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1 Marie-Thérèse El-aher et al non-polyq fragments and Huntington s disease The EMO Journal Expanded View Figures HTT-Q TEV rec αtub sh-htt cells 167/ /586 TEV-Q -Q23 Particle volume (µm³) 48 Tub 5 Empty 167/ TEV 167TEV 167/ 167/513TEV + + Nocodazole treated GM13 167/586 TEV-Q -Q23 -HTTQ -HTTQ -HTT N513 Tubulin (trafected) Tubulin % of survival Time (h) -Q -Q ell death ratio at 24 h (SNIPer/TR) 167/-Q fter 2 h washout 1 µm GM13 1 µm TEV 167TEV 167/ 167/513TEV α Nocodazole 2 h washout Figure EV1. Time- and site-specific control of huntingtin proteolysis. Immunoblotting analyses of HTT N-ter and -ter fragments generated after cleavage of the indicated cotructs expressed in HEK293T cells and using recombinant TEV system. no TEV referred to -Q cotructs, without TEVrcs. Survival curves of striatal cells expressing cotructs containing no TEV, one or two TEVrcs. Toxicity is expressed as the percentage of survival over time. cotructs with TEV sequence iertio at positio 167 and 586 are as functional as FL- HTT on the dynein-dependent Golgi reassembly assay. Upper panels show representative HeLa cells stably silenced for endogenous HTT and that re-express 167/586, HTT167/ -Q or -Q23 and treated with nocodazole for 2 h (left panels) and after 2-h washout (right panels). Western blotting shows the expression level of trafected in sh- HTT cell line. Graph summarising cell death ratio at 24 hof TEV-Q analysed in Fig 1. HTT proteolysis-induced toxicity is shown as a ratio of the percentage of cell death obtained in SNIPer- TEV condition over pcn condition. ata information: The graphs (mean SEM) display pooled data from three to four independent experiments (), from two independent experiments performed in duplicates () or from three to four independent experiments (). In (), total number of cells and number of independent experiments are as follows: -Q + pcn: 96 (n = 3) and FL- HTT-Q + pcn: 88 (n = 3) and FL- HTT167/-Q + pcn: 192 (n = 4). In (), total number of cells is as follows: nocodazole treated: 6, washout condition: 8. In (), total number of cells is as in Fig 1 with 14 cells for. Statistics were done by Kaplan Meier, log-rank test (), one-way NOV with onferroni s multiple comparison tests () and ordinary one-way NOV with Fisher LS test (). : non-significant, P <.5, P <.1, P <.1. ª 215 The uthors The EMO Journal EV1
2 The EMO Journal non-polyq fragments and Huntington s disease Marie-Thérèse El-aher et al HTT Q23 INPUTS / / IP IgG HTT Q INPUTS IP αtub -HTT -HTT -HTT+ -HTT+ -HTTQ -HTTQ -HTTQ tubulin ell death ratio at 24 h (Sp/TR) si-htt: / /586 TEV-Q si-htt: / /-Q + (trafected) WT HTT(endogenous) tubulin Figure EV2. Intramolecular interactio between proteolytic huntingtin fragments. Immunoprecipitatio upon cleavage of - by the SNIPer-TEV performed in HEK293T cell. indicates a non-specific band. Immunoprecipitatio of HeLa cell extracts expressing -N-HTTQ cotructs or and - or using anti- antibody. Immunoblotting analysis of the expression level of N- and -HTT alone or in combination with striatal cells. indicates a non-specific band. Toxicity in striatal cells trafected with 167/586 and 167/-Q and sirn targeted agait endogenous HTT (si-htt). Graph summarising cell death ratio at 24 h of proteolysis-induced toxicity is shown as a ratio of the percentage of cell death obtained in SNIPer-TEV condition over pcn condition. ownregulation of HTT expression was assessed by immunoblotting (right). The bar graphs (mean SEM) display pooled data from two to four independent experiments. Total number of cells is as follows: 167/586 without si-htt: 399 cells; with si-htt: 23 cells; 167/-Q without si-htt: 43 cells; with si-htt: 199 cells. Statistics were done by unpaired t-test. : non-significant. EV2 The EMO Journal ª 215 The uthors
3 Marie-Thérèse El-aher et al non-polyq fragments and Huntington s disease The EMO Journal GM13 GM TR 433 TR 433 +MSO +Z-EV-FMK H2O2 H2O2 +Z-EV-FMK 12 TGN38 rab-5 EE1 L3- TGN38 rab5- EE1 L3- E %ofcelldeath MSO +Z-V-FMK TUNEL sirn negative sirn beclin1 n1 sirn beclin1 n2 eclin 1 + sirn negative + sirn beclin1 n1 LMP-2 LMP-2 + sirn beclin1 n2 Figure EV3. Released -terminal HTT fragment elicits endoplasmic reticulum dilation, stress and cell death. Immunostainings of striatal cells trafected with -tagged fragment. Golgi apparatus compartments were immunostained with anti-gm13 (cis-golgi), anti-tr433 (medial-golgi) and anti-tgn38 (tra-golgi) antibodies. Early endosomes were either stained with anti-ee1 or labelled with Rab5-expressing cotructs. L3--labelled autophagosomes and lysosomes were immunostained with anti-lmp2. Left: Striatal cells trafected with were treated with Z-EV-FMK, a caspase-3 inhibitor and analysed for cell death. Right: Non-trafected cells were treated for 2 h with 1 mm of H 2 O 2 and Z-EV-FMK. Toxicity in striatal cells trafected with and treated with Z-V-FMK. ssessment of N fragmentation in -expressing striatal cells using a TUNEL assay. E Toxicity in striatal cells trafected with fragment and sirn targeted agait eclin-1 (two independent sirn n1 & n2). ownregulation of eclin-1 expression was assessed by immunoblotting. ata information: The bar graphs (mean SEM) display pooled data from three to five independent experiments in triplicates. For the quantification of cell death after H 2 O 2 treatment (), one experiment was performed in duplicate and more than 1, cells were analysed. Statistics were done by unpaired t-test (, ) or one-way NOV with onferroni s multiple comparison test. : Non-significant, P <.5. ª 215 The uthors The EMO Journal EV3
4 The EMO Journal non-polyq fragments and Huntington s disease Marie-Thérèse El-aher et al 48 ell death (%) N548-HTTQ N548-HTTQ128 -HTTQ23 -HTTQ -HTT N548-HTT Q128 N548-HTT Q -HTT tr 1 µm -HTT (143) IP -HTT (145) -HTT Figure EV4. -ter huntingtin causes toxicity in fly. nalysis of -HTT toxicity in striatal cells (left) and representative image of vacuolation in -HTT expressing cells (right). The graph (mean SEM) displays pooled data from three independent experiments in duplicate. Total number of cells is as follows: -HTTQ23: 2285, -HTTQ: 1698, -HTT : 483, : 78. Statistics were done by one-way NOV with onferroni s multiple comparison test, P <.1. ssessment of N548-HTT tragene expression on total brain lysates of adult flies (4 days of age) by immunoblotting. etection of -HTT tragenes in two independent fly strai (143, 145) by immunoprecipitation. ontrol flies (ctr) are W 1118 flies. EV4 The EMO Journal ª 215 The uthors
5 Marie-Thérèse El-aher et al non-polyq fragments and Huntington s disease The EMO Journal Ident Species ENS Human (Homo sapie) ENST ow (os taurus) ENSF og (anis familiaris) ENSPO Guinea Pig (avia porcellus) ENSR Zebrafish (anio rerio) ENSNO rmadillo (asypus novemcinctus) ENSOR Kangaroo rat (ipodomys ordii) ENSETE Lesser hedgehog tenrec (Echinops telfairi) ENSE Horse (Equus caballus) ENSF at (Felis catus) ENSGL hicken (Gallus gallus) ENSG Stickleback (Gasterosteus aculeatus) ENSLF Elephant (Loxodonta africana) ENSMMU Macaque (Macaca mulatta) ENSMI Mouse Lemur (Microcebus murinus) ENSMO Opossum (Monodelphis domestica) ENSMUS Mouse (Mus musculus) ENSMLU Microbat (Myotis lucifugus) ENSOU Rabbit (Oryctolagus cuniculus) ENSORL Medaka (Oryzias latipes) ENSOG ushbaby (Otolemur garnettii) ENSPTR himpanzee (Pan troglodytes) ENSPPY Orangutan (Pongo pygmaeus) ENSP Hyrax (Procavia capeis) ENSPV Megabat (Pteropus vampyrus) ENSRNO Rat (Rattus norvegicus) ENSSR Shrew (Sorex araneus) ENSSTO Squirrel (Spermophilus tridecemlineatus) ENSTRU Fugu (Takifugu rubripes) ENSTSY Tarsier (Tarsius syrichta) ENSTNI Tetraodon (Tetraodon nigroviridis) ENSTE Tree Shrew (Tupaia belangeri) ENSTTR olphin (Tursiops truncatus) % of traferrin uptaked/cell " 3" 7" 15" 45" 2' 6' Time Similarity Empty vector Empty vector+dynamin 1-K I: dynamin 1 Similarity plot of aligned sequences 2 3 Relative residue position Q N-586-HTTQ SS Q N-586-HTTQ Oligomeric form Monomeric form E F H ER-NM1-WT ER-NM1-K44 T E T E ER-NM1 () alnexin ynamin 1 ER-NM1-WT () dynamin 1 Oligomeric/ monomeric dynamin 1 12% % 8% 6% 4% 2% % IP Q -HTTQ Q -HTTQ -HTTQ Q eif2alpha Mitochondrial hsp7 nnexinv αtubulin ctin ER-dsred G ER-NM1-WT () ER-dsred 15 Input -HTTQ -HTTQ ER-NM1-WT ER-NM1-WT alnexin ER-NM1-K44() calnexin Figure EV5. -terminal HTT impairs the activity of ER-localised dynamin 1 that induces ER dilation and death. Identification of coerved regio in huntingtin. Search of primary sequence of HTT orthologues for highly coerved regio. nalysis of 32 orthologous sequences and generation of a residue coervation plot using the EMOSS package tool plocon. Smoothing was applied to the results revealing regio with higher degrees of identity. Pairwise comparison of human HTT versus. elega,. mellifera,. intestinalis,. rerio, F. rubripes or G. gallus Htt. Very N-ter part of HTT (residues 17 4) and regio starting from 12 to 3144 showed high similarity between the different species. ssessment of dynamin 1 activity by traferrin uptake in striatal cells. -tagged dynamin 1-K44 cotruct was used as a positive control for endocytosis inhibition. The curves indicate the percentage of traferrin uptake over time in s ( ) and min ( ). ynamin 1 oligomeric profile in HTT-expressing cells treated or not with SS, a non-cleavable cross-linker. HEK293T cells were trafected with the indicated HTT fragments and dynamin 1. Endogenous HTT was downregulated using sirn which do not target the HTT cotructs. E Localisation of engineered ER-targeted dynamin 1 (ER-NM1-WT or ER-NM1-K44) cotructs at ER by cellular fractionation in striatal cells. Total (T), cytosolic () and ER membrane (E) fractio. ER-NM1 was detected using anti-. F, G nalysis of localisation of engineered ER-targeted dynamin 1 (ER-NM1-WT or ER-NM1-K44) using anti- immunostaining in striatal cells. Note the artificial high co-localisation on ER network as shown by immunostaining with ER-dsRed and calnexin. H Striatal cells expressing the indicated -tagged HTT fragments and ER-NM1-WT were subjected to immunoprecipitation and analysed by immunoblotting. ata information: The graphs (mean SEM) display pooled data from three independent experiments in triplicate. In (), total number of cells is 1, cells per experiment. Statistics were done by unpaired t-test () or one-way NOV with onferroni s multiple comparison test (). P <.5, P <.1. ª 215 The uthors The EMO Journal EV5
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