AAN 2025 | What does a positive test for Parkinson’s ‘seeding’ mean? Fact and fiction

Hello, I’m Alberto Espay from the University of Cincinnati. Today I’m presenting what is fact and what is fiction from the alpha-synuclein seed amplification assay, which has become one the most important areas of endeavor in the last year or two. These are my disclosures and I do not think any of them are relevant to the topic and their discussion. First, the seeding. There are different definitions of seeding and it’s important to know that from a biophysical perspective, seeding is the precipitation of proteins along a thermodynamically favorable pathway of nucleation. That’s important because the key points I’m going to make with regards to that phenomenon underpinning the seed amplification assay is that this is a physical phenomenon that’s governed by nucleation, not replication. The pathology is a product of nucleation, not replication again. The seeding assay is a sensitive test to identify the presence of pathology, but it’s a qualitative test that cannot measure how much of that pathology there is, nor how little of the monomeric synuclein there remains in the tissue tested. So if you look at this brain and pay attention to nothing else, you’ll see that the brain changes, but you might have failed to recognize that something else changed too, and that is the environment in which that brain is. And that’s because to have clumps of protein, which we call pathology, there has to be a transition from low levels of pathological synuclein to high because monomeric synuclein has undergone a phase transition, which is shown here in an example of a water to ice transition, which can be, of course, catalyzed by seeding, as it is in this example. So a chunk of ice acts as a seed to accelerate the phase transition process. This is just a complicated slide, but it’s the only complicated slide that I wanted to put out here just to understand this issue of energy with regards to concentration. So what you see first is that under normal conditions, a protein assumes its thermodynamically favorable conformation, which is soluble and usually the monomeric state into which most of the proteins are. Now, by the way, this is the state of monomeric alpha-synuclein. So when the concentration increases and it becomes supersaturated, then a different energy equilibrium is obtained because there is a greater interaction between different proteins and therefore you force the aggregation of them into the amyloid state of proteins, which is a cross-beta configuration and is what we know as pathological alpha-synuclein. There is a barrier between monomeric and pathological, and that barrier is overcome or lowered by the addition of either heterogeneous nucleation, the case of, say, a viral infection, or a seed. A seed, which is kind of like the example I showed you in the prior video, an aggregated protein is going to catalyze that transition between monomeric and pathological. So this is what you typically see in a seeding assay. If seeding is to occur, there is a monomeric to fibular or amyloid state. We call that a process of crystallization just because it is where the physics have mostly been worked out, but in the case of proteins, we use the term polymerization. If you add an aggregated protein, a seed as we know it, you are catalyzing nucleation and that curve moves to the left. It’s a faster nucleation. So essentially, to go from a high monomeric state to a low monomeric state, there has been a transition in three different forms. We call them heterogeneous nucleation, seeded nucleation, or homogeneous nucleation. And you see in each of these conditions, protein concentration must be high. When it is low, the nucleation doesn’t happen. So again, this is a process of precipitation, nucleation, that is explained by these physical principles that I’ve mentioned before. And it’s not replication. It’s important to distinguish replication from nucleation because one is active replication, but the other one is passive. It’s a passive spread that’s governed by essentially this supersaturated state. It requires a few ingredients such as temperature, pH, and a surface catalyst in addition to supersaturation, not dependent on what kind of conformation of the amino acids any protein may have. Replication, on the other hand, must be done faithfully because it is a code that in an active dependent fashion, ATP dependent fashion, will create identical copies. What you end up with replication are exact copies of the original. What you end up with nucleation are polymorphs, which are infinitely variable formations of pathological states. So the synuclein seed amplification assay, therefore, is dependent on this idea that high concentration will create the conditions for nucleation to occur, but low concentration will not. And so you won’t see the seeding signal that is typically what we’re looking for to define if there is a positive outcome to the seed amplification assay. Now, normally, the concentration of the proteins in the brain is measured in picomolar range, essentially picograms per ml. That’s too low for the seeding assay to occur. So in the laboratory, we increase that concentration one million fold to micromolar. and in that case we magnify the signal, we make it more thermodynamically likely that proteins will undergo this phase transition. So what we see with a positive seed amplification assay in the laboratory is not an indication of pathogenesis in the human brain or pathogenesis in the individual that we’re testing, much less an indication of biology. Now, a positive seed amplification assay is really good for one thing, which is to identify the presence of pathological synuclein. It has a very high sensitivity and specificity for the clinical diagnosis of Parkinson’s. However, cannot discriminate between motor and non-motor forms of Parkinson’s, between whether there is cognitive impairment or not, etc. Because it is a qualitative test, present versus absence. It really is unable to quantify how much of the proteins there is either in the monomeric or pathological state and therefore it doesn’t correlate with severity of a patient it doesn’t predict what kind of progression anyone will have and is unable to help us with subtyping either clinically or biologically. And importantly, it cannot be used for monitoring response to treatment because the only way to turn from positive to negative would be to eliminate synuclein from the body, which would be not ideal at all. The consequences of a negative alpha-synuclein seed amplification assay is that it’s actually quite interesting. It tells us about a subpopulation of patients with LRRK-PD. About a third of them do not have synuclein. So this is a good feature of them. But then what do we do with these individuals? How do we think about them? Do they not have Parkinson’s? And it’s interesting that it is associated with a higher neurofilament-like level, which means that there probably is some degree of accelerated degeneration in the absence of synuclein aggregation, which has been conceived as the potential for helping neurons withstand the forces of apoptosis and other detrimental agents. And this is work that Bob Burke and colleagues did in Columbia University in the late 90s. Now, to end, what does a positive alpha-synuclein seed amplification assay mean? Well, it is a very sensitive test to identify pathology but cannot tell us anything about pathogenesis. Supports a clinical diagnosis of Parkinson’s, but it cannot make the diagnosis in an individual without symptoms. The seeding amplification assay is qualitative and therefore is incapable of quantifying how much we have of the pathology, how much less we have of the monomeric peptides from which it comes, cannot help for that reason with clinical or biological subtyping, with prognosis, with disease staging, or with treatment response. Therefore, it helps one thing and one thing only. It helps with defining the extent to which we support the clinical diagnosis of Parkinson’s but cannot be construed as meaning anything else. Thank you.

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