Test twice, treat once The PNE (polyneuritis) plan
We know more now than we did in 2012 when we met Keno, beloved by Polly Miller. He eventually succumbed to his disease, but we still remember him. In this photograph, he is ponying Cupcake during a period of remission. Polly sent us this picture saying “It was a great day with my ponies!”
Unfortunately, we didn’t keep a blood sample, we discard them after six months. But we would bet our bottom dollar we could measure neurofilaments to help us manage Keno’s disease. If we just had a time machine.
Making the point that species differences matter
Spinal ganglion neurons in rodents are subdivided on the basis of their phenotype when examined by electron microscopy into light and dark neurons. Horse spinal ganglion neurons show neurochemical differences when compared to those of rodents. We are interested in the role of neurofilaments, neuron-specific cytoskeletal proteins, as biomarkers for our work in amyotrophic lateral sclerosis (ALS) and equine neurological diseases. Hold that thought that there are differences between species.
What are neurofilaments?
Neurofilaments are structural proteins of neurons. They play a central role in the transport of molecules throughout the axon and contribute to the integrity of the neuronal cytoarchitecture. When cells are damaged neurofilaments are released. They are made up of three subunits, a heavy chain (NfH), a middle chain and a light chain (NfL). Interestingly, neurofilaments are the most abundant cytoskeletal molecule in myelinated neurons.
The levels of NfL differ between species in progressive disease, but the interpretation is similar
In humans, the level of neurofilament chains in the blood of ALS patients was associated with progression of disease and risk of death. More importantly, progression of NfL in the blood forecast the conversion from the presymptomatic to the symptomatic stage of disease and the time of survival. Rodent models of axon-associated disease also show a progression of neurofilament levels in blood, but maximum values differ from levels in humans. These important structural molecules are conserved between species but differences in neurochemicals mean the testing reagents need to be selected with care.
Neurofilament proteins are released when the neuron is damaged in disease, such as inflammation or trauma. Measuring neuronal degeneration or axon damage is desirable for monitoring chronic neurodegenerative diseases in people and horses. There are two interesting (to us) neurofilaments that can be measured, NfL and NfH.
The pNfH is a phosphorylated form of the protein found in the fluid in the central nervous system. There are studies that correlate pNfH with EMND and EDM when compared to clinically normal horses. Some laboratories use pNfH to definitively diagnose EDM in horses. The values are only informative if elevated “but a “normal” value of less than a nanogram in a milliliter of serum doesn’t rule the disease out.” The interpretation is that neurologic disease is “very likely with a CSF NfH value of 3 ng/ml or more suggesting EDM or cervical vertebral compressive myelopathy.” The original manuscript suggests better results could be obtained by developing more sensitive reagents for horses. No truer words are spoken!
NfL is useful in detecting and monitoring PNE
The NfL protein is detected in serum or CSF during disease using commercial kits for people and horses. We compared the levels of NfL in a set of diseased horse sera, clinically normal horse sera, and levels in sera from an ALS patient. Surprisingly, the horse kit was more sensitive for detecting human NfL! When we got to the root of the problem, we found the reagents in the equine kit were really human antibody specific! We purchased the MSD Mesoscale system that has validated NfL assays and repeated our work. The take home message is one needs experience and an inquisitive mind to interpret NfL test kits and values in diseased animals. Heads up researchers! Plasma exosomes may be a more sensitive source for NfL in horses.
NfL stands out for detection of PNE but not EPM or EMS
Horse sera was interrogated for serum/plasma NfL concentration starting in 2018. Serum/plasma values in horses with and without neurological disease that had a presumptive diagnosis of EPM due to Sarcocystis neurona were selected. Values >1 ng/ml in horses with clinical signs of disease were in 35% of the samples from suspect EPM cases. Surprisingly, only 1 clinically normal horse was positive for NfL.
It is important to recognize infections with Sarcocystis fayeri are related to equine neurodegenerative disease and may confound interpretations of neurofilament values in horses. Our paper showing a correlation between S. fayeri anti-toxin, measured by ELISA, and neurological disease is available. The data showed that 23% of horses with clinical signs of disease and seropositive for S. fayeri, had a NfL concentration of >1 ng/ml.
Read this paragraph twice!
Diseased horses seronegative for S. neurona or S. fayeri anti-toxin were tested and risk of death was accurately predicted in the horses with circulating MP2/MPP antibodies and an elevated NfL. Sixty-four percent of clinically ill horses with a presumptive diagnosis of PNE had an abnormal NfL value.
Said another way, the majority of EPM-suspect horses with clinical signs of disease and antibodies against S. neurona did not have circulating NfL (65% had values <1 ng/ml). Similarly, the majority of horses with EMS, equine muscular sarcocystosis, detected by S. fayeri anti-toxin antibodies did not have abnormal NfL, 77% were negative.