Journal: Frontiers in Cellular Neuroscience
Article Title: Neuronal activity drives PCDH9 cleavage and nuclear translocation to coordinate structural and functional remodeling
doi: 10.3389/fncel.2025.1736960
Figure Lengend Snippet: Neuronal activation triggers Matrix Metalloproteinase (MMP)-dependent PCDH9 cleavage in primary neurons. (A) Experimental scheme (top panel) and representative PCDH9 Western blot (bottom panels) of primary neurons treated with 20 μM NMDA for 30 min, 1 h or 2 h. At short exposures (bottom left panel), two full-length PCDH9 isoforms (PCDH FL; ~130 and ~180 kDa; indicated by arrowheads) are detected, as previously reported ( ; ). At longer exposure time (right bottom panel), additional lower molecular-weights bands become visible, including one above 30 kDa whose level changes with NMDA treatment (putative PCDH9 C-terminal fragment, PCDH9 CTF). GAPDH was used as a loading control. (B) Quantifications of PCDH9 FL and PCDH9 CTF levels from A. Protein levels were normalized to GAPDH. n = 3-6 independent cultures. (C) Representative WB and corresponding quantification of PCDH9 CTF levels in primary neurons pretreated with the MMP inhibitor GM6001 (5 h prior to NMDA exposure, 6 h total) before exposure to NMDA (1 h). n = 3 independent cultures. (D) Representative WB and corresponding quantification of PCDH9 CTF levels in primary neurons treated 1 h with glutamate (glu), bicuculline (BIC) or tetrodotoxin (TTX). n = 3-7 independent cultures. (E) Representative WB and corresponding quantification of PCDH9 CTF levels in rat primary neurons pretreated with the NMDAR antagonist APV (1 h prior to NMDA exposure, 2 h total) before exposure to NMDA (1 h). n = 3 independent cultures. (F) Representative WB and corresponding quantification of PCDH9 CTF levels in primary neurons pretreated with the NMDAR antagonist APV (1 h prior to glutamate exposure, 2 h total) before exposure to glutamate (1 h). n = 3 independent cultures. The bands corresponding to full-length (FL) PCDH9 are also displayed. WB quantifications are presented as protein levels normalized to GAPDH. Mean ± SD is shown. Statistical analysis was performed using Kruskal-Wallis test followed by Dunn's multiple comparisons test against untreated controls. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Article Snippet: DIV15 neurons were treated with the following reagents: NMDA (20 μM; Sigma- Aldrich), APV (100 μM; Merck), glutamate (50 μM; Tocris), bicuculline (40 μM; Sigma-Aldrich), tetrodotoxin (2 μM; Tocris), GM6001 (10 μM; Tocris).
Techniques: Activation Assay, Western Blot, Control
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