Author: Stephanie Shepheard
Shepheard, Stephanie, 2015 Neurotrophin receptor p75 extracellular domain as a novel urinary biomarker in Motor Neuron Disease, Flinders University, School of Medicine
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Motor neuron disease (MND) is a relentlessly progressive neurodegenerative disease, which is typically fatal within 3 years. Objective biomarkers indicative of underlying pathology are crucial for developing treatments, as no effective treatments exist, and the subjective ALS functional rating scale (ALSFRS-r) is the only validated disease progression measure. The common neurotrophin receptor p75 (p75NTR) binds all neurotrophins and is not normally expressed by motor neurons after birth. However, p75NTR is re-expressed in motor neurons of the SOD1G93A MND mouse model, and motor neurons and Schwann cells of people with MND. Crucially, the p75NTR extracellular domain (p75NTRECD) is cleaved from injured motor neurons and detected in rat urine after sciatic nerve injury. The aim of this study was to test p75NTRECD in urine as an objective biomarker of MND. The SOD1G93A MND mouse model was used to investigate urinary p75NTRECD levels in pre-clinical MND, and people with MND were recruited for urinary p75NTRECD investigation in the clinic. The study first addressed the feasibility of urinary p75NTRECD as a biomarker by using novel detection methods of Immunoprecipitation/Western blot and Immunoprecipitation/Mass Spectrometry. Using Immunoprecipitation/Western blot, urinary p75NTRECD was detected in SOD1G93A mice throughout life, but was not detected in healthy control mice until the equivalent of end stage disease. Similarly, urinary p75NTRECD was detected in people with MND, but not healthy individuals via Immunoprecipitation/Western Blot. Urinary p75NTRECD was detected in people with MND using Immunoprecipitation followed by Orbitrap or Triple-TOF Mass Spectrometry, confirming the presence of peptides from only the extracellular domain of p75NTR. A novel indirect sandwich ELISA was then developed to quantify urinary p75NTRECD levels in SOD1G93A mice, people with sporadic MND, and their respective controls. Urinary p75NTRECD was significantly higher in SOD1G93A mice than healthy age-matched controls prior to disease onset, and throughout disease progression to end stage disease. Additionally, urinary p75NTRECD changed in one of two treatments. Riluzole, the only FDA approved MND treatment, had no effect on urinary p75NTRECD levels or survival and behavioural measurements, whereas treatment with a potential therapy called c29 resulted in significantly decreased urinary p75NTRECD and a significant behavioural improvement. This is the first description of urinary p75NTRECD in SOD1G93A mice, showing promise as a biomarker of disease progression and treatment effectiveness. In people with MND, urinary p75NTRECD was significantly higher than healthy individuals and people with Multiple Sclerosis. In addition, MND urinary p75NTRECD trended higher than Parkinson’s disease. As determined by testing pre-symptomatic people carrying SOD1 mutations, urinary p75NTRECD was significantly increased from, but not prior, to symptom onset. In addition, urinary p75NTRECD increased with disease progression, and correlated to the ALSFRS-r scale, time from symptom onset, and diagnosis. This is the first time urinary p75NTRECD has been quantified in clinical MND, showing promise as a prognostic and disease progression biomarker. The results of this study support the hypothesis that urinary p75NTRECD is a biomarker of MND in SOD1G93A MND model mice and people with MND, and establishes a solid foundation for using urinary p75NTRECD as a biomarker in pre-clinical MND models, clinical trials, and the clinic.
Keywords: p75NTR, neurotrophin receptor p75, Motor Neuron Disease, Amyotrophic Lateral Sclerosis, biomarker, urine, SOD1, SOD1G93A mouse
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Dr. Mary-Louise Rogers