Presynaptic α ₂δ subunits are key organizers of glutamatergic synapses

Significance

Voltage-gated calcium channels are important regulators of neuronal functions, as for example synaptic transmission. Their auxiliary α ₂δ subunits are modulating the calcium currents. Beyond that they have emerged as modulators of synaptic functions. Here, we established a cellular triple knockout/knockdown model in cultured hippocampal neurons by knocking out or knocking down the expression of all three α ₂δ subunits expressed in brain. Our experiments demonstrate that the presynaptic loss of α ₂δ proteins leads to a severe defect in glutamatergic synapse formation, which could be rescued by reintroducing any of the three neuronal α ₂δ isoforms. Thus, our study suggests that α ₂δ proteins are critical regulators of excitatory synapse formation and thereby contributes to the understanding of basic nerve cell functions.

Abstract

In nerve cells the genes encoding for α ₂δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α ₂δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α ₂δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α ₂δ isoforms as synaptic organizers is highly redundant, as each individual α ₂δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α ₂δ-2 and α ₂δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α ₂δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α ₂δ implicates α ₂δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α ₂δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.