Autophagy and up-regulation of autophagy-associated proteins microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 have been shown to occur in spinal cord injury (SCI). Bcl-2/E1B-19 K-interacting protein 3 (BNIP3) and Nip-like protein X (NIX, also known as BNIP3L) are mitochondrial BH3-only proteins that are implicated in mitophagy. In this study, we show that mitophagy is activated in the injured neurons, and hypoxia-inducible proteins BNIP3, NIX, and p53 are upregulated after SCI. Numerous autophagosomes containing damaged mitochondria (mitophagosomes) were found in the injured neurons 24 h after SCI in rats by transmission electron microscopy; mitophagy, therefore, had occurred. Hypoxia-inducible proteins BNIP3, NIX, and p53 were upregulated in spinal cord neurons in both a rat model of SCI and cultured primary spinal neurons exposed to hypoxia. BNIP3 and NIX were transcriptionally regulated mainly by hypoxia-inducible factor-1α as well as p53 in cultured spinal cord neurons. This study provides direct morphological and biochemical evidence for mitophagy in the damaged neural tissue after SCI.
Autophagy and mitophagy have been shown to occur in spinal cord injury (SCI). Bcl-2/E1B-19KD-interacting protein 3 (BNIP3) and its homologue, NIX, have been implicated in the regulation of mitophagy. The aim of this work was to characterize the mechanisms and role of BNIP3 in SCI-associated mitophagy. Our data showed that BNIP3, targeted to mitochondria, interacted with microtubule-associated protein 1A/1B-light chain 3 (LC3), which is targeted to autophagosomes, thus forming a mitochondria-BNIP3-LC3-autophagosome complex and resulting in mitophagy. Downregulation of BNIP3 by RNA interference strengthened the mitochondrial function and decreased cell death in spinal cord neurons under hypoxia. Particularly, BNIP3 knockdown significantly improved neurological recovery and the number of neuronal nuclei-positive cells post-SCI in rats. The present study demonstrated that BNIP3 interacts with LC3 to induce mitophagy, whereas its inhibition provided protective neuronal effects in SCI rat models both in vivo and in vitro.
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