Behavioural and physiological phenotypes elicited by <i>Sirt2</i> knock-down and knock-out in R6/2 mice.

<p>(<b>A–B</b>) Mean body weight measurements in (A) female and (B) male mice. (<b>C–D</b>) Grip strength in (C) female and (D) male mice. (<b>E</b>) Rotarod performance. (<b>F</b>) Brain weight measured at 9 and 15 weeks of age. Error bars represent SEM. WT – wild type, HET – <i>Sirt2</i>HET, KO – <i>Sirt2</i>KO, R6/2 HET – <i>Sirt2</i>HETxR6/2, R6/2 KO – <i>Sirt2</i>KOxR6/2.</p

Expression of cholesterogenic enzymes at 15 week of age.

<p>The Taqman qPCR assay was used to measure the mRNA expression of the cholesterogenic enzymes in the cortex of 15 week old wild type (WT), <i>Sirt2</i>HET (HET), <i>Sirt2</i>KO (KO), R6/2, <i>Sirt2</i>HETxR6/2 (R6/2 HET) and <i>Sirt2</i>KOxR6/2 (R6/2 KO) mice. Values were normalised to the housekeeping genes <i>Atp5b</i> and <i>Canx</i> and expressed as fold change of WT ± SEM. n≥7/genotype.</p

Levels of soluble mHTT in various brain regions at 4, 9 and 15 weeks of age.

<p>Representative western blots from cortex, hippocampus and brain stem of (<b>A–B</b>) 4, (<b>C–D</b>) 9 and (<b>E–F</b>) 15 week old wild type (WT), <i>Sirt2</i>HET (HET), <i>Sirt2</i>KO (KO), R6/2, <i>Sirt2</i>HETxR6/2 (R6/2 HET) and <i>Sirt2</i>KOxR6/2 (R6/2 KO) mice probed with an anti-HTT antibody (S829) and tubulin (Tub) as loading control. Both soluble mHTT transprotein and aggregates retained in the stacking gel can only be detected in mice expressing the R6/2 transgene (A, C, E). All samples were run on the same gel. White lines indicate where lanes are not contiguous.</p

Expression of cholesterogenic enzymes in WT and R6/2 mice after an acute dose of [S]-35.

<p>(<b>A</b>) Structure of [S]-35. (<b>B</b>) Amount of [S]-35 present in brain samples from mice dosed with vehicle, 1 mg/kg or 3 mg/kg as analysed by LC/MS/MS. No compound was detected in mice dosed with vehicle alone. n≥8/genotype/dose/time point. Black line denotes the SIRT2 IC₅₀ concentration of 560 nM for [S]-35 as determined in <i>in vitro</i> assays. (<b>C–D</b>) Expression of cholesterogenic enzymes in the cortex of 12 week old wild type (WT) and R6/2 mice (<b>C</b>) 4 h and (<b>D</b>) 8 h after an acute dose of the [S]-35 SIRT1/SIRT2 inhibitor or vehicle. Values were normalised to the housekeeping genes <i>Atp5b</i> and <i>Canx</i> and expressed as fold change of WT Vehicle ± SEM. n≥7/genotype/treatment group.</p

Reduction of <i>Sirt2</i> mRNA and an absence of the SIRT2 protein in <i>Sirt2</i> knock-out mice.

<p>(<b>A</b>) Exon-intron structure of the <i>Sirt2</i> gene in mouse and the location of the insertion (light blue) in exon 11 (after nucleotide 18883) in <i>Sirt2</i>KO mice. The positions of the sequencing forward and reverse primers are shown. 1-<i>Sirt2</i> forward, 2-<i>Sirt2</i> forward Seq2, 3-<i>Sirt2</i> forward Seq3, A-<i>Sirt2</i> reverse KO, B-<i>Sirt2</i> reverse WT. (<b>B</b>) Cortical <i>Sirt2</i> mRNA levels in 4 week old <i>Sirt2</i>KO (KO), <i>Sirt2</i>HET (HET) and wild type (WT) mice. Expression levels were normalised to the housekeeping genes <i>Atp5b</i> and <i>Canx</i> and expressed as fold change of WT levels ±SEM. n = 8/genotype. (<b>C</b>) Western blotting of KO, HET and WT brain lysates with SantaCruz H-95 (upper panel) and Sigma S8447 (lower panel) antibodies. The S8447 probed blot was used to quantify SIRT2 levels (both bands) between HET and WT (right panel). Values were normalised to α-tubulin (Tub) and expressed as fold change of WT ±SEM. * denotes a non-specific band. (<b>D</b>) Western blotting of KO, HET and WT brain lysates with SantaCruz H-95 antibody (long exposure) demonstrating that the <i>Sirt</i>2 disrupting mutation does not result in the production of an N-terminal fragment of SIRT2. *denotes non-specific bands. (<b>E</b>) SIRT2 is localised to the cytoplasm. Purity of fractions was determined by measuring the expression of actin (C-cytoplasm) and H3 (N-nucleus).</p

Cerebellar Soluble Mutant Ataxin-3 Level Decreases during Disease Progression in Spinocerebellar Ataxia Type 3 Mice

<div><p></p><p>Spinocerebellar Ataxia Type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominantly inherited neurodegenerative disease caused by an expanded polyglutamine stretch in the ataxin-3 protein. A pathological hallmark of the disease is cerebellar and brainstem atrophy, which correlates with the formation of intranuclear aggregates in a specific subset of neurons. Several studies have demonstrated that the formation of aggregates depends on the generation of aggregation-prone and toxic intracellular ataxin-3 fragments after proteolytic cleavage of the full-length protein. Despite this observed increase in aggregated mutant ataxin-3, information on soluble mutant ataxin-3 levels in brain tissue is lacking. A quantitative method to analyze soluble levels will be a useful tool to characterize disease progression or to screen and identify therapeutic compounds modulating the level of toxic soluble ataxin-3. In the present study we describe the development and application of a quantitative and easily applicable immunoassay for quantification of soluble mutant ataxin-3 in human cell lines and brain samples of transgenic SCA3 mice. Consistent with observations in Huntington disease, transgenic SCA3 mice reveal a tendency for decrease of soluble mutant ataxin-3 during disease progression in fractions of the cerebellum, which is inversely correlated with aggregate formation and phenotypic aggravation. Our analyses demonstrate that the time-resolved Förster resonance energy transfer immunoassay is a highly sensitive and easy method to measure the level of soluble mutant ataxin-3 in biological samples. Of interest, we observed a tendency for decrease of soluble mutant ataxin-3 only in the cerebellum of transgenic SCA3 mice, one of the most affected brain regions in Spinocerebellar Ataxia Type 3 but not in whole brain tissue, indicative of a brain region selective change in mutant ataxin-3 protein homeostasis.</p></div

Western blot and TR-FRET analyses revealed an age dependent decrease of soluble mutant ataxin-3 levels in cerebellum.

<p>A–D) Two animals of the indicated genotypes per age were immunoblotted and detected with an ataxin-3 (clone 1H9) antibody. In all samples the endogenous ataxin-3 at 42 kDa was detected (indicated by an arrow head). In transgenic SCA3 mice a protein band at 60 kDa revealed the human ataxin-3 protein with 70Qs (arrow). Whole brain lysates showed similar expression levels of overexpressed human ataxin-3 in SCA3 transgenic mice at the age of 12 and 22 months (A and densitometric analysis in C; p = 0.6). In the cerebellum, one of the mainly affected brain areas in SCA3, less overexpressed ataxin-3 is detectable at the age of 22 months compared to 12 months of age (B). Densitometric analysis confirmed this observation (D, p = 0.3). As loading control actin is shown. E, F) Analysis of these samples by TR-FRET detection revealed similar levels of ataxin-3 in SCA3 transgenic mice in whole brain lysates at the age of 12 and 22 months (p = 0.52; E). In comparison in homogenates of the cerebellum the level of overexpressed ataxin-3 in SCA3 transgenic mice decreases in an age dependent manner, although this did not reach statistical significance (p = 0.19, F). Bars represent averages and standard deviation of biological triplicates.</p

Calbindin immunoreactivity showed reduced arborization of Purkinje cells with age independently from transgene expression.

<p>A) Double-immunofluorescence staining with calbindin (green) and ataxin-3 (clone 1H9, red) revealed aggregates in the granular layer of the cerebellum but not in the Purkinje cell layer of SCA3 transgenic mice. Calbindin staining of the Purkinje cells demonstrated shrinkage and loss of cells as well as a reduction of arborization of Purkinje cells with age in both, wildtype and SCA3 transgenic mice. B) Quantification of optical densitometry of calbindin showed a loss of immunoreactivity with age in both genotypes, respectively (*p<0.05; **p<0.01; a.u. = arbitrary units). Scale bar = 20 µm, ML = molecular layer, P = Purkinje cells and GL = granular layer.</p

The number of ataxin-3 positive aggregates is inversely correlated with the level of soluble ataxin-3 in Western blot and TR-FRET analyses.

<p>A) shows representative immunohistochemical staining with an antibody against ataxin-3 (clone 1H9) in SCA3 transgenic mice compared to sex- and age-matched wildtype controls at the age of 12 and 22 months. No aggregates are detectable in wildtype animals. However, in SCA3 transgenic mice increasing numbers of aggregates are found at the age of 22 months compared to 12 months (A), but this did not reach significance (p = 0.076) after counting three independent animals per genotype (B). C) Measuring the size of aggregates in the granular layer of the cerebellum on the other hand revealed significant larger aggregates with disease progression in SCA3 transgenic mice (***p<0.001). Scale bar = 20 µm, ML = molecular layer, P = Purkinje cells and GL = granular layer.</p

EM48 staining for aggregated mutant HTT appears to increase in amount and intensity with increasing age and disease progression.

<p>In 4 week old B6 110Q R6/2 mice, very few small, circular intranuclear inclusions characteristic of 110Q R6/2 mice can be identified. However, by 10 weeks of age such inclusions are readily observed neuronal nuclei. Intensity of EM48 staining appears increase from 4 and 10 weeks of age, further suggesting an increase in aggregate load.</p