SCA1-like disease in mice expressing wild-type ataxin-1 with a serine to aspartic acid replacement at residue 776

Abstract

Glutamine tract expansion triggers nine neurodegenerative diseases by conferring toxic properties to the mutant protein. In SCA1, phosphorylation of ATXN1 at Ser776 is thought to be key for pathogenesis. Here, we show that replacing Ser776 with a phosphomimicking Asp converted ATXN1 with a wild-type glutamine tract into a pathogenic protein. ATXN1[30Q]-D776-induced disease in Purkinje cells shared most features with disease caused by ATXN1[82Q] having an expanded polyglutamine tract. However, in contrast to disease induced by ATXN1[82Q] that progresses to cell death, ATXN1[30Q]-D776 failed to induce cell death. These results support a model where pathogenesis involves changes in regions of the protein in addition to the polyglutamine tract. Moreover, disease initiation and progression to neuronal dysfunction are distinct from induction of cell death. Ser776 is critical for the pathway to neuronal dysfunction, while an expanded polyglutamine tract is essential for neuronal death.

Figure 1. Atrophy of Purkinje cell dendrites in ATXN1[30Q]-D776 mice

Calbindin immunofluorescent confocal images taken at 60x with 150x enlargements (inserts) from the region of the molecular layer indicated by the white box. (A) Purkinje cell dendritic tree of 12-week-old wt/FVB mice having a molecular layer thickness of 184 μm, ± 2.6 (n = 6). (B) Purkinje cell dendritic tree of 12-week-old ATXN1[30Q]-S776 homozygous mice having a molecular layer thickness of 162 μm, ± 3.5 (n = 6). (C) Purkinje cell dendritic tree of 12-week-old ATXN1[82Q]-S776 mice having a molecular layer thickness of 157 μm, ± 2.7 (n = 5). (D) Purkinje cell dendritic tree of 12-week-old ATXN1[30Q]-D776 mice having a molecular layer thickness of 159 μm, ± 2.4 (n = 4).

Figure 2. Altered distribution of climbing fiber terminals on Purkinje cell dendrites in 12-week-old SCA1 mice

(A-D) Distribution of CF terminals assessed by VGLUT2 immunostaining. Left panel is calbindin immunofluorescence (green) of Purkinje cells and right panel is VGLUT2 immunostaining (red) depicting CF terminals. (A) wt/FVB mice showing a mean relative CF to molecular layer thickness height of 0.86 ± 0.006 (n = 6). (B) ATXN1[30Q]-S776 homozygous mice with a mean relative CF to molecular layer thickness height of 0.81 ± 0.009 (n = 6). (C) ATXN1[82Q]-S776 mice having a mean relative CF to molecular layer thickness height of 0.78 ± 0.012 (n = 5). (D) ATXN1[30Q]-D776 mice with a mean relative CFs to molecular layer thickness height of 0.69 ± 0.019 (n = 4). (E-H) Distribution of CF terminals assessed by anterograde olivocerebellar projection labeling. In each panel, Purkinje cells were identified by calbindin immunofluorescence (red) and climbing fiber tracts by the presence of biotin conjugated to Alexa Fluor 488 (green) following injection into the contralateral inferior olive. (E) Distribution of CF terminals on Purkinje cells in a 12-week-old wt/FVB mouse. (F) Distribution of CF terminals on Purkinje cells in a 12-week-old ATXN1[30Q]-S776 homozygous mouse. (G) Distribution of CF terminals on Purkinje cells in a 12-week-old ATXN1[82Q]-S776 mouse. (H) Distribution of CF terminals on Purkinje cells in a 12-week-old ATXN1[30Q]-D776 mouse.

Figure 3. Altered neurological status of ATXN1[30Q]-D776 mice

(A) Accelerating Rotarod performance on day four at 6 and 12 weeks-of-age. ATXN1[30Q]-S776/S776 (homozygous) mice, at 6 and 12 weeks (n = 9), were compared to age-matched wt/FVB mice, at 6 and 12 weeks (n = 10). ATXN1[30Q]-D776 mice at 6 weeks (n = 5) and at 12 weeks (n = 14), compared to age-matched littermate wt/FVB mice at 6 weeks (n = 9) and at 12 weeks (n =12; *p = 0.025 and **p = 0.0008, student t-test, two tailed equal variance). Performance of ATXN1[82Q]-S776 mice, at 6 weeks and 12 weeks (n = 15), compared to age-matched littermate wt/FVB mice, at 6 weeks and at 12 weeks (n = 8; *p = 0.006 and **p = 0.0005, student t-test, two tailed equal variance). (B) ATXN1[30Q]-D776 mice (n = 9, *p = 0.008 student t-test, two tailed equal variance) show a similar gait performance defect as ATXN1[82Q]-S776 mice (n = 20, **p = 0.005 student t-test, two tailed equal variance) compared to age-matched littermate wt/FVB mice (n = 9 &16, respectively) as assessed by hind stance width at 12 weeks-of-age. (C) Four day trial of accelerating Rotarod performance of mice at 30 weeks-of-age. Both ATXN1[30Q]-D776 (n=3) mice and ATXN1[82]-S776 (n=4) at 30 weeks-of-age were compared to age-matched wt/FVB (n=3) (p=0.04 and p=0.03).

Figure 4. Cerebellar pathology in year-old SCA1 mice

(A) Calbindin immunofluorescence of Purkinje cells in one-year-old mice expressing ATXN1[30Q]-D776 showing a molecular layer thickness of 148 ± 3.8 μm (n = 3). Insert shows the absence of nuclear inclusions in PC nuclei assessed by triple immunostaining for calbindin (blue), ATXN1 (green), ubiquitin (red), and calbindin (blue). Image is a single z-scan (180x). (B) Calbindin (red) and ATXN1 (green) immunofluorescence in Purkinje cells of 12-week-old mice expressing ATXN1[82Q]-D776. Average molecular thickness was 147 ± 2.1 μm. (C) Calbindin (red) and ATXN1 (green) immunofluorescence in Purkinje cells of 12-week-old mice expressing ATXN1[82Q]-S776. Average molecular thickness was 157 ± 2.8 μm. (D) Accelerating Rotarod performance on day four of 6 (n = 12) and 12-week-old ATXN1[82Q]-D776 (n = 5) compared to wt/FVB littermate controls. (n = 12 & 4, respectively), *p = 0.02, student t-test two tailed equal variance). (E) Calbindin immunofluorescence of Purkinje cells in one-year-old mice expressing ATXN1[82Q]-D776 with a molecular layer thickness of 97 ± 7.0 μm (n = 3). Insert depicts the presence of ubiquitin positive nuclear inclusions in PC nuclei as revealed by triple immunostaining for calbindin (blue), ATXN1 (green) and ubiquitin (red). Image is a single z-scan (180x).