TUBB3 Mutations Research Articles

Beta tubulin isoforms are not interchangeable for rescuing impaired radial migration due to Tubb3 knockdown

Over the last years, the critical role of cytoskeletal proteins in cortical development including neuronal migration as well as in neuronal morphology has been well established. Inputs from genetic studies were provided through the identification of several mutated genes encoding either proteins associated with microtubules (DCX, LIS1, KIF2A, KIF5C, DYNC1H1) or tubulin subunits (TUBA1A, TUBB2B, TUBB5 and TUBG1), in malformations of cortical development (MCD). We also reported the identification of missense mutations in TUBB3, the postmitotic neuronal specific tubulin, in six different families presenting either polymicrogyria or gyral disorganization in combination with cerebellar and basal ganglial abnormalities. Here, we investigate further the association between TUBB3 mutations and MCDs by analyzing the consequences of Tubb3 knockdown on cortical development in mice. Using the in utero-electroporation approach, we demonstrate that Tubb3 knockdown leads to delayed bipolar morphology and radial migration with evidence, suggesting that the neuronal arrest is a transient phenomenon overcome after birth. Silenced blocked cells display a round-shape and decreased number of processes and a delay in the acquisition of the bipolar morphology. Also, more Tbr2 positive cells are observed, although less cells express the proliferation marker Ki67, suggesting that Tubb3 inactivation might have an indirect effect on intermediate progenitor proliferation. Furthermore, we show by rescue experiments the non-interchangeability of other beta-tubulins which are unable to rescue the phenotype. Our study highlights the critical and specific role of Tubb3 on the stereotyped morphological changes and polarization processes that are required for initiating radial migration to the cortical plate.

Off the rails: axonal cargoes on the road to nowhere

EMBO J (2013) 32 10, 1352–1364. doi:10.1038/emboj.2013.59 The importance of the microtubule cytoskeleton for neuronal development, organization and maintenance is reflected in the finding that mutations in various tubulin genes cause a range of nervous system abnormalities. Mutations in the β-tubulin isotype-III gene, TUBB3, alter the interaction between microtubules and motor proteins leading to neurological impairments. Niwa et al (2013) now report that TUBB3 mutations lead to neuronal defects by impairing axonal transport.

Complication begets clarification in classification

As recently as 12 years ago, breakthroughs aided by laboratory neuroscience and modern neuroimaging had made differentiation and classification of malformations of cortical development seem straightforward (Barkovich et al. , 2001). Some resulted from abnormal neuronal proliferation (microcephalies, megalencephalies), others from abnormal neuronal migration (heterotopia and lissencephaly), and still others from abnormal cortical organization (mostly polymicrogyrias). The protein products of the few genes known to be associated with these disorders ( LIS1 and DCX with lissencephaly; FLNA with heterotopia) support these mechanistic concepts, as the lissencephaly genes are important in the migration of neurons (specifically in the extension of leading processes and nucleokinesis, the process by which the nucleus is able to follow the leading process); and the assumption that mutations of FLNA (which produces an actin cross-linking phosphoprotein important for cell locomotion) in neurons impair their migration is logical. Although details needed clarification, everything seemed to make sense. The discovery that tubulin mutations cause brain malformations was easily incorporated into existing malformation classifications. That some infants with lissencephaly had mutations of TUBA1A (Poirier et al. , 2007) did not dislodge existing concepts, as both LIS1 and DCX were known to be microtubule-associated proteins (MAPs), which complex with microtubules and/or microtubule motor proteins during cell migration. Reports that mutations of TUBB2B are associated with polymicrogyria (Jaglin et al. , 2009) posed more difficulties, but caused no great surprise, as polymicrogyria is hard to explain. This malformation of cortical development is associated with many different conditions, including prenatal injury (infection or ischaemia) and many mutations. The causative mechanisms are unknown, and the appearance on imaging varies greatly. Still, it was easy to accept that disruption of the complex (and poorly understood) processes of terminal neuronal migration and cortical organization could result from TUBB2B mutations. Similarly, the report that TUBB3 mutations are associated …

A novel syndrome caused by the E410K amino acid substitution in the neuronal β-tubulin isotype 3

Missense mutations in TUBB3, the gene that encodes the neuronal-specific protein β-tubulin isotype 3, can cause isolated or syndromic congenital fibrosis of the extraocular muscles, a form of complex congenital strabismus characterized by cranial nerve misguidance. One of the eight TUBB3 mutations reported to cause congenital fibrosis of the extraocular muscles, c.1228G>A results in a TUBB3 E410K amino acid substitution that directly alters a kinesin motor protein binding site. We report the detailed phenotypes of eight unrelated individuals who harbour this de novo mutation, and thus define the 'TUBB3 E410K syndrome'. Individuals harbouring this mutation were previously reported to have congenital fibrosis of the extraocular muscles, facial weakness, developmental delay and possible peripheral neuropathy. We now confirm by electrophysiology that a progressive sensorimotor polyneuropathy does indeed segregate with the mutation, and expand the TUBB3 E410K phenotype to include Kallmann syndrome (hypogonadotropic hypogonadism and anosmia), stereotyped midface hypoplasia, intellectual disabilities and, in some cases, vocal cord paralysis, tracheomalacia and cyclic vomiting. Neuroimaging reveals a thin corpus callosum and anterior commissure, and hypoplastic to absent olfactory sulci, olfactory bulbs and oculomotor and facial nerves, which support underlying abnormalities in axon guidance and maintenance. Thus, the E410K substitution defines a new genetic aetiology for Moebius syndrome, Kallmann syndrome and cyclic vomiting. Moreover, the c.1228G>A mutation was absent in DNA from ∼600 individuals who had either Kallmann syndrome or isolated or syndromic ocular and/or facial dysmotility disorders, but who did not have the combined features of the TUBB3 E410K syndrome, highlighting the specificity of this phenotype-genotype correlation. The definition of the TUBB3 E410K syndrome will allow clinicians to identify affected individuals and predict the mutation based on clinical features alone.

P.11 The phenotype in E410K Beta-tubulin isotype 3 mutations

IntroductionThe Congenital Cranial Dysinnervation Disorders are congenital, non-progressive disorders that result from developmental abnormalities of one or more cranial nerves/nuclei with primary or secondary dysinnervation and include Duane's syndrome, Möbius...

Evidence of an Asymmetrical Endophenotype in Congenital Fibrosis of Extraocular Muscles Type 3 Resulting fromTUBB3Mutations

Orbital magnetic resonance imaging (MRI) was used to investigate the structural basis of motility abnormalities in congenital fibrosis of the extraocular muscles type 3 (CFEOM3), a disorder resulting from missense mutations in TUBB3, which encodes neuron-specific beta-tubulin isotype III. Ophthalmic examinations in 13 volunteers from four CFEOM3 pedigrees and normal control subjects, were correlated with TUBB3 mutation and MRI findings that demonstrated extraocular muscle (EOM) size, location, contractility, and innervation. Volunteers included clinically affected and clinically unaffected carriers of R262C and D417N TUBB3 amino acid substitutions and one unaffected, mutation-negative family member. Subjects with CFEOM3 frequently had asymmetrical blepharoptosis, limited vertical duction, variable ophthalmoplegia, exotropia, and paradoxical abduction in infraduction. MRI demonstrated variable, asymmetrical levator palpebrae superioris and superior rectus EOM atrophy that correlated with blepharoptosis, deficient supraduction, and small orbital motor nerves. Additional EOMs exhibited variable hypoplasia that correlated with duction deficit, but the superior oblique muscle was spared. Ophthalmoplegia occurred only when the subarachnoid width of CN3 was <1.9 mm. A-pattern exotropia was frequent, correlating with apparent lateral rectus (LR) muscle misinnervation by CN3. Optic nerve (ON) cross sections were subnormal, but rectus pulley locations were normal. CFEOM3 caused by TUBB3 R262C and D417N amino acid substitutions features abnormalities of EOM innervation and function that correlate with subarachnoid CN3 hypoplasia, occasional abducens nerve hypoplasia, and subclinical ON hypoplasia that can resemble CFEOM1. Clinical and MRI findings in CFEOM3 are more variable than those in CFEOM1 and are often asymmetrical. Apparent LR innervation by the inferior rectus motor nerve is an overlapping feature of Duane retraction syndrome and CFEOM1. These findings suggest that CFEOM3 is an asymmetrical, variably penetrant, congenital cranial dysinnervation disorder leading to secondary EOM atrophy.

Human TUBB3 Mutations Perturb Microtubule Dynamics, Kinesin Interactions, and Axon Guidance

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.