pexpr(5605):pmex-5::cas9(cdna + syntron)::tbb-2 3’utr; cbr-unc-119  (Addgene inc)

Journal: Science Advances

Article Title: Glial-derived mitochondrial signals affect neuronal proteostasis and aging

doi: 10.1126/sciadv.adi1411

Figure Lengend Snippet: ( A ) Changes in gene expression in CEPsh glia expressing jmjd-1.2 as compared to N2 control animals are represented in a volcano plot (for RNA-seq analysis, data are compared using two biological replicates for N2 control and three biological replicates for hlh-17p::jmjd = 1.2). ( B ) Differentially expressed genes in glial jmjd-1.2 worms compared to their change in published datasets of mitochondrial stress induced by electron transport chain knockdown by RNAi ( cox-5B RNAi) or overexpression of jmjd-1.2 in all worm tissues . ( C ) The average change in gene expression of the indicated gene groups (see Methods) as compared to N2 control animals. UPR MT genes are shown in ( D ). HSR, heatshock response. ( E ) Overlap of induced genes in glial jmjd-1.2 worms with genes induced in paraquat stress. ( F ) Gene enrichment analysis was plotted using gProfiler . ( G ) Representative fluorescent micrographs of protein aggregation (Q44::YFP) in the intestine of worms expressing jmjd-1.2 under the CEPsh glia ( hlh-17) promoter at day 3 of adulthood. Scale bar, 250 μm. ( H ) Quantification of number of fluorescent foci (i.e., aggregates) per worm using Fiji local extrema analysis ( n < 30). ( I ) Representative fluorescent images of lipid droplets reporter animals ( DHS-3::GFP ), either in control animals or animals expressing jmjd-1.2 in CEPsh glia cells at day 1 of adulthood. Images of aligned whole worms ( n > 10) were acquired on a stereomicroscope (top) to visualize whole-animal changes in lipid droplet levels, and via high-resolution compound microscopy (bottom) to visualize size and morphology of lipid droplets. Quantification in fig. S3E.

Article Snippet: PHX3684 , sybIs2974[hlh-17p::NLS-FLP D5::tbb-2 3′UTR;Cbr-unc-119(+)] II; unc-119(ed3); syb3684 , SunyBiotech.

Techniques: Gene Expression, Expressing, Control, RNA Sequencing, Knockdown, Over Expression, Microscopy

Journal: Science Advances

Article Title: Glial-derived mitochondrial signals affect neuronal proteostasis and aging

doi: 10.1126/sciadv.adi1411

Figure Lengend Snippet: ( A ) Schematic of spatial mutation strategy. Expression of the flipase FLP D5 under the CEPsh glial-specific ( hlh-17p ) or pan-neuronal ( rgef-1p ) promoters, in combination with an independent FRT (FLP recognition target) allele of a gene of interest results in a tissue-specific mutation. ( B ) Representative fluorescent micrographs of the indicated strains at day 1 adult animals. Scale bar, 25 0 μm. ( C and D ) Median spatial profiles of the indicated animals (see Methods), for depletion in CEPsh glial cells (C) or neuronal cells (D) quantified with large particle biosorter ( n > 50). The integrated fluorescence intensity of the 30% most posterior portion of the animals was calculated and plotted in ( E ). One-way ANOVA with Tukey’s multiple comparisons test, *** P < 0.001 and **** P < 0.0001.

Article Snippet: PHX3684 , sybIs2974[hlh-17p::NLS-FLP D5::tbb-2 3′UTR;Cbr-unc-119(+)] II; unc-119(ed3); syb3684 , SunyBiotech.

Techniques: Mutagenesis, Expressing, Fluorescence

Journal: Science Advances

Article Title: Glial-derived mitochondrial signals affect neuronal proteostasis and aging

doi: 10.1126/sciadv.adi1411

Figure Lengend Snippet: ( A ) Thrashing of animals expressing the aggregating polyQ tract Q40, with and without glial jmjd-1.2 , measured using WormTracker ( n > 100). ( B ) Chemotaxis index of worms toward benzaldehyde ( n > 200). ( C ) Filter retardation assay for Q40::YFP , with and without glial jmjd-1.2 (top) and its quantification using integrated intensity measurements in Fiji (bottom). Data are representative of three independent biological replicates. ( D ) Representative fluorescent micrographs of Q40::YFP for the annotated genotypes on day 5 of adulthood. See fig. S6 for images of worms at day 1. WT, animals expressing wild-type copy of unc-13 ; e51, animals expressing loss of function mutant unc-13(e51) . ( E ) Integrated fluorescence intensity measurements of Q40::YFP in the head region of the animals ( n > 30) using Fiji. One-way ANOVA with Tukey’s multiple comparisons test; ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ( F ) Representative blot of neuronal Q40::YFP protein at days 1 and 5 of adulthood in control (N2) and glial jmjd-1.2 ( hlh-17p::jmjd-1.2 ) animals. Total Q40::YFP expression was measured via standard Western blots in whole worm lysates using a standard anti-GFP antibody. Signal intensity was quantified using integrated intensity measurements in Fiji in ( G ) and normalized to an H3 load control. Measurements in (G) were performed on two biological replicates, and data are presented as means ± SD. ( H ) Model of communication from glial cells to peripheral tissues. CEPsh glia use SCVs upon UPR MT activation to signal to neurons, which reduce protein aggregation and use DCVs, neuropeptide processing, and a WNT ligand to drive protein homeostasis and metabolic changes in the periphery.

Article Snippet: PHX3684 , sybIs2974[hlh-17p::NLS-FLP D5::tbb-2 3′UTR;Cbr-unc-119(+)] II; unc-119(ed3); syb3684 , SunyBiotech.

Techniques: Expressing, Chemotaxis Assay, Mutagenesis, Fluorescence, Control, Western Blot, Activation Assay

Journal: Science Advances

Article Title: Glial-derived mitochondrial signals affect neuronal proteostasis and aging

doi: 10.1126/sciadv.adi1411

Figure Lengend Snippet: Strains and plasmids used in this study. UTR, untranslated region.

Article Snippet: PHX3684 , sybIs2974[hlh-17p::NLS-FLP D5::tbb-2 3′UTR;Cbr-unc-119(+)] II; unc-119(ed3); syb3684 , SunyBiotech.

Techniques:

Journal: bioRxiv

Article Title: Glial-derived mitochondrial signals impact neuronal proteostasis and aging

doi: 10.1101/2023.07.20.549924

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: PHX3684 , sybIs2974[hlh-17p::NLS-FLP D5::tbb-2 3’UTR;Cbr-unc-119(+) ] II; unc-119(ed3); syb3684 , SunyBiotech.

Techniques:

Journal: microPublication Biology

Article Title: A histamine-gated channel is an efficient negative selection marker for C. elegans transgenesis

doi: 10.17912/micropub.biology.000349

Figure Lengend Snippet: A-D. Dose-response relationship and time-course for the paralysis of transgenic animals with HisCl1 expressed from the indicated promoters in response to increasing histamine concentrations and longer exposure. P tag-168 primarily drives expression in the nervous system. P mlc-2 is pan-muscular (El Mouridi et al. , 2020), P rpl-3 is a strong ribosomal promoter (likely ubiquitously expressed), and P snt-1 is widely expressed in the nervous system (Nonet et al. , 1993). Top panels: fed animals on OP50 bacteria. Bottom panels: starved L1 animals. Quantification: Four plates with 15 transgenic animals were scored by eye for paralysis for each promoter and condition (concentration and time-point) in two technical replicates. E. Schematic showing the use of histamine selection to identify Mos1-mediated Single-Copy transgene Insertions (MosSCI) (Frøkjær-Jensen et al. , 2008) using a red marker to identify arrays (P mlc-1 ::tagRFP-T), and a plasmid expressing histamine (P snt-1 :: ce-HisCl1 ) as a negative selection marker. Insertions were identified from starved plates with histamine or by “chunking” onto plates with histamine. F. Quantification of relative toxicity of histamine and hsp :: peel-1 selection. We used the frequency of stable F2 rescued lines relative to the number of plates with F1 rescue as a proxy for toxicity. G . Quantification of the MosSCI insertion frequency and the frequency of plates with “escapers” (moving animals with fluorescent array markers).

Article Snippet: pNP403 P tag-168::HisCl1::sl2::unc-54 3′ UTR (Pokala et al. ) pSEM236 P mlc-2::ce-HisCl1::rpl-3 3′ UTR (Addgene #159797) pSEM237 P rpl-3::ce-HisCl1::rpl-3 3′ UTR (Addgene #159796) pSEM238 P snt-1::ce-HisCl1::rpl-3 3′ UTR(Addgene #161515) pSEM233 P mlc-1 :: tagRFP-T :: cbr-tbb-2 3′ UTR (Addgene #159899) pCFJ1532 P smu-1 :: mosase(PATC) :: smu-1 3′ UTR (Addgene # 159807) All pSEM vectors are available at Addgene, but we recommend using pSEM238.

Techniques: Transgenic Assay, Expressing, Concentration Assay, Selection, Marker, Plasmid Preparation