A novel RNAi screen for neurotrophin receptor internalisation and trafficking in motor neurons

A primary focus of the Molecular Neuropathobiology laboratory is the investigation of the long range trafficking of neurotrophins and neurotrophin receptors in motor neurons (MNs). The goal of my project was to deepen our understanding of the nature of the cellular machinery controlling long-range neurotrophin trafficking in MNs, by discovering new players involved in this process. In order to achieve this goal I performed an siRNA screen in MNs derived from mouse ES cells to monitor the cell surface binding and internalization of two fluorescently tagged reporters: the binding fragment of the Tetanus neurotoxin (HC), which enters an axonal transport compartment shared with neurotrophins and their receptors, and an antibody directed against the extracellular domain of the neurotrophin receptor p75NTR. A high-throughput, lipidbased siRNA transfection method was optimised for ES cell-derived MNs and used to screen a library of siRNAs directed against a pool of genes involved in endocytosis and membrane trafficking. The primary candidate genes were subsequently validated, and one gene in particular, Bicaudal D homolog 1 (BICD1), was selected for further analyses. BICD1 is a member of the Bicaudal D family, whose members function as molecular motor adaptors with pleiotropic roles in intracellular trafficking. Bicd1 expression at E12.5 and 13.5 was restricted to the nervous system, suggesting an important role for BICD1 in neurons. I used gene-trapped ES cells to derive MNs depleted of the BICD1 protein (Bicd1gt/gt MNs), which, when challenged with either HC or the p75NTR antibody, displayed an increased intracellular accumulation of both probes. Furthermore, I found that the level of neurotrophin receptors exposed at the plasma membrane was increased in these cells compared to their wild type counterparts, suggesting that BICD1 might be involved in the regulation of neurotrophin receptor dynamics in mammalian neurons. I also found that TrkB signalling upon stimulation with the brain-derived neurotrophic factor (BDNF) in Bicd1gt/gt MNs, was impaired. This suggests that the depletion of BICD1 not only affects the trafficking of neurotrophin receptors, but also their signalling capabilities. In conclusion, this thesis work has demonstrated that the concept of high throughput screening can be applied to cells notoriously difficult to handle and transfect, such as MNs. This approach was successful in unravelling a new role for BICD1 in neurons, where it appears to regulate the intracellular trafficking and signalling of neurotrophin receptors. Taken together with the in vivo expression data, these data suggest that BICD1 plays an important role in the development and function of the nervous system

Compartmentalized Signaling in Neurons: From Cell Biology to Neuroscience

Neurons are the largest known cells, with complex and highly polarized morphologies. As such, neuronal signaling is highly compartmentalized, requiring sophisticated transfer mechanisms to convey and integrate information within and between sub-neuronal compartments. Here, we survey different modes of compartmentalized signaling in neurons, highlighting examples wherein the fundamental cell biological processes of protein synthesis and degradation, membrane trafficking, and organelle transport are employed to enable the encoding and integration of information, locally and globally within a neuron. Comparisons to other cell types indicate that neurons accentuate widely shared mechanisms, providing invaluable models for the compartmentalization and transfer mechanisms required and used by most eukaryotic cells

siRNA screen of ES cell-derived motor neurons identifies novel regulators of tetanus toxin and neurotrophin receptor trafficking

Neurons rely on the long-range transport of several signaling molecules such as neurotrophins and their receptors, which are required for neuronal development, function and survival. However, the nature of the machinery controlling the trafficking of signaling endosomes containing activated neurotrophin receptors has not yet been completely elucidated. We aimed to identify new players involved in the dynamics of neurotrophin signaling endosomes using a medium-throughput unbiased siRNA screening approach to quantify the intracellular accumulation of two fluorescently tagged reporters: the binding fragment of tetanus neurotoxin (HCT), and an antibody directed against the neurotrophin receptor p75NTR. This screen performed in motor neurons differentiated from mouse embryonic stem (ES) cells identified a number of candidate genes encoding molecular motors and motor adaptor proteins involved in regulating the intracellular trafficking of these probes. Bicaudal D homolog 1 (BICD1), a molecular motor adaptor with pleiotropic roles in intracellular trafficking, was selected for further analyses, which revealed that BICD1 regulates the intracellular trafficking of HCT and neurotrophin receptors and likely plays an important role in nervous system development and function

Bicaudal‐D1 regulates the intracellular sorting and signalling of neurotrophin receptors

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor‐containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain‐derived neurotrophic factor (BDNF)‐activated TrkB and p75 neurotrophin receptor (p75NTR) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co‐localisation of these neurotrophin receptors with retromer‐associated sorting nexin 1. The resulting re‐routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling‐competent TrkB isoforms and p75NTR available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand‐activated receptors

Sterigmatocystin Occurrence in Paddy and Processed Rice Produced in Italy in the Years 2014\u20132015 and Distribution in Milled Rice Fractions

The occurrence of sterigmatocystin (STC) in paddy and processed rice samples produced in Italy was surveyed. After extraction and purification, STC was analysed using HPLC-MS/MS. STC was detected in all paddy rice samples (n = 49), in the range 0.29\u201315.85 gkg1. As regards processed rice, a widespread contamination was found in brown and parboiled rice. All the brown rice samples were contaminated between 0.12 and 1.32 gkg1; for parboiled rice, the incidence was 90.9% and the maximum level was 1.09 gkg1. The contamination in white rice was significantly lower (p < 0.01). The STC distribution in different rice fractions, obtained by the de-hulling and polishing processes, was evaluated. After de-hulling, the STC percentage remaining in brown rice was in the range 21.2%\u201330.8%. The polishing process, from brown to white rice, caused another remarkable decrease of contamination; the STC remaining in white rice was 2.2%\u20138.3% of the amount found in paddy rice

Two stage fracture of a polyethylene post in a 9-year-old posterior-stabilized knee prosthesis: a case report

<p>Abstract</p> <p>Introduction</p> <p>Several cases of tibial post breakage are reported in the literature. To the best of our knowledge, only three cases of NexGen knee prosthesis (Zimmer, Warsaw, Indiana, USA) tibial post failure have been reported.</p> <p>Case presentation</p> <p>In November 1999, a 63-year-old Caucasian woman from Italy with a history of symptomatic left knee osteoarthritis underwent a total knee arthroplasty. In March 2008, while rising from a chair, she felt a sudden pain and instability in her left knee. She reported a fracture of the polyethylene post of the tibial insert. No malposition or malalignment of either the femoral or tibial components were identified. The polyethylene tibial insert was studied under light microscopy and scanning electron microscopy. The fracture was also noted to have occurred without any notable polyethylene wear.</p> <p>Conclusion</p> <p>Scanning electron microscopy revealed two different damage patterns that could be explained with a two-stage rupture of our patient's polyethylene post. This could have been caused by a non-optimal ligamentous balancing during first implant surgery. Her knee probably developed a varus instability that weakened the post, and then a posterior anterior stress finally broke the polyethylene.</p

Structural studies unravel the active conformation of apo RORγt nuclear receptor and a common inverse agonism of two diverse classes of RORγt inhibitors

The nuclear receptor retinoid acid receptor-related orphan receptor γt (RORγt) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. RORγt has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for RORγt. Here, we report the crystal structures for the ligand-binding domain of RORγt in both apo and ligand-bound states. We show that apo RORγt adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of RORγt in the active state in the absence of a ligand. The structures of ligand-bound RORγt reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of RORγt toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists

The fourth phase of the radiative transfer model intercomparison (RAMI) exercise : Actual canopy scenarios and conformity testing

The RAdiative transfer Model Intercomparison (RAMI) activity focuses on the benchmarking of canopy radiative transfer (RT) models. For the current fourth phase of RAMI, six highly realistic virtual plant environments were constructed on the basis of intensive field data collected from (both deciduous and coniferous) forest stands as well as test sites in Europe and South Africa. Twelve RT modelling groups provided simulations of canopy scale (directional and hemispherically integrated) radiative quantities, as well as a series of binary hemispherical photographs acquired from different locations within the virtual canopies. The simulation results showed much greater variance than those recently analysed for the abstract canopy scenarios of RAMI-IV. Canopy complexity is among the most likely drivers behind operator induced errors that gave rise to the discrepancies. Conformity testing was introduced to separate the simulation results into acceptable and non-acceptable contributions. More specifically, a shared risk approach is used to evaluate the compliance of RI model simulations on the basis of reference data generated with the weighted ensemble averaging technique from ISO-13528. However, using concepts from legal metrology, the uncertainty of this reference solution will be shown to prevent a confident assessment of model performance with respect to the selected tolerance intervals. As an alternative, guarded risk decision rules will be presented to account explicitly for the uncertainty associated with the reference and candidate methods. Both guarded acceptance and guarded rejection approaches are used to make confident statements about the acceptance and/or rejection of RT model simulations with respect to the predefined tolerance intervals. (C) 2015 The Authors. Published by Elsevier Inc.Peer reviewe