Dp71 Point Mutations Induce Protein Aggregation, Loss of Nuclear Lamina Integrity and Impaired Braf35 and Ibraf Function in Neuronal Cells

Figure 1. C272Y and E299del mutations provoke Dp71 aggregation in neuronal cells. (A) Schematic representation of Dp71 fused to GFP showing the location of C272Y and E299del mutations. The different Dp71 domains are depicted: WW, EF (EF hands), ZZ, SBD (syntrophin binding domain) and DBD (dystrobrevin binding domain). (B) SH-SY5Y and N1E-115 cells transiently expressing GFP, GFP-Dp71, Dp71-C272Y or Dp71-E299del were stained with DAPI to visualize nuclei, prior to being imaged by CLSM. Typical optical Z-sections are shown (scale bar = 10 μm). Percentage of (C) SH-SY5Y- or (D) N1E-115-transfected cells containing aggregates of Dp71 mutant variants is shown. (E) Quantification of the subcellular distribution of GFP-Dp71, Dp71-C272Y or Dp71-E299del aggregates in transfected N1E-115 cells. Nuclear, cells with aggregates observed in the nucleus; Non-nuclear, cells without aggregates observed in the nucleus. Results represent the mean +/- SEM of three independent experiments (n = 100). Significant differences were calculated using one-way ANOVA and Dunnett’s multiple comparison test (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with Dp71).

Figure 2. Structural analysis of C272Y and E299del mutations in Dp71. (A) Ribbon representation of the predicted Dp71 structures by I-TASSER server, illustrating the effect of C272Y and E299 mutations. (B) Superimposition of Dp71 (orange) with Dp71-C272Y (blue; upper panel) and Dp71 (orange) with Dp71-E299del (purple; bottom panel), highlighting the structural differences between Dp71 and its mutant variants. Protein models were visualized using Chimera v 1.16 (www.cgl.ucsf.edu/chimera/; accessed on 10 and 15 February 2022). (C) Upper panel: Ribbon representation of the effect of C272Y mutation on Dp71 structure. Amino acids are colored according to the vibrational entropy change upon mutation; yellow represents a rigidification of the structure in Dp72-C272Y mainly at positions 230 to 241 (zone 1), 265 to 280 (zone 2) and 295 to 300 (zone 3). Lower panels: Cys and Tyr amino acid residues at position 272 of wt and mutant Dp71, respectively, are colored in light green. In Dp71, hydrogen bonds are shown in red, while substitution by tyrosine in the C272 mutant causes a change in the surrounding interatomic interactions characterized by hydrophobic contacts (green) and weak hydrogen bonds…

Figure 3. Effect of C272Y and E299del aggregates on neuronal cell morphology. (A) N1E-115 cells transiently expressing GFP, GFP-Dp71, Dp71-C272Y or Dp71-E299del were stained with DAPI to visualize nuclei, prior to being imaged by CLSM, and single typical optical Z-sections are shown (scale bar = 5 μm). Morphometric analysis of the nucleus was carried out to measure (B) nuclear circularity and (C) nuclear area using Fiji software. (D) SH-SY5Y cells transiently expressing GFP, GFP-Dp71 or Dp71-C272Y and Dp71-E299del were stained with phalloidin to visualize the actin-based cytoskeleton, prior to being imaged by epifluorescence microscopy (scale bar = 20 μm). (E) The cellular area was measured using ImageJ 1.53a software. Results represent the mean +/− SEM of three independent experiments (n = 100 cells per experimental condition), with significant differences calculated by one-way ANOVA and Dunnett’s multiple comparison test (ns, not significant; *, p < 0.05; **, p < 0.01).

Figure 4. Neuronal cells expressing Dp71-C272Y and Dp71-E299del show the altered localization of β-DG. SH-SY5Y and N1E-115 cells transiently expressing GFP, GFP-Dp71, Dp71-C272Y or Dp71-E299del were immunostained for β-DG and counterstained with DAPI to visualize nuclei, prior to being imaged by CLSM. Representative optical Z-sections are shown (scale bar = 10μm). The polarized distribution of β-DG with aggregates is denoted by arrows.

Figure 5. Neuronal cells expressing Dp71-C272Y or Dp71-E299del exhibit the altered localization of nuclear lamins. SH-SY5Y and N1E-115 cells transiently expressing GFP, GFP-Dp71, Dp71-C272Y or Dp71-E299del were immunostained for lamin B1 (LB1) (A) or lamin A/C (LAC) (B) and counterstained with DAPI to visualize nuclei, prior to being imaged by CLSM. Typical optical Z-sections are shown (scale bar = 10 μm). Cytoplasmic aggregates of nuclear lamins are denoted by arrows, and their faint discontinuous labeling is denoted by asterisks. The percentage of SH-SY5Y-transfected cells showing the aberrant distribution of lamin B1 (C) or lamin A/C (D) is shown. Results represent the mean +/− SEM of three independent experiments (n = 100 cell per experimental condition), with significant differences calculated using one-way ANOVA and Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01).

Figure 6. Distribution of the heterochromatin marker H3K9me2 is altered in SH-SY5Y cells expressing Dp71 mutant variants. (A) SH-SY5Y cells transiently expressing GFP, GFP-Dp71, Dp71-C272Y or Dp71-E299del were immunostained for H3K9me2 and counterstained with DAPI to visualize nuclei. Cells were imaged by CLSM, and representative single optical Z-sections are shown (scale bar = 10 μm). (B) Quantification of H3K9me2 immunofluorescence intensity was carried out in transfected cells using Fiji software. Results represent the mean +/− SEM of three independent experiments (n = 100 cells per experimental condition). Statistical analysis was performed using one-way ANOVA and Dunnett’s multiple comparison test. (C) Line profile analysis of H3K9me2 immunolabeling was carried out using digitalized images and Image J 1.53a software (n = 25 cells per experimental condition).

Figure 7. Dp71 interacts with BRAF35 and colocalizes with BRAF35 and iBRAF during the neuronal differentiation of N1E-115 cells. (A) N1E-115 cell lysates were subject to immunoprecipitation (IP) using anti-BRAF35 antibodies and the immunoprecipitated proteins were analyzed by Western blotting using antibodies against Dp71 and BRAF35. (B) Lysates from N1E-115 cells transiently expressing GFP or GFP-Dp71 were immunoprecipitated using the GFP-Trap system. The captured proteins were subjected to Western blot analysis using antibodies against BRAF35 and GFP. Input corresponds to 5% of protein lysates prior to immunoprecipitation; IgG0, nonspecific antibody; Ub, unbound proteins; B, bound proteins. Undifferentiated (UD) and differentiated (DIF) N1E-115 cells were immunostained with (C) anti-BRAF35 or (D) anti-iBRAF antibodies and anti-Dp71 and were antibody and counterstained with DAPI to visualize nuclei. (E) Undifferentiated and differentiated N1E-115 cells were immunostained for the neuronal differentiation marker MAP2 and counterstained with DAPI to visualize nuclei. Cells were imaged by CLSM, and representative single optical Z-sections are shown (scale bar = 10 μm).

Figure 8. Cells expressing Dp71-C272Y or Dp71-E299del exhibit the altered localization of Braf35 and iBRAF. (A) SH-SY5Y cells transiently expressing GFP, GFP-Dp71, Dp71-C272Y or Dp71-E299del were immunostained for BRAF35 and counterstained with DAPI to visualize nuclei. (B) SH-SY5Y and N1E-115 cells transiently expressing GFP, GFP-Dp71, Dp71-C272Y or Dp71-E299del were immunostained for iBRAF and counterstained with DAPI to observe nuclei. Cells were imaged by CLSM, and representative single optical Z-sections are shown (scale bar = 10 μm). Cytoplasmic mislocalized BRAF35 or iBRAF is denoted by arrows.

Figure 9. Dp71 but not Dp71-C272Y induces the expression of synapsin in neuronal cells. N1E-115 cells transiently expressing GFP-Dp71 or Dp71-C272Y were induced to neuronal differentiation by 3 days of DMSO treatment. The expression of synapsin mRNA was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the transfected undifferentiated (UD) and differentiated (DIF) cells. TBP was used as endogenous control. Data correspond to the mean ± SEM of three independent experiments, with significant differences obtained by one-way ANOVA and Sidak’s multiple comparison test (ns, not significant; *, p < 0.05, ****, p < 0.0001).