WO2012103577A1

WO2012103577A1 - Biomarker for motor neuron disease (mnd)

Info

Publication number: WO2012103577A1
Application number: PCT/AU2012/000076
Authority: WO
Inventors: Robert Archer RUSH; Mary-Louise Rogers; Stephanie Ruth SHEPHEARD; Timothy CHATAWAY
Original assignee: Flinders University Of South Australia
Priority date: 2011-02-01
Filing date: 2012-01-31
Publication date: 2012-08-09
Also published as: AU2012212387A1

Abstract

A method of diagnosing or prognosing motor neuron disease (MND) is described which comprises detecting p75 neurotrophin receptor (p75NTR) or a fragment thereof (eg the extracellular domain; ECD) in a test body sample (eg whole blood and urine) from said subject, or otherwise detecting a change in the amount of p75NTR or a fragment thereof in a test body sample from said subject taken at two or more time points. Similar methods may be applied to the monitoring of MND progression or assessing the effectiveness of an MND therapy. A method of screening an agent that is capable of treating MND is also described.

Description

BIOMARKER FOR MOTOR NEURON DISEASE (MND)

FIELD OF THE INVENTION

The present invention relates to the field of neurological diseases, in particular motor neuron disease (MND). In accordance with the present invention, methods of diagnosing or prognosing MND are described along with methods for identifying and/or developing new therapeutic agents for MND.

INCORPORATION BY REFERENCE

This patent application claims priority from:

- Australian Provisional Patent Application No 201 1900312 titled "Biomarker for motor neuron disease" filed 1 February 2011.

The entire content of this application is hereby incorporated by reference.

BACKGROUND TO THE INVENTION

MND, also known as Lou Gehrig's disease and Amyotrophic Lateral Sclerosis (ALS), has been described as one the most incapacitating diseases of the human species (Ludolph AC, 2006).

Accordingly, MND diagnosis can be devastating for the individual concerned; the average life expectancy following diagnosis being just 36 months. At present, the only effective and FDA- approved treatment available is riiuzole, which extends patient survival for about three months (Chourdry RB & M Cudkowicz, 2005).

MND is the most common form of motor neuron degenerative disease (reviewed by Turner BJ & Talbot, 2008) and is characterised by progressive muscular paralysis, due to a combination of voluntary muscle weakness, atrophy and spasticity, which reflects the degeneration of motor neurons in the primary motor cortex (upper motor neurons), brain stem and spinal cord (lower motor neurons). MND usually affects adults in mid to late life, with a peak age of onset in the fifties to sixties. A 2007 review suggested that the worldwide incidence of MND to be 2 per 100,000 with a prevalence of 4 per 100,000 (Hirtz D et ai, 2007). MND Australia, the national peak body for MND care and research in Australia, reports that an estimated 1400 Australians have MND, and each day, at least one person dies and another is diagnosed (MND Australia 2010).

The cause of MND is mostly unknown and a significant volume of research has been conducted into possible environmental and lifestyle risk factors. Some risk factors that have, however, been implicated include the consumption of cycad plant-derived food (resulting.in exposure to milligrams of the neurotoxin cycasin per day), exposure to heavy metals such as lead, smoking, and exposure to electromagnetic fields. In addition, some rare genetic mutations have been identified as probable causing factors of MND. These include mutations in the copper, zinc superoxide dismutase (SOD1) gene, RNA-metabolising proteins TDP-43 and fused in sarcoma/translated in liposarcoma (FUS; Ilieva H et ai, 2009) and optineurin (OPTN; Maruyama H et ai, 2010), with the list likely to expand. It is therefore currently postulated that a complex genetic-environmental interaction is the causal factor for motor neuron degeneration in most forms of MND (Strong MJ, 2010).

Ten percent of MND cases are familial (fMND) and approximately 20% of fMND is caused by mutations in the copper, zinc superoxide dismutase (SODl) gene found on chromosome 21q21 (ALS1) (Strong MJ, 2010; Turner BJ & K Talbot, 2008) which codes for the SODl protein, a ubiquitously expressed, cytosolic metalloenzyme of 153 amino acids, encoded by five exons.

Mutations of the SODl gene are known to be responsible for 3% of the more common sporadic MND (sMND). In 1993, landmark research identified 1 1 missense mutations in the SODl gene across 13 families with fMND (Rosen DR et ai, 1993), and following research was directed towards determining the mechanism of SODl -mediated MND; over 150 mutations have now been identified throughout all of the five exons of this single gene (Andersen PM, 2006; Wroe R, 2010), the majority of these being point mutations of highly conserved amino acids such as alanine for glycine in position 93 (SODl⁰⁹³*) (Cleveland D & JD Rothstein 2001). This has allowed for the generation of transgenic mice carrying mutations of human SODl such as S0D1^G93A mice (Gurney ME et ai, 1994) that have been used to study MND.

Despite significant advances, the diagnosis of MND is still based on the presence of characteristic clinical findings, and the "ruling out" of similar diseases that share some of these characteristics. There is, accordingly, a great need for the identification of one or more biomarkers for diagnosing or prognosing MND. Further, such a biomarker(s) may assist in the identification and/or development of efficacious drugs for treating MND. Amongst possible MND biomarkers recently studied in MND model mice are decreased levels of 5-methyltetrahydrofolate found in the plasma, spinal cord and cortex in early stages of pre-symptomatic MND SODl^G93A mice (Zang AA et ai, 2010), increased levels of phosphorylated neurofilament subunit H (pNF-H) in serum of MND mice, which also showed promising preliminary results in MND patients (Boy lan K et ai, 2009), increased levels of tau, and decreased levels of SlOObeta and soluble CD 14 in the cerebrospinal fluid of MND patients (Sussmuth SD et ai, 2010). The present invention relates to the p75 neurotrophin receptor (p75NTR). p75NTR is a cell membrane receptor that is most well-known for its interaction with neurotrophins. Nerve growth factor (NGF), brain-derived neurotropic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5 are all bound by p75NTR with equally low affinity. The cellular role of p75NTR is contradictory and functions both to promote survival and induce cell death, dependent on the interaction of p75NTR with other receptors. Alone, p75NTR can induce apoptosis and cell death upon binding neurotrophins, and also causes apoptosis in a complex formed with sortilin, by binding to pro- neurotrophins. An interaction with Trk receptors and mature neurotrophins causes pro-survival signals, and interactions with the Nogo-66 receptor and its associated ligands NogoA, myelin associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMGP) induces inhibition of neurite outgrowth (Dupuis L et al, 2008; Lu B et al, 2005; Rogers ML et al, 2008; Teng KK & BL Hempstead, 2004). In rodents, p75NTR is highly expressed during developmental cell death and axon outgrowth and then decreases post-natally to 5% of neonatal levels by 4 weeks of age (Yan Q and EM Johnson Jr, 1987), and is also greatly reduced in different types of cells in adulthood such as motor neurons (Copray JCVM et al, 2003). Similar developmental regulation occurs with human p75NTR (Zupan A A et al, 1989) and multiple studies have found that its expression can be robustly induced by injury. Additionally, elevated p75NTR expression has been found in neurological diseases, deficits and syndromes such as Alzheimer's Disease, neural crest tumours, stroke, ischaemia and excitotoxicity, cerebellar Purkinje cell degeneration, schizophrenia, bronchial asthma and some autoimmune diseases (Schor NF, 2005). Of particular interest, is that p75NTR expression has been found to be upregulated in the spinal cord of MND patients post mortem (Seeburger JL et al, 1993) and in the SODl⁰⁹³* mouse model of MND (Copray JCVM et al, 2003). p75NTR has also been detected in the urinary protein of rats following sciatic nerve injury (Distefano PS & EM Johnson, 1988).

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method of diagnosing or prognosing motor neuron disease (MND) in a subject, the method comprising:

(i) detecting p75 neurotrophin receptor (p75NTR) or a fragment thereof in a test body sample from said subject; or

(ii) detecting a change in the amount of p75NTR or a fragment thereof in a test body sample from said subject taken at two or more time points.

In a second aspect, the present invention provides a method of screening an agent that is capable of treating motor neuron disease (MND) in a subject, wherein said method comprises the steps of; providing an animal model for MND;

administering a test agent to said animal; and

(i) detecting p75 neurotrophin receptor (p75NTR) or a fragment thereof in a test body sample from said animal; or

(ii) detecting a change in the amount of p75NTR or a fragment thereof in a test body sample from said animal taken at two or more time points.

In a third aspect, the present invention provides a method of monitoring motor neuron disease (MND) progression in a subject, the method comprising: (i) detecting p75 neurotrophin receptor (p75NTR) or a fragment thereof in a test body sample from said subject; or

In a fourth aspect, the present invention provides a method of assessing the effectiveness of a therapy applied to treat motor neuron disease (MND) in a subject, the method comprising:

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the results of a Western Blot (WB) test of p75NTR antibodies under different conditions. 10 μg of cell lysates of indicated species were separated by SDS-PAGE and subject to WB under reducing and non-reducing conditions, with either goat anti-mouse p75NTR, mouse anti- human p75NTR MLR2 or rabbit anti-human p75NTR as detection (n^ blots);

Figure 2 shows the results of a WB test of goat anti-mouse p75NTR with samples of recombinant p75NTR-Fc and baby hamster kidney fibroblasts (BSR) negative control lysates. Samples were separated by SDS-PAGE and subject to a WB under reducing conditions with goat anti-mouse p75NTR;

Figure 3 shows the results of a WB test with Sypro Ruby stain of urinary protein. 20 μg of precipitated urinary protein from SODl⁰⁹³* and B6 controls was separated by SDS-PAGE and subject to WB using goat anti-human p75NTR (A), prior to antibody treatment, Sypro Ruby total protein stain was used (B);

Figure 4 provides the results of a study to quantify p75NTR from SODl^G93A and B6 control and end- stage protein. 40 μg of urinary protein from SODl⁰⁹³" and B6 mice was separated by SDS-PAGE along with mouse p75NTR-Fc and transferred to nitrocellulose and subject to WB using goat anti- mouse. WB were performed, each containing two end-stage S0D1^G93A samples (lanes 3 and 4), two age-matched B6 control samples (lanes 1 and 20 and p75NTR-Fc as standard (lanes 6-9)). (A) shows the results from one blot and (B) the p75NTR standard curve from A where absorbance was measured in a set area, with all values corrected by subtracting an area without sample (lane 5). The amount of p75NTR in ng could be determined for each sample on the blot. The data from three such blots was pooled (n=6 of SOD1 ^³* and B6 samples) and related back to mg of urinary protein (C) or ml of mouse urine (D). There was significantly more p75NTR found in SOD1 mice urine than B6 age- matched controls (paired t-test, C: p=0.0016; D: p=0.0109);

Figure 5 shows the results of immunoprecipitation (IP) of p75NTR for mouse, human and rat cell lysates using MLR2 or MLR1 as pull down. 500 μg of cell lysates from the species indicated were subjected to IP using 5 μg of MLR2 (A and B) or MLR1 (C and D) and detected with goat anti-mouse p75NTR (A and C) or rabbit anti-human p75NTR (B and D). In addition, cell lysates from the indicated species were included as controls for WB (A⁰ - IP control with no cell lysate); Figure 6 provides the results of IP of mouse, human and rat cell lysates in addition to recombinant p75NTR-Fc using goat anti-mouse p75NTR as pull down. 500 μg of cell lysates form the species indicated were subject to IP using 5 μg of goat anti-mouse p75NTR and detected with mouse anti- human p75NTR MLR2 (A and B) or rabbit anti-human p75NTR (C and D) under reducing (A and C) or non-reducing (B and D) conditions. In addition, BSA was spiked with p75NTR-Fc (E);

Figure 7 provides the results of experimentation to optimise WB of urinary immunoprecipitation (IP). 500 μg (lane 10, 110 μg (lane 2) and 20 μg (lane 3) of urinary protein was tested with IP with 5 μg of pull down MLR2. A constant amount of urinary protein (250μg) was tested with 2.5 μg (lane 6) and 10 μg (lane 4) of antibody;

Figure 8 shows a silver stained gel of S0D1^G93A end-stage and B6 age-matched control urinary protein;

Figure 9 shows a silver stained 2D PAGE gel of precipitated S0D1^G93A end-stage urinary protein (100 ug);

Figure 10 shows the detection of p75NTR in the urine of SODl⁰⁹³¹^ mice from 60 days to end-stage. ΓΡ of 110 μg of urinary protein from SODl ⁰⁹³* and B6 control mice at 40, 60, 80, 100 days and end- stage was conducted using MLR2 as pull down and goat anti-mouse as detection. Bands

corresponding to p75NTR (box) are detected in the urinary protein of the S0D1^G93A mouse beginning at 60 days of age, but not in age-matched B6 control (until end stage);

Figure 11 provides graphical results of the detection of p75NTR in urinary protein from S0D1^G93A mice before onset of MND symptoms. (A) Disease onset was first detected by grip duration testing at 100 days of age. (B) Disease onset was detected by neurological scoring at 120 days of age. In contrast, p75NTR was detected by IP and subsequent WB in SOD 1^093/1 mouse urinary protein at 60 days of age and older; Figure 12 shows the detection of p75NTR in the urine of an MND patient. IP WB of 500 μ of a urinary protein sample was conducted using MLR2 (5 μg) as pull down and goat anti-p75NTR (4 μg; N5788) for detection, essentially as indicated in Figure 6. Bands corresponding to p75NTR (box) are detected in the urinary protein of the patient. Controls were 500 μg urinary protein samples from healthy individuals and protein from negative control fibroblast cells;

Figure 13 provides graphical results showing human and mouse p75NTR detection sensitivity by ELISA conducted using MLR2 for p75NTR capture and goat anti-p75NTR (4 μ& N5788) for p75NTR detection. Signal to noise (S/N) ratios were calculated for the mouse and human samples using mouse p75NTR-ECD (1 157-NR) and human p75NTR-ECD (PE-1237); and

Figure 14 provides graphical results showing significantly increased levels of p75NTR in the urine of (A) end stage SODl^G93A mice (n=4) relative to control mice, (B) MND patients (n=8) relative to healthy individuals (** = p<0.01, two-tailed t-test), and (C) MND patients at 0-6 months (n=4), 6-12 months (n=l) and 12-25 (n=3) months post-diagnosis.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "p75NTR" encompasses full length p75NTR polypeptides (-60-67 kDa) and multimers thereof from, for example, mammalian species (eg human, mouse and rat) as well as variants thereof which show substantially equivalent immunological and/or biological activity. The term "fragment thereof encompasses p75NTR fragments (eg degradation products of p75NTR) of, preferably, 20 or more amino acids in length such as the extracellular domain (ECD; ~50kDa) which, preferably, show substantially equivalent immunological and/or biological activity. Particularly preferred p75NTR fragments are those including an epitope sequence CEEIPGRWITRSTPPE (SEQ ID NO: 1), or a sequence substantially corresponding thereto. A sequence "substantially

corresponding" to the sequence of SEQ ID NO; 1 is to be understood as referring to a variant epitope sequence including one or more amino acid substitution (especially conservative amino acid substitutions such as G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Na- alkylamino acids), addition or deletion. The term "test body sample" as used herein refers to a sample of a body fluid, separated cells (ie cells taken from the body and at least partially separated from other body components), a tissue or an organ. Samples of body fluids can be obtained by methods well known to the person skilled in the art, and tissue or organ samples may be obtained from any tissue or organ by, for example, biopsy.

Separated cells may be obtained from a body fluid, tissue or organ by separating techniques such as centrifugation or cell sorting. Preferably, cell, tissue or organ samples are obtained from those cells, tissues or organs which express or produce p75NTR. The test body sample(s) for use in the method of the first aspect may, therefore, be preferably selected from urine, whole blood, blood plasma, serum, buffy coat, cerebrospinal fluid, seminal fluid, synovial fluid, a tissue biopsy and/or an organ biopsy. More preferably, the test body sample(s) is selected from the group consisting of urine, whole blood, blood plasma and serum. Most preferably, the test body sample(s) is urine.

The test body sample(s) may be pre-symptomatic (ie the test body sample(s) may be taken from the subject at a time point before any MND symptoms appear in the subject) and/or post-symptomatic (ie the test body sample(s) may be taken from the subject at a time point which coincides with one or more MND symptoms, especially one or more early MND symptoms such as stumbling due to weakness of leg muscles, difficulty holding objects due to weakness of hand muscles (ie which may be detected by grip duration and/or grip strength tests), weakness of the tongue and/or throat muscles, and cramps and muscle twitching (fasciculation)).

The subject will typically be a human, generally of a mid to late stage of life. The method of the first aspect may, however, be suitable for use in veterinary applications and, as such, the subject may be, for example, a livestock or thoroughbred animal, companion animal (eg dog or cat) or an exotic animal (eg a tiger or elephant).

In a first preferred embodiment, the method of the first aspect comprises detecting p75NTR or a fragment thereof in a test body sample. The p75NTR may be detected qualitatively or quantitatively. The method of such an embodiment is preferably conducted in vitro. The detection of p75NTR (or fragment thereof) in the test body sample or the detection of p75NTR (or fragment thereof) in an amount greater than would be otherwise expected in an equivalent body sample taken from a normal subject (ie a subject, preferably age-matched, showing no symptoms of MND and which is, preferably, also of good health, does not smoke and is of the same gender as the subject who has provided the test body sample), is indicative of MND in the subject. In an in vitro method, the p75NTR (or fragment thereof) may be detected in the test body sample by any suitable method including, for example, immunoassays such as enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry (eg with sectionalised samples of a tissue biopsy) using an anti-p75NTR antibody or fragment thereof (eg a polyclonal or monoclonal antibody or fragment thereof such as an Fv, Fab, F(ab)₂ fragment that is capable of binding p75NTR or a fragment thereof, and recombinant antibodies that bind p75NTR (or a fragment thereof) such as a single chain antibody (eg scFv antibodies)) or assays involving the use of other ligands that bind to p75NTR (or fragment thereof) such as, for example, peptides, polypeptides, nucleic acids or aptamers (eg nucleic acid or peptide aptamers). Particularly suitable methods for detecting p75NTR (or fragment thereof) in a test body sample are immunoassays utilising labelled molecules in various sandwich, competition, or other assay formats. Such immunoassays will develop a signal which is indicative of the presence or absence of p75NTR (or a fragment thereof)- Further, the strength of the signal generated by such immunoassays may be correlated directly or indirectly (for example, reversely proportional) to the amount of p75NTR (or fragment thereof) present in a sample(s). Preferably, such immunoassays utilise an anti-p75NTR antibody or fragment thereof that specifically binds to p75NTR (or fragment thereof).

As used herein, the term "specifically binds" means that the anti-p75NTR antibody (or fragment thereof) does not bind substantially to (that is, substantially "cross-react" with) another peptide, polypeptide or substance present in the test body sample. Preferably, specifically bound p75NTR (or fragment thereof) will be bound with at least 3 times higher, more preferably at least 10 times higher, and most preferably at least 50 times higher affinity than any other relevant peptide, polypeptide or substance. Non-specific binding may be tolerable, if it can still be distinguished and measured unequivocally, for example, according to its size on a Western Blot, or by the relatively higher abundance of the p75NTR (or a fragment thereof such as the extracellular domain (ECO) or other p75NTR fragment including the epitope sequence of SEQ ID NO: 1 or a sequence substantially corresponding thereto) in the sample.

Other particularly suitable methods for determining the amount of p75NTR (or a fragment thereof) present in a test body sample(s) are methods comprising the measurement of a physical or chemical property specific for p75NTR (or fragment thereof) such as a precise molecular mass or nuclear magnetic resonance (NMR) spectrum. Such methods may, therefore, be conducted using biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass spectrometers (eg by conducting mass spectroscopy sequencing of peptides generated from digesting p75NTR), NMR- analysers and chromatography devices. Further particularly suitable methods for determining the amount of p75NTR (or a fragment thereof) present in a test body sample(s) include microplate ELISA-based methods, fully-automated or robotic immunoassays, enzymatic Cobalt Binding Assay (CBA) and latex agglutination assays. Still further examples of particularly suitable methods for determining the amount of p75NTR (or a fragment thereof) present in a test body sample(s) include methods involving precipitation (eg immunoprecipitation), electrochemiluminescence (ie electro- generated chemiluminescence), electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry, latex-enhanced turbidimetry and nephelometry.

A particular example of a suitable method for detecting p75NTR in a test body sample(s), described in detail in the following example, involves the use of 2D gel electrophoresis. The presence of p75NTR (or a fragment thereof) is determined by the identification of a band corresponding to a molecular weight of -50 kDa or -60-67 kDa and an isoelectric point (PI) of about 4.5.

In a second preferred embodiment, the method of the first aspect comprises detecting a change in the amount of p75 TR or a fragment thereof in a test body sample from said subject taken at two or more time points. The method of such an embodiment is preferably conducted in vitro. The amount of p75NTR (or fragment thereof) may be determined using any of the suitable methods described above.

The "change in the amount of p75NTR (or fragment thereof)", for the purposes of the present invention, may be represented by an increase in the amount of p75NTR (or fragment thereof) that is detectable by serial measurements. For example, an increase in the amount of p75NTR (or fragment thereof) may be detected by comparing the amount of p75NTR (or fragment thereof) in a test body sample at a given time point with the amount of p75 TR (or fragment thereof) in the same test body sample taken at an earlier time point. Such an increase is indicative of MND in said subject and or indicates MND disease progression. The magnitude or rate of increase in the amount of p75NTR between time points may be used for MND prognosis (eg identifying rate of decline and survival period).

As mentioned above, the test body sample(s) used in the method of the first aspect is, most preferably, urine. Urine offers advantages over other kinds of body samples inasmuch as it is relatively abundant, samples can be acquired without invasive techniques, and multiple samples can be collected from a subject over time. However, it has been found that urine may contain amounts of proteinaceous materials which may obscure the detection of p75NTR or a fragment thereof (eg in a method involving 2D gel electrophoresis). Accordingly, it may be preferred that the urine sample or the urinary protein within the sample be subjected to a depletion treatment (ie a partial removal of protein, particularly proteins other than p75NTR or fragments thereof) using any of the methods well known to those skilled in the art (eg by passing the urine through Proteominer columns; Bio-Rad Laboratories, Inc, Hercules, CA, United States of America). It will be understood by those skilled in the art that the method of the first aspect may be used in combination with an independent analysis of one or more other biomarkers or potential biomarkers of MND such as, for example, decreased levels of 5-methyltetrahydrofolate in plasma, increased levels of phosphorylated neurofilament subunit H (pNF-H) in serum (Bo lan K et al, 2009), increased levels of serum metalloproteinase-9 (MMP-9) (Soon CPW et al. 2010), increased levels of tau, decreased levels of SlOObeta and soluble CD 14 in cerebrospinal fluid, increased levels of TDP-43 in cerebrospinal fluid, and genetic biomarkers such as the abovementioned known fMND- and sMND- 1 inked mutations in the SODl gene. Using a panel of tests, those skilled in the art could, for example, monitor the onset of motor neuron pathology in a subject developing MND and/or differentiate MND from other diseases.

The diagnosis or prognosis of MND in accordance with the method of the first aspect may provide an opportunity for early intervention and treatment of the disease in the said subject. Further, the ability to detect MND-associated p75NTR (or a fragment thereof) in pre-symptomatic test body samples may be particularly useful in the context of fMND, where members of families predisposed to MND may be regularly screened for MND-associated p75NTR, is a test body sample so as to allow for pre- symptomatic identification of those members who will develop MND and thereby provide an opportunity for early intervention and treatment.

Thus, where p75NTR (or a fragment thereof) is detected in a test body sample or there is a detected increase in the amount of p75NTR (or a fragment thereof) in a test body sample from said subject taken at two or more time points, that is associated with MND in the subject, and there has been, optionally, an independent and positive analysis of one or more other biomarkers or potential biomarkers of MND in the subject, the method of the first aspect may further comprise treating said subject (eg with an agent effective for the treatment of MND such as riluzole, or other suitable treatment) for MND.

Thus, it is to be understood that the present invention extends to a method for treating MND in a subject, wherein said method comprises diagnosing or prognosing MND in said subject by:

(ii) detecting a change in the amount of p75NTR or a fragment thereof in a test body sample from said subject taken at two or more time points; and

thereafter administering to said subject an effective amount of an agent for the treatment of MND, optionally in admixture with a pharmacologically-acceptable carrier and/or excipient. The agent for the treatment of MND may be selected from the group consisting of riluzole and other suitable agents for treating MND. The agent may be formulated into any suitable

pharmaceutical/veterinary composition or dosage form (eg compositions for oral, buccal, nasal, intramuscular and intravenous administration). Typically, such a composition will be administered to the subject in an amount which is effective to treat MND, and may therefore provide between about 0.01 and about 100 μg/kg body weight per day of the agent, and more preferably provide from 0.05 and 25 μg kg body weight per day of the agent. A suitable composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.

In a second aspect, the present invention provides a method of screening an agent that is capable of treating motor neuron disease (MND) in a subject, wherein said method comprises the steps of;

providing an animal model for MND;

administering a test agent to said animal; and

(ii) detecting a change in the amount of p75NTR or a fragment thereof in a test body sample from said animal taken at two or more time points. The test agent may be selected from known and novel compounds, complexes and other substances which may, for example, be sourced from private or publicly accessible agent libraries (eg the Queensland Compound Library (Griffith University, Nathan, QLD, Australia) and the Molecular Libraries Small Molecule Repository (NIH Molecular Libraries, Bethesda, MD, United States of America). The test agent may therefore comprise a protein, polypeptide or peptide, or a mimetic thereof (including so-called peptoids and retro-inverso peptides), or a small organic molecule, especially those which comply or substantially comply with Lipinski's Rule of Five for "druglikeness" (Lipinski CA et al., 2001). The test agent may also be selected on the basis of structural analysis of known or novel compounds or may otherwise be designed following structural analysis of p75NTR binding sites.

The test agent may be a composition comprising one or more active agents.

The animal model may be any suitable animal model of MND. Preferably, the animal model is a SODl⁰⁹³* mouse or another transgenic mouse or animal expressing a human SOD1 gene comprising an MND-iinked mutation(s). The test body sample(s) may be pre-symptomatic (ie the test body sample(s) may be taken at a time point before any MND symptoms appear in the animal) and/or post-symptomatic (ie the test body sample(s) may be taken from the subject at a time point which coincides with one or more MND symptoms (for example, in a mouse model, reduced performance on grip duration and/or grip strength tests). For the S0D1^G93A mouse, the test body sample(s) may be taken at, for example, one or more of 40, 60, 80, 100, 120 and 140 days. In such mice, MND symptoms are not observed before about 100 days of age (eg grip strength decrease appears at about 129 days of age). However, as described hereinafter, the present applicant has been able to detect MND-associated p75NTR (and fragments thereof) in test body sample(s) from the S0D1^G93A mouse at about 60 days of age. Further, it has been found that the amount of p75NTR (or fragments thereof) in the test body sample increases with MND disease progression in the SODl⁰⁹^ mouse.

Most preferably, the test body sample(s) is urine. A test agent which achieves a reduction in the amount of p75NTR present in a test body sample relative to the typical amount observed in an equivalent test body sample from an untreated MND animal model may be considered to show potential as the basis of a treatment of MND. Similarly, a ; test agent which achieves stabilisation or a reduction of the amount of, or even a reduction in the rate of increase in the amount of, p75NTR (or a fragment thereof) present in a test body sample from the MND model animal taken at two or more time points, may also be considered to show potential as the basis of a treatment of MND.

The method of the second aspect may utilise one or more of the suitable methods for quantitatively or qualitatively detecting p75NTR (or a fragment thereof) described above.

Those skilled in the art will appreciate that the method of the second aspect may also be adapted for the development or optimisation of an agent identified as being capable of treating MND (eg to assess optimum dosing or modifications to improve pharmacokinetics etc). Accordingly, the present invention also extends to such adapted methods.

Further, in a third aspect, the present invention provides a method of monitoring motor neuron disease (MND) progression in a subject, the method comprising:

(ii) detecting a change in the amount ofp75NTR or a fragment thereof in a test body sample from said subject taken at two or more time points. Moreover, in a fourth aspect, the present invention provides a method of assessing the effectiveness of a therapy applied to treat motor neuron disease (MND) in a subject, the method comprising:

A therapy which achieves a reduction in the amount of p75NTR (or a fragment thereof) present in a test body sample relative to the typical amount observed in an equivalent test body sample from an untreated MND patient or a patient treated with an alternative therapy (eg a treatment other than riluzole) may be considered as achieving therapeutic benefit (eg slowing disease progression).

Similarly, a test agent which achieves stabilisation or a reduction of the amount of, or even a reduction in the rate of increase in the amount of, p75NTR (or a fragment thereof) present in a test body sample from the MND patient taken at two or more time points, may also be considered as achieving therapeutic benefit in that subject.

The present invention is hereinafter further described by way of the following, non-limiting examples. EXAMPLES

Example 1

The SOD 1^{093 1} mouse model of MND is considered to be the standard model for testing possible therapeutics in pre-clinical trials. This example investigated whether the presence of p75NTR in the urine of SODl^G93A mice could be detected and used as a biomarker for MND and to monitor MND progression in this animal model (particularly, pre- and post-symptomatic).

Methods and Materials

Materials

Goat anti-p75NTR (# N5788) was from Sigma-Aldrich Pty Ltd (Sydney, NSW, Australia), rabbit anti-human p75NTR (# ANT-007) from Alomone Labs (Israel), and monoclonal mouse anti-human p75NTR (MLRl and MLR2) made in-house (see below). Secondary antibodies used for Western Blot (WB) were all from Jackson ImmunoResearch Laboratories Inc (West Grove, PA, United States of America). Mouse p75NTR-Fc was from Biosensis Pty Ltd (Th^'ebarton, SA, Australia). All common chemicals were from Sigma-Aldrich. Mouse colony maintenance and behavioral testing

A colony of high copy number S0D1^G93A mice was bred. For the purposes of this example and ethics requirements, end-stage of disease was determined by reaching a neurological score of 3 (see Table 1). The colony was maintained by crossing S0D1^G93A males with control B6 females (Jackson ImmunoResearch Laboratories) and resultant litters expected to contain 50% human mutant

SODl^G93A-carrying transgenic mice and 50% control B6 mice. The DNA profile of each mouse was tested by running DNA obtained from tail tips through polymerase chain reaction (PCR) to determine if mice carried copies of the human mutant SODl⁰⁹^ transgene (Leitner M et al., 2009). Weight

Mice weights were measured at 40, 60, 80 and 100 days (±2 days), and frequently at end-stage until mice were euthanised (based on neurological score of 3). Weekly weight measurements were recorded as part of normal colony maintenance and these were also used to track overall mice health and disease progression.

Neurological scoring

Neurological scores of the hind-limbs were measured at 40, 60, 80 and 100 days (±2 days) of age and two times a week at end stage, using the following criteria until mice were euthanised upon reaching a neurological score of 3 (Leitner M et al., 2009).

Grip duration tests

Mice were placed in a grip duration testing (hanging wire) apparatus (Miana-Mena FJ et al, 2005) at 40, 60, 80 and 100 days (± 2 days) and at end-stage. Mice were tested for time to fall from a wire cage lid when placed on the lid and the lid placed upside down. This was done 3 times, with a break of 2 minutes between trials, and a cut-off of 90 seconds to determine grip duration. Statistical analysis

Graph Pad Prism (v.4) was used to analyse behavioural and neurological data test scores from SODl⁰⁹³ and B6 control mice to determine the statistical significance of any differences observed. Two-way ANOVA tests were performed on the data obtained from neurological scores, grip duration testing and weight measurements to evaluate the different parameters over time. Percentage survival was determined by the creation of a Kaplan Meier survival curve. All data is presented as mean ± standard deviation, and the significance level was set to p < 0.05.

Urinary sample collection and preparation

Urinary samples were obtained from SOD1 ^³* and control B6 mice of 40, 60, 80 and 100 days (±2 days) of age and at end-stage of disease (-145-160 days, defined as a neurological score of 3).

Urinary samples were collected through the use of a metabolic cage except for end-stage samples, which were obtained directly from the bladder upon euthanasia. All samples were immediately placed in Eppendorf tubes on ice, containing 50μ1 of protease/phosphatase inhibitor cocktail made as per manufacturer's instructions (F Hoffmann-La Roche AG, Basel, Switzerland), before being transferred to long term storage at -80°C.

Urinary protein precipitation for Western Blot analyses

Urinary protein precipitation was performed using a method modified from Thongboortkerd et ai, 2006). Samples were spun at 12,000g, 4°C, with 9 x the sample volume of ethanol for optimal purification (Thongboonkerd V et ai, 2006). Spins at 15,000g were then performed with 13% trichloroacetic acid and two volumes of 100% acetone at 4°C. Precipitated samples were resuspended in 2 x sodium dodecyl sulphate (2 x SDS) using a sonicator when needed to aid dissolving. Urinary protein clean-up for immunoprecipitation

Diafiltration (exchanging liquid for IX PBS) of urinary protein samples for immunoprecipitation (IP) was performed using 5kDa cut-off spin columns. Briefly, samples were spun at 2,300g (Vivaspin4, 4 ml) or 12,000g (Vivaspin500, 500μΙ) at 4°C as per manufacturer's instructions (Sartorius AG, Goettingen, Germany) with equal volumes of PBS so that each sample was washed between ten and fifteen times by volume, and then concentrated down to approximately 60μ1.

Specific gravity

The specific gravity of mouse urine was tested through by MultiStix Urinalysis (F Hoffmann-La Roche AG) as per the manufacturer's instructions. Protein quantification

Urinary protein samples were quantified using the BioRad DC Protein Assay Kit Microplate Assay Protocol as per manufacturer's instructions (Bio-Rad Laboratories) in Costar 96 well assay plates. Assay results were read with a Perkin Elmer Victor X4 Multilabel Plate Reader at 750 nm, room temperature. Bovine Serum Albumin (BSA) standards of 1 mg ml, 0.8 mg ml, 0.4mg/ml, 0.2mg/ml, O.l mg ml and 0.05mg/ml created by serial dilution were used as a standard curve to plot sample absorbance levels and determine sample protein concentrations using Microsoft Excel.

Cell culture

Cells from different sources were used as controls for p75NTR in WB and IP experiments. A mouse neuroblastoma x motor neuron-enriched spinal cord cell line (NSC-34) (Cashman NR et al., 1992) was used as a mouse p75NTR positive control, and rat derived p75NTR in the form of C6 astrocytoma cells (Benda P et al., 1968) and human derived p75NTR in the form of A875 melanoma cells (Giard DJ et al., 1973) were also used. Samples of a baby hamster kidney fibroblast cell lysate (BSR; Rogers ML et al., 2006) were used as a negative control as they do not produce p75NTR. All cells were cultured in Dulbecco's Modified Eagle Medium (Invitrogen Corporation, Carlsbad, CA, United States of America) containing 1 % L-glutamine and 1 % PSG and either 10% foetal bovine serum (FBS)(NSC-34 and A875 cells) or 5% FBS and 10% normal horse serum (C6 cells). Lvsate preparation

Cells were spun down at 400g and washed twice with PBS containing protease/phosphatase inhibitor cocktail (F Hoffmann-LaRoche AG), and then after centrifugation, put into Lysis buffer (0.15 M NaCl, 1 raM EDTA, 10 mM Tris pH 7.2, 1% Triton X-100) containing protease/phosphatase inhibitor. Cells were disrupted with Retsch Tissue Lyser (setting 300, 3 minutes; Qiagen Inc, Germantown, MD, United States of America; Rogers ML et al, 2010) and then centrifuged at

14,000 g at 4° C to remove cell debris. Following quantification for protein amount, the supernatant was aliquoted and used as controls in SDS-PAGE, WB and IP.

Antibody preparation

Monoclonal anti-human p75NTR antibodies (MLR1 and MLR2) were purified from cell conditioned supernatant (supplied by Dr Rogers, Flinders University of South Australia, Bedford Park, SA, Australia) produced by culturing hybridoma cell lines (Rogers ML et al., 2006). Briefly, supernatant collected after culturing the hybridomas was pumped (using a peristaltic pump) over Protein G Agarose affinity columns as per manufacturer's instructions (Cat. # 16-266; Millipore Corporation, Bellerica, MA, United States of America) and recirculated over the column for 3 days at 4°C. After washing unbound protein with PBS, 0.1M Glycine made in PBS (pH 2.7) was then used to elute the protein and pH brought back to neutral using 2M Tris. HiTrap desalting columns (Cat. # 17-1408-01 , GE Biosciences, Schenectady, NY, United States of America) were then used for buffer exchange into PBS (pH 7.4) and antibody concentrated using Vivaspin4 100 kDa cut-off columns spun at 2,300g at 4° C (Sartorius). Producing polyclonal antibody to extracellular p75NTR

A peptide with a sequence (CCEEIPGRWITRSTPPE; SEQ ID NO:2) corresponding to amino acid residues 188-203 of human p75NTR (SwissProt accession P08138; CEEIPGRWiTRSTPPE; SEQ ID NO: 1 ) that was used to immunise rabbits that produced extracellular p75NTR antibody (Cat. # ANT- 007; Alomone Labs) was synthesised, and then conjugated with KLH to increase immunogenicity. Two rabbits were immunised with the KLH-conjugate over 2 months with three injections, and one site ELISA assays used to determine a positive immune response. Un-conjugated peptide was also obtained to make an affinity column for purification of anti-p75NTR from the serum of final bleeds.

ID SDS-PAGE and Western Blot

SDS-PAGE was performed using an Invitrogen XCell SureLock Mini-Cell system with either 10 or 12 well NuPAGE Novex 4-12% Bis-Tris Mini Gels. Precipitated samples and controls were prepared by boiling for 5 min at 95°C with SDS sample buffer, dithiothreitol (DTT) and bromophenol blue. Immunoprecipitated samples were mixed with bromophenol blue before being separated by SDS- PAGE, as samples were previously boiled to break the bonds formed with pull down antibodies and Protein G agarose beads during immunoprecipitation. Each gel used for WB was run with one lane containing 10 μ\ BioRad Precision Plus Dual-Colour marker (Cat # 161-0374) and those for Silver stain BioRad Precision Plus Unstained marker (Cat# 161-0363). Gels were run in lx Running Buffer (diluted from 20x Running Buffer) at 200V and 110mA until the marker and the bromophenol blue were about 0.5 cm from the bottom of the gel (approximately 1 hour). Following SDS-PAGE, samples were transferred from gels to nitrocellulose membranes (Invitrogen, 0.45μπι pore size, Cat# LC2001 ) using the transfer equipment compatible with the Invitrogen XCell SureLock Mini-Cell system. Transfers were run at 30V, 200mA for 1 hour and 7 minutes on ice using lx Transfer Buffer with 20% methanol. Gels were treated with Coomassie Blue stain to confirm that sufficient transfer of samples had occurred. Following transfer, WB membranes were treated with Sypro Ruby total protein stain as per manufacturer's instructions (Invitrogen) and imaged using a 605DF40 filter and IR setting of-0.85 on a Fuji Film Imager (LAS 4000). Membranes were then blocked for two hours with Tris buffered Saline Tween 20 (TBST) containing 7% skim milk and a primary antibody was added overnight in TBST containing 1% skim milk. Following 4 x 15 min wash steps with TBST 1% skim milk, secondary antibody was added for two hours (1/ 5000 in TBST 1% skim milk), membranes were washed 4 x again with TBST 1% skim milk and then with 1 x TBS, and an enzymatic chemiiuminescence substrate (ECL; GE Biosciences) was used to visualise protein bands using a Fuji Imager system (LAS 4000) and recorded using FujiFilm Global MultiGauge® electrophoretic software. p75NTR quantification from mouse urinary protein samples

40μg of precipitated urinary protein samples from SOD 1⁰⁹³ "end-stage (2 per blot) and B6 age- matched control mice (2 per blot) along with mouse p75NTR-Fc (Biosensis) were separated by SDS- PAGE and transferred to nitrocellulose as described above. After blocking with TBST 7% skim milk and exposure to goat anti-mouse p75NTR, then bovine anti-goat HRP, the blots were developed with ECL (GE Biosciences) and visualised using a Fuji Imager (LAS 4000). Three SDS-PAGE gels and their resulting nitrocellulose membranes (after transfer) had lanes containing 1 , 2, 5 and 10 ng of mouse p75NTR-Fc. FujiFilm Global MultiGauge® electrophoretic analysis software was used to quantify the fluorescence of bands on WB. This software was used to create a standard curve of p75NTR-Fc and determine the amount of p75NTR (in ng) in the urinary protein samples. The obtained values were then plotted and analysed for significance by t-Test using Prism (v.4).

Immunoprecipitation

Samples of urinary protein, cell lysates (500μg), BSA (500μg) and p75NTR-Fc (10 ng in 500μg BSA) were immunoprecipitated using different antibody combinations. Samples were pre-cleared to remove any non-specific binding between sample and Protein G Agarose. However, p75NTR-Fc was not pre- cleared with Agarose beads as the human Fc component of this protein binds Protein G (as per

Millipore's instructions). Samples to be pre-cleared were mixed with 20μ1 of Protein G Agarose beads (Millipore) and rotated for 2h. After centrifugation at lOOOg, samples were removed from Protein G, pull down antibody added (Table 2), and then samples were rotated overnight at 4°C. After this, samples were mixed with 20μ1 of Protein G Agarose for an hour at room temperature (rotating) to create a Protein G bead-antibody-sample complex. After centrifugation at 1 OOOg, supernatant was removed, and Protein G agarose beads were resuspended in 2 x SDS with 10 x DTT and heated at 100°C to break the bonds between the sample, antibody and Protein G Agarose. After centrifugation and Protein G Agarose bead removal, the resulting supernatant was subject to SDS-PAGE and WB. Resultant blots were visualised with a Fuji Film Imager, and then stripped with 0.1M Glycine in I xPBS and re-probed with different WB antibody combinations to obtain further results while minimising sample use. Antibody combinations used in immunoprecipitation

Results

Disease progression in the SODl^G93A mouse model of MNP

SODl⁰⁹^ mice are a model of MND, carrying 21 copies of the S0D1^G93A human mutant transgene and developing progressive disease from 120 days to end-stage. Behavioural and neurological tests were performed on a group of SODl^693- and B6 age-matched control mice. These tests aimed to show disease progression. Initially, data from male (n=5) and female (n=5) S0D1^G93A mice were analysed separately against B6 age- and gender-matched controls (n=5 per gender) to ensure that no statistically significant differences exist in disease progression based on gender (two-way ANOVA). After finding no statistical difference between the males and females, statistical analysis was performed between combined (male and female; n=l 0) data. It was found that SODl⁰⁹³* mice of both genders reached the end-stage of disease (as determined by a neurological score of 3) between 146 and 157 days of age, after which stage they were euthanased. The median survival in S0D1^G93A males (n=5) was 149 days, and in S0D1^G93A females (n=5), 153 days, whereas the B6 controls remained alive and healthy until the end of experimental procedures. When the survival of all the S0D1^G93A mice (n=10) was compared to that of the B6 age-matched controls (n=10), it was found that the decreased observed life span from 145 days in the SODl^G93A mice was statistically significant (p < 0.001 ; Kaplan Meier survival test).

S0D1^G93A mice of both genders displayed progressive hind-limb paralysis towards end-stage of disease, whereas B6 age-matched controls registered no signs of paralysis using neurological scoring. Hind limb paralysis was evident on average at 132 days of age in SOD1 ^G93A males and all males showed paralysis by 145 days (n=5). Females showed paralysis on average at 145 days and all displayed symptoms by 150 days (n=5). At 140 days of age, the neurological scores recorded for all SODl^G93A mice (n=10) were significantly higher than those of age-matched controls (n=l 0) (p < 0.001 , two-way ANOVA) and continued to be so at 145, 150, 155 and 160 days of age until euthanasia.

First signs of grip strength decrease were detected around 129 days of age in S0D1^G93A males (n=5), and around 135 days of age in SODl⁰⁹³'* females (n=5). The grip duration scores of all S0D1^G93A mice (n=10) were significantly lower than those of the age-matched controls (n=10) at 135 days and continued to be so at 145 and 150 days of age until euthanasia (p < 0.001, two-way ANOVA), whereas age-matched controls were able to hang from the wire mesh for over 90 seconds in each trial , until experiments were terminated.

Male SODl^G93A mice weights (n=5) were significantly lower than males of age-matched controls (n=5) at 145 days (p < 0.05) and at 150 days the difference in weight increased (p < 0.001). Female SODl^093- mice weights (n=5) followed a similar pattern to that observed in the SOD1 ^G93A males; female weights (n=5) were significantly lower than that of the female age-matched controls (n=5) at 145 days (p < 0.01) and decreased further at 150 days (p < 0.001, all tests two-way ANOVA).

Statistical significance was seen between the weights of pre-symptomatic male (n=5) and female SODl⁰⁹³"⁴¹ mice (n=5) (60 days p < 0.05; 80 days p < 0.01 , 100 days p < 0.01 ; two-way ANOVA) and so weight analyses were not pooled to determine weight change against B6 controls.

Optimising Western Blot for detection of mouse p75NTR

Western blotting (WB) was used to determine the abundance of p75NTR in SODl⁰⁹^ and B6 mouse urine. A number of commercial and in-house antibodies were tested, the aim being to detect both the full length (~60-67kDa) and extracellular domain (ECD; ~50kDa) of p75NTR. Initially, samples of mouse (NSC-34), rat (C6), human (A875) and mouse embryonic day 14 spinal cord cell lysates (known to contain p75NTR) were tested by WB.

WB using polyclonal goat anti-mouse p75NTR (Sigma-Aldrich) shows a band of 60 to 67kDa in the mouse cell lysates and embryonic spinal cord lysates of El 4 using goat anti-mouse p75NTR under reducing conditions (Figure 1 A, lanes 8 and 9). Lower molecular weight bands (~50kDa) represent the ECD of p75NTR. The goat anti-mouse p75NTR (Figure 1 A) also detected bands corresponding to mouse (lane 4), rat (lane 3) and human p75NTR (lane 2) under non-reducing conditions. No bands were detected in a control cell lysate subject to WB that does not contain p75NTR (Figure 1 A, lane 1). As previously described (Rogers ML et al, 2006), the monoclonal mouse anti-human p75NTR MLR2, cannot detect p75NTR under reducing conditions. MLR2 detects bands corresponding to human p75NTR under non-reducing conditions with a band at 60kDa (Figure IB, lane 2). There were no bands detected when MLR2 was used under non-reducing conditions in rat (Figure IB, lane 3) and mouse (Figure IB, lane 4) cell lysates, or in the control cell lysate (Figure IB, lane 1). Two bands were seen clearly at 60kDa and 50kDa in mouse cell lysates using rabbit anti-human p75NTR (Figure 1C). To verify that the goat anti-mouse p75NTR antibody was detecting mouse p75NTR, recombinant p75NTR-Fc was run on WB in addition to control cell lysates not known to contain p75NTR (Figure 2). This antibody can detect 5ng (Figure 2, lane 3) and lOng (Figure 2, lane 2) of mouse p75NTR-Fc as indicated by a band at 65kDa, but no bands were seen with BSR negative control cell lysates (Figure 2, lane 1). Also, no bands were visible when an identical blot was exposed to secondary bovine anti-goat-HRP alone (no primary antibody) confirming that the human Fc part of mouse p75NTR-Fc is not detected with secondary bovine anti-goat-HRP that has been cross-reacted against various species IgG including human {data not shown). Subsequently, goat anti-mouse p75NTR was used for the detection of p75NTR in mouse urinary protein using WB.

Detection of p75NTR in mouse urinary protein by Western Blotting

Western blotting (WB) using goat anti-mouse p75NTR was used to detect p75NTR in SOD1 °"^A mouse urine. 2(^g of urinary protein samples from SODl^G93A mice and B6 age-matched controls were subject to WB from 40 days to end-stage (Figure 3 A). No bands were visible in mouse urinary protein before end-stage (Figure 3 A, lanes 1-8). This was repeated four times, with broad p75NTR bands always detected in S0D1^G93A mouse urine that had reached end-stage disease (145-160 days; Figure 3 A, lane 9) and, as expected, p75NTR is visible in the positive control mouse cell lysates (NSC-34; Figure 3 A, lane 11). Sample loading detected by the total protein stain Sypro Ruby is shown in Figure 3B. WBs were then used to quantify the p75NTR in end-stage SOD1 ^G93A and B6 age-matched control mice. Two samples of S0D1^G93A end-stage urinary protein (Figure 4 A, lanes 3 and 4), and two from B6 control mice (Figure 4A, lanes 1 and 2) in addition to a standard curve of mouse p75NTR-Fc (Figure 4A, lanes 6-9) were subject to WB. Fuji Imager Multi-Gauge software was then used to graph a standard curve of p75NTR (Figure 4B) and the amount of p75NTR in the urinary samples was determined. This procedure was repeated for three different blots, with the amount of p75NTR detected graphed in relation to both the total protein concentration, and per ml of urine (Figure 5C and Figure 5D). In end-stage SODl^G93A mice, 12.80 ± 2.7ng (n=6) of p75NTR per mg of urinary protein was detected or 22.74 ± 6.4ng (n=6) of p75NTR per ml of urine. In comparison, B6 mice had 0.18 ± 0.18ng (n=6) of p75NTR per mg of urinary protein or 1.4 ± 1.2ng (n=6) of p75NTR per ml of urine. The large standard error in the B6 control samples reflected that p75NTR was only found in 1 of 6 samples.

Development of immunoprecipitation for detecting mouse p75NTR

In order to confirm the identity of p75NTR in the mouse urinary protein and to gain more sensitivity, an immunoprecipitation (IP) protocol was developed. Eight different combinations of antibodies and running conditions were tested on mouse (NSC-34), rat (C6) and human (A875) derived cell lysates, to determine the combination that would be best able to visualise mouse-derived p75NTR. When MLR1 or MLR2 was used as pull down antibody for IP and goat anti-mouse p75NTR as detection (Figure 5A and C), mouse and human p75NTR detection was enriched when compared to WB of cell lysates alone (Figure 5A, lane 3 and 4 compared to Figure 5A, lanes 6 and 7). 60 and 50kDa bands corresponding to mouse derived full length and ECD of p75NTR were present in IPs using MLR1 or MLR2 as pull down and goat anti-mouse p75NTR as detection (Figure 5A and C, lane 4).

However, there was a 150kDa band pulled down using MLR1 (Figure 5C, lane 4) not found in the corresponding lysates (Figure 5C, lane 7) indicating that this antibody may not be as effective as MLR2 in pulling down mouse p75NTR (Figure 5D, lane 4) where no such band is present. MLR1 and MLR2 as pull down and goat anti-mouse detection also detects p75NTR from human-derived A875 cell lysates (Figure 5A and C, lane 3) at 50, 65 and 150kDa. These same bands were also present in WB of human cell lysates (Figure 5 A and C, lane 6). MLR1 or MLR2 as pull down, with rabbit anti- human p75NTR antibody as detection shows the presence of p75NTR from human cell lysates (Figure 4B and D, lanes 3) but not mouse (Figure 4 A and D, lane 4) or rat (Figure 4A and D, lane 2). The control IPs (no sample) showed a band at 25kpa (Figure 4A, B, C and D, lane 1 ). This indicates that the secondary antibodies used in IP procedures, even though cross-reacted against mouse, rat, human, rabbit and goat IgG, are detecting the light chain of IgG. IP protocols using goat anti-mouse p75NTR for pull down and MLR2 as detection were not effective in cell lysates; however a band at 65kDa was present when BSA was spiked with 5ng p75NTR-Fc (Figure 6E, lane 1 ). Previous WB shows that this antibody was unable to detect p75NTR under reducing conditions (Figure 1 B, lane 6- 9) but able to detect human p75NTR in non-reducing conditions (Figure IB, lane 2). There were light bands at 60kDa (Figure 6A, lane 2-4) using MLR2 as detection but non-reducing conditions were messy (Figure 6B, lane 1-3). Dark bands at approximately 150kDa in all IP samples (Figure 6B, lane 1-3) suggest that non-reducing conditions may be ineffective for IP.

Rabbit anti-human p75NTR antibody as detection with goat-anti mouse as pull down, showed bands corresponding to multimeric p75NTR (150kDa), full length (60kDa) and ECD (50kDa) in human (Figure 6C, lane 3) and rat (Figure 6C, lane 2) cell lysates under reducing conditions. Interestingly, the strongest bands were seen in rat cell lysates (Figure 6C, lane 2). This combination of goat anti- mouse as pull down and rabbit anti-human p75NTR as detection was ineffective at pulling down mouse-derived p75NTR under reducing or non-reducing conditions (Figure 6C, lane 4, and Figure 6D, lane 3). Urine samples from end-stage S0D1 ^G93A mice were subject to the IP protocol with different amounts of protein and pull down antibody, to discern the most suitable combination for accurate detection of p75NTR (Figure 7). When 500μg (Figure 7, lane 1) or 1 10μg (Figure 7, lane 2) of urine was subject to IP and the amount of antibody used was 5 μg, a broad band near 50kDa was detected in urine by IP. However, when 20μg of urine was pulled down with the 5 g of antibody, there were no bands (Figure 7, lane 3). In addition, raising the amount of pull down antibody to l (^g, increased the number of probable non-specific bands (Figure 7, lane 4), whereas 5 μg of pull down antibody was effective at pull down (Figure 7, lane 5).

Urinary protein of SODl⁰⁹^ and B6 control mice

Preliminary investigations of urinary content from S0D1^G93A mice in comparison to B6 controls were performed to discern protein concentration and specific gravity. It was found that the average protein concentration determined for urine samples from SODl^G93A mice did not differ significantly from B6 control urinary protein across all ages (two-way ANOVA). The identical values obtained using a specific gravity test also showed no difference, suggesting that differences in p75NTR levels in the urinary protein are not due to a change in kidney/ bladder function in the SODl^G93A mice. Silver staining of urinary protein samples separated by SDS-PAGE was also performed, as shown in Figures 8 and 9. Silver Stain of S0D1^G93A end-stage and B6 age-matched control urinary protein (Figure 8, lane 1 and 2) indicated no obvious differences in protein bands. The Sypro Ruby total protein stain in Figure 3 also showed no obvious differences in the total protein of SODl^G93A and B6 control urine at any of the ages tested. Large bands present at 25kDa and below in Figure 3, lane 1-10 and Figure 8, lane 1 and 2 are likely to be the Major urinary proteins (MUPs) (Cavaggioni A & C Mucignat-Caretta, 2000).

The presence of p75NTR before MNP symptoms

After determining conditions for urinary p75NTR IP and showing disease progression in a sample population of SOD 1^093/11 and B6 control mice, tests were run to determine at what age of life p75NTR could be detected. Urinary protein from SODl^G93A and B6 age-matched controls from 40 (n=2), 60 (n=3), 80 (n=2) and 100 (n=2) days and end-stage (B6 n=2; βθϋΐ^093*11 n=5) (145-160 days) were therefore subjected to IP. Although 500μg of sample showed the most p75NTR in SODl^G93A end-stage urine, the lower but still detectable sample volume of 1 10μg (Figure 7, lane 2) was used because of the available urinary protein sample sizes.

The results are shown in Figures 10 and 11. In particular, Figure 10 shows that p75NTR was detectable in the urinary protein of SOD I⁰⁹³ mice at 60 (lane 4), 80 (lane 6), and 100 (lane 8) days of age, and also at end-stage (lane 10), whereas p75NTR was not detectable in the B6 age-matched control mouse urinary protein until older age (145-160 days; Figure 10, lane 9). No p75NTR was detected in SODl⁰⁹³¹^ mouse urine of 40 day old mice (lane 2). In contrast to when p75NTR was first detectable in the urinary protein of SOD 1 mice, symptom onset was first observed by the grip duration test at 100 days, and the difference in grip duration was not significant between SODl⁰⁹³* and B6 control mice at 100 days of age but was at 135 days (p < 0.001 , two-way ANOVA). Moreover, neurological scores were significant at 145 days (p < 0.001 , two-way ANOVA) in contrast to p75NTR detected at 60 days (Figure 1 1). Weight changes also showed significance at 145 days, well after p75NTR is detected in S0D1^G93A mouse urine.

Further, using sandwich ELISA analysis of diafiltered SOD l⁰⁹³* , and C57BL/6J (B6) mice urine (analysed at 10% in sample buffer), it was also found that there are higher levels of p75NTR in SODl⁰⁹^ mouse urine at 60 and 80 days of age and at end-stage (see Table 3).

Table 3 p75NTR measured by sandwich ELISA (pg/ml)

Discussion

It has been demonstrated that the neurotrophin receptor p75 (and fragments thereof) is detectable in the urinary protein of SOD^G93A mice and not B6 age-matched controls. No obvious differences were found between the total protein composition of urinary protein from SOD 1^{093 A} and B6 controls, as determined by studies of protein concentrations and specific gravity. Given that this indicates that kidney function is not altered in the SOD 1^{0 3 A} mouse, the presence of p75NTR in urine is not due to a change in the processing of urinary protein. p75NTR was first detected in the SOD1^{093 1} mouse at 60 days of age, which is earlier than the first detectable signs of paralysis shown in behavioural and neurological tests. These findings indicate that p75NTR could be used as a biomarker for MND diagnosis, prognosis and monitoring of MND disease progression. Further, while the SODl^G93A mouse model of MND is considered to be the standard model for testing possible therapeutics in pre-clinical trials, the hitherto lack of any biochemical biomarkers for determining disease progression in the SODl^{G 3A} mouse has presented a barrier to the successful translation between pre-clinical trials and human trials. Thus, the present finding that the presence of p75NTR (and fragments thereof) in the urine of S0D1^G93A mice could be detected and used as a biomarker to monitor MND progression in this animal model (particularly, pre- and post-symptomatic), indicates that p75NTR also represents a useful biomarker for assessing the effects of potential therapeutic agents for human MND. Example 2

This example investigated whether the presence of p75NTR in the urine of human patient samples could also be detected and used as a biomarker for MND and a tool for monitoring MND progression.

Methods and Materials

Urinary sample collection and preparation

Urinary samples were obtained from eight sporadic MND patients and five healthy individuals. MND patients were all assessed as having sporadic (non genetic) MND with bulbar or leg onset with an age range of 61± (41-78y at onset) and both upper and lower motor neuron damage that cannot be attributed to other causes. Urinary samples were collected and stored at -70°C within 2 hours of collection after centrifugation at lOOOg for 10 min (at 4°C) prior to diafiltration substantially as described above in Example 1.

Immunoprecipitation

A 500 μg sample of urinary protein from a single MND patient (with sporadic MND, bulbar onset and 23 months since diagnosis) was immunoprecipitated, and subject to Western blot as described for mouse samples above In Example 1. Protein (500 μg) from A875 human melanoma cells was used as a positive control, and protein (500 μg) from baby hamster kidney fibroblast cells was used as a negative control.

Assessment of signal to noise in p75NTR sandwich ELISA

A sandwich ELISA using MLR2 anti-human p75NTR monoclonal antibody for p75NTR capture and the goat anti-mouse polyclonal antibody (N5788) for p75NTR detection, was assessed for sensitivity and signal to noise (S/N) ratios. Samples comprising 3.12, 6.25, 12.5, 25, 50, 100, 200 and 400 pg ml of mouse and human p75NTR protein in sample buffer were tested in the ELISA and absorbance measured at 450nm. S N ratios were calculated by dividing the absorbance at 200 pg/ml by the background absorbance.

Detection of relative levels of p75NTR in MND and healthy samples

Urine samples from end stage S0D1^G93A mice (n=4) and age-matched C57BL/6J (B6) healthy control mice (n=4) were run in the mouse sandwich ELISA at 10% urine in sample buffer.

Additionally, urine samples from MND patients (n=8) and healthy individuals (n=6) were run in the human sandwich ELISA at 10% urine in sample buffer. The amounts of p75NTR (or fragment thereof) in the samples were measured using standards of mouse p75NTR-ECD (#1 157-NR; R&D Systems, Inc., Minneapolis, MN, United States of America)) or human p75NTR-ECD (#PE-1237; Biosensis).

Results

A urinary protein sample from an MND patient and healthy individuals was subjected to immunoprecipitation/Westera Blot (IP/ WB). The results are shown in Figure 12; the p75NTR extracellular domain (ECO; ~50kDa, highlighted by the black box) was clearly detectable in MND patient urine but was not detectable in healthy individual control urine or the negative control cell protein.

Next, a novel sensitive sandwich ELISA for urinary p75NTR was assessed for sensitivity and signal to noise (S N) ratios. The results are provided in Figure 13; it was found that both mouse and human derived forms of p75NTR are detectable to nanomolar sensitivity, with acceptable levels of signal to noise ratios (S/N < 3.5) achieved.

Additionally, urine samples from MND patients (n=8) and healthy individuals (n=6) were run in the human sandwich ELISA at 10% urine in sample buffer. The amounts of p75NTR (or fragments thereof) in the samples were measured by comparison with mouse p75NTR-ECD (1157-NR) and human recombinant p75NTR-ECD (PE-1237).

Using the ELISA for urinary p75NTR, an assessment was made of the relative amounts of p75NTR (or fragments thereof) in urine samples. Preliminary data (Figures 14A and B) shows significantly higher levels of p75NTR in the urine of end stage SODl^G93A mice in comparison to age-matched C57BL/6J (B6) healthy control mice (** = _/J<0.05, two-tailed t-test). Significantly higher levels of p75NTR-ECD were also detected in the urine of MND patients compared to that of urine of healthy individuals (** =p<0.05, two-tailed t-test). When urine samples were tested in the sandwich ELISA, it was also found that there were significantly (p=0.048) higher levels of p75NTR in urine from patients at 0-6 months (n=4) or 12-25 months (n=3) after MND diagnosis compared to healthy individuals (one-way ANOVA).

Discussion

It has been demonstrated that urine samples from MND patients, but not healthy individuals, showed the presence of the p75NTR-ECD by IP/ WB. Also, using a novel sandwich ELISA, it was also found that there are significantly higher p75NTR levels in the urine of S0D1^G93A mice and MND patients than controls. These findings provide strong evidence that p75NTR can be used as a biomarker for both the SODl^CT3 MND mouse model and human MND. The use of p75NTR (or a fragment thereof) as a biomarker, would enable progression towards rapid identification of new effective treatments and therapeutic agents for MND, as well as the faster exclusion of ineffective ones. Exam le 3

This example investigated whether the presence of p75NTR in the urine of SODl⁰⁹³* mice and human MND patients, could be detected using mass spectroscopy (MS) sequencing. Methods and Materials

Mass spectroscopy analysis for p75NTR

Urine samples (ie 1 ml end-stage S0D1^G93A mice and 5 ml of MND patient urine) were diafiltered with 1 x PBS (lOx volume) and, following protein estimation using a Lowry assay, an approximately 70(^g sample subjected to immunoprecipitation (IP) using mouse anti-human p75NTR (MLR2; 2.0 μg) followed by Protein G Agarose beads (5 μΐ) essentially as described above in Example 1). Beads were the washed four times with PBS 4x then placed into 25 μΐ of ammonium bicarbonate (pH 8.3) before being subjected to digestion with Proteomics-grade protease glutamyl endopeptidase (GluC; Sigma-Aldrich). The supernatant containing the digested peptides were analysed with a Thermo Orbitrap XL linear ion trap mass spectrometer fitted with a nanospray source (Thermo Electron Corporation, San Jose, CA, United States of America). Next, the samples were applied to a 300 mm i.d. x 5 mm CI 8 PepMap 100 precolumn (Dionex Corporation, Sunnyvale, CA, United States of America) and separated on a 75 mm x 150 mm CI 8 5μπι ΙΟθΑ column (Nikkyo Technos, Co., Ltd, Tokyo, Japan), using a Dionex Ultimate 3000 HPLC (Dionex) with a 55 minute gradient from 2% acetonitrile (CAN) to 45% ACN containing 0.1% formic acid at a flow rate of 200 nl/min followed by a step to 77% ACN for 9 minutes. The mass spectrometer was operated in positive ion mode with one FTMS scan of m/z 300- 2000 at 60,000 resolution followed by ITMS or FTMS product ion scans of the 6 most intense ions with dynamic exclusion of 15 seconds with 10 ppm low and high mass width relative to the reference mass, an exclusion list of 500 and collision-induced dissociation energy of 35%. Only multiply charged ions were selected for MS/MS of Glu-C digested peptides.

The spectra were searched with BSI (Bioinformatics Solutions Inc., Waterloo, ON, Canada), PEAKS Studio software against the Swissprot database. Searches were performed using a precursor mass tolerance of 10 ppm for identification of precursor ions. Mouse p75NTR was identified by checking peptides against mouse Q9Z0W1 (TNR16 MOUSE; mouse p75NTR) and human p75NTR using P08138 (TNR16_HUMAN; human p75NTR) from the UniProtKB/Swiss-Prot database

(http://www.uniprot.org/uniprot/).

Results

The mouse p75NTR was identified in the urine samples by mass spectroscopy sequencing of the peptides sequences:

1. CLGLQSMSAPCVE (SEQ ID NO:3), which corresponds to the sequence of amino acid residues 82-94 of mouse p75NTR (m/z 688.2995; mass 1352.5774 Da); and

2. DTERQLRE (SEQ ID NO:4), corresponding to the sequence of amino acid residues 165-172 of mouse p75NTR (m/z 524.2594; mass 1046.4993 Da).

The presence of human p75NTR was identified in human MND patient urine by mass spectroscopy sequencing of the peptide sequences:

1. RQLRECTRWADAECEE (SEQ ID NO:5), corresponding to amino acid residues 175-190 of human p75NTR (m/z 499.4715; mass 19993.83734 Da);

2. TRWADAECEEJPGRWITRSTPPE (SEQ ID NO:6), corresponding to amino acid residues 181-203 of human p75NTR (m/z 850.3738; mass 1698.3722 Da); and

3. GSDSTAPSTQEPEAPPE (SEQ ID NO:7), which corresponds to the sequence of amino acids 204-220 of human p75NTR (m/z 468.4878; mass 1869.9043 Da). Discussion

It has been demonstrated that detection of p75NTR in urine by mass spectroscopy sequencing (detection) is a viable alternative to immunoassay-based approaches. Mass spectroscopy sequencing may offer the advantages of increased sensitivity and specificity using either targeted MS for specific peptides or multiple reaction monitoring for quantitation of these peptides.

Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

The reference to any prior art in this specification is not, and should not be taken as, an

acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

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Claims

A method of diagnosing or prognosing motor neuron disease (MND) in a subject, the method comprising:

The method of claim 1, when used in combination with an independent analysis of one or more other biomarkers of MND selected from the group consisting of: decreased levels of 5- methyltetrahydrofolate in plasma, increased levels of phosphorylated neurofilament subunit H (pNF-H) in serum, increased levels of serum metalloproteinase-9 (MMP-9), increased levels of tau, decreased levels of SlOObeta and soluble CD14 in cerebrospinal fluid, increased levels of TDP-43 in cerebrospinal fluid, and fMND- and sMND-linked mutations in the SODl gene.

A method for treating motor neuron disease (MND) in a subject, wherein said method comprises diagnosing or prognosing MND in said subject by:

thereafter administering to said subject an effective amount of an agent for the treatment of MND, optionally in admixture with a pharmacologically-acceptable carrier and/or excipient.

A method of monitoring motor neuron disease (MND) progression in a subject, the method comprising:

A method of assessing the effectiveness of a therapy applied to treat motor neuron disease (MND) in a subject, the method comprising:

(i) detecting p75 neurotrophin receptor (p75NTR) or a fragment thereof in a test body sample from said subject; or (ii) detecting a change in the amount of p75NTR or a fragment thereof in a test body sample from said subject taken at two or more time points.

The method of any one of claims 1 to 5, wherein the method comprises detecting p75NTR extracellular domain (ECD) or a p75NTR fragment including the epitope sequence of SEQ ID NO: 1.

The method of any one of claims 1 to 5, wherein the method comprises detecting a change in the amount of p75NTR extracellular domain (ECD) or a p75NTR fragment including the epitope sequence of SEQ ID NO: 1.

The method of any one of claims 1 to 7, wherein the test body sample(s) is whole blood, blood plasma or serum.

The method of any one of claims 1 to 7, wherein the test body sample(s) is urine.

The method of any one of claims 1 to 9, wherein the subject is a human.

The method of any one of claims 1 to 10, wherein the p75NTR or a fragment thereof is detected by immunoassay.

The method of any one of claims 1 to 10, wherein the p75NTR or a fragment thereof is detected by mass spectroscopy sequencing.

The method of claim 12, wherein the detection of the p75NTR or a fragment thereof uses an antibody or fragment thereof that specifically binds p75NTR extracellular domain (ECD) or a p75NTR fragment including the epitope sequence of SEQ ID NO: 1.

A method of screening an agent that is capable of treating motor neuron disease (MND) in a subject, wherein said method comprises the steps of;

providing an animal model for MND;

administering a test agent to said animal; and

15. The method of claim 14, wherein the animal model is a SOD1 G93A mouse or another transgenic mouse or animal expressing a human SOD1 gene comprising an MND-linked mutation(s).

The method of claim 14 or 15, wherein the method comprises detecting p75NTR extracellular domain (ECD) or a p75NTR fragment including the epitope sequence of SEQ ID NO: 1.

The method of claim 14 or 15, wherein the method comprises detecting a change in the amount of p75NTR extracellular domain (ECD) or a p75NTR fragment including the epitope sequence of SEQ ID NO: 1.

The method of any one of claims 14 to 17, wherein the test body sample(s) is whole blood, blood plasma or serum.

The method of any one of claims 14 to 17, wherein the test body sample(s) is urine.

The method of any one of claims 14 to 19, wherein the p75NTR or a fragment thereof is detected by immunoassay.

The method of any one of claims 14 to 19, wherein the p75NTR or a fragment thereof is detected by mass spectroscopy sequencing.

The method of claim 21 , wherein the detection of the p75NTR or a fragment thereof uses an antibody or fragment thereof that specifically binds p75NTR extracellular domain (ECD) or a p75NTR fragment including the epitope sequence of SEQ ID NO: 1.