New ultrasensitive screens for Parkinson’s disease

David Walt (left) is a professor at Harvard Medical School, with appointments in the Department of Pathology at Brigham and Women’s Hospital (both MA, USA). His lab covers a wide range of topics, including diagnostics and the study of Parkinson’s disease and his recent work has led to an exciting potential new diagnostic test for the condition.

Here he reveals a new alpha-synuclein assay approach that could fast track the diagnosis of Parkinson’s disease, documented in a recent paper, and allow researchers to catch the condition at an earlier stage when more can be done to delay its progression. He also reveals his ongoing work with extracellular vesicles and their diagnostic potential for neurodegenerative diseases.

What are the current standard procedures for Parkinson’s disease diagnostics?

The current procedures encompass two different realms. The first, which is primarily used, relies on clinical symptoms; clinicians will observe key characteristics in their patients and then put them through a series of cognitive and functional tests over a period of time. This allows them to assess whether their patients’ performances are deteriorating with respect to their responses to these standard tests.

The other method is used if somebody is suspected of having Parkinson’s disease, and needs confirmation. In this case, there are imaging tests involving the injection of a radioactive tracer followed by a SPECT scan of the brain, which interrogates dopamine receptors in the brain.

Where do these current diagnostic practices fall short in the management of Parkinson’s disease?

Both of these tests essentially monitor the symptoms or later-stage manifestations of the disease, they don’t get down to molecular-level resolution. Therefore, they don’t get to the fundamental underlying biochemistry of the disease. As a result, you can’t catch the disease very early in its progression or use these tests to help investigate the pathophysiology of the condition.

The current tests are only carried out once symptoms begin to appear and at that point, the disease has already progressed significantly. The brains of patients who have passed away at different stages demonstrate that even at early symptom onset, there is already significant deterioration of brain tissue due to the formation of plaques.

How is your lab working to address these shortcomings and improve our diagnostic capabilities for Parkinson’s?

My lab has historically focused on developing new technologies that can address unmet clinical diagnostic needs. We do that by developing ultrasensitive methods for detection. The most well known of these is a single molecule array technology or ‘Simoa’, which is able to digitize the immunoassays that have been used for many years. This digitization enables us to detect proteins that are up to 10,000 times lower in concentration than traditional immunoassays used in clinical laboratories for the last four decades.

We have a new technology that we reported last year called Mosaic, which is an even more powerful technology able to detect very low concentrations of molecules in both cerebrospinal fluid (CSF) as well as in blood plasma. That’s where our focus lies now; in trying to detect these very low-concentration markers that are specific to neurodegenerative diseases. By detecting these low-level biomarkers, we hope to pick up very early indications that somebody’s going to develop Parkinson’s so they can be put on an interventional management plan early.

What molecules are you looking to detect for Parkinson’s disease?

One of them is alpha synuclein, the protein that generates fibrils in the brain that form deposits or plaques that are certainly a signature of the disease. What we are trying to do is detect alpha-synuclein fibrils circulating in the bloodstream, which indicates that there is some abnormal process generating these fibrils in patients who ultimately will develop Parkinson’s.

But there are other markers that we’re also focusing on. We’re looking at post-translationally modified proteins that indicate there is adverse metabolism taking place. We’re doing that by isolating extracellular vesicles, abbreviated as EVs, which are tiny packets containing cell contents wrapped in a section of the cell membrane that has split off from a cell. We hope we can isolate these EVs from the brain, pull them down, isolate their contents, and measure alpha-synuclein fibrils and other proteins that might indicate that there’s altered metabolism in the brains of these individuals.

What are the pros and cons of blood and cerebrospinal fluid (CSF) as sources for the detection of alpha synuclein and extracellular vesicles?

The difference between CSF and blood plasma is an important one. It is generally accepted that if you’re going to try to find biomarkers that are released from the brain, CSF is the ideal fluid because it’s in direct contact with the brain but no other tissues and organs in the body. The problem with using blood is that both EVs and alpha synuclein are present in lots of tissues. So either of these biomarkers, if detected in the blood, have the potential to originate from some other tissue besides the brain.

What’s more, the blood–brain barrier prevents things from crossing into the bloodstream, so the amount of material from the brain that gets into the blood is very low in concentration. This is why you need ultrasensitive methods to detect them. One of the reasons that we’re using EVs is that we can pull down EVs from the blood that have specific proteins on their surface that are only present in the brain. By doing that, we hope that we can burst those EVs and analyze their contents to see what is in the cells of the brain exclusively.

For the alpha-synuclein assay, our initial focus was on demonstrating its utility in CSF, where we could be more certain that it had come from the brain. As we develop these specific pull-down capabilities, increase their sensitivity and are able to detect and extract EVs from the brain in the blood and search for Parkinson’s specific proteins within them, we will then move to analyzing blood plasma. Blood plasma is obviously much more amenable to widespread use. People who are suspected of having Parkinson’s may be willing to give a spinal tap to extract CSF, but as a screening tool, it’s an invasive and uncomfortable procedure, so won’t be something that can be done for millions of people.

An important point to note is that, while alpha synuclein may be found in these EVs, we don’t necessarily need to use the EV approach to ensure their detection is specific to Parkinson’s. This is because if we can detect alpha synucleins are aggregated into fibrils in the blood, wherever they are coming from, it’s a bad sign that indicates the early stages of Parkinson’s. So if we can detect these alpha-synuclein aggregates using our ultrasensitive method, then we know that individual has the possibility of a Parkinson’s disease diagnosis.

How have you achieved this level of sensitivity with this assay?

It’s actually quite simple. We partition a sample of the patient’s CSF or blood into many different reactors, such as a microwell array with many ‘microwells’ that each contain a small volume of the sample.  The wells contain such a small volume that there can only be at most one molecule of the biomarker we are looking to detect in each microreactor. Most of the microreactors do not contain a biomarker molecule but occasionally there will be a single molecule present. We then seal the microreactors so the molecule can’t escape.

For the alpha-synuclein assay, we put monomers of alpha synuclein in those wells along with a dye that will bind to an alpha-synuclein fibril. Over time, if there is a single fibril trapped in one of these microreactors, it will react with other monomers of alpha synuclein and create larger fibrils, to which this dye will bind. Once bound the dye will fluoresce, creating a readout.

We can then count the number of microreactors that have a fluorescent signal, giving us a quantitative measure of the number of alpha-synuclein fibrils in the sample. The advantage is that if you’re trying to see if a patient is progressing, you can take samples six months apart, and you see there’s an increase in the number of alpha-synuclein fibrils present, then you know that patient is progressing and at what rate.

We also showed in our recent paper that we can use the test to identify inhibitors that prevent alpha-synuclein aggregation. If these inhibitors are eventually approved for drug use, then they could be used to treat individuals at much earlier stages when the alpha-synuclein fibrils are first detected in their blood. The hope is that these drugs would be efficacious in individuals at a much earlier stage of the disease than symptomatic patients who are currently enrolled in studies.

How can this assay be applied to the EV approach you mentioned earlier?

To answer this question I need to go back a little. A couple of years ago, we published a paper detailing the use of a marker called L1CAM that everyone in the community was using to pull down neuronal EVs. We showed that this marker was not actually specific to the brain or, in fact, even a transmembrane protein; it was just something that happened to get stuck to the outside of these membranes. We felt an obligation to the community to go on a search to find something that actually worked to pull down brain-derived EVs.

We’re currently in the final stages of validating a number of markers that we’ve discovered that will allow us to fulfill that obligation, which we will report soon. We have been able to use these markers in this assay to pull down neurospecific EVs, which we can break open and load into the microwells and then follow the same procedure as I described in answer to one of your prior questions.

If there was one piece of information or a fantasy technology that you could access, to better understand Parkinson’s disease and its diagnosis what would it be?

It’s a great question. My biggest wish would be to have a multiplexed panel of markers circulating in the bloodstream that would enable us to specifically identify a patient early in the disease process. Currently, most of the biomarkers identified are also present in almost all tissues. I’d love to find something that is unique and specific to an altered brain that we could measure at a very early stage and by identifying that marker, we would also be able to elucidate the underlying mechanism of the disease.

To me, it’s unclear whether alpha synuclein itself is a cause or a correlate of disease. I would love to have something that is a confirmed causative biomarker, that helps us not only detect and diagnose disease but also helps us understand the disease at the early stages, such that we could intervene therapeutically.