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Showing posts with label Schizophrenia. Show all posts
Showing posts with label Schizophrenia. 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. 


Abstract


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.  

Conclusion
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.







Monday 11 December 2017

Cognitive Loss/Impaired Sensory Gating from HCN Channels - Recovered by PDE4 Inhibition or an α2A Receptor Agonist

Today we have a complex dysfunction, but we have a plausible understanding of the detailed biological underpinnings and several therapeutic options. It is relevant to people with autism who have impaired sensory gating (they find noises like a clock ticking annoying), and perhaps those who struggle with complex thought. It is very likely to be disturbed in some people with ADHD and many with schizophrenia.

Trouble in the Pre-Frontal Cortex


For a recap on sensory gating, here is an earlier post:-

Sensory Gating in Autism, Particularly Asperger's


Today’s dysfunction relates to HCN channels located on those tiny dendritic spines in a part of the brain called the pre-frontal cortex. These are a type of voltage gated potassium channel found in your brain and heart, there are 4 types, it looks to me that HCN2 is the key one today.
The pre-frontal cortex (PFC) is seen as the part of the brain most affected by mental illness (schizophrenia, bipolar, ADHD etc.), although medicine’s current understanding looks rather medieval to me.
These HCN channels can open when they are exposed to cAMP (cyclic adenosine monophosphate). When open, the information can no longer flow into the cell, and thus the network (created by numerous interacting neurons) is effectively disconnected.
By keeping these channels closed, it is thought that you can improve working memory and reducing distractibility. Now you might think distractibility is an odd word, and it is not a word I expected to encounter, what it really means is impaired sensory gating. This is a core feature of Asperger’s, ADHD and schizophrenia.
One of the key risk genes for schizophrenia, DISC1, also affects HCN channels and this may account for some of the cognitive deficit found in schizophrenia. High level thinking is particularly affected.  It is thought that loss of DISC1 function in the PFC would likely prevent proper PDE4 function, leading to a dysregulated build-up of cAMP in dendritic spines resulting in excessive opening of HCN channels


I did wonder how nicotine fits in, since in earlier post we saw that α7 nAChR agonists, like nicotine, improve sensory gating and indeed that people with schizophrenia tend to be smokers. It turns out that nicotine is also an HCN channel blocker. For a change, everything seems to fit nicely together. There are different ways to block HCN channels, some of which are indirect. One common ADHD drug, Guanfacine, keeps these channels closed, but in a surprising way.
Alpha-2A adrenergic receptors near the HCN channels, on those dendritic spines, inhibit the production of cAMP and the HCN channels stay closed, allowing the information to pass through into the cell, connecting the network. These Alpha-2A adrenergic  receptors are stimulated by a natural brain chemical norepinephrine, or by drugs like Guanfacine.
Stress appears to flood PFC neurons with cAMP, which opens HCN channels, temporarily disconnects networks, and impairs higher cognitive abilities.
This would explain why stress makes people’s sensory gating problems get worse. So someone with Asperger’s would get more distracted/disturbed at exam time at school for example, or when he goes for a job interview. Reducing stress is another method to improve sensory gating and indeed cognition. In Monty, aged 14 with ASD, the only time he exhibits significantly impaired sensory gating, is when he has stopped all his Polypill therapies for several days. I think stress/anxiety is what has changed and this opens those HCN channels. Then even the sound of someone eating food next to him makes him angry.
Excessive opening of HCN channels might underlie many lapses in higher cognitive function.
While the researchers at Yale patented the idea of HCN blockers to improve cognition, we can see how other existing ideas to improve cognition may indeed have the same mechanism, most notably PDE4 inhibitors.
The University of Maastricht holds patents on the use of Roflumilast, a PDE4 inhibitor, to improve cognition; most interestingly, this takes effect at one fifth of the COPD dosage, for which it is an approved drug. At high doses PDE4 inhibitors have annoying side effects, but at low doses they tend to be trouble-free.
One effect of a PDE4 inhibitor is that it reduces cAMP. So a PDE4 inhibitor acts indirectly like an HCN blocker.
Not surprisingly recent research showed that low doses of Roflumilast improves sensory gating in those affected by this issue.
So rather than waiting for a brain selective HCN blocker, the potential exists to use a one fifth dose of Roflumilast today. This is something that should indeed be investigated across different types of cognitive dysfunction.
There are numerous dysfunctions that can impair cognition and they can occur in different diagnosis. For example impaired autophagy is a key feature of Huntington’s, impaired remyelination defines multiple sclerosis, low levels of nerve growth factor are a key feature of Rett syndrome. Less severe dysfunctions of these processes occur in entirely different conditions.
It is thought that people with Alzheimer’s might benefit from PDE4 inhibition. If it was me, I would try it in all types of dementia or cognitive loss of any kind.

PDE4 Inhibitors
There have been many mentions of PDE4 inhibitors elsewhere in this blog. They are broadly anti-inflammatory and anti-oxidant, but currently only widely used to treat asthma in Japan and COPD in Western countries. COPD is a kind of very severe asthma.
Traditionally a PDE4 inhibitor is thought of as drug used to block the degradative action of phosphodiesterase 4 (PDE4) on cyclic adenosine monophosphate (cAMP). That all sound complicated, just think of it as increasing cAMP.
Now cAMP is a messenger in many biological processes, one of which relates to PKA (Protein Kinase A). In autism we know that PKA, PKB and PKC are often disturbed. These PKs are very important because they have the ability to literally change the function of thousands of proteins in your body. This is similar to how epigenetic tags can switch on or switch off a particular gene. PKs, via a different mechanism we will look at in another post, change the function of proteins, so it is very important that you have the correct level of PKA, PKB and PKC.
We saw in a recent post that the Pitt Hopkins gene TCF4 is regulated by PKA and that under-expression of TCF4 is also a feature of some ID and schizophrenia. So more PKA, please.

You can use a PDE4 inhibitor to increase cAMP, which then increases PKA.

Other effects of PDE4 inhibitors
Today’s post is about sensory gating and the effect here of PDE4 inhibitors is via the effect of cAMP on those HCN channels in your tiny dendritic spines.
There are numerous other effects of PDE4 that may also be therapeutic. One interesting effect is that inhibition of PDE4 can mimic calorie restriction by activating AMPK/SIRT1 pathway.
Calorie restriction has just been shown in a large trial to be able to reverse type 2 diabetes, if initiated with a few years of the disease developing.
Humans have evolved based to periods of feast and famine. Periods of fasting may be therapeutic for many modern conditions.
Not surprisingly one side effect of PDE4 inhibitors is weight loss. Many psychiatric drugs cause troubling weight gain.

Acute administration of Roflumilast enhances sensory gating in healthy young humans in a randomized trial. 

Abstract

 

INTRODUCTION:

Sensory gating is a process involved in early information processing which prevents overstimulation of higher cortical areas by filtering sensory information. Research has shown that the process of sensory gating is disrupted in patients suffering from clinical disorders including attention deficit hyper activity disorder, schizophrenia, and Alzheimer's disease. Phosphodiesterase (PDE) inhibitors have received an increased interest as a tool to improve cognitive performance in both animals and man, including sensory gating.

METHODS:

The current study investigated the effects of the PDE4 inhibitor Roflumilast in a sensory gating paradigm in 20 healthy young human volunteers (age range 18-30 years). We applied a placebo-controlled randomized cross-over design and tested three doses (100, 300, 1000 μg).

RESULTS:

Results show that Roflumilast improves sensory gating in healthy young human volunteers only at the 100-μg dose. The effective dose of 100 μg is five times lower than the clinically approved dose for the treatment of acute exacerbations in chronic obstructive pulmonary disease (COPD). No side-effects, such as nausea and emesis, were observed at this dose. This means Roflumilast shows a beneficial effect on gating at a dose that had no adverse effects reported following single-dose administration in the present study.

CONCLUSION:

The PDE4 inhibitor Roflumilast has a favourable side-effect profile at a cognitively effective dose and could be considered as a treatment in disorders affected by disrupted sensory gating.


Background Information
Selective phosphodiesterase (PDE) inhibition has been considered as a very promising target for cognition enhancement.
Roflumilast is a PDE4 inhibitor that has been developed by Takeda for Chronic Obstructive Pulmonary Disease (COPD). In recent year, Maastricht University has been collaborating with Takeda to develop Roflumilast for cognitive impairments
In 2015 Takeda sold COPD indication of Roflumilast to AstraZeneca, and ownership of IP for treatment of cognitive impairment returned to Maastricht University.
Compelling clinical results
A single administration of Roflumilast improves episodic memory in mice, and in young and elderly healthy subjects at a non-emetic dose
As shown in the figure, healthy (A) and memory impaired (B) elderly subjects showed better performances in the delayed recall of the Verbal Learning Task after roflumilast

Key Features and Advantages
Opportunities to reposition a clinically-proven safe compound with a well-established pharmacology.
Compelling preclinical and clinical evidences showing that Roflumilast effectively deliver to the brain to produce robust cognitive enhancement.
Pro-cognitive effects at low dose (5 times lower than COPD indication), which allows to circumvent the emetic effects commonly observed with other PDE4 inhibitors
Maastricht University has a strong IP protection extending to at least 2033.

PDE inhibitors in psychiatry--future options for dementia, depression and schizophrenia?

Author information

Abstract

Phosphodiesterases are key enzymes in cellular signalling pathways. They degrade cyclic nucleotides and their inhibition via specific inhibitors offers unique 'receptor-independent' opportunities to modify cellular function. An increasing number of in vitro and animal model studies point to innovative treatment options in neurology and psychiatry. This review critiques a selection of recent studies and developments with a focus on dementia/neuroprotection, depression and schizophrenia. Despite increased interest among the clinical neurosciences, there are still no approved PDE inhibitors for clinical use in neurology or psychiatry. Adverse effects are a major impediment for clinical approval. It is therefore necessary to search for more specific inhibitors at the level of different PDE sub-families and isoforms.


The current study found that brain cells in PFC contain ion channels called hyperpolarization-activated cyclic nucleotide-gated channels (HCN) that reside on dendritic spines, the tiny protrusions on neurons that are specialized for receiving information. These channels can open when they are exposed to cAMP (cyclic adenosine monophosphate). When open, the information can no longer flow into the cell, and thus the network is effectively disconnected. Arnsten said inhibiting cAMP closes the channels and allows the network to reconnect.
Guanfacine can strengthen the connectivity of these networks by keeping these channels closed, thus improving working memory and reducing distractibility," she said. "This is the first time we have observed the mechanism of action of a psychotropic medication in such depth, at the level of ion channels."
Arnsten said the excessive opening of HCN channels might underlie many lapses in higher cognitive function. Stress, for example, appears to flood PFC neurons with cAMP, which opens HCN channels, temporarily disconnects networks, and impairs higher cognitive abilities.
The study also found alpha-2A adrenergic receptors near the channels that inhibit the production of cAMP and allow the information to pass through into the cell, connecting the network. These receptors are stimulated by a natural brain chemical  norepinephrine or by medications like guanfacine.
 “Guanfacine can strengthen the connectivity of these networks by keeping these channels closed, thus improving working memory and reducing distractibility,” she said. “This is the first time we have observed the mechanism of action of a psychotropic medication in such depth, at the level of ion channels.”
Yale has submitted a patent application on the use of HCN blockers for the treatment of PFC cognitive deficits based on the data reported in the Cell paper.

The full Yale paper:

The prefrontal cortex (PFC) is among the most evolved brain regions, contributing to our highest order cognitive abilities. It regulates behavior, thought, and emotion using working memory. Many cognitive disorders involve impairments of the PFC. A century of discoveries at Yale Medical School has revealed the neurobiology of PFC cognitive functions, as well as the molecular needs of these circuits. This work has led to the identification of therapeutic targets to treat cognitive disorders. Recent research has found that the noradrenergic α2A agonist guanfacine can improve PFC function by strengthening PFC network connections via inhibition of cAMP-potassium channel signaling in postsynaptic spines. Guanfacine is now being used to treat a variety of PFC cognitive disorders, including Tourette’s Syndrome and Attention Deficit Hyperactivity Disorder (ADHD). This article reviews the history of Yale discoveries on the neurobiology of PFC working memory function and the identification of guanfacine for treating cognitive disorders.

Molecular modeling suggests that, similarly to ZD 7288, nicotine and epibatidine directly bind to the inner pore of the HCN channels. It is therefore likely that nicotine severely influences rhythmogenesis and high cognitive functions in smokers.

Modulation of HCN channels in lateral septum by nicotine


Conclusion
I think many people stand to benefit from the drugs mentioned in today’s post, but for different biological reasons. A person with Pitt Hopkins may benefit from Roflumilast because it will upregulate PKA and then increase expression of their remaining TCF4 gene.
In a person with schizophrenia there are multiple reasons these drugs might help them and it will depend on which genes they have that are misexpressed (TCF4, DISC1 etc.).
In a person with idiopathic Asperger’s and impaired sensory gating it looks like the effect on HCN channels is what is important.
I think low dose Roflumilast has great potential for many. The Japanese drug Ibudilast very likely will provide similar benefits, but at what dosage?
PDE4 inhibitors do have side effects at higher doses in part because there are several different types of PDE4 (PDE4A, PDE4B, PDE4C etc) and different drugs effect different subtypes differently.
Ibudilast is used as a daily drug therapy for asthma in Japan and is being studied as a therapy for Multiple Sclerosis (MS) in the US.
Roflumilast is sold by Astra Zeneca as Daxas/Daliresp but at a high dose of 500mcg to treat flare ups of COPD (Chronic Obstructive Pulmonary Disease) it does cause troubling side effects, but it reduces your chance of dying from COPD.
The cognitive dose used in research is 100mcg. Higher doses had no cognitive/sensory gating benefit.
Further investigation of the ADHD drug Guanfacine should be made, because some of the people who benefit from a PDE4 inhibitor might get a similar effect from Guanfacine. People with Pitt Hopkins would not be in this category. A person with Asperger’s and impaired sensory dating should respond to Guanfacine, a cheap drug.
At the end of the day, choice of therapy will come down to side effects and cost. In the US, Roflumilast is expensive ($330), seven times more expensive than in some other countries; in the UK the price of the same 30 tablets is $50. One pack would be enough for 5 months at the suggested dose.