Abnormal cellular phenotypes of fibroblasts from the patient with <i>LMNA</i> p.R388P-associated L-CMD and lipodystrophy.

<p><b>A)</b> Phase-contrast images of control (passage 11) and patient (passage 9) cells. Scale bar, 100 μm. <b>B)</b> Measurement of nuclear area for control and patient fibroblasts at passages 13 and 11, respectively (mean ± s.e.m.). n > 100 nuclei/condition. <b>C)</b> Senescence assessment using the β-Galactosidase assay for control and patient fibroblasts at passages 9 and 6, respectively. <b>D)</b> Whole cell extracts of fibroblasts from control and patient at passages 10 and 14 (P10, P14) were analysed by western blot using antibodies against LA/C, LB1 or GAPDH. ECL signals were then scanned and quantified. The graph illustrates LA and LC ECL signal intensities as relative optical densities (O.D.) normalised versus GAPDH (n = 4) (mean ± s.e.m.). <b>E)</b> Human skin fibroblasts from control (passage 13) and patient (passage 11) were fixed, labelled with anti-LA/C or anti-LB1 antibodies, and observed by immunofluorescence microscopy. DNA was stained with Hoechst. Shown are images representative for normal nuclear morphology (Normal), nuclear dysmorphy (Dysm.) and lamina with a honeycomb pattern (Hon.). Asterisks indicate the honeycomb aspect of the lamin A/C network. Arrows indicate the regions with weak lamin B1 staining. The percentages of the different phenotypes are indicated on the pictures; n > 300 nuclei/condition. Scale bar, 10 μm.</p

A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus

<div><p>A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by <i>LMNA</i>. <i>LMNA</i> mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous <i>LMNA</i> p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient’s skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient’s mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other <i>LMNA</i> mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.</p></div

Changes in the lamin A in situ proximity with LAP2α and emerin in response to R388P-LA expression.

<p><b>A,E)</b> Whole cell extracts of C2C12 cells either control (Ctrl) or overexpressing wild-type (WT) or R388P (RP) LA, tagged with GFP (left panel) or FLAG (right panel) were analysed by western blot using anti-GFP, anti-FLAG, anti-LAP2α, anti-emerin and anti-GAPDH antibodies, as indicated. <b>B-D, F-H)</b> C2C12 cells overexpressing WT (upper panel) or R388P (lower panels) GFP-LA or FLAG-LA were fixed, labelled with anti-GFP and anti-LAP2α or anti-FLAG and anti-emerin antibodies, and processed either for immunofluorescence <b>(B,F)</b> or proximity ligation assay (PLAssay) <b>(C,G)</b>, before observation at the confocal microscope. Scale bar, 10 μm. <b>D,H)</b> Quantification of PLA signals per nucleus among GFP/PLA or FLAG/LA positive cells as shown in <b>C,G)</b>. The graphs show the median intensity of the total PLA signals detected per nucleus (upper panel) and the median frequency of the PLA signals detected at the intranuclear periphery (lower panel). Boxes show first and third quartiles, bars are put according to Tukey method for n = 59 (WT) and 31 (R388P) nuclei for <b>D</b> and n = 67 (WT) and 74 (R388P) nuclei for <b>H</b> *** p < 0.001 (Mann Whitney test).</p

Schematic illustration of the secondary structures of A-type lamins expressed by transfection with constructs.

<p>Shown are prelamin A (preLA) and/or mature Lamin A (mLA) either WT or R388P, with or without the L647R mutation tagged with the FLAG (circles) or GFP sequences (hexagons) when using the pCMV-tag2A or pEGFP vectors, respectively. Stars indicate the position of the mutation R388P. Of note, the L647R mutation disrupts the proteolytic cleavage site of prelamin A, inhibiting the maturation of preLA into mLA.</p

p.R388P mutation alters solubility and nuclear envelope targeting of lamin A.

<p><b>A)</b> Whole cell extracts of C2C12 cells overexpressing wild-type (WT), R388P (RP), L647R (LR) FLAG-LA or WT FLAG-mature LA (mLA) were analysed by western blot using anti-FLAG, anti-LA/C (which recognise ectopic and endogenous A-type lamins), anti-prelamin A (preLA) and anti-GAPDH antibodies. <b>B)</b> C2C12 cells overexpressing WT or R388P FLAG-LA were fixed, labelled with anti-FLAG antibodies, and immunofluorescence observed at the confocal microscope. Representative cells presenting either a strong FLAG-lamin A staining at the nuclear envelope (NE), a predominant nucleoplasmic staining (Nu.) or the presence of small aggregates with ring—like structures (Ag.) are shown. Scale bar, 10 μm. <b>C)</b> Percentage of the three phenotypes NE, Nu. and Ag. for cells expressing WT or R388P FLAG-LA, as indicated. Data are the mean ± s.e.m. of 5 independent experiments and 100–250 nuclei/condition, *** p < 0.001 (two-way ANOVA with Bonferroni post-tests). <b>D)</b> C2C12 cells overexpressing WT or R388P FLAG-LA underwent cell fractionation to enrich the insoluble nuclear matrix intermediate filament scaffold. Proteins from soluble (S1, S2, S3) and insoluble (Ins.) fractions as well as whole cell extracts (T) were analysed in parallel by western blot using anti-FLAG antibodies. Samples loaded in each lane correspond to an identical cell number. Red Ponceau staining of nitrocellulose membrane illustrates the protein specificity of each fraction. <b>E)</b> ECL signals obtained in <b>D)</b> were scanned and quantified. The graph illustrates the distribution of FLAG tagged proteins into the different fractions assuming that 100% of the signal correspond to the total of signals recovered in [S1 + S2 + S3 + Ins.]. Data are the mean ± s.e.m. of 3 independent experiments. ** p < 0.01 and * p < 0.05 (two-way ANOVA with Bonferroni post-tests).</p

Altering cytoskeletal element integrity or global histone acetylation pattern does not rescue nuclear shape in myoblasts expressing R388P LA.

<p>In <b>A, C</b> and <b>E</b>, immunofluorescently-labelled cells were observed under confocal microscopy; scale bars, 10 μm. <b>A)</b> C2C12 cells overexpressing WT or R388P FLAG-LA were treated with 0.05% DMSO (0) or with 5 to 10 μM Nocodazole (Noco.) for 3 h at 37°C. After fixation, cells were labelled with rabbit anti-FLAG (green) and mouse anti-α-tubulin antibodies (red). <b>B)</b> The graph illustrates the percentage (mean ± s.e.m.) of dysmorphic nuclei among cells treated as shown in <b>A)</b>, * p < 0.05 for n = 3 independent experiments and >100 nuclei/experiment (WT and R388P data were analysed separately by Kruskal-Wallis tests with pairwise comparisons of groups). <b>C)</b> C2C12 cells overexpressing WT or R388P FLAG-LA were treated with 0.2% DMSO (0) or with the actin depolymerising agent Cytochalasin D at 1 or 2 μM (Cyto.) for 3 h at 37°C. After fixation, cells were labelled with rabbit anti-FLAG antibodies and with phalloidin. <b>D)</b> Percentage (mean ± s.e.m.) of dysmorphic nuclei among cells treated as in <b>C)</b>, p>0.05 for n = 3 independent experiments and >100 nuclei/experiment (WT and R388P data were analysed separately by Kruskal-Wallis tests with the pairwise comparisons of groups). <b>E)</b> C2C12 cells overexpressing WT or R388P FLAG-LA were fixed and labelled with rabbit anti-FLAG (red) and mouse anti-desmin (green) antibodies. DNA was stained with Hoechst (blue). Representative cells illustrating nuclear shape in the absence or presence of endogenous desmin expression (left and right panels, respectively). <b>F)</b> The graph illustrates the mean ratio (± s.e.m.) of desmin-positive vs -negative cells for transfected cells versus untransfected control cells. Are considered the whole population of cells expressing WT or mutant (R388P) FLAG-LA and the subpopulation of cells expressing R388P FLAG-LA with dysmorphic nuclei (R388P Dysm.), p>0.05 for n = 4 independent experiments and >100 nuclei/experiment (Kruskal-Wallis tests with pairwise comparisons of groups). <b>G)</b> C2C12 cells, control (C) or expressing WT or R388P FLAG-LA were incubated in the absence (-) or presence (+) of anacardic acid (100 μM) or trichostatin A (100 nM), as indicated. Whole cell extracts were analysed by western blot using anti-FLAG, anti-H3K9ac (left panel) and anti-H3K27ac (right panel) antibodies. GAPDH and Actin detection were used as loading controls in the left and right panels, respectively. <b>H)</b> C2C12 cells expressing WT or R388P FLAG-LA were treated with drugs as in <b>G)</b>. The graph illustrates the percentage of dysmorphic nuclei for cell populations (mean ± s.e.m), * p < 0.05 for n = 3 independent experiments and >100 nuclei/experiment (Kruskal-Wallis test with pairwise comparisons of groups).</p

Different maturation intermediates of R388P prelamin A induce nuclear dysmorphy.

<p><b>A, D, G</b> and <b>J</b>, immunofluorescently-labelled cells observed under confocal microscopy; scale bars, 10 μm. <b>A)</b> C2C12 cells overexpressing WT (WT) or mutant (R388P) FLAG-LA were fixed and labelled with anti-FLAG antibodies. Representative nuclei with a normal shape (ovoid) or with dysmorphy are shown. <b>B)</b> Percentage of dysmorphic nuclei among cells expressing either WT FLAG-LA or R388P FLAG-LA, as evaluated by visual observation (mean ±s.e.m.), *** p < 0.001 for n = 8 independent experiments with >100 nuclei/experiment (Mann Whitney test). <b>C)</b> Whole cell extracts of C2C12 cells overexpressing WT or R388P FLAG-LA and treated with DMSO (0) or 10 μM (10) Mevinolin (Mev) for 18 h, were analysed by western blot using anti-FLAG antibodies. GAPDH was used as a loading control. <b>D)</b> C2C12 cells as described in <b>C)</b> were fixed and labelled with anti-FLAG antibodies. <b>E)</b> Percentage of dysmorphic nuclei of cell populations assessed by visual observation as in <b>D)</b> (mean ± s.e.m.), for n = 3 independent experiments and >100 nuclei/experiment (Kruskal-Wallis test with pairwise comparisons of groups). <b>F)</b> Whole cell extracts of C2C12 cells overexpressing FLAG-tagged R388P LA, R388P-L647R preLA, WT LA or L647R preLA were analysed by western blot using anti-FLAG and anti-GAPDH antibodies. <b>G)</b> C2C12 cells as described in <b>F)</b> were fixed, labelled with anti-FLAG antibodies. <b>H)</b> Percentage of dysmorphic nuclei for cell populations observed in <b>G)</b> (mean ± s.e.m.), p = 0.9 for n = 5 independent experiments with cells expressing R388P FLAG-LA and >100 nuclei/experiment (Mann Whitney test). n = 2 independent experiments with cells expressing R388P FLAG-LA and >100 nuclei/experiment. <b>I)</b> Whole cell extracts of C2C12 cells overexpressing R388P FLAG-LA, R388P FLAG-mLA or WT FLAG-mLA were analysed by western blot using anti-FLAG and anti-GAPDH antibodies. <b>J)</b> C2C12 cells as described in <b>I)</b> were fixed and labelled with anti-FLAG antibodies. <b>K)</b> Percentage of dysmorphic nuclei for cell populations observed as in <b>J)</b> (mean ± s.e.m.), * p < 0.05 for n = 4 independent experiments and >100 nuclei/experiment (Kruskal-Wallis test with pairwise comparisons of groups).</p

Correlation between MyoSets at baseline on dominant (D) and non-dominant (ND) sides, clinical and motor function parameters.

<p>* p-value < 0.05,</p><p>** p-value < 0.01.</p><p>Correlation between MyoSets at baseline on dominant (D) and non-dominant (ND) sides, clinical and motor function parameters.</p

MoviPlate correlations with grip and pinch strength.

<p>Correlations at baseline between the MoviPlate scores and grip and pinch strength for all SMA patients for the non-dominant hands (dark dots) and the dominant hands (clear dots).</p

Comparison of strength and motor function between the two groups on dominant (D) and non-dominant (ND) sides.

<p>* p-value < 0.05 for group effect,</p><p>** p-value < 0.01 for group effect.</p><p>Comparison of strength and motor function between the two groups on dominant (D) and non-dominant (ND) sides.</p