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. 2012 Dec;48(3):526-32.
doi: 10.1016/j.nbd.2012.07.024. Epub 2012 Aug 4.

LANP mediates neuritic pathology in Spinocerebellar ataxia type 1

Marija Cvetanovic  1 , Rupinder K Kular, Puneet Opal
Affiliations

LANP mediates neuritic pathology in Spinocerebellar ataxia type 1

Marija Cvetanovic et al. Neurobiol Dis. 2012 Dec.

Abstract

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease that results from a pathogenic glutamine-repeat expansion in the protein ataxin-1 (ATXN1). Although the functions of ATXN1 are still largely unknown, there is evidence to suggest that ATXN1 plays a role in regulating gene expression, the earliest process known to go awry in SCA1 mouse models. In this study, we show that ATXN1 reduces histone acetylation, a post-translational modification of histones associated with enhanced transcription, and represses histone acetyl transferase-mediated transcription. In addition, we find that depleting the Leucine-rich Acidic Nuclear Protein (LANP)-an ATXN1 binding inhibitor of histone acetylation-reverses aspects of SCA1 neuritic pathology.

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Figures

Fig. 1
Fig. 1
ATXN1 affects histone acetylation. a) The level of histone acetylation in cerebellar lysates of SCA1 mice is compared to wild-type littermates by staining with an antibody specific to acetylated histone H3. Staining with an actin-specific antibody serves as a loading control. The intensity of the lanes is quantified by densitometry where the histograms show the mean value of densitometry with error bars showing SE. n=9 pairs of adult mice. p=0.0347 using paired two-tailed t-test. b) Histone acetylation is decreased in cells expressing mutant ATXN1. HeLa cells transiently transfected with mutant ATXN1 84Q-GFP or wild-type ATXN1 2Q-GFP were sorted for GFP positive (transfected) and GFP negative (untransfected) cells. Western blot with an anti-GFP antibody shows the efficiency of sorting. Western blot with an anti-actin antibody serves as a loading control. Data is representative of three independent experiments. p=0.0122 for ATXN1 84Q (with asterisk) and p=0.3471 for ATXN1 2Q using paired two tailed t-test.
Fig. 2
Fig. 2
Synergistic transcriptional repression by ATXN1 and LANP. a) N2A cells were co-transfected with the CBP-Gal4 (100 ng/well) and increasing amounts (25, 50 and 100 ng) of LANP and ATXN1 2Q or 84Q-GFP to cause progressive repression of CBP activity when compared to cells transfected with CBP-Gal4 and empty vector. ANOVA p<0.05 comparing 50 or 100 ng of each construct (LANP, ATXN1 2Q or ATXN1 84Q) to CBP control. No statistical difference (p>0.05) was present when comparing repression caused by equal DNA amounts of ATXN1 2Q and ATXN 1 84Q. b) N2A cells were co-transfected with CBP-Gal4 and low levels (5 ng) of LANP and ATXN1 (2Q or 84Q) that by themselves do not cause any inhibition. When co-transfected LANP and ATXN1 84Q synergistically inhibit CBP activity (asterisk signifies p<0.05; Analysis of variance (ANOVA) with post-hoc t test comparing ATXN1 84Q to ATXN1 84Q and LANP). ATXN1 2Q did not show statistical synergism (p>0.05). The data in a. and b. is representative of three (a) or eight (b) independent experiments, Error bars=SE.
Fig. 3
Fig. 3
LANP depletion ameliorates the anti-neuritic effects of mutant ataxin-1 in PC12 cells. a) PC12 cells transfected with GFP (control) or GFP-tagged wild type (2Q) or mutant (84Q) ATXN1 were treated with NGF for 72 h. Cells displaying neurites were counted. Data is average of three independent experiments with error bars representing SE. Data was analyzed using ANOVA with Bonferroni post test, p<0.05 comparing GFP transfected control to ATXN1 2Q or ATXN1 84Q. b) and c) PC12 cells were transfected with one of the following constructs expressing: GFP (control), GFP-tagged wild type (2Q) or mutant (84Q) ATXN1 along with control and LANP siRNA as shown. Cells were treated with NGF for 72 h and cells displaying processes longer than one cell body length were counted (b). Length of the longest neurite in GFP positive cells was measured and plotted (c). Data is average of four (b) or three (c) independent experiments, with error bars representing SE. Data was analyzed using ANOVA with Bonferroni post test, for both b and c. p<0.05 comparing control siRNA ATXN1 84Q to LANP siRNA ATXN1 84Q; p>0.05 comparing control siRNA ATXN1 2Q to LANP siRNA ATXN1 2Q.
Fig. 4
Fig. 4
LANP deletion ameliorates pathology but not behavioral deficits in SCA1. a) Rotarod performance of 13 week old mice of indicated genotypes. Analyzed by two way ANOVA with Bonferroni post test p<0.05 comparing WT to SCA1 or WT to SCA1;LANP−/−, and p>0.05 comparing SCA1 to SCA1;LANP−/−. b-e) Cerebella of 1 year old wild type, SCA1, and SCA1;LANP−/− mice were stained with Purkinje cell specific marker calbindin or VGLUT2, labeling presynaptic terminals of climbing fibers. b) Average intensity of z-stacks was measured and normalized to the intensity of wild-type littermates (n=5). Paired t-test values were p=0.0185 and p=0.09 comparing SCA1 mice to wild-type and SCA1; LANP−/− mice respectively. c) An average width from at least six mice of each genotype is shown with error bars showing SE. Statistical analysis was performed using paired t-test with p=0.0367 and p=0.1211 comparing SCA1 mice to wild-type and SCA1;LANP−/− mice respectively. d) Quantification of VGLUT2 staining demonstrating retraction of dendritic arbor in SCA1 mice and rescue of this effect in SCA1;LANP−/− mice. n=3 mice of each genotype. Statistical analysis was performed using paired t-test with p<0.05 comparing SCA1 mice to wild-type or SCA1 mice to SCA1;LANP−/− mice. e) Representative images of VGLUT2 staining.
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