6-hydroxydopamine (6-OHDA) Oxidative Stress Assay for Observing Dopaminergic Neuron Loss in Caenorhabditis elegans

The nematode Caenorhabditis elegans is a powerful genetic model that can be used to investigate neuronal death. Research using C. elegans has been crucial to characterize cell death programmes that are conserved in mammals. Many neuronal signaling components, such as those mediating dopaminergic neurotransmission, are preserved as well. Dopaminergic neurons are progressively lost in Parkinson's disease and an important risk factor to develop this disease appears to be oxidative stress, the increased occurrence of highly reactive oxygen species. Oxidative stress-induced dopaminergic neurodegeneration is mimicked in animal models by treatment with 6-hydroxydopamine (6-OHDA), a dopamine analog, which is specifically taken up into dopaminergic neurons. After exposing C. elegans to 6-OHDA, the loss of fluorescently labeled dopaminergic neurons can be easily monitored. An organisms' sensitivity to oxidative stress is thought to be influenced by basal levels of intrinsic oxidative stress and the ability to counteract oxidative stress and oxidative stress-induced damage. The C. elegans '6-OHDA model' led to the discovery of novel genes that are required to protect dopaminergic neurons and it has helped to determine the effects of conserved cell death and cell engulfment pathways in dopaminergic neurodegeneration. Here, we describe a simple protocol that allows for the easy detection of dopaminergic neuron loss after 6-OHDA treatment in C. elegans

Tetraspanin (TSP-17) Protects Dopaminergic Neurons against 6-OHDA-Induced Neurodegeneration in <i>C. elegans</i>

Parkinson's disease (PD), the second most prevalent neurodegenerative disease after Alzheimer's disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling

Enhancing precision in human neuroscience

Human neuroscience has always been pushing the boundary of what is measurable. During the last decade, concerns about statistical power and replicability – in science in general, but also specifically in human neuroscience – have fueled an extensive debate. One important insight from this discourse is the need for larger samples, which naturally increases statistical power. An alternative is to increase the precision of measurements, which is the focus of this review. This option is often overlooked, even though statistical power benefits from increasing precision as much as from increasing sample size. Nonetheless, precision has always been at the heart of good scientific practice in human neuroscience, with researchers relying on lab traditions or rules of thumb to ensure sufficient precision for their studies. In this review, we encourage a more systematic approach to precision. We start by introducing measurement precision and its importance for well-powered studies in human neuroscience. Then, determinants for precision in a range of neuroscientific methods (MRI, M/EEG, EDA, Eye-Tracking, and Endocrinology) are elaborated. We end by discussing how a more systematic evaluation of precision and the application of respective insights can lead to an increase in reproducibility in human neuroscience

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

Lekmäns återupplivning, upplevelser vid hjärt - och lungräddning

Syfte med denna studie var att utreda lekmäns upplevelser vid hjärt- och lungräddning (HLR). Arbetet har gjorts i samarbete med uppdragsgivaren samt en medicine studerande vid Helsingfors Universitet. Medicine studeranden har utfört en kvantitativ forskning an-gående lekmäns utförande av hjärt- och lungräddning. Hans forskningsresultat presente-ras inte i detta arbete. Studiens centrala frågeställningar: 1) Vilka svårigheter upplevde lekmännen vid HLR och HLR+D? 2) Upplever lekmännen att det behöver mera kunskap för att kunna agera vid en riktig återupplivning? I denna studie deltog 28 lekmän, förstaårsstuderanden inom vårdsektionen vid yrkeshög-skolan Arcada. Alla deltagarna deltog i ett prehospitalt återupplivningsscenario under hösten 2014. Simuleringen skedde parvis. Dryga hälften av informanterna fick instrukt-ioner av nödcentralen att använda en defibrillator i samband med hjärt- och lungräddning. Resten av lekmännen fick instruktioner att endast utföra hjärt- och lungräddning. Infor-manterna svarade genast efter simuleringen på en webbenkät samt deltog i en kort struk-turerad intervju. Deltagarna avgränsades på basis av att de inte hade tidigare utbildning inom vård. Lekmännen hade endast blivit lärda hjärt- och lungräddning men inte defi-brillering. Undersökningen utfördes med en kombination av kvantitativa och kvalitativa forskningsmetoder. Tidigare forskningar har avgränsats till lekmannaåterupplivning. Som teoretisk bakgrund till examensarbete beskrivs hjärt- och lungräddning samt hur man kan mäta återupplivningens kvalitet. Resultatet påvisar att igenkännande av en livlös människa, agerande vid hjärtstillestånd samt användningen av hjärtstartare upplevdes svårt. Majoriteten tvekar på att de klarar av en riktig återupplivning men har en positiv inställning till att öva hjärt- och lungräddning. Därmed konstateras att mera kunskap behövs samt regelbundet övande för att kunna hantera en återupplivning

<i>glit-1</i> is expressed in the pharynx, the intestine and in several cells in the head including dopaminergic neurons.

<p>(A) L1 stage larva expressing Ex[P<i>glit-1</i>::<i>gfp</i>::<i>glit-1</i>]. (B) L4 stage larva expressing Ex[P<i>glit-1</i>::<i>gfp</i>::<i>glit-1</i>]. This image was generated from three individual images in ImageJ using the Stitching plugin [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007106#pgen.1007106.ref060" target="_blank">60</a>]. (C)–(F) Close up of dopaminergic neurons of the same L4 stage larva. The green channel shows expression of Ex[P<i>glit-1</i>::<i>gfp</i>::<i>glit-1</i>]. The red channel shows expression of Is[P<i>dat-1</i>::NLS::<i>rfp</i>;P<i>ttx-3</i>::<i>mCherry</i>] for labelling of dopaminergic neuron nuclei, as well as the pharynx muscle marker Ex[P<i>myo-2</i>::<i>mCherry</i>] and the body muscle marker Ex[P<i>myo-3</i>::<i>mCherry</i>] that were used for injections.</p

<i>glit-1</i> and <i>tsp-17</i> exhibit increased dopamine-induced paralysis.

<p>(A) Percentage of moving young adults on plates with indicated concentrations of dopamine. Error bars = StDev of 2 technical replicates, each with 25 animals per strain and condition. Total number of animals per condition n = 50 (***p<0.001, *p<0.05, n.s. p>0.05; two-tailed t-test comparing wild-type and mutant animal data at 25 mM dopamine). (B) Percentage of moving young adults on plates containing 50 mM dopamine. Indicated with grey symbols are the 2–4 biological replicates, each performed with 50 animals per strain. Total number of animals per condition n = 100–200 (***p<0.001, *p<0.05; two-tailed t-test).</p

Single-copy expression of <i>glit-1</i> alleviates neurodegeneration in <i>glit-1</i> mutants and <i>glit-1</i> likely functions in a pathway with the tetraspanin <i>tsp-17</i>.

<p>(A) Effect of a single-copy Is[P<i>glit-1</i>::<i>glit-1</i>] construct on dopaminergic neurodegeneration after treatment with 10 mM 6-OHDA. Error bars = SEM of 2 biological replicates, each with 100–105 animals per strain. Total number of animals per condition n = 200–205 (****p<0.0001, **p<0.01; G-Test). (B) Effect of single-copy Is[P<i>dat-1</i>::<i>glit-1</i>] and Is[P<i>pha-4</i>::<i>glit-1</i>] constructs on dopaminergic neurodegeneration after treatment with 10 mM 6-OHDA. Error bars = SEM of 2 biological replicates, each with 100–105 animals per strain. Total number of animals per condition n = 200–205 (n.s. p>0.05; G-Test). (C) Effect of a single-copy Is[P<i>elt-2</i>::<i>glit-1</i>] construct on dopaminergic neurodegeneration after treatment with 10 mM 6-OHDA. Error bars = SEM of 2 biological replicates, each with 95–100 animals per strain. Total number of animals per condition n = 195–200 (n.s. p>0.05,; G-Test). (D) Dopaminergic head neurons in wild-type and <i>glit-1</i> and <i>tsp-17</i> single and double mutants 24, 48 and 72 hours after treatment with 0.75 mM 6-OHDA and 72 hours after control treatment with ascorbic acid only (‘72 h Ctr’). Error bars = SEM of 2–3 biological replicates, each with 60–115 animals per strain. Total number of animals per condition n = 180–350 (*p<0.05, n.s. p>0.05; G-Test). The ‘72 h Ctr’ was conducted twice with 30 animals per strain, resulting in a total n = 60.</p

<i>glit-1(gt1981)</i> exhibits increased 6-OHDA-induced dopaminergic neurodegeneration.

<p>(A) GFP-labelled <i>C</i>. <i>elegans</i> dopaminergic head neurons– 4 CEP neurons and 2 ADE neurons–in BY200 wild-type animals. (B) Remaining dopaminergic head neurons in BY200 wild-type animals and <i>gt1981</i> mutants 48 hours after treating L1-L4 larval stages or adult animals with 10 mM 6-OHDA. Animals possessing all neurons were scored as ‘ADE + CEP’ (white bar), those with partial loss of CEP but intact ADE neurons as ‘ADE + partial CEP’ (light grey bar), those with complete loss of CEP but intact ADE neurons as ‘only ADE’ (dark grey bar) and those with complete loss of dopaminergic head neurons as ‘no ADE + CEP’ (black bar). Error bars = SEM of 2 biological replicates, each with 25–40 animals per stage and strain. Total number of animals per condition n = 50–80 (****p<0.0001, n.s. p>0.05; G-Test comparing BY200 wild-type and mutant data of the same lifecycle stages). (C) Dopaminergic head neurons 24, 48 and 72 hours after treatment with 10 mM 6-OHDA and 72 hours after treatment with ascorbic acid only (‘72h Ctr’) for BY200 wild-type or <i>glit-1</i> mutant animals. Error bars = S.E.M. of 2 biological replicates for <i>glit-1(ok237)</i> and 3 biological replicates for all the other strains, each with 60–115 animals per strain and concentration. Total number of animals for the ‘72h Ctr’ experiment n = 30–100 and for all other conditions n = 130–340 (****p<0.0001; G-Test comparing BY200 wild-type and mutant data of the same time point). (D) GLIT-1 protein structure prediction based on homology modelling with acetylcholinesterase (PDB ID: 2W6C). The <i>gt1981</i> point mutation leads to a proline to glycine conversion (P113G) and is indicated in red. The amino acids replacing the acetylcholinesterase catalytic triad are indicated in blue. (E) <i>glit-1</i> gene structure with positions of the <i>glit-1(gt1981)</i> point mutation, the <i>glit-1(gk384527)</i> splice site mutation and the <i>glit-1(ok237)</i> deletion. For the point mutations the nucleotide and amino acid changes are indicated in brackets. The <i>ok237</i> deletion spans 5’UTR and first exon of <i>glit-1</i> and 5’UTR and first exons of <i>dnj-14</i> (DNaJ domain (prokaryotic heat shock protein)) and is indicated with a red bar. Also, <i>glit-1</i> is located in an operon (grey bar) with the ribosomal protein <i>rpl-25</i>.<i>1</i> (Ribosomal Protein, Large subunit).</p