Inhibition of Calpain Prevents Manganese-Induced Cell Injury and Alpha-Synuclein Oligomerization in Organotypic Brain Slice Cultures

Abstract

Overexposure to manganese has been known to promote alpha-synuclein oligomerization and enhance cellular toxicity. However, the exact mechanism of Mn-induced alpha-synuclein oligomerization is unclear. To explore whether alpha-synuclein oligomerization was associated with the cleavage of alpha-synuclein by calpain, we made a rat brain slice model of manganism and pretreated slices with calpain inhibitor II, a cell-permeable peptide that restricts the activity of calpain. After slices were treated with 400 μM Mn for 24 h, there were significant increases in the percentage of apoptotic cells, lactate dehydrogenase release, intracellular [Ca2+]i, calpain activity, and the mRNA and protein expression of calpain 1 and alpha-synuclein. Moreover, the number of C- and N-terminal fragments of alpha-synuclein and the amount of alpha-synuclein oligomerization also increased. These results also showed that calpain inhibitor II pretreatment could reduce Mn-induced nerve cell injury and alpha-synuclein oligomerization. Additionally, there was a significant decrease in the number of C- and N-terminal fragments of alpha-synuclein in calpain inhibitor II-pretreated slices. These findings revealed that Mn induced the cleavage of alpha-synuclein protein via overactivation of calpain and subsequent alpha-synuclein oligomerization in cultured slices. Moreover, the cleavage of alpha-synuclein by calpain 1 is an important signaling event in Mn-induced alpha-synuclein oligomerization.

Untangling the Manganese-α-Synuclein Web

“…The C-terminal end undergoes phosphorylation at multiple sites and is believed to be integral in the formation of fibrils but the mechanisms are unclear. Early intermediary oligomers of α-Syn appear to be the pathogenic species, rather than the mature fibrils (Xu et al, 2015 ). Of particular interest to this review, α-Syn exhibits an affinity for metals (Uversky et al, 2001 ; Binolfi et al, 2006 ) and it appears as though protein aggregation and cross-linking can be triggered by the presence of metals such as Aluminum (Al), Copper (Cu), Cadmium (Cd), Iron (Fe), Mn, and Zinc (Zn; Paik et al, 1999 ).…”

“…It has been hypothesized that α-Syn aggregation occurs through oligomerization of fragmented α-Syn. Calpain I was shown to play an important signaling role in Mn-induced α-Syn oligomerization (Xu et al, 2015 ). Xu and colleagues treated organotypic rat brain slices with 400 μM Mn 2+ for 24 h and observed increases in the number of apoptotic cells (by up to 29.6%), lactate dehydrogenase release, intracellular calcium concentrations, calpain activity, mRNA and protein expression of calpain 1, and α-Syn.…”

“…Xu and colleagues treated organotypic rat brain slices with 400 μM Mn 2+ for 24 h and observed increases in the number of apoptotic cells (by up to 29.6%), lactate dehydrogenase release, intracellular calcium concentrations, calpain activity, mRNA and protein expression of calpain 1, and α-Syn. Calpain inhibitor II was used to pretreat cells prior to Mn exposure in an effort to explore the role of calpain in α-Syn oligomerization following Mn exposure (Xu et al, 2015 ). Pretreatment with calpain inhibitor II (4 μM) decreased the number of C- and N-terminal fragments of α-Syn and decreased overall oligomerization of α-Syn, suggesting that calpain-cleaved α-Syn fragments promote α-Syn oligomerization (Xu et al, 2015 ).…”

“…Calpain inhibitor II was used to pretreat cells prior to Mn exposure in an effort to explore the role of calpain in α-Syn oligomerization following Mn exposure (Xu et al, 2015 ). Pretreatment with calpain inhibitor II (4 μM) decreased the number of C- and N-terminal fragments of α-Syn and decreased overall oligomerization of α-Syn, suggesting that calpain-cleaved α-Syn fragments promote α-Syn oligomerization (Xu et al, 2015 ). This study provided further evidence that suggests that calpain I interacts with α-Syn in the cytoplasm of neuronal cells (Xu et al, 2015 ).…”

Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism–dystonia

“…Furthermore, polymorphisms in ATP13A2 are associated with increased susceptibility to Mn toxicity 16 . Alpha-synuclein is also suggested to act as an intracellular Mn store 17 , and Mn exposure promotes alpha-synuclein oligomerization and enhances cellular toxicity 18 19 20 .…”