[Sciences/BBB] #RareDiseases Day 2017: a focus #GLUT1 Deficiency and “LoveSome1withGlut1” fundraising

Today is February 28th. It is also named “Rare Diseases Day” that normally takes place on February 29th but since that day is also rare, we celebrate it on February 28th.
Why celebrate “Rare Diseases Day”? Because as their name suggest, there are diseases that affect only few individual and therefore are classified rare.
Because they are rare, they are under less attention from major healthcare actors at different levels.

From the basic scientist standpoint, these are diseases that are so rare that the interest to fund these diseases are almost zero because budgets are tight and priority is given to research for diseases with a much more prevalence.

From the pharmaceutical company standpoint, these are investment that are risky by their failure and also by the prospect of seeing investment on return. It costs a lot to validate the therapeutic efficacy and ensure the production of these therapeutics into a cGMP grade (pure and safe enough for being used in humans). Therefore, pharmaceutical companies stay away from these diseases and put their bet on safer projects.

From the clinical standpoint, clinicians may encounter only one or two cases during their whole professional career. For families, it is devastating to not put a name on the condition affecting their child for years, only to hear at the end that there is no curative treatment for.

One of these rare diseases is the GLUT1 deficiency syndrome (GLUT1 DS).

Glucose is the number 1 source of energy for eukaryotic cells, from yeasts to human cells. It enters the cells via a transporter (a protein present in the cell membrane). Glucose uptake is indeed ensured by different transporters all belonging to the solute carriers (SLCs) super-family. We have some of these carriers that are sodium-independent (GLUTs) and some sodium-dependent (SGLTs).

The brain is particularly avid of glucose: 25% of our daily glucose uptake is directed strictly to the brain. Considering the small size of the brain (3lbs organ out of a 140lbs individual), you can understand how important glucose is for nerve cells.

The uptake of glucose to the brain is ensured by the presence of several transporters at the blood-brain barrier (BBB), with GLUT1 being the most abundant glucose transporters. There are also reports of other glucose transporters (GLUT3, GLUT4, SGLT1 and SGLT6) but their contribution is less well understood (Meireles et al. 2013). In the brain, astrocytes and neurons also express some glucose transporters (GLUT1 and GLUT3 in astrocytes; GLUT3 in neurons) but it is considered that upon crossing of the BBB that astrocytes reduce such glucose into lactate (a fermentation byproduct of glucose degradation). Such lactate is believed to serve as a rapid source of fuel for neurons.

GLUT1 deficiency syndrome (GLUT1DS) was firstly reported by Pr. Darryl DeVivo (Columbia University, New York, USA) in 1991 (De Vivo et al. 1991) and was characterized by the presence of a low level of glucose in the cerebrospinal fluid (the fluid in which the brain baths in) despite a normal blood glucose level. It was later identified that GLUT1 (encoded by the gene SLC2A1) was responsible of such clinical presentation (Seidner et al. 1998). This condition is affecting children in their infancy resulting in the presence of epileptic seizures that are classified as refractory or “drug-resistant”. Indeed, a recent study suggests that GLUT1 DS is the second most abundant genetic form of childhood epilepsies (Afawi et al. 2016).

Until now, there are no treatment for this condition. The only common symptomatic treatment and intervention is forcing these children bodies to switch their brain metabolism from a carb-inclusive normal diet into a ketogenic diet (similar to the Atkins diet). By removing carbs, you are forcing the brain to fast and solicit lipids from fat storage as a source of energy. These fatty acids can be broken down into ketone bodies (acetoacetate, acetone and beta hydroxybutyrate). These ketone bodies can be transported into the brain via the monocarboxylate transporter 1 (MCT1, another member of the SLC super-family) and serve as alternative fuel. Yet, such treatment is only a temporary fix as it requires an extreme observance of this diet as well non-negligible side effects on the body.

My current research focuses on trying to develop a patient-specific model of the BBB using patient-derived induced pluripotent stem cells (iPSCs) in order to understand how glucose is uptaken at the BBB and how such mutations impact such uptake. I am still new in the field and it takes time and money to validate such models, as we have to move to incremental steps to validate such models.  However, little things can make changes and it takes a village to raise someone.

Today, I would like to give my thanks for the GLUT1 Deficiency Foundation (http://www.g1dfoundation.org) to support part of my research through their research grant program aimed as a first step to better understand the diseases. Some of my current research would not have been possible without the effort of families that have been actively fundraising and raising awareness about this disease.

The fund I have been awarded through the foundation is simply an amazing effort and reward, as I am grateful for this fund raised through cookie sales, lemonade stands or simply generous donations from anonymous donors.

If everything goes well, the GLUT1 Deficiency Foundation is due for a bi-annual meeting (this July) involving scientists, clinicians, patients and families discussing about the recent research and outcomes obtained both at the bench and the bedside. I am very excited to have my research project moving on and being able to present a poster to this meeting and share how this research grant has been able to help to shed new lights on glucose transport at the BBB and hopefully moving to a diseased model, one little step at the time.

This is why I am thankful to the GLUT1 Deficiency Foundation and please help me support their campaign by helping them to raise funds through their “Love Some1 with Glut1”: http://www.g1dfoundation.org/ways-to-give/donate/.

References:

Afawi, Z., Oliver, K. L., Kivity, S. et al. (2016) Multiplex families with epilepsy: Success of clinical and molecular genetic characterization. Neurology, 86, 713-722.

De Vivo, D. C., Trifiletti, R. R., Jacobson, R. I., Ronen, G. M., Behmand, R. A. and Harik, S. I. (1991) Defective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay. N Engl J Med, 325, 703-709.

Meireles, M., Martel, F., Araujo, J. et al. (2013) Characterization and modulation of glucose uptake in a human blood-brain barrier model. J Membr Biol, 246, 669-677.

Seidner, G., Alvarez, M. G., Yeh, J. I. et al. (1998) GLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrier. Nat Genet, 18, 188-191.

 

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[Movies/SciFi] Turbo Kid (80%)

Sometimes a good surprise can await you around the corner. This was the case of “Turbo Kid”, as it popped as a suggestion on my digital service.
Turbo Kid is a French-Canadian movie written and directed by François Simard, Anouk Whissell, and Yoann-Karl Whissell. Not much famous actors in the cast except with the exception of Michael Ironside (known for his role as Ham Tyler in the original “V” series).
“Turbo Kid” is not totally new to me, as the first iteration known as “T for Turbo” was the prototype for it and frankly it created a huge buzz:

It rightfully made a loud buzz by its VHS feeling of an 80’s post-nuke movie, a kind of genre I have been bottle-fed thanks to the Italian moviemaker. All the ingredients were already in: the post-nuke wasteland, the abandoned quarrel, the Bontempi synthesizers, the cheap gore effect, and the BMX ride. Even the inspiration of the “Cannon Pictures” gives this huge nostalgic vibe.

The plot is fairly simple but fairly fun. The plot takes place in a post-nuke 1997, in a quarrel posing as a wasteland. Water is scarce and became a precious commodity. In a complete anarchy, the law of the jungle prevails: the survival of the fittest.
“The Kid”, the main protagonist, lives on scavenging on his BMX bike and fomenting revenge to Zeus (played by Michael Ironside), as Zeus killed his mom in front of him. Taking courage from reading “Turbo Rider”, a comic book.
One day, the Kid finds Apple, an android girl that quickly becomes attached to him. This only starts the trouble once Apple get kidnapped by Zeus and fleeing from his minions, the Kid ends up in an abandoned vehicle and finds the remain of a certain “Turbo Rider”….

The movie is not aiming very high but it hits hard on the GenXers like me. You could almost believe you have seen this movie as a kid, as the feeling is so genuine to 80’s Sci-Fi B-movies, especially Italian post-nukes: They were cheap, the plot was quite often cheesy. But oh gosh they were great and fun to watch!

Here is the official trailer, you can catch the full movie on Netflix:

[Neurosciences/BBB] Journal Club – Blood–brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders

An interesting paper that was recently published by Fiorentino and colleagues entitled “Blood–brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders”  in Molecular Autism journal. The article is in open-access so you can download the full-article here: (https://molecularautism.biomedcentral.com/articles/10.1186/s13229-016-0110-z).

In this paper, the used postmortem brain tissue and duodenum tract biopsies samples from patients diagnosed with autism spectrum disorders (ASD), schizophrenia (SCZ) and compared to controls. They measured changes in tight junctions and inflammation at mRNA and protein levels using qPCR and Western-blot respectively.
One aspect that I found missing in the sample used is the size of the male population, largely bigger than the female population. It would have been neat to have the same population weight (as many male samples than female samples) but you cannot choose the samples you get. Also, the ethnicity is predominantly Caucasian.
The data is interesting but also a bit confusing in terms of statistical analysis, with some p-hacking and data misleading by showing the standard error mean (SEM) instead of standard deviation (SD). The SEM (SEM=√SD/number of samples) has little meaning when your sample size below 100. it just makes the graph nicer, but it does not help the reader to assess the statistics.
At mRNA levels, the authors found an increase in claudin-3, claudin-5, claudin-12 and tricellulin (TRIC) mRNAs in ASD patients compared to SCZ. Now here what bothers me is their selective statistical analysis, almost cherry-picking it. For instance, claudin-3 is increased in both the SCZ and ASD groups. They observed a statistical difference between ASD and control, but mentioned nothing about the SCZ versus control, despite showing similar mean +/- SEM. Now Claudin-5, claudin-12 and tricellulin (TRIC) were selectively increased in ASD patients compared to control, but again they failed to compare SCZ to control samples (side-note: the authors failed to provide the primer sequences for TRIC. Bad job!). Still it is interesting, tight junction (TJ) complexes seem to be up-regulated in ASD patients compared to SCZ and controls. Does it mean the BBB is compromised? At this point, no. Seems even the BBB maybe better.
In the next figure (Fig.2) there are some interesting data but also some dishonest statistics done. The authors consistently compared ASD to SCZ instead of controls. Thats some scientific dishonesty that should have been caught by reviewers. Shame on you reviewers!
Still there are some interesting trends. The authors observe an increase in matrix metalloproteinases (MMP-2 and MMP-9). These are known proteases at the BBB are they are commonly associated with a loss of the BBB during stroke, these MMPs can cleave the basement membrane supporting the BBB and also the TJ complexes. But not much for the other markers used, except maybe a down-regulation in SCZ patients compared to controls. The same sort of p-hacking occurred for looking at inflammatory cytokines (IL-1beta, IL-6 and IL-8). There is not much differences between ASD versus control, but by using the Texas sharpshooter fallacy (by using the SCZ as a “control”) you bring statistical differences where there is not. This is the big problem of this paper: trying to oversell the data using a p-hacking method.
Now expression at mRNA level is not 100% translated in changes at protein levels. in Figure 5 and 6, the authors looked at the changes at protein levels for claudin-5 and claudin-12 The authors used two protein controls: alpha smooth muscle actin (SMA) and beta-actin (ACTIN). ACTIN is a housekeeping gene, it is expressed in all cells at very high level. This is why we use ACTIN as a loading control, to be sure we are observing differences solely based on protein expression not due to differences in amount of total proteins between samples. The use of SMA is smart because it can help determine the amount of medium and large blood-vessels. Capillaries (the vessels carrying on the BBB phenotype) are poor in SMA expression, as they have few smooth muscle cells (SMA is a smooth muscle cell marker). In vessels with higher diameter, you have a intermediate layer (called media) rich in smooth muscle cells. The blots are interesting but I thought the normalization was not great. The authors normalized Claudin-5 and -12 expression against SMA. I would have first show the normalization of these proteins and SMA against actin to have an overall view and show that our samples are not biased (if our samples are random, so should be SMA. However, we are expecting to see a relative similar levels of SMA between the different groups). Interestingly, Claudin-5 protein level is higher in ASD compared to control, whereas claudin-12 is lower.
Figure 7 is in my opinion interesting and confusing in the same time. The reason it is confusing it the experimental design. I have no clues on what the author try to show us. They show a panel of different mRNA expression of different markers for each patients separately. They should have kept the same representation (the average expression of each individuals plotted as dot-plot) as the previous figure. My speculation is that once average, they were facing a important variability in the sample distribution that nothing was likely to come out (likely no observable differences compared to healthy controls). Almost feel like the authors have these piece of extra data with no where to go and used to fill up the paper. It may also explain why the authors failed to show any proteins for these data.
Now, the authors are a bit over-optimistically concluding that there is a leaky BBB in ASD patients. That’s not exactly true for two reasons: claudin-5 levels are higher and they fail to provide adequate data to support this claim. Post-mortem immunostaining against TJ complexes can let measure changes in TJ strands in microvessels. By measuring the average length of these strands, you can speculate a disrupted BBB. In addition, non-invasive MRI (with gadolinium tracer) would help refine this claim and demonstrate evidence of this claim.
The idea of a disrupted BBB in ASD patients is interesting and some of these data are interesting. Yet this study has some issues to convince a BBB expert about the presence of a leaky BBB. Dont let get fooled by some snake oil sellers that will use this paper to support some “leaky brain” or “leaky gut” in ASD patients. This paper show anything except these bogus claims.

[Symphonic Metal] Xandria – Theater of Dimensions (92%)

It has been a while since I did not posted a review on a metal album. Xandria nicely reminded me that it was long due by releasing their latest album “Theater of Dimensions”. Well, what I can tell about Xandria. I really liked their album “Neverworld’s End” fronted by Manuella Kraller. The release of “Sacrificium” and the change in line-up with Dianne Von Giersbergen (ex-Libris) now fronting was letting me skeptical. Don’t take me wrong on this one. Dianne with her prog metal band is simply awesome but I found her a bit in retreat and soft in “Sacrificium”.
I purchased the digital album containing 13 tracks for 75 minutes of some awesomess. There is a deluxe version with four acoustic if you have the extra $$$ for it. I have been running in a loop for the last 72 hours. What I can tell, we have the perfect blend of Xandria taking the best of “Neverworld’s End” and “Sacrificium”. That gives a detonating mixture.
We start with “Where The Heart is Home” bringing the bang and the bombastic symphonics with the metal, with Dianne perfect as ever unleashing her voice into this engaging first track. We rapidly follow with “Death To The Holy” that infuse Celtic influence. This is the main influence of the album, a certain Celtic feeling but also feeling like a Pirate song. Very engaging song and one of my favorites of the album.
“Forsaken Love” dig further into the Celtic influence, nice and very enjoyable ballad to listen. “Call of Destiny” brings on the same tempo than “Nightfall” and “Sacrificium”, fast-paced and heavy. “We Are Murderers” push aside the symphonic elements and put own the heaviness. “Dark Night of the Soul” brings on the enjoyment of the ballad, swinged by Dianne powerful voice. “When The Walls Came Down” brings on the heavy riffs back and the kick that make the Symphonic Metal powerful.
“Ship of Doom” was nice if you are in the mood of some pirates song, arghhh!  If you really like the Celtic tones, jump on “Ceili” blending it with metal and let chill your spine. Very engaging and refreshing to listen to it. “Song For Sorrow and Woe” was okay but not more than that in my opinion. Same as “Burn Me”. “Queen of Hearts Reborn” brings on the Xandria signature: a very delicate dosage of choirs, symphonic and heaviness with Dianne flying high pitches with a grace and an elegance.
Finally, the last track “A Theater of Dimensions”, the piece of resistance (14:24). A very nice song in three acts starting as an acoustic ballad, raising the tone with the heavy elements and riffs and adding the symphonic and getting into the same tones than Dark Sarah, giving this kind of Tim Burtonesque feel to it. A damn good song, that rides like a rollercoaster.
Xandria is back with a bang. Nicely crafting powerful melodics, choirs, heavy riffs and soprano vocals. This album settles Dianne as the perfect singer for the band, only unleashing her impressive voice. If you have never listen to Dianne singing, you have never listen to Symphonic metal. Xandria rightfully earned the title “album of the month” and makes a bang into 2017.