Neurodegenerative disease biomarkers

Abstract

The invention relates to neurodegenerative disorders, and particularly to diagnostic and prognostic methods, apparatus and kits for neurodegenerative disorders, such as mild cognitive impairment (MCI), Alzheimer's disease, Parkinson's disease, Huntington's disease or Motor Neurone disease. The invention also extends to methods of detecting novel biomarkers, and methods for treating patients diagnosed or prognosed with a neurodegenerative disorder. A mathematical correlation between the concentrations of Tau protein and T14 peptide is found in samples from healthy subjects without a mood disorder, but not in samples from subjects having neurodegenerative disorders or a predisposition thereto.

Description

[0001]Neurodegenerative Disease The invention relates to neurodegenerative disorders, and particularly to diagnostic and prognostic methods, apparatus and kits for neurodegenerative disorders, such as mild cognitive impairment (MCI), Alzheimer's disease, Parkinson's disease, Huntington's disease or Motor Neurone disease. The invention also extends to methods of detecting novel biomarkers, and methods for treating patients diagnosed or prognosed with a neurodegenerative disorder. Neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease or Motor Neurone disease, are some of the biggest socioeconomic burdens in the world with a growing worldwide incidence rate of almost 10 million new cases of dementia each year. Since both the incidence and prevalence of AD increases with age, the number of patients is growing significantly with an aging population. In 2015, there were over 46 million people living with dementia with an estimated socioeconomic cost of $800 billion annually and it is expected that the number of patients will rise to over 130 million by 2050 costing society over $2 trillion annually. AD was recently announced as the UK’s leading cause of death in the over 65’s, and in the US it is now the 6th leading cause of death across all ages. At present, there exists no single test that can be carried out to diagnose Alzheimer’s disease, or to prognose cognitive decline. Clinical classification of AD relies on a combination of subjective reporting, medical history evaluation, cognitive function tests, and costly brain imaging scans with no true classification possible until a post-mortem examination of the brain can be conducted. All of these tests require the involvement of expert medical practitioners, but are still of variable accuracy. They also take time, and are, therefore, slow and expensive. There is, therefore, a need to provide improved methods and kits for diagnosing disease in subjects suspected of having a neurodegenerative disorder, and especially Alzheimer’s disease, as well as for prognosing disease aetiology or progression in subjects already diagnosed with the condition. There is also a need to provide methods and apparatuses for screening, and detecting the risk of developing a neurodegenerative disorder in subjects who are pre-symptomatic for such conditions, or suspected of being so, namely the entire adult population. Ideally, such improved methods and apparatuses are quick, quantitative (or at least semi-quantitative or qualitative), accurate, painless and cheap. Above all, given the scale of screening required, any test would need to be independent of specialist supervision. The inventors have continued their previous work in this area, and have focused on the toxic peptide, “T14”, as well as Tau, a primary pathological marker of Alzheimer’s disease. T14 is derived from the C-terminus of acetylcholinesterase (AChE), and is present as a naturally occurring bioactive molecule in brain tissue. WO 2016 / 156803 describes an antibody raised against the AChE-derived peptide (T14) peptide, and it has previously been shown that T14 found in cerebrospinal fluid (CSF) or in venous blood samples can act as a robust biomarker for diagnosing neurodegenerative disorders, such as Alzheimer’s disease. However, as discussed in the Examples, the inventors have now surprisingly demonstrated that there is an unexpectedly strong correlation between the T14 peptide and Tau protein levels in healthy individuals, which is not apparent in subjects suffering from neurodegenerative disease. Such a correlation with tau has not been reported for any other brain molecule. Therefore, the inventors believe that this correlation (or absence thereof) can be used as a reliable diagnostic or prognostic biomarker of neurodegenerative disorder, and in particular, early stage (i.e. mild cognitive impairment, MCI) neurodegenerative disease. Hence, according to a first aspect of the invention, there is provided a method for diagnosing a subject suffering from a neurodegenerative disorder, or a pre- disposition thereto, or for providing a prognosis of the subject’s condition, the method comprising: (i) determining a plurality of values based on a sample obtained from a subject, the values comprising a concentration of Tau protein in the sample, and a concentration of a peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof in the sample; (ii) determining an expected value, wherein the expected value is based on a mathematical correlation between the concentration of Tau protein and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, in a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto, and the expected value is further based on either the determined concentration of Tau protein or the determined concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and (iii) comparing the expected value with a corresponding determined value, wherein the expected and determined values are: (a) expected and determined concentrations of Tau protein; (b) expected and determined concentrations of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and / or (c) an expected and a determined mathematical relation between concentrations of Tau protein and the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof based upon a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto; and when the determined value deviates from the expected value by more than a first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis. In a second aspect, there is provided a kit for diagnosing a subject suffering from a neurodegenerative disorder, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, the kit comprising: (i) means for determining, in a sample obtained from a test subject, a plurality of values based on a sample obtained from a subject, the values comprising a concentration of Tau protein in the sample, and a concentration of a peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof in the sample; and (ii) means for determining an expected value, wherein the expected value is based on a mathematical correlation between the concentration of Tau protein and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, in a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto, and the expected value is further based on either the determined concentration of Tau protein or the determined concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; wherein, when comparing the expected value with a corresponding determined value, the expected and determined values are: (a) expected and determined concentrations of Tau protein; (b) expected and determined concentrations of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and / or (c) an expected and a determined mathematical relation between concentrations of Tau protein and the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof based upon a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto; and wherein when the determined value deviates from the expected value by more than a first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis. In a third aspect, there is provided a method of treating a subject suffering from a neurodegenerative disorder, or a pre-disposition thereto, the method comprising: (i) determining a plurality of values based on a sample obtained from a subject, the values comprising a concentration of Tau protein in the sample, and a concentration of a peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof in the sample; (ii) determining an expected value, wherein the expected value is based on a mathematical correlation between the concentration of Tau protein and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, in a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto, and the expected value is further based on either the determined concentration of Tau protein or the determined concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and (iii) comparing the expected value with a corresponding determined value, wherein the expected and determined values are: (a) expected and determined concentrations of Tau protein; (b) expected and determined concentrations of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and / or (c) an expected and a determined mathematical relation between concentrations of Tau protein and the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof based upon a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto; when the determined value deviates from the expected value by more than a first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis; and (iv) administering, or having administered to the subject, a therapeutic agent that treats, prevents, reduces or delays progression of the neurodegenerative disorder, or the pre-disposition thereto. Advantageously, as demonstrated in the Examples, and as illustrated in Figure 11, the inventors have demonstrated that the correlation between T14 and Tau levels can be used as a reliable predictor of neurodegenerative disease with a high degree of sensitivity, and therefore allows routine diagnosis of a neurodegenerative disorder and appropriate intervention with therapeutic treatment. Advantageously, this diagnostic method is not dependent on the age, gender, medication or indeed any other individual subject circumstances, given that the person would effectively be providing their own benchmark (i.e. internal control). It will be appreciated that the method according to the first aspect is useful for enabling a clinician to make decisions with regards to the best course of treatment for a subject who is suffering from a neurodegenerative disorder. In addition, the method of the first aspect is useful for monitoring the efficacy of a putative treatment for a neurodegenerative disorder. Hence, the kit according to the second aspect is useful for providing a prognosis of the subject’s condition, such that the clinician can carry out the treatment according to the third aspect. Preferably, the kit is configured to carry out the method according to the first or third aspect. The kit may also be used to monitor the efficacy of a putative treatment for a neurodegenerative disorder. The method and the kit are therefore very useful for guiding a neurodegenerative disorder treatment regime for the clinician, and to monitor the efficacy of such a treatment regime. Furthermore, pharmaceutical companies would be able save vast amounts of time and costs in their drug development if an accurate measure of disease progression were readily available, in terms of timescales, smaller sample groups, and other value metrics, because the patient would effectively serve as their own control, as it could be monitored how much they had, or had not, deteriorated since an earlier test (e.g. a day, week or month previously). Therefore, preferably the methods and kits are used in a clinical trial to monitor the activity or efficacy of a drug administered to the test subject, and preferably determine how much the subject has, or has not, deteriorated since an earlier test. Examples of suitable therapeutic agents which may be administered to the subject to prevent or treat the neurodegenerative disorder include, but are not limited to, acetylcholinesterase inhibitors, such as Rivastigmine, Galantamine, and Donepezil, and / or N-methyl-D-aspartate (NMDA) antagonists, such as Memantine. Alternative therapies include the use of a cyclic peptide as described in WO2015 / 004430, a linear peptide as described in WO2015 / 053601, or a peptidomimetic as described in WO2018 / 033724, the contents of these three patent applications all being incorporated herein by reference. As demonstrated in the Examples, and as shown in Figures 14 and 15, the inventors have demonstrated that there is a significant correlation between T14 levels and tau proteins (p-Tau and t-Tau) in cognitively healthy (CH) individuals, which was not observed in those with mild cognitive impairment (MCI), and Alzheimer's disease (AD). Further investigation within the CH group indicated that this correlation was predominantly present in individuals without mood disorders, but disappeared in those with mood disorders, resembling the pattern seen in MCI and AD patients. These findings reinforce the newly discovered relationship between T14 and tau pathology, and suggest that the ratio of T14 / P-Tau or T-Tau levels in CH individuals can serve as a distinguishing factor between those with or without mood disorders. This observation supports a link between T14 and mood regulation in the IC nuclei, which are known to be particularly susceptible in AD. In one embodiment, therefore, the methods and kits of the invention may diagnose a subject suffering from a neurodegenerative disorder, or a pre-disposition thereto, or provide a prognosis of the subject’s condition, where the subject is cognitively healthy. It will be appreciated that the term “cognitively healthy” can mean that the subject can clearly think, learn, and remember, and / or does not suffer from any cognitive impairment, such as mild cognitive impairment (MCI). In another embodiment, the methods and kits of the invention may diagnose a subject suffering from a neurodegenerative disorder, or a pre-disposition thereto, or provide a prognosis of the subject’s condition, where the subject has at least one mood disorder and / or a history of at least one mood disorder. The mood disorder may be selected from a list of mood disorders consisting of: bipolar disorder, cyclothymia, depression, hypomania, major depressive disorder, disruptive mood dysregulation disorder, persistent depressive disorder, and premenstrual dysphoric disorder. It will be appreciated that when the determined value deviates from the expected value by more than the first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis. It may be appreciated that the determined value may be greater or less than the expected value. Accordingly, the first threshold amount may be understood to define a range, wherein an upper limit of the range is the expected value plus the first threshold amount, and a lower limit of the range is the expected value minus the first threshold amount, and when the determined value falls outside the range, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis. Preferably, the first threshold amount is 200% or less of the expected value, 100% or less of the expected value, 80% or less of the expected value or 60% or less of the expected value. Preferably, the first threshold amount is 55% or less or 52% or less of the expected value. More preferably, the first threshold is 50% or less, 45% or less, or 40% or less of the expected value. Even more preferably, the first threshold is 35% or less, 30% or less or 25% or less of the expected value. More preferably, the first threshold is 20% or less, 15% or less or 10% or less of the expected value. Most preferably, the first threshold is 5% or less of the expected value. Accordingly, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis, whenever the determined value deviates by more than any of these amounts from the expected value. Conversely, preferably, when the determined value deviates from the expected value by less than a second threshold amount, the subject does not suffer from a neurodegenerative disease, or have a pre-disposition thereto, or has a positive prognosis, and when the determined value deviates from the expected value by greater than the first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis. The second threshold amount may be understood to define a range, wherein an upper limit of the range is the expected value plus the second threshold amount, and a lower limit of the range is the expected value minus the second threshold amount, and when the determined value falls inside the range, the subject does not suffer from a neurodegenerative disease, or have a pre-disposition thereto, or has a positive prognosis, and when the determined value falls outside the range, the subject suffers from a neurodegenerative disease, or has a pre- disposition thereto, or has a negative prognosis. Preferably, the second threshold amount is 200% or less of the expected value, 100% or less of the expected value, 80% or less of the expected value or 60% or less of the expected value. Preferably, the second threshold amount is 55% or less or 52% or less of the expected value. More preferably, the second threshold is 50% or less, 45% or less, or 40% or less of the expected value. Even more preferably, the second threshold is 35% or less, 30% or less or 25% or less of the expected value. More preferably, the second threshold is 20% or less, 15% or less or 10% or less of the expected value. Most preferably, the second threshold is 5% or less of the expected value. Accordingly, the subject does not suffer from a neurodegenerative disease, or have a pre-disposition thereto, or has a positive prognosis whenever the determined value deviates from the expected value by less than any of these amounts from the expected value. Preferably, the first and second threshold amounts are the same. Preferably, the mathematical correlation between the concentration of Tau protein and the concentration of said peptide is given by the formula y=mx+c, wherein y is the determined or expected concentration of Tau protein, x is the determined or expected concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, and m and c are constants. Preferably, the determined mathematical relation is a value of one or both of said constants. In one embodiment, the determined mathematical relation comprises a ratio between the concentration of Tau protein and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof. The determined mathematical relation may comprise a ratio between the concentration of Tau protein minus a constant and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof. Alternatively, the determined mathematical relation may comprise a ratio between the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof minus a constant and the concentration of the Tau protein. In an alternative embodiment, the determined mathematical relation may comprise the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof minus the concentration of the Tau protein multiplied by a constant. Alternatively, the determined mathematical relation may comprise the concentration of Tau protein minus the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, multiplied by a constant. The neurodegenerative disease may be selected from a group consisting of: Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); schizophrenia; Lewy-body dementia; Frontotemporal Dementia; and Mild Cognitive Impairment. It is preferred, however, that the neurodegenerative disease is selected from a group consisting of Alzheimer's Disease, Parkinson's Disease; Motor Neuron Disease and Mild Cognitive Impairment, and preferably Alzheimer's Disease, Parkinson's Disease, and Mild Cognitive Impairment. Most preferably, however, the neurodegenerative disease is Alzheimer's Disease. Even more preferably, however, the neurodegenerative disease comprises mild cognitive impairment (MCI) presenting prior to any of the conditions described above, and in particular, the very early stages of Alzheimer's Disease. MCI is a definite condition, and not a state where one may or may not suffer from dementia later. Thus, the invention advantageously enables screening healthy subjects from those who are not healthy as they suffer from MCI. Following on from their previous studies, the inventors continued their research with acetylcholinesterase, and their antibody which exhibits immunospecificity to specific regions in the C-terminus of this enzyme, as described in WO 2016 / 156803. Acetylcholinesterase is a serine protease that hydrolyses acetylcholine, and is well- known to the skilled person. The major form of acetylcholinesterase which is found in the brain is known as tailed acetylcholinesterase (T-AChE). The protein sequence of one embodiment of human tailed acetylcholinesterase (Gen Bank: AAA68151.1) is 614 amino acids in length, and is provided herein as SEQ ID No:1, as follows: 1 mrppqcllht pslaspllll llwllgggvg aegredaell vtvrggrlrg irlktpggpv 61 saflgipfae ppmgprrflp pepkqpwsgv vdattfqsvc yqyvdtlypg fegtemwnpn 121 relsedclyl nvwtpyprpt sptpvlvwiy gggfysgass ldvydgrflv qaertvlvsm 181 nyrvgafgfl alpgsreapg nvglldqrla lqwvqenvaa fggdptsvtl fgesagaasv 241 gmhllsppsr glfhravlqs gapngpwatv gmgearrrat qlahlvgcpp ggtggndtel 301 vaclrtrpaq vlvnhewhvl pqesvfrfsf vpvvdgdfls dtpealinag dfhglqvlvg 361 vvkdegsyfl vygapgfskd neslisraef lagvrvgvpq vsdlaaeavv lhytdwlhpe 421 dparlreals dvvgdhnvvc pvaqlagrla aqgarvyayv fehrastlsw plwmgvphgy 481 eiefifgipl dpsrnytaee kifaqrlmry wanfartgdp neprdpkapq wppytagaqq 541 yvsldlrple vrrglraqac afwnrflpkl lsatdtldea erqwkaefhr wssymvhwkn 601 qfdhyskqdr csdl [SEQ ID No:1] The amino acid sequence of T30 (which corresponds to the last 30 amino acid residues of SEQ ID No:1) is provided herein as SEQ ID No:2, as follows:- KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL [SEQ ID No:2] The amino acid sequence of T14 (which corresponds to the 14 amino acid residues located towards the end of SEQ ID No: 1, and lacks the final 15 amino acids found in T30) is provided herein as SEQ ID No: 3, as follows:- AEFHRWSSYMVHWK [SEQ ID No: 3] Preferably, therefore, the peptide of SEQ ID No: 3, or a variant or fragment thereof is T14. However, fragments of T14 (SEQ ID No: 3) are also detectable in the methods and kits of the invention, and can act as diagnostic or prognostic markers used in accordance with the invention. Thus, in one embodiment, a fragment of SEQ ID No: 3 preferably comprises an amino acid sequence of SEQ ID No: 4 (i.e. T7), i.e. SYMVHWK. In another embodiment, a fragment of SEQ ID No: 3 preferably comprises an amino acid sequence of SEQ ID No: 5 (i.e. T8), i.e. SSYMVHWK. In another embodiment, a fragment of SEQ ID No: 3 preferably comprises an amino acid sequence of SEQ ID No: 6 (i.e. T9), i.e. WSSYMVHWK. In another embodiment, a fragment of SEQ ID No: 3 preferably comprises an amino acid sequence of SEQ ID No: 7 (i.e. T10), i.e. RWSSYMVHWK. In another embodiment, a fragment of SEQ ID No: 3 preferably comprises an amino acid sequence of SEQ ID No: 8 (i.e. T11), i.e. HRWSSYMVHWK. In another embodiment, a fragment of SEQ ID No: 3 preferably comprises an amino acid sequence of SEQ ID No: 9 (i.e. T12), i.e. FHRWSSYMVHWK. In another embodiment, a fragment of SEQ ID No: 3 preferably comprises an amino acid sequence of SEQ ID No:10 (i.e. T13), i.e. EFHRWSSYMVHWK. In other words, although detection of T14 (i.e. SEQ ID No: 3) is preferred, the invention may also rely on detection of one or more of any of T7-T13 (i.e. SEQ ID No: 4-10). The amino acid sequence of Tau is provided herein as SEQ ID No: 11, as follows:- MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSD AKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQEPESGK VVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRHAPELLKHQLLGD LHQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFP AEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQDAPLEFTFHVEITPNVQKEQAHSEEHLGRA AFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDD KKAKTSTRSSAKTLKNRPCLSPKHPTPGSSDPLIQPSSPAVCPEPPSSPKYVSSVTSRTGSSGAK EMKLKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSP GTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKH QPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPG GGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPV VSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL [SEQ ID No: 11] Preferably, therefore, Tau protein comprises or consists of SEQ ID No: 11, or a variant or fragment thereof. As shown in Figure 10, robust correlation was observed between T14 concentrations and total Tau levels in healthy subjects. However, as shown in Figure 9, an even more surprisingly strong correlation was observed between T14 concentrations and phosphorylated Tau levels in healthy subjects. Accordingly, the Tau protein detected in the methods and kits of the invention may comprise total Tau protein and / or phosphorylated Tau protein. More preferably, the Tau protein detected comprises total Tau protein and phosphorylated Tau protein. Most preferably, the Tau protein detected comprises phosphorylated Tau protein, and preferably not total Tau. The subject may be a vertebrate, mammal, or domestic animal. Most preferably, however, the subject is a human being, who may be male or female. The subject may be a child or adult. The age of the subject may be at least 20, 30, 40, 50, 60 or 70 years old. The subject may be at least 80 or 90 years old. The subject may be less than 90, 80, 70, 65, 60 or 50 years old. Preferably, the subject is tested before any symptoms of neurodegenerative disorder are apparent. In one embodiment, the methods and kit are used on a subject already diagnosed with a neurodegenerative disorder. Accordingly, the subject is preferably symptomatic and showing signs of cognitive decline or dementia. Thus, the methods and kit may be prognostic and monitor disease progression. In another embodiment, the methods and kit are used on a subject who is suspected of having a neurodegenerative disorder. The subject may be symptomatic for a neurodegenerative disorder. Thus, the methods and apparatus may be diagnostic. In a preferred embodiment, the methods and apparatus are used on a subject who is not suspected of having a neurodegenerative disorder. Preferably, the subject is tested before any symptoms of neurodegeneration, cognitive decline or neurodegenerative disorder are apparent. Preferably, the subject is pre- symptomatic. Thus, the methods and apparatus may be diagnostic. The methods according to the invention may be carried out in vivo, in vitro or ex vivo. Preferably, however, the methods are carried out in vitro. Preferably, the sample comprises a biological sample. The sample may be any biological material that is obtainable from the subject from which SEQ ID No:3 (T14), or a variant or fragment thereof, and Tau protein is obtainable. The sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof. Preferably, the sample comprises blood, urine, tissue, and / or CSF. In one preferred embodiment, the sample comprises CSF, most preferably lumber CSF. In another preferred embodiment, the sample comprises a peripheral tissue sample. Preferably, the sample comprises nasal discharge or nasal fluid. The kit may comprise a sample collection container for receiving the extracted sample. Blood samples and CSF from lumbar puncture may be assayed for T14 / Tau levels immediately. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before the T14 / Tau assay is conducted. Detection of T14 / Tau may be carried out on whole blood. Preferably, however, the blood sample comprises blood serum. Preferably, the blood sample comprises blood plasma. The blood may be further processed before the T14 / Tau assay is performed. For instance, an anticoagulant, such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Thus, the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting. Alternatively, the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction, which may be used for analysis. Hence, it is preferred that the T14 / Tau is analysed or assayed in a blood plasma or a blood serum sample. It is especially preferred that T14 / Tau concentration is measured in vitro from a blood serum sample or a plasma sample taken from the subject. Preferably, the kit and / or methods according to the invention are used to determine the concentration of T14 and Tau protein in the sample. The means for determining the T14 and Tau concentration, may comprise an assay adapted to detect the presence and / or absence of T14-positive cells (i.e. cells comprising SEQ ID No: 3) and Tau-positive cells in the sample. The kit or method may comprise the use of a positive control and / or a negative control against which the assay may be compared. T14 (SEQ ID No: 3) and Tau protein may be assayed by a number of ways known to one skilled in the art. For example, preferably an immunoassay is employed to measure T14 and / or Tau peptide levels. However, it will be appreciated that non- immuno based assays may be employed, for example, labelling a compound having affinity with a ligand of the T14 and / or Tau peptide molecule, and then assaying for the label. T14 and / or Tau peptide may also be determined with Western Blot analysis, which may be used to determine the total protein level of T14 and / or Tau peptide. T14 and / or Tau peptide concentration may therefore be detected by enzyme-linked immunosorbent assay (ELISA), fluorometric assay, chemiluminescent assay, or radioimmunoassay analyses. In an embodiment, T14 and / or Tau peptide concentration may therefore be detected using an ELISA-like format or a Lateral Flow Test format. The means for determining, in the sample obtained from the test subject, the concentration of T14 and Tau may comprise an anti-T14 antibody or antigen- binding fragment thereof, i.e. a T14-neutralising antibody, and an anti-Tau antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be polyclonal or monoclonal. The antibody or antigen-binding fragment thereof may be generated in a rabbit, mouse or rat. Preferably, the anti-T14 antibody or antigen-binding fragment thereof specifically binds to SEQ ID No:3. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acid in the C-terminus of SEQ ID No:3. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acid in SEQ ID No: 4 (i.e. SYMVHWK, which are the C-terminal amino acids numbers 7-14 of SEQ ID No: 3). Preferably, the antibody or antigen- binding fragment thereof specifically binds to a C-terminal lysine (K) residue in the epitope. The inventors have surprisingly observed that the C-terminal amino acid sequence VHWK in SEQ ID No: 3, which is described herein as SEQ ID No.12 (i.e. the C-terminal amino acids numbers 10-14 of SEQ ID No: 3), acts as an epitope for the antibody or antigen-binding fragment thereof. Accordingly, more preferably the antibody or antigen-binding fragment thereof specifically binds to one or more amino acid in SEQ ID No: 12. Most preferably, the antibody or antigen-binding fragment thereof specifically binds to SEQ ID No: 12. Hence, it will be appreciated that the epitope to which the antibody binds comprises or consists of SEQ ID No: 12. Thus, the antibody or antigen-binding fragment thereof binds specifically to SEQ ID No: 3, or a fragment or variant thereof, and can be used as or in the detection means. Preferably, the anti-T14 antibody or antigen-binding fragment thereof does not bind to SEQ ID No: 2 (i.e. T30). Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No: 13 (i.e. T15), i.e. NQFDHYSKQDRCSDL. Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No: 14 (i.e. β-amyloid (Aβ), i.e. DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. The kit of the second aspect may further comprise a label which may be detected. The term “label” can mean a moiety that can be attached to the means for determining, in the sample obtained from the test subject, the concentration of (i) T14 and (ii) Tau. Moieties can be used, for example, for therapeutic or diagnostic procedures. Therapeutic labels include, for example, moieties that can be attached to the antibody or fragment thereof described herein, and used to monitor the binding of the antibody to the T14 peptide (i.e. SEQ ID No: 3, or fragment or variant thereof) and the Tau protein (i.e. SEQ ID No: 11, or fragment or variant thereof). Diagnostic labels include, for example, moieties which can be detected by analytical methods. Analytical methods include, for example, qualitative and quantitative procedures. Qualitative analytical methods include, for example, immunohistochemistry and indirect immunofluorescence. Quantitative analytical methods include, for example, immunoaffinity procedures such as radioimmunoassay, ELISA or FACS analysis. Analytical methods also include both in vitro and in vivo imaging procedures. Specific examples of diagnostic labels that can be detected by analytical means include enzymes, radioisotopes, fluorochromes, chemiluminescent markers, and biotin. T14 peptide and / or Tau peptide concentrations may be measured by double- antibody sandwich ELISA, which will be known to the skilled technician. The ELISA may comprise using a suitable antibody for coating a microtiter plate. For example, such a suitable antibody for T14 may comprise an affinity-purified polyclonal anti- T14 peptide antibody described herein (WO 2016 / 156803). Antibodies for detecting Tau are known (e.g. Lumipulse® G pTau 181 kit with antibody detecting phosphorylated tau at site 181). Furthermore, the ELISA may comprise using a suitable antibody for detection. For example, such a suitable antibody may comprise peroxidase-labelled monoclonal mouse anti human T14 peptide antibody. Human T14 peptide, which may be purified from plasma, and which then may be quantified by amino acid analysis, may be used to calibrate a plasma standard using standard techniques known to the skilled technician. A label can be attached directly to the antibody, or be attached to the secondary binding agent that specifically binds T14. Such a secondary binding agent can be, for example, a secondary antibody. A secondary antibody can be either polyclonal or monoclonal, and of human, rodent or chimeric origin. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms “substantially the amino acid / nucleotide / peptide sequence”, “variant” and “fragment”, can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID Nos: 1-14, and so on. Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein. The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment. Preferably, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N / T)*100. Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45ºC followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65ºC. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in, for example, SEQ ID Nos: 1-15. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids. All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:- Figure 1 shows the intensity of T14 peptide (SEQ ID No: 3) normalised to total protein stain in human CSF from control (CTRL), mild cognitive impairment (MCI), and Alzheimer’s Disease (AD) patients. n=22, one-way ANOVA p=0.3298, outlier exclusion > mean + 3SD. Figure 2 shows (A) the intensity of T14 (SEQ ID No: 3) normalised to total protein stain in human CSF from control (CTRL) patients across ages (age range 50-78). n=7, p(two-tailed) = 0.2411, R=0.2611, outlier exclusion > mean + 3SD. (B) shows the intensity of T14 normalised to total protein stain in human CSF from mild cognitive impairment (MCI) patients across ages (age range 59-75). n= 11, p(two- tailed) =0.0209, R=0.4645, outlier exclusion > mean + 3SD. Figure 3 shows the correlation matrix of T14, LC (locus coeruleus), noradrenaline, Abeta and Tau in CTRL, MCI and AD patients. Figure 4 shows the correlation matrix of T14, LC, noradrenaline, Abeta and Tau in MCI and AD patients. Figure 5 shows the correlation matrix of T14, LC noradrenaline Abeta and Tau in CTRL patients. Figure 6 shows the correlation of T14 with pTau in AD patients, n=4, P(two-tailed) = 0.1057, Pearson’s r = 0.8943. Figure 7 shows the correlation of T14 with pTau in MCI patients, n=11, P(two- tailed) = 0.4286, Pearson’s r = 0.2663. Figure 8 shows the correlation of T14 with pTau in AD&MCI patients, n=15, P(two- tailed) = 0.2716, Pearson’s r = 0.3034. Figure 9 shows the strong correlation of T14 with (phosphorylated) pTau in healthy (CTRL) patients, n=7, P(two-tailed) = 0.0008, Pearson’s r = 0.9559. Figure 10 shows the strong correlation of T14 with (total) Ttau in healthy (CTRL) patients, n=7, P(two-tailed) = 0.0017, Pearson’s r = 0.9394. Figure 11 is a flowchart of one embodiment of a method of the invention. Figure 12 shows the distribution of T14 in cerebrospinal fluid from living humans, across groups. Levels of T14 in human CSF across cognitive impairment progression expressed as a percentage of control (healthy patients), normalised to Ponceau, total protein stain. ns = non-significant. Figure 13 shows correlations of T14 with t-Tau and p-Tau181 in cerebrospinal fluid from living subjects, across groups. A) Correlation between t-Tau (pg / ml) and T14 (A.U.) in human CSF from cognitively healthy controls, n = 22. B) Correlation between p-Tau181 (pg / ml) and T14 (A.U.) in human CSF from cognitively healthy controls, n = 22. C) Correlation between t-Tau (pg / ml) and T14 (A.U.) in human CSF from patients with MCI, n = 27. D) Correlation between p-Tau181 (pg / ml) and T14 (A.U.) in human CSF from patients with MCI, n = 27. E) Correlation between t- Tau (pg / ml) and T14 (A.U.) in human CSF from patients with AD, n = 23. F) Correlation between p-Tau181 (pg / ml) and T14 (A.U.) in human CSF from patients with AD, n = 23. G) Correlation between t-Tau (pg / ml) and T14 (A.U.) in human CSF from patients with FTD, n = 20. H) Correlation between p-Tau181 (pg / ml) and T14 (A.U.) in human CSF from patients with FTD, n = 20. A.U. - arbitrary units, normalised to Ponceau, total protein stain. Figure 14 shows common AD markers (t-Tau and p-Tau181) and ratios of them with T14 in live human cerebrospinal fluid (CSF) in cognitively healthy controls with and without mood disorder. A) Levels of t-Tau (pg / ml) in CSF in cognitively healthy controls with no mood disorder (n = 12) and mood disorder (n = 10). B) Levels of p-Tau181 (pg / ml) in CSF in cognitively healthy controls with no mood disorder (n = 12) and mood disorder (n = 10). C) Levels of T14 normalised to total protein stain in CSF from cognitively healthy controls with no mood disorder (n = 12) and mood disorder (n = 10). D) Ratio of t-Tau (pg / ml) and T14 (A.U.) in CSF in cognitively healthy controls with no mood disorder (n = 12) and mood disorder (n = 10). E) Ratio of p-Tau181 (pg / ml) and T14 (A.U.) in CSF in cognitively healthy controls with no mood disorder (n = 12) and mood disorder (n = 10). A.U. - arbitrary units, normalised to Ponceau, total protein stain. Figure 15 shows correlations of T14 with AD markers t-Tau and p-Tau181 and the ROC curves of the T14 with t-Tau and p-Tau181 ratios in cognitively healthy controls with and without mood disorder. A) Correlation between t-Tau (pg / ml) and T14 (A.U.) in human CSF from cognitively healthy controls without mood disorder, n = 12. B) Correlation between p-Tau181 (pg / ml) and T14 (A.U.) in human CSF from healthy controls without mood disorder, n = 12. C) Correlation between t-Tau (pg / ml) and T14 (A.U.) in human CSF from cognitively healthy controls with mood disorder, n = 10. D) Correlation between p-Tau181 (pg / ml) and T14 (A.U.) in human CSF from cognitively healthy controls with mood disorder, n = 10. E) ROC curve of T14 and t-Tau (pg / ml) ratio in CTRL with and without mood disorder. F) ROC curve of T14 and p-Tau181 (pg / ml) ratio in CTRL with and without mood disorder. A.U. - arbitrary units, normalised to Ponceau, total protein stain. Examples The inventors have observed that a correlation (or absence of a correlation) between T14 and Tau levels can be used as a reliable predictor of neurodegenerative disease. Materials and methods Samples 200 µl of CSF samples were obtained through a collaboration with Dr Neus Falgas, Hospital Clinic de Barcelona. Initially, 22 samples (controls n=7 and AD- dementia / AD-MCI n=15) were tested for any correlation between T14 peptide (SEQ ID No: 3), Total Tau protein (TTau), phosphorylated Tau protein (pTau) and noradrenaline (the major source of which is the locus coeruleus, LC), amyloid-beta and the volume of the LC brain region (i.e. the size of the locus coeruleus was measured using imaging technology). To finalise the assessment, a total of 74 samples were used: -Alzheimer's Disease (AD, n = 52): All participants met the criteria of highest likelihood of AD biomarker pathology based on the National Institute on Aging and Alzheimer's Association (NIA-AA) diagnostic criteria (Alberts et al., 2011; McKhann et a., 2011). Our diagnostic protocol included a comprehensive neurological and neuropsychological evaluation, neuroimaging (CT or MRI scan), and a lumbar puncture. Based on the Clinical Dementia Rating (CDR), the AD participants were further classified as MCI (CDR 0.5, n = 28) or Dementia (CDR 1, n = 24). From now on we will refer to MCI due to AD as ‘MCI’ and AD dementia as ‘AD’. Cognitively healthy (CH, n = 22): Control participants had no cognitive complaints, presented normal cognition in a comprehensive neuropsychological testing, normal TC or MRI scan, and negative amyloid beta status in CSF.Mood disorders were noted based on clinical records and revealed n=10 with mood disorders, n=12 without. Detection of T14 peptide The primary antibody used to detect T14 was Ab20 A2 lyophilised antibody, reconstituted in sterile dH20 according to manufacturer’s instructions to the final concentration of 0.89mg / ml (peptide hapten; ‘N’ – CAEFHRWSSYMVHWK [SEQ ID No: 15], protein carrier – keyhole limpet hemocyanin, antigen- Cys-SH conjugate to protein carrier). Each gel had a secondary antibody only section to control for the bands being non-specific and caused by the secondary antibody. The secondary antibody was goat-anti-rabbit IgG H + L Horseradish peroxidase (HRP) conjugated antibody (1:10000, Thermo Fisher Scientific, cat. G-21234). Detection of Tau Total tau (tTau) was detected using: LUMIPULSE G System: https: / / www.fujirebio.com / en / products-solutions / lumipulse-g-total-tau. G fluid (CSF). This CSF biomarker is intended to be used as an aid for diagnosis in patients with cognitive impairment who are being evaluated for Alzheimer's Disease and other causes of cognitive decline. Phosphorylated tau (pTau) was detected using: Lumipulse® G pTau 181 - CLEIA / CLIA based assay for fully automated IVD immunoassay testing (fujirebio.com). Lumipulse® G pTau 181 - CLEIA / CLIA based assay for fully automated IVD immunoassay testing (fujirebio.com). Immunoreaction cartridges for in vitro diagnostic (IVD) use the LUMIPULSE G System for the quantitative measurement of Tau at threonine 181 (pTau 181) in human cerebrospinal fluid (CSF). This CSF biomarker is intended to be used as an aid for diagnosis in patients with cognitive impairment who are being evaluated for Alzheimer's Disease (AD) and other causes of cognitive decline. Detection of noradrenaline The inventors measured the free catecholamine noradrenaline concentrations by a non-competitive enzyme-linked immunosorbent assay (CatCombi ELISA, IBL international). After sample extraction using 300 microL of CSF in a borated affinity gel-coated extraction plate, chemical and enzymatic derivatization and immunological reactions were performed according to the manufacturer's protocol. Similar results were obtained using high-performance liquid chromatography with electrochemical detection (Chromsystems) and the Catecholamines in a plasma- HPLC kit (Chromsystems) after analytes were extracted from the CSF matrix by adsorption on alumina. The intra- and inter-coefficient variations were lower than 10%. Detection of amyloid-beta Amyloid beta was detected in the sample using the https: / / www.fujirebio.com / en / products-solutions / lumipulse-g-beta-amyloid-1-42 use with in human cerebrospinal fluid. This CSF biomarker is intended to be used as an aid for diagnosis in patients with cognitive impairment who are being evaluated for Alzheimer's Disease and other causes of cognitive decline. Tris-Glycine Western Blot and statistical analysis 10 µg of protein in all human CSF samples was mixed with 4X Laemmli Sample buffer (BioRad, United Kingdom, 101610747), 10% (v / v) of 2-mercaptoethanol and dH20 to make 48 µl total volume. The protein sample was then heated at 60°C for 10 minutes and loaded onto a 4-20% Mini-PROTEAN TGX Precast Gel 10 well (BioRad, United Kingdom, 4561093). Proteins were separated by electrophoresis 35mA and transferred from gels to polyvinylidene fluoride (PVDF) membranes. The Ponceau S Solution (ab270042, Abcam) was used according to the manufacturer’s protocol for 10 min to stain for total protein transferred to the membrane. It was then washed with water, and total proteins were visualized using a CCD Camera and G-Box software (Syngene, Cambridge, UK). The membrane was then blocked at room temperature for 1 hour in 5% defatted milk in Tris-buffered saline plus 0.05% 20 Tween 20 (TBS-T). The membranes were then incubated in the primary antibody against T14 (Ab20 A2, 1:1000, Genosphere), diluted in blocking solution, overnight at 4°C with gentle agitation. After primary incubation overnight, the membranes were washed three times with TBS-T for 5 minutes and incubated with the secondary goat-anti-rabbit IgG H + L Horseradish peroxidase (HRP) conjugated antibody (1:10000, Thermo Fisher Scientific, cat. G-21234), diluted in blocking solution, for 1 hour at room temperature, with gentle agitation. The membranes were then washed three times with TBS-T for 5 minutes, followed by one wash with TBS for 5 minutes. After washing, proteins were visualized using an enhanced chemiluminescence-based detection kit following manufacturer protocol (BioRad, United Kingdom, 1705061) and G-Box (Syngene, Cambridge, UK) gel system. Band intensity of the scanned blots were analysed using ImageJ and target proteins were normalised to total protein levels provided by the Ponceau S stain. GraphPad Prism (GraphPAD software, San Diego, CA) was used to obtain and analyse graphs. To perform correlations between T14, pTau and tTau in control (CTRL), mildly cognitively impaired (MCI) and Alzheimer’s (AD), Pearson’s correlation test (two- tailed) was used and to exclude any outliers 3xSD+mean was used. Lines were produced using simple linear regression for visual inspection of the data. Results Referring first to Figure 1, one can see that the amount of T14 peptide is lower in mild cognitive impairment (MCI) and Alzheimer’s Disease (AD) patients compared to the control (CTRL). Only the T14 is normalised in the figures, and tau (pTau or tTau) is in raw concentration. Figure 2 shows the relationship between T14 levels and patient age in control and MCI subjects. As can be seen, T14 levels tend to increase with age in healthy subjects, but decrease with age in MCI patients. Figures 3-5 are checkerboard plots showing: (a) the correlation matrix of T14, LC, noradrenaline, Abeta and Tau in CTRL, MCI and AD patients (Figure 3); (b) the correlation matrix of T14, LC, noradrenaline, Abeta and Tau in MCI and AD patients (Figure 4); and (c) the correlation matrix of T14, LC noradrenaline Abeta and Tau in CTRL patients (Figure 5). Figure 3 shows that there is no significant correlation between T14 and any of the parameters measured in CSF. Figure 4 shows that there is no significant correlation between T14 and any of the parameters measured in CSF when MCI and AD populations are combined. Figure 5, however, surprisingly shows that there is not only a strong correlation between pTau and T14, but an even stronger correlation between tTau and T14. Furthermore, it shows that although there is a good correlation between Abeta and T14, it is not as strong as for pTau or tTau. Referring to Figure 6, there is shown the correlation of T14 with pTau in AD patients. Figure 7 shows the correlation of T14 with pTau in MCI patients. In view of the low Pearson’s r = 0.2663, this is very low correlation. Figure 8 shows the correlation of T14 with pTau in AD&MCI patients. In view of the low Pearson’s r = 0.3034, this is very low correlation. Figure 9 shows the strong correlation of T14 with (phosphorylated) pTau in healthy (CTRL) patients. In view of the surprisingly high Pearson’s r = 0.9559, this is strong correlation. Figure 10 shows the strong correlation of T14 with (total) tTau in healthy (CTRL) patients. In view of the high Pearson’s r = 0.9394, this is strong correlation. Referring to Figure 12, it is demonstrated that levels of T14 showed a trend of increase across the cognitive impairment (between Cognitively Healthy (CH) (mean = 100), MCI (mean = 169.537) and AD (mean = 188.493), albeit not significant (ordinary one-way ANOVA: F(3,88) = 0.7821, p = 0.5071 with Dunnett’s multiple comparisons test CH vs MCI p = 0.7011, CH vs AD p = 0.5620. Figure 13 shows that T14 significantly correlated with t-Tau (Pearson’s correlation: r = 0.7082, p = 0.0002) and p-Tau181 (Pearson’s correlation: r = 0.6985, p = 0.0003) in CH (Fig 2 A-B). However, in MCI (t-Tau - Pearson’s correlation: r = - 0.1007, p = 0.6172; p-Tau181 - Pearson’s correlation: r = - 0.06821, p = 0.7353) and AD (t-Tau - Pearson’s correlation: r = 0.1334, p = 0.5439; p-Tau181 - Pearson’s correlation: r = 0.08027, p = 0.7158) that correlation was lost (Fig 2 C- F). Moreover, in FTD, T14 did not correlate with t-Tau - Pearson’s correlation: r = 0.4065, p = 0.0753 (Fig 2 G), p-Tau181 - Pearson’s correlation: r = - 0.2971, p = 0.2033, (Fig 2 H) or amyloid beta (Pearson’s correlation, p = 0.4398, n = 20). Further, there was no correlation between mood vs non-mood disorder in either MCI or AD, see Table 5 below. Table 5. Summary of correlations between T14, t-Tau, and p-Tau 181, with and without mood disorder in MCI and AD. r95%R numb inctoenrfviadence p valueer lsquaredof pairs MCI no T14 vs t-Tau -0.1064 -0.6029 to 0.4496 0.01131 0.7175 14 mood disorder T14 vs p-Tau -0.08004 -0.5857 to 0.4705 0.006406 0.7856 14 MCI T14 vs t-Tau -0.1522 -0.6488 to 0.4353 0.02317 0.6196 13 mood disorder T14 vs p-Tau -0.1003 -0.6172 to 0.4770 0.01007 0.7443 13 AD no T14 vs t-Tau 0.3407 -0.1674 to 0.7057 0.116 0.1809 17 mood disorder T14 vs p-Tau 0.2624 -0.2498 to 0.6598 0.06885 0.3089 17 AD T14 vs t-Tau -0.07552 -0.8359 to 0.7841 0.005703 0.8869 6 mood disorder T14 vs p-Tau 0.018 -0.8053 to 0.8176 0.0003238 0.973 6 Since mood disorders are associated with early neuropathological changes of AD due to the IC core disruption when AD CSF biomarkers might not still be altered, the inventors explored whether any of the biochemical measurements could be associated with this behavioural feature. Referring to Figure 14, although scoring equally on cognitive scores and having negative levels of established AD biomarkers, subjects within the control group could be differentiated between those with a history of mood disorders (n = 10) from those without (n = 12). For both t- Tau and p-Tau181, levels were indistinguishable in subjects with or without mood disorders: Figure 14 A; t-tau: t(20) = 0.3113, p = 0.7588, n = 22, Figure 14 B; p- Tau181: t(20) = 0.0276, p = 0.9783, n = 22. On the other hand, there was a non- significant trend towards reduction in T14 levels in the mood disorder subgroup, compared to those with no mood disorder (t(20) = 1.617, p = 0.1215, n = 22, as shown in Figure 14 C. A more emphatic effect emerged when T14 and Tau were calculated as a ratio of one to the other; there was a significant reduction in the ratio of T14 to t-Tau in patients with mood disorders compared to those without, t(20) = 2.837, p = 0.0102, as shown in Figure 14 D, and a trend towards an effect in the ratio of T14 to p-Tau181 (t(20) = 1.788, p = 0.0890, as shown in Figure 14 E. Referring to Figure 15, it was demonstrated that there was an increased correlation between T14 and t-Tau (Pearson’s correlation: r = 0.9250, p < 0.0001) and p- Tau181 (Pearson’s correlation: r = 0.8858, p = 0.0001) in CH samples only from the subgroup with no mood disorder, as shown in Figures 15 A and B), whereas the correlation seen previously within the cognitively healthy group, now disappeared when including only samples from the subgroup with mood disorder (t-Tau - Pearson’s correlation: r = 0.3363, p = 0.3420; p-Tau181 - Pearson’s correlation: r = 0.3691, p = 0.2939), as shown in Figures 15 C and D. ROC curves for t-Tau, showed a high significance (Area under curve = 0.8250, p = 0.0101, 95% Cl = 0.627-1), as shown in Figure 15 E), whereas for p-Tau181 the trend was similar though not significant (Area under curve = 0.7208, p = 0.0806, 95% CI = 0.5005- 0.9412), as shown in Figure 15 F. Graph Analysis The mathematical correlation between the concentration of Tau protein and the concentration of the T14 peptide in Figures 9 and 10 is given by the formula y=mx+c, wherein y is the determined or expected concentration of Tau protein, x is the determined or expected concentration of T14 peptide, and m and c are constants. When the data points of the subjects represented in Figure 9 are specifically analysed (i.e. T14 against pTau), the formula for the straight line is: [pTau] = 331.03 [T14] + 42.042, wherein y is [pTau], x is [T14], and 331.03 and 42.042 are constants. Similarly, when the data points of the subjects represented in Figure 10 are specifically analysed (T14 against tTau), the formula for the straight line is: [tTau] = 2123 [T14] + 141.51, wherein y is [tTau], x is [T14], and 2123 and 141.51 are constants. It will be appreciated that these formulae are specific to the units used to measure the concentrations of pTau, tTau and T14, and if one were to use different units, or had a larger sample size then the numbers would likely change slightly, and so the formulae can be genericised to: [Tau] = a[T14] + c, wherein a and c are constants. In order to determine if a person has neurodegenerative disorder, such as Alzheimer’s Disease, one can measure the concentration of Tau and T14, and then: 1. Put the value of the concentration of [T14] into the above formula, and thereby calculate an expected concentration of Tau (pTau and / or tTau), compare the expected concentration of Tau to the measured concentration, and if the measured concentration is within a certain range, the subject does not have Alzheimer’s Disease. 2. Put the value of the concentration of Tau (pTau and / or tTau), into the following formula: [T14] = ([Tau] - c) / a. One can thereby calculate an expected concentration of T14, compare the expected concentration of T14 to the measured concentration and if the measured concentration is within a certain range, then the subject does not have Alzheimer’s. 3. Put the numbers the concentration of Tau (pTau and / or tTau), and T14 into the following equation d = a[T14] – [Tau], then compare the value one gets for d to the value of c calculated for a population of subjects who do not suffer from Alzheimer’s disease or have a pre-disposition thereto, and if the value of d is within a certain range, then the subject does not have Alzheimer’s. 4. Put the numbers the concentration of Tau (pTau and / or tTau), and T14 into the following equation e = ([Tau] – c) / [T14], then compare the value one gets for e to the value of a calculated for a population of subjects who do not suffer from Alzheimer’s disease or have a pre-disposition thereto, and if it is within a certain range, then the subject does not have Alzheimer’s. For example, the expected concentration of pTau and tTau can be calculated for the healthy control subjects in Figures 9 and 10, respectively, using the formulae provided above. The results are provided below. Table 1: Difference between expected and determined concentration of pTau in subjects who do not suffer from Alzheimer’s or have a predisposition thereto (see Figure 9) Expected Difference Amount Determined concentration between determined concentration Determined of pTau based determined concentration of T14 concentration upon the concentration differs from (normalised of pTau / determined and expected the expected to total pg / ml concentration concentration concentration protein) of T14 / of pTau / / % of pg / ml pg / ml expected concentration 0.000571911 45 42 3 6.56% 0.329105812161151 10 6.63% 0.048921865 50 58 8 14.14% 0.1565853897994 15 15.85% 0.033227685 51 53 2 3.85% 0.0168752696748 19 40.67% 0.1176745477481 7 8.64% As can be seen in Table 1, using pTau, the determined concentration is within about 3-16% from the expected concentration in the majority of subjects. Only one sample is about 41% from the expected concentration. Table 2: Difference between expected and determined concentration of tTau in subjects who do not suffer from Alzheimer’s or have a predisposition thereto (see Figure 10) Expected Difference Amount Determined concentration between determined concentration Determined of tTau based determined concentration of T14 concentration upon the concentration differs from (normalised of tTau / determined and expected the expected to total pg / ml concentration concentration concentration protein) of T14 / of tTau / / % of pg / ml pg / ml expected concentration 0.000571911212143 69 48.54% 0.329105812935840 95 11.28% 0.048921865 228 245 17 7.08% 0.156585389308474 166 35.01% 0.033227685215212 3 1.39% 0.016875269241177 64 35.90% 0.117674547344391 47 12.10% As can be seen in Table 2, using tTau, the determined concentration is no more than 48% from the expected concentration. Although the correlation between tTau and T14 is not quite as strong as it is between pTau and T14, the inventors believe that there is still significant enough correlation for use in diagnosing MCI and / or Alzheimer’s disease, as discussed below. For example, the expected concentration of pTau and tTau can be calculated for the MCI and AD subjects in Figures 7 and 8, respectively, using the formulae provided above. The results are provided below. Table 3: Difference between expected and determined concentration of pTau in subjects who suffer from Alzheimer’s Dementia (first four subjects) or Alzheimer’s Mild Cognitive Impairment (remaining subjects) Expected Difference Amount Determined concentration between determined concentration Determined of pTau based determined concentration of T14 concentration upon the concentration differs from (normalised of pTau / determined and expected the expected to total pg / ml concentration concentration concentration protein) of T14 / of pTau / / % of pg / ml pg / ml expected concentration 0.056807513 190 61 129 212.26% 0.035579369 100 54 46 85.81% 0.0323422838853 35 66.83% 0.0084287057345 28 62.83% 0.0057457111944 75 170.80% 0.0365211928054 26 47.79% 0.13309991 258 86 172 199.64% 0.038348115 111 55 56 102.79% 0.014382639 113 47 66 141.44% 0.01845329 97 48 49 101.45% 0.0150594915847 111 235.98% 0.0503701137559 16 27.73% 0.025528027550 25 48.54% 0.209603333 90 111 21 19.23% 0.008599488 100 45 55 122.77% As can be seen in Table 3, using pTau, in the vast majority of cases, the determined concentration is significantly different to the expected concentration. For example, most subjects have a determined concentration which is more than 50% different from the expected concentration, and many are significantly more different, such as over 100% different. Table 4: Difference between expected and determined concentration of tTau in subjects who suffer from Alzheimer’s Dementia (first four subjects) or Alzheimer’s Mild Cognitive Impairment (remaining subjects) Expected Difference Amount Determined concentration between determined concentration Determined of pTau based determined concentration of T14 concentration upon the concentration differs from (normalised of tTau / determined and expected the expected to total pg / ml concentration concentration concentration protein) of T14 / of pTau / / % of pg / ml pg / ml expected concentration 0.056807513 1248 262 986 376.13% 0.035579369 636 217 419 193.03% 0.032342283 558 210 348 165.50% 0.008428705 509 159 350 219.31% 0.00574571 677 154 523 340.45% 0.036521192 711 219 492 224.59% 0.13309991 1257 424 833 196.41% 0.038348115 749 223 526 235.99% 0.014382639 775 172 603 350.47% 0.01845329 640 181 459 254.20% 0.01505949 1016 173 843 485.65% 0.050370113 504 248 256 102.86% 0.02552802 479 196 283 144.75% 0.209603333 555 586 31 5.37% 0.008599488 552 160 392 245.50% As can be seen in Table 4, using tTau, in the vast majority of cases, the determined concentration is significantly different to the expected concentration, and even more different from the values seen when using pTau as the biomarker. For example, most subjects have a determined concentration which is more than 100% different from the expected concentration, and many are significantly more different, such as over 200% or 300% different. Conclusions As shown in the data, there is an unexpectedly strong positive correlation between Tau (both pTau and tTau) and T14. In addition, there is likely a correlation between AD and T14. In addition, the inventors’ study revealed a significant correlation between T14 levels and tau proteins (p-Tau and t-Tau) in cognitively healthy (CH) individuals, which was not observed in those with mild cognitive impairment (MCI), and Alzheimer's disease (AD). Further investigation within the CH group indicated that this correlation was predominantly present in individuals without mood disorders, but disappeared in those with mood disorders, resembling the pattern seen in MCI and AD patients. These findings reinforce the newly discovered relationship between T14 and tau pathology, as disclosed herein, and suggest that the ratio of T14 / P-Tau or T-Tau levels in CH individuals can serve as a distinguishing factor between those with or without mood disorders. This observation supports a link between T14 and mood regulation in the IC nuclei, which are known to be particularly susceptible in AD. Accordingly, the inventors have demonstrated that the correlation between T14 and Tau levels can be used as a reliable predictor of neurodegenerative disease with a high degree of specificity and sensitivity, and therefore allows routine diagnosis of a neurodegenerative disorder and appropriate intervention with therapeutic treatment. A simple flowchart of an embodiment of the method is shown in Figure 11. Advantageously, this diagnostic method is not dependent of age, gender, medication or indeed any other individual circumstances, given that the person would effectively be providing their own benchmark.

Claims

Claims 1. A method for diagnosing a subject suffering from a neurodegenerative disorder, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, the method comprising: (i) determining a plurality of values based on a sample obtained from a subject, the values comprising a concentration of Tau protein in the sample, and a concentration of a peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof in the sample; (ii) determining an expected value, wherein the expected value is based on a mathematical correlation between the concentration of Tau protein and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, in a population of subjects who do not suffer from a neurodegenerative disorder or have a pre- disposition thereto, and the expected value is further based on either the determined concentration of Tau protein or the determined concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and (iii) comparing the expected value with a corresponding determined value, wherein the expected and determined values are: (a) expected and determined concentrations of Tau protein; (b) expected and determined concentrations of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and / or (c) an expected and a determined mathematical relation between concentrations of Tau protein and the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof based upon a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto; and when the determined value deviates from the expected value by more than a first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis.

2. The method according to claim 1, wherein the first threshold amount is 200% or less of the expected value, 100% or less of the expected value, 80% or less of the expected value or 60% or less of the expected value.

3. The method according to either claim 1 or claim 2, wherein the first threshold amount is: (i) 55% or less or 52% or less of the expected value; (ii) 50% or less, 45% or less, or 40% or less of the expected value; and / or (iii) 35% or less, 30% or less, or 25% or less of the expected value.

4. The method according to any preceding claim, wherein the first threshold amount is: (i) 20% or less, 15% or less or 10% or less of the expected value; and / or (ii) 5% or less of the expected value.

5. The method according to any preceding claim, wherein when the determined value deviates from the expected value by less than a second threshold amount, the subject does not suffer from a neurodegenerative disease, or have a pre-disposition thereto, or has a positive prognosis, and when the determined value deviates from the expected value by greater than the first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis.

6. The method according to claim 5, wherein the second threshold amount is 200% or less of the expected value, 100% or less of the expected value, 80% or less of the expected value or 60% or less of the expected value.

7. The method according to either claim 5 or claim 6, wherein the second threshold amount is: (i) 55% or less or 52% or less of the expected value; (ii) 50% or less, 45% or less, or 40% or less of the expected value; (iii) 35% or less, 30% or less or 25% or less of the expected value; (iv) 20% or less, 15% or less or 10% or less of the expected value; and / or (v) 5% or less of the expected value.

8. The method according to any one of claims 5-7, wherein the first and second threshold amounts are the same.

9. The method according to any preceding claim, wherein the mathematical correlation between the concentration of Tau protein and the concentration of said peptide is given by the formula y=mx+c, wherein y is the determined or expected concentration of Tau protein, x is the determined or expected concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, and m and c are constants.

10. The method according to claim 8, wherein the determined mathematical relation is a value of one or both of said constants.

11. The method according to any preceding claim, wherein: (i) the determined mathematical relation comprises a ratio between the concentration of Tau protein and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; (ii) the determined mathematical relation comprises a ratio between the concentration of Tau protein minus a constant and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, or wherein the determined mathematical relation comprises a ratio between the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof minus a constant and the concentration of the Tau protein; and / or (iii) the determined mathematical relation comprises the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof minus the concentration of the Tau protein multiplied by a constant, or wherein the determined mathematical relation comprises the concentration of Tau protein minus the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, multiplied by a constant.

12. The method according to any preceding claim, wherein the neurodegenerative disease is selected from a group consisting of: Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; AmyotrophicLateral Sclerosis (ALS); schizophrenia; Lewy-body dementia; Frontotemporal Dementia; and Mild Cognitive Impairment.

13. The method according to any preceding claim, wherein the neurodegenerative disease is Alzheimer's Disease.

14. The method according to any preceding claim, wherein the neurodegenerative disease comprises mild cognitive impairment (MCI) presenting prior to any of the conditions described above, and in particular, the very early stages of Alzheimer's Disease.

15. The method according to any preceding claim, wherein the Tau protein comprises or consists of SEQ ID No: 11, or a variant or fragment thereof.

16. The method according to any preceding claim, wherein the Tau protein detected comprises total Tau protein or phosphorylated Tau protein.

17. The method according to any preceding claim, wherein the Tau protein detected comprises total Tau protein and phosphorylated Tau protein.

18. The method according to any preceding claim, wherein the age of the subject is at least 20, 30, 40, 50, 60, 70, 80 or 90 years old.

19. The method according to any preceding claim, wherein the subject is tested before any symptoms of neurodegenerative disorder are apparent.

20. The method according to any preceding claim, wherein the sample is blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof.

21. The method according to any preceding claim, wherein the sample comprises blood, nasal discharge or fluid, urine, tissue, and / or CSF.

22. The method according to any preceding claim, wherein the sample comprises CSF, preferably lumber CSF.

23. The method according to any preceding claim, wherein the sample comprises nasal discharge or nasal fluid.

24. A kit for diagnosing a subject suffering from a neurodegenerative disorder, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, the kit comprising: (i) means for determining, in a sample obtained from a test subject, a plurality of values based on a sample obtained from a subject, the values comprising a concentration of Tau protein in the sample, and a concentration of a peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof in the sample; and (ii) means for determining an expected value, wherein the expected value is based on a mathematical correlation between the concentration of Tau protein and the concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof, in a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto, and the expected value is further based on either the determined concentration of Tau protein or the determined concentration of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; wherein, when comparing the expected value with a corresponding determined value, the expected and determined values are: (a) expected and determined concentrations of Tau protein; (b) expected and determined concentrations of the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof; and / or (c) an expected and a determined mathematical relation between concentrations of Tau protein and the peptide comprising or consisting of SEQ ID No: 3 (T14), or a variant or fragment thereof based upon a population of subjects who do not suffer from a neurodegenerative disorder or have a pre-disposition thereto; and wherein when the determined value deviates from the expected value by more than a first threshold amount, the subject suffers from a neurodegenerative disease, or has a pre-disposition thereto, or has a negative prognosis.

25. The kit according to claim 23, wherein the kit is configured to carry out the method according to any one of claims 1-22.

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