Codon modification of Tuba1a alters mRNA levels and causes a severe neurodevelopmental phenotype in mice.

The tubulinopathies are an umbrella of rare diseases that result from mutations in tubulin genes and are frequently characterised by severe brain malformations. The characteristics of a given disease reflect the expression pattern of the transcript, the function of a given tubulin gene, and the role microtubules play in a particular cell type. Mouse models have proved to be valuable tools that have provided insight into the molecular and cellular mechanisms that underlie the disease state. In this manuscript we compare two Tuba1a mouse models, both of which express wild-type TUBA1A protein but employ different codon usage. We show that modification of the Tuba1a mRNA sequence results in homozygous lethality and a severe neurodevelopmental phenotype. This is associated with a decrease in the number of post-mitotic neurons, PAX6 positive progenitors, and an increase in the number of apoptotic cells. We attribute this to a decrease in the stability of the modified Tuba1a transcript, and the absence of compensation by the other neurogenic tubulins. Our findings highlight the importance of maintaining the wild-type coding sequence when engineering mouse lines and the impact of synonymous genetic variation.

A proteomic survey of microtubule-associated proteins in a R402H TUBA1A mutant mouse.

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs.

A high sensitivity ZENK monoclonal antibody to map neuronal activity in Aves.

The transcription factor ZENK is an immediate early gene that has been employed as a surrogate marker to map neuronal activity in the brain. It has been used in a wide variety of species, however, commercially available antibodies have limited immunoreactivity in birds. To address this issue we generated a new mouse monoclonal antibody, 7B7-A3, raised against ZENK from the rock pigeon (Columba livia). We show that 7B7-A3 labels clZENK in both immunoblots and histological stainings with high sensitivity and selectivity for its target. Using a sound stimulation paradigm we demonstrate that 7B7-A3 can detect activity-dependent ZENK expression at key stations of the central auditory pathway of the pigeon. Finally, we compare staining efficiency across three avian species and confirm that 7B7-A3 is compatible with immunohistochemical detection of ZENK in the rock pigeon, zebra finch, and domestic chicken. Taken together, 7B7-A3 represents a useful tool for the avian neuroscience community to map functional activity in the brain.

A Putative Mechanism for Magnetoreception by Electromagnetic Induction in the Pigeon Inner Ear.

A diverse array of vertebrate species employs the Earth's magnetic field to assist navigation. Despite compelling behavioral evidence that a magnetic sense exists, the location of the primary sensory cells and the underlying molecular mechanisms remain unknown [1]. To date, most research has focused on a light-dependent radical-pair-based concept and a system that is proposed to rely on biogenic magnetite (Fe3O4) [2, 3]. Here, we explore an overlooked hypothesis that predicts that animals detect magnetic fields by electromagnetic induction within the semicircular canals of the inner ear [4]. Employing an assay that relies on the neuronal activity marker C-FOS, we confirm that magnetic exposure results in activation of the caudal vestibular nuclei in pigeons that is independent of light [5]. We show experimentally and by physical calculations that magnetic stimulation can induce electric fields in the pigeon semicircular canals that are within the physiological range of known electroreceptive systems. Drawing on this finding, we report the presence of a splice isoform of a voltage-gated calcium channel (CaV1.3) in the pigeon inner ear that has been shown to mediate electroreception in skates and sharks [6]. We propose that pigeons detect magnetic fields by electromagnetic induction within the semicircular canals that is dependent on the presence of apically located voltage-gated cation channels in a population of electrosensory hair cells.

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

Lidocaine is a nocebo treatment for trigeminally mediated magnetic orientation in birds.

Even though previously described iron-containing structures in the upper beak of pigeons were almost certainly macrophages, not magnetosensitive neurons, behavioural and neurobiological evidence still supports the involvement of the ophthalmic branch of the trigeminal nerve (V1) in magnetoreception. In previous behavioural studies, inactivation of putative V1-associated magnetoreceptors involved either application of the surface anaesthetic lidocaine to the upper beak or sectioning of V1. Here, we compared the effects of lidocaine treatment, V1 ablations and sham ablations on magnetic field-driven neuronal activation in V1-recipient brain regions in European robins. V1 sectioning led to significantly fewer Egr-1-expressing neurons in the trigeminal brainstem than in the sham-ablated birds, whereas lidocaine treatment had no effect on neuronal activation. Furthermore, Prussian blue staining showed that nearly all iron-containing cells in the subepidermal layer of the upper beak are nucleated and are thus not part of the trigeminal nerve, and iron-containing cells appeared in highly variable numbers at inconsistent locations between individual robins and showed no systematic colocalization with a neuronal marker. Our data suggest that lidocaine treatment has been a nocebo to the birds and a placebo for the experimenters. Currently, the nature and location of any V1-associated magnetosensor remains elusive.

Mutations in Either TUBB or MAPRE2 Cause Circumferential Skin Creases Kunze Type.

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a ß-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.

TUBB5 and its disease-associated mutations influence the terminal differentiation and dendritic spine densities of cerebral cortical neurons.

The microtubule cytoskeleton is critical for the generation and maturation of neurons in the developing mammalian nervous system. We have previously shown that mutations in the ß-tubulin gene TUBB5 cause microcephaly with structural brain abnormalities in humans. While it is known that TUBB5 is necessary for the proper generation and migration of neurons, little is understood of the role it plays in neuronal differentiation and connectivity. Here, we report that perturbations to TUBB5 disrupt the morphology of cortical neurons, their neuronal complexity, axonal outgrowth, as well as the density and shape of dendritic spines in the postnatal murine cortex. The features we describe are consistent with defects in synaptic signaling. Cellular-based assays have revealed that TUBB5 substitutions have the capacity to alter the dynamic properties and polymerization rates of the microtubule cytoskeleton. Together, our studies show that TUBB5 is essential for neuronal differentiation and dendritic spine formation in vivo, providing insight into the underlying cellular pathology associated with TUBB5 disease states.

High resolution anatomical mapping confirms the absence of a magnetic sense system in the rostral upper beak of pigeons.

The cells that are responsible for detecting magnetic fields in animals remain undiscovered. Previous studies have proposed that pigeons employ a magnetic sense system that consists of six bilateral patches of magnetite containing dendrites located in the rostral subepidermis of the upper beak. We have challenged this hypothesis arguing that clusters of iron-rich cells in this region are macrophages, not magnetosensitive neurons. Here we present additional data in support of this conclusion. We have undertaken high resolution anatomical mapping of iron-rich cells in the rostral upper beak of pigeons, excluding the possibility that a conserved six-loci magnetic sense system exists. In addition we have extended our immunohistochemical studies to a second cohort of pigeons, confirming that iron rich cells in the upper beak are positive for MHCII and CD44, which are expressed by macrophages. We argue that it is important to critically assess conclusions that have been made in the past, while keeping an open mind as the search for the magnetoreceptor continues.

An iron-rich organelle in the cuticular plate of avian hair cells.

Hair cells reside in specialized epithelia in the inner ear of vertebrates, mediating the detection of sound, motion, and gravity. The transduction of these stimuli into a neuronal impulse requires the deflection of stereocilia, which are stabilized by the actin-rich cuticular plate. Recent electrophysiological studies have implicated the vestibular system in pigeon magnetosensation. Here we report the discovery of a single iron-rich organelle that resides in the cuticular plate of cochlear and vestibular hair cells in the pigeon. Transmission electron microscopy, coupled with elemental analysis, has shown that this structure is composed of ferritin-like granules, is approximately 300-600 nm in diameter, is spherical, and in some instances is membrane-bound and/or organized in a paracrystalline array. This organelle is found in hair cells in a wide variety of avian species, but not in rodents or in humans. This structure may function as (1) a store of excess iron, (2) a stabilizer of stereocilia, or (3) a mediator of magnetic detection. Given the specific subcellular location, elemental composition, and evolutionary conservation, we propose that this structure is an integral component of the sensory apparatus in birds.

Mutations in the ß-tubulin gene TUBB5 cause microcephaly with structural brain abnormalities.

The formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly (TUBA1A), polymicrogyria (TUBA1A, TUBB2B, TUBB3), and an ocular motility disorder (TUBB3). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperone-dependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly.

Clusters of iron-rich cells in the upper beak of pigeons are macrophages not magnetosensitive neurons.

Understanding the molecular and cellular mechanisms that mediate magnetosensation in vertebrates is a formidable scientific problem. One hypothesis is that magnetic information is transduced into neuronal impulses by using a magnetite-based magnetoreceptor. Previous studies claim to have identified a magnetic sense system in the pigeon, common to avian species, which consists of magnetite-containing trigeminal afferents located at six specific loci in the rostral subepidermis of the beak. These studies have been widely accepted in the field and heavily relied upon by both behavioural biologists and physicists. Here we show that clusters of iron-rich cells in the rostro-medial upper beak of the pigeon Columbia livia are macrophages, not magnetosensitive neurons. Our systematic characterization of the pigeon upper beak identified iron-rich cells in the stratum laxum of the subepidermis, the basal region of the respiratory epithelium and the apex of feather follicles. Using a three-dimensional blueprint of the pigeon beak created by magnetic resonance imaging and computed tomography, we mapped the location of iron-rich cells, revealing unexpected variation in their distribution and number--an observation that is inconsistent with a role in magnetic sensation. Ultrastructure analysis of these cells, which are not unique to the beak, showed that their subcellular architecture includes ferritin-like granules, siderosomes, haemosiderin and filopodia, characteristics of iron-rich macrophages. Our conclusion that these cells are macrophages and not magnetosensitive neurons is supported by immunohistological studies showing co-localization with the antigen-presenting molecule major histocompatibility complex class II. Our work necessitates a renewed search for the true magnetite-dependent magnetoreceptor in birds.

Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria.

Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. Here we report de novo mutations in a beta-tubulin gene, TUBB2B, in four individuals and a 27-gestational-week fetus with bilateral asymmetrical polymicrogyria. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to impaired formation of tubulin heterodimers. These observations, together with previous data, show that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly and pachygyria, but also polymicrogyria malformations.