Fig 2: Decrease of Cav2.2 accumulations and spontaneous Ca2+ transients in growth cones of trkBTK-/- motoneurons corresponds to altered axon growth on laminin-221. (A) Representative images of axonal growth cones of wild type motoneurons cultured on laminin-221/211 for 5 days in vitro in the presence of BDNF and CNTF. TrkB receptors (green) and Cav2.2 calcium channels (magenta) were closely localized in growth cone protrusions, highlighted by white arrowheads (scale bar: 5 µm). (B) In trkBTK-/- growth cones Cav2.2 accumulation (magenta) was affected in growth cone tips, whereas tau levels (green) were not altered (scale bar: 5 µm). Statistical analysis of Cav2.2 immunoreactivity in trkBTK-/- axonal growth cones (0.54 ± 0.1, Q2 0.47, n = 4, N = 66) in comparison to wild type controls (1.00 ± 0.04, Q2 1.01, n = 6, N = 78) revealed a significant difference (p = 0.0015). Similar findings were obtained by normalizing Cav2.2 against the internal reference protein tau (trkBTK+/+ 1.00 ± 0.08, Q2 0.95; trkBTK-/- 0.65 ± 0.06, Q2 0.67; p = 0.0118) (C) The structural phenotype was accompanied by significant differences in the frequency of spontaneous calcium transients between control and trkBTK-/- growth cones (Control 0.92 ± 0.13, Q2 0.60, N = 72; trkBTK-/- 0.55 ± 0.09, Q2 0.30, N = 64; p = 0.0337). Representative recordings of control (upper trace, blue arrowheads) and trkBTK-/- (lower trace, magenta arrowheads) growth cones showed a reduced number of calcium spikes when trkB signaling is impaired. (D) Wild type and trkBTK-/- motoneurons were cultured on laminin-221/211 for 7 days in vitro in the presence of BDNF and CNTF and stained against tau (scale bar: 150 µm). TrkB mutant cells (316.4 ± 12.65 µm, Q2 314.5 µm, n = 7, N = 298) grew longer axons than controls (262.9 ± 11.79 µm, Q2 278.8 µm, n = 7, N = 283, p = 0.0093) (E) Treatment with 30 nM ?-conotoxin (CTX) caused enhanced axonal elongation in wild type motoneurons, but not in trkBTK-/- cells.

Fig 3: PRRT2 directly interacts with P/Q-type Ca2+ channels(A) Left: representative immunoblots of the expression levels of Cav2.1 in total cortical lysates of WT and PRRT2 KO mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) immunoreactivity was included as a control of equal loading. Right: quantification of Cav2.1 expression in WT and PRRT2 KO mice normalized on GAPDH expression and expressed in percentage of the mean WT immunoreactivity (means ± SEMs with superimposed individual values; n = 3 independent preparations).(B) Representative immunoblots of cell surface biotinylation performed in primary hippocampal WT and PRRT2 KO neurons (left panel). Total cell lysates (input), biotinylated (extracellular), and non-biotinylated (intracellular) fractions were analyzed by immunoblotting. Na/K-ATPase 1 and actin were included as markers of plasma membrane and cytosolic fractions, respectively. Individual values and means ± SEMs of total cell, extracellular, and intracellular Cav2.1 expression normalized on Na/K-ATPase 1 and actin expression (n = 5 independent preparations).(C) Co-immunoprecipitation of PRRT2 with Cav2.1. Detergent extracts of mouse brain were immunoprecipitated (IP) with Cav2.1 antibodies or with an anti-GFP antibody, as indicated. After electrophoretic separation of the immunocomplexes and western blotting, membranes were probed with anti-Cav2.1 antibodies to test the immunoprecipitation efficiency, as well as with anti-PRRT2 antibodies to probe the interaction. Left: a representative immunoblot is shown. Right: quantification of the PRRT2 immunoreactive signal in the IP samples, normalized to the binding of the anti-GFP control (means ± SEMs with superimposed individual values, n = 6 independent experiments). Immunoglobulin G (IgG) HC, antibody heavy chain. Input, 10 µg total extract.(D) Specificity of PRRT2 for Cav2.1 over Cav2.2. Extracts of HEK293 cells transiently expressing HA-tagged PRRT2 and either Cav2.1 or Cav2.2 were subjected to affinity precipitation with anti-HA agarose beads. Left panel: representative immunoblot showing input and pulled-down fractions from extracts of Cav2.2-expressing HEK293 cells. Center panel: representative immunoblot showing input and pulled-down fractions from extracts of Cav2.1-expressing HEK293 cells. VGCC (top) and PRRT2-HA (bottom) immunoblots are shown. Right panel: quantitative evaluation of the amounts of Cav2.1 and Cav2.2 immunoreactivities bound to PRRT2-HA, expressed in percentage of the respective input (means ± SEMs with superimposed individual values, n = 3 independent experiments).(E) Proteomic screen using the C-terminal domain of the Cav2.1 as a bait. Left: volcano plot combining fold change (FC in log2) with statistical significance (p value in l-log10). PRRT2 is purified with FC > 4 versus control with a corresponding p = 0.012. Right: primary amino acid sequence of mouse PRRT2 with the exclusive spectra sequences identified by mass spectrometry analysis highlighted in yellow.(F) Pull down of PRRT2 by the C-terminal domain of Cav2.1. A SNAP fusion protein of the C-terminal domain of Cav2.1 was used as a bait for pulling down PRRT2 from an extract of PRRT2-expressing HEK293 cells. Pull down with SNAP alone was used as an internal control. Left: a representative immunoblot is shown. Right: quantification of the PRRT2 immunoreactive signal in the pulled-down samples, expressed in percentage of the respective input (means ± SEMs with superimposed individual values, n = 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t test/Mann-Whitney U test.

Fig 4: BDNF signaling is sufficient to induce Cav2.2 clustering and local Ca2+ transients at growth cone protrusions of embryonic motoneurons on laminin-221. (A) Representative images of axonal growth cones of non-pulsed and BDNF-pulsed axonal growth cones on laminin-221/211 stained against Cav2.2 (magenta) and APP (green) (scale bar: 5 µm). Upon BDNF pulse Cav2.2 channels clustered at growth cone tips as highlighted by white arrowheads. In the non-pulsed condition potential accumulations of Cav2.2 channels were rather detected in central growth cone areas as indicated by white circles. Cav2.2 levels revealed a significant increase upon BDNF pulse (no pulse 1.00 ± 0.04, Q2 1.00, n = 9, N = 221; 5' BDNF 1.93 ± 0.24, Q2 1.75, n = 9, N = 245; p = 0.0014). The ratio of Cav2.2 and APP immunoreactivities yielded similar results. (B) These structural changes upon treatment with BDNF were matched by significantly enhanced frequencies of spontaneous Ca2+ transients in comparison to non-pulse controls (no pulse 0.16 ± 0.08, Q2 0, IQR 0, N = 32; 5' BDNF 0.30 ± 0.08, Q2 0, IQR 2, N = 32; p = 0.0104). (Right panel) Identical growth cones were imaged prior to and 2 min after BDNF pulse. Representative recordings of non-pulsed and BDNF-pulsed traces showed increased numbers of Ca2+ spikes (magenta arrowheads) in response to BDNF. Upon BDNF the total number of calcium spikes almost doubled and the percentage of active growth cones displaying at least one spike per recording was greatly increased. (C) Representative images of non-pulsed and BDNF-pulsed trkBTK+/+ and trkBTK-/- growth cones stained against Cav2.2 (magenta) and synaptophysin (green) (scale bar: 5 µm). In wild type cells the acute application of BDNF resulted in increased Cav2.2 immunoreactivity at growth cone protrusions (indicated by white arrowheads). This effect was not visible in trkBTK-/- axonal growth cones, where Cav2.2 immunoreactivity rather occurred in central growth cone regions as emphasized by white circles. Synaptophysin appeared comparable in each condition serving as internal reference protein. (Right panel) Statistical significance was determined by the ratio of BDNF-pulsed vs. non-pulsed growth cones of trkBTK+/+ and trkBTK-/- motoneurons with respect to Cav2.2 alone (trkBTK+/+ 1.00 ± 0.06, Q2 1.00, n = 7, N(no pulse) = 88, N(5' BDNF) = 102; trkBTK-/- 0.75 ± 0.07, Q2 0.79, n = 7, N(no pulse) = 100, N(5' BDNF) = 106; p = 0.0184) and Cav2.2 signal intensities normalized against synaptophysin highlighting the reduced responsiveness of trkBTK-/- growth cones to acute BDNF application.

Fig 5: Mutual regulation of neuronal excitability and ß-actin presence in axonal growth cones of embryonic motoneurons on laminin-221. (A) Representative images of growth cones of embryonic motoneurons cultured on laminin-221/211 for 5 days in vitro with or without 30 nM CTX and stained against ß-actin (green) and tau (magenta) (scale bar: 5 µm). CTX-treated motoneurons developed smaller growth cones (Control 26.14 ± 1.13 µm2, Q2 25.4 µm2, n = 13, N = 226; 30 nM CTX 19.51 ± 1.17 µm2, Q2 19.96 µm2, n = 17, N = 274; p = 0.0005) with ß-actin deficits (Control 1.00 ± 0.05, Q2 1.00; 30 nM CTX 0.71 ± 0.07, Q2 0.68; p = 0.0037). Tau levels were comparable between both conditions. (B) Representative images of axonal growth cones of ß-actin depleted and control motoneurons cultured on laminin-221/211 for 5 days in vitro and stained against ß-actin (magenta) and tau (yellow) (scale bar: 5 µm). Cells were selected by GFP expression (green) indicating successful lentiviral transduction. In comparison to shLUC-transduced control motoneurons ß-actin shRNA-transduced cells revealed significantly smaller growth cones (shLUC 22.02 ± 1.68 µm2, Q2 16.89 µm2, IQR 13.07 µm2, N = 76; shActß 14.85 ± 1.18 µm2, Q2 11.70 µm2, IQR 11.45 µm2, N = 74) and highly decreased ß-actin signals (shLUC 1.00 ± 0.09, Q2 0.90, IQR 0.92; shActß 0.38 ± 0.05, Q2 0.27, IQR 0.53). The microtubule cytoskeleton represented by tau appeared not affected. Re-expression of ß-actin (“Rescue”) was able to rescue growth cone size (23.54 ± 2.44 µm2, Q2 17.09 µm2, IQR 13.15 µm2, N = 62) and ß-actin immunoreactivity (0.98 ± 0.09, Q2 0.96, IQR 0.82) validating the obtained ß-actin based growth cone phenotype. (C) Representative images of ß-actin depleted growth cones on laminin-221/211 stained against Cav2.2 (magenta) and synaptophysin (yellow) (scale bar: 5 µm). Knockdown of ß-actin resulted in reduced Cav2.2 signals (shLUC 1.00 ± 0.09, Q2 0.84, IQR 0.75, N = 54; shActß 0.70 ± 0.06, Q2 0.52, IQR 0.46, N = 63), whereas synaptophysin levels were not altered. Re-entry of ß-actin was able to rescue the detected phenotype (Rescue 1.04 ± 0.07, Q2 1.05, IQR 0.90, N = 64).