Abstract
Our aim was to elucidate whether osteopontin (OPN) is involved in the onset of mineralisation and progression of extracellular calcification in striatal lesions due to mitochondrial toxin 3-nitropropionic acid exposure. OPN expression had two different patterns when observed using light microscopy. It was either localised to the Golgi complex in brain macrophages or had a small granular pattern scattered in the affected striatum. OPN labelling tended to increase in number and size over a 2-week period following the lesion. Ultrastructural investigations revealed that OPN is initially localised to degenerating mitochondria within distal dendrites, which were then progressively surrounded by profuse OPN on days 7–14. Electron probe microanalysis of OPN-positive and calcium-fixated neurites indicated that OPN accumulates selectively on the surfaces of degenerating calcifying dendrites, possibly via interactions between OPN and calcium. In addition, 3-dimensional reconstruction of OPN-positive neurites revealed that they are in direct contact with larger OPN-negative degenerating dendrites rather than with fragmented cell debris. Our overall results indicate that OPN expression is likely to correlate with the spatiotemporal progression of calcification in the affected striatum, and raise the possibility that OPN may play an important role in the initiation and progression of microcalcification in response to brain insults.
Highlights
Excess mitochondrial calcium can lead to intramitochondrial calcification, which is proposed to act as calcium deposit nucleation[22,30,31,32,33]
Consistent with this hypothesis, the induction of OPN expression is associated with degenerating mitochondria in the striatal neurons of rats treated with the mycotoxin 3-nitropropionic acid (3-NP)34. 3-NP, which is a natural mitochondrial toxin, irreversibly inhibits the mitochondrial respiratory chain complex II, which leads to an increase in electron leakage from mitochondria and the production of reactive oxygen species[35,36,37]
No significant immunoreactivity for OPN was detected in the cortex or striatum of saline-treated control rats or in the un-lesioned cortex of rats injected with 3-NP (Fig. 1a), as reported previously[3,4,5,23,25]
Summary
Excess mitochondrial calcium can lead to intramitochondrial calcification, which is proposed to act as calcium deposit nucleation[22,30,31,32,33] These observations may lead us to speculate that OPN is involved in the onset of mineralisation, including in intracellular calcification, which occurs in degenerating neurons. 3-NP, which is a natural mitochondrial toxin, irreversibly inhibits the mitochondrial respiratory chain complex II, which leads to an increase in electron leakage from mitochondria and the production of reactive oxygen species[35,36,37] Taken together, these observations indicate that the 3-NP model is a good model for better understanding the mechanisms underlying the intracellular calcification elicited by brain insults. We used focused ion beam milling (FIB)-scanning electron microscopy (SEM) combined with 3-dimensional (3D) reconstruction, which is a useful method for the study of brain ultrastructure and is used in correlative light and electron microscopic studies[39,40,41]
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