6-OHDA-induced dopaminergic neurodegeneration in <i>Caenorhabditis elegans</i> is promoted by the engulfment pathway and inhibited by the transthyretin-related protein TTR-33

<div><p>Oxidative stress is linked to many pathological conditions including the loss of dopaminergic neurons in Parkinson’s disease. The vast majority of disease cases appear to be caused by a combination of genetic mutations and environmental factors. We screened for genes protecting <i>Caenorhabditis elegans</i> dopaminergic neurons from oxidative stress induced by the neurotoxin 6-hydroxydopamine (6-OHDA) and identified the <u>t</u>rans<u>t</u>hyretin-<u>r</u>elated gene <i>ttr-33</i>. The only described <i>C</i>. <i>elegans</i> transthyretin-related protein to date, TTR-52, has been shown to mediate corpse engulfment as well as axon repair. We demonstrate that TTR-52 and TTR-33 have distinct roles. TTR-33 is likely produced in the posterior arcade cells in the head of <i>C</i>. <i>elegans</i> larvae and is predicted to be a secreted protein. TTR-33 protects <i>C</i>. <i>elegans</i> from oxidative stress induced by paraquat or H₂O₂ at an organismal level. The increased oxidative stress sensitivity of <i>ttr-33</i> mutants is alleviated by mutations affecting the KGB-1 MAPK kinase pathway, whereas it is enhanced by mutation of the JNK-1 MAPK kinase. Finally, we provide genetic evidence that the <i>C</i>. <i>elegans</i> cell corpse engulfment pathway is required for the degeneration of dopaminergic neurons after exposure to 6-OHDA. In summary, we describe a new neuroprotective mechanism and demonstrate that TTR-33 normally functions to protect dopaminergic neurons from oxidative stress-induced degeneration, potentially by acting as a secreted sensor or scavenger of oxidative stress.</p></div

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Epidermal control of axonal attachment via β-spectrin and the GTPase-activating protein TBC-10 prevents axonal degeneration

Neurons are subjected to strain due to body movement and their location within organs and tissues. However, how they withstand these forces over the lifetime of an organism is still poorly understood. Here, focusing on touch receptor neuron-epidermis interactions using Caenorhabditis elegans as a model system, we show that UNC-70/β-spectrin and TBC-10, a conserved GTPase-activating protein, function non-cell-autonomously within the epidermis to dynamically maintain attachment of the axon. We reveal that, in response to strain, UNC-70/β-spectrin and TBC-10 stabilize trans-epidermal hemidesmosome attachment structures which otherwise become lost, causing axonal breakage and degeneration. Furthermore, we show that TBC-10 regulates axonal attachment and maintenance by inactivating RAB-35, and reveal functional conservation of these molecules with their vertebrate orthologs. Finally, we demonstrate that β-spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/β-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration

A multi-channel device for high-density target-selective stimulation and long-term monitoring of cells and subcellular features in C. elegans

Selective cell ablation can be used to identify neuronal functions in multicellular model organisms such as Caenorhabditis elegans. The optogenetic tool KillerRed facilitates selective ablation by enabling light-activated damage of cell or subcellular components in a temporally and spatially precise manner. However, the use of KillerRed requires stimulating (5 min-1 h), culturing (~24 h) and imaging (often repeatedly) a large number of individual animals. Current manual manipulation methods are limited by their time-consuming, labor-intensive nature, and their usage of anesthetics. To facilitate large-scale selective ablation, culturing, and repetitive imaging, we developed a densely-packed multi-channel device and used it to perform high-throughput neuronal ablation on KillerRed-expressing animals. The ability to load worms in identical locations with high loading efficiency allows us to ablate selected neurons in multiple worms simultaneously. Our device also enables continuous observation of animals for 24 h following KillerRed activation, and allows the animals to be recovered for behavioural assays. We expect this multi-channel device to facilitate a broad range of long-term imaging and selective illumination experiments in neuroscience. This journal i

EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway

Functional regeneration after nervous system injury requires transected axons to reconnect with their original target tissue. Axonal fusion, a spontaneous regenerative mechanism identified in several species, provides an efficient means of achieving target reconnection as a regrowing axon is able to contact and fuse with its own separated axon fragment, thereby re-establishing the original axonal tract. Here we report a molecular characterization of this process in Caenorhabditis elegans, revealing dynamic changes in the subcellular localization of the EFF-1 fusogen after axotomy, and establishing phosphatidylserine (PS) and the PS receptor (PSR-1) as critical components for axonal fusion. PSR-1 functions cell-autonomously in the regrowing neuron and, instead of acting in its canonical signalling pathway, acts in a parallel phagocytic pathway that includes the transthyretin protein TTR-52, as well as CED-7, NRF-5 and CED-6 (refs 9, 10, 11, 12). We show that TTR-52 binds to PS exposed on the injured axon, and can restore fusion several hours after injury. We propose that PS functions as a 'save-me' signal for the distal fragment, allowing conserved apoptotic cell clearance molecules to function in re-establishing axonal integrity during regeneration of the nervous system

The diabetes drug liraglutide ameliorates aberrant insulin receptor localisation and signalling in parallel with decreasing both amyloid-β plaque and glial pathology in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) has been shown to involve desensitised insulin receptor (IR) signalling. Liraglutide, a novel glucagon-like peptide 1 (GLP-1) analogue that facilitates insulin signalling, is currently approved for use in type 2 diabetes mellitus. In the present study, we show that distinctive alterations in the localisation and distribution of the IR and increased levels of insulin receptor substrate (IRS)-1 phosphorylated at serine 616 (IRS-1 pS(616)), a key marker of insulin resistance, are associated with amyloid-β plaque pathology in the frontal cortex of a mouse model of AD, APPSWE/PS1dE9. Altered IR status in APPSWE/PS1dE9 is most evident in extracellular deposits with the appearance of dystrophic neurites, with significantly increased IRS-1 pS(616) levels detected within neurons and neurites. The IR and IRS-1 pS(616) changes occur in the vicinity of all plaques in the APPSWE/PS1dE9 brain, and a significant upregulation of astrocytes and microglia surround this pathology. We show that liraglutide treatment for 8 weeks at 25 nmol/kg body weight i.p. once daily in 7-month-old mice significantly decreases IR aberrations in conjunction with a concomitant decrease in amyloid plaque load and levels of IRS-1 pS(616). Liraglutide also induces a highly significant reduction in astrocytosis and microglial number associated with both plaques and IR pathology. The amelioration of IR aberrations and attenuation of IRS-1 pS(616) upregulation, plaque and glial activation in APPSWE/PS1dE9 mice treated with liraglutide support the investigation of the therapeutic potential of liraglutide and long-lasting GLP-1 agonists in patients with AD

<i>ttr-33</i> mutants develop at a normal speed and 6-OHDA must enter dopaminergic neurons to cause dopaminergic neurodegeneration.

<p>(A) TTR-33 protein structure prediction based on homology modelling using the structure of TTR-52 (PDB ID: 3UAF). The overlay of the TTR-33 and the TTR-52 structures is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007125#pgen.1007125.s002" target="_blank">S2B Fig</a>. The <i>ttr-33(gt1983)</i> point mutation leading to a glycine to glutamate (G27E) conversion and the <i>ttr-33</i>(<i>gk567379)</i> mutation leading to a leucine to phenylalanine (L72F) conversion are both indicated in red. (B) Dopaminergic head neurons in wild-type animals and <i>ttr-33</i> mutants 48 hours after treatment of L1-L4 larval stages or adult animals with 10 mM 6-OHDA. Error bars = SEM of 2 biological replicates, each with 25–45 animals per stage and strain. Total number of animals per condition n = 50–80 (****p<0.0001, **p<0.01, n.s. p>0.05; G-Test comparing BY200 wild-type and mutant animals data of the same lifecycle stages). (C) Developmental stages of wild-type and <i>ttr-33</i> mutant L1 stage larvae 48 hours after treatment with and without 10 mM 6-OHDA. <i>C</i>. <i>elegans</i> L1 stage larvae develop via the L2 (in red), L3 (in orange), L4 (green) and young adult stage (in light blue) into adults (in dark blue). Error bars = SEM of 3 biological replicates, each with 60–175 animals per treatment and strain. Total number of animals per condition n = 205–350 (n.s. p>0.05; G-Test). (D) Effect of <i>dat-1</i> mutation on dopaminergic neurodegeneration after treatment with 10 mM 6-OHDA. Error bars = SEM of 2 experiments, each with 90–115 animals per strain. Total number of animals per strain n = 200–535 (****p<0.0001; G-Test).</p

Evidence that <i>ttr-33</i> is expressed in the posterior arcade cells and in the pharynx.

<p>(A) Head region of L1 stage larvae expressing the extrachromosomal transcriptional reporter Ex[P<i>ttr-33</i>::<i>gfp</i>]. Arrows indicate strongly expressing cells. (B) L1 stage larva expressing the transcriptional reporter Ex[P<i>ttr-33</i>::<i>gfp</i>]. (C) Pretzel stage embryo expression the transcriptional reporter Ex[P<i>ttr-33</i>::<i>gfp</i>]. The speckled signal is caused by intestinal autofluorescence.</p

Interference with the <i>jnk-1</i> MAPK pathway increases 6-OHDA sensitivity in <i>ttr-33</i> mutants.

<p>(A) Effect of p38 and JNK stress response pathway mutations on dopaminergic neurodegeneration after treatment with 2.5 mM 6-OHDA. Error bars = SEM of 2–4 biological replicates, each with 100–120 animals per strain and concentration. Total number of animals per condition n = 200–425 (*p<0.05, n.s. p>0.05; G-Test). (B) Effect of p38 and JNK stress response pathway mutations on dopaminergic neurodegeneration after treatment with 25 mM 6-OHDA. Error bars = SEM of 3 biological replicates, each with 100–110 animals per strain. Total number of animals per strain n = 310–330 (n.s. p>0.05; G-Test). (C) Effect of <i>pmk-1</i> mutation on dopaminergic neurodegeneration after treatment with 25 mM 6-OHDA. Error bars = SEM of 3 biological replicates, each with 100–135 animals per strain. Total number of animals per strain n = 350–360 (n.s. p>0.05; G-Test).</p