Presynaptic α2δ subunits are key organizers of glutamatergic synapses

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.

In synapses neurotransmitter release is triggered by the entry of calcium through voltage-gated calcium channels (VGCCs). Neuronal VGCCs consist of an ion-conducting α₁ subunit and the auxiliary β and α₂δ subunits. α₂δ subunits, the targets of the widely prescribed antiepileptic and antiallodynic drugs gabapentin and pregabalin, are membrane-anchored extracellular glycoproteins, which modulate VGCC trafficking and calcium currents (1–5). In nerve cells α₂δ subunits have been linked to neuropathic pain and epilepsy (4) and they interact with mutant prion proteins (6) and regulate synaptic release probability (7). Importantly, all α₂δ isoforms are implicated in synaptic functions. Presynaptic effects of α₂δ-1, for example, may be mediated by an interaction with α-neurexins (8) or N-methyl-D-aspartate receptors (e.g., refs. 9 and 10). In contrast, postsynaptic α₂δ-1 acts as a receptor for thrombospondins (11) and promotes spinogenesis via postsynaptic Rac1 (12). α₂δ-2 is necessary for normal structure and function of auditory hair cell synapses (13); it has been identified as a regulator of axon growth and hence a suppressor of axonal regeneration (14) and was recently shown to control structure and function of cerebellar climbing fiber synapses (15). A splice variant of α₂δ-2 regulates postsynaptic GABA_A receptor (GABA_AR) abundance and axonal wiring (16). In invertebrates, α₂δ loss of function was associated with abnormal presynaptic development in motoneurons (17, 18) and in mice the loss of α₂δ-3 results in aberrant synapse formation of auditory nerve fibers (19). Finally, α₂δ-4 is required for the organization of rod and cone photoreceptor synapses (20, 21).Despite these important functions, knockout mice for α₂δ-1 and α₂δ-3 show only mild neurological phenotypes (5, 10, 22–25). In contrast, mutant mice for α₂δ-2 (ducky) display impaired gait, ataxia, and epileptic seizures (26), all phenotypes consistent with a cerebellar dysfunction due to the predominant expression of α₂δ-2 in the cerebellum (e.g., ref. 15). Hence, in contrast to the specific functions of α₂δ isoforms (discussed above) the phenotypes of the available knockout or mutant mouse models suggest a partial functional redundancy in central neurons. Moreover, detailed mechanistic insights into the putative synaptic functions of α₂δ subunits are complicated by the simultaneous and strong expression of three isoforms (α₂δ-1 to -3) in neurons of the central nervous system (27).In this study, 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, loss of presynaptic calcium channels and presynaptic vesicle-associated proteins, and a reduced size of the presynaptic active zone (AZ). Lack of presynaptic α₂δ subunits also induces a failure of postsynaptic PSD-95 and AMPA receptor (AMPAR) localization and a thinning of the postsynaptic density (PSD). Each individual α₂δ isoform (α₂δ-1 to -3) could rescue the severe phenotype, revealing the highly redundant role of presynaptic α₂δ isoforms in glutamatergic synapse formation and differentiation. Together our results show that α₂δ subunits regulate presynaptic differentiation as well as the transsynaptic alignment of postsynaptic receptors and are thus critical for the function of glutamatergic synapses.