Mitochondrial uncoupling protein-2 protects the immature brain from excitotoxic neuronal death

Abstract

Excitotoxic cell death is the fundamental process responsible for many human neurodegenerative disorders, yet the basic mechanisms involved are not fully understood. Here, we exploited the fact that the immature brain is remarkably resistant to seizure-induced excitotoxic cell death and examined the underlying protective mechanisms. We found that, unlike in the adult, seizures do not increase the formation of reactive oxygen species or result in mitochondrial dysfunction in neonatal brain, because of high levels of the mitochondrial uncoupling protein (UCP2). UCP2 expression and function were basally increased in neonatal brain by the fat-rich diet of maternal milk, and substituting a low-fat diet reduced UCP2, restored mitochondrial coupling, and permitted seizure-induced neuronal injury. Thus, modulation of UCP2 expression and function by dietary fat protects neonatal neurons from excitotoxicity by preventing mitochondrial dysfunction. This mechanism offers novel neuroprotective strategies for individuals, greater than 1% of the world's population, who are affected by seizures.

Fig 1

Neuronal injury in seizure-sensitive limbic regions of mature and postnatal day 10 (P10) rats after systemic administration of kainic acid., (A, B) Hippocampal CA3 from adult rats demonstrates neurons with silver affinity within the pyramidal cell layer (green arrows), indicating their injury.,,,, Such cells are not found in the P10 rat CA3 (C) including the pyramidal (sp), radiatum (sr), or oriens (so) cell layers. (D, E) Low and higher magnification views of the perirhinal cortex of mature rat show excitotoxicity in both deep and superficial layers of this seizure-vulnerable limbic region, whereas the corresponding region from a P10 rat (F) is free of silver-stained cells. (G–I) The piriform cortex, a highly seizure-vulnerable region, demonstrates seizure-injured neurons in adult (G, H) but not immature (I) brain.

Fig 2

Uncoupling protein (UCP) function and UCP2 expression in seizure-vulnerable limbic brain regions of immature (P10) compared with adult rat. (A) Mitochondrial fractions from hippocampus (top) and perirhinal/piriform cortex (bottom) of the P10 rat are highly uncoupled on exposure to the UCP-activating free fatty acid (FFA) palmitate, when respiration is compared with the maximum respiration induced by the chemical uncoupler FCCP (bottom inset). Adult hippocampal and perirhinal/piriform cortex mitochondria respond modestly to FFA (indicating little UCP available for activation), whereas mitochondria from immature rats are completely uncoupled in the presence of palmitic acid (bars represent group means ± SEM; asterisk indicates p < 0.02). (B) When brain sections from immature and adult rats are processed for UCP2 immunocytochemistry, fewer UCP2-immunoreactive neurons (arrows) are evident in adult CA3 (8.7 ± 0.9) compared with the corresponding region of the P10 rat hippocampus (17.6 ± 0.6; C, D). (E, F) Compared with the rare UCP2-immunoreactive neurons in adult perirhinal cortex (E, arrows), dense staining is found in the limbic cortex of immature rats (F, arrows).

Fig 3

Differential effects of severe seizures on reactive oxygen species (ROS) production in mitochondria from immature and adult rat limbic neurons. (A) Evaluation of adult rat ROS production showed a significant increase at 6 and 24 hours after kainic acid administration, whereas (B) infant rat ROS production was not enhanced by these severe seizures. (C) Prolonged seizures significantly increased the number of UCP2-expressing neurons in the CA3 hippocampal region of mature rats. (D) Concomitantly, adult rat mitochondria demonstrated a significant increase in fatty acid–induced respiration (a measure of UCP function) 24 hours after the seizures. For all graphs, bars represent group means ± SEM; asterisks indicate p < 0.05).

Fig 4

Reduction of dietary fat by substitution of an isocaloric, low-fat diet to immature rats reduces UCP function and promotes ROS production as well as seizure-induced excitotoxicity. (A) UCP function, measured as fatty acid–induced respiration, is significantly reduced in neonatal rats fed low-fat diet compared with maternal milk–fed littermates and resembles those in adult mitochondria. (inset) Basal ROS production in the presence of oligomycin (to maximize membrane potential) in isolated mitochondria from the group fed a low-fat diet are significantly increased compared with milk-fed littermates, approaching the basal levels found in adult mitochondria. (B) Energetic demand induced by severe seizures provokes striking increases in ROS production in UCP-suppressed (low-fat diet fed) neonatal rats, but not in those maintained on maternal milk. Bars represent means ± SEM; asterisks indicate p < 0.05). (C–H) Seizures provoke neuronal injury (visualized using Fluro-Jade) in several highly seizure-vulnerable regions of infant rats with suppressed UCP function. In adults, both perirhinal (C) and piriform (D) cortex demonstrated excitotoxic injury (arrows), whereas none was evident in the corresponding limbic regions of P10 rats on a “normal” high-fat diet (E, F). In striking contrast, excitotoxic injury occurred in perirhinal (G) and piriform (H) cortex of the low-fat diet fed infant rats (arrows).