Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centro-somal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regu-lated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor g-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in g-tubulin.ISSN:0896-6273ISSN:1097-419

A placebo-controlled randomised trial of budesonide for PBC following an insufficient response to UDCA

Background & Aims: In patients with primary biliary cholangitis (PBC), the efficacy of budesonide, a synthetic corticosteroid displaying high first-pass metabolism, is unresolved. In a placebo-controlled, double-blind trial, we evaluated the added-value of budesonide in those with PBC and ongoing risk of progressive disease despite ursodeoxycholic acid (UDCA) treatment. Methods: We evaluated 62 patients with PBC who had histologically confirmed hepatic inflammatory activity, according to the Ishak score, and an alkaline phosphatase (ALP) >1.5x upper limit of normal (ULN), after at least 6 months of UDCA therapy. Participants were randomly assigned 2:1 to receive budesonide (9 mg/day) or placebo once daily, for 36 months, with UDCA treatment (12-16 mg/kg body weight/day) maintained. Primary efficacy was defined as improvement of liver histology with respect to inflammation and no progression of fibrosis. Secondary outcomes included changes in biochemical markers of liver injury. Results: Recruitment challenges resulted in a study that was underpowered for the primary efficacy analysis. Comparing patients with paired biopsies only (n = 43), the primary histologic endpoint was not met (p>0.05). The proportion of patients with ALP = 15% decrease in ALP and normal bilirubin was higher in the budesonide group than in the placebo group at 12, 24, and 36 months (p Conclusion: Budesonide add-on therapy was not associated with improved liver histology in patients with PBC and insufficient response to UDCA; however, improvements in biochemical markers of disease activity were demonstrated in secondary analyses. Lay summary: Around one-third of patients with primary biliary cholangitis (PBC) needs additional medical therapy alongside ursodeoxycholic acid (UDCA) treatment. In this clinical trial, the addition of the corticosteroid budesonide did not improve liver histology; there were however relevant improvements in liver blood tests. (C) 2020 European Association for the Study of the Liver. Published by Elsevier B.V.Peer reviewe

How Morphological Constraints Affect Axonal Polarity in Mouse Neurons

Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations

Aspergillus Myosin-V Supports Polarized Growth in the Absence of Microtubule-Based Transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV

Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation

Neuronal Polarization: The Cytoskeleton Leads the Way

The morphology of cells is key to their function. Neurons extend a long axon and several shorter dendrites to transmit signals in the nervous system. This process of neuronal polarization is driven by the cytoskeleton. The first and decisive event during neuronal polarization is the specification of the axon. Distinct cytoskeletal dynamics and organization of the cytoskeleton determine the future axon while the other neurites become dendrites. Here, we will review how the cytoskeleton and its effectors drive axon specification and neuronal polarization. First, the role of the actin cytoskeleton and microtubules in axon specification will be presented. Then, we will discuss the role of the centrosome in axon determination as well as how microtubules are generated in axons and dendrites. Finally, we will discuss potential mechanisms leading to axon specification, such as positive feedback loops that could be a coordinated interaction between actin and microtubules. Together, this review will present the recent advances on the role of the microtubules and the actin cytoskeleton during neuronal polarization. We will pinpoint the upcoming challenges to gain a better understanding of neuronal polarization on a fundamental intracellular level. Finally, we will outline how reactivation of the intrinsic polarization program may help to induce axon regeneration after CNS injury. (C) 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 430-444, 201

Neuronal Polarization: The Cytoskeleton Leads the Way

The morphology of cells is key to their function. Neurons extend a long axon and several shorter dendrites to transmit signals in the nervous system. This process of neuronal polarization is driven by the cytoskeleton. The first and decisive event during neuronal polarization is the specification of the axon. Distinct cytoskeletal dynamics and organization of the cytoskeleton determine the future axon while the other neurites become dendrites. Here, we will review how the cytoskeleton and its effectors drive axon specification and neuronal polarization. First, the role of the actin cytoskeleton and microtubules in axon specification will be presented. Then, we will discuss the role of the centrosome in axon determination as well as how microtubules are generated in axons and dendrites. Finally, we will discuss potential mechanisms leading to axon specification, such as positive feedback loops that could be a coordinated interaction between actin and microtubules. Together, this review will present the recent advances on the role of the microtubules and the actin cytoskeleton during neuronal polarization. We will pinpoint the upcoming challenges to gain a better understanding of neuronal polarization on a fundamental intracellular level. Finally, we will outline how reactivation of the intrinsic polarization program may help to induce axon regeneration after CNS injury. (C) 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 430-444, 201

Fabrication of implantable microelectrode arrays by laser cutting of silicone rubber and platinum foil

A new method for fabrication of microelectrode arrays comprised of traditional implant materials is presented. The main construction principle is the use of spun-on medical grade silicone rubber as insulating substrate material and platinum foil as conductor (tracks, pads and electrodes). The silicone rubber and the platinum foil are patterned by laser cutting using an Nd:YAG laser and a microcontroller-driven, stepper-motor operated x-y table. The method does not require expensive clean room facilities and offers an extremely short design-to-prototype time of below 1 day. First prototypes demonstrate a minimal achievable feature size of about 30 ?m