Figure 1.
Human p75NTR extracellular domain (p75NTRECD) was detected in urine of humans living with ALS by immuno-precipitation/western blot.
Human p75NTRECD 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 p75NTRECD (∼50–55 kDA) and full-length p75NTR (p75NTRFL, ∼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.
Table 1.
Identification of p75NTRECD peptides after Mass Spectrometry of Urine subject to IP.
Figure 2.
p75NTRECD was identified by IP/WB of urine from SOD1G93A mice before motor symptoms were detected.
A. Mouse p75NTRFL 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 p75NTRECD is not present (BSR, 500 µg. lane 3). B. Symptoms of amyotrophic lateral sclerosis disease were found in SOD1G93A 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 SOD1G93A mice. C. p75NTRECD was detected after IP/WB of urinary protein from 60d (lane 4) and 80d (lane 6) pre-symptomatic SOD1G93A 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.
Table 2.
Characteristics of Individual ALS Patients and healthy Controls Included in the study.
Figure 3.
There are significant amounts of p75NTRECD in the urine of ALS patients compared to healthy controls or people with other neurological diseases.
A. p75NTRECD 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 p75NTRECD than bulbar onset but this was not significant. B. Levels of p75NTRECD 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 p75NTRECD 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 p75NTRECD.
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 p75NTRECD/mg creatinine).
Figure 5.
There are significant amounts of p75NTRECD in the urine of rapidly progressing compared to slowly progressing ALS patients.
Scatter plots show urinary p75NTRECD 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).
Table 3.
Characteristics of Patients in ALS Progression Study.