Does Aspirin protect against Alzheimer’s?

For this week I had decided to read and summarise a paper published in J Neuroscience, about the protective role of aspirin in Alzherimer’s Disease (AD). I saw a link highlighting this paper on Facebook and could not immediately get access to the full text (paywalls). I did not even read the full title, but I already had 1 question: “did they check by correlation studies if there’s less people chronically taking aspirin, among those with AD? When I finally saw the full title of the paper, I realised that the study was more precise: “Aspirin induces Lysosomal biogenesis and attenuates Amyloid plaque pathology in a mouse model of Alzheimer’s disease via PPARα”.

Here I got a bit doubtful if I should still read the paper for this blog. But I persisted. My main concern being: why this particular pathway? The answer, which gets obvious mid-paper is: the authors seem to have worked on this one molecule before. But the rational to have chosen Aspirin as treatment seems to have a logical argumentation.

Stepping back: Alzheimer’s disease is caused by accumulation of Amyloid-beta (Abeta). This is a protein that can aggregate with others of the same type in such unbreakable structures that the aggregates are known as plaques. In general, protein aggregates would normally be broken down by the cell machinery: e.g. proteasome and lysosomes, but in AD it seems that this machinery is not working, or is not efficient enough. The authors point out that aspirin has been shown to increase the production of lysosomes.

For this study, the authors follow up on this increase of production of lysosomes by aspirin.

They started by adding Abeta to cells and then aspirin and check that indeed, cells treated with aspirin have less Abeta because 1) they take in less Abeta from the solution and 2) they get rid of the previously taken in Abeta, faster. To check the cause of this clearance, the authors checked for lysosome numbers and saw that it was increased in cells treated with aspirin. Moreover, they hypothesised this was due to new production of lysosomes and therefore looked into known transcription factors (TF) that promote lysosome biosynthesis. Having identified TFEB, they looked for enhancers of this TF and this was when they identified the PPRE site, and went on to study PPARs (Peroxisome proliferator-activated receptor), the pathway they have been familiar with. The next step in this paper is to distinguish between PPAR alpha, beta and gamma, by coupling Abeta-incubation with aspirin treatment in the presence and absence of inhibitors of these 3 molecules: meaning that the authors were able to see that when PPAR alpha was blocked, then aspirin treatment did not lead to increase lysosome production and Abeta was not broken down efficiently.

Pathway-studies done in cells is very useful to pin-point all the players in a certain process but it is not always a good model of the real situation. Therefore, the authors have used a mouse model of AD to test aspirin treatment in vivo: mice with 5 mutations in the Abeta production pathway, that are known to cause familiar AD in humans. These mice develop AD “spontaneously” after a certain age. Thus, once the mice had developed AD, the authors have treated these mice with aspirin, as well as a new set of mice that have the 5 mutations associated with AD and are deficient for PPARalpha. The conclusion is that in the group of mice that are deficient for PPARalpha, the accumulation of Abeta plaques is higher, because the same aspirin treatment is not effective anymore.

So, my question remains: is there a correlation in humans between those taking aspirin and decrease risk of Alzheimer’s disease? This study does not address that.

What this study points at is to a link between PPAR alpha mediated increased lysosome production after treatment with aspirin.

 

Notes & References

Transcription Factor (TF) – protein capable to bind to a sequence of DNA and therefore regulate gene expression.

Enhancer region – sequence of DNA to which TF bind.

How I chose it

I am (still) interested in the role of the immune system as the poisonous environment in which ageing and neurodegenerative diseases (e.g. Alzheimer’s disease) develop. Thus, to me it was very important to do a PhD in the field of Neuroimmunology. There are a few places in the world where this field thrives, and I had excluded outside Europe (the daily live seems to be quite different from here) and South Europe (don’t like the weather). Scandinavia is too dark in winter, and I am a person that tends to see the world through darker glasses anyway. Central Europe comes as solution.

But I refused to work one more day for free (did that for 10 months) or for a stipend: I wanted to pay taxes! Paying taxes makes me believe I have a proper job, a job that will secure my retirement, my illnesses and even my maternity leave. This is only possible in a few countries: Switzerland is one of them.

There’s a hub for neuroimmunology in Zurich, so I applied here.

When I came for the interview I did not ask

  • How many PhD/master students are you supervising?
  • What do you expect from me in the next 4 years?
  • What do you expect from this project for the next 4 years?
  • How big do you expect the lab to become?
  • What techniques can I learn that will distinguish me from the competition when applying for a new job?
  • What can I bring to this lab, from all the things I have learned?
  • Who is my “go-to” person in case I have questions with data analysis?
  • How reliable is this model? How appropriate is this model to study this condition?

I just read the project description. I talked to the Professor, the 2 junior PIs, and the PhD student that I would replace. I was very happy with what I heard and I had a very positive feeling that our way to go about life matched. I chose the lab because it was quite small (but it tripled in 2 years) and because the project was pretty solid (actually, the model was not working), and just needed some molecular characterisation that I could do (still haven’t done it) and eventually I could starting exploring other angles and putting together bits and pieces.

For some of those questions I got to see/experience the answers in real life and the others I have eventually asked. Other important questions are not to be asked, but to be answered by the PhD candidate:

  • What do you want from your PhD?
  • Do you want to supervise a master student?
  • What do you want to learn that will make you a suitable candidate in your next job? This could be a super-fancy technique, a coding language, or organizational skills.
  • What do you have in your portfolio that you are confident with, and can use as a “reliable, always working method”?

In my case, I wanted to learn immunology, specially about the immune system. This is difficult on a daily basis because the project tends to be way more narrow than the general knowledge in immunology I would like to acquire. I don’t mind supervising master students, but I do not have a structured project they can pursue on their own. I would like to acquire good knowledge of FACS and CyTOF and my go to method is qPCR. It always works.

Adventures around the water bath

The expression of proteins in cellular models or bacteria is quite common in order to study their function and structure or for large scale production.

To make a bacteria express a certain protein, the cells must take up the DNA sequence that encodes for that protein. Because proteins can be modified after production by some cell-specific mechanisms, the final protein produced by a bacteria is not necessarily exactly the same as we would find in the original environment.

Not all bacteria can take up DNA, the bacteria who can do this are called competent bacteria, and to the process of inserting foreign DNA into a bacteria, we call transformation.

About a year ago, I had to do transformation of some competent bacteria. I did not want to express the protein, I needed to be able to distinguish a sequence of DNA that had been mutated from its normal version. We were hoping that in every four bacteria: one would have taken up the mutated version, one would have the control version and the other two would have  a mix of these two sequences. Because bacteria grow very fast, overnight I would have more than 100 colonies, from which I could select 10-20 and hoping “Mendel was on my side” send this for sequencing and voilà: I would know the exact mutation I had introduced in that DNA.

I have discovered the hard-way that even competent bacteria are not so competent if the scientist is not bright. After two months of failing to see bacterial colonies, asking for help from everyone I could find in the lab, making them read my protocol, comparing it to their protocol to no avail, the issue started bugging the professor. On a hallway conversation he says “Are you sure the water bath is at 42 C?” To which I reply “which water bath? I am using the thermo-block.”. It so seems that there is a preference for water baths over heating-blocks among the older generation of scientists. Is this well supported? The short answer is: of course not!

But there are differences between water-baths and heating blocks

Water baths need a very big amount of water to function, thus once they reach a certain temperature they will be very stable in maintaining it constant throughout the entire surface. This means that every single sample will be exposed to the same temperature as the one next to it and that every single particle of a given sample will be at that temperature.

Heating blocks on the other hand are constantly giving away heat, the temperature along its surface varies a bit (I never measured but I wouldn’t expect it to be much more than a few tenths of degree), also the temperature from the bottom of the tube to the upper part should vary a bit.

Is this sufficient to make a difference in getting colonies of bacteria growing?

In our lab, we will never know: people that used to use water bath still use them, people who used heating blocks still use them. And I, I have converted! Working experiments are better than non-working ones!

In any case, how does putting bacteria at 42 C help them taking up DNA?

Basically, we “heat-shock” them: the bacteria are on ice for 30 minutes, all cold and stiff. Then they go directly into 42 C, which makes them more comfortable. Suddenly, after 50 seconds, they go back to ice. As bacteria membranes are made of lipids, and lipids are very fluid, the changes in temperature allow for pores to be open in the cell membrane, through which DNA can move in without resistant.

The one that dances

I am the one who dances.

I have been interested in life sciences research since I have first seen movies about Ebola epidemics. I dreamt of being the one to be called and save the world. I grew up and aware of the difficulty behind saving the world. I haven’t quite given up yet. After doing my bachelor studies in Cellular and Molecular Biology, from FCT/UNL, I went on for a masters in Neuroscience. My stay in Rotterdam (Erasmus MC) made me realise people around the world are more similar than different. I also start taking what is good and I like from the places where I live and try to ignore what is missing. Since then I have also lived in the UK, where I worked in a pharmaceutical company. The shock of moving from academia to industry was only surpassed by the shock of moving back to academia. I have been doing my PhD in Immunology for two years now, studying Multiple Sclerosis in Zurich. I have deleted the genes coding for pattern recognition receptors from mice and now study the effect of this deletion in the onset of experimental autoimmune encephalomyelitis (the mouse equivalent of MS).

The last two years have been a journey of discovery: a PhD project takes time to materialise, research is slow, life is stressful and results are often frustrating. We have a team of about 15 people, they are all working in slightly (or completely) different topics, I learn new thing everyday just by having coffee with my colleagues, and this really keeps me going. I have discovered I like the inter-phases: when talking with someone from a different area, we need to ask more questions, be more precise, adapt vocabulary, stop assuming everyone has the same level of understanding or access to the same information.