The therapeutic potential of calorie restriction and the ketogenic diet have been repeatedly demonstrated in clinical settings and in various animal models of neurological disease. The underlying mechanisms involve an improvement in mitochondrial function, a decrease in the expression of apoptotic factors and an increase in the activity of neurotrophic factors. Clinical applications of ketogenic diets have been significantly hampered however by poor tolerability and potentially serious side-effects. Recent research aimed at identifying a mediator that can reproduce the neuroprotective effects of calorie restriction with less demanding changes to dietary intake suggests that ketone bodies might represent an appropriate candidate. Ketone bodies protect neurons against multiple types of neuronal injury and the underlying mechanisms are similar to those of calorie restriction and of the ketogenic diet. The present review describes the neuroprotective effects of calorie restriction, the ketogenic diet and ketone bodies and compare the molecular mechanisms of action of these interventions.

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...

Ketone bodies (KB) have been shown to prevent neurodegeneration in models of Parkinson's and Alzheimer's diseases, but the mechanisms underlying these effects remain unclear. One possibility is that KB may exert antioxidant activity. In the current study, we explored the effects of KB on rat neocortical neurons exposed to hydrogen peroxide (H 2 O 2 ) or diamide -a thiol oxidant and activator of mitochondrial permeability transition (mPT). We found that: (i) KB completely blocked large inward currents induced by either H 2 O 2 or diamide; (ii) KB significantly decreased the number of propidium iodide-labeled cells in neocortical slices after exposure to H 2 O 2 or diamide; (iii) KB significantly decreased reactive oxygen species (ROS) levels in dissociated neurons and in isolated neocortical mitochondria; (iv) the electrophysiological effects of KB in neurons exposed to H 2 O 2 or diamide were mimicked by bongkrekic acid and cyclosporin A, known inhibitors of mPT, as well as by catalase and DLdithiothreitol, known antioxidants; (v) diamide alone did not significantly alter basal ROS levels in neurons, supporting previous studies indicating that diamide-induced neuronal injury may be mediated by mPT opening; and (vi) KB significantly increased the threshold for calcium-induced mPT in isolated mitochondria. Taken together, our data suggest that KB may prevent mPT and oxidative injury in neocortical neurons, most likely by decreasing mitochondrial ROS production.

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Previous studies have shown that ketone bodies (KB) exert antioxidant effects in experimental models of neurological disease. In the present study, we explored the effects of the KB acetoacetate (ACA) and β-hydroxybutyrate (BHB) on impairment of hippocampal long-term potentiation (LTP) in rats by hydrogen peroxide (H2O2) using electrophysiological, fluorescence imaging and enzyme assay techniques. We found that: (1) a combination of ACA and BHB (1 mM each) prevented impairment of LTP by H2O2 (200 μM); (2) KB significantly lowered intracellular levels of reactive oxygen species (ROS) — measured with the fluorescent indicator carboxy-H2DCFDA — in CA1 pyramidal neurons exposed to H2O2; (3) the effect of KB on LTP was replicated by the protein phosphatase 2A (PP2A) inhibitor fostriecin; (4) KB prevented impairment of LTP by the PP2A activator C6 ceramide; (5) fostriecin did not prevent the increase in ROS levels in CA1 pyramidal neurons exposed to H2O2, and C6 ceramide did not increase ROS levels; (6) PP2A activity was enhanced by both H2O2and rotenone – a mitochondrial complex I inhibitor that increases endogenous superoxide production; and (7) KB inhibited PP2A activity in protein extracts from brain tissue treated with either H2O2 or ceramide. We propose that oxidative impairment of hippocampal LTP is associated with PP2A activation, and that KB prevent this impairment in part by inducing PP2A inhibition through an antioxidant mechanism.

Mouse models with physiological and behavioral differences attributable to differential plasticity of hippocampal and amygdalar neuronal networks are rare. We previously generated ataxin-2 (Atxn2) knockout mice and demonstrated that these animals lacked obvious anatomical abnormalities of the CNS, but showed marked obesity and reduced fertility. We now report on behavioral changes as a consequence of Atxn2-deficiency. Atxn2-deficiency was associated with impaired long-term potentiation (LTP) in the amygdala, but normal LTP in the hippocampus. Intact hippocampal plasticity was associated behaviorally with normal Morris Water maze testing. Impaired amygdala plasticity was associated with reduced cued and contextual fear conditioning. Conditioned taste aversion, however, was normal. In addition, knockout mice showed decreased innate fear in several tests and motor hyperactivity in open cage testing. Our results suggest that Atxn2-deficiency results in a specific set of behavioral and cellular disturbances that include motor hyperactivity and abnormal fear-related behaviors, but intact hippocampal function. This animal model may be useful for the study of anxiety disorders and should encourage studies of anxiety in patients with spinocerebellar ataxia type 2 (SCA2).

Apolipoprotein E ε4 allele (ApoE4) has been associated with increased risk of sporadic Alzheimer's disease (AD) and of conversion from mild cognitive impairment to AD. But the underlying mechanism of ApoE4 affecting brain atrophy and cognition is not fully understood. We investigated the effect of ApoE4 on amyloid beta (Aβ) protein burden and its correlation with the structure change of hippocampus and cortex, cognitive and behavioral changes in ApoE4 transgenic mice. Male ApoE4 transgenic mice and age-matched control mice at age 12 months and 24 months were tested in the Morris Water Maze (MWM). Brain volume changes (including whole brain, hippocampus, cortex, total ventricles and caudate putamen) were assessed by using small animal 7T-MRI. Aβ level was assessed by immunohistochemistry (IHC) and immunoprecipitation/western blot. In MWM, escape latency was longer and time spent in the target quadrant was shorter in aged ApoE4 mice (12- and 24-month-old), suggesting age- and ApoE4-dependent visuospatial deficits. Atrophy on MRI was prominent in the hippocampus (p=0.039) and cortex (p=0.013) of ApoE4 mice (24-month-old) as compared to age-matched control mice. IHC revealed elevated Aβ deposition in the hippocampus. Consistently, both soluble and insoluble Aβ aggregates were increased in aged ApoE4 mice. This increase was correlated inversely with hippocampal atrophy and cognitive deficits. These data give further evidence that ApoE4 plays an important role in brain atrophy and memory impairment by modulating amyloid production and deposition.