Mutation of the α-tubulin Tuba1a leads to straighter microtubules and perturbs neuronal migration.

Brain development involves extensive migration of neurons. Microtubules (MTs) are key cellular effectors of neuronal displacement that are assembled from α/ß-tubulin heterodimers. Mutation of the α-tubulin isotype TUBA1A is associated with cortical malformations in humans. In this study, we provide detailed in vivo and in vitro analyses of Tuba1a mutants. In mice carrying a Tuba1a missense mutation (S140G), neurons accumulate, and glial cells are dispersed along the rostral migratory stream in postnatal and adult brains. Live imaging of Tuba1a-mutant neurons revealed slowed migration and increased neuronal branching, which correlated with directionality alterations and perturbed nucleus-centrosome (N-C) coupling. Tuba1a mutation led to increased straightness of newly polymerized MTs, and structural modeling data suggest a conformational change in the α/ß-tubulin heterodimer. We show that Tuba8, another α-tubulin isotype previously associated with cortical malformations, has altered function compared with Tuba1a. Our work shows that Tuba1a plays an essential, noncompensated role in neuronal saltatory migration in vivo and highlights the importance of MT flexibility in N-C coupling and neuronal-branching regulation during neuronal migration.

Clonally Related Forebrain Interneurons Disperse Broadly across Both Functional Areas and Structural Boundaries.

The medial ganglionic eminence (MGE) gives rise to the majority of mouse forebrain interneurons. Here, we examine the lineage relationship among MGE-derived interneurons using a replication-defective retroviral library containing a highly diverse set of DNA barcodes. Recovering the barcodes from the mature progeny of infected progenitor cells enabled us to unambiguously determine their respective lineal relationship. We found that clonal dispersion occurs across large areas of the brain and is not restricted by anatomical divisions. As such, sibling interneurons can populate the cortex, hippocampus striatum, and globus pallidus. The majority of interneurons appeared to be generated from asymmetric divisions of MGE progenitor cells, followed by symmetric divisions within the subventricular zone. Altogether, our findings uncover that lineage relationships do not appear to determine interneuron allocation to particular regions. As such, it is likely that clonally related interneurons have considerable flexibility as to the particular forebrain circuits to which they can contribute.

A foot in the door: how the chromatin modifier Brg1 and Pax6 jointly potentiate adult neurogenesis.

Transcription factors and chromatin modifiers are known to contribute to cell specification. However, whether and how they jointly act is poorly understood. In this issue of Cell Stem Cell, Ninkovic et al. show that Pax6 and Brg1 cooperate to modulate gene expression in order to direct neurogenic fate in the olfactory bulb.

The origin of neocortical nitric oxide synthase-expressing inhibitory neurons.

Inhibitory neurons are critical for regulating effective transfer of sensory information and network stability. The precision of inhibitory function likely derives from the existence of a variety of interneuron subtypes. Their specification is largely dependent on the locale of origin of interneuron progenitors. Neocortical and hippocampal inhibitory neurons originate the subpallium, namely in the medial and caudal ganglionic eminences (MGE and CGE), and in the preoptic area (POA). In the hippocampus, neuronal nitric oxide synthase (nNOS)-expressing cells constitute a numerically large GABAergic interneuron population. On the contrary, nNOS-expressing inhibitory neurons constitute the smallest of the known neocortical GABAergic neuronal subtypes. The origins of most neocortical GABAergic neuron subtypes have been thoroughly investigated, however, very little is known about the origin of, or the genetic programs underlying the development of nNOS neurons. Here, we show that the vast majority of neocortical nNOS-expressing neurons arise from the MGE rather than the CGE. Regarding their molecular signature, virtually all neocortical nNOS neurons co-express the neuropeptides somatostatin (SST) and neuropeptide Y (NPY), and about half of them express the calcium-binding protein calretinin (CR). nNOS neurons thus constitute a small cohort of the MGE-derived SST-expressing population of cortical inhibitory neurons. Finally, we show that conditional removal of the transcription factor Sox6 in MGE-derived GABAergic cortical neurons results in an absence of SST and CR expression, as well as reduced expression of nNOS in neocortical nNOS neurons. Based on their respective abundance, origin and molecular signature, our results suggest that neocortical and hippocampal nNOS GABAergic neurons likely subserve different functions and have very different physiological relevance in these two cortical structures.

Mutations in the neuronal ß-tubulin subunit TUBB3 result in malformation of cortical development and neuronal migration defects.

Mutations in the TUBB3 gene, encoding ß-tubulin isotype III, were recently shown to be associated with various neurological syndromes which all have in common the ocular motility disorder, congenital fibrosis of the extraocular muscle type 3 (CFEOM3). Surprisingly and in contrast to previously described TUBA1A and TUBB2B phenotypes, no evidence of dysfunctional neuronal migration and cortical organization was reported. In our study, we report the discovery of six novel missense mutations in the TUBB3 gene, including one fetal case and one homozygous variation, in nine patients that all share cortical disorganization, axonal abnormalities associated with pontocerebellar hypoplasia, but with no ocular motility defects, CFEOM3. These new findings demonstrate that the spectrum of TUBB3-related phenotype is broader than previously described and includes malformations of cortical development (MCD) associated with neuronal migration and differentiation defects, axonal guidance and tract organization impairment. Complementary functional studies revealed that the mutated ßIII-tubulin causing the MCD phenotype results in a reduction of heterodimer formation, yet produce correctly formed microtubules (MTs) in mammalian cells. Further to this, we investigated the properties of the MT network in patients' fibroblasts and revealed that MCD mutations can alter the resistance of MTs to depolymerization. Interestingly, this finding contrasts with the increased MT stability observed in the case of CFEOM3-related mutations. These results led us to hypothesize that either MT dynamics or their interactions with various MT-interacting proteins could be differently affected by TUBB3 variations, thus resulting in distinct alteration of downstream processes and therefore explaining the phenotypic diversity of the TUBB3-related spectrum.

Disease-associated mutations in TUBA1A result in a spectrum of defects in the tubulin folding and heterodimer assembly pathway.

Malformations of cortical development are characteristic of a plethora of diseases that includes polymicrogyria, periventricular and subcortical heterotopia and lissencephaly. Mutations in TUBA1A and TUBB2B, each a member of the multigene families that encode alpha- and beta-tubulins, have recently been implicated in these diseases. Here we examine the defects that result from nine disease-causing mutations (I188L, I238V, P263T, L286F, V303G, L397P, R402C, 402H, S419L) in TUBA1A. We show that the expression of all the mutant proteins in vitro results in the generation of tubulin heterodimers in varying yield and that these can co-polymerize with microtubules in vitro. We identify several kinds of defects that result from these mutations. Among these are various defects in the chaperone-dependent pathway leading to de novo tubulin heterodimer formation. These include a defective interaction with the chaperone prefoldin, a reduced efficiency in the generation of productive folding intermediates as a result of inefficient interaction with the cytosolic chaperonin, CCT, and, in several cases, a failure to stably interact with TBCB, one of five tubulin-specific chaperones that act downstream of CCT in the tubulin heterodimer assembly pathway. Other defects include structural instability in vitro, diminished stability in vivo, a compromised ability to co-assemble with microtubules in vivo and a suppression of microtubule growth rate in the neurites (but not the soma) of cultured neurons. Our data are consistent with the notion that some mutations in TUBA1A result in tubulin deficit, whereas others reflect compromised interactions with one or more MAPs that are essential to proper neuronal migration.

Tubulin-related cortical dysgeneses: microtubule dysfunction underlying neuronal migration defects.

The fine tuning of proliferation and neurogenesis, neuronal migration and differentiation and connectivity underlies the proper development of the cerebral cortex. Mutations in genes involved in these processes are responsible for neurodevelopmental disorders, such as cortical dysgeneses, which are usually associated with severe mental retardation and epilepsy. Over the past few years, the importance of cytoskeleton components in cellular processes crucial for cortical development has emerged from a body of functional data. This was reinforced by the association of mutations in the LIS1 and DCX genes, which both encode proteins involved in microtubule (MT) homeostasis, with cerebral cortex developmental disorders. The recent discovery of patients with lissencephaly and bilateral asymmetrical polymicrogyria (PMG) carrying mutations in the alpha- and beta-tubulin-encoding genes TUBA1A and TUBB2B further supports this view, and also raises interesting questions about the specific roles played by certain tubulin isotypes during the development of the cortex.

A pachygyria-causing alpha-tubulin mutation results in inefficient cycling with CCT and a deficient interaction with TBCB.

The agyria (lissencephaly)/pachygyria phenotypes are catastrophic developmental diseases characterized by abnormal folds on the surface of the brain and disorganized cortical layering. In addition to mutations in at least four genes--LIS1, DCX, ARX and RELN--mutations in a human alpha-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative alpha-tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA-E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of alpha-tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype.

The fragile X mental retardation protein is a molecular adaptor between the neurospecific KIF3C kinesin and dendritic RNA granules.

Fragile X mental retardation 1 protein (FMRP) is an RNA-binding protein whose absence results in the fragile X syndrome, the most common inherited form of mental retardation. FMRP contains multiple domains with apparently differential affinity to mRNA and interacts also with protein partners present in ribonucleoprotein complexes called RNA granules. In neurons, these particles travel along dendrites and axons to translocate mRNAs to specific destinations in spines and growth cones, where local synthesis of neuro-specific proteins is taking place. However, the molecular mechanisms of how RNA granules are translocated to dendrites remained unknown. We report here the identification and characterization of the motor protein KIF3C as a novel FMRP-interacting protein. In addition, using time-lapse videomicroscopy, we studied the dynamics and kinetics of FMRP-containing RNA granules in dendrites and show that a KIF3C dominant-negative impedes their distal transport. We therefore propose that, in addition to modulate the translation of its mRNA targets, FMRP acts also as a molecular adaptor between RNA granules and the neurospecific kinesin KIF3C that powers their transport along neuronal microtubules.

The nuclear microspherule protein 58 is a novel RNA-binding protein that interacts with fragile X mental retardation protein in polyribosomal mRNPs from neurons.

The fragile X syndrome, the leading cause of inherited mental retardation, is due to the inactivation of the fragile mental retardation 1 gene (FMR1) and the subsequent absence of its gene product FMRP. This RNA-binding protein is thought to control mRNA translation and its absence in fragile X cells leads to alteration in protein synthesis. In neurons, FMRP is thought to repress specific mRNAs during their transport as silent ribonucleoparticles (mRNPs) from the cell body to the distant synapses which are the sites of local synthesis of neuro-specific proteins. The mechanism by which FMRP sorts out its different mRNAs targets might be tuned by the intervention of different proteins. Using a yeast two-hybrid system, we identified MicroSpherule Protein 58 (MSP58) as a novel FMRP-cellular partner. In cell cultures, we found that MSP58 is predominantly present in the nucleus where it interacts with the nuclear isoform of FMRP. However, in neurons but not in glial cells, MSP58 is also present in the cytoplasmic compartment, as well as in neurites, where it co-localizes with FMRP. Biochemical evidence is given that MSP58 is associated with polyribosomal poly(A)+ mRNPs. We also show that MSP58, similar to FMRP, is present on polyribosomes prepared from synaptoneurosomes and that it behaves as an RNA-binding protein with a high affinity to the G-quartet structure. We propose that this novel cellular partner for FMRP escorts FMRP-containing mRNP from the nucleus and nucleolus to the somato-dendritic compartment where it might participate in neuronal translation regulation.