintenance and plasticity. Synaptic cell adhesion molecules and neuronal activity are critically involved in these processes. While synaptic cell adhesion molecules govern molecular target recognition, structural integrity and plasticity of a synaptic contact, neuronal activity is one major factor underlying dynamic and adaptive responses of neurons and neuronal networks. Neuronal activity and synaptic cell adhesion molecules jointly contribute to the establishment of normal brain function. However, little is known about their interaction and mutual dependence. Here I show that Lrrtm1 and Lrrtm2, two recently identified synaptic cell adhesion molecules, are regulated by neuronal activity in cultured hippocampal neurons of the mouse. I found that their responsiveness to neuronal activity crucially depends on nuclear calcium signalling. In addition Lrrtm2 is bound and controlled by CREB, an important factor in activity-mediated gene transcription. Using inhibitors of several calcium-dependent pathways, I demonstrate that the expression of Lrrtm1 and Lrrtm2 is mediated by calcium/calmodulin-dependent kinases. Further I show that Lrrtm1 and Lrrtm2 mRNA levels increase during development, which correlates with the maturation of the neuronal network. I can further show that knock-down of Lrrtm2 does not influence spine density, contrary to what has been reported in the literature. It does, however, influence neuronal network activity, as I demonstrate in collaboration with H.E. Freitag using microelectrode array recordings. Similar changes appear under Lrrtm1 knock-down conditions. The network behavior in these cultures reverts to nearly normal by overexpression of Lrrtm1 protein. Similar to the changes observed in Lrrtm1 and Lrrtm2 knock-down cultures, overexpression of MeCP2 causes a desynchronization of the neuronal bursting activity. MeCP2 is a transcriptional regulator which is found mutated in Rett syndrome, a rare but severe neurodevelopmental disorder in humans. I could show that the expression of endogenous Lrrtm2 is deregulated in cultures overexpressing MeCP2. This suggests that the network changes observed in MeCP2 overexpressing cultures are caused by deregulation of Lrrtm2 by MeCP2. However, overexpression of Lrrtm2 protein failed to rescue the MeCP2-phenotype. A further aim of my studies was to analyze the function of the activity-responsiveness of Lrrtm1 and Lrrtm2. Using different methods I attempted to visualize AMPA receptor trafficking to the neuronal surface and the impact of Lrrtm knock-down thereon. However, the applied methods were too insensitive to detect changes in synaptic AMPA receptor surface expression. Together, these findings connect Lrrtm1 and Lrrtm2, respectively, two members of the group of synaptic cell adhesion molecules, to synaptic activity, nuclear calcium signalling and CBP/CREB, all of which are important mediators of sustained adaptive changes in the central nervous system. The findings also give the impetus to further explore the role of the Lrrtm1 and Lrrtm2 activity-responsiveness in neurons in vitro and in vivo.