The Extracellular Domain of Neurotrophin Receptor p75 as a Candidate Biomarker for Amyotrophic Lateral Sclerosis

Abstract

Objective biomarkers for amyotrophic lateral sclerosis would facilitate the discovery of new treatments. The common neurotrophin receptor p75 is up regulated and the extracellular domain cleaved from injured neurons and peripheral glia in amyotrophic lateral sclerosis. We have tested the hypothesis that urinary levels of extracellular neurotrophin receptor p75 serve as a biomarker for both human motor amyotrophic lateral sclerosis and the SOD1G93A mouse model of the disease. The extracellular domain of neurotrophin receptor p75 was identified in the urine of amyotrophic lateral sclerosis patients by an immuno-precipitation/western blot procedure and confirmed by mass spectrometry. An ELISA was established to measure urinary extracellular neurotrophin receptor p75. The mean value for urinary extracellular neurotrophin receptor p75 from 28 amyotrophic lateral sclerosis patients measured by ELISA was 7.9±0.5 ng/mg creatinine and this was significantly higher (p<0.001) than 12 controls (2.6±0.2 ng/mg creatinine) and 19 patients with other neurological disease (Parkinson's disease and Multiple Sclerosis; 4.1±0.2 ng/mg creatinine). Pilot data of disease progression rates in 14 MND patients indicates that p75NTRECD levels were significantly higher (p = 0.0041) in 7 rapidly progressing patients as compared to 7 with slowly progressing disease. Extracellular neurotrophin receptor p75 was also readily detected in SOD1G93A mice by immuno-precipitation/western blot before the onset of clinical symptoms. These findings indicate a significant relation between urinary extracellular neurotrophin receptor p75 levels and disease progression and suggests that it may be a useful marker of disease activity and progression in amyotrophic lateral sclerosis.

Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment

“…Notably, both of these conditions trigger increased cleavage of p75 NTR (DiStefano et al, 1993;Perlson et al, 2009), and we observed increased p75 NTR ECD levels with SOD1 G93A disease progression, as previously observed in human amyotrophic lateral sclerosis (Shepheard et al, 2014). As a result of c29 treatment, we observed a significant improvement in motor neuron survival after axotomy and in SOD1 G93A animals, and in the latter model, reduced levels of p75 NTR ECD, and a coincident improvement in rotarod performance and a delay in disease-induced weight loss, suggesting that the functional outcomes could be the result of reduced p75 NTR activation and cell death signaling.…”

“…For analysis, we pooled data obtained from consecutive samples such that many data points contain data derived from two samples from the same animals but collected a week or less apart. A sandwich ELISA was used to measure the extracellular domain fragment of p75 NTR ( p75 NTR ECD) in urine as previously described (Shepheard et al, 2014). Briefly, ELISA plates (96-well plates, Costar Corning) were coated for 18 h with anti-p75 NTR ECD MLR1 (4 µg/ml in 25 mM sodium carbonate 25 mM, sodium hydrogen carbonate, 0.01% thimerosol, pH 9.6) at 4°C.…”

“…The intracellular domain contains two regions that mediate intracellular signaling through protein-protein interactions: the juxtamembrane domain, and a tumor-necrosis-factor-receptorlike death domain (Roux and Barker, 2002;Skeldal et al, 2011). Increased metalloprotease cleavage of p75 NTR has been associated with the death of sciatic neurons following injury (DiStefano et al, 1993;Murray et al, 2003), and evidence of increased levels of p75 NTR proteolytic cleavage has been reported using urine analysis in both SOD1 transgenic mice and people living with amyotrophic lateral sclerosis (Shepheard et al, 2014). Elevated levels of the C-terminal and intracellular domain fragments have also been reported in the degenerating motor axons of SOD1 G93A transgenic mice (Perlson et al, 2009), and both p75 NTR cleavage fragments have been linked to death signaling (Skeldal et al, 2011).…”

Fluid-Based Biomarkers for Amyotrophic Lateral Sclerosis

“…Recently, the extracellular domain of p75NTR, which is a regulator of cell survival and death, was shown to exhibit increased levels in urine from ALS patients with limb or bulbar onset as compared with healthy controls [152]. Similar trends were also observed in SOD1 G93A mice [152].…”

“…To the best of our knowledge, only neurotrophin receptor p75 (p75NTR) [152], glucosylgalactosyl hydroxylysine (GluGal Hyl) [153], type IV collagen [154], and 8-hydroxyl-2'-deoxyguanosin (8OH2'dG) [155,156] have been explored as potential biomarkers warranting further studies of urine for ALS biomarkers. Recently, the extracellular domain of p75NTR, which is a regulator of cell survival and death, was shown to exhibit increased levels in urine from ALS patients with limb or bulbar onset as compared with healthy controls [152].…”