IGF-1 as a Potential Therapy for Spinocerebellar Ataxia Type 3

Abstract

Although the effects of growth hormone (GH) therapy on spinocerebellar ataxia type 3 (SCA3) have been examined in transgenic SCA3 mice, it still poses a nonnegligible risk of cancer when used for a long term. This study investigated the efficacy of IGF-1, a downstream mediator of GH, in vivo for SCA3 treatment. IGF-1 (50 mg/kg) or saline, once a week, was intraperitoneally injected to SCA3 84Q transgenic mice harboring a human ATXN3 gene with a pathogenic expanded 84 cytosine-adenine-guanine (CAG) repeat motif at 9 months of age. Compared with the control mice harboring a 15 CAG repeat motif, the SCA3 84Q mice treated with IGF-1 for 9 months exhibited the improvement only in locomotor function and minimized degeneration of the cerebellar cortex as indicated by the survival of more Purkinje cells with a more favorable mitochondrial function along with a decrease in oxidative stress caused by DNA damage. These findings could be attributable to the inhibition of mitochondrial fission, resulting in mitochondrial fusion, and decreased immunofluorescence staining in aggresome formation and ataxin-3 mutant protein levels, possibly through the enhancement of autophagy. The findings of this study show the therapeutic potential effect of IGF-1 injection for SCA3 to prevent the exacerbation of disease progress.

Keywords: Purkinje cells; autophagy; insulin-like growth factor-1; locomotor function; mitochondrial function; spinocerebellar ataxia type 3.

Figure 1

IGF-1 prevented impairment of the motor function in the SCA3 mice. (a) Latency to fall (time in seconds for which the mice persisted on the rotarod) for the SCA3 15Q mice and the saline- and IGF-1-treated SCA3 84Q mice during the 9 months of treatment. (b) Within the same group, the latency to fall at pretreatment was normalized to 100%. (c) EthoVision XT 7.0 software was used to analyze trajectories of the mice in the behavioral test. (d) The distance of movement, time of movement, frequency of zone change, and average velocity were included in transformed indices. (e) Captured images of the single stance for each paw. (f) Catwalk parameters included the step cycle, stride length, stand, and average speed. The data are presented as the means ± SEM. Note: # p < 0.05 denotes statistical significance in the saline-treated SCA3 84Q mice compared with the SCA3 15Q mice; * p < 0.05 indicates a significant difference.

Figure 2

IGF-1 prevented the death of the PCs in the cerebellum of the SCA3 84Q mice. (a) The arrows indicate the PCs located at the PCL (right panel). The average number of the PCs per 100 μm in the posterior lobules of the cerebellum is presented in the bar graph (mean ± SEM) (left panel). SCA3 15Q, n = 6; SCA3 84Q, n = 8; SCA3 84Q + IGF-1, n = 8. (b) Western blot analysis of calbindin (left panel). Relative expression levels of calbindin in the cerebellum (mean ± SEM) (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. (c) The lines indicate the distance from the tip of the granular layer (GL) to the white matter (right panel). Histogram showing the thickness of the GL (mean ± SEM) (left panel). SCA3 15Q, n = 5; SCA3 84Q, n = 6; SCA3 84Q + IGF-1, n = 8. (d) The frames are the sampling area of the Figure and the lines refer to the distance from the PCL to the edge of the molecular layer (ML) (right panel). Histogram showing the thickness of the ML (mean ± SEM) (left panel). SCA3 15Q, n = 5; SCA3 84Q, n = 6; SCA3 84Q + IGF-1, n = 8. Note: * p < 0.05 indicates a significant difference.

Figure 3

IGF-1 reduced the ataxin-3 protein level in the cerebellum of the SCA3 84Q mice. (a) Immunochemical staining of ataxin-3 in the cerebellum. The black arrows indicate PCs (right panel). Histograms show the means ± SEM (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (b) Western blot confirming ataxin-3 expression in the mouse cerebellum (left panel). Quantification of the ataxin-3 level relative to the total protein level (mean ± SEM) (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (c) Slices of the cerebellum of two mice in each group were selected and double-labeled using an aggresome detection kit (red) and an Alexa 488-conjugated secondary IgG against the anti-ataxin-3 antibody (green), and fluorescence intensities of 30–40 PCs in each mouse were examined using the ImageJ software. The white arrows indicate PCs. SCA3 15Q, n = 2; SCA3 84Q, n = 2; SCA3 84Q + IGF-1, n = 2. Note: * p < 0.05 indicates a significant difference.

Figure 4

Expression of the autophagic influx in the SCA3 mice. Representative Western blots of the autophagy-related markers (right panel). Quantitative results of the autophagy-related proteins were normalized to those of total protein (mean ± SEM) (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. Note: * p < 0.05 indicates a significant difference.

Figure 5

Expression of the mitochondrial function in the SCA3 mice. (a) Typical trace of respirometry measurements recorded using an Oroboros O2k with 2 mg/mL of the cerebellum. The blue curve indicates the oxygen concentration in the sealed chamber, whereas the red curve shows the oxygen consumption of tissue cells (left panel). Oxygen consumption of cells at different mitochondrial stages was corrected for ROX, and the respiratory capacities in the routine, OXPHOS, Max-Ox, and Max-U states were plotted as the means ± SEM (right panel). SCA3 15Q, n = 3; SCA3 84Q, n = 3; SCA3 84Q + IGF-1, n = 2. (b) The 8-OHdG protein expression in the cerebellum sections by IHC staining analysis; the arrows indicate PCs (left panel). Histogram shows the mean ± SEM (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 5; SCA3 84Q + IGF-1, n = 5. (c) Western blot was performed to analyze the expression of mitochondrial dynamics-related proteins (right panel). Quantification of mitochondrial dynamics-related proteins (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. Note: * p < 0.05 indicates a significant difference.

Figure 6

Plasma concentration of Nf-L. SCA3 15Q, n = 6; SCA3 84Q, n = 8; SCA3 84Q + IGF-1, n = 8. Note: * p < 0.05 was considered a statistically significant difference.

Figure 7

Tissue sections of the liver, lung, and kidney. No significant histopathological findings of the kidneys, liver, and lungs were observed in the SCA3 15Q, saline-treated SCA3 84Q, and IGF-1-treated SCA3 84Q mice. n = 3 in all the groups.