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

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

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

Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

<div><p>Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.</p></div