Synapses and disease in Ca2+ channel alpha2delta mouse models

In the central nervous system (CNS) the second messenger calcium regulates a variety of neuronal functions including neurotransmitter release, gene regulation, and neuronal plasticity. Voltage-gated calcium channels, which are composed of a pore forming alpha1 and the auxiliary alpha2/delta and beta subunits, provide a major calcium entry pathway in the membrane of neurons. While the primary function of beta subunits in membrane trafficking is well established, the roles of the largely extracellular alpha2/delta subunits are less well defined. Recent studies proposed an involvement of individual alpha2/delta isoforms in synapse formation, central pain processing, and extracellular matrix interactions. Whether and how alpha2/delta subunits also perform these functions in neurons of the central nervous system, which simultaneously express three distinct isoforms, is not known. We hypothesize that individual alpha2/delta subunits exert partially redundant and partially specific neuronal functions. With the aim of studying alpha2/delta subunits in loss-of-function approaches we have established breeding colonies of knockout mice for all neuronal isoforms (1, 2, and 3). Unlike for isoforms -2 (ducky) and -3, the consequences of alpha2/delta-1 knockout for CNS disease have not yet been addressed. Because this isoform is the primary target for the anti-epileptic and anti-allodynic drugs gabapentin and pregabalin, we hypothesize that its loss will reduce epileptic seizure threshold and affect anxiety-like behavior. These questions will be addressed in behavioral experiments. To isolate the contribution of the individual alpha2/delta subunits to neuronal functions we propose to generate three different alpha2/delta double-knockout mouse models. This limits expression to a single isoform and thus excludes mutual compensation. Immunocytochemsitry, FM dye release quantification, and electrophysiology in cultured hippocampal neurons from double-knockout mice will allow characterizing the roles of the individual isoforms for synapse formation, synaptic transmission, and calcium currents. Ultimately the results of these experiments shall be compared with those of a total knockdown, either by breeding triple-knockout mice or by siRNA knockdown in double-knockout neurons. Whether alpha2/delta isoforms differ in their pre- and postsynaptic targeting properties and colocalization with distinct alpha1 subunits will be analyzed by expressing epitope-tagged alpha2/delta subunits in hippocampal neurons. Finally, expression of extracellular epitope-tagged alpha2/delta-1 in hippocampal neurons will provide a paradigm for the proteomic identification of novel interaction partners. How alpha2/delta subunits mediate specific processes and contribute to neuronal disease are eminent questions of cellular and applied neurosciences. The proposed experiments will strongly contribute to our understanding of neuronal alpha2/delta subunit-dependent functions. Furthermore the outcomes of this study will guide future research directions and will shed light onto the mechanisms of central nervous system diseases.