Mutation of the variant alpha-tubulin TUBA8 results in polymicrogyria with optic nerve hypoplasia

Abstract

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.

Figure 1

Pedigrees of Two Families with PMG and Optic Hypoplasia In each case, the proband is indicated by an arrow.

Figure 2

Magnetic Resonance Images of Affected Individual IV:1 from Family 2 (A) The corpus callosum is absent, as is the cingulate gyrus (hollow white arrow). Disorganized gyral folds radiate toward a high-riding third ventricle. A prominent massa intermedia is seen (black arrow). There is malformation of the brainstem, with loss of demarcation of the pontomedullary junction (solid white arrow). (B) The cortex is thickened and the gyral pattern is abnormal. There is generalized PMG (black arrows), and agenesis of the corpus callosum has resulted in colpocephaly (white arrows). (C) Coronal section at the level of the third ventricle, demonstrating absence of the corpus callosum with a “Viking helmet” configuration of the frontal horns and a high-riding third ventricle.

Figure 3

Splicing Mutation of TUBA8 in PMGOH-Affected Individuals (A) Schematic to show site and extent of 14-bp deletion mutation. (B) Reverse transcription PCR analysis of LCL RNA. The skipped exon 2 is shaded light gray, coding regions of other exons are shown in dark gray. The following abbreviations are used: N, normal control; H, heterozygote; and P, patient (homozygous).

Figure 4

Tuba8 Expression Studied with In Situ Hybridization during Mouse Brain Development (A–D) Coronal sections at E13.5, E15.5, E18, and P0. (E) Sagittal section at P8. In (A)–(E), dashed boxes refer to areas shown enlarged in (F)–(J). (K–N) Hippocampus at E15, E18, P0, and P8. (O) Cerebellum at P8. (A and F) At E13.5 there is generally stronger expression in the postmitotic layers of the telencephalon, such as dorsal cortex and cingulate cortex (CiC), ganglionic eminence (GE). There is also evidence of expression in migratory cells of the SVZ/mantle zone of the ganglionic eminence (GE). (B and G) By E15.5 (B) the strongest expression is in the emerging cortical plate of the dorsal cortex, in perirhinal cortex (PRh), CiC and GE. High magnification (G) reveals most intense expression in cortical plate (CP), but there is also expression in the subventricular zone (SVZ), and intermediate zone (IZ). (C and H) At E18.5 (C) the cortical expression becomes more intense in the upper layers and subplate, with relatively less expression in putative layer VI. The VZ has stronger expression at this age (H) than at E15.5 (G). The dorsal and perirhinal cortices have stronger expression than piriform cortex (Pir). (D and I) At P0 the strongest cortical expression is in the layer of still migrating cortical neurons of the dense cortical plate (DCP) and subplate (SP). There is continued expression in the perirhinal cortex (PRh). The mediodorsal nuclei (md) of the thalamus (Th) have strong Tuba8 expression. (E and J) By P8, the cortical lamination is close to completion. The strongest expression of Tuba8 is in layers II–III and V and the subplate. The expression in the dorsomedial thalamus has been maintained, and in the olfactory bulb (OB) expression can be seen in the mitral cell layer (MiL). (K) The pyramidal layer of the hippocampus also starts to express Tuba8 from E15. (L) At E 18 there is strong expression of Tuba8 in the CA1-3 region of the hippocampus, with lower expression in dentate gyrus (DG). (M) At P0 there is strong Tuba8 expression in the hippocampus, more intense in regions CA1-3 than the dentate gyrus. (N) At P8 in the hippocampus there is especially strong expression in area CA3, but there is expression in other regions, including the dentate gyrus (DG). (O) Tuba8 expression is seen in the inner part of the external granular layer and possibly molecular layer (EGL/ML) and less intensely in the internal granular layer (IGL). Scale bars represent 150 μm in (A), 500 μm in (B)–(D), 1 mm in (E), 100 μm in (F)–(M), and 250 μm in (N) and (O). The following abbreviations are used: PP, preplate; MZ, marginal zone; and WM, white matter.

Figure 5

GFP-TUBA8 Incorporates into Microtubules within Cells Individual frames from a time-lapse series of MDCKII cells stably transfected with GFP-TUBA8. The fusion protein incorporates weakly into microtubules (A). Microtubule incorporation was consequently most evident in cellular structures containing bundled microtubules, such as the midbody in telophase cells (B, arrow) or the mitotic spindle (metaphase cell shown in C). Images in (A) and (B) are shown in inverted grayscale for improving image contrast, whereas (C) is shown in grayscale. Transfected cells were also fixed and immunostained with antibodies to α-tubulin (D and E, red) and GFP (E, green). Nuclei were counterstained with DAPI (E, blue). (E) shows a merged image. No obvious abnormalities in microtubule organization were observed in cells expressing higher levels of GFP-TUBA8. Scale bars in (A), (B), and (E) represent 20 μm. The scale bar in (C) represents 10 μm.