Oxygen Glucose Deprivation in Rat Hippocampal Slice Cultures Results in Alterations in Carnitine Homeostasis and Mitochondrial Dysfunction

Thomas F Rau,Mark S Wainwright,Xutong Sun,Michael Kavanaugh,Qing Lu,Matthew L Beckman,David J Poulsen,Shruti Sharma,Gregory Leary,Stephen M Black,Yali Hou

doi:10.1371/journal.pone.0040881

Abstract

Mitochondrial dysfunction characterized by depolarization of mitochondrial membranes and the initiation of mitochondrial-mediated apoptosis are pathological responses to hypoxia-ischemia (HI) in the neonatal brain. Carnitine metabolism directly supports mitochondrial metabolism by shuttling long chain fatty acids across the inner mitochondrial membrane for beta-oxidation. Our previous studies have shown that HI disrupts carnitine homeostasis in neonatal rats and that L-carnitine can be neuroprotective. Thus, this study was undertaken to elucidate the molecular mechanisms by which HI alters carnitine metabolism and to begin to elucidate the mechanism underlying the neuroprotective effect of L-carnitine (LCAR) supplementation. Utilizing neonatal rat hippocampal slice cultures we found that oxygen glucose deprivation (OGD) decreased the levels of free carnitines (FC) and increased the acylcarnitine (AC): FC ratio. These changes in carnitine homeostasis correlated with decreases in the protein levels of carnitine palmitoyl transferase (CPT) 1 and 2. LCAR supplementation prevented the decrease in CPT1 and CPT2, enhanced both FC and the AC∶FC ratio and increased slice culture metabolic viability, the mitochondrial membrane potential prior to OGD and prevented the subsequent loss of neurons during later stages of reperfusion through a reduction in apoptotic cell death. Finally, we found that LCAR supplementation preserved the structural integrity and synaptic transmission within the hippocampus after OGD. Thus, we conclude that LCAR supplementation preserves the key enzymes responsible for maintaining carnitine homeostasis and preserves both cell viability and synaptic transmission after OGD.

Highlights

Neonatal hypoxic-ischemic encephalopathy (HIE) is a pathological condition that occurs in 1–6 per 1000 live births and results in significant neurodevelopmental disabilities in affected infants [1,2]
Using hippocampal slice cultures exposed to oxygen glucose deprivation (OGD), we initially examined the effect of OGD on the protein levels of these three important mitochondrial proteins involved in carnitine metabolism
Our data indicate that OGD significantly decreases both Carnitine palmitoyl transferase 1 (CPT1) (,40% decrease, Fig. 1 A) and carnitine palmitoyl transferase 2 (CPT2) (,50% decrease, Fig. 1 B) protein levels and this decrease was ameliorated by LCAR treatment (Fig. 1 A & B)

Summary

Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) is a pathological condition that occurs in 1–6 per 1000 live births and results in significant neurodevelopmental disabilities in affected infants [1,2]. HIE will occur leading to seizures, blindness, severe cognitive deficits, and cerebral palsy characterized by hemiplegia, paraplegia, or quadriplegia [1]. Neonatal ischemia increases glutamate release activating NMDA, AMPA, and kainate receptors elevating cytosolic Ca2+. As Ca2+ levels increase mitochondrial buffering is overwhelmed, Ca2+ is spontaneously released, and the mitochondrial permeability transition pore (MPTP) is activated [3]. MPTP activation depolarizes the mitochondria resulting in a loss of ATP production, an increase in reactive oxygen species (ROS) and damage to cytochromes in the electron transport chain [4]. The increased presence of ROS and Ca2+ within the mitochondria disrupts key enzymes and transporters further exacerbating ATP depletion leading to necrotic and/or apoptotic cell death [2,3,4,5]

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Conclusion