Cerebral Folate Deficiency – increasing cerebral folate without increasing plasma/blood folate, via activating the reduced folate carrier (RFC)

 

Source: https://autism.fratnow.com/blog/folate-transport-systems-i-transmembrane-carriers/

Two readers of this blog have been telling me about the fundamental role of brain energy and metabolism in autism. Marco sent me a book called Brain Energy by a psychiatrist at the Harvard Medical School. He stumbled upon this subject when he encouraged a patient to lose weight using the ketogenic diet. As well as losing weight, the patient’s decades-long psychiatric disorders seemed to vanish. The author, Dr Palmer, now believes that many of his patients actually have metabolic disorders as the underlying basis of their psychiatric symptoms. 

Our reader Natasa is approaching with a similar idea, essentially that autism features a brain running on empty.

Today’s post is about increasing the level of folate within the brain, by targeting similar metabolic pathways to those that will boost “brain energy.”

Low levels of folate within the brain will cause varying degrees of neurological disorder.

There are three ways folate can cross into the brain.

1.     Folate receptor alpha (FRA)

2.     Proton-coupled folate transporter (PCFT)

3.     Reduced folate carrier (RFC)

Autoantibodies to the FRA have been linked to neurodevelopmental diseases, particularly cerebral folate deficiency, schizophrenia and autism. Recent studies have shown that these neurodevelopmental disorders can be treated with folinic acid (leucovorin).

Dr Frye, Professor Ramaekers and others are targeting the problem of low folate in the brain by supercharging the level of folate in the bloodstream and hoping more squeezes through the blood brain barrier.

In my previous post I mentioned that Agnieszka has pointed out the idea of using the supplement PQQ. This targets the third transport mechanism above, it is aiming to get more folate across via  the Reduced Folate Carrier (RFC).

Somebody recently wrote their PhD thesis on exactly this topic:- 

Regulation of Folate Transport at the Blood-Brain Barrier: A Novel Strategy for the Treatment of Childhood Neurological Disorders Associated with Cerebral Folate Deficiency

Camille Alam, Department of Pharmaceutical Sciences, University of Toronto 

Additionally, we provided in vitro and in vivo evidence that RFC expression and transport activity is inducible by another transcription factor, NRF-1. These findings demonstrate that augmenting RFC functional expression through interaction with specific transcription factors could constitute a novel strategy for enhancing brain folate delivery. Modulating folate uptake at the BBB may have clinical significance due to the lack of established optimal therapy for neurometabolic disorders caused by loss of FRα or PCFT function. 

What Camille is saying is that if folate transport mechanism number 1 and/or number 2 are not working, we can reinvigorate mechanism number 3.

So if you have Dr Frye’s folate receptor antibodies, or PCFT isn’t working then you might focus on Reduced Folate Carrier (RFC).

The good news is that we have lots of ways to target Reduced Folate Carrier (RFC).

We do not, it seems, have any clever ways to target PCFT. 

NRF-1 and PGC1-alpha

There is a lot in this blog about PGC1-alpha, because it is the master regulator for biogenesis of mitochondria.

All those people with impaired “brain energy” would love to activate PGC1-alpha.

NRF-1 is an activator of mitochondrial respiratory chain genes. NRF-1 specifically targets genes encoding subunits of the mitochondrial respiratory chain complexes, particularly complexes I, III, and IV. By binding to their promoters, NRF-1 directly stimulates their transcription, leading to increased synthesis of these critical protein components and enhanced oxidative phosphorylation (OXPHOS) capacity.

Synergy between NRF-1 and PGC-1alpha

PGC-1alpha acts as the upstream regulator. Various stimuli, such as exercise, cold exposure, and certain hormones, can trigger PGC-1alpha expression. Once activated, PGC-1alpha directly interacts with and co-activates NRF-1, enhancing its binding to target gene promoters and amplifying its transcriptional activity.

NRF-1 as the downstream effector.  NRF-1 fine-tunes the expression of specific mitochondrial genes, ensuring a balanced and efficient OXPHOS system. This synergy between PGC-1alpha and NRF-1 optimizes mitochondrial function and cellular energy production.

So for Natasa, trying to boost energy production in the brain and in the rest of the body, it would be ideal to have more NRF-1 and more PGC-1alpha

What has optimized mitochondrial function got to do with more folate in the brain?

It turns out that you can increase expression of Reduced Folate Carrier (RFC) via activating NRF-1 and/or PGC1alpha.

So what is good for your brain energy is likely to also be good for your brain folate.

Nuclear respiratory factor 1 (NRF-1) upregulates the expression and function of reduced folate carrier (RFC) at the blood-brain barrier

Folates are important for neurodevelopment and cognitive function. Folate transport across biological membranes is mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate transport primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems results in suboptimal folate levels in the cerebrospinal fluid (CSF) causing childhood neurological disorders. Our group has reported that upregulation of RFC at the blood-brain barrier (BBB) through interactions with specific transcription factors, that is, vitamin D receptor (VDR) could increase brain folate delivery. This study investigates the role of nuclear respiratory factor 1 (NRF-1) in the regulation of RFC at the BBB. Activation of NRF-1/PGC-1α signaling through treatment with its specific ligand, pyrroloquinoline quinone (PQQ), significantly induced RFC expression and transport activity in hCMEC/D3 cells. In contrast, transfection with NRF-1 or PGC-1α targeting siRNA downregulated RFC functional expression in the same cell system. Applying chromatin immunoprecipitation (ChIP) assay, we further demonstrated that PQQ treatment increased NRF-1 binding to putative NRF-1 binding sites within the SLC19A1 promoter, which encodes for RFC. Additionally, in vivo treatment of wild type mice with PQQ-induced RFC expression in isolated mouse brain capillaries. Together, these findings demonstrate that NRF-1/PGC-1α activation by PQQ upregulates RFC functional expression at the BBB and could potentially enhance brain folate uptake.

The hugely simple intervention mentioned above is to just take vitamin D. This has nothing to do with brain energy.

Upregulation of reduced folate carrier by vitamin D enhances brain folate uptake in mice lacking folate receptor alpha

Folates are critical for brain development and function. Abnormalities in brain folate transport have been implicated in a number of childhood neurodevelopmental disorders, including cerebral folate deficiency syndrome, hereditary folate malabsorption, and autism spectrum disorders. These disorders have devastating effects in young children, and current therapeutic approaches are not sufficiently effective. In this study, we demonstrate that functional expression of the folate transporter, reduced folate carrier, at the blood–brain barrier and its upregulation by the vitamin D nuclear receptor can remarkably increase folate transport to the brain. These findings provide a strategy for enhancing brain folate delivery for the treatment of neurometabolic disorders caused by folate transport defects.

 Low vitamin D correlates with poor health, dementia, and death from all causes

Taking vitamin D has become popular in recent years.

A correlation does not guarantee causality.  It was thought that vitamin D might be the silver bullet to improved health in older people. It has not proved to be.

Low vitamin D also correlates with less time outdoors, doing some physical activity. Taking vitamin D does not mean you will live longer, but we know for sure that exercise improves many medical concerns that will improve healthy life expectancy.

The concern many people now have regarding skin cancer leads to some healthy active people having low vitamin D. Put on that sunscreen and your exposed skin will not be able to produce your vitamin D.

Vitamin D is important to health and is easy to maintain in the normal range, but it is just one element of good health. It might be one way to increase folate in the brain, for those who need it. 

 

Conclusion

How do you increase folate in the brain?

The obvious way is to put more folate in your blood, this is the standard therapy. You either take calcium folinate tablets or, very rarely, the more potent infusions.

If you have antibodies blocking transport via FRA, you could follow the hypothesis that these antibodies are from a reaction to cow’s milk and try going dairy-free. There is a complex relationship between milk and folate receptor alpha antibodies (FRAA), but direct evidence of milk causing FRAA production is limited.

Milk, particularly cow's milk, contains proteins similar to folate receptor alpha found in humans. Some individuals, mainly those with a genetic predisposition, could develop FRAA that cross-react with these milk proteins. This cross-reactivity would not necessarily mean the milk directly caused FRAA production but might trigger an existing immune response. Some studies, though not all, have found an association between higher milk consumption and increased FRAA levels.

If you want to increase folate transport via our third mechanism, Reduced Folate Carrier (RFC) you have many options:

The obvious first step is to take a vitamin D supplement to raise levels to the high end of normal. This can be done by taking a larger supplement just once a week, because vitamin D has a long half-life.

As you can see from the study below in children there is a correlation between low vitamin D and low folate in children.

 

Evaluation of correlation between vitamin D with vitamin B₁₂ and folate in children

The present study reported a positive correlation between vitamin D and vitamin B12 and folate levels. Regular measurement of these two micronutrient levels in children with vitamin D deficiency is important for public health.

Vitamin D is low in much of the population, even more so in wintertime. It seems particularly low in children with autism, perhaps because they are spending less time playing outside than other children.

Activate NRF-1 and/or PGC1alpha:

1.     Exercise, particularly endurance training

2.     PQQ supplement

3.     Perhaps resveratrol/pterostilbene

4.     Butyric acid / sodium butyrate

5.     The very safe old drug Metformin

6.     Other type 2 diabetes drugs like Pioglitazone

Metformin has been shown to raise IQ in Fragile-X by about 10 points and has a range of metabolic benefits and even cancer preventative effects. This common diabetes medication primarily targets AMPK, an energy sensor molecule upstream of PGC-1alpha. By activating AMPK, metformin indirectly stimulates PGC-1alpha and subsequently NRF1, leading to enhanced mitochondrial function.

Pioglitazone has been researched in autism and is my choice for peak risk spring/summer aggression and self-injury. Pioglitazone can potentially upregulate PGC-1alpha expression through several pathways:

•                    Pioglitazone activates AMPK, an important energy sensor molecule. AMPK can then stimulate PGC-1alpha expression through various signaling pathways.

•                    Pioglitazone activates PPAR-gamma and PPAR-gamma directly interacts with PGC-1alpha, potentially increasing its activity.

I think Metformin has a better safety profile than Pioglitazone and so better for every day use.

Butyric acid does have the potential to activate PGC-1alpha. Butyric acid is produced in the gut by fermentation. You need “good” bacteria and fiber. People with healthy diet naturally produce it. You can also buy it as a supplement (sodium butyrate) since it has numerous benefits – everything from gut health, bone health to a tight blood brain barrier.

According to a doctor I was talking to recently, nobody wants to hear that exercise is a key part of health. It is free and the side effects are generally all good ones. Endurance exercise will boost NRF1 and PGC1alpha. Many people with autism are overweight, often due to the psychiatric drugs they have been put on.

Sirtuin activators boost NRF1 and PGC1 alpha. There are drugs and foods which can do this, but a potent way is through exercise.

I hope Dr Frye is checking his patients’ vitamin D levels and supplementing to the safe upper limit.

Those taking I/V calcium folinate might want to look at the more potent ways to activate NRF1 and/or PGC1alpha.

 

Mutations in CACNA2D1 plus KDM6B -- Gabapentin and Calcium Folinate? Perhaps PQQ? Perhaps BHB?

 

A little research can sometimes be eye opening

I was recently sent genetic results from several parents and surprisingly some have multiple potentially highly causal genes. Some are mutations that are extremely rare and one was unique.

Today I am looking at one case with two genes highlighted in whole exome sequencing (WES), one is a calcium ion channel and the other is a gene extremely close to the one causing Kabuki syndrome.  Interestingly, two possible interventions did very quickly appear.

The report states:

UNCLEAR RESULT

Variants of uncertain significance (VUS) identified

Based on current evidence, the clinical relevance of the detected variants remains unclear.

Kabuki syndrome is caused by mutations in KMT2D or KDM6A.

KDM6A and today’s gene KDM6B both target trimethylation on lysine 27 of histone H3 (H3K27me3), a mark associated with gene silencing. By removing this mark, they activate gene expression. So, mutations in either gene will cause a cascade of effects on numerous other genes.

The old post below suggested the use of HDAC inhibitors to correct the mis expressed genes. In particular, BHB from the ketogenic diet was discussed.

Notably, histone deacetylase inhibition rescued structural and functional brain deficits in a mouse model of Kabuki syndrome.

 

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

           

The calcium channel involved today is not one we have previously looked at, but it is the target of the very well-known drug Gabapentin. This drug is used to treat epilepsy and neuropathic pain. The child does have abnormal EEG and seizures, plus autism, ADHD and absent speech.

Mutations of the KDM6B causing autism were first described only in 2019. In 2022 mutations in this gene were found in several patients with cerebral folate deficiency (CFD), one of the authors is our old friend Dr Ramaekers.

We know a lot about CFD, thanks to our reader Roger, Dr Frye, Dr Ramaekers, and now Agnieszka and Stephen. Over in the US one of the founders of an autism organisation told me her son was diagnosed in adulthood with CFD, when he finally had a spinal tap.

Interestingly, Agnieszka has pointed out a novel way to potentially increase folate in the brain using an OTC supplement called PQQ.

 

Protective effects of pyrroloquinoline quinone in brain folate deficiency

Results

Folate deficiency resulted in increased expression of inflammatory and oxidative stress markers in vitro and in vivo, with increased cellular ROS levels observed in mixed glial cells as well as a reduction of mitochondrial DNA (mtDNA) content observed in FD mixed glial cells. PQQ treatment was able to reverse these changes, while increasing RFC expression through activation of the PGC-1α/NRF-1 signaling pathway.

Conclusion

These results demonstrate the effects of brain folate deficiency, which may contribute to the neurological deficits commonly seen in disorders of CFD. PQQ may represent a novel treatment strategy for disorders associated with CFD, as it can increase folate uptake, while in parallel reversing many abnormalities that arise with brain folate deficiency.

 

PQQ is a relatively common OTC supplement that looks helpful in older people and those with mitochondrial dysfunctions (most older people, plus many with autism).  It can also improve sleep.  The common 20mg dose seems to be based on what was used in a clinical trial in Japanese adults. Japanese drugs are dosed to reflect the size of Japanese people. American women on average weigh 40% more than Japanese women.

PQQ is present in mother’s milk, so it is not some scary artificial compound.

CFD looks like another nexus point where may different genetic variants produce a downstream meeting point.  This means numerous different underlying autisms will share a common beneficial therapy. It will not be a cure, but it should improve the outcome.

The only way to access I/V calcium folinate looks to be via confirmation of very low levels in spinal fluid, so a spinal tap would be necessary.  This is not easy, as Agnieszka has found out.  For some people oral calcium folinate is not sufficiently potent to reverse CFD.

KDM6B

Mutations of the KDM6B gene causing autism were first described only in 2019. In 2022 mutations in this gene were found in several patients with cerebral folate deficiency (CFD).

 

Genetic variants in the KDM6B gene are associated with neurodevelopmental delays and dysmorphic features

Lysine-specific demethylase 6B KDM6B demethylates trimethylated lysine-27 on histone H3. The methylation and demethylation of histone proteins affects gene expression during development. Pathogenic alterations in histone lysine methylation and demethylation genes have been associated with multiple neurodevelopmental disorders. We have identified a number of de novo alterations in the KDM6B gene via whole exome sequencing (WES) in a cohort of 12 unrelated patients with developmental delay, intellectual disability, dysmorphic facial features, and other clinical findings. Our findings will allow for further investigation in to the role of the KDM6B gene in human neurodevelopmental disorders.

 

Layman’s guide to the KDM6B gene

https://www.simonssearchlight.org/research/what-we-study/kdm6b/

 

12% of people with CFD studied in the paper below had mutations in KDM6B. So clearly all people with a mutation in this gene should be tested for CFD vis a spinal tap.

 

KDM6B Variants May Contribute to the Pathophysiology of Human Cerebral Folate Deficiency

Cerebral folate deficiency syndrome (CFD) was defined as any neurological condition that was associated with low concentrations of 5-methyltetrahydrofolate in the cerebrospinal fluid. Previous clinical studies have suggested that mutations in the folate receptor alpha FOLR1 gene contribute to CFD. In this study, we identified six genetic variants in histone lysine demethylase 6B (KDM6B) in 48 CFD cases. We demonstrated that these KDM6B variants decreased FOLR1 protein expression by manipulating epigenetic markers regulating chromatin organization and gene expression. In addition, FOLR1 autoantibodies were identified in CFD patients’ serum. To the best of our knowledge, this is the first study to report that KDM6B may be a novel CFD candidate gene in humans.

The way to confirm CFD, with certainty, is via a spinal tap.  This can then open the door to intravenous therapy with calcium folinate.

There is a blood test which then would lead to oral calcium folinate therapy.  This is now very common in children with autism in the US. It improves speech.

www.fratnow.com

The problem is that some people need the more potent intravenous therapy and without a spinal tap there is not enough proof to get the therapy.

 

CACNA2D1

The CACNA2D1 gene encodes voltage-dependent calcium channel subunit alpha-2/delta-1. 

Different types of mutation will have different effects and varying degrees of severity.

Some mutations in this gene are associated with a condition called “Developmental and Epileptic Encephalopathy 110”.

Developmental and epileptic encephalopathy-110 (DEE110) is an autosomal recessive disorder characterized by profound global developmental delay and hypotonia apparent in infancy followed by onset of seizures in the first months or years of life. Affected individuals achieve almost no developmental milestones and show impaired intellectual development, poor or absent speech, inability to walk or grasp objects, peripheral spasticity, and poor eye contact. Brain imaging shows hypoplastic corpus callosum and cortical atrophy.

CACNA2D1 is also a novel Brugada Syndrome susceptibility gene.

Brugada syndrome may be a major cause of sudden cardiac death in men under 40. People with Brugada syndrome on average die between the ages of 26 to 56 years, with an average age of 40 years. If treated appropriately, patients can have a normal lifespan.

A pediatric cardiologist should be consulted.

Fortunately the Alpha-2/delta proteins are believed to be the molecular target of the gabapentinoids gabapentin and pregabalin, which are used to treat epilepsy and neuropathic pain.

This means that an obvious path to investigate is whether the drug gabapentin has a positive effect. Mutations could produce either gain of function of loss of function.

Gabapentin binds to a the α2δ subunit. This binding does not directly block or open the channel, but it influences its overall activity.

The exact mechanism of action is still not fully understood, but it is believed that gabapentin:

·       Reduces the release of certain neurotransmitters involved in pain signaling, such as glutamate and substance P.

·       Alters the trafficking and function of the calcium channels themselves.

·       Therefore, gabapentin's action is more complex than simply "blocking" or "opening" channels. 

Gabapentin is not guaranteed to help in this case, but certainly might do.

Conclusion

The take home is really that if you invest thousands of dollars/euros/pounds in genetic testing, it is well worth your time spending some time on the internet looking up any flagged genes.

People expect too much from the geneticist writing the report.

Double check these things yourself.  Take your findings to an open-minded neurologist, who reads the research literature.

Be aware that the same mutation can be present in one or even both parents, with no noticeable negative effect, but be disease causing in their child. Genetics is often about the probability of something happening, rather the certainty. 

Look at partially-effective or sometimes-effective interventions in the research. For example, one reader is looking at mutations in NF1 plus a gene affecting epigenetics. He might want to try Lovastatin.  NF1 causes an increase in RAS, which is a pro-growth signal, this leads to RASopathies which can cause intellectual disability (ID). Lovastatin reduces RAS and it was trialled to reduce ID in NF1 - the results were mixed. It probably matters at what age you start trying to reduce RAS.