1. The striatal long noncoding RNA Abhd11os is neuroprotective against an N-terminal fragment of mutant huntingtin in vivo 
Laetitia Francelle, Laurie Galvan, Marie-Claude Gaillard, Fanny Petit, Benoît Bernay, Martine Guillermier, Gilles Bonvento, Noëlle Dufour, Jean-Marc Elalouf, Philippe Hantraye, Nicole Déglon, Michel de Chaldée, Emmanuel Brouillet 
Neurobiology of Aging 
Volume 36, Issue 3, Pages 1601.e e16 (March 2015)
DOI: /j.neurobiolaging
Copyright © 2015 The Authors Terms and Conditions

2. Fig. 1
Downregulated expression of Abhd11os in genetic mouse models of HD. Expression of Abhd11os was measured by RT-qPCR in 9-month-old BACHD mice (A) and 13-month-old KI140CAG (B). Controls (CTRL) are age-matched wild-type littermates. Results are presented as mean ± standard error of the mean. HET, heterozygous (140Q/+); HOM, homozygous (140Q/140Q) knock-in mice carrying an expanded CAG repeat. *p < 0.05, n = 6 mice per group, unpaired Student t test; **p < 0.001, n = 5 mice per group, 1-way ANOVA and Bonferroni post hoc. Abbreviations: ANOVA, analysis of variance; HD, Huntington disease.
Neurobiology of Aging  , 1601.e e16DOI: ( /j.neurobiolaging )
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3. Fig. 2
Efficiency of the lentiviral vectors (LV) developed for Abhd11os overexpression and knock-down. (A) Mice were injected with LV-Abhd11os mixed with LV-GFP in a 4:1 ratio in the right striatum and with a lentiviral vector expressing beta-galactosidase (LV-LacZ; control) mixed with LV-GFP in the left striatum. A second group of mice was injected with LV-shAbhd11os-GFP (bicistronic construct also expressing GFP) in the right striatum and with a lentiviral vector expressing a shRNA directed against luciferase (LV-shLuc-GFP; control) in the left striatum. Six weeks later, the striatal regions expressing GFP (B) were dissected out from fresh slices using a punch (C) and analyzed by RT-qPCR (D). Results are expressed relative to controls as mean ± standard error of the mean (n = 5 to 7 mice per group). *p < , paired Student t test. Abbreviations: GFP, green fluorescent protein; shRNA, small hairpin RNA.
Neurobiology of Aging  , 1601.e e16DOI: ( /j.neurobiolaging )
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4. Fig. 3
Effect of the overexpression of Abhd11os on the toxicity induced by mHtt. Adult male mice received a bilateral intrastriatal injection of a mixture containing LV-Htt171-82Q with either LV-LacZ (LacZ, control) or LV-Abhd11os. Six weeks after infection, brains were processed for histologic evaluation using anti-DARPP32, anti-NeuN, and anti-ubiquitin-immunohistochemistry. Left panel: typical coronal mouse brain sections displaying representative areas with depleted staining. Right panel: histograms representing quantitative determination of the volume with depleted staining in the striatum. Results are expressed as mean ± standard error of the mean (n = 6–11 mice/group). *p < 0.05; **p < 0.02, unpaired Student t test. Scale bars: NeuN, DARPP32, 100 μm; ubiquitin, 50 μm; and (lower images) 20 μm. Abbreviations: LV, lentiviral vectors; mHtt, mutant huntingtin.
Neurobiology of Aging  , 1601.e e16DOI: ( /j.neurobiolaging )
Copyright © 2015 The Authors Terms and Conditions

5. Fig. 4
Effect of the knock down of Abhd11os on the toxicity induced by mHtt. Adult male mice received a bilateral intrastriatal injection of a mixture containing LV-Htt171-82Q with either LV-shLuc (control) or LV-shAbhd11os. Six weeks after infection, brains were processed for histologic evaluation using anti-DARPP32, anti-NeuN, and anti-ubiquitin-immunohistochemistry. Left panel: typical coronal mouse brain sections displaying representative areas with depleted staining. Right panel: histograms representing quantitative determination of the volume with depleted staining in the striatum. Results are expressed as mean ± standard error of the mean (n = 7–12 mice/group). *p < 0.05, unpaired Student t test. Scale bars: NeuN, DARPP32, 100 μm; ubiquitin, 50 μm; and (lower images) 20 μm. Abbreviations: LV, lentiviral vectors; mHtt, mutant huntingtin.
Neurobiology of Aging  , 1601.e e16DOI: ( /j.neurobiolaging )
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6. Supplementary Fig. 1
Abhd11os RNA expression in HEK293T cells does not lead to a peptide production. (A) Representation of DNA constructs containing the full-length sequence of Abhd11os, for lentiviral-mediated expression in mice (construct ORF1 and 2) or containing either ORF1 (construct ORF1) or ORF2 (construct ORF2) and a C-terminal HA tag to permit their potential detection in transfected HEK293T. (B) Validation of the plasmids used for biochemical studies done by molecular biology analysis. Construct ORF1 and ORF2 with or without ATG start codon (construct without start codon is used as control of nonexpression) were digested by restriction enzymes and separated in agarose gel to reveal the DNA fragments. Bands obtained correlate with the expected sizes (numbers in white upper the gel) according molecular weight marker (1 kbp). (C) Attempt to detect a putative peptide corresponding to Abhd11os by Western blot analysis of total protein extracts in HEK293T cells transfected with plasmids coding ORF1 and ORF2 constructs, with or without ATG start codon. Compared with protein expression of a control plasmid (Dclk3-HA), Abhd11os plasmids did not lead to the production of a detectable peptide. Actin and active caspase 3 were used to control protein load in each lane. Duplicate experiments are presented with medium (left) and low (right) molecular weights markers. (D) Detection of a putative peptide corresponding to Abhd11os by immunofluorescence of the HA tag in HEK293T cells. ORF1 and ORF2 constructs, with or without ATG start codon were used for transfection. As negative control, HEK293T cells were transfected with an empty vector. As positive control, HEK293T cells were transfected with a plasmid expressing the striatal protein Dclk3 with an HA tag. (E) In vitro assessment of the efficiency of the constructs used for Abhd11os overexpression and downregulation. Transfection of HEK293T cells with full-length Abhd11os resulted in a significant overexpression of Abhd11os (taken as 100% of expression in these experiments) as compared with nontransfected cells. Co-transfection with sh-Abhd11os led to a significant downregulation of Abhd11os RNA expression as compared with cells transfected with Abhd11os alone. **p < 0.01, n = 3, as compared with N.T. *p > 0.05, n = 3, as compared with Abhd11os, 1-way ANOVA and Bonferroni post hoc. Abbreviations: ANOVA, analysis of variance; HA, hemagglutinin; N.T., non-transfected cells.
Neurobiology of Aging  , 1601.e e16DOI: ( /j.neurobiolaging )
Copyright © 2015 The Authors Terms and Conditions

7. Supplementary Fig. 2
Impact of overexpression of Abhd11os on endogenous Abhd11 expression. Mice were injected with a lentiviral vector expressing beta-galactosidase (LV-LacZ; control) mixed with LV-GFP in the left striatum, and with LV-Abhd11os mixed with LV-GFP in the right striatum. Results are expressed relative to controls as mean ± standard error of the mean (Lacz, n = 5; Abhd11os, n = 7). Endogenous Abhd11 level expression in mice injected with LacZ compared with mice injected with Abhd11os. *p < 0.02, n = 7 mice/group, paired Student t test. Abbreviations: GFP, green fluorescent protein; LV, lentiviral vector.
Neurobiology of Aging  , 1601.e e16DOI: ( /j.neurobiolaging )
Copyright © 2015 The Authors Terms and Conditions