Differential requirements of tubulin genes in mammalian forebrain development

Abstract

KEY WORDSTubulin human forebrain development ABSTRACT Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations results from de novo heterozygous mutations in tubulin genes. However, the absolute requirement for most of these genes in development and disease has not been previously tested in genetic, loss of function models. Here we present two novel pathogenic tubulin alleles: a human TUBA1A missense variant with a phenotype more severe than most tubulinopathies and a mouse ENU allele of Tuba1a. Furthermore, we directly test the requirement for Tuba1a, Tuba8, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tuba8, Tubb2a or Tubb2b does not lead to cortical malformation phenotypes or impair survival. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. Thus, despite their functional similarity, the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.

Microtubule Dysfunction: A Common Feature of Neurodegenerative Diseases

“…Using real-time PCR experiments, Breuss and colleagues have demonstrated lower expression of tubb2a during brain development, which reached its highest level at E14.5, E16.5, and around the time of birth [ 78 ]. This finding suggests that this tubulin carries out relatively minor functions during brain development, in accordance with the mild brain phenotypes observed in patients harboring TUBB2A mutations [ 91 , 92 , 93 ] and the recent finding that the loss of tubb2a leads to relatively mild cortical malformations in mice [ 10 ]. Accordingly, we have recently described a novel TUBB2A mutation associated with a phenotype presenting peripheral sensory motor polyneuropathy without cortical malformation [ 94 ].…”

“…In many organisms, both α and β tubulins are encoded by multiple genes at different loci [ 8 , 9 ]. Most of them, such TUBB2A , TUBB2B , and TUBA1A , TUBA1B , TUBA1C , are clustered in the genome and are presumably the products of genomic duplications [ 10 ]. In vitro experiments have shown that tubulin isotypes have different effects on MT dynamics [ 11 , 12 , 13 , 14 ], suggesting that they can differentially regulate the MT network in vivo.…”

“…Expression studies on its mRNA have shown that that this isotype is the most prevalent α-tubulin expressed in embryonic nervous system, representing more than 95% of total α-tubulin mRNA [ 106 , 107 ], consistent with its pathogenic role in migration disorders such as pachygyria, lissencephaly, and polymicrogyria [ 104 , 108 , 109 , 110 , 111 , 112 , 113 , 114 ]. The loss of Tuba1a in mouse is perinatal lethal and leads to significant forebrain dysmorphology [ 10 ].…”

Tubulin mutations in brain development disorders: Why haploinsufficiency does not explain TUBA1A tubulinopathies

“…Insight to mechanistic questions can be gained from examining available mammalian models of Tuba1a mutations. Homozygous Tuba1a deletion results in severe brain defects including disruption of cortical layering, enlargement of cerebral and third ventricles, and a reduction in the size of the basal ganglia (Bittermann et al, ). In contrast, no brain defects were noted in heterozygous Tuba1a knock‐out mice (Bittermann et al, ).…”

Reduced TUBA1A Tubulin Causes Defects in Trafficking and Impaired Adult Motor Behavior

“…To overcome the technical limitations to studying the specific impact of TUBA1A in adult 76 neurons, we use the Tuba1a ND mutant mouse which harbors an asparagine to aspartic acid substitution at 77 amino acid residue 102 (Gartz Hanson et al, 2016). Homozygous Tuba1a ND/ND mice are neonatal lethal 78 and have severe brain malformations consistent with homozygous Tuba1a null and Tuba1a quas mutant mice 79 (Bittermann et al, 2019) and similar to phenotypes observed in human patients with heterozygous 80 TUBA1A mutations (Aiken et al, 2017;Gartz Hanson et al, 2016). The corresponding ND substitution in 81 the primary -tubulin in yeast decreased -tubulin protein levels, altered microtubule dynamics, reduced 82 incorporation of the mutant -tubulin into the lattice, and impaired mitotic division (Gartz Hanson et al, 83 2016).…”