Research ArticleNEURODEGENERATIVE DISEASE

mGluR5 antagonism increases autophagy and prevents disease progression in the zQ175 mouse model of Huntington’s disease

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Science Signaling  19 Dec 2017:
Vol. 10, Issue 510, eaan6387
DOI: 10.1126/scisignal.aan6387

Figures

  • Fig. 1 Chronic administration of CTEP improves motor impairments in zQ175 mice.

    (A and B) Mean ± SEM of grip strength [gram-force (gf)] after acute (1 week; A) and chronic (12 weeks; B) treatment with vehicle or CTEP (2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine) (2 mg/kg) in heterozygous zQ175 (Q175/+), homozygous zQ175 (Q175/Q175), and wild-type (+/+) mice (n = 12 for all groups). (C and D) Mean ± SEM of latency to fall from accelerating rotarod after acute (1 week; C) and chronic (12 weeks; D) treatment with vehicle or CTEP in Q175/+, Q175/Q175, and wild-type mice (n = 12 for all groups). (E and F) Mean ± SEM of velocity (E) and distance traveled (F) in open field after chronic (12 weeks) treatment with vehicle or CTEP in Q175/+, Q175/Q175, and wild-type mice (n = 12 for all groups). *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) comparisons.

  • Fig. 2 Administration of CTEP improves performance on the ladder rung test in zQ175 mice.

    (A and B) Mean ± SEM of percent error (% error) in limb placement while completing the horizontal ladder task after acute (1 week; A) and chronic (12 weeks; B) treatment with vehicle or CTEP (2 mg/kg) in Q175/+, Q175/Q175, and wild-type (+/+) mice (n = 12 for all groups). (C and D) Mean ± SEM of the time required to complete the horizontal ladder task after acute (1 week; C) and chronic (12 weeks; D) treatment with vehicle or CTEP in Q175/+, Q175/Q175, and wild-type mice (n = 12 for all groups). **P < 0.01 and ***P < 0.001 by two-way ANOVA and Fisher’s LSD comparisons.

  • Fig. 3 Administration of CTEP improves cognitive deficits in zQ175 mice.

    (A and B) Mean ± SEM of the recognition index, for exploring a novel object versus a familiar object on the second day of novel object recognition test, after acute (1 week; A) and chronic (12 weeks; B) treatment with vehicle in heterozygous zQ175 (Q175/+), homozygous zQ175 (Q175/Q175), and wild-type (+/+) mice (n = 12 for all groups). (C and D) Mean ± SEM of the recognition index, for exploring a novel object versus a familiar object in the second day of novel object recognition test, after acute (1 week; C) and chronic (12 weeks; D) treatment with CTEP (2 mg/kg) in Q175/+, Q175/Q175, and wild-type mice (n = 12 for all groups). **P < 0.01 and ***P < 0.001 by two-way ANOVA and Fisher’s LSD comparisons.

  • Fig. 4 Chronic administration of CTEP enhances the clearance of mutant huntingtin aggregates in zQ175 mice.

    (A to C) Representative images of staining for mutant Htt using the antibody EM48 (A) and quantification of the number (B) and size (C) of huntingtin aggregates in striatal brain slices from heterozygous zQ175 (Q175/+) and homozygous zQ175 (Q175/Q175) mice after chronic (12 weeks) treatment with vehicle or CTEP (2 mg/kg). (D to F) Representative images of EM48 staining and quantification of the number (E) and size (F) of huntingtin aggregates in cortical brain slices from Q175/+ and Q175/Q175 mice after chronic treatment with vehicle or CTEP. (G to I) Representative images of EM48 staining (G) and quantification of the number (H) and size (I) of huntingtin aggregates in hippocampal brain slices from Q175/+ and Q175/Q175 mice after chronic treatment with vehicle or CTEP. Images are representative of six independent experiments (scale bars, 50 μm). Data are means ± SD of five different 900-μm2 regions from six brain slices of different regions in five to six mice per group. **P < 0.01 and ***P < 0.001 by two-way ANOVA and Fisher’s LSD comparisons.

  • Fig. 5 Treatment with CTEP reduces ERK1/2 phosphorylation, caspase-3 activity, and cell death.

    (A) Representative Western blots and mean ± SD showing the fold change in pERK1/2 as a percent of ERK1/2 (extracellular signal–regulated protein kinases 1 and 2) in brain lysates from wild-type (+/+), heterozygous zQ175 (Q175/+), and homozygous zQ175 (Q175/Q175) mice after 12 weeks of treatment with either vehicle or CTEP (2 mg/kg) expressed as the fraction of the vehicle-treated wild-type control (n = 5 to 6). (B) Caspase-3 activity measured in brain lysates from vehicle- and CTEP-treated wild-type, Q175/+, and Q175/Q175 mice (n = 3). RFU, relative fluorescence unit. (C and D) Representative confocal microscopy images of in situ BrdU-Red DNA Fragmentation [terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL)] staining (C) and quantification of TUNEL-positive cells counterstained with Hoechst (D) in striatal slices from vehicle- and CTEP-treated wild-type, Q175/+, and Q175/Q175 mice (n = 6). Scale bar, 50 μm. (E) Quantification of the number of neuronal nuclei (NeuN)–positive neurons in striatal brain slices from vehicle- and CTEP-treated wild-type, Q175/+, and Q175/Q175 mice. Data are quantified from five different 300-μm2 regions from five to six independent mouse brains for each group. Data are means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA and Fisher’s LSD comparisons.

  • Fig. 6 Chronic CTEP treatment activates an autophagy pathway in zQ175 mice.

    (A) Representative Western blots of pGSK3β-Ser9, ZBTB16, ATG14, and p62 with the corresponding loading controls in brain lysates from heterozygous zQ175 (Q175/+), homozygous zQ175 (Q175/Q175), and wild-type (+/+) mice after chronic treatment with either vehicle or CTEP (2 mg/kg). GSK3β, glycogen synthase kinase 3β. (B to E) Quantification of blots represented in (A), presented as fold change in pGSK3β-Ser9 (B), ZBTB16 (C), ATG14 (D), and p62 (E) band intensity relative to each in vehicle-treated wild-type samples (n = 5 to 6). Data are means ± SD. (F and G) Representative confocal images of EM48 (green) and p62 (red) colocalization in striatal slices from vehicle-treated (F) and CTEP-treated (G) Q175/Q175 mice. Images are representative of three independent experiments. Scale bars, 50 μm. (H) Quantification of p62 immunostaining shown as mean ± SD of the number of fluorescent p62 aggregates in five 900-μm2 regions from six brain slices from three different vehicle- and CTEP-treated wild-type, Q175/+, and Q175/Q175 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA and Fisher’s LSD comparisons.

  • Fig. 7 Chronic CTEP treatment activates classical autophagic pathways that regulate autophagosome biogenesis.

    (A to D) Representative Western blots (A) and mean ± SD for fold change in pULK1-Ser757 (B), pATG13-Ser355 (C), and Beclin1 (D) with the corresponding loading controls in brain lysates from heterozygous zQ175 (Q175/+), homozygous zQ175 (Q175/Q175), and wild-type (+/+) mice after chronic treatment with either vehicle or CTEP (2 mg/kg) and expressed as a fraction of the vehicle-treated wild-type samples (n = 5 to 6). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA and Fisher’s LSD comparisons.

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