Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy

Abstract

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with impact on neuronal function and survival in the developing brain.

Figure 1

Clinical Features of Subjects with Biallelic TBCD Mutations (A) Features of affected subjects. Note microcephaly in subjects F118_346, F118_347, 3641284, and 1455707 and tetraplegia in F118_346 and F18_347. (B) Brain MRIs of individuals F118_347 (8 months old; top left three panels) and F118_346 (2 years old; middle left three panels) showing axial, sagittal, and coronal views (from the left). F118_347 exhibits diffuse prominence of the cortical sulci with thinned corpus callosum and white matter volume loss with hypomyelination; the eldest sib exhibits more severe MRI features, with diffusely widened cortical sulci and cerebellar foliae and enlargement of lateral and third ventricles. MRIs of subjects CMH445 (16 years old; top right two panels) and CMH444 (13 years old; middle two right panels). In the former, diffuse prominence of the cortical sulci and cerebellar foliae with mild enlargement of the lateral and third ventricles and thinned corpus callosum are documented. In the latter, a milder pattern is observed, documenting a thin corpus callosum and partial white matter hypomyelination and mild cortical atrophy. MRIs of subjects 3641284 (3.5 years old; bottom three left panels) and 6215546 (17 years old; bottom right two panels). In the former, enlarged ventricles and generalized atrophy of gray matter, thin corpus callosum, cerebellar vermis atrophy, and abnormal white matter T2 and FLAIR signal hyperintensity due to severe secondary hypomyelination are observed. In the latter, slightly enlarged posterior lateral ventricles, mild cortical atrophy, hypoplastic splenium of the corpus callosum, and ectopic neuropituitary are noted.

Figure 2

Disease-Causing TBCD Mutations Affect TBCD Stability and Function (A) Western blot (WB) analysis showing decreased level of TBCD in fibroblasts from affected individuals 3641284, 6215546, and F118_346 compared to control cells (WT) (above). Levels of Myc-tagged TBCD^p.Thr374Met (374), TBCD^p.Arg377Gln (377), TBCD^p.Ala626Thr (626), and TBCD^p.Pro1122Leu (1,122) mutants were confirmed to be variably reduced compared to wild-type TBCD (WT) by transient expression experiments in HeLa and HEK293T cells (below). (B) TBCD^p.Thr374Met (374) and TBCD^p.Arg377Gln (377) mutants display defective TBCD:β-tubulin:ARL2 complex formation. Constructs encoding the GST-tagged TBCD proteins were co-expressed with ARL2 in HEK293T cells and affinity purified, followed by GST cleavage. Proteins were resolved by SDS-PAGE and stained with Coomassie blue.

Figure 3

Defective TBCD Function Impacts Microtubule Dynamics (A) Increased levels of polymerized α-tubulin in fibroblasts with biallelic mutations in TBCD. Representative western blots (WB) of total (T), soluble (S), and polymerized (P) pools of α-tubulin in untreated cells during interphase (left) and synchronized cells during mitosis (right). Equal amounts of proteins from both fractions were loaded onto SDS-PAGE and analyzed by WB probing with anti-α-tubulin antibody. Densitometry is reported. Data are expressed as means ± SD; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (B) Accelerated microtubule re-polymerization in primary fibroblasts obtained from subjects with biallelic TBCD mutations. Representative confocal microscopy analysis of fibroblasts from affected subjects after nocodazole treatment showing accelerated microtubule re-polymerization compared to control cells (left). Fixed cells were stained with anti-α-tubulin antibody (red) and DAPI (blue). The scale bar (47 μm) is the same for all panels. Quantification of total fluorescence normalized on cell number, referred to the exponential phase of re-polymerization (5 to 20 min), is also shown (right).

Figure 4

Defective TBCD Function Affects the Mitotic Spindle Confocal microscopy analysis was performed in synchronized skin fibroblasts from subjects with biallelic TBCD mutations and control cells. Images are representative of each stage of the cell cycle. Cells were stained using antibodies against pericentrin (red, centrosome marker) and β-tubulin (green, marker for microtubules and mitotic spindle); chromosomes are DAPI stained (blue). Scale bars represent 6 μm.