Rebel without a Cause - Searching for the Origins of Parkinson's Disease with Wes

"It’s had a significant impact on our research... we get double the amount of data in a third of the time."

- Dr. Mattia Volta, Senior Researcher, EURAC Research

Dr. Mattia Volta

More than 10 million people worldwide suffer from Parkinson’s disease, a brain disorder that impairs movement. A burning question at the center of Parkinson’s disease research is its origin, which is needed to better design therapeutic strategies. Dr. Mattia Volta, a Senior Researcher at the Institute for Biomedicine at EURAC Research has dedicated his career to understanding Parkinson’s disease.

In the race to identify a cause, Dr. Volta was one of the earliest adopters of Wes™, a Simple Western™ instrument from ProteinSimple. Wes is a capillary-based automated immunoassay platform that generates high quality Western blot data in only 3 hours. Additionally, Wes...

• needs only 3 μL of sample
• has pg-level sensitivity
• is open to any antibody for detection
• runs up to 25 samples per run
• has built-in analysis software for immediate quantification

Here, Dr. Volta speaks candidly about why he uses Wes to shed light on the origins of Parkinson’s disease.

WHAT’S THE IMPORTANCE OF YOUR RESEARCH INTO PARKINSON’S DISEASE?

My research is focused on trying to elucidate mechanisms relevant to Parkinson’s disease pathogenesis – ideally, we want to understand how the disease begins. This is a very important question because if we know how Parkinson’s disease starts, we can better design therapeutic strategies. For these to be effective you need to know what to target— what is dysfunctional—in order to try to correct it.

To study pathogenesis, we take information from genomics and genetics. We use genetic models—there are a few forms of Parkinson’s disease that are linked to specific mutations in specific genes. These mutations can serve to represent the majority of Parkinson’s disease patients in terms of clinical and neuropathological presentation. Then, we use genomic information which tells us about genetic risk factors. These are not causal mutations, but are associated with Parkinson’s disease – their presence increases the risk of developing the disease by a small percentage. This expands our information from a single protein (causal genetics) to a cellular pathway that might be involved (genetic risk factor information).

We are focused on leucine-rich repeat kinase 2 (LRRK2) and alpha-synuclein as a window into Parkinson’s disease. We then use the information from genomics to identify which pathways these Parkinson’s disease proteins might play common roles—for example in autophagy—which is the main focus of my group’s research.

It’s well known that LRRK2 and alphasynuclein regulate autophagy and that they affect and impair it in the presence of a Parkinson’s disease-linked mutation. We are trying to find other players that might explain how LRRK2 and alpha-synuclein modulate and impair autophagy.

HOW DO YOU USE WES FOR PROTEIN QUANTIFICATION IN YOUR PATHOGENESIS WORK?

We use Wes for relative quantitation of specific protein abundances in some lysates, for example tissue lysates, and to study phospho-proteins in order to get a picture of signaling pathways. We’re most interested in signaling pathways, and for this we specifically look for phosphorylated proteins, as these tell us if a pathway is activated. For example, p38 phosphorylation is a marker of activation of this kinase.

There is a particular protein in the LRRK2 pathway that we only use Wes for and that’s Rab10. This is a key protein in our research because it is directly phosphorylated by LRRK2. It tells us if LRRK2 is active or not which is very, very important for us.

We use Wes to look for Rab10 in both in its native and phosphorylated form because Wes is very sensitive and gives us very clean results – it’s just perfect.

Wes analysis of levels of phosphorylated Rab10 in (from left) naïve SH-SY5Y cells, cells expressing WT LRRK2, cells expressing LRRK2-G2019S

Figure 1.  Wes analysis of levels of phosphorylated Rab10 in (from left) naïve SH-SY5Y cells, cells expressing WT LRRK2, cells expressing LRRK2-G2019S and then the latter with application of a LRRK2 kinase inhibitor at 2 different concentrations, showing the reduction of phospho-Rab10.

HAS WES’ ENHANCED WESTERN BLOTTING-LIKE RESULTS IMPACTED THE SPEED AND SCALE OF YOUR PARKINSON’S DISEASE RESEARCH?

I would say it’s had a significant impact on our research. I think the main advantage for us is the time that we save because it’s much faster than traditional Western blot. The speed with which results can be obtained means that we can run, for example, two runs or 44 samples a day - with Western blot if you push it you can get 17 samples in two and a half to three days. We get double the amount of data in a third of the time.

There’s also the added advantage that during the three hours of the run, the operator is free to do other things. So, the gain in time is much larger than just the difference in operating time. This also means we can let it run overnight and get the results the next morning which is very useful.

The simplicity of use is really great. Anyone can be trained on Wes in just half a day. There are students who just started in the lab who were proficient on Wes before they were proficient in Western blot because it’s so easy and very, very intuitive. I also personally like the software a lot. It’s very simple and young students don’t have any problems getting on the system or with the data processing.

Depending on the protein target, we also get better resolution with Wes and better quantitation. It does depend on the antibody, but when we have a good antibody, Wes is much better than Western blot. Also, for a Western blot, you need substantial amounts of protein to be able to clearly see changes or even to identify the protein of interest. With Wes that can be reduced a lot - you don’t need massive overexpression to visualise a specific protein because the machine is much more sensitive.

HAVE YOU COME ACROSS ANY IMMUNOASSAY TESTING OR QUANTIFICATION ISSUES WITH WES?

No, we haven’t really had any. We go through a setting up stage whenever we probe for a new protein—for example, in a new matrix or a new cell line—but that just takes a day and then usually, immediately after the first run, we normally find the right conditions straight away. Then, we can just process the samples for the experiments right after.

Of course, it’s an immunoassay so if the antibodies aren’t great, then the results aren’t great, but that’s irrespective of the technique. However, I would say, that even if it’s not a great antibody then Wes is still greatly superior to Western blot. We haven’t had any problems with sensitivity or specificity.

A good example of when Wes outperformed Western blot was with p38. The Western blot for the phosphorylated active form of p38 is terrible because p38 has four different forms, which are basically the same molecular weight and overlap a lot. The chemiluminescence images were on top of one another, and for some reason, on membranes the antibodies seem to lose a lot of specificity.

When we ran the same antibody on Wes with its capillary system the peaks were beautiful and separated. This means that the antibody works well, but not on Western blot. It convinced everyone there and then that the machine was a great asset!

HOW DO YOU SEE WES FITTING INTO YOUR FUTURE PATHOGENESIS WORK AS YOU MOVE FORWARD?

I think we will be able to use Wes for any aspect of our project because to understand cell biology, we need to clarify signaling mechanisms. When we investigate a signaling mechanism, we go down to single proteins to quantify and see how proteins are different in abundance or in phosphorylation or post translational modifications.

To do that you need a technique like Western blot or Wes. For us Wes is doing a much better job and it allows us to do more experiments and faster. I think that’s a pretty good deal for us.

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