Betaine (also known as TMG, or trimethylglycine) is a methyl derivative of glycine, first isolated from sugar beet and hence its name.
Today’s post
was prompted by our reader, and Covid home-school instructor, AJ. He raised the question of whether betaine can
be used like Bumetanide to normalize chloride levels in neurons.
I am combing
this idea with news from Genoa in Italy, where they have developed gene therapy
as an alternative to Bumetanide and in their words :-
“This sets the stage for the development of a gene therapy
approach to overcome the shortcomings of bumetanide treatment.”
The interesting thing is that
neither of these ideas come from autism research. The idea to use Betaine was stumbled upon and
was then written up in a Norwegian case study about Creatine transporter deficiency. The Italians are trying to improve cognition
in brain disorders and their model of choice was Down syndrome.
As we have seen time and again,
elevated chloride within neurons is a common feature of many types of brain
disorders from some idiopathic autism, to Down syndrome, to adult conditions
such as Parkinson’s disease. Today we
learn that it is may well be a feature of Creatine Transporter Deficiency.
I have been rather wary of
writing about any kind of gene therapy, because it seemed either too far ahead
of its time, or just absurdly expensive.
There are some new $1+ million treatments.
This may be about to change
given that the Biontech (AKA Pfizer vaccine), Moderna, Janssen (Johnson &
Johnson) and Oxford AstraZeneca vaccines for Covid 19 are all based on gene
therapy.
The Biontech people are really
clever and were already trying to treat various kinds of cancer and other
condition using gene therapy, before they developed their highly successful Covid
vaccine.
The Italians in Genoa used an
adeno-associated virus (AAV)-mediated RNA interference (RNAi) to target and
reduce neuronal NKCC1 expression, rescue neuronal Cl- homeostasis, GABAergic transmission, and
cognitive deficits. The benefit was
still there 6 months after the injection.
Don’t worry if the above
paragraph makes little sense. Just read on.
The same
type of adeno-associated virus (AAV)
vector is the platform for gene therapy delivery used in the Astra Zeneca,
Janssen and the Russian Sputnik covid vaccines.
The virus is just the delivery
system (vector) to get some genetic code into cells.
The Oxford-AstraZeneca COVID-19 vaccine uses a
chimpanzee adenoviral vector. It delivers the gene that encodes the SARS-CoV-2
spike protein, to our cells. Our cells
then transcribe this gene into messenger RNA, or mRNA, which in turn prompts
our cellular machine to make the spike protein in the main body of the cell. The
mRNA molecule behaves essentially like a recipe. Then our cells present the spike protein on
the cell surface, prompting our immune system to make antibodies and
mount T cell responses.
Biontech and Moderna are pioneers
of mRNA vaccines, which bypass one step in the above process. They do not
require our cells to make the messenger
RNA, or mRNA. They have already made it
for you.
Gene therapy for autism?
Single gene autisms are all
potential candidates for gene therapy.
The problem is that most autism
and all Down syndrome is polygenic, there can be hundreds of miss-expressed
genes.
But the researchers in Italy show
us that even polygenic autism and Down syndrome can benefit from therapy targeting
a single gene. You just have to select
the right one.
The problem is the price. Covid
vaccines are made in huge quantities and are cheap.
Customized gene therapy is ultra
expensive, in part because each therapy has to be approved individually.
An NKCC1 Gene Therapy?
The Italians have already made
the NKCC1 Gene Therapy. The question is will
it ever going be available to humans with Down Syndrome, Autism or even
Parkinson’s disease?
A common feature of
diverse brain disorders, is the alteration of GABA-mediated inhibition due to
aberrant intracellular chloride homeostasis induced by changes in the
expression and/or function of chloride transporters. Notably, pharmacological
inhibition of the chloride importer NKCC1 is able to rescue brain-related core
deficits in animal models of these pathologies and some human clinical studies.
Here, we show that
reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down
syndrome (DS) restores intracellular chloride concentration, efficacy of
GABA-mediated inhibition and neuronal network dynamics in vitro and ex vivo.
Importantly, AAV-mediated neuron-specific NKCC1 knockdown in vivo rescues
cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a
mechanistic link between NKCC1 expression and behavioral abnormalities in DS
mice, and establish a molecular target for new therapeutic approaches,
including gene therapy, to treat brain disorders characterized by neuronal
chloride imbalance.
This sets the stage for the development of
a gene therapy approach to overcome the shortcomings of bumetanide treatment.
This
highlights a causative role of NKCC1 upregulation in learning and memory
deficits in adult Ts65Dn mice, thus also validating brain NKCC1 as a target for
ameliorating cognitive disabilities in DS. Furthermore, our neuro-specific
knockdown approach points to neurons as major players in the NKCC1- dependent
cognitive impairment in DS mice. Nevertheless, we cannot exclude that other
cell types which also express NKCC1 (e.g. glial cells) could still play a role
in the overall cognitive impairment that characterizes DS.
Despite
the very large and fast-increasing literature both on animal models and patients
indicating positive outcomes upon bumetanide treatment, there is not yet a
strong demonstrated direct link between NKCC1 inhibition, restoration of Cl-
homeostasis and full GABAergic inhibitory signaling, and rescue of brain
deficits. Moreover, bumetanide has strong
diuretic activity, triggering ionic imbalance, and potential ototoxicity
25,26. This hampers its use for clinical
applications in lifelong treatments4,27 and may strongly jeopardize treatment compliance
along years of treatment. Moreover, bumetanide
was given systemically in most studies, and the suboptimal brain pharmacokinetic
profile of the drug28 raises questions on its mechanism of action29. Here, we demonstrate that adeno-associated virus (AAV)-mediated RNA
interference (RNAi) to target (and reduce) neuronal NKCC1 expression rescues neuronal
Cl- homeostasis, GABAergic transmission, and cognitive deficits in the Ts65Dn
mouse model of Down syndrome. This sets the stage for the development of a gene therapy approach to overcome
the shortcomings of bumetanide treatment.
“Thus, our results indicate the efficacy
of long-term AAV9-mediated neuro-specific NKCC1 knockdown in rescuing cognitive
deficits in Ts65Dn mice.”
“Besides establishing a causal
link between NKCC1 upregulation and cognitive impairment in DS, our data also provide
a proof-of-concept for a neuro-specific RNAi gene therapy approach to restore
hippocampus-dependent cognitive behaviors in adult animals specifically in the
brain, and without affecting peripheral organs (e.g., the kidney). This is
particularly relevant in the context of the current clinical trials repurposing
the strong diuretic bumetanide to treat brain disorders with impaired chloride
homeostasis3. Importantly, we achieved a comparable degree of
long-term cognitive rescue with two different amiR sequences against NKCC1,
underlining the specificity of our approach.”
Gone Fishing
If a trip to Italy for gene
therapy is not realistic, this takes us back to AJ’s idea, which is to use Betaine. The correct version is TMG or glycine betaine, and confusingly not Betaine HCl.
Fish love the taste of betaine.
Betaine was
first isolated from sugar beet.
I recall from my time at the sugar
factory, when I was 18, that once you have sliced up the sugar beet and
extracted as much sugar as possible you are left with the pulp. This pulp is dried, molasses is added back
and then it is made into pellets. The
pellets are fed to cattle and horses.
They taste pretty bad in my opinion.
To humans it tastes bad because of the
beet molasses by-product.
The molasses by-product from sugar
cane tastes great to humans. That is why
they make rum in the Caribbean, and not in England or Canada.
Brown sugar from a sugar beet factory
is made by adding sugar cane molasses to white sugar from beet. It is a cheat really.
Cows love sugar beet by-products.
It
turns out that fish love betaine HCl.
Betaine HCl is an excellent natural attractor
that stimulates a strong, prolonged feeding response from carp and many other
coarse fish.
Betaine HCl is now used to induce feeding in the
fish farming industry
As our reader Tyler has highlighted, Betaine HCl, that fish like and is available is a cheap supplement is not the same as the Betaine used in the medical case study. Confusingly, the original Betaine (TMG, or called glycine betaine) gave way to a class of compounds all called betaines. One of these betaines is betaine HCL.
In most cases, in the medical literature when they refer to Betaine, they mean glycine betaine, also known as TMG.
Betaine HCl is used to increase acidity in your stomach. The effect of betaine compounds other than glycine betaine/TMG on NKCC1 is unknown.
Glycine Betaine (TMG) and NKCC1
It seems that betaine reduces
your level of NKCC1 RNA.
In your DNA are the instructions
to make the NKCC1 transporter. To go from these instructions to actually making
the transporters you need RNA.
In some autism there are too many
NKCC1 transporters, so put simply there was too much NKCC1 RNA. So, if you can
find a substance that reduces NKCC1 RNA, you might well solve the problem.
The caveat is that the substance
must not also increase KCC2 RNA. This
appears to be what taurine does.
Here, finally, is AJ’s paper:
Background
Creatine transporter
deficiency (CTD) is an X-linked form of intellectual disability (ID) caused by
SCL6A8 mutations. Limited information exists on the adult course of CTD, and
there are no treatment studies in adults.
Methods
We report two half-brothers with CTD, 36 and 31 years at
intervention start. Their clinical phenotypes were consistent with CTD, and
intervention was indicated because of progressive disease course, with
increased difficulties speaking, walking and eating, resulting in fatigue, and malnutrition. We therefore performed treatment trials with arginine,
glycine and a proprietary product containing creatine and betaine, and then a
trial supplementing with betaine alone. Results In the older patient, glycine
and arginine were accompanied by adverse effects, while betaine containing
proprietary product gave improved balance, speech and feeding. When
supplementation stopped, his condition deteriorated, and improved again after
starting betaine supplement. Betaine supplementation was also beneficial in the
younger patient, reducing his exhaustion, feeding difficulties and weight loss,
making him able to resume his protected work.
Discussion &
conclusion
We report for the first time that betaine supplement was
well tolerated and efficient in adults with CTD, while arginine and/or
glycine were accompanied by side effects. Thus, betaine is potentially a new useful treatment for CTD
patients. We discuss possible underlying treatment mechanisms. Betaine has been reported to
have antagonistic effect on NKCC1 channels, a mechanism shared with bumetanide,
a medication with promising results in both in autism and epilepsy. Further
studies of betaine's effects in well-designed studies are warranted.
The mechanism of betaine’s assumed favorable effect
is unknown. We do not know whether betaine influences the cell creatine content
in itself or its effects are more aspesific. However, we would like to present
some hypotheses. First, betaine may have effect in CTD by modulating
GABA-transmission. Betaine has been reported to have an antagonistic effect on
NKCC1 channels, which also influences GABAergic neurotransmission. Inhibiting
NKCC1 is a mechanism shared with bumetanide, a well-known diuretic medication
that in recent years has been found to influence GABAergic transmission, and
thereby it has been found promising in treatment of several brain conditions,
including autism, and epilepsy. NKCC1 inhibition by bumetanide has also been tried with
success in other rare neurodevelopmental disorders fragile X syndrome and
tuberous sclerosis. Second,
betaine’s properties as an osmolyte may be of importance, as betaine has
similarities with creatine in being an osmolyte. Osmotic properties are thought
to be one of the central mechanism behind bumetanide’s efficacy in treating
brain disorders. Thus, it could be speculated that the lack of
intracellular creatine in CTD may result in inefficient osmolyte regulation,
and that betaine supplementation replaces the lacking creatine and thereby
improves the neuronal adaption to salinity changes, edema or cellular
dehydration. Betaine has osmolyte properties that even makes it act as a
“chemical chaperone” increasing the stability of cell and membrane proteins.
Fourth, it is possible that betaine has some effect through modifying
methylation. Methylation of GAA by GAMT to form creatine is a rate-limiting
step in the creatine synthesis by neurons. Betaine could stimulate this by
donating methyl groups to SAMe, which donates a methyl group to GAA to form
creatine. This might reduce the burden when body demands more methyl groups for
creatine synthesis. Similar mechanisms may be responsible for a beneficial
effect of both betaine and s-adenosyl methionine (SAMe). However, as creatine
and GAA share the same transporter, one would not expect GAA to enter the
GAMTexpressing cells in patients suffering from CTD. Still, it cannot be
excluded that there is some rest function in the creatine transporter, and that
increased endogenous synthesis improves the condition slightly. Furthermore, it
is possible that CTD increases the need for methylation agents in general, as
creatine supplementation has been found to reduce the need for other
methylation agents [34]. Thus, it is likely that betaine may have a positive
effect in CTD by improving methylation capacity for other reactions than those
directly involved in creatine production. Betaine’s effect on muscle may be
also of importance, as animal studies have shown that muscles growth improves
with betaine [35], which potentially could have had a positive impact on our
patients fatigue and weight loss. To summarize, betaine has several properties that make it likely that it
will have a beneficial effect in CTD, especially the properties as an osmolyte,
a down regulator of the NKCC1 channel and an influencer of GABAergic
transmission. These
properties are similar to the properties of bumetanide, a promising new medication for treatment of autism and
epilepsy, which are common symptoms of CTD. Further research is needed, however, to elucidate the role
of betaine in CTD.
If you
read the detail of the old paper that is referred to in the above paper, you
see that betaine is not blocking the NKCC1 channels as suggested, but it seems
to be reducing the number of them. The
net effect may be the same, but the process is very different.
The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in
different liver cell types. NKCC1 was found in rat liver parenchymal and
sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1
expression in rat hepatic stellate cells increased during
culture-induced transformation in the myofibroblast-like phenotype. NKCC1
inhibition by bumetanide increased α1-smooth muscle actin expression
in 2-day-cultured hepatic stellate cells but was without effect on basal and
platelet-derived-growth-factor-induced proliferation of the 14-day-old cells.
In perfused rat liver the NKCC1 made a major contribution to volume-regulatory
K+ uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by
elevation of extracellular NaCl or raffinose concentration but not
hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA
and protein expression. This was antagonized by the
compatible organic osmolytes betaine or taurine. The data suggest a role of
NKCC1 in stellate cell transformation, hepatic
volume regulation, and long-term adaption to dehydrating conditions.
Aha! Glycine Betaine
and Taurine – not so fast
You have to check the effect on
both NKCC1 and KCC2. One lets chloride into
neurons and the lets it out. You want to
block NKCC1 and not KCC2, otherwise you undo all the good you have done.
Both glycine betaine (TMG) and taurine are
already used as autism supplements at low doses. The paper below suggest that Taurine is not a
good idea for people with high levels of chloride within neurons.
GABA inhibits mature neurons and conversely
excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2)
expression. We observed that ectopically expressed KCC2 in embryonic cerebral
cortices was not active; however, KCC2 functioned in newborns. In vitro studies
revealed that taurine increased KCC2 inactivation in a
phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2
were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated,
and the inhibitory effect of taurine was not observed. Exogenous taurine
activated the with-no-lysine protein kinase 1 (WNK1) and downstream
STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response
1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the
absence of taurine. Phosphorylation of SPAK was consistently higher in
embryonic brains compared with that of neonatal brains and down-regulated by a
taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration
was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which
may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to
embryonic neuronal Cl(-) homeostasis, which is required for normal brain
development.
So, it is likely only Glycine Betaine (TMG) may
be of potential benefit, in the case of lowering chloride.
Glycine Betaine
in the broader research
Betaine in
Inflammation: Mechanistic Aspects and Applications
Betaine is known as
trimethylglycine and is widely distributed in animals, plants, and
microorganisms. Betaine is
known to function physiologically as an important osmoprotectant and methyl
group donor. Accumulating evidence has shown that betaine has
anti-inflammatory functions in numerous diseases. Mechanistically, betaine
ameliorates sulfur amino acid metabolism against oxidative stress, inhibits
nuclear factor-κB activity and NLRP3 inflammasome activation, regulates energy
metabolism, and mitigates endoplasmic reticulum stress and apoptosis.
Consequently, betaine has beneficial actions in several human diseases, such as
obesity, diabetes, cancer, and Alzheimer’s disease.
Betaine
is a stable and nontoxic natural substance. Because it looks like a glycine
with three extra methyl groups, betaine is also called trimethylglycine . In addition,
betaine has a zwitterionic quaternary ammonium form [(CH3)3N+ CH2COO−]
(Figure 1). In the nineteenth century, betaine was first identified in
the plant Beta vulgaris. It was then found at high concentrations
in several other organisms, including wheat bran, wheat germ, spinach, beets,
microorganisms, and aquatic invertebrates. Dietary betaine intake plays a
decisive role in the betaine content of the body. Betaine is safe at a daily intake of 9–15 g for human and
distributes primarily to the kidneys, liver, and brain. The accurate
amount of betaine intake generally relies on its various sources and cooking methods.
Besides dietary intake, betaine can be synthesized from choline in the body.
Studies report that high concentrations of betaine in human and animal neonates
indicate the effectiveness of this synthetic mechanism.
Many psychiatric drugs act on the receptors or
transporters of certain neurotransmitters in the brain. However, there is a
great need for alternatives, and research is looking at other targets along the
brain's metabolic pathways. Lack of glycine betaine contributes to brain
pathology in schizophrenia, and new research shows that betaine supplementation
can counteract psychiatric symptoms in mice.
Conclusion
Early on in
the Covid saga, I saw interviews with both the Moderna researchers and the
Oxford (AstraZeneca) researchers. Both claimed that they designed their
vaccines over a weekend. This was made
possible by the Chinese releasing the DNA code of the virus.
When you
think about gene therapy for autism and Down syndrome, the same likely applies;
much could be achieved over a weekend.
The
expensive and time-consuming part is the testing and approval process.
In the Covid
pandemic the approval process was modified to allow for emergency use. Perhaps this should also be the case for all gene
therapies?
What use is
a $2 million therapy for autism or Down syndrome?
In theory,
if you gave your gene therapy prior to birth or shortly thereafter, it might be
fully curative. Realistically, by the
time you get the therapy it is just going to be beneficial and you will still
need other ongoing therapies.
Note that
gene therapy normally applies to just one gene.
In Down syndrome people have a third copy of all, or just part, of
Chromosome 21. This results directly in
the miss-expression of hundreds of genes from that chromosome.
The gene
that encodes NKCC1 is on Chromosome 5, which has nothing directly to do with
Down syndrome.
The NKCC1
transporter is over-expressed in Down syndrome as a down stream consequence of
the disorder. It is caused by the “faulty GABA switch”, referred to in earlier
posts.
The Italian
gene therapy to lower chloride in neurons and so raise cognition, has numerous
applications, in people currently of all ages, so there is a big potential
market.
Why not gene
therapy for all single gene autisms? It
could be a highly productive use of the researcher’s weekends, for a year or
two.
The issue is
who would pay for the $20 to $30 million approval process, for each gene?
Maybe some
of the billions in profit from clever Covid vaccines could be used for pro bono
gene therapy? Highly unlikely.
Biontech,
who are the brains behind the Pfizer vaccine, do have plans to develop gene
therapy for other medical conditions. I
think these will be ultra expensive,
That brings
me back to Glycine Betaine (TMG), is 10g a day of this supplement really going to reduce the expression of NKCC1
transporters in neurons and so lower chloride within neurons? It seems to work in creatine transporter
deficiency, is all we can say.
Glycine betaine, at
much lower doses, has been used by DAN and now MAPS doctors for decades. They
use it as a “methyl-donor”. There is a combination
of real science and hocus-pocus surrounding DNA methylation.
