Skip to content

horseontrailFinding biomarkers that reflect the amount of peripheral nerve damage (peripheral neuropathies) and that the biomarker will quickly drop in value in response to to effective treatment are desired goals.  The tools we need for developing biomarkers for equine neurodegenerative diseases are not available.  These tools include a laboratory model for each neurodegenerative disease, putative treatments, and a money bin.

There is an alternate path leading to biomarker development and that is the horizon we are chasing.  The biomarker quest project identifies natural cases of disease with neurodegeneration followed by evaluating the data from those cases. Sifting through the data is a process of eliminating the negative, selecting the positive, and interpreting the in-between.  I hear a jingle in there somewhere! Generally diseases follow a typical course, or pathogenesis.  Interpreting enough cases points toward the direction we should take and where to concentrate our assets. Often clues to a direction come from previous researchers.

Our assay to detect anti-myelin protein antibodies was described in the literature over 25 years ago.  Researchers used data from horses with polyneuritis equi (PNE) to study human neurodegenerative diseases. Since then novel developments in molecular biology, such as learning how to fold proteins and identifying sequences (genetic code) that are key elements in inflammation, refined the bioassay’s.  The anti-myelin protein antibody tests affirm a specific disease pathogenesis for PNE in humans and horses that involves myelin degeneration, exposure of the immune system to the myelin protein, and production of anti-myelin protein antibodies.  A boon to our work is being able to glean information from human literature and apply it to our horse cases.  An appropriate application from new human neurodegenerative research is an assay for serum or plasma neurofilament light, NfL.

Neurofilaments are the major cytoskeletal proteins of neurons in both the central nervous system (CNS) and peripheral nervous system (PNS).  Neurofilaments form a structure (lattice) composed of light, medium, and heavy chains. Damage to nerves releases fragments of the neurofilament proteins into the central nervous system fluid (CSF) or circulatory system (plasma or serum). The elevation of neurofilaments in the CNS was observed in patients with amyotrophic lateral sclerosis over 20 years ago. Other neurodegenerative diseases also result in the release of NfL.  Abnormal levels of neurofilaments are associated with the disease process and are not necessarily specific for the etiology.

What is interesting is that human patients with demyelinating and axonal forms of an inherited neuropathy exhibit a slowly progressive, axonal degeneration at a constant rate.  Patients with the inherited disease were monitored for NfL over time and the plasma concentration of NfL increased when values were compared to age matched, healthy controls. It was noted that plasma NfL (pNfL) concentration increases with advancing age in some normal subjects.  An increase in disease severity was correlated with pNfL and pNfL discriminated between patients with the disease versus healthy controls. There are sub-types of this inherited, human neurodegenerative disease and NfL was elevated in all forms of disease. In humans, NfL is elevated in several other neurodegenerative diseases.  Because NfL isn’t specific to etiology it wont’ be useful by itself for a diagnosis; however, because it is a dynamic measure of axonal damage, NfL promises utility for monitoring  a response to treatment. There’s a path we intend to follow!

NfL may be an important biomarker when evaluating a disease known to show no CNS involvement.  In these cases,  changes in concentration of NfL would be attributed to peripheral neuropathies. One caveat is that NfL may be elevated in a T-cell proliferative disorder because neurofilaments are expressed in human T lymphocytes. In human studies, NfH (the heavy chain) was not correlated with disease severity. One proposed reason for the lack of correlation between disease and NfH concentration is that NfH aggregates form and these protein clumps lower detected levels of NfH in fluids.

In humans, and horses with polyneuritis equi,  the gold standard for measuring disease severity for patients is a clinical score. There are several limitations to the clinical score including the scale and a ceiling effect for the most severely affected individuals. It is worth mentioning that a therapeutic benefit for neurodegenerative diseases is to stop progression of disease and is most useful early in the disease process.

A blood biomarker may be more sensitive to multifocal peripheral nerve disease, such as polyneuritis equi.  If proven, that means that when neurofilaments are detected in the serum and disease is supported by the clinical exam, treatment can begin before severe irreparable damage occurs. The most useful interpretation of NfL bioassay is a change in an individual over time or with treatment because intrasubject variability is not expected. The intersubject variability in NfL concentration is a factor we are examining between treated and not treated horses.  Our data may indicate how one horse responds is more meaningful than how groups of horses respond.  That’s why we have our biostatistician on instant redial!

Our goal is to determine the responsiveness of NfL concentration in the clinical course of PNE and relate the assay to demyelination using a second bioassay that detects antibodies against exposed myelin proteins. We have some data to discuss if it relates to a specific case you are working with, the data we have will be at least a year from publication.  If we can help give us a call, we can guide your test selection.

Sometimes we are asked what leads us down a specific road or what inspires an idea.  It is generally the need to find a solution to a problem that is an “outlier”.  An outlier is a case or situation that doesn’t fit the rest of the data.  Everything isn’t a bell-shaped curve, although that is where we start.  As the Covid19 Pandemic sweeps the country, everyone is becoming savvy with interpreting clinical disease and seropositive test results. We thought it would be fun to let you interact with our data as we develop our newest idea.

Our recent work with amyotrophic lateral sclerosis (ALS)  led us to neurofilament light, NfL, a possible biomarker for disease in equine neurodegenerative disease. Serum NfL is a validated marker for ALS as well as a potential pharmacodynamic biomarker that is relevant to ALS therapy development.  It is possible that we could measure NfL in horses and use the result to select candidates for a field study as well as determine a response to treatment.  NfL can change in days with an effective therapy.

We started the analysis of banked serum from some of the cases on which we consulted and will look for patterns.  Our objective is to identify disease conditions and clinically validate serum NfL as a prognostic in our work. When we are satisfied we will send all the data to our biostatistician, the real number cruncher. Of course, he always says we have a biased data set-we look at horses that veterinarians suspect equine protozoal myeloencephalitis or polyneuritis equi.  We have some “gold standard” negative sera and even sera from other countries in which some diseases aren’t present.

What does early, exciting work look like? Here is the result.  Nine sera were selected and examined against many controls.  We detected NfL in horses. A high value in our clinical case analysis was 46.04 ng/ml and the lowest 2.78 ng/ml.  We will select more cases and update our graph so you can follow along! Most likely the graph will get more complicated as we add horses with and without disease, and before and after treatment.

image

If we selected a sample from a horse that you submitted you already got the result by email.  If you would like us to add your sample to our early analysis, please give us a call at the lab.  We are all here hunkered down and continuing to offer you cutting edge work.