Cilantro (Coriander leaves) for sound sensitivity? cGPMax for some Pitt Hopkins and Rett syndrome. Plus, microdeletion of 2P16.3 NRXN1 and mutations in GPC5

 

Today’s post combines a very simple therapy for sound sensitivity that landed in my inbox from New Zealand with two more genes that I was recently asked about.

Before I get started I would like to thank our reader Daniel who is trying to spread that word that the IGF-1 targeting therapy cGPMax works in some Rett syndrome (half a capsule daily). I did go into the science of IGF-1 related therapies at the recent conference in Abu Dhabi. In that presentation I pointed out that the cGPMax therapy might well be helpful in Pitt Hopkins syndrome. I saw today that Soko, an 8 year old girl with Pitt Hopkins, had already made a trial and her parents are impressed:-

“Equally significant has been the positive shift in Soko's emotional well-being. Her struggles with irritability and mood fluctuations feel like are not as frequent and we feel like there is more often a sense of calm and emotional regulation. This has had a profound ripple effect on our little family and our stress levels.

Perhaps most striking has been the accelerated rate at which Soko acquires new skills. CGP Max has seemingly unlocked hidden potentials within her. This rapid skill acquisition has been very exciting for us. In the last year she has gone from being unable to walk to walking unassisted and even tackling steps no handed!”

I did some checking and some other parents have tried cGPMax for Pitt Hopkins. For Rett syndrome Daniel found that a lower dose was more beneficial than a higher dose. It is always best to start with low doses and gradually increase them.

This does link to today’s post because a  microdeletion of NRXN1 can cause Pitt Hopkins Like Syndrome 2 (PHLS2). In theory all these syndromes are untreatable, but try telling that to Soko’s parents.

 

Back to sound sensitivity

Today’s sound sensitivity is the type that is moderated by Ponstan (mefenamic acid) and indeed Diclofenac. It might well include those whose sound sensitivity responds to a simple potassium supplement.

If you want to look into the details, you can see from previous posts how potassium and potassium ion channels play a fundamental role in both hearing and its sensory processing. They also play a key role in excitability of neurons and so can play a key role in some epilepsy and some intellectual disability.

It turns out that Cilantro/Coriander leaves contains a chemical that activates the ion channels  KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3). This effect is shared by Ponstan and Diclofenac.

In the case of Andy from New Zealand the effect of a 425mg Cilantro supplement lasts very much longer than taking a low dose of Ponstan or Diclofenac.

So, if your child responds well to Ponstan and can then happily take off his/her ear defenders, but you do not want to medicate every day, then a trial of Cilantro could be interesting.

I was curious as to why the effect would last so much longer than Ponstan/Diclofenac.  All of these drugs lower potassium levels within neurons.  Is the beneficial effect coming from lowering potassium levels and so reducing neuronal excitability?  Or, is the effect coming directly from a specific ion channel?

Nobody can tell you the half-life of the active component of cilantro,  (E)-2-dodecenal, in humans.  Andy thinks it must have a long half-life.

 

Cilantro (Coriander leaves)

If you live in North America you will know what cilantro is, for everyone else it means coriander leaves. Coriander seeds are the dried fruit of the coriander plant and, confusingly, in American English coriander means coriander seeds.

The medicinal properties of the leaves and seeds are not the same.

Cilantro leaves contain a compound called (E)-2-dodecenal, which has been shown to activate a specific family of potassium ion channel called KCNQ, otherwise known as Kv7 . These channels are found in neurons, and they play an important role in regulating the electrical activity of the brain.

When (E)-2-dodecenal binds to KCNQ/Kv7 channels, it causes them to open, which allows potassium ions to flow out of the neuron. This outflow of potassium ions helps to stabilize the neuron's membrane potential and makes it less likely to fire an action potential.

The level of potassium inside neurons is much higher than the level outside. Having it too high, or indeed too low, would affect the excitability of the neuron.

I am wondering if the problem with potassium is mirroring the problem we have with chloride; perhaps both are elevated inside neurons. That would be nice and simple.

The discovery that cilantro can activate KCNQ channels helps to explain its potential anticonvulsant properties.  KCNQ channel dysfunction has been linked to certain types of epilepsy, and drugs that activate these channels are being investigated as potential treatments for these conditions.

Research suggests cilantro's active compound, (E)-2-dodecenal, targets multiple KCNQ channels, particularly:

• KCNQ2/KCNQ3: This is the most common type of KCNQ channel found in neurons.
• KCNQ1 in complex with KCNE1: This form is mainly present in the heart. KCNE1 acts as a regulatory subunit that influences KCNQ1 channel function.

 

Cilantro leaf harbors a potent potassium channel-activating anticonvulsant

Herbs have a long history of use as folk medicine anticonvulsants, yet the underlying mechanisms often remain unknown. Neuronal voltage-gated potassium channel subfamily Q (KCNQ) dysfunction can cause severe epileptic encephalopathies that are resistant to modern anticonvulsants. Here we report that cilantro (Coriandrum sativum), a widely used culinary herb that also exhibits antiepileptic and other therapeutic activities, is a highly potent KCNQ channel activator. Screening of cilantro leaf metabolites revealed that one, the long-chain fatty aldehyde (E)-2-dodecenal, activates multiple KCNQs, including the predominant neuronal isoform, KCNQ2/KCNQ3 [half maximal effective concentration (EC50), 60 ± 20 nM], and the predominant cardiac isoform, KCNQ1 in complexes with the type I transmembrane ancillary subunit (KCNE1) (EC50, 260 ± 100 nM). (E)-2-dodecenal also recapitulated the anticonvulsant action of cilantro, delaying pentylene tetrazole-induced seizures. In silico docking and mutagenesis studies identified the (E)-2-dodecenal binding site, juxtaposed between residues on the KCNQ S5 transmembrane segment and S4-5 linker. The results provide a molecular basis for the therapeutic actions of cilantro and indicate that this ubiquitous culinary herb is surprisingly influential upon clinically important KCNQ channels

Activation of KCNQ5 by cilantro could also contribute to its gut stimulatory properties, as KCNQ5 is also expressed in gastrointestinal smooth muscle, and its activation might therefore relax muscle, potentially being therapeutic in gastric motility disorders such as diabetic gastroparesis.

The KCNQ activation profile of (E)-2-dodecenal bears both similarities and differences to that of other KCNQ openers. We recently found that mallotoxin, from the shrub Mallotus oppositifolius that is used in African folk medicine, also activates KCNQ1-5 homomers, prefers KCNQ2 over KCNQ3, and in docking simulations binds in a pose reminiscent to that predicted for (E)-2-dodecenal, between (KCNQ2 numbering) R213 and W236 In addition to the widespread use of cilantro in cooking and as an herbal medicine, (E)-2-dodecenal itself is in broad use as a food flavoring and to provide citrus notes to cosmetics, perfumes, soaps, detergents, shampoos, and candles (59).

Our mouse seizure studies suggest it readily accesses the brain, and it is likely that its consumption as a food or herbal medicine (in cilantro) or as an added food flavoring would result in KCNQ-active levels in the human body; we found the 1% cilantro extract an efficacious KCNQ activator, and (E)-2-dodecenal itself showed greater than half-maximal opening effects on KCNQ2/3 at 100 nM (.10 mV shift at this concentration) (EC50, 60 6 20 nM). We anticipate that its activity on KCNQ channels contributes significantly to the broad therapeutic spectrum attributed to cilantro, which has persisted as a folk medicine for thousands of years throughout and perhaps predating human recorded history.

 

From the University of California: 

How cilantro works as a secret weapon against seizures

In a new study, researchers uncovered the molecular action that enables cilantro to effectively delay certain seizures common in epilepsy and other diseases.

The study, published in FASEB Journal, explains the molecular action of cilantro (Coriandrum sativum) as a highly potent KCNQ channel activator. This new understanding may lead to improvements in therapeutics and the development of more efficacious drugs.

“We discovered that cilantro, which has been used as a traditional anticonvulsant medicine, activates a class of potassium channels in the brain to reduce seizure activity,” said Geoff Abbott, Ph.D., professor of physiology and biophysics at the UC Irvine School of Medicine and principal investigator on the study.

“Specifically, we found one component of cilantro, called dodecenal, binds to a specific part of the potassium channels to open them, reducing cellular excitability.”

 

KCNQ channels and autism

There is a growing body of research suggesting a connection between KCNQ channels and autism.

·        KCNQ channel mutations: Genetic studies have identified mutations in several KCNQ channel genes (including KCNQ2, KCNQ3) in individuals with ASD. These mutations might disrupt the normal function of KCNQ channels, leading to abnormal brain activity.

• Neuronal excitability: KCNQ channels help regulate the electrical activity of neurons by controlling the flow of potassium ions. Mutations or dysfunction in KCNQ channels could lead to increased neuronal excitability, which has been implicated in ASD. 

• Shared features: Epilepsy is a common comorbidity with autism. Interestingly, KCNQ channel dysfunction is also linked to certain types of epilepsy. This suggests some shared mechanisms between these conditions.

 

KCNQ Dysfunction and Intellectual Disability

Mutations in certain KCNQ genes can lead to malfunctions in the corresponding potassium channels. These malfunctions can disrupt normal neuronal activity and contribute to intellectual disability.

• KCNQ2/3 Mutations: Research suggests increased activity in KCNQ2 and KCNQ3 channels, due to mutations in their genes, might be associated with a subset of patients with intellectual disability alongside autism spectrum disorder. 

• KCNQ5 Mutations: Studies have identified mutations in the KCNQ5 gene, leading to both loss-of-function and gain-of-function effects on the channel. These changes in KCNQ5 channel activity can contribute to intellectual disability, sometimes accompanied by epilepsy.

 

The other naming system

KCNQ channels belong to a larger potassium channel family called Kv7. So, you might see them referred to as Kv7.1 (KCNQ1), Kv7.2 (KCNQ2), and so on, based on their specific gene and protein sequence.

 

Mefenamic acid and Kir channels (inwards rectifying potassium ion channels)

Ponstan (mefenamic acid) affects Kir channels and KCNQ channels.

Different Kir channel subtypes contribute to various brain functions, including:

• Neuronal excitability: Kir channels help regulate the resting membrane potential of neurons, influencing their firing activity.
• Potassium homeostasis: They play a role in maintaining the proper balance of potassium ions within and outside neurons, crucial for normal electrical signaling.
• Synaptic inhibition: Some Kir channels contribute to inhibitory neurotransmission, which helps balance excitatory signals in the brain.

Kir Channels are primarily inward rectifiers, meaning they allow potassium ions to flow more easily into the cell than out. They play a role in setting the resting membrane potential of cells, influencing their excitability.

KCNQ Channels can be voltage-gated or regulated by other factors. They contribute to various functions like regulating neuronal firing in the brain,

 

Other effects of Cilantro

It is certainly could be just a coincidence that Cilantro and Ponstan affect KCNQ channels. Cilantro has many other effects.

Coriandrum sativum and Its Utility in Psychiatric Disorders

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10385770/

Recent research has shown that Coriandrum sativum offers a rich source of metabolites, mainly terpenes and flavonoids, as useful agents against central nervous system disorders, with remarkable in vitro and in vivo activities on models related to these pathologies. Furthermore, studies have revealed that some compounds exhibit a chemical interaction with γ-aminobutyric acid, 5-hydroxytryptamine, and N-methyl-D-aspartate receptors, which are key components in the pathophysiology associated with psychiatric and neurological diseases. 

 

Bioactivities of isolated compounds from Coriandrum sativum by interaction with some neurotransmission systems involved in psychiatric and neurological disorders.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10385770/table/molecules-28-05314-t002/?report=objectonly

 

 

Understanding 2p16.3 (NRXN1) deletions

One parent contacted me to ask about the genetic test results they had received for their child.

To understand what happens when parts of the NRXN1 gene are missing you need to read up on neurexins and neuroligins.

 

Neurexins and Neuroligins

Neurexins ensure the formation of proper synaptic connections, fine-tune their strength, and contribute to the brain's adaptability. Understanding their role is crucial for understanding brain development, function, and various neurological disorders.

Neurexins and neuroligins are cell adhesion molecules that work together to ensure proper synapse formation, function, and ultimately, a healthy and functioning brain.

Neuroligins are located on the postsynaptic membrane (receiving neuron) of a synapse.

Neurexins are located on the presynaptic membrane (sending neuron) of a synapse.

Mutations in either neurexin or neuroligin genes have been linked to various neurodevelopmental disorders, including autism.

A comprehensive presentation for families is below:

 

Understanding 2p16.3 (NRXN1) deletions

https://www.rarechromo.org/media/information/Chromosome%20%202/2p16.3%20(NRXN1)%20deletions%20FTNW.pdf

 

A microdeletion in the NRXN1 gene on chromosome 2p16.3 can cause a condition similar to Pitt-Hopkins syndrome, but referred to as Pitt-Hopkins like syndrome 2 (PHLS2).

 

NRXN1 Gene:

• NRXN1 codes for a protein called neurexin 1 alpha, which plays a critical role in the development and function of synapses, the junctions between neurons in the brain.
• Neurexin 1 alpha helps neurons connect with each other and transmit signals.

Microdeletion:

• A microdeletion is a small deletion of genetic material from a chromosome.
• In PHLS2, a microdeletion occurs in the NRXN1 gene, removing some of the genetic instructions needed to produce functional neurexin 1 alpha protein.

Pitt-Hopkins Like Syndrome 2 (PHLS2):

• PHLS2 is a genetic disorder characterized by intellectual disability, developmental delays, and various neurodevelopmental features.
• Symptoms can vary depending on the size and specific location of the NRXN1 microdeletion.
• Common features include:
• Intellectual disability (ranging from mild to severe)
• Speech and language impairments
• Developmental delays in motor skills
• Stereotypies (repetitive movements)
• Seizures
• Behavioral problems (e.g., hyperactivity, anxiety)
• Distinctive facial features (not always present)

 

What has this got to do with Pitt Hopkins syndrome (loss of TCF4)?

“TCF4 may be transcribed into at least 18 different isoforms with varying N-termini, which impact subcellular localization and function. Functional analyses and mapping of missense variants reveal that different functional domains exist within the TCF4 gene and can alter transcriptional activation of downstream genes, including NRXN1 and CNTNAP2, which cause Pitt-Hopkins-like syndromes 1 and 2.”

 

NRXN1 interactions with other genes/proteins

Given the function of neurexins and neuroligins, you would expect that the common interactions of NRXN1 are with neuroligins. We see below the NLGNs (neuroligin genes/proteins)

Our more avid readers may recall that neuroligins are one mechanism for regulating the GABA switch. This is the developmental switch that should occur in all humans about two weeks after birth.  If it does not occur, the brain cannot develop and function normally. Autism and intellectual disability are the visible symptoms.

 

An unexpected role of neuroligin-2 in regulating KCC2 and GABA functional switch

https://molecularbrain.biomedcentral.com/articles/10.1186/1756-6606-6-23#:~:text=Novel%20function%20of%20neuroligin%2D2,expression%20level%20was%20significantly%20decreased.

 

We report here that KCC2 is unexpectedly regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses. The expression of NL2 precedes that of KCC2 in early postnatal development. Upon knockdown of NL2, the expression level of KCC2 is significantly decreased, and GABA functional switch is significantly delayed during early development. Overexpression of shRNA-proof NL2 rescues both KCC2 reduction and delayed GABA functional switch induced by NL2 shRNAs. Moreover, NL2 appears to be required to maintain GABA inhibitory function even in mature neurons, because knockdown NL2 reverses GABA action to excitatory. 

Our data suggest that in addition to its conventional role as a cell adhesion molecule to regulate GABAergic synaptogenesis, NL2 also regulates KCC2 to modulate GABA functional switch and even glutamatergic synapses. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.

 

It would seem plausible that in the case of microdeletions of the NRXN1 gene there will be a direct impact on the expression of NLGN2 gene that encodes neuroligin 2.

So plausible therapies to trial for microdeletions of the NRXN1 gene would include bumetanide, as well as cGPMax, due to the link with Pitt Hopkins.

 

GPC5 gene 

Finally, we move on to our last gene which is GPC5.

The protein Glpycan 5/GPC5 plays a role in the control of cell division and growth regulation.

Not surprising, GPC5 acts a tumor suppressor, making it a cancer gene. Because of this it is also an autism gene. It also plays a role in Alzheimer’s disease.

I was not sure I would be able to say anything about how you might treat autism caused by a mutation in GPC5.

 

Glycan susceptibility factors in autism spectrum disorders

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556687/

 

I am assuming the mutation causes a loss of function, meaning there is a reduced level of the protein Glpycan 5.

Since one role of this gene is to suppress Wnt/beta-catenin signaling, you might want to replace this action.

This is actually covered in my blog in various places. One way is via a GSK-3β inhibitor.

GSK-3β inhibitor include drugs designed to block GSK-3β activity, examples include lithium (used for bipolar disorder), kenpaullone, and tideglusib. Certain natural compounds like curcumin and quercetin have been shown to possess GSK-3β inhibitory effects.

Atorvastatin, which my son has taken for 10 years, is indirectly a GSK-3β inhibitor

Some natural compounds like fisetin (found in fruits and vegetables) have been shown to promote beta-catenin phosphorylation, leading to its degradation.

In previous posts I pointed out that the cheap kids’ anthelmintic medication Mebendazole is indirectly another Wnt inhibitor. This is because it reduces TNIK. TNIK promotes Wnt signaling by stabilizing beta-catenin, a key player in the pathway. By reducing TNIK levels, mebendazole indirectly disrupts Wnt signaling. Mebendazole is therefore a novel cancer therapy and is being investigated to treat brain cancers, colon cancer, breast cancers etc.

Unlike what is says in the literature about GPC5, there actually are many options that can be safely trialed.

Note that you may not know for sure that any mutation is actually causal/pathogenic. Some people have several “likely pathogenic” mutations, some likely are not.

 

Conclusion

We have covered the potassium ion channel Kv7.1 previously. In Pitt Hopkins syndrome this ion channel is over expressed and so you would want to inhibit it. Do not take Cilantro, it would have the opposite effect to what you want.

It looks like cGPMAX is one thing you need to trial for Pitt Hopkins syndrome and Rett syndrome. For idiopathic autism it may, or may not help. Try a low dose first, observe the effect, then try a higher dose.

In Rett syndrome we know that people with have as much NKCC1 RNA — a molecule that carries the instructions to make the protein — as healthy individuals. However, their levels of KCC2 RNA are much lower, potentially disrupting the excitation/inhibition balance of nerve cell signaling. This will result in elevated chloride in neurons. This is correctable today using bumetanide.

People with NRXN1 microdeletions do seem to have treatment options, as do people with GPC5 mutations.

Note that out reader Janu, treating a mutation in GABRB2, reports success with a combination of the SSRI drug Lexapro and sodium valproate.

I am a fan of low dose Ponstan for sound sensitivity, it has numerous potentially beneficial mechanisms. It has been even shown to protect against Alzheimer’s disease.  There is no reason not to give cilantro a try as an alternative or complement to improve sound sensitivity.

Dried coriander is normally made from the seeds and is not what you need. In your supermarket you can buy fresh coriander leaves (Cilantro). The fresh herb is about 90% water, but when you dry the herb you will lose at lot of the active substance because it is volatile and will evaporate. My guess is that you will need 2-3 g of the fresh herb to equal Andy’s 425mg supplement.  You can eat the stalks as well as the leaves, it all has the same pungent taste.

Wnt, TCF4 and Pre-myelinating Oligodendrocytes

Cartoons in art class - Monty is getting ready for Easter break, but not in the Maldives

Today’s post may sound very complicated and narrow, but it is very relevant to people with the following: - 

·        Pitt Hopkins Syndrome (insufficient expression of the Transcription Factor #4  TCF4 gene)

·        Multiple Sclerosis

·        Some Mental Retardation/Intellectual Disability (MR/ID)

·        Schizophrenia

·        Impaired Wnt signalling

·        Perhaps PAK1 inhibitor responders

I do feel that Multiple Sclerosis could be treated very much better if some effort was made to translate the existing science, freely available to all, into therapy. You could greatly improve many people’s lives just by repurposing cheap existing drugs.

In simple terms, to produce myelin that you need to coat axons in your brain, you need a type of cell called an oligodendrocyte (OL).  You need a lot of these cells and you need them to get busy. They place tiny pieces of white insulation along axons of your brain cells, this produces the so called “white matter”.  These pieces of insulation are needed to make electrical signals flow correctly in your brain.

It has been shown that in some people the oligodendrocyte precursors (OLPs) do not “mature” and instead get stuck as premyelinated oligodendrocytes (pre-OL). That means reduced myelination and loss of white matter.

It is clearly shown in the graphic below: -

Tcf4 is expressed in oligodendrocyte lineage in human developmental white matter and in active areas of MS lesions. (A) Tcf4 is expressed in white matter tracts during myelination of human developmental brain at postnatal age 1 mo, 3.5 mo, and 16 mo, but is not expressed by 7 yr. Tcf4 colocalizes with Olig2 when expressed in the developing human corpus callosum. (B) Tcf4 protein expression is evident in active MS lesions, but it is not seen in normal-appearing white matter (NAWM) or in the core of chronic MS lesions. An illustrative MS case is shown with several lesion types present. NAWM stains with Luxol Fast Blue (LFB) and contains sparse LN3(HLA-DR)-positive inflammatory cells, organized SMI-31 axon fibers, and no Tcf4-positive cells. Chronic plaques have sparse LFB staining and LN3-positive cells, intact axons, but no Tcf4-positive cells. In contrast, Tcf4-positive cells are present in active areas of plaques with abundant LN3-positive cells and intact demyelinated axons. Tcf4 expression in active lesions colocalizes (open arrowheads) with a subset of Olig2 cells.


Don’t worry if you don't follow everything. There is nothing wrong with your white matter.

We come back to Wnt signalling that we covered in depth in older posts. This is a complex signalling pathway implicated in autism, some cancers and other conditions. You can both increase and reduce Wnt signalling, which will affect the transcription of numerous genes.

TCF4 is the Pitt Hopkins gene. We have across this syndrome several times, while it is rare, a milder miss-expression of the gene is actually quite common.  Reduced expression of TCF4 is a common feature of MR/ID very broadly. TCF4 has been found to be over-expressed in schizophrenia.

People with Multiple Sclerosis (MS) have been found to have oligodendrocytes “stuck” as non-myelinating (premyelinated oligodendrocytes, pre-OL). Inhibiting the Wnt pathway might play a role in treatment during periods of acute demyelination, when there is a lack of newly minted myelin-producing oligodendrocytes. The study below does refer to Wnt inhibitors in the pipeline as potential cancer therapies.  It looks to me that safe Wnt inhibitors like the cheap drugs widely used to treat children with parasites (Mebendazole/ Niclosamide) could be repurposed to treat the acute phase of multiple sclerosis.

Mebendazole/ Niclosamide are safe and dirt cheap, whereas the (slightly) disease changing MS drugs currently cost $50,000+ a year.

TCF4 links everything together

Wnt signalling needs to be active to block premyelinated oligodendrocytes into transforming into oligodendrocytes (OL). So by inhibiting Wnt signalling you may remove one of the problems in MS; you probably only need to do this during relapses of MS.  

There actually is a finally stage to getting the oligodendrocytes (OL) to myelinate many axons and not be lazy.

In the jargon “dysregulation of Wnt–β-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination”.

A miss-expression of TCF4 is clearly also going to affect myelination and its does in both Pitt Hopkins and MS.

One feature of Pitt Hopkins (caused by haploinsufficiency of the transcription factor 4) is indeed delayed myelination measured via MRI at the age of 1. By the age of 9 white matter (the myelin-coated part of your brain) appears normal. This fits with what I highlighted in red under figure 6 above.

Nothing is simple. Activating Wnt signalling is known to increase expression of TCF4.  

Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS

The progressive loss of CNS myelin in patients with multiple sclerosis (MS) has been proposed to result from the combined effects of damage to oligodendrocytes and failure of remyelination. A common feature of demyelinated lesions is the presence of oligodendrocyte precursors (OLPs) blocked at a premyelinating stage. However, the mechanistic basis for inhibition of myelin repair is incompletely understood. To identify novel regulators of OLP differentiation, potentially dysregulated during repair, we performed a genome-wide screen of 1040 transcription factor-encoding genes expressed in remyelinating rodent lesions. We report that ∼50 transcription factor-encoding genes show dynamic expression during repair and that expression of the Wnt pathway mediator Tcf4 (aka Tcf7l2) within OLPs is specific to lesioned—but not normal—adult white matter. We report that β-catenin signaling is active during oligodendrocyte development and remyelination in vivo. Moreover, we observed similar regulation of Tcf4 in the developing human CNS and lesions of MS. Data mining revealed elevated levels of Wnt pathway mRNA transcripts and proteins within MS lesions, indicating activation of the pathway in this pathological context. We show that dysregulation of Wnt–β-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination, based on (1) conditional activation of β-catenin in the oligodendrocyte lineage in vivo and (2) findings from APC^Min mice, which lack one functional copy of the endogenous Wnt pathway inhibitor APC. Together, our findings indicate that dysregulated Wnt–β-catenin signaling inhibits myelination/remyelination in the mammalian CNS. Evidence of Wnt pathway activity in human MS lesions suggests that its dysregulation might contribute to inefficient myelin repair in human neurological disorders 

Potential Tcf4-catenin activities in oligodendrocyte development

The pattern of Tcf4 protein expression, from P1 to P30 and during remyelination after injury, defines the window of potential canonical Wnt pathway functions. Within this context, we observed that Tcf4 expression marked ∼15%–20% of OLPs at any given stage assessed. These findings were consistent with two possibilities. First, Tcf4 expression could demarcate a subset of OLPs. Second, it was possible that Tcf4 expression transiently marks all (or the vast majority) of OLPs during development. Our functional evidence strongly supports the latter conclusion, based on the fact that activity of activated β-catenin is Tcf-dependent (van de Wetering et al. 2002), coupled with the robust phenotype in DA-Cat and APC^Min animals, in which we observe pervasive effects of Wnt pathway dysregulation on myelin production throughout the CNS. Interestingly, although Tcf4 proteins are coexpressed with nuclear Olig1 proteins, Tcf4 segregated from cells expressing Olig1 mRNA transcripts, consistent with the possibility that Tcf4 is expressed at a transition stage when nuclear Olig1 proteins become down-regulated during remyelination.

Previous work has suggested inhibitory functions of Tcf4 on myelin basic protein gene expression in vitro (He et al. 2007), and our studies indicate that Tcf4 interactions with β-catenin inhibit myelination in vivo. Additional studies are warranted to rule out possible β-catenin-independent roles for Tcf4 in oligodendrocyte development. Although Wnt pathway activation has conventionally been thought of as activating gene targets, recent work has identified novel Tcf–β-catenin DNA regulatory binding sites that repress targets (Blauwwkamp et al. 2008). In this regard, one intriguing candidate target is HYCCIN (DRCTNNB1A), a Wnt-repressed target (Kawasoe et al. 2000) with essential roles in human myelination (Zara et al. 2006), which is expressed in rodent oligodendrocytes and down-regulated in Olig2cre/DA-Cat mice (Supplemental Fig. 8). Further studies are needed to better understand Tcf4–catenin function and its direct gene targets during oligodendrocyte lineage progression.

Wnt pathway dysregulation in OLPs as a mechanism leading to chronic demyelination in human white matter diseases

Therapeutic opportunities might arise from an enhanced understanding of the process regulating normal kinetics of remyelination. How might the negative regulatory role of the canonical Wnt pathway help to explain the pathology of demyelinating disease? Delayed remyelination due to Wnt pathway dysregulation in OLPs could lead to chronic demyelination by OLPs then missing a “critical window” for differentiation (Miller and Mi 2007; Franklin and Ffrench-Constant 2008). This “dysregulation model” of remyelination failure requires the Wnt pathway to be active during acute demyelination, as suggested by data from our animal systems and human MS tissue.

Canonical WNT signaling has been implicated in a variety of human diseases (Nelson and Nusse 2004), and gain-of-function mutations in β-catenin are etiologic in several cancers including the majority of colon adenocarcinomas. Approaches for treating Wnt-dependent cancers by promoting differentiation (and hence cell cycle arrest or apoptosis) using pharmacological inhibitors of the pathway are under development (Barker and Clevers 2005). It is possible that such antagonists might play a role in the therapeutic enhancement of remyelination by normalizing the kinetics of myelin repair. If so, the animal models described here (e.g., APC^+/−) should be useful in preclinical testing. However, it is important to note that while dysregulation of a pathway might delay remyelination, it is overly simplistic to expect that inhibition of the same pathway would accelerate repair in the complex milieu of an MS lesion in which several inhibitory pathways might be active, compounded by the presence of myelin debris (Kotter et al. 2006). Indeed, because of the need to synergize with other processes (e.g., those associated with inflammation), accelerated differentiation might negatively affect repair (Franklin and Ffrench-Constant 2008). Further work is needed to comprehensively understand interactions of regulatory networks required for optimal remyelination and how these may be dysregulated in human demyelinating diseases.

Neurologic and ocular phenotype in Pitt-Hopkins syndrome and a zebrafish model.

Abstract

In this study, we performed an in-depth analysis of the neurologic and ophthalmologic phenotype in a patient with Pitt-Hopkins syndrome (PTHS), a disorder characterized by severe mental and motor retardation, carrying a uniallelic TCF4 deletion, and studied a zebrafish model. The PTHS-patient was characterized by high-resolution magnetic resonance imaging (MRI) with diffusion tensor imaging to analyze the brain structurally, spectral-domain optical coherence tomography to visualize the retinal layers, and electroretinography to evaluate retinal function. A zebrafish model was generated by knockdown of tcf4-function by injection of morpholino antisense oligos into zebrafish embryos and the morphant phenotype was characterized for expression of neural differentiation genes neurog1, ascl1b, pax6a, zic1, atoh1a, atoh2b. Data from PTHS-patient and zebrafish morphants were compared. While a cerebral MRI-scan showed markedly delayed myelination and ventriculomegaly in the 1-year-old PTHS-patient, no structural cerebral anomalies including no white matter tract alterations were detected at 9 years of age. Structural ocular examinations showed highly myopic eyes and an increase in ocular length, while retinal layers were normal. Knockdown of tcf4-function in zebrafish embryos resulted in a developmental delay or defects in terminal differentiation of brain and eyes, small eyes with a relative increase in ocular length and an enlargement of the hindbrain ventricle. In summary, tcf4-knockdown in zebrafish embryos does not seem to affect early neural patterning and regionalization of the forebrain, but may be involved in later aspects of neurogenesis and differentiation. We provide evidence for a role of TCF4/E2-2 in ocular growth control in PTHS-patients and the zebrafish model. 


Conclusion  

If you have a myelinating disease, you might want to read up on TCF4 and Wnt signalling. Probably not what the Minions take to read on the beach in the Maldives.

We also should recall the importance of what I am calling the "what, when and where" in neurological disorders. This is important for late onset disorders like schizophrenia, since the symptoms often develops in late teenage years and so it is potentially preventable, if identified early enough.

Today we see that TCF4 is expressed in white matter only in early childhood. If you knew what changes take place in the brains of children who go on to develop schizophrenia, you might well be able to prevent its onset.

Preventing some autism is already possible, as has been shown in mouse models, but in humans it is more complicated because of the "when" and quite literally the "where". There will be a post showing how the brain overgrowth typical of autism can be prevented using bumetanide, before it occurs, at least in mice.


  

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.

Histone deacetylase inhibition rescues structural and functional brain deficits in a mouse model of Kabuki syndrome

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.

A ketogenic diet rescues hippocampal memory defects in a mouse model of 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 (29⇓–31). 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).                   

Autism spectrum disorder in Kabuki syndrome: clinical, diagnostic and rehabilitative aspects assessed through the presentation of three cases.

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 promotes the expression of brain derived neurotrophic factor (BDNF) through the action of the ketone body β-hydroxybutyrate.

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.

Ketone beta-hydroxybutyrate up-regulates BDNF expression through NF-κB as an adaptive response against ROS, which may improve neuronal bioenergetics and enhance neuroprotection

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:

Ketones and Autism Part3 - Niacin Receptor HCA2/GPR109A in Autism, Colonic Inflammation, Psoriasis and Multiple Sclerosis

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. 

Ketogenic diet improves the spatial memory impairment caused by exposure to hypobaric hypoxia through increased acetylation of histones in rats

 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   

Suppression of Oxidative Stress by β-Hydroxybutyrate, an Endogenous Histone DeacetylaseInhibitor

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. 

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

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

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.