Age-dependent increase of α-Syn immunoreactivity in the ileum
As growing evidence suggesting that PD also affects the gut and the incidences of the debilitating digestive symptoms may partly be explained by the presence of inclusions and debris immunoreactive for α-Syn in the enteric nervous system [26], the α-Syn accumulation was evaluated in the enteric nervous system with aging and immunofluorescence analysis of samples from the 1-, 6-, 12-, and 18-month-old mice was performed. As shown in Figure 1, the amount of α-Syn immunoreactivity in the ileum was systematically increased with aging. Furthermore, there was a significant difference in the levels of ileum α-Syn immunoreactivity in samples from the 18-month-old group compared to the 1-month-old group (p<0.05, Figure 1).
Age-dependent increase of α-Syn immunoreactivity in the thoracic spinal cord
In order to investigate whether α-Syn immunoreactivitychanges in the thoracic spinal cord with aging, immunofluorescence analysis of samples from the 1-, 6-, 12-, and 18-month-old mice was performed. The analysis showed that the levels of α-Syn immunoreactivity in the thoracic spinal cord increased with aging. There was a significant difference in levels of the thoracic spinal cord α-Syn immunoreactivity from samples of 18-month-old group compared to samples from the 1-month-old group (p<0.05, Figure 2). Furthermore, predominant location of α-Syn immunoreactivity in the thoracic spinal cord was changed with aging. In the 1-month- and 6-month-old groups, the α-Syn immunoreactivity was predominantly presented in white matter of the thoracic spinal cord. However, the α-Syn immunoreactivity was predominantly displayed in grey matter of the thoracic spinal cord in the 12-month- and 18-month-old group.
Age-dependent increase of α-Syn immunoreactivity in the midbrain
To investigate variation of the α-Syn immunoreactivity in the midbrain with aging, immunofluorescence was performed using samples from at 1-, 6-, 12-, and 18-month-old mice. This analyses revealed that the levels of α-Syn immunoreactivity in the midbrain were increased with aging, while there was a trend of age-related decreases in the number of dopminegic neuron. There was a significant difference in the levels of the midbrain α-Syn immunoreactivity from 18-month-old group compared to the those of the 1-month-old group (p<0.05, Figure 3). However, there was no significant difference in the number of dopaminergic neuron among all groups.
Age-dependent increase in the levels of α-Syn monomers and oligomers in the ileum, the thoracic spinal cord, and the midbrain
To examine whether the levels of α-Syn aggregation are changed with aging in vivo, α-Syn monomers and oligomers in the ileum, thoracic spinal cord, and midbrain were detected by Western blotting in samples from at 1-, 6-, 12-, and 18-month-old mice. This analysis revealed that the levels of α-Syn monomers and oligomers in the ileum, thoracic spinal cord, and midbrain were increased with aging (Figure 4A). The levels of α-Syn monomers in the ileum, thoracic spinal cord, and midbrain obtained from 18-month-old group were significantly higher than those in the corresponding tissues from the 1-month-old group (p<0.05, Figure 4B). Meanwhile, there was no significant difference between the 1-month-old group and the 12-month-old group. Besides, the levels of α-Syn oligomers in the ileum, thoracic spinal cord, and midbrain obtained from the 18-month-old group were also significantly increased in the corresponding tissues when compared to those form the 1-month-old group. Interestingly, the significant increase was also detected at the 12-month in the thoracic spinal cord and midbrain as well (p<0.05, Fig 4C). Furthermore, the ratios of oligomers to monomers in every group was analyzed. The results showed that the ratios of oligomers to monomers in the ileum, thoracic spinal cord, and midbrain obtained from 18-month-old group were all significantly higher than obtained from the 1-month-old group (p<0.05, Fig 4D). Moreover, the ratios in the ileum and midbrain from the 12-month-old group were increased significantly when compared with the 1-month-old group (p<0.05, Fig 4D).
Age-dependent decrease in the α-Syn mRNA expression levels in the ileum, thoracic spinal cord, and midbrain
To further confirmed changes in the expression of α-Syn with aging, the levels of α-Syn mRNA were quantified at 1, 6, 12, and 18 month in the ileum, thoracic spinal cord, and midbrain respectively. As shown in Figure 5, there was no significant change in the levels of α-Syn mRNA with age among all groups, although α-Syn mRNA expression decreased gradually with aging (Figure 5).
Simultaneous age-dependent increase in the caspase-1 levels in the ileum, thoracic spinal cord, and midbrain
Cellular stress triggers caspase-1 activation and caspase-1-mediated release of interleukin-1 (IL-1) and IL-18, which triggers an inflammatory response. In neurodegenerative diseases, the continued accumulation of misfolded protein aggregates can trigger and maintain the activation of inflammasome, which drives CNS inflammation and neuropathology(Gordon et al., 2018). Previous studies have suggested the possibility of activated caspase-1 directly cleaving a-Syn into smaller fragments(Wang et al., 2016). To test whether the levels of caspase-1 vary with aging as well, caspase-1 expression in the ileum, thoracic spinal cord, and midbrain at 1, 6, 12, and 18 months was quantified by Western blotting analysis. This analysis indicated that the levels of caspase-1 increased gradually with the age. Furthermore, the leveles of caspase-1 in the 18-month-old group were significantly higher than those in the 1-month-old group (p<0.05, Figure 6).
Inhibition of caspase-1 activation induces decrease in the α-Syn accumulation
To see whether caspase-1 is involved in the α-Syn accumulation, we treated SH-SY5Y cells with the caspase-1-specific inhibitor VX765 and monitored its effects on the aggregation of α-Syn. Western blot analysis showed that both 100 nM and 200 nM rotenone treatment increased the levels of α-Syn monomers and oligomers in the SH-SY5Y cells. Furthermore, the levels of α-Syn monomers and oligomers in 200 nM rotenone group were significantly higher than in the control group. However, VX765 significantly reduced the levels of both oligomers and monomers of α-Syn in 200 nM rotenone group (p<0.05, Figure 7). Futhermore, immunofluorescence staining also demonstrated that caspase-1 inhibitor VX765 could inhibit the α-Syn accumulation promoted by rotenone (Figure 8).