α2δ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions

Abstract

The α₂δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α₂δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α₂δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α₂δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α₂δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α₂δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α₂δ-1-3 isoforms (α₂δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α₂δ proteins. In contrast to this redundant presynaptic function, α₂δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α₁-α₂δ protein-protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α₂δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α₂δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α₂δ and Cachd1.

Keywords: Cachd1; presynaptic calcium imaging; synapse formation; synaptic differentiation; voltage-gated calcium channels; α2δ subunits.

Figure 1

α₂δ-4 and Cachd1 can be expressed on the somato-dendritic and axonal surface. (A) Live cell surface staining of wildtype primary hippocampal neurons overexpressing soluble eGFP together with HA-tagged constructs of α₂δ subunits or Cachd1. (A) The somatic expression pattern of the anti-HA labelling revealed that all α₂δ subunits and Cachd1 can be expressed on the neuronal surface. (B) Anti-HA and synapsin immunofluorescent labelling revealed that all α₂δ subunits and Cachd1 show synaptic and dendritic membrane targeting (white arrowheads). Neurons overexpressing α₂δ-1 displayed a weaker expression pattern on the somatic, dendritic, and axonal surface compared to α₂δ-2, α₂δ-4, and Cachd1. (C) Quantification of the average HA fluorescent intensities illustrated that the surface expression of α₂δ-1 was reduced compared to those of the other subunits. α₂δ-2 expression in soma, dendrites, and axons was higher compared to α₂δ-1, α₂δ-4, and Cachd1. Quantification data are shown as values for individual cells (dots) and means ± SEM. ANOVA with Tukey’s multiple comparison test (C) 16–22 cells per condition from three independent culture preparations. F_{(3, 60)} = 53.6, p < 0.0001. Significances of post hoc test (*** p < 0.001, ** p < 0.01, * p < 0.05) in comparison to α₂δ-1, unless otherwise indicated, are indicated with asterisks. Scale bars, 5 µm.

Figure 2

α₂δ-4 and Cachd1 can target presynaptic membranes. An analysis of axonal varicosities illustrated a presynaptic localization pattern for all α₂δ subunits and Cachd1. This can be inferred from the color overlay and the co-localization of the linescan peaks of HA-α₂δ (red), synapsin (blue) and eGFP (green). Axonal varicosities were identified by their eGFP expression and are outlined by a dashed line. α₂δ-4 specifically accumulated in the perisynaptic membrane around a central synapsin cluster. Overall, α₂δ-2 and α₂δ-4 displayed higher axonal expression compared to α₂δ-1 and Cachd1 (for a quantitative analysis, see Figure 1C). Scale bar, 1 µm.

Figure 3

All α₂δ subunits and Cachd1 rescue glutamatergic synapse formation. (A) Immunofluorescent labelling of synapsin and Ca_V2.1 in α₂δ TKO/KD neurons (α₂δ-2/3 double knockout neurons transfected with shRNA-α₂δ-1/eGFP) illustrated a severe defect in glutamatergic synapse formation and differentiation, the absence of synapsin and Ca_V2.1 clusters in presynaptic varicosities. Boutons and axons were identified by their eGFP expression and are outlined by a dashed line. The reintroduction of α₂δ-1, α₂δ-4, or Cachd1 could rescue the accumulation of Ca_V2.1 and synapsin in the presynaptic boutons. This is supported in the color overlay, qualitative linescan analysis, and the quantification of the fluorescent intensities of Ca_V2.1 (B) and synapsin 1 (C). Quantification is represented as values for individual cells (dots) and means ± SEM. ANOVA with Tukey’s multiple comparison test with 39–46 cells per condition from three independent culture preparations. (B) F_{(3, 169)} = 25.85, p < 0.0001, post hoc: *** p< 0.0001. (C) F_{(3, 169)} = 19.25, p < 0.0001, post hoc: *** p < 0.0001. Significance of the TKO/KD in comparison to rescue conditions is indicated in the graphs with asterisks. Scale bar, 1 µm.

Figure 4

All α₂δ subunits and Cachd1 can restore presynaptic calcium signaling in α₂δ TKO/KD neurons. Mean sample traces (A), cumulative frequency distribution blots (B), and quantification (C) of presynaptic calcium imaging (SynGCaMP6f) in α₂δ TKO/KD neurons. (A,C) Presynaptic calcium signals were significantly reduced in TKO/KD neurons (black) in response to 1 action potential (AP), 3 AP, and 10 AP stimulation compared to double heterozygous controls (green). The introduction of α₂δ-1 (orange), α₂δ-4 (blue), or Cachd1 (purple) restored presynaptic calcium transients in all stimulation patterns. (B) The fraction (see text) and mean of responding synapses in TKO/KD neurons was strongly reduced compared to control and rescue conditions. ANOVA with Tukey’s multiple comparison test: 1 AP: F_{(4, 2504)} = 30.7, p < 0.0001; 3 AP: F_{(4, 2504)} = 38.6, p < 0.0001; 10 AP: F_{(4, 2504)} = 95.0, p < 0.0001. Significances of post hoc tests compared to the TKO/KD condition are indicated in the graphs by asterisks (*** p < 0.001). (C) Quantification shows values for individual cells (dots) and means ± SEM. 21–23 cells per condition were obtained from three independent culture preparations (number of synapses analyzed: control, 483; TKO/KD, 506; rescue α₂δ-1, 462; rescue α₂δ-4, 484; rescue Cachd1, 484). ANOVA with Tukey’s multiple comparison test: 1 AP: F_{(4, 105)} = 4.5, p = 0.0021; 3 AP: F_{(4, 105)} = 11.6, p < 0.0001; 10 AP: F_{(4, 105)} = 35.9, p < 0.0001. Significances of post hoc tests of TKO/KD compared to the other conditions are indicated in the graphs by asterisks (*** p < 0.001).

Figure 5

Overexpression of α₂δ-4 or Cachd1 does not increase presynaptic clustering of Ca_V2.1 channels. (A) Immunofluorescence analysis of axonal varicosities from wildtype neurons overexpressing eGFP and α₂δ or Cachd1 constructs, labelled against synapsin and Ca_V2.1. Micrographs show immunofluorescent signals of Ca_V2.1 channels at presynaptic boutons, identified by eGFP expression (outlined with a dashed line) and presynaptic synapsin labelling along untransfected dendrites (see also qualitative linescan analysis). Contrary to neurons overexpressing α₂δ-1, which display two times higher Ca_V2.1 intensity, neurons overexpressing α₂δ-4 and Cachd1 exhibited Ca_V2.1 levels similar to those of the eGFP control. Quantification of Ca_V2.1 (B) and synapsin 1 (C) shows values for individual cells (dots) and means ± SEM. Cells were obtained from three independent culture preparations. ANOVA with Tukey’s multiple comparison test. (B) 38 cells per condition, F_{(3, 148)} = 41, p < 0.0001. (C) 38 cells per condition, F_{(3, 148)} = 1.4, p = 0.25. Significance of post hoc test in comparison to α₂δ-1 is indicated by asterisks (*** p < 0.001). Scale bar, 1 µm.

Figure 6

Overexpression of α₂δ-1, α₂δ-4, or Cachd1 does not increase clustering of N-type channels. (A) Immunofluorescence analysis of axonal varicosities from wildtype neurons overexpressing eGFP and α₂δ or Cachd1 constructs, labelled against synapsin and Ca_V2.2. Micrographs show immunofluorescent signals of Ca_V2.2 channels at presynaptic boutons, identified by eGFP expression (outlined with a dashed line) and presynaptic synapsin labelling along untransfected dendrites (see also qualitative linescan analysis). Neurons overexpressing α₂δ-1, α₂δ-4, or Cachd1 exhibited Ca_V2.2 levels similar to those of the eGFP control. Quantification of Ca_V2.2 (B) and synapsin 1 (C) shows values for individual cells (dots) and means ± SEM. Cells were obtained from three independent culture preparations. ANOVA with Tukey’s multiple comparison test. (B) 24–39 cells per condition, F_{(3, 110)} = 1.58, p = 0.20. (C) 24–39 cells per condition, F_{(3, 110)} = 0.99, p = 0.40. Scale bar, 1 µm.

Figure 7

Overexpression of α₂δ-4 or Cachd1 does not alter the composition of glutamatergic synapses. (A) Co-transfection of soluble eGFP and α₂δ subunits or Cachd1 in combination with immunofluorescent labelling of presynaptic vGlut1 and postsynaptic GABA_A-receptors was used to detect the formation of mismatched synapses. Only the overexpression of α₂δ-2 led to the formation of mismatched synapses, as detected by postsynaptic GABA_A receptor clusters opposite vGlut1 positive glutamatergic terminals (A, α₂δ-2). Overexpression of α₂δ-4 or Cachd1 did not induce the formation of mismatched synapses and did not alter the molecular composition of glutamatergic synapses. Quantifications of immunofluorescence intensities of vGlut1 (B), GABA_A receptor (C), gephyrin (D), GluR1 (E), vGAT (F) and the bouton size as identified by eGFP fluorescence area (G) show values for individual cells (dots) and means ± SEM. Cells were obtained from three independent culture preparations. ANOVA with Tukey’s multiple comparison test was performed on 29–45 cells per condition. (B) F_{(4, 203)} = 0.58, p = 0.68. (C) F_{(4, 203)} = 103.6, p < 0.0001. (D) F_{(3, 81)} = 135.2, p < 0.0001. (E) F_{(3, 108)} = 28.8, p < 0.0001. (F) F_{(3, 104)} = 0.30, p = 0.88. (G) F_{(3, 306)} = 1.0, p = 0.51. Significances of post hoc test in comparison to α₂δ-2 are indicated by asterisks (*** p < 0.0001). Scale bar, 1 µm.

Figure 8

α₂δ subunits and Cachd1 differentially affect presynaptic calcium transients. Mean sample traces (A), cumulative frequency distribution blots (B), and quantification (C) of presynaptic calcium signals (SynGCaMP6f) in wildtype neurons overexpressing either α₂δ or Cachd1 constructs. Overexpression of α₂δ-4 led to a reduction in presynaptic calcium signals in response to stimulation with 1 AP, 3 AP and 10 AP (blue) compared to control (green). In contrast, overexpression of α₂δ-1 (orange) and Cachd1 (purple) increased calcium transients in all stimulation paradigms, as indicated in the mean sample traces and the maximal responses. Quantification shows values for individual cells (dots) and means ± SEM. 37–53 cells were obtained from four independent culture preparations (number of synapses analyzed: SynGCaMP6f, 1257; α₂δ-1, 1122; α₂δ-4, 1806; Cachd1, 1650). ANOVA with Tukey’s multiple comparison test: 1 AP: F_{(3, 244)} = 6.4, p = 0.0004; 3 AP: F_{(3, 185)} = 4.7, p = 0.0036; 10 AP: F_{(3, 183)} = 8.7, p < 0.0001. Significances of post hoc tests between conditions are indicated in the graphs by asterisks (*** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 9

Structural interface analysis revealed preferences in complex formation. (A) Cryo-EM structure of the human Ca_V2.2 calcium channel complex (PDB code: 7MIY), with the α₁ subunit (light blue) and α₂δ-1 (green.) Next to the membrane view, the complex is opened up to show the interaction interface between α₂δ-1 (top) and α₁ (bottom). Residues engaging in the subunit interactions are labelled and shown in sphere representation with the respective charges (blue, positive and red, negative). (B) Models of opened complexes (α₂δ-1, top and α₁, bottom) of the Ca_V2.1 α₁ subunit with mouse α₂δ-1, human α₂δ-4, and mouse Cachd1. The predicted interacting residues are labelled and shown in sphere representation in the respective charges (blue, positive, and red, negative); additional interacting residues are labelled in bold. Refer to text for description.