Julian Ik-Tsen Heng

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.

Molecular layers underlying cytoskeletal remodelling during cortical development

During neural development, the cytoskeleton of newborn neurons undergoes extensive and dynamic remodelling to facilitate the sequential steps of neurogenesis, cell migration and terminal differentiation. It is clear from studying the mechanisms that precipitate these functions that different configurations of the cytoskeleton prefigure the correct execution of each step and define cohorts of proteins the functions of which are indispensable for the control of neuronal migration but not terminal differentiation. These combinatorial protein functions are also predetermined by regulated gene expression and the precise subcellular localisation of their protein products. Here, we expand on this view in the context of recent data on how the cytoskeleton is regulated during the maturation of cortical neurons within the developing brain.

WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression

Summary The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase–anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3

We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole‐exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator‐like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging–negative focal epilepsy. ANN NEUROL 2016;79:132–137

Familial cortical dysplasia caused by mutation in the mTOR regulator NPRL3

We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole‐exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator‐like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging–negative focal epilepsy. ANN NEUROL 2016;79:132–137

COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression

During corticogenesis, late-born callosal projection neurons (CPNs) acquire their laminar position through glia-guided radial migration and then undergo final differentiation. However, the mechanisms controlling radial migration and final morphology of CPNs are poorly defined. Here, we show that in COUP-TFI mutant mice CPNs are correctly specified, but are delayed in reaching the cortical plate and have morphological defects during migration. Interestingly, we observed that the rate of neuronal migration to the cortical plate normally follows a low-rostral to high-caudal gradient, similar to that described for COUP-TFI. This gradient is strongly impaired in COUP-TFI–/– brains. Moreover, the expression of the Rho-GTPase Rnd2, a modulator of radial migration, is complementary to both these gradients and strongly increases in the absence of COUP-TFI function. We show that COUP-TFI directly represses Rnd2 expression at the post-mitotic level along the rostrocaudal axis of the neocortex. Restoring correct Rnd2 levels in COUP-TFI–/– brains cell-autonomously rescues neuron radial migration and morphological transitions. We also observed impairments in axonal elongation and dendritic arborization of COUP-TFI-deficient CPNs, which were rescued by lowering Rnd2 expression levels. Thus, our data demonstrate that COUP-TFI modulates late-born neuron migration and favours proper differentiation of CPNs by finely regulating Rnd2 expression levels.

Molecular mechanisms of projection neuron production and maturation in the developing cerebral cortex

The cerebral cortex is a brain structure unique to mammals and highly adapted to process complex information. Through multiple developmental steps, the cerebral cortex is assembled as a huge diversity of neurons comprising a complex laminar structure, and with both local and long-distance connectivity within the nervous system. Key processes must take place during its construction, including: (i) regulation of the correct number of neurons produced by progenitor cells, (ii) temporal and spatial generation of neuronal diversity, and (iii) control of neuron migration and laminar positioning as well as terminal differentiation within the mature cortex. Here, we seek to highlight recent cellular and molecular findings underlying these sequential steps of neurogenesis, cell fate specification and migration during cortical development, with particular emphasis on cortical projection neurons.

Insights into the Biology and Therapeutic Applications of Neural Stem Cells

The cerebral cortex is essential for our higher cognitive functions and emotional reasoning. Arguably, this brain structure is the distinguishing feature of our species, and yet our remarkable cognitive capacity has seemingly come at a cost to the regenerative capacity of the human brain. Indeed, the capacity for regeneration and neurogenesis of the brains of vertebrates has declined over the course of evolution, from fish to rodents to primates. Nevertheless, recent evidence supporting the existence of neural stem cells (NSCs) in the adult human brain raises new questions about the biological significance of adult neurogenesis in relation to ageing and the possibility that such endogenous sources of NSCs might provide therapeutic options for the treatment of brain injury and disease. Here, we highlight recent insights and perspectives on NSCs within both the developing and adult cerebral cortex. Our review of NSCs during development focuses upon the diversity and therapeutic potential of these cells for use in cellular transplantation and in the modeling of neurodevelopmental disorders. Finally, we describe the cellular and molecular characteristics of NSCs within the adult brain and strategies to harness the therapeutic potential of these cell populations in the treatment of brain injury and disease.

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.

Transcriptional regulation of intermediate progenitor cell generation during hippocampal development

During forebrain development, radial glia generate neurons through the production of intermediate progenitor cells (IPCs). The production of IPCs is a central tenet underlying the generation of the appropriate number of cortical neurons, but the transcriptional logic underpinning this process remains poorly defined. Here, we examined IPC production using mice lacking the transcription factor nuclear factor I/X (Nfix). We show that Nfix deficiency delays IPC production and prolongs the neurogenic window, resulting in an increased number of neurons in the postnatal forebrain. Loss of additional Nfi alleles (Nfib) resulted in a severe delay in IPC generation while, conversely, overexpression of NFIX led to precocious IPC generation. Mechanistically, analyses of microarray and ChIP-seq datasets, coupled with the investigation of spindle orientation during radial glial cell division, revealed that NFIX promotes the generation of IPCs via the transcriptional upregulation of inscuteable (Insc). These data thereby provide novel insights into the mechanisms controlling the timely transition of radial glia into IPCs during forebrain development. Summary: The Nfix and Nfib transcription factors are required for the timely transition of radial glia into intermediate progenitor cells during mouse forebrain development.