Cerebellar soluble mutant ataxin-3 level decreases during disease progression in Spinocerebellar Ataxia Type 3 mice

Abstract

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.

Figure 1. Establishing a TR-FRET based immunoassay to detect soluble ataxin-3 levels.

A) Schematic illustration of antibody binding sites in context of the ataxin-3 protein and the principal of the TR-FRET immunoassay. By using this antibody combination (1H9 and MW1) only ataxin-3 with more than 6 CAG repeats can be detected (no detection of wildtype mouse ataxin-3) since the MW1 antibody is specific against an expanded polyQ-stretch. B) Measuring mutant ataxin-3 levels in HEK293T cells transiently transfected with ataxin-3 with different polyQ-lengths (15Q, 77Q, 148Q) revealed a detection of mutant ataxin-3 with 15, 77 and 148 polyglutamine repeats. Furthermore, ataxin-3 with a highly elongated polyQ-repeat (77 or 148Q) can be detected more efficiently than ataxin-3 with a normal, non-expanded repeat (15Q, ***p<0.001). Formation of aggregates was only found in cells transfected with ataxin-3-148Q but not with ataxin-3-77Q, which is associated with a significant reduction of soluble ataxin-3-148Q compared to ataxin-3-77Q (*p<0.05). C) To validate detectable protein amounts in mouse brain homogenates different concentrations of protein levels were tested. Whereas in wildtype lysates only a background noise signal was measured, in SCA3 transgenic whole brain lysates a protein concentration dependent TR-FRET signal was detected. D, E) To validate the specificity of the used antibody combinations for the disease protein ataxin-3 (1H9– MW1) or huntingtin (2B7– MW1) a detection of protein in ataxin-3 transgenic mice (SCA3), huntingtin transgenic mice (R6/2) and wildtype mice was carried out. The antibody combination for ataxin-3 (1H9– MW1) showed only a specific signal in SCA3 transgenic mice, whereas the antibodies tailored for huntingtin (2B7– MW1) only detect in R6/2 huntingtin transgenic mice. Bars represent averages and standard error of the mean n = 6.

Figure 2. Western blot and TR-FRET analyses revealed an age dependent decrease of soluble mutant ataxin-3 levels in cerebellum.

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.

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

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.

Figure 4. Calbindin immunoreactivity showed reduced arborization of Purkinje cells with age independently from transgene expression.

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.