03 Mar 2017

In the February 22 Neurology online, scientists reported that a fragment of the p75 neurotrophin receptor could mark progression for amyotrophic lateral sclerosis (ALS). Researchers led by Mary-Louise Rogers, Flinders University Centre of Neuroscience in Adelaide, Australia, and Michael Benatar, University of Miami, Florida, confirmed that ALS patients shed more of the extracellular domain of this receptor into their urine than healthy controls do, and that their p75^ECD levels rise as disease worsens. The researchers propose that p75^ECD could become the first marker of ALS progression, and that it could prove useful in clinical trials.

“The difference between the controls and ALS patients is dramatic, and the increase over time seems compelling,” said Mark Bothwell, University of Washington, Seattle, who was not involved in the study.

No fluid-based biomarker has emerged to reliably track ALS. Blood levels of light and heavy chains of neurofilament distinguish ALS patients from controls, but they do not change as disease progresses (see Jun 2015 news; Boylan et al., 2009). 

Steady climb: In the urine, p75^ECD levels went up as disease progressed in ALS patients. [Courtesy of Shepheard et al., 2017. ©Neurology.] 

P75 receptor is highly expressed during development, downregulated, then re-emerges in injured or diseased neurons. Signaling through this receptor contributes to both survival and apoptotic processes. In patients with ALS, the receptor appears on the surface of apoptotic motor neurons and Schwann cells. It also appears on hippocampal neurons in people with Alzheimer’s disease (Seeburger et al., 1993; Kerkhoff et al., 1991; Ito et al., 2016). As part of the protein’s normal processing, the extracellular domain sheds from the cell surface and winds up in the urine (Smith et al., 2015). Previously, Rogers and others reported that p75^ECD levels are higher in patients’ urine than in controls (Shepheard et al., 2014).

In SOD1 G93A ALS models, urine p75^ECD climbs as the mice deteriorate, but it was unknown if that happens in ALS patients, too (Matusica et al., 2016). To find out, first author Stephanie Shepheard at Flinders analyzed samples from the South Australian Motor Neuron Disease Clinic in Adelaide, and from the Kessenich Family ALS Center at the University of Miami. She used ELISA to measure p75 in the urine of 54 ALS patients who averaged 64 years of age, as well as 45 of their healthy spouses and friends, average age 50. The researchers normalized urine p75^ECD measurements to the levels of creatinine, a byproduct of muscle metabolism, in order to account for people’s hydration level.

ALS patients had an average 5.6 ng of urinary p75^ECD per mg creatinine, compared to 3.6 in controls (see image below). The assay yielded the same measurements for patients regardless of what time of day the samples were taken, whether the samples underwent freeze-thaw cycles, or were stored at varying temperatures. This suggests the assay is robust, wrote the authors.

Of the 54 ALS patients, 31 gave up to five additional urine samples, each taken a few months apart. As their disease worsened, p75^ECD levels went up by about 0.19 ng/mg creatinine per month. Spaghetti plots showed that levels rose steadily in almost every patient tested during a period of 30 to 50 months. By comparison, cross-sectional data from the controls suggested an age-related p75^ECD increase of 0.32 ng/mg creatinine per decade.

The increase in ALS patient p75^ECD correlated with a worsening on the ALS Functional Rating Scale of about 0.82 points per month. Higher p75^ECD levels at diagnosis also predicted shorter survival.

Results suggest that p75^ECD could join a few other potential ALS biomarkers and be the first to track with disease progression, claim the authors. However, given that urine levels of p75^ECD overlap with those of controls, it is unlikely to work as a diagnostic marker, Benatar said. Since p75^ECD is a general marker of neurodegeneration, it will not be specific for ALS, either.

Being able to assay urine instead of blood offers a distinct advantage, however, he said. Interestingly, p75^ECD has been proposed to protect against Aβ toxicity and has been found at low levels in the CSF and high levels in serum of AD patients (Yao et al., 2015; Jiao et al., 2015). 

“It is exciting that there may be a credible biomarker for ALS with the potential for monitoring disease progression and effectiveness of treatments,” said Elizabeth Coulson, University of Queensland, Australia, who was not involved in this study but has collaborated with this group on a previous p75 paper. She added that since other groups have associated p75ECD with Alzheimer’s disease, researchers could look at this biomarker in a range of diseases.

If the concentration of this protein fragment changes dynamically in response to treatments that slow neurodegeneration, that might make a potential pharmacodynamic indicator, said Benatar. He acknowledged that the healthy controls in this study were significantly younger than the patients, and the current analysis only cross-sectional. However, since p75^ECD appears to change only slowly in healthy controls, he expects it will barely change over a period of 18 months, the timeframe of most ALS trials.

Benatar and Rogers are working with the Clinical Research in ALS and Related Disorders for Therapeutic Development (CReATe) consortium to reproduce this data in a larger cohort. The consortium’s Phenotype-Genotype-Biomarker study collects urine, blood, and CSF samples from ALS patients every three to six months while clinically assessing cognitive and behavioral symptoms. With 700 patients examined longitudinally, the study will be able to determine whether p75^ECD can predict future rates of functional decline, Benatar said.

Bothwell added that p75^ECD might be a useful antecedent marker in people with suspected familial ALS. “With ALS, about half of the motor neurons can be lost before a major functional deficit is detected,” he said. He also noted it would be interesting to determine whether p75^ECD comes from Schwann cells or motor neurons, and whether it reflects higher expression or faster cleavage of p75.—Gwyneth Dickey Zakaib

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References

News Citations

1. Blood Marker May Predict ALS Progression 4 Jun 2015

Paper Citations

1. Boylan K, Yang C, Crook J, Overstreet K, Heckman M, Wang Y, Borchelt D, Shaw G. Immunoreactivity of the phosphorylated axonal neurofilament H subunit (pNF-H) in blood of ALS model rodents and ALS patients: evaluation of blood pNF-H as a potential ALS biomarker. J Neurochem. 2009 Dec;111(5):1182-91. PubMed.
2. Seeburger JL, Tarras S, Natter H, Springer JE. Spinal cord motoneurons express p75NGFR and p145trkB mRNA in amyotrophic lateral sclerosis. Brain Res. 1993 Sep 3;621(1):111-5. PubMed.
3. Kerkhoff H, Jennekens FG, Troost D, Veldman H. Nerve growth factor receptor immunostaining in the spinal cord and peripheral nerves in amyotrophic lateral sclerosis. Acta Neuropathol. 1991;81(6):649-56. PubMed.
4. Ito S, Ménard M, Atkinson T, Brown L, Whitfield J, Chakravarthy B. Relative expression of the p75 neurotrophin receptor, tyrosine receptor kinase A, and insulin receptor in SH-SY5Y neuroblastoma cells and hippocampi from Alzheimer's disease patients. Neurochem Int. 2016 Dec;101:22-29. Epub 2016 Sep 29 PubMed.
5. Smith KS, Rush RA, Rogers ML. Characterization and changes in neurotrophin receptor p75-Expressing motor neurons in SOD1(G93A) G1H mice [corrected]. J Comp Neurol. 2015 Aug 1;523(11):1664-82. Epub 2015 Apr 30 PubMed.
6. Shepheard SR, Chataway T, Schultz DW, Rush RA, Rogers ML. The extracellular domain of neurotrophin receptor p75 as a candidate biomarker for amyotrophic lateral sclerosis. PLoS One. 2014;9(1):e87398. Epub 2014 Jan 27 PubMed.
7. Matusica D, Alfonsi F, Turner BJ, Butler TJ, Shepheard SR, Rogers ML, Skeldal S, Underwood CK, Mangelsdorf M, Coulson EJ. Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment. J Cell Sci. 2016 Feb 1;129(3):517-30. Epub 2015 Oct 26 PubMed.
8. Yao XQ, Jiao SS, Saadipour K, Zeng F, Wang QH, Zhu C, Shen LL, Zeng GH, Liang CR, Wang J, Liu YH, Hou HY, Xu X, Su YP, Fan XT, Xiao HL, Lue LF, Zeng YQ, Giunta B, Zhong JH, Walker DG, Zhou HD, Tan J, Zhou XF, Wang YJ. p75NTR ectodomain is a physiological neuroprotective molecule against amyloid-beta toxicity in the brain of Alzheimer's disease. Mol Psychiatry. 2015 Nov;20(11):1301-10. Epub 2015 Apr 28 PubMed.
9. Jiao SS, Bu XL, Liu YH, Wang QH, Liu CH, Yao XQ, Zhou XF, Wang YJ. Differential levels of p75NTR ectodomain in CSF and blood in patients with Alzheimer's disease: a novel diagnostic marker. Transl Psychiatry. 2015 Oct 6;5:e650. PubMed.

External Citations

1. Phenotype-Genotype-Biomarker study

Further Reading

Papers

1. Smith KS, Rush RA, Rogers ML. Characterization and changes in neurotrophin receptor p75-Expressing motor neurons in SOD1(G93A) G1H mice [corrected]. J Comp Neurol. 2015 Aug 1;523(11):1664-82. Epub 2015 Apr 30 PubMed.
2. Matusica D, Alfonsi F, Turner BJ, Butler TJ, Shepheard SR, Rogers ML, Skeldal S, Underwood CK, Mangelsdorf M, Coulson EJ. Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment. J Cell Sci. 2016 Feb 1;129(3):517-30. Epub 2015 Oct 26 PubMed.

News

1. New Spectroscopy Method Detects Myelin Changes in Early ALS 12 Nov 2016
2. Blood NfL Looks Good as Progression and Outcome Marker 9 Jun 2016
3. More Data, More Progress: ALS Researchers Debut Shared Database 17 Oct 2014
4. Biomarker Panel Predicts Slow or Fast ALS Course 13 Dec 2013
5. News Brief: Muscle-Monitoring Method Earns $1 Million ALS Prize 8 Feb 2011