Martin Breuss

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 β-Tubulin Gene TUBB5 Cause Microcephaly with Structural Brain Abnormalities

Summary 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.

Microtubules and Neurodevelopmental Disease: The Movers and the Makers

The development of the mammalian cortex requires the generation, migration and differentiation of neurons. Each of these cellular events requires a dynamic microtubule cytoskeleton. Microtubules are required for interkinetic nuclear migration, the separation of chromatids in mitosis, nuclear translocation during migration and the outgrowth of neurites. Their importance is underlined by the finding that mutations in a host of microtubule associated proteins cause detrimental neurological disorders. More recently, the structural subunits of microtubules, the tubulin proteins, have been implicated in a spectrum of human diseases collectively known as the tubulinopathies. This chapter reviews the discovery of microtubules, the role they play in neurodevelopment, and catalogues the tubulin isoforms associated with neurodevelopmental disease. Our focus is on the molecular and cellular mechanisms that underlie the pathology of tubulin-associated diseases. Finally, we reflect on whether different tubulin genes have distinct intrinsic functions.

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 β-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.

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death. Highlighted article: New mouse models reveal that human disease-causing mutations in the β-tubulin gene Tubb5 lead to defects in mitotic progression and massive cell death in the cortex.

The expression of tubb2b undergoes a developmental transition in murine cortical neurons

The development of the mammalian brain requires the generation, migration, and differentiation of neurons, cellular processes that are dependent on a dynamic microtubule cytoskeleton. Mutations in tubulin genes, which encode for the structural subunits of microtubules, cause detrimental neurological disorders known as the tubulinopathies. The disease spectra associated with different tubulin genes are overlapping but distinct, an observation believed to reflect functional specification of this multigene family. Perturbation of the β‐tubulin TUBB2B is known to cause polymicrogyria, pachygyria, microcephaly, and axon guidance defects. Here we provide a detailed analysis of the expression pattern of its murine homolog Tubb2b. The generation and characterization of BAC‐transgenic eGFP reporter mouse lines has revealed that it is highly expressed in progenitors and postmitotic neurons during cortical development. This contrasts with the 8‐week‐old cortex, in which Tubb2b expression is restricted to macroglia, and expression is almost completely absent in mature neurons. This developmental transition in neurons is mirrored in the adult hippocampus and the cerebellum but is not a universal feature of Tubb2b; its expression persists in a population of postmitotic neurons in the 8‐week‐old retina. We propose that the dynamic spatial and temporal expression of Tubb2b reflects specific functional requirements of the microtubule cytoskeleton. J. Comp. Neurol. 523:2161–2186, 2015.

Cytoarchitectural disruption of the superior colliculus and an enlarged acoustic startle response in the Tuba1a mutant mouse

The Jenna mutant mouse harbours an S140G mutation in Tuba1a that impairs tubulin heterodimer formation resulting in defective neuronal migration during development. The consequence of decreased neuronal motility is a fractured pyramidal cell layer in the hippocampus and wave-like perturbations in the cerebral cortex. Here, we extend our characterisation of this mouse investigating the laminar architecture of the superior colliculus (SC). Our results reveal that the structure of the SC in mutant animals is intact; however, it is significantly thinner with an apparent fusion of the intermediate grey and white layers. Birthdate labelling at E12.5 and E13.5 showed that the S140G mutation impairs the radial migration of neurons in the SC. A quantitative assessment of neuronal number in adulthood reveals a massive reduction in postmitotic neurons in mutant animals, which we attribute to increased apoptotic cell death. Consistent with the role of the SC in modulating sensorimotor gating, and the circuitry that modulates this behaviour, we find that Jenna mutants exhibit an exaggerated acoustic startle response. Our results highlight the importance of Tuba1a for correct neuronal migration and implicate postnatal apoptotic cell death in the pathophysiological mechanisms underlying the tubulinopathies.

Tubulins and brain development – The origins of functional specification

The development of the vertebrate central nervous system is reliant on a complex cascade of biological processes that include mitotic division, relocation of migrating neurons, and the extension of dendritic and axonal processes. Each of these cellular events requires the diverse functional repertoire of the microtubule cytoskeleton for the generation of forces, assembly of macromolecular complexes and transport of molecules and organelles. The tubulins are a multi-gene family that encode for the constituents of microtubules, and have been implicated in a spectrum of neurological disorders. Evidence is building that different tubulins tune the functional properties of the microtubule cytoskeleton dependent on the cell type, developmental profile and subcellular localisation. Here we review of the origins of the functional specification of the tubulin gene family in the developing brain at a transcriptional, translational, and post-transcriptional level. We remind the reader that tubulins are not just loading controls for your average Western blot.

Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/β-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of β-tubulin to fold or become assembled into the α/β-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.