H-ABC- and dystonia-causing TUBB4A mutations show distinct pathogenic effects

Abstract

Mutations in the brain-specific β-tubulin 4A (TUBB4A) gene cause a broad spectrum of diseases, ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Currently, the mechanisms of how TUBB4A variants lead to this pleiotropic manifestation remain elusive. Here, we investigated whether TUBB4A mutations causing either DYT-TUBB4A (p.R2G and p.Q424H) or H-ABC (p.R2W and p.D249N) exhibit differential effects at the molecular and cellular levels. Using live-cell imaging of disease-relevant oligodendrocytes and total internal reflection fluorescence microscopy of whole-cell lysates, we observed divergent impact on microtubule polymerization and microtubule integration, partially reflecting the observed pleiotropy. Moreover, in silico simulations demonstrated that the mutants rarely adopted a straight heterodimer conformation in contrast to wild type. In conclusion, for most of the examined variants, we deciphered potential molecular disease mechanisms that may lead to the diverse clinical manifestations and phenotype severity across and within each TUBB4A-related disease.

Fig. 1.. Microtubule incorporation assay of GFP-tagged WT and mutant TUBB4A in U-2 OS cells.

(A) Immunofluorescence staining of U-2 OS cells overexpressing either GFP or TUBB4A-GFP variants (inverted gray scale) against pan-α-tubulin (inverted gray scale). While the fluorescence signals of GFP and α-tubulin do not clearly overlap in the control, all TUBB4A forms show a prominent microtubule staining in both TUBB4A-GFP and α-tubulin channels and robust colocalization (merge, yellow). Scale bars, 20 and 5 μm (magnified panels). Immunoblots against TUBB4A-GFP and pan-α-tubulin (B) or pan-β-tubulin (C) show no alterations in tubulin composition or TUBB4A stability. Uncropped immunoblots are shown in fig. S3. In sum, H-ABC–linked but not dystonia-linked TUBB4A mutations show impaired microtubular integration.

Fig. 2.. Data distribution of microtubule growth parameters from whole-cell lysates containing WT or mutant TUBB4A.

(A) TIRF microscopy time-lapse images at different time points (0:30, 3:00, and 6:35 min:s) of whole-cell lysates, showing EB3-GFP (green)–decorated growing microtubules from GMPCPP-stabilized and rhodamine-labeled seeds (red). Representative kymographs are shown to the right of the corresponding image sequences (white bars, 2 μm). (B to E) Data distribution and statistical analysis of EB3 comet (B) velocity, (C) growth events per microtubule, (D) run length, and (E) run time. Run length was defined as the microtubule growth distance between two pause events and run time as the growth duration between two pause events. The corresponding microtubule growth rate (velocity) was determined for each growth step. As growth event, every microtubule regrowth was counted [see enlargements in (A)]. The boxes show the quartiles of the dataset, the whiskers represent 1.5× of the interquartile range, the median values are indicated as horizontal lines inside the boxes, and data points outside the whiskers are marked as black diamonds. The mean values of the complete biological replicates are shown as filled circles on the right side of each boxplot. Statistical analysis of four to five independent experiments (transfection, cell culture, lysate preparation, and TIRF imaging) was applied by one-way analysis of variance (ANOVA) and post hoc Dunnett test. In total, 56 to 76 microtubules per TUBB4A form were analyzed. In sum, H-ABC–linked TUBB4A mutants display reduced microtubule growth in all parameters but elevated growth events, whereas p.Q424H led to faster and longer microtubule growth, with fewer growth interruptions. MT, microtubule.

Fig. 3.. Live-cell imaging of EB3-GFP comets in primary oligodendrocytes expressing either WT or mutant forms of TUBB4A.

(A) Differentiation scheme for primary murine oligodendrocytes overexpressing WT or disease-causing TUBB4A mutations. (B) Example of tracked EB3 comets (red lines) in an oligodendrocyte either in the soma or along with processes. The region of interest (purple dotted line) either includes (soma) or excludes (processes) comet detection. Scale bar, 10 μm. (C) Representative kymographs display EB3-GFP comet trajectories for all TUBB4A forms in processes. Please note the declining comet speed at distal ends. Scale bars, 5 μm. Data distribution of microtubule growth velocity (D), EB3-GFP comet displacement (E), and run time (F). Data derived from the oligodendroctye processes are shown hatched. Statistical analysis: One-way ANOVA followed by Dunnett post hoc test; black horizontal lines inside each box indicate the median; whiskers represent 1.5× of the interquartile range. Outliers are visualized as black diamonds. Two independent experiments in total of 15 to 16 cells per TUBB4A variant. DIV, days in vitro. In sum, disease-causing TUBB4A mutations disrupt microtubule growth in oligodendrocytes in congruence with the severity of the corresponding disease phenotype.

Fig. 4.. In silico determination of tubulin heterodimer conformation.

(A) Position of the modeled and simulated TUBB4A mutations in the intradimer region of a tubulin heterodimer. For better visualization, the nucleotides guanosine triphosphate and guanosine diphosphate are not shown. (B) Illustration of the main conformational changes of the heterodimer. (C) For the bending angle, the data distribution for WT and p.D294N is bimodal. For p.R2G and p.R2W, however, the data distribution is unimodal. Furthermore, all mutants show a right shift of the data compared to WT, which indicates that the mutant formed a rather curved heterodimer conformation. (D) The twist angle data distribution is unimodal for WT, p.R2G, and p.D294N, whereas it is bimodal for p.R2W. In sum, TUBB4A mutations located in the intradimer region cause differential bending and twisting conformations of the tubulin heterodimer.

Fig. 5.. Relative interaction frequency of salt bridges and hydrogen bonds within the intradimer contact site.

(A) Schematic representation of interaction cluster positions in the heterodimer (side view). (B) Heatmap for salt bridges and hydrogen bond contact frequencies. Determining the relative salt bridge and hydrogen bond frequency revealed a destabilization in the luminal intradimer region in all mutants. The p.R2W mutant showed a stabilization in the surface cluster and a destabilization in the central intradimer (intra) cluster. In contrast, the p.D249N mutant displayed stabilization in the surface and intraclusters. (C to F) Occurrence of three salt bridges during conformal changes of the heterodimers. Salt bridges (TUBA1A:TUBB4A): lumen (p.R79:p.E45), central intradimer (p.E71:p.K252), and surface (p.K427:p.K401). In sum, the extent to which the luminal heterodimer interactions destabilize and the surface/central intradimer interactions stabilize is consistent with disease phenotype severity.

Fig. 6.. Interpretation of the observed effects of TUBB4A mutants on microtubular dynamics, heterodimer flexibility, and oligodendrocytic fate.

(A) WT TUBB4A can incorporate into microtubules, leading to elongation. (B) MD analysis showed that the WT heterodimer adopts a range of straight and curved conformations. (C) H-ABC–causing mutants demonstrated reduced incorporation into microtubules and slower growth. Because of the changes in microtubule dynamics over time, it is tempting to speculate that the mutants accumulate in the free tubulin pool. Ultimately, although similar tubulin concentrations could be present compared to WT, the amount of incorporable tubulin might be reduced, slowing microtubule growth. (D) The in silico simulation of intradimer mutations revealed that the straight conformation is less likely adopted, possibly leading to sterical hindrances during microtubule polymerization. (E) We hypothesize that TUBB4A mutations severely affecting microtubule dynamics disrupt proliferating oligodendrocytes. This results in fewer myelin-forming cells, leading to hypomyelination shortly after birth. (F) Moderate to severe TUBB4A mutations presumably enable the formation of myelinating oligodendrocytes. However, the myelination efficiency is markedly reduced because of microtubule alterations, displayed by hypomyelination at subtle later time points. In contrast, dystonia-related TUBB4A mutations might only lead to minor changes within oligodendrocytes, putatively resulting in altered signal transduction, while MRI scans of the brain show no apparent loss of white matter.