Figure 7.
Proteasome and NKCC1 activities modulate AIS dynamics via Ecm29. (A) Ecm29 loss alters developmental AIS dynamics. (A1) Representative images of cortical neurons treated with or without 10 µM BUM and immunostained with AnkG antibodies at indicated time points. Scale bar, 20 µm. (A2) Scatter plot showing shorter distances from cell bodies of AIS start positions (upper panel) in Ecm29 KO (Ecm29−/−) cortical neurons relative to wild-type (Ecm29+/+) neurons in ∼5–7-DIV cultures. AIS start and end positions were estimated based on positions of 50% peak AnkG intensity at both tails, with the distance between these positions defining AIS length (lower panel). *, P < 0.05; ****, P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons post hoc test. (A3) Cumulative probability plot showing that NKCC1 inhibition by 10 µM BUM treatment (+BUM; added at 4 DIV) restored the abnormally accelerated proximal shift in AIS position (upper panel) in 7-DIV Ecm29 KO neurons. Data from experiments similar to those described in A1 and A2. ****, P < 0.0001; ns, not significant, two-way ANOVA with Dunnett’s post hoc test. (B) Blockade of proteasome activity causes an early proximal shift in AIS position in 7-DIV cortical neurons. (B1) Representative images of 7-DIV cortical neurons treated with or without proteasome inhibitors (MG132 or lactacystin; 2.5 µM, 8 h), and ectopically expressing SC-siRNA or Rpt1 targeting-siRNA (Rpt1-siRNA A-D), and/or control GFP or Ecm29-IRES-GFP, followed by immunostaining with AnkG antibodies (red) or Rpt1 antibody (gray). Scale bars, 20 μm. (B2 and B3) Averaged AnkG intensity along axons (n ≥ 57 per group; B2) and summary scatter plot (n = 7–68; B3) showing that proteasome inhibition and Ecm29 loss significantly shorten the AIS start position relative to that seen in wild-type neurons (±SEM; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant, one-way ANOVA followed by Dunnett’s post hoc test). Note that AIS integrity, as reflected by the peak AnkG intensity (upper panel, B2), is comparable in wild-type and Ecm29 KO neurons. (B4) Histogram (n = 7–68 per group; ****, P < 0.0001; ns, not significant, one-way ANOVA followed by Dunnett’s post hoc test) showing effects of proteasome and NKCC1 inhibition on the changes in the AIS start position and length. (C) Similar to A, except that 7-DIV cortical neurons were costained with antibodies against pan-voltage-gated Na+ channels (Pan-NaV; C1–C3). Scale bar, 20 µm. Note that both pan-NaV and AnkG start positions in Ecm29 KO neurons exhibit an accelerated proximal shift in AIS position (C2 and C4). Data represent mean ± SEM, n > 60 cells per group from three independent experiments. ****, P < 0.0001; ns, not significant by one-way ANOVA followed by Dunnett’s post hoc test. (D) Schematic illustrating proposed AIS-associated, Ecm29−/− proteasome-dependent modulation of GABA-evoked excitability properties in maturing neurons, requiring developmental NKCC1 down-regulation and proper subcellular localization of proteasomes.

Proteasome and NKCC1 activities modulate AIS dynamics via Ecm29. (A) Ecm29 loss alters developmental AIS dynamics. (A1) Representative images of cortical neurons treated with or without 10 µM BUM and immunostained with AnkG antibodies at indicated time points. Scale bar, 20 µm. (A2) Scatter plot showing shorter distances from cell bodies of AIS start positions (upper panel) in Ecm29 KO (Ecm29−/−) cortical neurons relative to wild-type (Ecm29+/+) neurons in ∼5–7-DIV cultures. AIS start and end positions were estimated based on positions of 50% peak AnkG intensity at both tails, with the distance between these positions defining AIS length (lower panel). *, P < 0.05; ****, P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons post hoc test. (A3) Cumulative probability plot showing that NKCC1 inhibition by 10 µM BUM treatment (+BUM; added at 4 DIV) restored the abnormally accelerated proximal shift in AIS position (upper panel) in 7-DIV Ecm29 KO neurons. Data from experiments similar to those described in A1 and A2. ****, P < 0.0001; ns, not significant, two-way ANOVA with Dunnett’s post hoc test. (B) Blockade of proteasome activity causes an early proximal shift in AIS position in 7-DIV cortical neurons. (B1) Representative images of 7-DIV cortical neurons treated with or without proteasome inhibitors (MG132 or lactacystin; 2.5 µM, 8 h), and ectopically expressing SC-siRNA or Rpt1 targeting-siRNA (Rpt1-siRNA A-D), and/or control GFP or Ecm29-IRES-GFP, followed by immunostaining with AnkG antibodies (red) or Rpt1 antibody (gray). Scale bars, 20 μm. (B2 and B3) Averaged AnkG intensity along axons (n ≥ 57 per group; B2) and summary scatter plot (n = 7–68; B3) showing that proteasome inhibition and Ecm29 loss significantly shorten the AIS start position relative to that seen in wild-type neurons (±SEM; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant, one-way ANOVA followed by Dunnett’s post hoc test). Note that AIS integrity, as reflected by the peak AnkG intensity (upper panel, B2), is comparable in wild-type and Ecm29 KO neurons. (B4) Histogram (n = 7–68 per group; ****, P < 0.0001; ns, not significant, one-way ANOVA followed by Dunnett’s post hoc test) showing effects of proteasome and NKCC1 inhibition on the changes in the AIS start position and length. (C) Similar to A, except that 7-DIV cortical neurons were costained with antibodies against pan-voltage-gated Na+ channels (Pan-NaV; C1–C3). Scale bar, 20 µm. Note that both pan-NaV and AnkG start positions in Ecm29 KO neurons exhibit an accelerated proximal shift in AIS position (C2 and C4). Data represent mean ± SEM, n > 60 cells per group from three independent experiments. ****, P < 0.0001; ns, not significant by one-way ANOVA followed by Dunnett’s post hoc test. (D) Schematic illustrating proposed AIS-associated, Ecm29−/− proteasome-dependent modulation of GABA-evoked excitability properties in maturing neurons, requiring developmental NKCC1 down-regulation and proper subcellular localization of proteasomes.

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