D-beta-hydroxybutyrate rescues mitochondrial respiration and mitigates features of Parkinson disease

Abstract

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of the nigrostriatal dopaminergic neurons accompanied by a deficit in mitochondrial respiration. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that causes dopaminergic neurodegeneration and a mitochondrial deficit reminiscent of PD. Here we show that the infusion of the ketone body d-beta-hydroxybutyrate (DbetaHB) in mice confers partial protection against dopaminergic neurodegeneration and motor deficits induced by MPTP. These effects appear to be mediated by a complex II-dependent mechanism that leads to improved mitochondrial respiration and ATP production. Because of the safety record of ketone bodies in the treatment of epilepsy and their ability to penetrate the blood-brain barrier, DbetaHB may be a novel neuroprotective therapy for PD.

Figure 1

Brain levels of DβHB and β-hydroxybutyrate dehydrogenase (βHBD) under different treatments. (a) One day after implantation of pumps containing DβHB, animals were injected intraperitoneally with saline (Sal), MPTP, or 3-NP as described in Methods, and brain levels of DβHB were measured at 0 days (90 minutes after the fourth injection), 2 days, and 7 days thereafter. The utilization of DβHB was increased when cells were under metabolic stress induced by these toxins. n = 4–6; *P < 0.05 and **P < 0.01 compared with the respective control saline groups. (b) Western blot analysis of ventral midbrains from MPTP-intoxicated mice shows upregulation of this enzyme as early as day 0. n = 4–5 per group; *P < 0.05 compared with the control saline group. β-Actin is used to normalize βHBD values.

Figure 2

Protective effect of DβHB against MPTP-induced neurodegeneration. (a–h) TH-positive neurons in SNpc, and (i–p) TH-positive terminals in striatum. Animals were infused subcutaneously with vehicle (saline; a, e, i, and m), DβHB (1.6 mmol/kg/d; b, d, f, h, j, l, n, and p), or LβHB (1.6 mmol/kg/d; c, g, k, and o) 1 day before receiving intraperitoneal injections of either saline (a–d and i–l) or MPTP (18 mg/kg; e–h and m–p). There is an extensive loss of TH-positive neurons (e) and terminals (m) in MPTP-injected animals. This loss is attenuated by DβHB (f and n) but not by its inactive isomer LβHB (g and o). The complex II inhibitor 3-NP was given intraperitoneally (15 mg/kg) daily for the entire period of DβHB infusion. In the presence of 3-NP, DβHB does not confer neuroprotection. Scale bars: 500 μm (a–h) and 1 mm (i–p). Please refer to Table 1 for quantification of neurons and terminals in each animal group.

Figure 3

Protective effect of DβHB against motor deficit in MPTP-treated mice. Animals were infused subcutaneously with either vehicle (saline) or DβHB (1.6 mmol/kg/d) 1 day before receiving intraperitoneal injections of either saline or MPTP (18 mg/kg). Pumps were removed at day 7, and animals were allowed to recover from surgery and dehydration for an additional 7 days before their Rotarod performance was assessed. Motor deficit is observed in the MPTP-treated animals, but DβHB significantly improves this impairment. DβHB does not affect base-line motor function in saline-injected mice. n = 4–13; *P < 0.05 compared with the saline-vehicle group; ^#P < 0.05 compared with the MPTP-vehicle group.

Figure 4

DβHB increases oxygen consumption in purified brain mitochondria. Mitochondria (300 μg) were incubated in the absence or presence of MPP⁺ (5 minutes; a) or rotenone (2.5 minutes; b) at 30°C, and then 5 mM DβHB was added to induce oxygen consumption. DβHB attenuated inhibition of mitochondrial respiration induced by MPP⁺ (a) or rotenone (b) at indicated concentrations, which blocked about 25–90% of oxygen consumption when glutamate and malate were used as NADH-linked substrates (data not shown). (c) The improvement of oxygen consumption by DβHB is stereospecific and is blocked by 10 mM 3-NP, a complex II inhibitor. (d) DβHB increases oxygen consumption in a dose-dependent and saturable fashion as seen with succinate, a complex II substrate, although not as efficiently as succinate does on an equimolar basis. n = 3–4.

Figure 5

Dose-response study of NADH in complex I activity (a and b) and brain levels of succinate (c). In mitochondria lysed by freeze-thawing, when the inhibition of complex I activity was titrated with different concentrations of MPP⁺ (a) or rotenone (b), different amounts of NADH did not produce different responses in complex I activity (n = 4 per group). (c) Levels of succinate were measured in the brains of animals treated with 18 mg/kg/d MPTP or 15 mg/kg 3-NP, or both. Levels of succinate in the group that received DβHB (1.6 mmol/kg/d) are significantly increased in the presence of 3-NP. n = 3–10 per group; *P < 0.05 compared with the control saline group; ^#P < 0.05 compared with the 3-NP group. (d) Histochemical analysis in striatal sections shows that when animals were treated with 3-NP (right panel) at this concentration for 8 days, there was approximately 40% reduction in complex II activity in the striatum compared with that in the group treated with saline (left panel). n = 5 per group; **P < 0.01. Scale bar: 500 μm.