Abstract
Nelfinavir (NLF), an antiretroviral agent, preserves mitochondrial membranes integrity and protects mature brain against ischemic injury in rodents. Our study demonstrates that in neonatal mice NLF significantly limits mitochondrial calcium influx, the event associated with protection of the brain against hypoxic-ischemic insult (HI). Compared to the vehicle-treated mice, cerebral mitochondria from NLF-treated mice exhibited a significantly greater tolerance to the Ca2+-induced membrane permeabilization, greater ADP-phosphorylating activity and reduced cytochrome C release during reperfusion. Pre-treatment with NLF or Ruthenium red (RuR) significantly improved viability of murine hippocampal HT-22 cells, reduced Ca2+ content and preserved membrane potential (Ψm) in mitochondria following oxygen-glucose deprivation (OGD). Following histamine-stimulated Ca2+ release from endoplasmic reticulum, in contrast to the vehicle-treated cells, the cells treated with NLF or RuR also demonstrated reduced Ca2+ content in their mitochondria, the event associated with preserved Ψm. Because RuR inhibits mitochondrial Ca2+ uniporter, we tested whether the NLF acts via the mechanism similar to the RuR. However, in contrast to the RuR, in the experiment with direct interaction of these agents with mitochondria isolated from naïve mice, the NLF did not alter mitochondrial Ca2+ influx, and did not prevent Ca2+ induced collapse of the Ψm. These data strongly argues against interaction of NLF and mitochondrial Ca2+ uniporter. Although the exact mechanism remains unclear, our study is the first to show that NLF inhibits intramitochondrial Ca2+ flux and protects developing brain against HI-reperfusion injury. This novel action of NLF has important clinical implication, because it targets a fundamental mechanism of post-ischemic cell death: intramitochondrial Ca2+ overload → mitochondrial membrane permeabilization → secondary energy failure.
Highlights
Neonatal hypoxic-ischemic brain injury (HI) is a leading cause of permanent neurological deficit in children
At 0 minute of reperfusion, immediately at the end of HI, brain mitochondria isolated from mice pre-treated with NLF demonstrated significantly decreased Ca2+ content compared to the vehicle-treated littermates (Fig. 1A, B)
There are two important results: (1) pre-treatment with NLF significantly reduced the HI-brain injury in neonatal mice and (2) this neuroprotection was associated with attenuation of Ca2+ influx into cerebral mitochondria during HI-insult and reperfusion
Summary
Neonatal hypoxic-ischemic brain injury (HI) is a leading cause of permanent neurological deficit in children. There are two types of membranes permeabilization in post-ischemic mitochondria: Bax/Bak dependent outer membrane permeabilization and Ca2+ triggered, cyclophilin D-sensitive, inner membrane permeabilization, known as mitochondrial permeability transition pore (mPTP). An opening of Bax/Bak sensitive outer mitochondrial membrane pore causes a release of proapoptotic proteins. Studies in mature animals demonstrated that following cerebral and cardiac ischemia irreversible cell injury takes place when mitochondria open the cyclophilin-D sensitive mPTP [5,6,7] [8,9], the pathogenic significance of mPTP opening in the immature HI-brain is uncertain. In neonatal rats and mice subjected to a global hypoxia-ischemia-reperfusion injury, post-treatment with cyclosporine A markedly potentiated neuroprotective effect of Ca2+ channel antagonist, nimodipine [14]
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