Recent evidence suggests that intracellular Zn 2؉ accumulation contributes to the neuronal injury that occurs in epilepsy or ischemia in certain brain regions, including hippocampus, amygdala, and cortex. Although most attention has been given to the vesicular Zn 2؉ that is released into the synaptic space and may gain entry to postsynaptic neurons, recent studies have highlighted pools of intracellular Zn 2؉ that are mobilized in response to stimulation. One such Zn 2؉ pool is likely bound to cytosolic proteins, like metallothioneins. Applying imaging techniques to cultured cortical neurons, this study provides novel evidence for the presence of a mitochondrial pool distinct from the cytosolic protein or ligand-bound pool. These pools can be pharmacologically mobilized largely independently of each other, with Zn 2؉ release from one resulting in apparent net Zn 2؉ transfer to the other. Further studies found evidence for complex and potent effects of Zn 2؉ on isolated brain mitochondria. Submicromolar levels, comparable to those that might occur on strong mobilization of intracellular compartments, induced membrane depolarization (loss of ⌬ m), increases in currents across the mitochondrial inner membrane as detected by direct patch clamp recording of mitoplasts, increased O2 consumption and decreased reactive oxygen species (ROS) generation, whereas higher levels decreased O2 consumption and increased ROS generation. Finally, strong mobilization of proteinbound Zn 2؉ appeared to induce partial loss of ⌬ m, suggesting that movement of Zn 2؉ between cytosolic and mitochondrial pools might be of functional significance in intact neurons.

Dietary protocols that increase serum levels of ketones, such as calorie restriction and the ketogenic diet, offer robust protection against a multitude of acute and chronic neurological diseases. The underlying mechanisms, however, remain unclear. Previous studies have suggested that the ketogenic diet may reduce free radical levels in the brain. Thus, one possibility is that ketones may mediate neuroprotection through antioxidant activity. In the present study, we examined the effects of the ketones β-hydroxybutyrate and acetoacetate on acutely dissociated rat neocortical neurons subjected to glutamate excitotoxicity using cellular electrophysiological and single-cell fluorescence imaging techniques. Further, we explored the effects of ketones on acutely isolated mitochondria exposed to high levels of calcium. A combination of β-hydroxybutyrate and acetoacetate (1 mM each) decreased neuronal death and prevented changes in neuronal membrane properties induced by 10 μM glutamate. Ketones also significantly decreased mitochondrial production of reactive oxygen species and the associated excitotoxic changes by increasing NADH oxidation in the mitochondrial respiratory chain, but did not affect levels of the endogenous antioxidant glutathione. In conclusion, we demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD + /NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress. Keywordsglutamate; neurotoxicity; diet; mitochondria; oxidation; stress Address correspondence to: Jong M. Rho, MD., Neurology Research, NRC 4 th Fl., Barrow Neurological Institute and St. Joseph's Hospital & Medical Center, 350 W. Thomas Road, Phoenix, AZ 85013, Email: jong.rho@chw.edu. Section Editor: Molecular Neuroscience W. Sieghart, Brain Research Institute, University of Vienna, Division of Biochemistry and Molecular Biology, Spitalgasse 4, A-1090 Vienna, Austria Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Calorie restriction can decrease the risk of neurodegenerative disease and protect the brain against acute insults such as stroke (Mattson et al, 2002). Similarly, the ketogenic diet, a highfat, low-carbohydrate diet created to mimic the effects of calorie restriction, is an extremely efficacious treatment for medically intractable epilepsy Vining et al, 1998). Several metabolic changes occur during calorie restriction and the ketogenic diet, notably an increase in seru...

A truncated form of the Huntington's disease (HD) protein that contains the polyglutamine repeat, Httex1p, causes HD-like phenotypes in multiple model organisms. Molecular signatures of pathogenesis appear to involve distinct domains within this polypeptide. We studied the contribution of each domain, singly or in combination, to sub-cellular localization, aggregation and intracellular Ca2+ ([Ca2+]i) dynamics in cells. We demonstrate that sub-cellular localization is most strongly influenced by the first 17 amino acids, with this sequence critically controlling Httex1p mitochondrial localization and also promoting association with the endoplasmic reticulum (ER) and Golgi. This domain also enhances the formation of visible aggregates and together with the expanded polyQ repeat acutely disrupts [Ca2+]i levels in glutamate-challenged PC12 cells. Isolated cortical mitochondria incubated with Httex1p resulted in uncoupling and depolarization of these organelles, further supporting the idea that Httex1p-dependent mitochondrial dysfunction could be instrumental in promoting acute Ca2+ dyshomeostasis. Interestingly, neither mitochondrial nor ER associations seem to be required to promote long-term [Ca2+]i dyshomeostasis.