D-β-Hydroxybutyrate Is Protective in Mouse Models of Huntington's Disease

Abnormalities in mitochondrial function and epigenetic regulation are thought to be instrumental in Huntington's disease (HD), a fatal genetic disorder caused by an expanded polyglutamine track in the protein huntingtin. Given the lack of effective therapies for HD, we sought to assess the neuroprotective properties of the mitochondrial energizing ketone body, D-β-hydroxybutyrate (DβHB), in the 3-nitropropionic acid (3-NP) toxic and the R6/2 genetic model of HD. In mice treated with 3-NP, a complex II inhibitor, infusion of DβHB attenuates motor deficits, striatal lesions, and microgliosis in this model of toxin induced-striatal neurodegeneration. In transgenic R6/2 mice, infusion of DβHB extends life span, attenuates motor deficits, and prevents striatal histone deacetylation. In PC12 cells with inducible expression of mutant huntingtin protein, we further demonstrate that DβHB prevents histone deacetylation via a mechanism independent of its mitochondrial effects and independent of histone deacetylase inhibition. These pre-clinical findings suggest that by simultaneously targeting the mitochondrial and the epigenetic abnormalities associated with mutant huntingtin, DβHB may be a valuable therapeutic agent for HD

Drp1 inhibition attenuates neurotoxicity and dopamine release deficits in vivo

Mitochondrial dysfunction has been reported in both familial and sporadic Parkinson’s disease (PD). However, effective therapy targeting this pathway is currently inadequate. Recent studies suggest that manipulating the processes of mitochondrial fission and fusion has considerable potential for treating human diseases. To determine the therapeutic impact of targeting these pathways on PD, we used two complementary mouse models of mitochondrial impairments as seen in PD. We show here that blocking mitochondrial fission is neuroprotective in the PTEN-induced putative kinase-1 deletion (PINK1−/−) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse models. Specifically, we show that inhibition of the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) using gene-based and small-molecule approaches attenuates neurotoxicity and restores pre-existing striatal dopamine release deficits in these animal models. These results suggest Drp1 inhibition as a potential treatment for PD

DβHB extends life expectancy and stabilizes glucose levels of Tg-R6/2 mice.

<p>Animals from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024620#pone-0024620-g004" target="_blank">Figure 4</a> were monitored twice daily for their survival rate (<b>A</b>). These mice were considered to reach their end stage if they were unable to right themselves after being placed on their back or died overnight. DβHB significantly extended the survival time in Tg-R6/2 mice. <i>n</i> = 12 (Ntg-saline), <i>n</i> = 7 (Tg-saline), <i>n</i> = 7 (Tg-DβHB). <i>p</i><0.001 Kaplan-Meier analysis between Tg-sal and Tg- DβHB groups. These animals were also assessed weekly for glucose levels after six hours of fasting (<b>B</b>). One animal from the Tg-saline group survived up to 14 weeks and exhibited an elevated glucose level.</p

DβHB prevents histone deacetylation induced by mhtt.

<p>Animals recovered from the survival study were perfused and striatal sections were obtained and immunostained for histone H4 acetylation. Immunoreactivity was visualized by incubation in 3,3′-diaminobenzidine . There was less immunoreactivity of histone H4 acetylation in the transgenic animals as compared to the Ntg mice. DβHB treatment restored H4 acetylation in the Tg animals. <i>n</i> = 2 representative animals per group.</p

DβHB and LβHB do not inhibit HDAC activities.

<p>Hela nuclear extract was used as a source of class I and II HDAC activity (<b>Panel A</b>) where as recombinant human Sir1 was used as a source of class III HDAC activity (<b>Panel B</b>). HDAC activities were assessed in the absence (control, “C”) or presence of 4 mM DβHB (“D”), LβHB (“L”) or sodium butyrate (“Na”, an HDAC I and II inhibitor), 1 µM trichostatin A (“TA”, an HDAC I and II inhibitor) or 1 mM nicotinamide (“Nic”, an HDAC III inhibitor). Activities were quantified based on fluorescence intensity and expressed as arbitrary fluorescence unit (AFU). <i>n</i> = 6–16 per group from 5 independent experiments.</p

DβHB attenuates motor deficits in Transgenic-R6/2 mice.

<p>Six week old male transgenic (Tg)-R6/2 mice and non-transgenic (Ntg) littermates were infused with either saline vehicle or DβHB as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024620#pone-0024620-g002" target="_blank">Figure 2</a>. Osmotic minipumps were replaced every two weeks for the entire study period. Locomotor activities were assessed using infrared photobeams chambers. Tg-R6/2 mice displayed significant impairment in locomotor movements. In contrast, these abnormalities were delayed and less severe in the Tg group treated with DβHB (<i>n</i> = 7). DβHB did not affect locomotor function of Ntg mice (not shown). Units expressed as % control of Ntg-Saline group at seven weeks old (<i>n</i> = 12). *<i>p</i><0.05 as compared to the respective Tg-saline groups (<i>n</i> = 7) at the same time points.</p

Metabolic pathways of DβHB in mitochondria.

<p>The metabolism of DβHB in mitochondria is stereospecific to the D isoform. Under normal physiological conditions, the level of DβHB is low but becomes dramatically elevated during starvation, increased fatty acid metabolism or pathological conditions such as diabetes. From its site of production in the liver, DβHB is released into the blood and circulated for utilization by other tissues. In general, the rate of ketone body usage in the brain is proportional to the concentration in the circulation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024620#pone.0024620-Sokoloff1" target="_blank">[59]</a>. Circulating DβHB readily crosses the blood-brain barrier and enters brain mitochondria where it is metabolized by mitochondrial β-hydroxybutyrate dehydrogenase to acetoacetate, which is subsequently converted to acetyl-coenzyme A to feed into the tricarboxylic acid cycle (TCA) cycle. The intermediate products generated from this cycle, NADH and succinate, in turn feed into the electron transport chain to subsequently generate ATP at complex V. Through this metabolic pathway, DβHB is an excellent alternative source of energy in the brain when glycolysis is not operative or when glucose supply is depleted such as during starvation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024620#pone.0024620-Sokoloff1" target="_blank">[59]</a>.</p