Elsevier

Neurobiology of Aging

Volume 39, March 2016, Pages 25-37
Neurobiology of Aging

Regular article
Ketones block amyloid entry and improve cognition in an Alzheimer's model

https://doi.org/10.1016/j.neurobiolaging.2015.11.018Get rights and content

Abstract

Sporadic Alzheimer's disease (AD) is responsible for 60%–80% of dementia cases, and the most opportune time for preventive intervention is in the earliest stage of its preclinical phase. As traditional mitochondrial energy substrates, ketone bodies (ketones, for short), beta-hydroxybutyrate, and acetoacetate, have been reported to provide symptomatic improvement and disease-modifying activity in epilepsy and neurodegenerative disorders. Recently, ketones are thought as more than just metabolites and also as endogenous factors protecting against AD. In this study, we discovered a novel neuroprotective mechanism of ketones in which they blocked amyloid-β 42, a pathologic hallmark protein of AD, entry into neurons. The suppression of intracellular amyloid-β 42 accumulation rescued mitochondrial complex I activity, reduced oxidative stress, and improved synaptic plasticity. Most importantly, we show that peripheral administration of ketones significantly reduced amyloid burden and greatly improved learning and memory ability in a symptomatic mouse model of AD. These observations provide us insights to understand and to establish a novel therapeutic use of ketones in AD prevention.

Introduction

Alzheimer's disease (AD), the most common cause of late-onset dementia, has a high global economic impact (Mayeux and Stern, 2012) and takes an incalculable toll on patients and their families. Remarkable advances in unraveling the biological underpinnings of AD have been made, but little progress in the development of clinical treatments has been achieved (Holtzman et al., 2012). Recent evidence indicates that amyloid-β (Aβ) is absorbed into and transported through distal axons, accumulates to mitochondria of cell bodies, and is further transferred to neighboring neurons (Song et al., 2014). Mitochondrial dysfunction has been associated with the preclinical and clinical stages of AD (Caldeira et al., 2013, Eckert et al., 2012), and an accumulation of Aβ 1–42 causes mitochondrial failure (Calkins et al., 2011). Furthermore, intracellular accumulated Aβ impairs mitochondrial function (Lustbader et al., 2004, Szatmari et al., 2013, Umeda et al., 2011) by binding to mitochondrial proteins (Borger et al., 2011, Borger et al., 2013, Szatmari et al., 2013). Mitochondrial dysfunction increases the formation of reactive oxygen species (ROS), and there is strong evidence linking oxidative stress with AD (Leuner et al., 2012, Lin and Beal, 2006).

Traditionally, beta-hydroxybutyrate (BHB) and acetoacetate (ACA), 2 main ketone bodies that we named ketones, have been regarded as energy carriers (Dedkova and Blatter, 2014). Ketones are consumed by brain as the major energy sources when glucose is limited (Cunnane et al., 2011, Seyfried and Mukherjee, 2005). As mitochondrial energy substrates, ketones have been reported reducing amyloid neurotoxicity and its pathology, protecting neurons, and improving memory ability (Kashiwaya et al., 2000, Kashiwaya et al., 2013, Newport et al., 2015). However, ketones are more than just metabolites (Newman and Verdin, 2014, Shimazu et al., 2013) and are also endogenous neuroprotective factors, but the mechanisms are not understood (Rahman et al., 2014).

We hypothesized that learning and memory deficits in AD could be prevented by ketones through a blockade of amyloid entry into the cell and a reduction of oxidative stress. Our in vitro experimental data showed that ketones prevented oligo-Aβ42–induced membrane disruption, neuronal injury, mitochondrial dysfunction, and ROS formation. Furthermore, ketones reduced intracellular level of Aβ42 and protected synaptic plasticity against oligo-Aβ42 toxicity. In vivo experiments in an AD mouse model revealed that ketones improved mitochondrial function by restoring complex I activity and reducing soluble Aβ42 level. Finally, although ketones did not affect wild-type (WT) animals, they drastically improved memory performance in the AD mouse model. These observations provide a pharmacological foundation for ketones and a new insight on its targets in AD prevention.

Section snippets

Oligomeric Aβ42 preparation

To prepare soluble oligomeric Aβ42, human synthetic Aβ42 (rPeptide) was treated in 20 μM ammonium acetate in distilled water (pH = 8, ionic strength = 0.25 M), incubated for 30 minutes at room temperature, lyophilized, and stored in −80 °C. The pretreated Aβ42 was dissolved in artificial cerebrospinal fluid (aCSF) or culture media at room temperature immediately before use. The presence and stability of oligomers were tested by electron microscopy and Western blot techniques.

Animals

All experiments

Characterization of oligo-Aβ42

To obtain stable Aβ42 oligomers, synthetic human Aβ42 was solubilized in ammonium acetate, lyophilized, and redissolved in aCSF. Several lines of evidence support the notion that ammonium acetate induced the generation of oligomers. First, electron microscopic analysis revealed different sizes of annular Aβ42 oligomers (Fig. 1A–C, arrows) only when Aβ42 was pretreated with ammonium acetate (Fig. 1A and C). Second, we used Western blot to detect Aβ42 forms. There were monomers, dimers, trimmers,

Discussion

In this study, we addressed the question whether ketones could alleviate acute and chronic deficits in models of AD. After acute exposure to exogenous oligo-Aβ42, we found intracellular exogenous Aβ42 level increased, stronger oxidative stress, mitochondrial complex I dysfunction, and inhibition of long-term plasticity in hippocampal slices. Pre-exposure of the cells or brain slices to ketones blocked oligo-Aβ42 entry and protected against its neurotoxicity. This “acute exposure” model might

Disclosure statement

The authors report no conflict of interest.

Acknowledgements

This work is supported by the Mary S. Easton Center for Alzheimer's Disease Research at UCLA, the Arizona Alzheimer's Disease Consortium AG019610 to EMR, and the Barrow Neurological Foundation BNF 3031880 to JS.

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    This article is dedicated to Nicole Maalouf in memory of her late husband Dr. Marwan Maalouf who was killed by a reckless driver on October 15, 2012.

    1

    These authors contributed equally to this work.

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