[Sciences/BBB] About the Thanksgiving tryptophan comatose and the BBB

Happy Thanksgiving everyone, I hope you are enjoying your family gathering. I know many of you are dreading to meet the family and extended family to discuss about controversial topics and differences in opinion.
But the other big menace coming in, that is particularly feared by the Black Friday shoppers: “The Thanksgiving turkey comatose” myth. This myth is perpetuating the idea that the Thanksgiving feast will induce a lethargic state attributed to the tryptophan present in turkey. Lets use this time to talk about tryptophan, turkey and of course the BBB in all that.

1. What is tryptophan?

Tryptophan is one of the 22 amino acids forming the building bricks of each of our proteins. It belongs to one of the few amino acids that our body cannot produce and therefore has to get it from our food supply.
In addition to its role in proteins, tryptophan is also an interesting molecule for the central nervous system, because it serves as a precursor for serotonin (a neurotransmitter also known as 5-hydroxytryptamine) and melatonin (commonly known as the “clock hormone”). You can see the similarities in structure of these molecules below:
Picture1

Tryptophan is particularly enriched in meat. According to the USDA, turkey meat contains the highest level of tryptophan from all foods, followed by white eggs, soybean and seaweeds. This partly support the claim of turkey being rich in tryptophan.

2. How does the tryptophan enters the central nervous system?

Like you expect, the blood-brain barrier is impermeable to any charged molecule. This is the case of many amino acids circulating in the blood (pH=7.4). Thus amino acids can enter the brain only by using special “revolving door” called solute carriers (SLCs). Tryptophan is transported by a particular amino acid transporter called large amino acid transporter 1 (LAT1). LAT1 is a particular transporter because it is formed by two subunits named SLC3A2 (also named CD98) and SLC7A5.
LAT1 is not specific to tryptophan, it also allows the transport of other aromatic amino acids like phenylalanine and tyrosine, but also chained amino acids such as leucine or arginine.

The impact of dysfunction in LAT1 remains poorly understood, however a study by Mykkaenen and colleagues noted several point mutations in SLC7A7 with a rare disease named lysinuric protein intolerance, a rare autosomal disease primarily described in patients from Finnish and Japanese origin marked by the impaired transport and elimination of basic amino acids following a protein-rich diet.

3. What is the function of tryptophan in the brain?

As I have previously mentioned, tryptophan is the precursor of two major neuromediators: serotonin and melatonin.
Serotonin is produced by a certain type of neurons named “serotoninergic neurons”. Like other neurons expressing a particular neurotransmitter other than glutamate or gamma-aminobutyrate (GABA), these neurons are restricted to a certain localization usually referred as “nucleus” (kernel, core). These neurons can project their axons all through the brain via a process called projections, allowing these neurons to interact with far-fetched neurons localized in a remote location.

In the case of serotoninergic (5-HT) neurons, these neurons are located in a structure called “raphe nucleus” and project to areas in which such neurotransmitter interact with 5-HT receptors. Through the interactions with the receptor, serotonin plays an important role in the modulation of several behavior including appetite, emotional (depression, anxiety), cognitive (schizophrenia) motor and autonomous (for instance emesis, the scientific term of “puking“).

In addition to the biological effects on the brain, the serotonin system is also linked to the circadian rhythm system (what we can call the “biological clock”) as depicted in the picture below:

sadserotoninfigure

We are diurnal animals as our main activity occurs during daylight and concludes with our sleep cycle during the dark period. In opposite, some animals like rodents are nychthemeral animals (active during dark phase and sleeping during daylight).

The light/dark cycle phase is determined by our eyes and retina. Such retina will transmit the presence of light to a particular nucleus named “suprachiasmatic nucleus” (SCN) . This nucleus is consisted by cells and nuclei functioning as oscillators. You can think about a pendulum in perpetual movement or a ticking clock. When darkness settles, the retina start to slowdown the information coming to the SCN.
In turn, the SCN becomes less active and relieve the blockade of the activity of the pineal gland. The pineal gland in turn start to secrete melatonin (aka the sleep hormone) that act as a “negative feedback loop” further shutting down the SCN and stimulate the production of serotonin via the raphe nucleus. All these events ultimately giving us the feeling of being sleepy and the process of sleeping.

4. So why we claim the “turkey comatose” is real?

As you can see in this myth, we are facing a post-hoc ergo fallacy. “I feel sleepy after Thanksgiving dinner. I ate large amount of turkey meat at Thanksgiving dinner. Turkey contains tryptophan and sleep is controlled by melatonin (a tryptophan derivative). Thus the tryptophan contained in the turkey meat is responsible of the food comatose”.

As you have seen, this does not make sense as the sleep/wake cycle is driven by the light exposure. This is also explaining partly why some people feel more tired and less motivated during winter times.

One explanation we can discuss is the particular food intake we all face during Thanksgiving that exceed our usual amount of food. We rarely experience such a feast and copious meal during the year. The table is furnished with so different plates, rich in proteins and carbohydrates.
This create a spike in food intake and food digestion that will likely create a urge of blood flow towards the gastrointestinal tract. This physiological phenomenon is named “postpandrial torpor”, making you feel sleepy and tired after a large meal, even if the meal was completely turkey-free.

So in conclusion, if you want to avoid “the turkey comatose”, don’t blame it on the turkey. Blame it on your eyes having a bigger appetite that your stomach can sustain. Keep it in moderation and now you know about the tryptophan transport at the BBB.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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[Sciences/BBB] Acute Necrotizing Encephalitis (of childhood), a blood-brain barrier perspective.

This is a blog post following a request by a page follower on my Facebook account to provide an “layman” perspective on acute necrotizing encephalitis (ANE), also referred as acute necrotizing encephalitis of childhood (ANEC). This is a very short and surely incomplete summary but it should be a great starter to give the current perspective of this condition through the lens of the blood-brain barrier.

It is a condition that was firstly discovered by Mizuguchi and colleagues in 1995 firstly described in infants and toddlers (http://jnnp.bmj.com/content/jnnp/58/5/555.full.pdf). It was firstly described in patients from Asian origin (Japan). It was initially described to occur during the winter period, in particular with region that had experienced an influenza A outbreak. The main clinical feature of the disease marked by the presence in the magnetic resonance imaging (MRI) of increased water content inside the brain, mostly associated with edema (brain swelling). This increased water content can only be explained by the opening of the blood-brain barrier.

Water diffusion between the blood and the brain is tightly regulated by the blood-brain barrier (BBB). The BBB provides two kinds of barrier: a physical barrier (by the presence of tight junctions) and a chemical barrier (by the presence of solute carriers and drug efflux pumps). The case of water as a molecule (H2O) is very interesting. Water is a very small molecule (the molecular weight is 18g/mol or also 18 Daltons) but also a very polarized molecule. Hydrogens and the oxygen atoms forming H2O are not completely neutral, hydrogen carries a tiny positive-charge and oxygen carries two tiny-negative charges (we refer in chemistry as electronegative charges). Think about having a tiny magnet. In the opposite, cell membranes are made of phospholipids. As their name say, they are lipids by definition or what we commonly call them as “fatty acids”. Lipids have a distinct composition, they are mostly formed by carbons and hydrogens. Carbon is not much a magnet atom, it neither likes to carry positive charges nor negative charges. This is why lipids are commonly referred as apolar molecules. Now, polar and apolar molecules behave like water and oil mixed together: they simply do not mix and will sequestrate themselves, usually forming a oil droplet surrounded by water. Water entrance inside the brain is believed to occur mostly via paracellular route, as depicted in the picture below (source: http://www.nature.com/nrn/journal/v7/n1/full/nrn1824.html?foxtrotcallback=true).

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Tight junctions are very tights, letting water fall through the cracks only in a tiny amount. Imagine having a very good rooftop that only let water fall through one drop every hour. The problem with the opening of the BBB following various factor is the massive entrance of water. Think about having a hole in your rooftop and facing a tropical storm shower outside: you are facing now a massive and unregulated entrance of water inside the brain, leading to a brain swelling.
In peripheral tissue, edema (swelling) formation can naturally expand, resulting in a swollen tissue. The problem with the brain is its anatomical structure: it is encased inside a rigid shell (skull) that has no exit route for the penetrating water. This results in an increased pressure inside the brain (we usually referring as increase in intracranial pressure or ICP). This increased pressure induce a mechanical stress, crushing brain cells via mechanical stress and ultimately neuronal cell death. Such swelling appears to occur in specific brain regions, with a primary lesion site in the gray matter (neurons), with persistent deposition of hemosiderin and white matter (axon fibers) cysts during and after the recovery phase. Until now, we don’t exactly know what cause such disease, but appears as following a viral infection including flu (influenza A and B, swine flu (H1N1), parainfluenza virus), varicella, measles, rubella and various herpesviruses (HHV-6, HHV-7) (https://www.hindawi.com/journals/mi/2015/792578/#B1), although the presence of such viral agents (detection by polymerase chain reaction) in spinal tap as well as post-mortem signs of brain inflammation remains anecdotal.

Interestingly, it seems that patients suffering from ANE undergo a very severe immune response commonly referred as “cytokine storm”, as several studies noted an increase in inflammatory markers (in particular interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha (TNF-alpha) making this phenomenon the most prevalent hypothesis.

Immune cells communicate to each other via a common language called “cytokines”. Cytokines are like a “RED ALERT” system, they signal some breach in security or incoming danger.
Brain microvascular endothelial cells (BMECs) lining the blood side of the BBB can also understand the “cytokine” language and understand such signal as “RED ALERT – OPEN THE BBB SIGNAL” as depicted in the picture below (source: https://www.researchgate.net/profile/Nicolas_Weiss/):

Now where are these cytokines coming from and how they are triggered? It is a very good question. This is where the viral infection comes in. I will not details much about the immune response to viruses, but you can ask @TheMadVirologist for any questions related to viruses. For this I will use a figure that resume the immune response to viruses (source: https://www.researchgate.net/profile/Francoise_Stoll-Keller/).

figure-1-function-of-dendritic-cells-in-the-immune-response-to-virusesfollowing-the

Upon infection, infected cells will display viral particles on the cell surface and will also secrete a protein called “interferon-gamma”. This is a sort of cellular “SOS Danger” to the immune system. Natural killer cells, dendritic cells and macrophages may start the early response, also known as “innate immunity” to contain the viral infection. In addition, free circulating viruses can be spotted by B cells through their array of surface antibodies and trigger what we refer to as “acquired immunity”. Viral infection will trigger an immune response and we can think that maybe an overactive immune system may exaggerate the danger resulting into the “cytokine storm”. Another hypothesis is that such cytokine storm maybe triggered by natural killer (NK) cells.

This hypothesis is further supported by the presence of a higher count of natural killer leukocytes in ANE patients during the recovery phase. Natural killer (NK) cells are immune cells normally targeting cancer cells and cells infected by viruses.  This “cytokine storm” maybe the causative agent of the blood-brain barrier disruption (BBB) by different mechanisms (source: http://stroke.ahajournals.org/content/strokeaha/42/11/3323/).

but appears to occurs via an matrix-metalloproteinase (MMPs) dependent pathway. Under the stimulation of such cytokines, brain endothelial cells and astrocytes may increase the production and releases of MMPs locally. These MMPs act as little scissors that can chop the extracellular matrix supporting brain endothelial cells and astrocytes end-feet processes. In addition, these MMPs can also chop tight junction proteins that are involved in tight junction (TJ) complexes. These TJs are very important as they provide the barrier limiting the diffusion of water and solutes between the blood and the brain.

In addition to the cytokine storm hypothesis, it seems that other factors maybe involved in the pathophysiology of the disease. Until now, Ran binding protein 2 (RANBP2) (http://www.cell.com/ajhg/fulltext/S0002-9297(08)00630-7). RANBP2 is a protein involved in the nuclear pore complex, yet the relevance of this mutation at the blood-brain barrier remains unknown. In neurons, it is associated with cellular structures different from the cell nucleus, in particular it is associated with mitochondria (power house of cells) and microtubules.

Another protein of interest associated is EphB2, a receptor for ephrins (https://www.ncbi.nlm.nih.gov/pubmed/?term=ephb2+blood-brain+barrier). Ephrins play an important role in brain wiring during development (axon guidance) but also play a role in the formation of the vascular tree.

The function of EphB2 and ephrins at the blood-brain barrier remains unclear. However, a recent study identified the expression of EphB2 at the cell surface of endothelial cells including primary human non-BBB (HUVECs) and BBB (HBMECs) endothelial cells. Furthermore, a case report from a patient suffering from systemic lupus erythromatous (SLE), an autoimmune disorder, presenting the case of ANE showed the presence of antibodies in the serum capable to bind selectively to EphB2.

Yet, at this point we don’t know if this antibody binding is enough to trigger the BBB disruption or it requires the recruitment of immune cells to trigger such disruption.

 

 

 

 

 

 

 

 

 

[BBB/Junk Sciences] Polysorbate 80 and the BBB or how to put anti-vaxxers into a blowing cognitive dissonance

Here we go again, anti-vaxxers keeping on moving the goalpost to fit their belief instead to change to adjust it to the facts. First it was mercury, then it was formaldehyde, then aluminum, today the “ingredient du jour” is polysorbate 80 and tomorrow they will blame it to PBS saline solution.

The latest fad as I have seen is to blame polysorbate 80 as a source of “vaccine-injury” with the bold claim that it breaks down the blood-brain barrier (BBB). Lets put the fact straight and debunk this one for all. But what is even better is the “what if” counter-argument. What if polysorbate 80 was indeed a good ingredient? I will come to that later.

Polysorbate (aka Tween 80) is a amphiphile compound   as you can see the molecular structure below (source Wikipedia):
1200px-polysorbate_80

You can see the structure made of a lipophilic (loves fat) tail and a series of hydrophilic  (loves water) tails, loaded with oxygen and hydroxyl groups. This is a typical structure of a detergent: one side will mix well with water, the other will mix very well with fat and oils. The result? You can form microspheres that can dissolve well in water and dissolve fat into water. This is how a detergent works, it helps to breakdown fats into small spheres and dissolve them in the drain water.
Polysorbate 80, due to this property, is very good to dissolve drugs and medicines that under normal condition would barely dissolve into biological fluids. This is why we have it in vaccines, but we also have it in medicines. Thats the job of biopharmaceutics: finding formulations to dissolve drugs into the body and allow them to reach a concentration high enough to display their therapeutic activity.

The use of polysorbate 80 in drug delivery of anti-cancerous drug is probably the first and foremost main driving factor on investigating its effect on the BBB. Brain tumors (primary and metastatic alike) are up until now one of the most dreaded and deadliest form of cancer. For instance, the average expected lifespan upon diagnosis of a grade IV glioma (aka glioblastoma multiforme) is grim: 18-months, with less than 5% survival after 5 years. The major issue is being able to deliver drugs and chemotherapy across the BBB. As reported by Pr. William Partridge (UCLA) the BBB remains the bottleneck in drug development for the treating neurological disorders (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC539316/?fref=gc&dti=873247819461536)

The first report of the investigation of polysorbate 80 on the BBB is probably by Spiegelman and colleagues in 1984 (http://thejns.org/doi/pdf/10.3171/jns.1984.61.4.0674), investigating the effect of the solvent used in etoposide solution for treating cancer. According to their  result, they noted a statistical difference in the BBB permeability  (using Evans Blue and 99mTc as tracers) following the injection of 1.125ml/kg. According to their paper, 5mL solution contained 400mg of polysorbate 80 or a concentration of 80mg/mL. Based on this, we can assume that the BBB effect was observed for a dose of 90mg/kg. Thats a very huge dose.
If we go back to the manure anti-vaxxers say, the amount injected via vaccines is enough to cause a barrier opening. According to John Hopkins University Institute of Vaccine Safety (http://www.vaccinesafety.edu/components-DTaP.htm), the expected concentration of polysorbate is lesser or equal to 100mcg or micrograms. Thats 0.1mg per dose. If we assume such dose is injected to a newborn (average weight ~3 kgs), then the amount injected is about 0.033mg/kg. Thats 2700 times less than what has been reported to induce a BBB disruption. Also you have to factor the bioavailability of polysorbate (that is 100% upon IV route) making this number a very optimistic number.
Now, the interesting twist about polsyorbate 80 is its use to enhance some drug carriers and its widely used for finding novel formulation to enhance the delivery of anti-cancerous drugs across the BBB. You can find a list of publications on Pubmed about that aspect (https://www.ncbi.nlm.nih.gov/pubmed/?term=polysorbate+80+blood-brain+barrier). What if polysorbate 80 not only will not injure your brain, but actually may help deliver drugs to help your brain fight disease?

 

Keep in mind that polysorbate 80 is good at dissolving lipid in water solutions but it is not good to let charged molecules accross the BBB, just in case someone comes with the claims that it conjugates with aluminum. Thats some high-school chemistry level.

 

 

[Neurosciences/BBB] 8th GLUT1 Deficiency Conference – Summary

IMG_0272

Today wrapped the second and last day of the 8th GLUT1 Deficiency conference that was held in Nashville, TN this year. It was my second time I am attending this conference and honored to be a guest speaker this year.

 

The whole conference took place at the Inn at Opryland, part of the Gaylord Resort at Opryland. It is a fairly impressive complex with shuttle to the Opry Mills outlet shopping center and, the Gaylord Resort & Convention Center (in which the AACP is also holding a meeting starting today but I am just attending one day meeting there).

According to the organizers, we had about 220 attendees, with 68 families present. What I liked this year was the blending between parents, healthcare providers and scientists. In the previous conference, the first day was family and healthcare providers and the second day was the professional day. This allowed a unique interactions, questions & answers and discussion.

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It was also a very good time for updating my knowledge on the disease. Not much on the basic science, but more on the current treatment and dietary intervention with various experts of the field including Pr. Jorg Klepper (University of Essen, Germany); Pr. Juan Pascual (UT Southwestern, Dallas, TX); Pr. Eric Kossoff (John Hopkins University, Baltimore, MD) and other scientific experts.
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My learning from the conference is that the disease in an evolutive disorder. We learn more about the disease as we learn from the patients growing in. As the patient grows, he or she displays different symptoms: “funny eyes movements” during infancy, presence of absence seizures during toddler times and learning attention and deficit during early school age, presence of movement disorders in both during childhood and adulthood and migraines, hemiplegia and “writers hand fatigue” syndrome. This seems to be linked by an impaired glucose uptake in the cerebral cortex and the thalamus.  It also seems that there is at some point in the disease the presence of a sexual dimorphism, as female patients seems to experience in their teenage years a “paroxysmal dystonia” that seems triggered by moderate and vigorous exercise. So, the GLUT1DS is not a static disorder. It is a disorder evolving over time with its clinical manifestations evolving as well.
The second thing I learned is the variety of “ketogenic diets”. There is not one single “keto diet” but several variants with different dosages and variety, including a Modified Atkins Diet.

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It seems there is not a “one size fits all” but rather different types of diets that also seems to vary with age.

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The younger age appears to need the following of a strict keto diet and as the patients age, some softening and flexibility can be introduced. It seems the critical time for the keto diet is infancy and childhood. The earlier the child is introduced, the better. There are also several companies providing cookbooks, supplements like keto powders or kets-friendly products aimed for patients.

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In terms of diagnosis, some interesting news came from a French biotech startup that can measure GLUT1 levels in RBC within 24 hours using a proprietary cell assay (that looks like an antibody assay) using a flow cytometry-based approach.
Another interesting result is the outcome of the ketogenic diet for GLUT1DS patients. For the vast majority of GLUT1DS patients (95% of patients), the keno diet significantly decrease the number of seizures by at least 50%. In contrast, other types of epilepsies combined only show a 50% of patients showing a responsive outcome to keto diet. Still, 5% of GLUT1DS do not respond to keto diet and there is a fraction of patients that show a normal glucose CSF levels and/or GLUT1 expression. We certainly have a lot of patients that undergo undiagnosed or misdiagnosed for years as “drug-refractory epilepsies”. But it seems that some patients maybe falsely diagnosed as GLUT1DS. Hopefully, with the decrease in price for DNA testing (it seems 23andMe can detect some GLUT1 SNPs) may help to broaden the diagnosis and identification of patients.
Some interesting topics presented at the conference was some possible drug adverse effects reported in G1D heterozygous mice in particular to diazepam and phenobarbital but also other drugs. Some parents noted the anecdotical adverse reactions following certain treatment. However, the absence of studies directly investigating such drug adverse effects in G1D patients most of the time go under the radar, with the health practitioner attributing it to the disease condition rather than some particular drug adverse effects. Having from screening tools can greatly help.
Another interesting presentation is the study of G1D heterozygous mice. These mice seems to display a lower brain vascular density compared to wild-type. This is not surprising considering the recent work of Pr. Peter Carmeliet (Universidaed Leuwen, Belgium) on endothelial cell metabolism. According to Pr. Carmeliet, brain endothelial cells highly depend on glycolysis to function despite being in presence of plenty amount of oxygen levels.
There have been also discussion of trying to setup a comprehensive guide for parents for a consensus on GLUT1DS diagnosis and management that can help them as a source for documentation during their visit with their doctors. There is also a discussion of improving the community outreach to professionals and politicians to improve the funding and the recognition of GLUT1DS as a condition, discussing about supporting open-access options for certain papers allowing parents a free-access to these new studies and also finding ways to support GLUT1DS awareness and management among minority populations and in other geographic areas (especially South America).
The person missing at this meeting by his presence was certainly Pr. Daryl DeVivo (Columbia University, New York, NY). Little patients left him some very kind words and their name on a paper board. I found it was a very cute gesture and remembered us that his absence was felt.
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The interesting silver lining comes from Europe, as they have set now a sister association that held their first European GLUT1 meeting last fall and plan to hold it in London in 2018 and in Paris in 2020.
For me, I am looking forward to attend the 2019 meeting in Washington DC and hopefully bring on some more breaking news from my lab there.

 

 

 

[Stem Cells/BBB] Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier

Vatine et al. show that human iPSC-based modeling can pinpoint the origin of a neuronal disorder in the brain as a defect in transport of thyroid hormone across the blood-brain barrier, rather than in the neurons themselves.

Source: Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier

[Stem Cells/BBB] Huntington’s Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits

Lim et al. show that HD iPSCs-derived brain microvascular endothelial cells have impaired angiogenic and barrier properties. Transcriptomic analysis provides mechanistic insights into pathways that underlie dysfunction, and WNT inhibition prevents angiogenic deficits. This system also suggests strategies to reduce disease burden and assess BBB penetration of drugs for HD.

Source: Huntington’s Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits

[Sciences/BBB] Endothelial TLR4 and the microbiome drive cerebral cavernous malformations (Tang et al., Nature 2017)

You may have heard about this study that showed how your gut bacteria were responsible for stroke. Of course headline news always love to stretch scientific findings as much as I use to stretch my Stretch Armstrong when I was a kid. However, the paper cited was indeed published in Nature and can be found here:
https://www.nature.com/nature/journal/vaop/ncurrent/full/nature22075.html

It is a very interesting paper to read, because a lot of it sounds like a serendipity and lucky strikes. This paper investigated changes in two mouse models of cerebral cavernoma (Ccms). Ccms are a particular type of hemorrhagic stroke because they are mostly genetics (there are three Ccm genes described, in this study they focused on Krit1 and Ccm2) and most of the time go unnoticed. Mutations in those genes result in some alterations in brain microvessels, making some tiny anatomical abnormalities resulting in a higher susceptibility in some of these micro vessels to spontaneously burst and bleed.

The authors of this study have been developing Cre/Lox mice colonies for Ccm2 and Krit1 to better understand the pathology of this disease. The advantage of Cre/Lox is you can knockout a gene in a specific place at a specific time, just by injecting or providing a molecule (usually tamoxifen) that will induce it.

They have been breeding mice that were deficient in Ccm2 or Krit1 and were as expected developing brain micro bleeds (usually around their first two weeks of postnatal age). Following some changes in the animal facility, they observed that a small fraction of their mice colonies suddenly became resistant to cerebral micro bleeds: they still carried the mutations but they fail to develop these microbleeds. Therefore some non-genetic factors were influencing this resistance pattern.
Things became even more interesting as they found that among some of these resistant mice, some developed again the microbleeds within a same littler. The only difference between those developing the microbleeds and those which did not were apparently related to the intraperitoneal (i.p.) injection of tamoxifen. Have the authors provided the tamoxifen through the drinking water, that would have ended the story here.
The authors indeed found that those who reversed their phenotype from resistant to susceptible developed a bacterial infection at the site of the i.p. injection suggesting that such micro bleed was driven by some bacterial factor. They showed that similar results were obtained if they injected LPS (a common Gram-negative antigen) to these mice.

They identified two receptors known to play a role in cellular response to pathogens (we refer such signaling pathways as Pathogen Associated Molecular Patterns or PAMPs): TLR4 (toll-like receptor 4) and CD14 (TLR4 co-receptor). By knocking down these receptors in their Ccm-resistant animals, they were capable to block such bacteria-induced response. The possible interactions of Gram-negative bacteria with these two receptors at the blood-brain barrier maybe enough to trigger the cerebral micro bleeds.

What is also interesting is that mutations in these two genes (some single nucleotide polymorphism or SNP) in patients known to have an history of Ccm also resulted in a higher probability to have brain microbleeds.

I will not spoil the rest of the story but it confirms the presence of a brain-gut axis in Ccm, suggesting the possible effect of the gut microbiota as a risk factor to increased microbleeds in Ccm patients. Let it be clear, these bacteria WILL NOT induce Ccm in normal invididuals. It increase the risk of bleeds in patients already at risk of Ccm.

Another limitation is that in vitro data to confirm the presence of TLR4/CD14 at the BBB and fails to explain how these receptors are triggered by the gut microbiota. The authors suggested a bacteremia (circulating bacteria from gut to the brain via bloodstream) but I remain skeptical about it.

Nevertheless it is a very good paper that worth being read.