Showing posts with label PKB. Show all posts
Showing posts with label PKB. Show all posts

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.

Tuesday 23 February 2016

Therapeutic Epigenetics in Autism and Junk DNA

Today’s post takes another dip into the genetics of autism and currently existing therapies that could be re-purposed for autism.  We also see that many secrets remain beyond the 3% of your DNA that usually gets all the research attention.  The remaining 97% is not junk after all.

There was an earlier post on this blog that introduced Epigenetics.  It is not such a complicated subject, just think about it as little tags on your DNA that turn genes on/off usually when they should not be, but there remains the possibility to use epigenetics for good.  In people with under-expression of an important gene you could “tag it” and then increase its expression.

The exome is the part of your DNA that encodes the various proteins needed to build your body.  The remaining 97% of your DNA was once thought to be just junk; we saw in recent post that one part contains enhancers and silencers that control expression of the genes in the 3% that is the exome.

A recent study of gene expression in neurological conditions including autism showed just how broadly disturbed gene expression is.

(A) Consistent fold enrichments were found for each cell type across fourteen cortical and three subcortical brain regions of Alzheimer's patients. The box plots mark the distribution of cellular fold enrichments across all the brain regions examined. Asterisks mark that the fold enrichment for each cell type that was found to be significantly non-zero with p < 0.05. (B) Two independent autism studies show the same cellular phenotypes, including upregulation of glial cells and downregulation of neurons. Asterisks mark those cell types found to be significantly differential with p < 0.05 after BH correction over all groups.

Here I am making the point that even though only a handful of genes may have an identifiable dysfunction, a much broader range of genes seem to be affected, as we see in the wide range of over and under expressed genes.

While it would be logical to think about a specific dysfunction needing a therapy that targets just that gene, this appears not to be necessary.

It appears that downstream processes may be the most damaging/relevant, for example disturbances in Protein Kinase A and C (PKA and PKC) may play a key role in many cases of regressive autism, and this will feature in its own post, because it would be treatable today. 

Reduced activity of protein kinase C in the frontal cortex of subjects with regressive autism: relationship with developmental abnormalities.

Brain Region–Specific Decrease in the Activity and Expression of Protein Kinase A inthe Frontal Cortex of Regressive Autism


Both the above papers are by Abha and Ved Chauhan.  I put Abha on my Dean’s list long ago.  I did have a discussion with her a while back.  She is clearly a very nice person and intellectually towers over the Curemark lady (Joan Fallon) who gets $40 million to play with her pancreatic enzymes, but never publishes anything except very superficial patents.

I think for $40 million Abha and Ved could figure it all out.

PKB, otherwise known as Akt is also very relevant to some types of autism.

Tamoxifen, recently shown to reverse autism in a SHANK3 mouse model, is a PKC inhibitor.

Another epigenetic drug, Theophylline activates PKA.

Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer.

If you could identify if a particular person was hypo/hyper in PKA, PKB and PKC, this might well open the door to an effective treatment.

Research on PKB, also known as AKT

Dysregulation of theIGF-I/PI3K/AKT/mTOR signaling pathway in autism spectrum disorders.

And a paper from the clever Japanese:-

Autism spectrum disorder is a set of neurodevelopmental disorders in terms of prevalence, morbidity and impact to the society, which is characterized by intricate behavioral phenotype and deficits in both social and cognitive functions. The molecular pathogenesis of autism spectrum disorder has not been well understood, however, it seems that PI3K, AKT, and its downstream molecules have crucial roles in the molecular pathogenesis of autism spectrum disorder. The PI3K/AKT signaling pathway plays an important role in the regulation of cell proliferation, differentiation, motility, and protein synthesis. Deregulated PI3K/AKT signaling has also been shown to be associated with the autism spectrum disorder. Discovery of molecular biochemical phenotypes would represent a breakthrough in autism research. This study has provided new insight on the mechanism of the disorder and would open up future opportunity for contributions to understand the pathophysiology

For those who favour dietary intervention:-

Based on the above chart curcumin should likely be good for my N=1 case of autism. Time will tell.

Consequences of upstream dysfunctions

So it might be better to consider autism as a disease of wider downstream gene expression, rather than necessarily of “faulty” genes.  Modulating the resulting wider gene expression may be much more realistic than fixing individual genes.

It is certainly plausible that the body has its own protective self-repair mechanism that might be somehow re-energized. Some people have pondered why so many highly intelligent mathematicians and computer scientists seem have relatives with autism.  The clever genes do associate with a type of autism plus ID/MR.  It was suggested that protective genetic changes might be in play, so that the people with the most genetic variance are actually the family members without the autism.

This does remain conjecture, but as more whole genome data is collected we are seeing some interesting findings.

A fascinating very recent study that looked at a group of 53 families with autism using the traditional approach of whole exome sequencing and also microarray. 

Using these methods, that are the current gold standard, the researchers found very little.  Dysfunctions in the 700 known autism genes were not detected.

However using more expensive whole genome sequencing, dysfunctions were identified in the “DNA junk” zone very close beside the known autism genes.  The researchers were then able to identify the genetic cause of 30% of the cases, a big improvement on 0%.  I expect if they looked a little harder the 30% would be higher.

“We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism.”

“For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES).

Comparing the sequences of the individuals with autism and those of their unaffected siblings, the researchers found that people with autism are more likely to have genetic variants — either single base-pair changes in the sequence or small CNVs — in swaths of DNA abutting known autism genes. But the researchers only found the variants after they restricted their search to regions of the genome already implicated in autism, and even then the statistical significance is modest.

Sequencing whole genomes could reveal the genetic cause of autism in as much as 30 percent of people for whom faster and cheaper sequencing methods come up short

“It’s increasing power even in areas that are supposed to be covered by whole-exome sequencing,” says Peixoto. “It seems that it’s clear that whole-genome sequencing will become the standard.”

One specific microRNA has strong links to autism spectrum disorder, say TSRI scientists


Many diseases have an epigenetic component. The severe progressive asthma that is COPD is a well-known example.  It appears that smoking in middle age often leads to permanent epigenetic changes that come back to haunt often then non-smokers in old age.  Even though they have not smoked for twenty years, there oxidative stress response has been permanently modified.  This results in a kind of steroid resistance, so that usually reliable drug therapies fail to work. 

It is thought that autism has an epigenetic component.  This would do some way to explaining 30-40% of the increase in prevalence in recent years that is not explained by ever widening diagnostic criteria.

Because epigenetic changes can be heritable and can be accumulated from all kinds of exposures, even simple ones like severe emotional stress and pollution, you can reconcile autism as being primarily a genetic condition even though incidence has clearly risen within one or two generations. So you can have an “epigenetic epidemic”, so to speak.

Epigenetics as a therapy

While much is written about epigenetic change being bad, it could also be good.

There are many known substances that affect gene expression; some are very target specific which is useful.

This answers a recent issue raised by a reader of this blog who did exome sequencing. What is the point of discovering a genetic dysfunction if there is no therapy? Medicine is some decades behind science, better to know what gene is affected because you well be able to affect its expression, you just need some help from Google.

Epigenetic therapy could be used to remove unwanted tags, but it could also be used to leave new ones to upregulate under-expressed genes.

Such epigenetic therapy is already a reality in COPD and is being considered for rare single autisms where one copy of the gene is not functional, so turn up the volume on the remaining copy.

As we saw in the post on epigenetics, one potential category of drugs are HDAC inhibitors, these would affect one epigenetic mechanism.

There are many such HDAC inhibitors and most have other modes of action, so you cannot be sure what is giving the noted effect.


This epilepsy drug has numerous effects including as a HDAC inhibitor.  Given to mothers during pregnancy it can cause autism in the offspring, but when given to the affected offspring the autism can be reduced.

Valproate is given off label to treat autism even when no epilepsy is present.

As we saw in the comments section, long term valproate se can have side effects.


This substance derived from broccoli and patented by Johns Hopkins, is another HDAC inhibitor.  It also upregulates Nrf2, which turns on the oxidative response genes.  This was proposed as a COPD therapy by Professor Barnes.

We saw in a post that for Nrf2 to have its full effect there needed to be enough of a protein called DJ-1.  You can increase DJ-1 expression with cinnamon (sodium benzoate).

That was one reason to think that cinnamon would complement Sulforaphane as a therapy for both COPD and some autism.

Sodium Butyrate

Sodium Butyrate is an HDAC inhibitor that is available as a supplement. We came across it in an earlier post as a precursor to butyric acid.  Butyric acid plays a role in the permeability of the gut and the Blood Brain Barrier (BBB).  It also seems to protect from auto immune disease.

Butyrate is fed to millions of farm animals every day to increase their resistance to auto-immune disease.

Butyric acid is produced naturally in the gut by the bacteria living there, however the amount can be increased by the uses of a particular probiotic-bacteria.

This would support the uses of sodium butyrate and the Miyari 588 bacteria.

I have on my to-do-list to investigate higher doses of Miyari 588, but having read the comment by Alli that 500 mg of sodium butyrate is effective, I will try that first.  She also found higher doses ineffective, which was the same in a mouse study published last November,

The study below highlights which genes were down-regulated and which were up-regulated, the overall effect was beneficial

Sodium butyrate attenuate ssocial behavior deficits and modifies the transcription ofinhibitory/excitatory genes in the frontal cortex of an autism model.


The core behavioral symptoms of Autism Spectrum Disorders (ASD) include dysregulation of social communication and the presence of repetitive behaviors. However, there is no pharmacological agent that is currently used to target these core symptoms. Epigenetic dysregulation has been implicated in the etiology of ASD, and may present a pharmacological target. The effect of sodium butyrate, a histone deacetylase inhibitor, on social behavior and repetitive behavior, and the frontal cortex transcriptome, was examined in the BTBR autism mouse model. A 100 mg/kg dose, but not a 1200 mg/kg dose, of sodium butyrate attenuated social deficits in the BTBR mouse model. In addition, both doses decreased marble burying, an indication of repetitive behavior, but had no significant effect on self-grooming. Using RNA-seq, we determined that the 100 mg/kg dose of sodium butyrate induced changes in many behavior-related genes in the prefrontal cortex, and particularly affected genes involved in neuronal excitation or inhibition. The decrease in several excitatory neurotransmitter and neuronal activation marker genes, including cFos Grin2b, and Adra1, together with the increase in inhibitory neurotransmitter genes Drd2 and Gabrg1, suggests that sodium butyrate promotes the transcription of inhibitory pathway transcripts. Finally, DMCM, a GABA reverse agonist, decreased social behaviors in sodium butyrate treated BTBR mice, suggesting that sodium butyrate increases social behaviors through modulation of the excitatory/inhibitory balance. Therefore, transcriptional modulation by sodium butyrate may have beneficial effects on autism related behaviors.



Theophylline is an old asthma drug that is an HDAC inhibitor.

At low doses it is now being trialled as an epigenetic add-on therapy in COPD.  It pretty obviously does work, but data needs to be collected to measure how effective it is and what is the best dose.

It shows how the COPD researchers/clinicians like Professor Barnes are doing a good job and not frightened to experiment.

Would a similar low dose of theophylline benefit a sub-group of those with autism/schizophrenia?  I think it is quite likely.

COPD and autism/schizophrenia share the same impaired oxidative stress response.

Chronic Obstructive Pulmonary Disease (COPD) is a progressive lung disease characterised by progressive airflow limitation. In the UK, it affects around 3 million people, is the fifth leading cause of death and costs the NHS approximately £1 billion annually. Exacerbations of COPD account for 60% of NHS COPD costs and are associated with accelerated rate of lung function decline, reduced physical activity, reduced quality of life, increased mortality and increased risk of co-morbidities. COPD treatment guidelines recommend inhaled corticosteroids (ICS) to reduce exacerbations and improve lung function. However, in COPD, airway inflammation is relatively insensitive to the anti-inflammatory effects of ICS and even high doses fail to prevent exacerbations. Preclinical and pilot studies demonstrate that low dose theophylline may increase the sensitivity of the airway inflammation to ICS, and thus when used with ICS will reduce the rate of COPD exacerbation. In this study we will determine the clinical effectiveness and cost-effectiveness of adding low dose theophylline to ICS therapy in patients with COPD. The primary outcome is the number of exacerbations. The primary economic outcome is the cost-per-QALY gained during the one year treatment period. We will recruit 1424 participants from primary and secondary care across seven areas of the UK. Participants will be randomised to theophylline (200 mg once or twice daily depending on smoking status and weight) or placebo for 12 months. We will follow participants up at six and twelve months to assess the number of exacerbations. We will also collect data on adverse events, health care utilisation, quality of life and breathlessness, and lung function. Low dose theophylline is cheap (10p/day) and, if shown to make current ICS therapy more effective in a cost effective manner, it will improve the quality of life of COPD patients and reduce the burden of COPD on the NHS.

At large doses, Theophylline has long been a therapy for asthma and COPD, but as with Sodium Butyrate, it is quite possible that larger doses of Theophylline produce a different result.  In other words the epigenetic effect fortunately comes from the low dose.

Low doses mean less chance of side effects.

For example, in anyone predisposed to reflux/GERD/GORD many asthma drugs pose a problem because at the same time as opening the airways in your lungs they will relax the lower esophageal sphincter and allow stomach acid to rise upwards.

We saw in an earlier post that in some types of autism something called mGluR5 is dysfunctional in the brain. By chance mGluR5 is also involved in closing the lower esophageal sphincter.  In people with reflux/GERD/GORD a mGluR5 inhibitor was found to have promise for the management of their symptoms.

Randomised clinical trial:effects of monotherapy with ADX10059, a mGluR5 inhibitor, on symptoms and reflux events in patients with gastro-oesophageal reflux disease.

So it is not surprising that many people with autism also have reflux/GERD/GORD. 

But the dysfunction with mGluR5 in autism can be both hyper and hypo, so the therapy might be a positive allosteric modulator (PAM), or a negative allosteric modulator (NAM).  

In someone with autism + reflux/GERD/GORD  it would be reasonable to think a NAM, like ADX10059, might help both conditions.

Gene Repression and Genome Stability

There is another epigenetic process that may be disturbing gene expression in some people and may be treatable.

I have been trying to find why so many people with autism can benefit from biotin; I think I have found a plausible explanation.

“Biotinylation of histones plays a role in gene repression and repression of transposable elements, thereby maintaining genome stability”

I think in some people with autism and no clinical deficiency of biotin the continued “overdosing” of biotin might be having an effect on gene expression, bringing things a little closer to where they should be.

Rather beyond the scope of this blog, it appears that in some people the impaired genome stability, reversible with biotin(ylation), this might be a significant cancer risk.

In essence, for most people supraphysiological concentrations of biotin will do absolutely nothing, but in a sub-group it might do a lot of good.  It is epigenetic, but you do not have to understand it to benefit from it.  It is complicated.

Transposable elements such as long terminal repeats (LTR) constitute 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events, and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila melanogaster enhances retrotransposition in the germline. Importantly, we demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles and transposition events and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk.

Here, we provide evidence for the existence of a novel diet-dependent epigenetic mechanism that represses retrotransposons. Importantly, we demonstrated that depletion of biotinylated histones in biotin-deficient cells increases LTR transcript levels, production of viral particles, and retrotransposition events, and ultimately decreases chromosomal stability. Both biotin deficiency and supplementation are prevalent in the US. For example, moderate biotin deficiency has been observed in up to 50% of pregnant women (35,36). About 20% of the US population reports taking biotin supplements (37), producing supraphysiological concentrations of vitamin in tissues and body fluids (23,28,35). The findings presented here suggest that altered biotin status in these population subgroups might affect chromosomal stability and cancer risk. 

Biotin and biotinidase deficiency

Biotin requirements for DNA damage prevention



I never got round to writing part 2 of my epigenetics post, but my experience of HDAC inhibitors to date has been very positive.

I would be the first to admit that this is rather hit and miss.  It was only when reading the paper on potential therapies for Pitt Hopkins, that was openly musing about HDAC inhibitors, in an equally hit and miss approach, that I thought I would write further about it.

It really seems totally haphazard, because you cannot predict the effect with any level of certainty.  If there is a self-repair mechanism trying to maintain homeostasis of the genome, haphazard may be good enough.

10mg of biotin twice a day does have a mild but noticeable stabilizing effect; is this caused by better maintaining genome stability? I have no idea. 

I will try sodium butyrate and if it works I will have to establish what dose of Miyari 588 produces the same effect.  Both are used in animal feed to reduce inflammatory disease, so you are already indirectly exposed to them if you eat meat.

Theophylline should also be investigated.  This is a very well understood drug and small doses really do seem to help people with COPD.

PKA, PKB and PKC are likely at the core of most people’s autism.  Many existing therapies can modify their expression.

Whole genome sequencing, carried out at great precision, is clearly the only satisfactory genetic testing method.  The other, cheaper, methods are just missing key data and giving many false negative results, i.e. saying there are no identifiable genetic dysfunctions, when this is not true.