Altered tubulin detyrosination due to SVBP malfunction induces cytokinesis failure and senescence, underlying a complex hereditary spastic paraplegia

Abstract

Senescence, marked by permanent cell cycle arrest may contribute to the decline in regenerative potential and neuronal function, thereby promoting neurodegenerative disorders. In this study, we employed whole exome sequencing to identify a previously unreported biallelic missense variant in SVBP (p.Leu49Pro) in six patients from three unrelated families. These affected individuals present with a complex hereditary spastic paraplegia (HSP), peripheral neuropathy, verbal apraxia, and intellectual disability, exhibiting a milder phenotype compared to patients with nonsense SVBP mutations described previously. Consistent with SVBP's primary role as a chaperone necessary for VASH-mediated tubulin detyrosination, both patient fibroblasts with the p.Leu49Pro mutation, and HeLa cells harboring an SVBP knockdown exhibit microtubule dynamic instability and alterations in pericentriolar material (PCM) component trafficking and centrosome cohesion. In patient fibroblasts, structural abnormalities in the centrosome trigger mitotic errors and cellular senescence. Notably, premature senescence characterized by elevated levels of p16INK4, was also observed in patient peripheral blood mononuclear cells (PBMCs). Taken together, our findings underscore the critical role of SVBP in the development and maintenance of the central nervous system, providing novel insights associating cytokinesis failure with cortical motor neuron disease and intellectual disability.

Keywords: HSP; SVBP; centrosome; cytokinesis failure; microtubule detyrosination; senescence.

FIGURE 1

Clinical features of families with a novel SVBP mutation. (a) Pedigree for families A, B, and C. (b) Axial and sagittal T1 MRI sequences of patient P2 showing diffuse cerebellar atrophy, enlarged ventricles, thin corpus callosum, and diffuse cerebellar atrophy (c) Sagittal T1 and axial FLAIR MRI of patient P4 showing cerebellar vermis atrophy, ears of the lynx sign (white arrow), an asymmetrical ventricle enlargement. (d) Axial T2 and sagittal T1 MRI sequences from patient P6 showing ears of the lynx sign (white arrow), enlarged ventricles, and vermis cerebellar atrophy.

FIGURE 2

Pathogenicity of SVBP variant on the VASH1 secretion and MT detyrosination activity. (a) Close‐up views of the VASH1–SVBP interface, with interacting residues shown as sticks. VASH1 residues are colored blue and labeled with blue letters, while SVBP residues are colored orange and labeled with orange letters. (b) HeLa cells transfected with vectors directing the expression of FLAG‐tagged SVBP^WT, SVBP^L49P and VASH1, or combinations thereof, were subjected to immunoblot analysis with anti‐Flag antibody. Total amounts of β‐actin were used as a loading control. (c) Control (CTL) and patient (P2 and P4) fibroblasts were stained with anti‐SVBP (red) and anti‐α‐tubulin (α‐tub; green) antibodies and DAPI (blue). Scale bars: 10 μm. (d) Control (CTL) and patient (P2 and P4) fibroblasts were subjected to immunoblot analysis using the anti‐SVBP antibody. Total amounts of α‐tubulin were used as a loading control. (e) Control (CTL) and patient (P2 and P4) fibroblasts were treated with vehicle or paclitaxel and stained with anti‐α‐tubulin (α‐tub; red) and anti‐detyrosinated tubulin (detyr‐tub; green) antibodies and DAPI (blue). Scale bars: 50 μm. (f) Control (CTL) and patient (P2 and P4) fibroblasts were treated with vehicle or paclitaxel and subjected to immunoblot analysis using antibodies directed against detyrosinated (detyr‐tubulin) and α‐tubulin. Total amounts of β‐actin were used as a loading control. The relative ratios of detyrosinated versus total ⍺‐tubulin levels are indicated (n = 3).

FIGURE 3

SVBP mutant induces centrosome cohesion deficit, mitotic spindle abnormalities, cytokinesis failure, and chromosome instability. (a) Interphase control (CTL) and patient (P2 and P4) fibroblasts were stained with alpha‐tubulin (α‐tub; green) and anti‐pericentrin (Peric.; red) antibodies and DAPI (blue) at cell passages <10 (inset shows enlargement of the PCM, asterisk (*) mark the position of centrosome). Scale bars, 10 μm. Percentage of control (CTL) and patient fibroblasts (P2 and P4) with split centrosomes and centrosomal pericentrin intensity were quantified. n ≥ 50 cells/100 centrosomes; mean ± SD; *p < 0.05; **p < 0.01 by one‐way ANOVA with Tukey post hoc tests. (b) Representative picture of control fibroblasts (CTL) treated with vehicle (Veh.) or parthenolide (PN, 5 μM, 24 h) and stained with anti‐pericentrin (Peric.; green) antibodies and DAPI (blue) (white arrows indicate the position of the centrosome). Scale bars, 20 μm. Percentage of cells treated or not with parthenolide (PN) was quantified. n ≥ 20 cells/40 centrosomes; mean ± SD; **p < 0.01 by two‐tailed t test. (c) Control (CTL) and patient (P2 and P4) fibroblasts were stained at different mitotic phases (prophase, metaphase, and cytokinesis) with alpha‐tubulin (α‐tub; green) and anti‐pericentrin (Peric.; red) antibodies and DAPI (blue) at cell passages <10 (inset shows enlargement of the PCM, asterisk (*) mark the position of centrosome). Scale bars, 5 μm. Mitotic defects (spindle abnormalities and cytokinesis defects) were quantified in control (CTL) and patient (P2 and P4 fibroblasts). n ≥ 10 cells for each mitotic phase; Mean ± SD; *p < 0.05; *** < p < 0.001 by one‐way ANOVA with Tukey post hoc tests. (d) Representative pictures of control (CTL) and patient (P2 and P4) fibroblasts at cell passages >10 stained with anti‐α‐tubulin (α‐tub; green) and DAPI (blue), showing the presence of micronuclei and binucleated cells (white arrows) (insert shows enlargement of the indicated area). Scale bars, 50 or 10 μm. The percentage of cells with micronuclei and binucleated cells were quantified. n ≥ 50; Mean ± SD; *p < 0.05; **p < 0.01 by one‐way ANOVA with Tukey post hoc tests.

FIGURE 4

CRISPR/Cas9 knockout of SVBP in HeLa cells induces centrosome abnormalities and aberrant mitosis. (a) Interphase Wild‐type (WT) and SVBP‐KO (SVBP ^ko) HeLa cells were stained with alpha‐tubulin (α‐tub; green) and anti‐pericentrin (Peric.; red) antibodies and DAPI (blue) (inset shows enlargement of the PCM; the asterisk (*) marks the position of the centrosome). Scale bars, 10 μm. Percentage of wild‐type (WT) and SVBP‐KO (SVBP ^ko) HeLa cells with split centrosomes and centrosomal pericentrin intensity were quantified. n ≥ 30 cells/60 centrosomes per condition; Mean ± SD; **p < 0.01 by two‐tailed t test. (b) Representative image of wild‐type (WT) and SVBP‐KO (SVBP ^ko) expressing PACT‐RFP (asterisk (*) mark the position of centrosome). Twenty‐four hours after transfection, cells were fixed and stained for DNA content (DAPI; blue). Scale bars, 10 μm. Percentage of wild‐type (WT) and SVBP‐KO (SVBP ^ko) HeLa cells expressing PACT‐RFP with split centrosomes was quantified. n ≥ 10 cells/20 centrosomes per condition; Mean ± SD; **p < 0.01 by two‐tailed t test. (c) Wild‐type (WT) and SVBP‐KO (SVBP ^ko) HeLa cells were stained at different mitotic phases (prophase, metaphase, and cytokinesis) with alpha‐tubulin (α‐tub; green) and anti‐pericentrin (Peric., red) antibodies and DAPI (blue) (inset shows enlargement of the PCM; the asterisk (*) marks the position of the centrosome). Mitotic defects (spindle abnormalities and cytokinesis defects) in wild‐type (WT) and SVBP‐KO (SVBP ^ko) HeLa cells were quantified. n ≥ 10 cells for each mitotic phase; Mean ± SD; *p < 0.05; *** < p < 0.001 by two‐tailed t test. (d) Representative pictures of wild‐type (WT) and SVBP‐KO (SVBP ^ko) HeLa cells stained with anti‐alpha‐tubulin (α‐tub, green) and DAPI (blue) at passages >10, showing the presence of multinucleated cells (white arrows indicate multinucleated cells); insert shows enlargement of the indicated area. Scale bars, 50 or 10 μm. The percentage of multinucleated and micronucleated wild‐type (WT) and SVBP‐KO (SVBP ^ko) HeLa cells were quantified. n ≥ 50; Mean ± SD; *p < 0.05; **p < 0.01 by two‐tailed t test.

FIGURE 5

SVBP mutant induces cell cycle arrest and senescence. (a) Control (CTL) and patient (P2 and P4) fibroblasts at cell passage >10 were subjected to immunoblot analysis using anti‐p53 antibodies. Total amounts of α‐tubulin were used as a loading control. (b) Quantitative RT‐PCR analysis of p53 gene expression in control (CTL) and patient (SVBP ^mut) fibroblasts at cell passage >10. Two independent experiments were performed. n = 3–2, Mean ± SD; **p < 0.01 by two‐tailed t test. (c) Representative histogram from flow cytometry of control (CTL) and patient fibroblasts (SVBP ^mut) positive for Annexin V‐APC at cell passage >10. Two independent experiments were performed. n = 3–1, Mean ± SD; *p < 0.05; **p < 0.01 by one‐way ANOVA with Tukey post hoc tests. (d) Representative flow cytometry profiles of control (CTL) and patient (SVBP ^mut) fibroblasts labeled with EdU at cell passage >10. (e) Quantification of EdU positive cells. Two independent experiments were performed. n = 3–2, Mean ± SD; *, p < 0.05 by two‐tailed t test. (f) Representative flow cytometry profiles of control (CTL) and patient (SVBP ^mut) fibroblasts labeled with H3Ser28P at cell passage >10. (g) Quantification of H3Ser28P positive cells. Two independent experiments were performed. n = 3–2, Mean ± SD; *, p < 0.05 by two‐tailed t test. (h) Representative pictures of control (CTL) and patient (P2 and P4) fibroblasts at cell passage >10 stained for SA‐β‐gal activity. The percentage of SA‐β‐gal‐positive cells was quantified. Two independent experiments were performed. n = 3–1, Mean ± SD; **p < 0.01; *** < p < 0.001 by one‐way ANOVA with Tukey post hoc tests. (i) Quantitative RT‐PCR analysis of Lmnb1, p21, p16, and IL‐6 gene expression in control (CTL) and patient (SVBP ^mut) fibroblasts at cell passage >10. Two independent experiments were performed. n = 3–2, Mean ± SD; *, p < 0.05; *** < p < 0.001 by two‐tailed t test. (j) Control (CTL) and patient (P2 and P4) fibroblasts at cell passage >10 were subjected to immunoblot analysis using anti‐γH2AX and anti‐H2AX antibodies. (k) Quantitative RT‐PCR analysis of, p16 gene expression in control (CTL) and patient (SVBP ^mut) PBMC. Two independent experiments were performed. n = 17–20, Mean ± SD; *p < 0.05; **p < 0.01; *** < p < 0.001 by two‐tailed t test.