Wednesday 19 September 2018

Ketones and Autism Part 5 - BHB, Histone Acetylation Modification, BDNF Expression, PKA, PKB/Akt, Microglial Ramification, Depression and Kabuki Syndrome

Child displaying elongated eyelids typical of Kabuki syndrome
Source: Given by Parents of children pictured with purpose of representing children with kabuki on Wikipedia. 

The syndrome is named after its resemblance to Japanese Kabuki makeup.

As we have discovered in this blog, autism is just a condition where certain genes are over-expressed and other genes are under-expressed. Put like that makes it sound quite simple.

Methylation of histones can either increase or decrease transcription of genes. The subject is highly complex, but we can keep things simple.

The child in the photo above has Kabuki syndrome and is likely to exhibit features of autism.  In most cases this is the result of a lack of expression of the KMT2D/MLL2 gene which encodes a protein called Histone-lysine N-methyltransferase.  Unfortunately, this is quite an important protein, because it promotes the “opening of chromatin”.  It adds a “trimethylation mark to H3K4”, just think of it as a pink post-it on your DNA. 
We get H3K4me3, which is an epigenetic marker (me3, because it is trimethylation). H3K4me3 promotes gene activation and it can cause a relative imbalance between open and closed chromatin states for critical genes. It has been suggested that it may be possible to restore this balance with drugs that promote open chromatin states, such as histone deacetylase inhibitors (HDACi).
What all this means is that people with Kabuki start with under-expression of just one gene, but this leads to the miss-expression of numerous other genes. Because science has figured out what the KMT2D/MLL2 gene does, we can find ways of treating this syndrome.

BHB as an HDAC inhibitor and a treatment for Kabuki syndrome

HDAC inhibitors (HDACi) are also suggested as therapies for other single gene syndromes. We saw in an earlier post that in Pitt Hopkins syndrome people lack Transcription Factor 4 (TCF4). Too little TC4 is not good, but too much TC4 is one feature of schizophrenia.
We saw in the research that we can increase expression of TCF4 using a class 1 HDAC inhibitor and we can also activate the Wnt pathway, which can also be achieved by inhibiting GSK3 (all themes covered in this blog).
So, Pitt Hopkins therapies include: -
·        Wnt activation (covered extensively in this blog) this includes statins and GSK3 inhibitors like Lithium

·        HDAC inhibitors like valproic acid, some cancer drugs, sodium butyrate and indeed the ketone BHB
This also means that people with schizophrenia, and likely too much TCF4, might benefit from the opposite gene expression modification, so a Wnt inhibitor, these include some cheap, safe, drugs used to treat children with parasites (Mebendazole/ Niclosamide etc) and of course GSK3 activators.
It is interesting that after 500 posts of this amateur blog you can start to fit the science together and identify rational therapies for complex disorders and  note that these therapies have much wider application, either to milder conditions or discovering avenues to treat the opposite genetic variation.  The underlying biological themes are all reoccurring in different types of autism/schizophrenia/ bipolar and you do wonder why more has not been done by professionals to apply this knowledge. 500 posts may sound a lot, but for autism researchers this is their paid, full-time job, not just a hobby pastime.

But then again, Simon Baron-Cohen, Head of Cambridge University's Autism Research Centre, recently published an article in which he wrote:

"We at the Autism Research Centre have no desire to cure, prevent or eradicate autism ... As scientists, our agenda is simply to understand the causes of autism." 

Whose team is he playing for?

My conclusion is that perhaps Baron-Cohen has Asperger's himself, because he does not realize that a disorder, severe enough for a medical/psychiatric diagnosis, is a bad thing that should be minimized and ideally prevented, just like any other brain disorder. His cousin the actor Sacha gives a very good impression of someone with bipolar, so perhaps they both need a Wnt activator?

Would a mother with Multiple Sclerosis (MS) want her daughter to also develop MS to share the experience? I think not. If it is just "quirky autism", it does not warrant a medical diagnosis, because it is perfectly okay to be quirky. 

This blog does have many Aspie readers who do want pharmacological therapy and that is their choice; I am fully supportive of them and wish them well.

Back to Kabuki
There is more than one type of HDAC and so there are different types of HDACi.  There are actually 18 HDAC enzymes divided into four classes
The ketone BHB inhibits HDAC class I enzymes called HDAC2 and HDAC3
The good news is that the ketogenic diet, which produces BHB, does indeed show merit as a therapy for Kabuki.

Kabuki syndrome is caused by haploinsufficiency for either of two genes that promote the opening of chromatin. If an imbalance between open and closed chromatin is central to the pathogenesis of Kabuki syndrome, agents that promote chromatin opening might have therapeutic potential. We have characterized a mouse model of Kabuki syndrome with a heterozygous deletion in the gene encoding the lysine-specific methyltransferase 2D (Kmt2d), leading to impairment of methyltransferase function. In vitro reporter alleles demonstrated a reduction in histone 4 acetylation and histone 3 lysine 4 trimethylation (H3K4me3) activity in mouse embryonic fibroblasts from Kmt2d+/βGeo mice. These activities were normalized in response to AR-42, a histone deacetylase inhibitor. In vivo, deficiency of H3K4me3 in the dentate gyrus granule cell layer of Kmt2d+/βGeo mice correlated with reduced neurogenesis and hippocampal memory defects. These abnormalities improved upon postnatal treatment with AR-42. Our work suggests that a reversible deficiency in postnatal neurogenesis underlies intellectual disability in Kabuki syndrome.

Intellectual disability is a common clinical entity with few therapeutic options. Kabuki syndrome is a genetically determined cause of intellectual disability resulting from mutations in either of two components of the histone machinery, both of which play a role in chromatin opening. Previously, in a mouse model, we showed that agents that favor chromatin opening, such as the histone deacetylase inhibitors (HDACis), can rescue aspects of the phenotype. Here we demonstrate rescue of hippocampal memory defects and deficiency of adult neurogenesis in a mouse model of Kabuki syndrome by imposing a ketogenic diet, a strategy that raises the level of the ketone beta-hydroxybutyrate, an endogenous HDACi. This work suggests that dietary manipulation may be a feasible treatment for Kabuki syndrome.
 Although BHB has previously been shown to have HDACi activity (7, 21), the potential for therapeutic application remains speculative. Here, we show that therapeutically relevant levels of BHB are achieved with a KD modeled on protocols that are used and sustainable in humans (22, 23). In addition, we demonstrate a therapeutic rescue of disease markers in a genetic disorder by taking advantage of the BHB elevation that accompanies the KD.
Our findings that exogenous BHB treatment lead to similar effects on neurogenesis as the KD support the hypothesis that BHB contributes significantly to the therapeutic effect. In our previous study (6), the HDACi AR-42 led to improved performance in the probe trial of the MWM for both Kmt2d+/βGeo and Kmt2d+/+ mice (genotype-independent improvement). In contrast, KD treatment only led to improvement in Kmt2d+/βGeo mice (genotype-dependent improvement). This discrepancy may relate to the fact that AR-42 acts as an HDACi but also affects the expression of histone demethylases (24), resulting in increased potency but less specificity. Alternatively, because the levels of BHB appear to be higher in Kmt2d+/βGeo mice on the KD, the physiological levels of BHB might be unable to reach levels in Kmt2d+/+ mice high enough to make drastic changes on chromatin.
In addition to the effects seen on hippocampal function and morphology, we also uncovered a metabolic phenotype in Kmt2d+/βGeo mice, which leads to both increased BHB/AcAc and lactate/pyruvate ratios during ketosis; an increased NADH/NAD+ ratio could explain both observations. This increased NADH/NAD+ ratio may relate to a previously described propensity of Kmt2d+/βGeo mice toward biochemical processes predicted to produce NADH, including beta-oxidation, and a resistance to high-fat-diet–induced obesity (27). If this exaggerated BHB elevation holds true in patients with KS, the KD may be a particularly effective treatment strategy for this patient population; however, this remains to be demonstrated. Alterations of the NADH/NAD+ ratio could also affect chromatin structure through the action of sirtuins, a class of HDACs that are known to be NAD+ dependent (28). Advocates of individualized medicine have predicted therapeutic benefit of targeted dietary interventions, although currently there are few robust examples (2931). This work serves as a proof-of-principle that dietary manipulation may be a feasible strategy for KS and suggests a possible mechanism of action of the previously observed therapeutic benefits of the KD for intractable seizure disorder (22, 23).                   
Kabuki syndrome (KS) (Kabuki make-up syndrome, Niikawa-Kuroki syndrome) is a rare genetic disorder first diagnosed in 1981. Kabuki make-up syndrome (KMS) is a multiple malformation/intellectual disability syndrome that was first described in Japan but is now reported in many other ethnic groups. KMS is characterized by multiple congenital abnormalities: craniofacial, skeletal, and dermatoglyphic abnormalities; intellectual disability; and short stature. Other findings may include: congenital heart defects, genitourinary anomalies, cleft lip and/or palate, gastrointestinal anomalies including anal atresia, ptosis and strabismus, and widely spaced teeth and hypodontia. The KS is associated with mutations in the MLL2 gene in some cases were also observed deletions of KDM6A. This study describes three children with autism spectrum disorders (ASDs) and KS and rehabilitative intervention that must be implemented.

So what?
Unless you know someone with Kabuki syndrome, you might be wondering what does this matter to autism.
What is shows is that BHB/KD is sufficiently potent to be a viable HDAC inhibitor. 
We know that some cancer drug HDAC inhibitors are effective in some mouse models of autism. But these drugs usually have side effects. 

HDAC Inhibitors for which Cancer/Autism? 

BHB is safe endogenous substance, so it is a “natural” HDACi. 

The effect of HDAC2 and HDAC3 on BDNF 
Brain derived neurotropic factor (BDNF) is like brain fertilizer. In some types of autism, you would like more BDNF.
When you exercise you produce BHB and that goes on to trigger the release of BDNF. This process also involves NF-kB activation

Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite β-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of β-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of β-hydroxybutyrate. Electrophysiological measurements indicate that β-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF.

Results: ROS was significantly increased in neurons after 6 hours of ketone incubation. However, after 24 hours, neurons show improved efficiency in ATP productions, upregulated expressions of antioxidant enzyme SOD2, and enhanced resistance to excitotoxicity. These effects were significantly abolished in neurons after treatment with TrkB inhibitor. More interestingly, ROS scavengers or blocking ROS-dependent NF-kB activation significantly decreased ketone-dependent BDNF-upregulation in neurons, suggesting that ROS may have increased BDNF expressions to improve mitochondrial respiration as adaptive responses.
Conclusions: 3OHB initially generates ROS and poses oxidative stress. However, ROS appears to trigger adaptive responses against oxidative stress by upregulating BDNF through NF-kB activation, which can improve mitochondrial oxidative capacity and ultimately enhance neuroprotection
BHB/KD promotes PKA/CREB activation 
Another clever way to change the function/expression of multiple genes in one single step is to use a protein kinase.  Up to 30% of all human proteins may be modified by kinase activity.  
A protein kinase is an enzyme that modifies other proteins by chemically adding phosphate groups to them (phosphorylation). Phosphorylation usually results in a functional change of the target protein.
In the autism research you may well have come across PKA, PKB (Akt) and PKC. They clearly are disturbed in much autism.
The research shows that BHB activates PKA.
If you want good myelination you need PKA.
This might be another reason why BHB/KD is helpful in people with Multiple Sclerosis.
In much autism the myelin coating is found to be abnormally thin. 

BHB, Microglial Ramification and Depression (yes, depression)
I am increasingly impressed by research from China. The paper below by Chao Huang et al is excellent and I think we need a Chinese on the Dean’s List of this blog, it looks like he is the first.
Nantong, China on the Yangtze River and home to Chao Huang and more than 7 million other people 
Source: Wikipedia Dolly 442

The ketone body metabolite β-hydroxybutyrate induces an antidepression-associated ramification of microglia via HDACs inhibition-triggered Akt-small RhoGTPase activation. 


Direct induction of macrophage ramification has been shown to promote an alternative (M2) polarization, suggesting that the ramified morphology may determine the function of immune cells. The ketone body metabolite β-hydroxybutyrate (BHB) elevated in conditions including fasting and low-carbohydrate ketogenic diet (KD) can reduce neuroinflammation. However, how exactly BHB impacts microglia remains unclear. We report that BHB as well as its producing stimuli fasting and KD induced obvious ramifications of murine microglia in basal and inflammatory conditions in a reversible manner, and these ramifications were accompanied with microglial profile toward M2 polarization and phagocytosis. The protein kinase B (Akt)-small RhoGTPase axis was found to mediate the effect of BHB on microglial shape change, as (i) BHB activated the microglial small RhoGTPase (Rac1, Cdc42) and Akt; (ii) Akt and Rac1-Cdc42 inhibition abolished the pro-ramification effect of BHB; (iii) Akt inhibition prevented the activation of Rac1-Cdc42 induced by BHB treatment. Incubation of microglia with other classical histone deacetylases (HDACs) inhibitors, but not G protein-coupled receptor 109a (GPR109a) activators, also induced microglial ramification and Akt activation, suggesting that the BHB-induced ramification of microglia may be triggered by HDACs inhibition. Functionally, Akt inhibition was found to abrogate the effects of BHB on microglial polarization and phagocytosis. In neuroinflammatory models induced by lipopolysaccharide (LPS) or chronic unpredictable stress (CUS), BHB prevented the microglial process retraction and depressive-like behaviors, and these effects were abolished by Akt inhibition. Our findings for the first time showed that BHB exerts anti-inflammatory actions via promotion of microglial ramification. 

NOTE:  Ramified Microglia = Resting Microglia

The brain microglia play important roles in sensing even subtle variations of their milieu. Upon moderate activation, they control brain activity via phagocytosis of cell debris and production of pro-inflammatory mediators and reactive oxygen species. However, a persistent activation would make the microglia transfer into a status with an amoeboid morphology tightly associated with neuronal damage and pro-inflammatory cytokine overproduction.

Unlike the activated microglia, the un-stimulated microglia are in a ramified status with extensively branched processes, an contribute to brain homeostasis via regulation of synaptic remodeling and neurotransmission. The ramified microglia has been shown to be associated with the induction of M2 polarization. A study by McWhorter et al. showed that elongation of macrophage by control of cell shape directly increases the expression of M2 markers and reduces the secretion of proinflammatory cytokines, suggesting that induction of microglial ramification may be a mechanism for regulation of microglial function. Methods that trigger microglial ramification may help treat brain disorders associated with neuroinflammation.
In this study, we found that BHB induces a functional ramification of murine microglia in both basal and inflammatory conditions in vitro and in vivo. The pro-ramification effects of BHB are associated with the change in microglial polarization and phagocytosis as well as the antidepressant-like effects of BHB in LPS- or chronic unpredictable stress (CUS)-stimulated mice. The ramified morphology in microglia is also induced by two BHB-producing stimuli fasting and KD, as well as two other HDACs inhibitors valproic acid (VPA) and trichostatin A (TSA). Given that microglial overactivation can mediate the pathogenesis of depression, induction of microglial ramification by BHB may have therapeutic significance in depression. 

These data confirm that BHB has an ability to transform the activated microglia back to their ramified and resting status in inflammatory conditions.

Recall the recent post about BHB and the Niacin Receptor HCA2/GPR109A in Autism:

The Chinese paper continues:

It is HDACs inhibition but not GPR109A activation that mediates the pro-ramification effect of BHB in microglia Akt inhibition abrogates the effects of BHB on microglial ramification, polarization, and phagocytosis
Akt inhibition prevents the antidepressant-like effects of BHB in acute and chronic depression models

Note that Akt is another name for Protein Kinase B (PKB)

One of the major findings in the present study is that the ketone body metabolite BHB as well as its producing stimuli fasting and KD induced reversible ramifications of murine microglia in vitro and in vivo, and these ramifications were not altered by pro-inflammatory stimuli. The ramified morphology induced by BHB seems to be a signal upstream of microglial polarization, and may mediate the antidepressant-like effect of BHB in depression induced by neuroinflammatory stimuli. Since the regulating effect of BHB in disorders associated with neuroinflammation has been well-documented, our findings provide a novel mechanism for the explanation of the neuroprotective effect of BHB in neurodegenerative and neuropsychiatric disorders from the aspect of the feedback regulation of microglial function by microglial ramification.
Induction of microglial ramification, a strategy neglected by most scientists for a long time, may have more important therapeutic significance than that of regulation of microglial polarization alone at the molecular level.

In experiments in vivo, we showed that BHB ameliorated the depressive-like behaviors induced by two neuroinflammatory stimuli LPS and CUS. These results are in accordance with previous reports, which showed that the BHB-producing stimuli, caloric restriction and fasting, produce potential antidepressant-like activities in both animals and humans. Thus, together with the pro-ramification effect of BHB in microglia in vitro, we speculate that the microglial shape change may be an independent signal that determines microglial function.

Our further analysis showed that the BHB-induced microglial ramification was mediated by the Rac1-Cdc42 signal, as BHB markedly increased the activity of Rac1 and Cdc42, and Rac1/Cdc42 inhibition attenuated the pro-ramification effect of BHB. The PI3K-Akt signal has been shown to mediate the activation of Rac1/Cdc42, and once accepting the signal from Akt, the Rac1-Cdc42 will be mobilized to promote lamellipodia/filopodia formation and cell shape change (Huang et al., 2016a). We showed that the BHB-induced microglial ramification was mediated by the Akt signal, as Akt inhibition suppressed the induction of microglial ramification by BHB. As a functional evidence for the involvement of Akt in the pro-ramification effect of BHB, Akt inhibition was found to block the functional changes in BHB-treated microglia in vitro and in vivo, including blockage of the anti-inflammatory and prophagocytic activity of BHB and abrogation of the antidepressant-like effects of BHB. Since the ramified morphology determines the anti-inflammatory phenotype in macrophages (McWhorter et al., 2013), our data suggest that there may exist a causal relationship between the ramified morphology and microglial function after BHB treatment, and this relationship may evidence the clinical significance of our findings, as the microglial process retraction has been shown to mediate the development of neurodegenerative and neuropsychiatric disorders.

Furthermore, considering the serum level of BHB in humans begin to rise to 6 to 8 mM with prolonged fasting (Cahill, 2006), investigation of whether the pro-ramification effect of BHB exists in human individuals should be of great value for the application of BHB in disease therapy. 

 Exposure to hypobaric hypoxia causes neuron cell damage, resulting in impaired cognitive function. Effective interventions to antagonize hypobaric hypoxia-induced memory impairment are in urgent need. Ketogenic diet (KD) has been successfully used to treat drug-resistant epilepsy and improves cognitive behaviors in epilepsy patients and other pathophysiological animal models. In the present study, we aimed to explore the potential beneficial effects of a KD on memory impairment caused by hypobaric hypoxia and the underlying possible mechanisms. We showed that the KD recipe used was ketogenic and increased plasma levels of ketone bodies, especially β-hydroxybutyrate. The results of the behavior tests showed that the KD did not affect general locomotor activity but obviously promoted spatial learning. Moreover, the KD significantly improved the spatial memory impairment caused by hypobaric hypoxia (simulated altitude of 6000 m, 24 h). In addition, the improving-effect of KD was mimicked by intraperitoneal injection of BHB. The western blot and immunohistochemistry results showed that KD treatment not only increased the acetylated levels of histone H3 and histone H4 compared to that of the control group but also antagonized the decrease in the acetylated histone H3 and H4 when exposed to hypobaric hypoxia. Furthermore, KD-hypoxia treatment also promoted PKA/CREB activation and BDNF protein expression compared to the effects of hypoxia alone. These results demonstrated that KD is a promising strategy to improve spatial memory impairment caused by hypobaric hypoxia, in which increased modification of histone acetylation plays an important role

Exogenous BHB prevents spatial memory impairment induced by hypobaric hypoxia

To further verify whether ketone body, a product of KD, has direct improving effect, we chose the most stable physiologic ketone body, BHB, for the subsequent experiment. In order to mimic the effect of KD as above described, the rats were pre-treated with BHB (at a dose of 200mg/kg/day) for 2 weeks and then submitted to Morris water maze test. Since intraperitoneal injection would allow substances to be absorbed at a slower rate and intraperitoneal injection would produce marginal effect during behavioral tests [16], we used the intraperitoneal injection of BHB, which has been applied in published reports [17, 18]. Although the rats in the control and BHB groups learned to find the platform with the same pattern during 5 days of acquisition training (Fig 4B), BHB could significantly improve the memory impairment induced by hypobaric hypoxia, represented by more crossing number, more time in the target quadrant, and decreased latency to first entry to platform compared to hypobaric hypoxia treatment alone (Fig 4C–4F). These results demonstrated that BHB has a direct memory-improving effect and served as the main executor of KD beneficial effects.

KD increases histone acetylation modification in the hippocampus

A previous study found that BHB is an endogenous HDAC inhibitor, and the KD recipe in our study substantially increased plasma levels of BHB. Then, we detected the effect of KD on histone acetylation in the hippocampus, which is responsible for learning and memory. As shown in Fig 5, the acetylated histone H3 (K9/K14), acetylated histone H3 (K14), and acetylated histone H4 (K12), were all increased in the hippocampus of the KD rats. Although the histone acetylation modifications listed above are decreased in hypoxia-treated rats, KD treatment could reverse the decreased levels of histone acetylation. The same pattern was displayed in the immunohistochemical staining, in which the hypoxia-induced decrease in acetylated histone H3 and acetylated histone H4 in the CA1 region of the hippocampus was reversed by KD treatment  

KD activates PKA/CREB signaling in the hippocampus

To explore a possible underlying mechanism of the beneficial effect of KD treatment on cognition, the activity of the PKA/CREB pathway in the four groups was also evaluated by western blot (Fig 7A). KD treatment was shown to not only increase the levels of PKA substrates and p-CREB (KD vs STD) but also reverse the decline in PKA substrates, p-CREB and CREB (KD-Hy vs STD-Hy). Although KD pre-treatment produced a partial restoration of PKA activity, p-CREB is nearly completely restore to its basic levels, which is may be account for its other upstream kinases, like calmodulin-dependent kinases [19]. Interestingly, the hypoxia-induced down-regulation of BDNF, a well-known neurotrophic factor involved in learning and memory formation processes, was also up-reregulated by KD treatment. These results demonstrated that KD treatment promoted PKA/CREB activation and BDNF protein expression. In order to detect whether KD promoted BDNF expression at mRNA levels, qRT-PCR assays were performed using BDNF specific primers. We found that KD-pretreatment significantly increased mRNA levels compared with that in hypobaric hypoxia group (Fig 7B). Next, we used ChIP-PCR to test if there might be increased enrichment of acetylated histones on the promoter of BDNF gene. We focused on the promoter I of BDNF gene, which response to neuronal activity [20). ]. The results showed that there is increased binding of acetylated histone H3 to the promoter I of BDNF gene (Fig 7C   

Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress. 
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.  

At the end of this fifth post on ketones and autism, I think we have established beyond any doubt that ketones can do some amazing things for numerous dysfunctions and diseases.
The question remains how much you need to achieve the various possible benefits. 
The next question, already put to me by one parent, is how do you measure such a benefit.  Some people’s idea of treating autism is just to eradicate disturbing behaviours like SIB and ensure a placid, cooperative child when out in public.  Other people notice small cognitive and speech changes, because they spend hours a day teaching their child. Small but significant cognitive improvement may not show up on autism rating scales.
You would expect a dose dependent response, so the more ketones the bigger the response, which suggests that the full Ketogenic Diet (KD) is the ultimate option.
A lot does seem to be possible just with BHB and C8 (caprylic acid) as supplements to a regular diet.
Adults with Alzheimer’s, or Huntington’s, or Multiple Sclerosis (MS) all stand to potentially benefit from ketone supplements.
Children/adults with certain single-gene autisms, not limited to Kabuki and Pitt Hopkins potentially should benefit from ketone supplements.
Interestingly, another benefit of BHB is on mood; it seems to make some people just feel much better, apparently all due to the effect on microglia. So perhaps autism parents who take antidepressants should try BHB instead.


  1. Hi Peter,

    Hope all is well.

    I haven't been commenting as much lately (I've been super busy) but I've certainly been reading your great posts, and this 5 part series has been outstanding.

    I'm still waiting on some news from the researchers we're working with, but will be meeting with them by end of year for an update (they are doing some indepth research on my daughter's gene).

    Due to some items you've noted in your ketone series that may be relevant to my daughter's mutation, I will be trialing C8 / BHB. I'll keep you and the community posted.

    By the way, on an unrelated note, I thought you may find the following paper I just found of interest:

    Vitexin may have some utility in NKCC1 inhibition.

    Thanks again for a tremendous series of posts!


    1. Thanks AJ.

      Vitexin (also known as Vitex, Chasteberry) is widely used as a supplement by women due to its effect on prolactin and other hormones. It also has an effect on dopamine. It is not expensive.

      We could do with more ways to block NKCC1, so it is an interesting idea.

    2. I agree with AJ, this ketone series is really outstanding!

      As you both are mentioning NKCC1 I have to say that the KCC2-NKCC1-bumetanide topic still is confusing. I have the feeling that there is more to this than just chloride in or out, but every time I try to understand the mechanism I fail. For some reason, furosemide keeps popping up and not bumetanide on my hit list.

      It's like this paper from 2012 ("old") which doesn't mention cognition but instead memory and depression:

      "both furosemide and bumetanide could affect long-term potentiation by effects on the brain renin-angiotensin system"
      "the effect of furosemide and bumetanide on angiotensin could enhance CREB-BDNF signalling since angiotensin II elevates CREB levels by 100%"
      "Also, the antioxidant, phosphodiesterase inhibiting and
      anti-apoptotic effects of furosemide could have additive or synergistic effects with those of sertraline to enhance cAMP-CREB- BDNF-ERK 1/2-Bcl-2 signalling."
      "Evidence has been shown that furosemide administration down-regulates the dopamine transporter"

      ..and then the discussion mentions a lot of properties of furosemide that could explain the results - some of these properties are shared with bumetanide and others are not.

      I realize that this paper doesn't reflect what we know today, but it does sound as if furosemid also has a lot of interesting properties for neurological conditions. Maybe it would actually be a good alternative to bumetanide in cases where GABA acts as it should.

      Or? Oh, please take me out of this confused misery!


    3. Ling, I can say that if furosemide downregulates DAT, would be disastrous here, as DAT cleares excess dopamine from the synapses.

    4. Well, I don't know.
      First I thought that it might have something to do with the recent discovery that Bumetanide can help in Parkinson, but this link explained the complicated matter in very simple words, and it doesn't seem to have anything to do with dopamine:

      I wonder what PDEs furosemide (and bumetanide?) inhibit.


    5. Peter, if we assume the paper above is right, it would be interesting to know how bumetanide/furosemide modulates angiotensin. As you have written before in the post on Angiotensin II, there is a lot of research being done on this topic for Alzheimers, and we still wait for that "Compound 21" to go through clinical trials.

      Interestingly, estrogen has a part to play in this system:
      "preclinical studies show that rodent female brains have more angiotensinogen-positive neurons than males and is further upregulated by estrogen supplementation. Ovariectomy (OVX) is a surgical procedure performed to evaluate the effects of estrogen depletion in rodents. OVX upregulated Angiotensin 1 receptors (ATR1) and ACE and downregulated Angiotensin 2 receptors (ATR2) and was reversed upon estrogen supplementation. When comparing intact females to males, only females experienced cognitive deficits and neuronal loss upon AT2R knockout and enhanced ACE2 expression levels upon restoration of AT2R signaling"

      The same paper points to BDNF as the main path to enhance cognition (CREB):
      "The neuroprotective effects of AT1R blockers and AT2R agonists are associated with increased BDNF release. Modulation of the angiotensin system to enhance BDNF production may improve cognition"

      Oh, and the interaction with the AT1 and AT2 was also mentioned:
      "Chronic blockade of the AT1R increases the production of Ang IV. Overexpressed Ang IV can activate AT1R signaling. When AT1R is blocked Ang IV, even in high concentration, binds solely to AT4R receptors. Ang IV/AT4R signaling increases the concentration of Ang III to bind to activate AT2Rs, NOS to exert anti-inflammatory effects, and enhances synaptic transmission and LTP. It is reasonable to suggest that the effects of losartan, and possibly other ARBs, could also be mediated by facilitating the activation of the AT4R leading to enhancing cognition [98]. Indeed, concurrent administration of losartan with an AT4R antagonist reversed losartan’s beneficial effects on spatial learning and memory"


    6. Ling, you will find that everything has multiple effects, like Telmisartan also affects PPARs. My investigation of Angiotensin II ended up making Monty sing, which while cute, is not really a breakthrough result.

      I would look further at estradiol and also see what is known about modifying SATB2 with HDAC inhibition. It seems that many single gene autism may benefit from an HDAC inhibitor and change the expression of 200 genes in one step. This change can be long lasting.

    7. Making my daughter sing would be a real breakthrough... But I understand what you mean. It is of course all very individual which treatments work and which doesn't, but I have no obvious connection to angiotensin II specificially.

      If you have the possibility to check if HDAC inhibition has any known connection to SATB2 it would be very kind of you.
      If you find any connection to TBR1 instead it would also be very interesting, since this gene probably can rescue most of the things I'm dealing with.

      Btw, I didn't knew suramin was an HDAC inhibitor.


    8. Sodium valproate is another HDAC inhibitor,it was life changing for my son, may be this is one of the main reasons among others.

    9. Ling, TBR1 is very much involved in epigenetic changes. It is not simple.

      The Epigenetic Factor Landscape of Developing Neocortex Is Regulated by Transcription Factors Pax6→ Tbr2→ Tbr1

    10. Thank you Peter, that is a very dense paper. It does mention a conncetion between Tbr1 and Arid1b that i wasn't aware of before. Valuable knowledge!


  2. Here is another paper that is not directly about autism as it is about plasticity changes during aging, but nevertheless is applicable to autism with intellectual disability as hyperplasticity and an inability to retain learned skills (regression) is a very common problem:

    Press Release:


    The conclusion of the researches was that contrary to conventional wisdom on the subject, older brains were even more plastic than younger brains, however, any learned changes to an older brain were quickly lost (i.e. they would not stick which would imply an inability to learn new information). Hyperplasticity and regressing learned skills is very common in autism with intellectual disability so I found this parallel striking. The problem with aged brains turned out to be decreased levels of GABA which both decreased plasticity to appropriate levels and helped maintain the learning of new skills.

    Improving GABA levels and GABA functionality in autism has been a hallmark of this blog, but this research may give some insight into one of the specific learning disabilities many with autism face as they are sometimes able to learn a new skill, but quickly forget the skill. For those who suffer these types of learning disabilities, focusing on GABAergic solutions may give a significant boost to learning.

  3. The next gene on my list of things to attenuate (If I can't upregulate SATB2 or TBR1) is a heavily downregulated AUTS2 gene. "Autism Susceptibility Candidate 2" sounds like an autism gene, but it also causes intellectual dysfunction. I don't think it has been mentioned on this blog before.

    "Patients have borderline to severe ID/developmental delay, 83-100% have microcephaly and feeding difficulties. Congenital malformations are rare [..] Behaviour is marked by a friendly outgoing social interaction. Specific features of autism (like obsessive behaviour) are seen frequently (83%%), but classical autism was not diagnosed in any."

    AUTS2 is important for neuronal migration and neuritogenesis.
    A downregulation means a lost balance with too little Rac1 (Rac1 is important for rescue) and too much Cdc42 (which is a risk factor for cancer). AUTS2 is also a transcription factor, so once again a gazillion of genes downstreams. Too much AUTS2 is implied in pancreatic cancer and too little in ALL/leukemia.

    What I've found out so far is that PAK1 is downregulated (so we don't want that bee propolis), that "Deficiency of Rac1 Blocks NADPH Oxidase Activation" (not sure about that) and that GLUT-4 translocation is downregulated (with treatment options like lycopene, taurine + fish oil, cinnamon, ginseng, Urtica dioica, muscle stretching).

    I am still looking desperately for ways to enhance AUTS2 or Rac1 (or eventually P-Rex1 and Elmo2/Dock180 that mediates the effect of AUTS2 on Rac1 as per


    1. Enter - surprisingly - sphingolipids:

      "Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo‐ to ganglio‐series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process.
      We find that globo‐series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn [..] promote[s ..] the synthesis of gangliosides.
      The decrease of globo‐series glycosphingolipids is required for AUTS2 induction and for stem cell differentiation to neural cells."

      I think this is important in the wider autism perspective, where neuronal migration and development is dysfunctional. The big question here is how to inhibit the globo-variant of this type of lipids.


    2. The gangliosides are important in neuroblastoma (NB) too:

      "One pharmacological agent that is well known to alter cellular ganglioside metabolism and successfully used in the oral maintenance therapy of disseminated NB is retinoic acid. Previous in vitro studies have shown that morphological signs of neuronal differentiation in response to retinoic acid are accompanied by an increase in total ganglioside content and a relative increase in the expression of certain complex gangliosides of both the ‘a’ and ‘b’ pathway in NB cells.
      In this context, the retinoic acid-induced increase in complex ‘b’ pathway gangliosides (CbG) expression in NB cells represents a transition into a ganglioside pattern which is associated with (i) neuronal differentiation in normal cells and (ii) clinically less-aggressive NB tumours"

      This one caught my eye mainly because the retinoic acid signalling pathway is one of my main clues to verbal enhancement.


    3. Sorry, I forgot to add the link:


    4. Ganglioside intake can reach the brain:

      "The ganglioside-enriched lipid diet significantly increased total gangliosides in the intestinal mucosa, plasma and brain compared with the control diet [in rats]."

      Gangliosides are abundant in human MFGM/milk:

      "Gangliosides are [..] present in mammalian milk, where they are almost exclusively associated with the membrane fraction of the fat globule. In human milk, the content and individual distribution of gangliosides changes during lactation, GD3 being the most abundant ganglioside in colostrum, while in mature milk, GM3 is the major individual species."

      Also, gangliosides enhance Bifido-strains in the gut and regulate intestinal immunity:

      "Gangliosides function as 'unintended' target receptors for bacterial adhesion in specific tissues. [..] this being the main mechanism by which these compounds can prevent infection. Ganglioside-supplemented infant formula has been reported to modify the intestinal ecology of preterm newborns, increasing the Bifidobacteria content and lowering that of Escherichia coli. In addition, the influence of dietary gangliosides on several parameters related to the development of intestinal immune system, such as cytokine and intestinal IgA production, has also been described in animal models."

      Gangliosides promote eyesight, like fish oil:

      "Animals fed dietary ganglioside increased in total retinal ganglioside and GD3 content during retinal development, with a concomitant alteration of phospholipid metabolism. Feeding animals dietary long-chain polyunsaturated fatty acids also affected ganglioside metabolism in the developing retina, suggesting a new mechanism by which these dietary lipids may promote maturation of photoreceptor cells"

      Intake of gangliosides are generally low in modern diets:

      "Dietary ganglioside intake is very low unless consuming whole-organ foods (i.e., brain), whole milk, buttermilk, or colostrum in high quantities. [..] The estimated average intake of ganglioside in a healthy population is well below levels believed to bear therapeutic benefit"

      And yes, while retinoic acid upregulates gangliosides it also upregulates AUTS2 in neuroblastoma cells:
      "retinoic acid treatment of LA-N-5 cells resulted in either modest, or significant upregulation of the target genes RB1, AUTS2 and ROR1"
      (Google 04-05-2013-JABSOM-2013-Aaron-Mel-Micon and you'll find the poster)

      So, maybe MFGM and retinoic acid would treat AUTS2-induced autism?


  4. My adult friend in her 50s who is on the KD says her sleep markedly changed for the better when she went on the KD. I would like one of my teenagers to try but think that supplement trial will allow you to see if you respond before fully committing? My child was a responder to sodium butyrate. Thanks for great posts! MH

    1. MH, what dose of sodium butyrate do you use and what was the effect? There are some overlaps between the ketone BHB and butyrate and some research suggests ketone supplements may be more effective when taken with butyrate.

      I think it is well worth giving ketone supplements a trial.

    2. Hi Peter, My son was taking BodyBio Sodium Butyrate in capsules. Just 1 capsule which is 600 mg. He is not taking it currently as away at university and can only get him to take so much :) BHB sounds like a potentially bigger win for him and might see if I could get him to trial it when home. Thanks again for your wonderful blog. We would have been lost without it MH

    3. Sorry, I didn't explain the benefits, but I think that his gut seemed to improve on the sodium butyrate which just generally just improved his engagement. His issues are relatively mild. The biggest winners have been NAC, brocooli and anti inflammatory things. We haven't tried some things, like bumet. but hope to in in the future. MH

    4. MH, thanks for the feedback. I think many people might benefit from sodium butyrate, but very few people try it. That seems odd, given that it is fed to millions of animals every day to improve their immune health.

  5. Off topic - for those who see emotional and mental health as a huge piece - so glad to finally see attention paid . I have been saying this for years.

    “Among autistic people, though, less extreme experiences — fire alarms, paperwork, the loss of a family pet, even a stranger’s offhand comment — can also be destabilizing. They can also be traumatized by others’ behavior toward them.”

    1. Hi Tanya,glad to hear from you, i was wondering how was your experience with Bimuno.I tried inulin and was a horrendous experience, he has chronic constipation SIBO. Do you think that probiotics are safer than prebiotics for Pans kids?

    2. Hola Valentina! espero que todo bien - a pesar de SIBO , es muy dificil :(
      I am going to try Bimuno again, small amounts spaced many days apart, but never again with probiotics. I think they are irritants for my son. I’m not sure which is better for Pans kids. And inulin is a big trigger for those with SIBO. He does well if he avoids foods that trigger his gut like high fructose,high lactose and high saturated fats. Even high inulin foods like asparagus if he eats too much it causes discomfort. Pepto Bismol helps for acute pain if he has too much. Milk of Magnesia might help your son with constipation? Avoiding food triggers has helped him the most . I know it’s not getting to root cause but for now it’s what we can do that helps most and doesn’t harm.

    3. Probably the reason why we get prescribed SSRI's, which will only add to the problem of the hyperserotonin state.

      Oddly psilocybin (5ht2a agonist obviously) helps my depression in a way very unlike regular SSRI's, namely by confronting one self with the problem in order to fix it, rather than masking the problem (through amygdala dulling effect of SSRI's).


      "In an evaluation of the safety and abuse research on the drug in hallucinogenic mushrooms, Johns Hopkins researchers suggest that if it clears phase III clinical trials, psilocybin should be re-categorized from a schedule I drug—one with no known medical potential—to a schedule IV drug such as prescription sleep aids, but with tighter control. Their analysis is summarized in the October print issue of Neuropharmacology."

      The abuse potential of medical psilocybin according to the 8 factors of the Controlled Substances Act

      Worthy read, even for those without autism but problems such as treatment resistant depression/atypical depression.

    4. Forgot to add, 5ht2a and 5ht1a are now also basically considered opposing to eachother with regards to many things.

    5. Aspie, could you explain your comment above (ketone series part 5)? Maybe you even have a link to the statement? I would be very interested.


  6. Valentina, I found this list of SIBO relief options you might find helpful. Many things on this list we have tried and a lot we still do that really helps. One thing on this list that is interesting to me that I haven’t done in a long time is cod liver oil - for the vitamin A. I wonder how high dose vitamin A maybe just once a week helps for those with SIBO/gut infections/pans etc?

  7. Thanks Tanya! I think that both vitamin A and D are important for healing SIBO.Iam struggling with my son´s constipation, we are on magnesium citrate and triphala and use neem as antimicrobial, also cayenne pepper. Iam trying to introduce insoluble fiber. I wonder if a possible loss of potassium from bumetanide could be worsening the problem.

  8. Valentina I remember how challenging that was for my son.. During day time, does your son have at least 3 hours between eating? If you keep trying all of the other more natural remedies and not much relief, maybe you could try low dose erythromycin as a prokinetic? We never had to use prescriptions for motility, so I can’t tell you from personal experience, but I know it is recommended by the GI doctors. Also as a prokinetic: LDN and a drug called “prucalopride”? Warm water with fresh lemon juice first thing in the morning used to help my son.also provides more potassium. In the end, In my son’s case, the antimicrobials never helped. I like potassium bicarbonate. I hope just adding in more potassium helps your son- that would be an easy solution . Good luck!

    1. Tanya, I was thinking of using 5HTP as a mild prokinetic, I prefer not to use Azithromycin.
      I will add extra potassium, he is taking 250 mg potassium citrate with 1mg bumetanide,may be could add 100 mg potassium bicarbonate, apart from bananas.

    2. Good idea!
      And erythromycin has a different mechanism of action than azith - these doctors who are using it as a prokinetic, use at lower dose than what is used for antibiotic purposes. But I understand completely why you would prefer not to use it. I’m sending you best wishes and hope - it can be overcome, you will figure out what is best for son. My son is doing so much better with the gut stuff. Just a few foods that bother him but really it is no big deal.

    3. Thanks Tanya, this is all part of his autoimmune condition,it must be fixed above all because its negative impact is huge. So,I will do the best to fix this problem, will watch bumetanide closer.

  9. Anxiolytic Effect of Exogenous Ketone Supplementation Is Abolished by Adenosine A1 Receptor Inhibition in Wistar Albino Glaxo/Rijswijk Rats.

    Thought this might be interesting for those who are either on keto or supplement with BHB. Also this shows that if you benefit from keto theres a chance you might respond also to adenosine raising supplements such as cordyceps.

  10. Roger, "growth factors" upregulate PI3K which in turn upregulates PKB (also known as Akt). There are feedback loops involved.

    There are many growth factors, including insulin.

    It is very complicated, as you can see here:-

    Nutrients will also have the same effect as growth factors. So if you over-eat you will activate PKB. If you starve yourself you would have the opposite effect.

    So I think you will find the ketogenic diet will, if anything, reduce PKB.

    In much disease you want less PKB/Akt.


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