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 SOD1(G93A) 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 p75NTR(ECD) 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 SOD1(G93A) 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.

Figure 1. Human p75NTR extracellular domain (p75NTR^ECD) was detected in urine of humans living with ALS by immuno-precipitation/western blot.

Human p75NTR^ECD was detected after immuno-precipitation (IP) of 500 µg urinary protein from ALS patients 1 and 2 (lane 4, lane 6) but not from healthy controls A and B (lane 5, lane 7). Human p75NTR^ECD (∼50–55 kDA) and full-length p75NTR (p75NTR^FL, ∼70–75 kDA) from A875 melanoma cells (5 µg lane 3) was enriched by IP of 500 µg of cell lysate (lane 2). No p75NRTR was detected after IP of control cells lacking p75NTR (BSR, 500 µg, lane 1). The light chain of IgG (25 kDa band) was detected after western blot (WB) of samples subject to IP.

Figure 2. p75NTR^ECD was identified by IP/WB of urine from SOD1^G93A mice before motor symptoms were detected.

A. Mouse p75NTR^FL and ^ECD from motor neuron-like NSC34 cells (5 µg, lane 1) was enriched by the IP/WB (500 µg, lane 2) and not detected in control IP where p75NTR^ECD is not present (BSR, 500 µg. lane 3). B. Symptoms of amyotrophic lateral sclerosis disease were found in SOD1^G93A mice from 100 days by the hanging wire grip duration test (n = 10) in comparison to none in C57BL/6J (B6) control mice (n = 10). End stage (ES) was at 150 days for SOD1^G93A mice. C. p75NTR^ECD was detected after IP/WB of urinary protein from 60d (lane 4) and 80d (lane 6) pre-symptomatic SOD1^G93A mice and also 100d (lane 8) and end stage (150d, lane 10). No p75NTR is seen in age matched C57BL/6J control mice until end stage (150d, lane 9). 110 µg of urinary protein was used for each IP of mouse urine and all IP/WB were repeated 3 times with similar results. The light chain of IgG (25 kDa band) was detected after WB of samples subject to IP.

Figure 3. There are significant amounts of p75NTR^ECD in the urine of ALS patients compared to healthy controls or people with other neurological diseases.

A. p75NTR^ECD levels measured by ELISA were significantly higher in all ALS patients (****; p<0.001, n = 28), including limb (ALS-L; n = 16) and bulbar (ALS-B; n = 12) onset in comparison to healthy controls (n = 12). ALS patients with limb onset disease trended to higher mean levels of p75NTR^ECD than bulbar onset but this was not significant. B. Levels of p75NTR^ECD detected in patients with other neurological conditions (OND; n = 19) are not significantly different to that seen in healthy individuals and are significantly lower (***p<0.001, n = 12) than that seen in ALS patients. All samples were assayed four times in quadruplicate. Levels of urinary p75NTR^ECD were standardised to urinary creatinine and data was analysed with one-way ANOVA and Bonferroni's multiple comparison post-hoc test, with significance at alpha  = 0.01 (1% level).

Figure 4. Receiver Operating Characteristic curves for distinguishing ALS patients from A. healthy controls and B. patients with other neurological conditions (OND) using p75NTR^ECD.

A The area under the curve (AUC) indicated ALS patients are distinguished from controls 100% of time (AUC: 1.0) with a 95% confidence interval of 1. B. In comparison, ALS was distinguished from OND (Parkinson's and Multiple Sclerosis) 96% of time, with a 95% confidence interval of 0.9 to 1.1. The cut-off value for distinguishing ALS patients from healthy controls, using the Youden index (Baker and Krame, 2007) was >4.8 and from OND >4.8 (ng p75NTR^ECD/mg creatinine).

Figure 5. There are significant amounts of p75NTR^ECD in the urine of rapidly progressing compared to slowly progressing ALS patients.

Scatter plots show urinary p75NTR^ECD concentrations in ‘slowly progressive ALS’ (n = 7) and ‘rapidly progressive ALS’ (n = 7). The horizontal lines represent the mean, with 95% confidence interval. Progression rate is decline in ALSFRS-r per month. *Mann–Whitney test (p = 0.004).