[Neurosciences/Cancer] About Sen. McCain brain tumor……and glioblastoma multiforme

You may have heard the tragic news that broke hell under the feet of Senator McCain (R-AZ) and his family on Wednesday. According to several sources, Senator McCain biopsy taken from his recent medical examination revealed to be classified as “glioblastoma multiform” (or GBM) for short.
I am not a brain cancer specialist but I have been doing some collaboration with a research group focused on GBM and I know all too well what does it mean and what is the prognosis. This is a type of tumor I would not wish my fiercest archenemy to get. I thought it would maybe help me to make a lay summary on GBM and explain why the BBB in that case is one of our fiercest challenge for drug delivery.

1. What is glioblastoma multiforme?
Glioblastoma multiforme (aka GBM) is a primary brain tumor characterized by its heterogeneity. However, we assume that GBM is originating from tumor astrocytes. Astrocytes are an important cell type of the macroglia, outnumbering neurons from 3:1 to 5:1. For a long time, astrocytes were considered as “glue cells”, playing only a function of scaffold and nourishing cells to neurons.
However, in the last 50 years, astrocytes have been shown to play much more important roles including the induction of the blood-brain barrier phenotype, regulation of the cerebral blood flow, modulation of neuronal cell activity, ability to form a parallel signaling network and also to play an important function in terms of protection of the brain during diseases.
The World Health Organization (WHO) classify GBM as a grade IV brain tumor (https://link.springer.com/article/10.1007/s00401-016-1545-1), meaning this type of cancer is classify as highly aggressive. Because the brain is a very soft tissue, tumor cells can easily proliferate, migrate and invade the surrounding healthy tissues.
The cause of GBM remains unclear, however we know that some GBMs are evolved from other types of brain tumors that have a lesser malignancy like lower-grade astrocytomas (grade II) or anaplastic astrocytomas (grade III). GBM is considered the most common type of primary tumor (not caused by metastatic cells) but also remains pretty rare with a case of 2-3 new patients diagnosed with the condition for every 100’000 inhabitants. There is a possible sexual dimorphism, as men are more likely to be affected than women (3:2 ratio), with an increased risk with age (https://www.ncbi.nlm.nih.gov/pubmed/17373878).
There is no particular risk factor associated with GBM. So far, we assume it has a strong genetic background, as several genes have been associated with GBM including some abnormalities (including loss of DNA in a chromosome domain) on the chromosome 10, mutations in various genes including TP53 (tumor suppressor gene, its function is to repair cell DNA or to induce cell death by apoptosis if it fails to repair), MDM2 (pro-survival gene, its function is to promote cell survival), EFGR and PDGFRα (these are two receptors that induce cell growth, cell proliferation and cell survival upon stimulation by growth factors).
Also noteworthy, there has been speculation and a perpetuated myth that wireless cell phones activity are associated with an increased risk of developing brain tumors. There is no reliable studies (both on epidemiological standpoint and on animal models) that can show an association between the use of cell phones with increased risk of brain tumors.

2. What are the treatments and prognosis for patients with GBMs?

This is where I cannot have much optimism. GBM is a very aggressive type of cancer. The average survival rate is about 18 months, with less than 5% of patients making through the 5-year milestone.
Like any type of cancer, there are different options proposed: radiation therapy, surgery and chemotherapy.
Surgery is commonly practiced but have several challenges: Firstly, it is very hard to identify GBM tissue from the healthy tissue by naked eye during surgery. The neurosurgeon has to rely on the MRI cliches to resect the tumor tissue. Secondly, the neurosurgeon wants to maximize the removal of tumor tissue but also he/she wants to limit the damage to the surrounding healthy tissue to not induce further brain damage. Thirdly, GBM is prone to form glioblastoma stem cell-like cells (GSCs) that share several features with stem cells. These cells can tolerate very aggressive environment and can rapidly proliferate. This is one of the common complication occurring in GBM patients. After you remove the tumor and see no trace of it under the MRI, you conclude it got eliminated. Only to find out three months later that the tumor grew back in size and started to invade more brain tissues.
Chemotherapy arsenal for GBM is very limited. So far, temozolomide is the way to go for GBM. However, 50% of the patients will not respond to temozolomide due to a mutation in the MGMT gene capable to inactivate it (http://www.sciencedirect.com/science/article/pii/S2352304216300162). Other anti cancerous agents including EGFR inhibitors (e.g. lapatinib) fail to show any activity to the presence of a pathological form of the blood-brain barrier (BBB) called “brain-tumor barrier” (BTB). This abnormal form of the BBB involve interactions with brain tumor cells. For a long time, the scientific community thought that BBB surrounding brain tumors was leaky and therefore accessible to chemotherapeutics. However, we know that indeed there is a BTB that can act as a barrier for the penetration and delivery of drugs into the tumor region.

There are some new avenues and approaches to target GBM but they are still very experimental. Amongst them, the possibility to use oncolytic viruses like a modified form of the polio virus capable to set brain tumor cells into “auto-destruction” mode. The second avenue explored is possible use of immunotherapy. The rationale behind is to help the immune system “to learn” about the tumor cells as foreign agents and strike them. There are some success using antibodies targeting tumors and also by reprogramming patients own cells (CAR-T cell therapy).

The diagnosis of GBM is probably one of the most difficult one a neurologist or neurosurgeon has to set, as it has a very poor prognosis. Let’s be honest, it is not looking good and for someone like Senator McCain that has been facing death several times during his military duties this is probably the toughest one to overcome.

3. Concluding remarks

This is why we need to foster research in brain tumors, this is why we need funding to help research findings, this is why we need clinical trials to pick the most promising drug candidate to fight this type of cancer, this is why we need to have a public health policy that ensure healthcare coverage for everyone can have access to treatment to beat the odds and not have to decline treatment because of the huge costs associated that health insurance may simply refuse to share the burden.

I am so embarrassed to say that right now the only thing we can provide Senator McCain and anyone with GBM and their relatives are our sympathies and our wishful thinking. This is why I have colleagues, peers working days and nights, weekends to bring on a “silver bullet” capable to annihilate such condition.

If you are looking to help, the best I can advise is to support research by donating to association like ABTA (http://www.abta.org) that focuses on funding research on brain tumors. Also considerate to let your voice heard and support healthcare policies that ensure an universal coverage of the population regardless of their age, gender, socio-economic status. Because refusing treatment by fear of letting your most loved ones with a humongous amount of debt should be the last of your worry.







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


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.



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.

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.


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


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.

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.

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.




[Science/Neurosciences] Mole rats running on…..fructose!

You may have heard about this paper from Park TJ and colleagues (Park TJ et al., Science 2017) on how mole rats were showing extreme compliance to anoxic (0% oxygen) level, no? It made the news these last couple of weeks and finally was able to put my hand on. You can access to it here (need to have a Science subscription though) but I read it and it is really interesting for many reasons, especially because I try to think how can we translate it as a therapeutical strategies for hypoxemic pre-terms babies or even as a stroke fighting-drug.

First, mole rats. Oh mole rats! Not the prettiest mammals out there. They are naked, they have long teeth and look all wrinkled. But they are underground dwelling animals like moles. Underneath, oxygenation is scarce and these animals have developed formidable adaption to hypoxia. We as humans can barely survive 8% oxygen (thats about the Mount Everest). At 6% oxygen (thats what would happen if a aircraft cabin undergo a depressurization), you die within minutes.

In this experiment, they went fairly extreme, they put the animals into anoxia (0% O2) and looked how long the animals would survive. They used a common mouse strain as a control. Mice rapidly died at 100% rate at 5% O2 and died twice faster (based on the number of breaths) at 0% O2. In opposite, mole rats went 30 times longer than mice and still were doing fine (0% deaths). Were mice died within 60 seconds, molerats died over 1000 seconds of anoxia. One possible reason is their ability of their heart to beat much longer than mice.

Now what is interesting is how the authors came to fructose. Mammalian cells run on glucose through the following biochemical pathway (see below):


I will spare you the Krebs cycle but this is what every since healthcare and life scientist have to learn. Glucose is broken down into many intermediates and at the end becomes pyruvate. From pyruvate, you can enter the Krebs Cycle and produce a significant amount of ATP (the fuel cell of every living organism) needed to provide energy for any biological process. Krebs cycle is very good at it and provides an ATP yield of 36ATP/glucose consumed. However, the Krebs cycle stall under hypoxia and forces the cell to adapt. In particular, it needs to regenerate NAD+ (from NADH) in order to keep the system flowing and producing energy. One way mammalian cells solved it is by converting pyruvate into lactate. Thats allow cells to produce some energy (2ATP/glucose) and regenerate its NAD+. However lactate has tendency to accumulate and develop adverse effect (the famous muscle cramps any runners have experienced).

Fructose is not much different from glucose, it has the same composition but just a little difference in the molecular structure.  We get fructose from our daily diet made of fruits and vegetables, but also from refined sugar (sucrose or HFCS, same deal).


Now fructose can bypass and feed the glycolysis at different steps:

Fructose can produce glyceraldehyde-3-P (GA3P) and dihydroxyacetone-P (DHAP) and enter the rest of the glycolysis. Now like glucose, fructose needs a transporter to enter inside the cells. Glucose has a myriad of glucose transporters (GLUTs and SGLTs) that can provide glucose inside the cells. But not fructose. These transporters have very poor affinity for fructose. In that case, fructose has one transporter called GLUT5 that prefers fructose over glucose.
Now this is where it becomes interesting, mole rats show much higher levels of fructose than mice during anoxia in many organs and in blood. Now the interesting fact is the high prevalence of it as fructose-1-P in the brain, only this form. How it goes in? I don’t know but mole rat brains have a higher GLUT5 expression than mice. Where this transporter is expressed? I don’t know either but it would interesting to look at this transporter at the BBB.

What is interesting is the difference in how mole rats  brain and heart differ from mice in terms of fructose activity. When administered fructose over glucose, mole rats organs know to switch between the two sugars to gets its energy. In the other hand, mice organs fail to switch and result in decrease their activity.

Now the question I have (since I am working on glucose transport across the BBB and its impact in kids suffering from GLUT1 deficiency) is: does human express GLUT5? If yes, which brain cells express it and if these cells can adopt fructose as a source of energy?

[Neurosciences/Alzheimer] Structural variation in amyloid-β fibrils from Alzheimer’s disease clinical subtypes

Interesting study published in Nature about how differences in Abeta fibrils have an implication on the clinical symptoms.
A quick refresher for those not much familiar (it is also a bit of a headache as I just dive in the Alzheimer’s research). Alzheimer’s disease is characterized by the formation of senile plaques and tangles, such structures have been considered as the causative agents in neuronal cell death.
These plaques are clumps of a peptide called Abeta (for amyloid beta) peptides. We know how these peptides are formed (by cleavage of the amyloid precursor protein or APP) and we know there are different “flavors” of Abeta that have been described: Abeta 1-40, Abeta 1-42…..
We also know that these peptides are “sticky”. They are released as monomers (single peptide) and because they are hydrophobic (hates water, like oil hates water) they will try to bind together and form oligomers (think about little sticky balls). These oligomers then can form fibrils.
It is a very “dry” study because it is heavy on structural biology and computational biology but in the same time very interesting.
It shows us that plaque formation is not a linear processes, there are different combinations possible (imagine like comparing snowflakes) that have a direct impact on the clinical presentation and outcomes in patients.
That’s maybe providing another way of thinking in targeting Abeta that can help us learn from the failure of previous clinical trials.

Abstract and link to the original paper

Aggregation of amyloid-β peptides into fibrils or other self-assembled states is central to the pathogenesis of Alzheimer’s disease. Fibrils formed in vitro by 40- and 42-residue amyloid-β peptides (Aβ40 and Aβ42) are polymorphic, with variations in molecular structure that depend on fibril growth conditions. Recent experiments suggest that variations in amyloid-β fibril structure in vivo may correlate with variations in Alzheimer’s disease phenotype, in analogy to distinct prion strains that are associated with different clinical and pathological phenotypes. Here we investigate correlations between structural variation and Alzheimer’s disease phenotype using solid-state nuclear magnetic resonance (ssNMR) measurements on Aβ40 and Aβ42 fibrils prepared by seeded growth from extracts of Alzheimer’s disease brain cortex. We compared two atypical Alzheimer’s disease clinical subtypes—the rapidly progressive form (r-AD) and the posterior cortical atrophy variant (PCA-AD)—with a typical prolonged-duration form (t-AD). On the basis of ssNMR data from 37 cortical tissue samples from 18 individuals, we find that a single Aβ40 fibril structure is most abundant in samples from patients with t-AD and PCA-AD, whereas Aβ40 fibrils from r-AD samples exhibit a significantly greater proportion of additional structures. Data for Aβ42 fibrils indicate structural heterogeneity in most samples from all patient categories, with at least two prevalent structures. These results demonstrate the existence of a specific predominant Aβ40 fibril structure in t-AD and PCA-AD, suggest that r-AD may relate to additional fibril structures and indicate that there is a qualitative difference between Aβ40 and Aβ42 aggregates in the brain tissue of patients with Alzheimer’s disease.

Source: Structural variation in amyloid-β fibrils from Alzheimer’s disease clinical subtypes : Nature : Nature Research

[Neurosciences/BBB] Alpha-Synuclein pre-formed fibrils impair tight junction protein expression without affecting cerebral endothelial function

Hi everyone, today I am experimenting a journal club on a blog, sharing my thought on some recent publications in the BBB field.
The paper I will be discussing today is a study recently published by Dr. Roger A Barker (University of Cambridge, Cambridge, UK) in the journal Experimental Neurology (IF=4.5) titled “Alpha-Synuclein pre-formed fibrils impair tight junction protein expression without affecting cerebral endothelial function” (http://www.sciencedirect.com/science/article/pii/S0014488616302710).
Why did I choose this paper? Because this paper is investigating the interaction between alpha-synuclein (aSyn) and the blood-brain barrier. I have recently developed interests to see how the BBB behave in neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s disease and in particular how the BBB let peptides involved in such diseases go in and out. In this study, they have used the hCMEC/D3 immortalized human brain endothelial cell line (Weksler et al., FASEB 2005) as well as primary human neurons and astrocytes co-cultures. For the hypothesis, they have used monomeric aSyn as well as what they referred as preformed fibrils (pff).
One of the caveat of this study is the use of hCMEC/D3 cells that are notoriously known for their poor barrier properties. This poor barrier properties was indeed displayed in the paper as the authors reported values of only 15 Ohms*cm2 that is very low. I was also surprised that the authors reported the use of FITC-dextran of 10kDa size as this compound is big enough to poorly cross the BBB. But the use of such tracer also make sense as it can also a comparison to aSyn. aSyn (as pff) did not have much effect on the barrier function, we can even observe an increase in the TEER and decrease in the permeability suggesting a possible induction of the barrier. Another piece of data presented purposively show a immunostaining for ZO1, a protein adapter for tight junction complexes. The immunolocalization of ZO1 was suboptimal, making difficult the interpretation of the negative effect of pff on ZO1. There is a increase  in immunoreactivity following pff treatment. Astrocytes co-cultures improved the barrier function and again no effect of pff was noted on the barrier function. The authors also showed no effects of pff on astrocytes GFAP expression.
The interesting but also the data that raised some skepticism is the experiments involving hCMEC/D3 cells co-cultured with primary neurons. The TEER is lower than the monocultures and astrocyte co-cultures (~8 Ohms.cm2) yet they display permeability values for FITC-dextran 10kDa that are 200X lower than the monoculture. Aside from this issue (that should have been noted by the peer-reviewers), there were also disprecancies between TEER and permeability. If we consider the relative permeability to untreated group accurate, we can note a 50% increase in permeability following treatment with pff or aSyn monomers. Again the immunostaining was pointless as the staining for ZO1 looked poor but also the representative pictures are displaying different cell densities (as noted by  DAPI-cell nuclei density per field).
To better understand the impact of pff on neurons, the authors treated some fetal cortical cells with aSyn monomers or pff using a TUNEL assay (a common technique to observe cell death in vitro) but this was using an immunohistochemistry approach (HRP with DAB stain) instead of the classical immunofluorescence.
Finally, the authors showed some quantitative protein expression analysis from the in vitro cultures and from post-mortem tissues obtained from PD patients. An interesting feature observed was the increase in ZO1, claudin-5 expression following neuronal co-culture, but also a very strong regulation of tricellulin and MarvelD3 proteins (their detection was weak if not negative in monocultures). Surprisingly, the pff treatment decreased occludin and ZO-1 expression at protein levels. The PD Western blots were showing much inter-individuals variability that makes hard to translate from in vitro to in vivo.
In conclusion, the title was very attracting and interesting but I felt the data was poorly supporting the claims. The hCMEC/D3 model is not the best model for modeling the BBB in vitro, especially considering the barrier values reported were below what are commonly reported. I was left on my hunger, it had some interesting data but also some data with lesser quality and foremost did not address if aSyn, as its monomeric or pff form can cross or not the BBB.


[Neurosciences/BBB/ASD] Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder

Very interesting study that came out, maybe the first study that directly link the blood-brain barrier dysfunction to a certain form of autism spectrum disorder (at least in a rodent model).
In this study, Tarlungeanu and colleagues identified the solute carrier SLC7A5, encoding for the large aminoacid transporter-1 (LAT1) as the causative agent. LAT1 usually transport branched aminoacids (BCAA) like valine, leucine and isoleucine inside the brain.
The first piece of interesting data is the aminoacid temporal profile compared to wt mice at different developmental stages (embryonic vs. postnatal versus adulthood. BCAA levels went down, but surprisingly phenylalanine and histidine levels went up.
SLC7A5 KO mice had a neurological signature similar to other autism models. They also validated their findings by whole-genome sequencing genes from patients (from Lybia and Turkey) from co-sanguine marriages displaying various neurological disorders and identified two key mutations impairing SLC7A5 activity.
You can find more about this study by clicking to the source: Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder

[Neurosciences] ….and another Alzheimer clinical trial bites the dust :(

….And another Alzheimer’s disease clinical trial bites the dust. This one is a Phase III trial (that means hundreds of volounteers). Eli Lilly new candidate, an antibody targeting amyloid beta plaques formation failed to show any improvement compared to placebo in their latest clinical trial.
That means Lilly will stop injecting money (we are talking about already $100 million spent in R&D considered lost, as the company will not be able to make return on investment) and bring us back to the blackboard: is amyloid beta the real culprit or are we missing it? Thats tilt in the favor of the tau team (that consider tau hyperphosphorylation the main culprit).
Thats some disappointing news for everyone and gives an idea why new treatments are so expensive once they enter the market upon FDA approval.

Source for this post: ACS Chemical & Engineering News (image: Lilly)
Lilly Alzheimer’s drug fails | November 23, 2016 Issue – Vol. 94 Issue 47 | Chemical & Engineering News