D-beta-hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease

Abstract

The heroin analogue 1-methyl-4-phenylpyridinium, MPP(+), both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Abeta(1-42), is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-beta-hydroxybutyrate protected cultured mesencephalic neurons from MPP(+) toxicity and hippocampal neurons from Abeta(1-42) toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

Figures

Figure 1

Anti-TH stain of day 7 of rat mesencephalic neuronal culture exposed to MPP+ and ketones for 2 days. (A) Control culture of anti-TH-stained mesencephalic neuronal cultures. (B) Cultures after addition of 5 μM MPP+, (C) after addition of MPP+ and 4 mM ketone bodies, and (D) after addition of 4 mM ketone bodies alone. Addition of 5 μM MPP+ to mesencephlic neuronal cultures resulted in a decrease in TH+ cells, a disappearance of neurites, and a shrinkage of cell body volume. Addition of 4 mM Na d-β-hydroxybutyrate to cultures containing 5 μM MPP+ reversed most of the effects of MPP+. The cell number and cell body volume did not differ significantly from control. (Scale bar = 20 μm.)

Figure 2

Time course of the effects of 5 μM Aβ1–42, 4 mM ketones, or the combination on the survival of hippocampal neurons in culture. ●, The mean control cell number/mm2 with error bar indicating the SEM where n = 12. All statistical tests performed were Mann–Whitney U tests, and significance was taken to be P < 0.05. ○, The mean cell number after exposure to Aβ1–42; ▴ after exposure to 5 μM Aβ1–42 + 4 mM d-β-hydroxybutyrate and ▵ after exposure to 4 mM d-β-hydroxybutyrate alone. Exposure to 5 μM Aβ1–42 significantly decreased the cell number compared with controls at 8 and 14 h as indicated by #. Addition of 4 mM d-β-hydroxybutyrate to cells exposed to 5 μM Aβ1–42 increased the cell number compared with exposure of Aβ1–42 alone at 8, 14, and 36 h as indicated by *. Addition of ketone bodies alone increased the cell number compared with controls as indicated by +. Our study therefore confirms the previous reports of the toxicity of Aβ1–42 to cultured hippocampal neurons (28). In addition we show that ketones not only reverse the toxicity of Aβ1–42, but act as a growth factor for neurons in culture.

Figure 3

The effects on cultured rat hippocampal cells of Aβ1–42, ketones, or the combination. (A) The 6-day control cultures of 18-day embryonic rat hippocampal tissue; (B) after 14 h exposure to 5 μM Aβ1–42, (C) after exposure to both Aβ1–42 and 4 mM d-β-hydroxybutyrate, and (D) the effects of ketone bodies alone. Addition of Aβ1–42 resulted in a decrease in neuronal number and number of neurites (B versus A). Addition of ketones to cells exposed to Aβ1–42 showed no decrease in neuron or neurite number, indicating that ketones act as neuroprotective agents against the toxicity of Aβ1–42 on cultured hippocampal neurons (C versus B).

Figure 4

The hypothesized effects of ketones on metabolic blocks induced by Aβ1–42 and MPP+. Usually brain entirely depends for energy on the mitochondrial metabolism of pyruvate produced from glucose by the glycolytic pathway. Aβ1–42 is reported to stimulate the phosphorylation of the E1a subunit of PDH by glycogen synthase kinase 3β (28). Phosphorylation of PDH blocks the conversion of pyruvate to acetyl CoA, which is required to fuel the TCA cycle, which provides mitochondrial NADH needed to power electron transport. Ketones provide the only alternative source of acetyl CoA for brain during inhibition of the PDH multienzyme complex. In so doing, ketone bodies not only increase mitochondrial acetyl CoA, citrate, and the first 1/3 of TCA cycle metabolites but also reduce the free mitochondrial NAD couple and oxidize the mitochondrial coenyzme Q couple, causing an increase in the ΔG of ATP hydrolysis (21). The oxidation of the coenzyme Q couple by ketones would tend to decrease the major source of mitochondrial reactive oxygen species, the semiquinone form of coenzyme Q (27), while at the same time relieving product inhibition of NADH dehydrogenase (EC 1.6.5.3), accounting for the ability of ketones to decrease MPP+ toxicity.