Abstract
In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ 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 α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-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 α2δ 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 α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.
Highlights
Results in aberrant synapse formation of auditory nerve fibers [19]
Three isoforms of the calcium channel α2δ subunit are expressed in hippocampal neurons [27], yet until today it is unclear whether all three isoforms contribute to specific neuronal and synaptic functions
Α2δ-3 shows a preferential expression in the axon. Despite these apparent overall differences all α2δ isoforms are expressed on the surface of axons and presynaptic membranes (SI Appendix, Fig. S1B), suggesting that, in principle, all three isoforms can contribute to synaptic functions
Summary
Results in aberrant synapse formation of auditory nerve fibers [19]. α2δ-4 is required for the organization of rod and cone photoreceptor synapses [20, 21]. By transfecting cultured hippocampal neurons from α2δ-2/-3 double-knockout mice with short hairpin RNA (shRNA) against α2δ-1, we developed a cellular α2δ subunit triple-knockout/knockdown model. Excitatory synapses from these cultures show a severe failure of synaptic vesicle recycling associated with severely reduced presynaptic calcium transients, Significance. Voltage-gated calcium channels are important regulators of neuronal functions, as for example synaptic transmission. Their auxiliary α2δ subunits are modulating the calcium currents. Our study suggests that α2δ proteins are critical regulators of excitatory synapse formation and thereby contributes to the understanding of basic nerve cell functions
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