α2δ-2 Protein Controls Structure and Function at the Cerebellar Climbing Fiber Synapse

Abstract

␣2␦ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, ␣2␦-2 (Cacna2d2), we used PCs as a model system to examine roles of ␣2␦ in excitatory synaptic function in male and female Cacna2d2 knockout (KO) mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked EPSCs. CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2 ϩ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wild-type as a result of increased glutamate reuptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of ␣2␦ proteins.

Action potential-coupled Rho GTPase signaling drives presynaptic plasticity

“…We could not use the more traditional method of recording miniature excitatory postsynaptic currents (mEPSCs), due to the need to measure quantal events from only the defined presynaptic WT or KO neurons. Although strontium-evoked quantal events are not equivalent to mEPSCs, they have been commonly used in other contexts to estimate quantal parameters from specific cell types and circuits ( Beeson et al, 2020 ; Ding et al, 2008 ; Gil et al, 1999 ; Hull et al, 2009 ; Wan et al, 2014 ; Zhang et al, 2015 ). Here, presynaptic Rac1 deletion did not affect the amplitude or frequency of strontium-induced quantal events ( Figure 4D ).…”

Presynaptic α 2 δ subunits are key organizers of glutamatergic synapses

“… 5 and 33 ). However, the severity of the phenotype of specific α 2 δ loss-of-function models strongly correlated with the expression level of the particular isoform in the affected cells or tissues: Knockdown of α 2 δ-1 affected synapse formation in retinal ganglion cells ( 11 , 12 ), lack of α 2 δ-2 causes pre- and postsynaptic defects in hair cells of the inner ear ( 13 ) and affected cerebellar climbing fiber synapses ( 15 ), knockout of α 2 δ-3 alters presynaptic morphology of auditory nerves ( 19 ), and in invertebrates loss of function of the homologous subunit resulted in abnormal presynaptic development in motoneurons ( 17 , 18 ). Finally, the predominant expression of α 2 δ-4 in the retina ( 38 ) is mirrored by retinal defects and consequences on the organization of rod and cone photoreceptor synapses ( 20 , 21 , 39 ).…”

“…In contrast, postsynaptic α 2 δ-1 acts as a receptor for thrombospondins ( 11 ) and promotes spinogenesis via postsynaptic Rac1 ( 12 ). α 2 δ-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 α 2 δ-2 regulates postsynaptic GABA A receptor (GABA A R) abundance and axonal wiring ( 16 ).…”

“…In contrast, mutant mice for α 2 δ-2 (ducky) display impaired gait, ataxia, and epileptic seizures ( 26 ), all phenotypes consistent with a cerebellar dysfunction due to the predominant expression of α 2 δ-2 in the cerebellum (e.g., ref. 15 ). Hence, in contrast to the specific functions of α 2 δ isoforms (discussed above) the phenotypes of the available knockout or mutant mouse models suggest a partial functional redundancy in central neurons.…”

Neuronal α2δ proteins and brain disorders

“…As heterologous co-expression of distinct α 2 δ-2 splice variants with various α 1 subunits caused similar effects on calcium current densities and activation/inactivation kinetics [45], these data suggest that the trans-synaptic function of α 2 δ-2 variants lacking exon 23 is independent of their role as a calcium channel subunit. Interestingly, α 2 δ-2 is also necessary for the proper spatial alignment of presynaptic L-type calcium channels and postsynaptic AMPA receptors in hair cell synapses of the inner ear [37], as well as for the structure and function of cerebellar climbing fiber synapses [5]. While the distinct signaling pathways remain to be determined, it is thus tempting to speculate that glutamatergic and GABAergic synapses may express a specific set of presynaptic α 2 δ isoforms and even splice variants in order to regulate synaptic connectivity and postsynaptic receptor abundance during development (Fig.…”