The neuroprotective effects of DβHB have been demonstrated in various models of neurological disorders 
. We previously showed that in the MPTP mouse model of Parkinson's disease, DβHB attenuated loss of dopaminergic neurons and functional deficits in a dose-dependent and stereospecific manner via
its bioenergetic effect 
. In the present study, we report that DβHB is neuroprotective in both mitochondrial toxin induced striatal neurodegeneration and a genetic mouse model of HD. It is likely that the bioenergetic effects of DβHB contributed to the protection in these models. Although the epigenetic effects observed in the present genetic model may also play a role, it requires further studies.
In addition to its adverse effects on mitochondria, mhtt induces transcriptional dysregulation by binding to the cAMP-responsive element-binding protein (CREB)-binding protein (CBP), which also functions as a histone acetyltransferase (HAT) 
. The expanded glutamine stretch in mhtt has been shown to bind to either the polyQ domain of CBP and sequester it in nuclear inclusions 
or bind to the acetyltransferase domain (outside the polyQ domain) in CBP 
, leading to the inhibiton of the HAT activity of CBP and ultimately, global histone deacetylation 
. This reduction in histone acetylation, in turn, leads to gene silencing 
. By increasing histone acetylation, histone deacetylase (HDAC) inhibitors improve motor dysfunction and brain pathologies, as well as increases life expectancy in animal models of HD 
. Based on its similar chemical structure to the HDAC inhibitors sodium butyrate and phenylbutyrate, we initially hypothesized that DβHB would attenuate abnormal levels of histone deacetylation induced by mhtt via
HDAC inhibition. Our subsequent studies demonstrated that although DβHB did prevent histone deacetylation induced by mhtt, the mechanism was not mediated by HDAC inhibition. Because DβHB is a well-established mitochondrial substrate, we assessed whether the observed epigenetic effect was mediated through mitochondria. The metabolism of DβHB to acetoacetate in mitochondria by β-hydroxybutyrate dehydrogenase is stereospecific 
. Although cytosolic enzymes have been reported to metabolize LβHB in chicken liver 
and sheep kidney 
, there is no evidence to support the existence of either a mitochondrial L-3-hydroxybutyrate dehydrogenase or a racemase that interconverts the L- and D-isoforms of β-hydroxybutyrate 
. Consistent with this, in our previous study using polarography we confirmed that DβHB, but not LβHB, could support mitochondrial respiration leading to higher ATP production 
. Therefore, our data suggest that DβHB did not restore histone acetylation via
a mitochondrial mechanism, because its mitochondrially inactive isomer, LβHB, produced the same epigenetic changes.
The precise mechanism by which DβHB prevents histone deacetylation induced by mhtt requires additional investigations. In general, there are a few potential mechanisms by which DβHB can increase histone acetylation in the presence of mhtt: 1) Inhibit HDAC activity, 2) Increase general histone acetyltransferase activity, 3) Reduce mhtt-induced protein aggregation, leading to less sequestration of CBP, and 4) Block the binding of mhtt to the active site of CBP, maintaining the normal HAT activity of this protein. We have eliminated the first possibility by demonstrating that DβHB did not inhibit any of the three classes of HDAC. Regarding the second possibility, we reasoned that if DβHB increased a general HAT activity, we would have detected an increase in histone acetylation when HttQ25
cells were treated with this compound. This rationale is further supported by the fact that when these cells were treated with a general HDAC inhibitor (sodium butyrate), histone acetylation was increased. A general mechanism such as inhibiting HDAC or stimulating HAT should lead to an increase in histone acetylation, regardless of the presence of mhtt or normal htt. The fact that we only observed a DβHB-mediated increase in histone acetylation in cells with mhtt and that this increase was only to restore acetylation to the baseline level rather than the dramatic increase seen with sodium butyrate, led us to the remaining two possibilities. However, based on our immunohistochemical observations in both stable PC12 cells and R6/2 mice, DβHB does not appear to reduce protein aggregation (data not shown). Therefore, we are left with our last hypothesis. That is, this effect of DβHB is specific to mhtt and it may be mediated through blocking the binding of mhtt to CBP, leaving the HAT activity of this protein intact. It is worth noting that in addition to CBP, other proteins with HAT activity such as p300 and P/CAF may also interact with mhtt 
. Thus, the effect of DβHB may not be restricted to just CBP.
Although DβHB extended the life span of R6/2 mice quite dramatically, DβHB did not produce a similarly striking improvement in motor deficits in these animals. This lack of robust effect may be a result of the experimental regimen. Because the half-life of DβHB is only about 1.64 h 
, it had to be delivered through osmotic minipumps. The large size of these pumps necessitated waiting until mice were six weeks old to implant them and begin treatment. However, R6/2 mice start to exhibit motor deficits as early as three-four weeks old and by six weeks already exhibit marked locomotor deficits 
. Thus, beginning DβHB infusion at six weeks was likely too late to get the maximum benefits of this molecule. Consistent with this theory, when these mice were treated beginning at seven weeks old, we did not detect such a clear trend of improvement in motor deficits (data not shown). As discussed in other reviews 
, an early HD neurological phenotype, probably as a result of neuronal dysfunction, occurs before the overt appearance of protein aggregations or neurodegeneration. This neuronal dysfunction is likely mediated by interactions between mhtt and other proteins 
. Relevant to our working hypothesis is the binding of mhtt to CBP, leading to histone hypoacetylation. The effects of gene silencing as a consequence of histone deacetylation very likely play a major role in early neurological deficits in HD patients and R6/2 mice. The ability of DβHB to prevent histone deacetylation induced by mhtt leads us to hypothesize that if given early enough and at a sufficient concentration to prevent the interactions between mhtt and CBP, DβHB may further attenuate neurological deficits in these animals. Future studies using mouse models with slower onset of symptoms or developing a means of delivering DβHB in mice younger than six weeks old are necessary to address this issue.
The short half-life of DβHB raises the question of whether this compound would be a feasible treatment for human diseases. We believe the first crucial step in drug discovery is to identify an active compound. Once such a compound has been identified, current pharmaceutical technology is readily available to improve such unfavorable pharmacokinetic profiles as a short half-life. For example, the compound of interest can be encapsulated in a polymer matrix facilitating slow release over time. This strategy is widely utilized by pharmaceutical companies and has been applied to control the release of levodopa (Sinemet® CR) for the treatment of Parkinson's disease. Relevant to the present study, orally active forms of DβHB with more favorable pharmacoketics are currently being developed by KetoCytonyx Inc, a biotechnology company dedicated to the use of ketone bodies for the treatment of various human diseases 
. Similar, Accera Inc. has recently marketed Axona™, a medium chain triglyceride to generate ketone bodies, for the treatment of Alzheimer's disease. In addition to the pharmaceutical form of DβHB, non-pharmacological approaches such as a ketogenic diet (KD) and caloric restriction can also produce higher levels of this molecule in the body. Although not palatable and hyperlipidimic, KD has been used successfully for almost a century for the treatment of refractory epilepsy in children. KD has also been demonstrated to be beneficial in transgenic mouse models of Alzheimer's disease 
and Amyotrophic Lateral Sclerosis 
as well as in patients with Parkinson's disease 
and Alzheimer's disease 
. Caloric restriction (e.g., alternate day fasting) leading to higher serum DβHB concentrations is also neuroprotective 
In summary, the present study adds HD to a growing list of neurological disorders in which DβHB may confer neuroprotection. In addition to its well established mitochondrial effect, we may also unravel a novel epigenetic function of this molecule, leading to new insights into the mechanism by which it confers neuroprotection in treating epilepsy and other neurological disorders. Numerous studies have demonstrated the beneficial effects of increasing histone acetylation in HD 
. Although we were unable to precisely elucidate the epigenetic mechanism of DβHB or to fully establish a direct link between its restoration of histone acetylation and neuroprotection, we believe the overall positive results in the present study serve as a starting point that encourages further investigations into the potential clinical relevance of this compound for HD.