Induction of Cell Stress in Neurons from Transgenic Mice Expressing Yellow Fluorescent Protein: Implications for Neurodegeneration Research

Peer reviewedPublisher PD

Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

<p>Abstract</p> <p>Background</p> <p>In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A). It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear.</p> <p>Results</p> <p>In this study, we have used a combination of <it>in vitro </it>and <it>in vivo </it>approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using <it>in vitro </it>motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in <it>Netrin-1 </it>mutant mice and in chick embryos expressing dominant-negative <it>Unc5a </it>receptors, indicating an <it>in vivo </it>role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK) and myosin light chain kinase (MLCK), which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function <it>in vivo </it>causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points.</p> <p>Conclusions</p> <p>Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.</p

YFP expression activates cell stress responses in neurons <i>in vivo</i>.

<p>A – 3D bar chart showing fold-differences in mRNA expression levels for 84 cell stress related genes comparing spinal cord from YFP-16 mice with wild-type mice (N = 3 samples, each consisting of pooled tissue from 3 mice). Note that we only observed increased expression of cell stress related genes. B – Representative fluorescent western blots for cell stress proteins in the spinal cord of wild-type and YFP-16 mice. Both caspase 1 (Casp1) and CCL3 had increased expression in YFP-expressing tissue, whereas STI1 (a stress protein not on the array) remained at the same levels found in wild-type mice and YFP (FP) was only present in YFP-16 tissue. C - Bar chart (mean ± s.e.m.) showing quantification of protein expression levels in YFP-16 spinal cord (normalised to wild-type: fluorescence intensity ratio of 1 = identical to wild-type), confirming increased expression levels of CCL3 and caspase 1 (N = 3 samples, each consisting of pooled tissue from 3 mice). D/E - Representative fluorescent western blots and bar chart showing caspase 1 expression in the spinal cord of wild-type, YFP-H (low YFP expression) and YFP-16 (high YFP expression) mice. Increased levels of caspase 1 correlated with the amount of YFP present. F-I - Representative confocal micrographs showing caspase 1 immunohistochemistry (red = caspase 1; yellow = YFP; blue = TO-PRO) in the spinal cord of a YFP-H mouse. Increased caspase 1 immunolabelling was restricted to YFP-positive neurons. Scale bar = 20 µm.</p

YFP expression in motor neurons subtly disrupts normal neuronal morphology and alters responses to dying-back pathology but not Wallerian degeneration.

<p>A–D – Representative confocal micrographs of NMJs in the levator auris longus muscle from a YFP-H mouse labelled for neurofilaments (NF: red) and postsynaptic motor endplates (BTX: blue). Note that all motor endplates were innervated, but only a small proportion of motor axons were YFP-positive in these mice (A). Panels B–D show high power micrographs of NMJs identified in panel A. Note abnormal accumulations of NF only in the motor nerve terminals of YFP-positive NMJs (B = NF accumulation score of 0, C = 3, D = 5). E – Bar chart (mean ± s.e.m.) showing quantification of NF accumulation in motor nerve terminals from YFP-H mice (0 = no accumulation; 5 = large abnormal accumulation), revealing a significant increase in NF accumulation in YFP-positive terminals (N = 5 mice, n = 9 muscles; *** P<0.001 Mann-Whitney test). F–G – Representative confocal micrographs of intramuscular axons supplying the transversus abdominis muscle from YFP-H mice (also labelled for NFs; red), before (F) and 20 hours after (G) intercostal nerve lesion. The presence of YFP did not alter the rate or morphological appearance of Wallerian degeneration after nerve injury, with NF fragmentation occurring in YFP-positive and –negative axons in all nerves examined (N = 6 mice, n = 6 nerves). H–K - Representative confocal micrographs of two NMJs in the levator auris longus muscle from a late-symptomatic (P24) <i>wasted</i>/YFP-H mouse labelled to reveal NFs (red) and postsynaptic motor endplates (BTX: blue). Note how the motor axon with YFP (bottom NMJ) remained intact whereas the motor axon without YFP (top) was undergoing retraction, characteristic of a dying-back pathology. L - Bar chart showing quantification of dying-back pathology at the NMJ in late-symptomatic (P24) <i>wasted</i>/YFP-H mice, revealing a significant attenuation of dying-back pathology in motor nerve terminals where YFP was present (i.e. a retention of endplates fully occupied by overlying NFs and a reduction in the numbers of partially occupied endplates; N = 4 mice, n = 7 muscles; *** P<0.001 Mann-Whitney test). Scale bars = 80 µm (A), 40 µm (B–D), 30 µm (F–G), 50 µm (H–K).</p

Mouse SuperArray data showing greater than 1.5 fold cell stress RNA expression changes in the spinal cord of YFP-16 mice compared with wild-type controls (*array cell refers to the location on the 3D bar chart in Fig. 1A).

<p>Mouse SuperArray data showing greater than 1.5 fold cell stress RNA expression changes in the spinal cord of YFP-16 mice compared with wild-type controls (*array cell refers to the location on the 3D bar chart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017639#pone-0017639-g001" target="_blank">Fig. 1A</a>).</p

The UK clinical eye research strategy: refreshing research priorities for clinical eye research in the UK.

ObjectivesTo validate and update the 2013 James Lind Alliance (JLA) Sight Loss and Vision Priority Setting Partnership (PSP)'s research priorities for Ophthalmology, as part of the UK Clinical Eye Research Strategy.MethodsTwelve ophthalmology research themes were identified from the JLA report. They were allocated to five Clinical Study Groups of diverse stakeholders who reviewed the top 10 research priorities for each theme. Using an online survey (April 2021-February 2023), respondents were invited to complete one or more of nine subspecialty surveys. Respondents indicated which of the research questions they considered important and subsequently ranked them.ResultsIn total, 2240 people responded to the survey (mean age, 59.3 years), from across the UK. 68.1% were female. 68.2% were patients, 22.3% healthcare professionals or vision researchers, 7.1% carers, and 2.1% were charity support workers. Highest ranked questions by subspecialty: Cataract (prevention), Cornea (improving microbial keratitis treatment), Optometric (impact of integration of ophthalmic primary and secondary care via community optometric care pathways), Refractive (factors influencing development and/or progression of refractive error), Childhood onset (improving early detection of visual disorders), Glaucoma (effective and improved treatments), Neuro-ophthalmology (improvements in prevention, diagnosis and treatment of neurodegeneration affecting vision), Retina (improving prevention, diagnosis and treatment of dry age-related macular degeneration), Uveitis (effective treatments for ocular and orbital inflammatory diseases).ConclusionsA decade after the initial PSP, the results refocus the most important research questions for each subspecialty, and prime targeted research proposals within Ophthalmology, a chronically underfunded specialty given the substantial burden of disability caused by eye disease

The UK clinical eye research strategy: refreshing research priorities for clinical eye research in the UK

Objectives To validate and update the 2013 James Lind Alliance (JLA) Sight Loss and Vision Priority Setting Partnership (PSP)’s research priorities for Ophthalmology, as part of the UK Clinical Eye Research Strategy. Methods Twelve ophthalmology research themes were identified from the JLA report. They were allocated to five Clinical Study Groups of diverse stakeholders who reviewed the top 10 research priorities for each theme. Using an online survey (April 2021-February 2023), respondents were invited to complete one or more of nine subspecialty surveys. Respondents indicated which of the research questions they considered important and subsequently ranked them. Results In total, 2240 people responded to the survey (mean age, 59.3 years), from across the UK. 68.1% were female. 68.2% were patients, 22.3% healthcare professionals or vision researchers, 7.1% carers, and 2.1% were charity support workers. Highest ranked questions by subspecialty: Cataract (prevention), Cornea (improving microbial keratitis treatment), Optometric (impact of integration of ophthalmic primary and secondary care via community optometric care pathways), Refractive (factors influencing development and/or progression of refractive error), Childhood onset (improving early detection of visual disorders), Glaucoma (effective and improved treatments), Neuro-ophthalmology (improvements in prevention, diagnosis and treatment of neurodegeneration affecting vision), Retina (improving prevention, diagnosis and treatment of dry age-related macular degeneration), Uveitis (effective treatments for ocular and orbital inflammatory diseases). Conclusions A decade after the initial PSP, the results refocus the most important research questions for each subspecialty, and prime targeted research proposals within Ophthalmology, a chronically underfunded specialty given the substantial burden of disability caused by eye disease