WO2009044119A1 - Prognostic or diagnostic test - Google Patents

Abstract

The application provides in vitro methods for identifying a subject predisposed to, or suffering from, a neurodegenerative disease. The method comprises obtaining a peripheral body fluid sample from a subject and examining TDP-43 levels in the sample from a test subject and comparing TDP-43 levels with a reference derived from an individual who does not suffer from a neurodegenerative disease, wherein raised concentration of TDP-43 in the sample from the test subject suggests that the subject is suffering from a neurodegenerative disease or is predisposed to developing a neurodegenerative disease. The application also methods of screening a compound for efficacy for treating or preventing neurodegenerative diseases based on the level of TDP-43.

Description

Prognostic or Diagnostic Test

The present invention relates to prognostic and diagnostic tests for neurodegenerative disease and also to kits for use in such tests.

Neurodegenerative diseases are a major clinical problem that may manifest in a number of forms. For Example frontotemporal lobar degeneration, Alzheimer's disease, motor neuron disease, Lewy body diseases , Parkinson Disease and the like.

Many neurodegenerative diseases are accompanied by dementia. Dementia is the progressive decline in cognitive function due to damage or disease in the brain beyond what might be expected from normal aging. Particularly affected areas may be memory, attention, language, and problem solving. Especially in the later stages of the condition, affected persons may be disoriented in time (not knowing what day of the week, day of the month, month, or even what year it is), in place (not knowing where they are), and in person (not knowing who they are). The prevalence of dementia is rising as the global life expectancy is rising. Particularly in Western countries, there is increasing concern about the economic impact that dementia will have in future, older populaces. Dementia is a non-specific term encompassing many disease processes. At present there is no cure for any type of dementia.

Frontotemporal lobar degeneration (FTLD) is a pathologic process involving degeneration of gray matter in the frontal lobe and anterior portion of the temporal lobe of the cerebrum, with sparing of the parietal and occipital lobes. FTLD is the second most common form of dementia after Alzheimer's disease and is therefore a major cause of neurological problems in the elderly. The syndrome of FTLD encompasses the clinical subgroups of frontotemporal dementia (FTD), FTD with motor neuron disease, semantic dementia and primary progressive aphasia, and is characterized by changes in behaviour, personality and language with relative preservation of memory and perception.

Pathologically, there are two main histological profiles associated with FTLD. One of these is tauopathy, the accumulation of hyperphosphorylated tau in neurons and occasionally in glia. However, the most common neuropathology associated with FTLD, accounting for well over half of all cases, is that known as FTLD-U, in which there are neuronal cytoplasmic inclusions and neurites that are immunoreactive for ubiquitin (ub-ir) but not for tau. FTLD pathology of this type was first described in patients with motor neuron disease (MND) and dementia but has subsequently been recognized as a common neuropathological feature of FTLD in patients without motor symptoms. This ub-ir pathology is characteristically found in granule cells of dentate fascia of the hippocampus and in neurons of layer 2 of the frontal and temporal neocortex.

Significant progress in our understanding of one of the genetic causes of FTLD-U was made in 2006 when null-mutations of the gene encoding the pleiotropic growth factor progranulin (PGRN) was demonstrated to be significant cause of this disease subtype.

A second major advancement in FTLD-U research was also made in 2006 when TAR DNA binding protein, TDP-43 was identified as the major ubiquitinated protein in the pathological lesions in FTLD-U and in motor neurone disease (MND)¹"². Prior to this discover little was known of the function of this 414 amino acid nuclear protein beyond it serving a role in nuclear transcription in relationship to alternative splicing or exon skipping.³'⁴ The involvement of TDP-43 in the ubiquitinated lesions of FTLD-U and MND has now been amply confirmed.^5"9 While ubiquitinated TDP-43 lesions are characteristically seen in those patients with inherited forms of FTLD associated with mutations in progranulin gene (PGRN),¹'^10'15 in other patients with FTLD, such as those with mutations in tau gene (MAPT), or in those with a Pick body-type histology, where the underlying histology is based on the accumulation of aggregated tau proteins, no such TDP-43 pathological changes are seen.⁷ Moreover, other studies have shown that TDP-43 pathological changes can also occur in about 20% of patients with Alzheimer's disease (AD),¹⁶ and are widely present in patients with Lewy body diseases¹⁷ or with parkinsonism-dementia complex of Guam . ^{! 8} It is therefore state of the art that TDP-43 may be linked to neurodegenerative diseases (including FTLD-U and MND). In clinical terms, about 70% of patients with

FTLD show behavioural and personality changes which are collectively described as frontotemporal dementia (FTD).¹⁹ Prospective and retrospective pathological studies have indicated that about half of all cases with FTD have a tau-positive, TDP-43- negative pathology, and half have ubiquitin-positive, TDP-43 -positive pathology. '

²⁴ Nevertheless, in patients with FTD, it is not possible to predict, in life, what the underlying histological changes might be solely on the basis of clinical phenotype, and it is only when mutations in MAPT or PGRN mutations are present that a tau or ubiquitin/TDP-43 based histology, respectively, can be inferred.

The differential diagnosis of dementia syndromes is clinical and radiological, and there are no litmus tests based on measures in blood or plasma to specify diagnosis. It will therefore be appreciated that a biochemical marker for neurodegenerative diseases would be desirable. Furthermore it would be desirable to develop a marker that that could distinguish between AD and FTLD, and between the histological subtypes of FTLD. Such a marker would not only be of great diagnostic value, but would be highly pertinent to the future development of drugs aimed at preventing or removing the intraneuronal accumulation of tau or TDP-43 proteins in these degenerative disorders.

Conceptually, the measurement of TDP-43 might be considered to be a useful biomarker. However, there are significant pathophysiological factors that would lead a skilled person to discount TDP-43 as a candidate marker for neurodegenerative diseases. These include:

(a) TDP-43 is a nuclear protein that a skilled person would not expect to be normally released from cells. A skilled person would therefore only expect to be able to measure TDP-43 in life by taking a tissue biopsy. This is obviously undesirable or impractical for brain tissue and TDP-43 would therefore be considered to be inaccessible for assay.

(b) The recent discovery that TDP-43 is found in ubiquitinated lesions within the cytoplasm of nerve cells from certain patients with neurodegenerative disease would not make a skilled person believe that TDP-43 was any more accessible for assay. The lesions found in such patients are in the form of insoluble inclusion bodies within neurons. Such cells are effectively "clogged" with these inclusions and they would not be expected to be released from the cells. Accordingly, even in the diseased state, a skilled person would only expect to measure TDP-43 in a tissue sample. This expectation is reinforced by the fact that studies to date have only been based on autopsy tissue samples. It is clearly pointless trying to develop a prognostic or diagnostic test when relevant tissue samples can only be obtained after death or by invasive tissue sampling.

(c) The view that TDP-43 is inaccessible for assay is reinforced when the blood-brain barrier is considered. TDP-43 would not be expected to be released across the cytoplasmic membrane of neurons. However, even if it was unexpectedly released, a skilled person would not expect a 414 amino acid protein to be able to cross the blood-brain barrier and enter peripheral circulation.

It is therefore clear, from (a) -(c) above, that a skilled person would not expect TDP-43 to be accessible and would not even consider it as being a useful prognostic or diagnostic marker.

There is a need to develop assays that may be used as diagnostic or prognostic tools when assessing a patient's condition or the likelihood of a subject developing dementia. The limitations of sensitivity and reliability of existing assays mean that patients with an increased risk of developing dementia, or patients in the early stages of the disease are not necessarily identified using existing tests. The inability to identify such patients may mean that opportunities for therapeutic intervention prior to the appearance of debilitating symptoms of disease are lost. It will be appreciated that a prognostic test, and also diagnostic tests for early disease, are ideally performed before any major symptoms or anatomical changes in the brain may be detected. Accordingly a skilled person would have even less reason to expect to detect TDP-43 in peripheral body fluid samples of presymptomatic subjects or subjects in the early stages of disease.

Also, it has been reported that plasma levels of biomarkers for neurogenerative diseases are not as predictive for disease as the CSF levels of those markers. This can be seen from documents which discuss the development of biomarkers as predictive indicators for neurogenerative diseases. For example, McCorquodale et al (2008)

Biomakers Med. 2(3) 209-214 state that the most studied biomarkers for Alzheimer's disease are AB and Tau, with a preference to measuring levels of these proteins in CSF, not plasma. Sunderland et al (2006) J. Geriatric Psychiatry and Neurology 19,

172-179 reach a similar conclusion, noting that the major biomarker of interest for diagnosing Alzheimer's disease are CSF levels of AB and Tau.

In view of the above, the inventors endeavoured to develop a prognostic and diagnostic test for neurodegenerative disease by testing peripheral body fluid samples from control subjects and subjects with neurodegenerative disease.

According to a first aspect of the invention there is provided an in vitro method for identifying a subject predisposed to, or suffering from, a neurodegenerative disease, the method comprising obtaining a peripheral body fluid sample from the subject and examining TDP-43 levels in the sample from a test subject and comparing TDP-43 levels with a reference derived from an individual who does not suffer from a neurodegenerative disease, wherein raised concentration of TDP-43 in the sample from the test subject suggests that the subject is suffering from a neurodegenerative disease or is predisposed to developing a neurodegenerative disease.

The invention has been based on research conducted by the inventors (see the Example) in which unexpectedly, and to their surprise, they found TDP-43 present within the plasma of subjects with neurodegenerative disease.

The presence of TDP43 protein in plasma and its use as a diagnostic marker for neurodegenerative disease, particularly dementia, is unexpected. It is usual when proteins such as TDP43 accumulate that they form insoluble inclusions and/or insoluble aggregates so that they do not remain free in cytoplasm. It is therefore very surprising that TDP43 is liberated from cells, even in the case of cell degeneration, due to its lack of solubility in the pathological state. This is particularly the case when it is considered that the extraction of TDP43 from brain tissue requires the tissue homogenate to be put through a sequential detergent extraction procedure. Pathological TDP-43 is found in the most insoluble fraction, and 8M urea is required to solublize it. Against that background, the presence of measurable levels of TDP43 in plasma, and that such levels are diagnostic for neurodegenerative disease, is very surprising.

Their studies lead them to appreciate that TDP-43 polypeptide would have useful applications in methods of testing for neurodegenerative diseases and particularly such diseases that are characterised by dementia.

By "TDP-43 polypeptide" is included any TDP-43 polypeptide or fragment thereof, or any variant thereof, that has sequence identity (as discussed below) with TDP-43 or is immunologically reactive with a TDP-43 antibody (e.g. the antibody discussed below).

Further information on the TDP-43 gene is provided in the NCBI Entrez data base. An examples of a human TDP-43 polypeptide has Genbank Accession number NP 031401 (gene accession is NM 007375). The protein sequence is provided below as SEQ ID NO:!.

MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRG VRLVEGILHAPDAGWGNLVYWNYPKDNKRKMDETD ASSAVKVKRAVQKT SDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEY ETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDE LREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAE PKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGM NFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQ REPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGW

GM

(SEQ ID NO: 1) Searching databases may identify further TDP-43 polypeptides. For example, BLAST searching (http://www.ncbi.nlm.nih.gOv/B LASTΛ may identify other TDP-43 polypeptides. It will be appreciated that such polypeptides are encompassed by the definition of "TDP-43 polypeptide" given above.

We also include homologues of TDP-43 polypeptide present in other species. These polypeptide are also included within the scope of the term "TDP-43 polypeptide" when referred to herein.

To be considered a TDP-43 polypeptide ot the invention, a polypeptide may have at least 50%, 60% to 70% and more preferably 70% to 80%, 80 to 90%, 90 to 95%, 96%, 97%, 98%, 99% or more sequence identity with a TDP-43 polypeptide sequence provided herein, for example as given in one of the listed accession numbers above or that of SEQ ID NO: 1.

A "fragment" of the TDP-43 polypeptide can be considered to be an TDP-43 polypeptide that may comprise, for example, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more or of the polypeptide sequence of full length TDP-43 polypeptide.

Also, it has been determined that some fragments of TDP-43 are associated with neurodegenerative diseases. For example, C-terminal breakdown or cleavage fragments of TDP-43 polypeptide are associated with disease and can be considered to be pathological forms of TDP-43 polypeptide. These C-terminal fragments can comprise polypeptide fragments of from about 24 kDa to about 26 kDa, for example C-terminal breakdown or cleavage TDP-43 polypeptides of around 25kDa. Hence a fragment of TDP-43 polypeptide includes these fragments. Analysis of such fragments have shown that one corresponds to amino acid residues 252-263, 276-293 and 409-414 of TDP-43 polypeptide. Hence a fragment of TDP-43 polypeptide also includes these fragments. Hence the method of the invention also includes measuring the presence of such fragments in the sample. A "variant" will have a region that has at least 50% (preferably 60%, 70%, 80%, 90%, 95%, 86%, 97%, 98%, 99% or more) sequence identity with a TDP-43 polypeptide as described herein. The percentage identity may be calculated by reference to a region of at least 50 amino acids (preferably at least 60, 75, or 100) of the candidate variant molecule, allowing gaps of up to 5%. By "variants" we also include insertions, deletions and substitutions, either conservative or non- conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the protein activity or ability to bind to particular binding partners, as appropriate.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. 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. Therefore by "conservative substitutions" is intended to include combinations such as GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.

The three-letter or one letter amino acid code of the IUPAC-IUB Biochemical Nomenclature Commission is used herein.

Calculation of percentage identities between different amino acid/polypeptide/nucleic acid sequences may be carried out as follows. A multiple alignment is first generated by the ClustalX program (pairwise parameters: gap opening 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delay divergent sequences 30%, DNA transition weight 0.5, negative matrix off, protein matrix gonnet series, DNA weight IUB; Protein gap parameters, residue-specific penalties on, hydrophilic penalties on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off). The percentage identity is then calculated from the multiple alignment as (N/T)*100, where N is the number of positions at which the two sequences share an identical residue, and T is the total number of positions compared. Alternatively, percentage identity can be calculated as (N/S)*100 where S is the length of the shorter sequence being compared. The amino acid/polypeptide/nucleic acid sequences may be synthesised de novo, or may be native amino acid/polypeptide/nucleic acid sequence, or a derivative thereof.

By "variants" we also include hyperphosphorylated TDP-43 polypeptide. It has recently been demonstrated that neurodegenerative disease is associated with hyperphosphorylation of the TDP-43 polypeptide (Hasegawa et al (2008) Ann.

Neurol. 64, 60-70). It has been shown that at least five sites are phosphorylated in

TDP-43 polypeptide in subjects with neurodegenerative diseases: Ser 370; Ser

403/404; and Ser 409/401. Hence the method of the invention also includes measuring the presence of TDP-43 polypeptide that is phosphorylated in one or more of these residues in the sample.

By the term "peripheral body fluid sample", we mean any body fluid that lies outside the central nervous system. For example the sample may be urine, sputum or lymph. However, it is preferred that the body fluid is blood or derived therefrom. The method of the first aspect of the invention is most preferably performed on a sample of serum or plasma.

It will be appreciated that determining whether a sample of body fluid contains a certain level of TDP-43 may be diagnostic of a neurodegenerative disease or it may be used by a clinician as an aid in reaching a diagnosis. Levels of TDP-43 may be monitored over time in a patient that has developed a neurodegenerative disease to assess how the disease develops. In most instances, an increase in TDP-43 levels over time will suggest to the clinician that the health of the subject is deteriorating. Accordingly the method has prognostic value in connection with subjects that already suffer from the disease. The methods of the invention may also be used for presymptomatic screening of a subject who may be in a risk group for developing a neurodegenerative disease, e.g. a patient having a family history of such diseases. Hence the methods of the invention may also be used to screen individuals who are asymptomatic. Raised TDP- 43 levels may then lead a clinician to recommend prophylactic treatment or even just life style changes.

By "raised concentration of TDP-43 in the bodily sample" we mean that the level of TDP-43 polypeptide which can be considered to be an indicator of a neurodegenerative disease may be, for example, at least 1 Vi fold higher, or it may be at least 2-fold, or 3-fold, 5-fold, 7-fold, 11 -fold, 13-fold or even higher, in the sample than the level of TDP-43 in a sample taken from an individual who is not suffering from a neurodegenerative disease. It is preferred that the levels are increased 7-fold when assessing subjects who may have FTD and that the levels are increased 13 -fold when assessing subjects who may have AD.

The inventors have found that, when measuring Optical Density (OD) as a function of the concentration of TDP-43 in an ELISA based assay (see below) that the cut off point for a normal (i.e. non-diseased) subject was an OD values of about 0.11. Accordingly a raised concentration of TDP-43 in the bodily sample may be considered to be an OD value of greater than 0.11 when TDP-43 levels are being measured by ELISA (e.g. according to the preferred methods described in the Example)

It has been noted by the inventors that TDP-43 is usually absent or found in very low levels in the serum of control subjects. Accordingly the mere presence of TDP-43 in the sample is considered to be instructive for a clinician. Therefore the term "raised concentration of TDP-43 in the bodily sample" may also be considered to include the mere presence of TDP-43 over a control level that may be expected to be around zero. This also includes the presence of fragments of the TDP-43 polypeptide or the hyperphosphorylated forms of the TDP-43 polypeptide discussed above. The method of the first aspect of the invention may be used to test for many forms of neurodegenerative diseases. For Example, Frontotemporal Lobar Degeneration, Alzheimer's disease, Motor Neuron disease, Lewy body disease, Parkinson Disease and other related disorders.

The method is particularly useful for testing for neurodegenerative diseases that are accompanied by dementia. It will be useful in identifying during their lifetime the presence of TDP-43 based pathological changes within the brains of patients with neurodegenerative disease, though because such pathological changes may occur in more than one clinical disorder it will not necessarily constitute a "one stop" diagnostic test which will completely distinguish, for example, people with Alzheimer's disease from those with other forms of dementia. However the methods of the first aspect of the invention are very useful for identifying whether or not a subject has, or may develop, a form of dementia. Furthermore the method is particularly useful for identifying those patients with a neorodegeneative disease (e.g. Alzheimer's disease) with TDP-43 pathological changes from those without such changes. The method is most preferably used for testing for frontotemporal lobar degeneration because it may distinguish those patients with TDP-43-based pathology (ie FTLD-U) from those with tau-based pathology. Presently, this cannot be determined unless patients also carry rare mutations in MAPT or PGRN genes that are known to be associated with AD or FTLD-U respectively. Such knowledge may have potential value for drug therapy or patient management. Because plasma TDP-43 levels may change over time, it is also possible that these may predict stage of disease.

According to one embodiment of the invention TDP-43 polypeptide levels may be measured. When this is the case, the sample may be any bodily sample into which the polypeptide may be secreted, e.g. it may be lymph or interstitial fluid. The sample may be a urine sample. TDP-43 polypeptide is preferably measured or assayed in a blood sample. The blood sample may be venous or arterial. Blood samples may be assayed immediately. Alternatively, the blood may be stored in a fridge before the assay is conducted. Measurement may be made in whole blood. However, in preferred embodiments of the invention, the blood may be further processed before an assay is performed. For instance, an anticoagulant, such as heparin, citrate, EDTA, and others may be added. It is most preferred that the blood sample is centrifuged or filtered to prepare a plasma or serum fraction for further analysis. It is most preferred that the sample is plasma. The plasma may be used immediately after it has been separated from blood cells or, alternatively it may be refrigerated or frozen before assay.

TDP-43 levels may be measured by a number of ways known to one skilled in the art. It will be appreciated that the polypeptide may be detected by labelling a compound having affinity for TDP-43. Antibodies, aptamers and the like may be labelled and used in such an assay.

TDP-43 polypeptide may be detected by non-immuno based assays. Such non- immuno based assays may utilise fluorometric or chemiluminescent labels. However, it is preferred that immunoassays are employed to detect TDP-43 polypeptide concentration in the sample. Examples of immunoassays include immunofluorescence techniques known to the skilled technician, immunohistochemistry, radioimmunoassay analyses and in particular enzyme-linked immunosorbent assay (ELISA).

Hence, a preferred method of measuring TDP-43 polypeptide comprises carrying out an ELISA on the sample. It may be required to first separate the proteins in the sample, for example, using isoelectric focussing before the ELISA step. As will be appreciated, such techniques are routine laboratory methods and are well known to the skilled person.

The methods of the first aspect of the invention may need a "reference sample". This would be the amount and/or activity of TDP-43 polypeptide in a sample of protein taken from a subject that does not have a neurodegenerative disease and preferably has no family history of developing such diseases.

Antibodies used in the ELISA may be produced as polyclonal sera by injecting TDP-43 antigen into animals. Preferred polyclonal antibodies may be raised by inoculating an animal (e.g. a rabbit) with antigen using techniques known to the art. Alternatively the antibody may be monoclonal. Conventional hybridoma techniques may be used to raise such antibodies. The antigen used to generate monoclonal antibodies for use according to the present invention may be the same as would be used to generate polyclonal sera.

Furthermore, antibodies can be readily raised to the, C-terminal breakdown or cleavage fragments of TDP-43 polypeptide that are associated with disease as described further herein. Such antibodies can be of use for the detection of the presence of the fragments of TDP-43 in the samples, and can be prepared using techniques well known in the art. Monoclonal or polyclonal antibodies can be used.

Antibodies that can specifically bind to the phosphorylated forms of TDP-43 associated with neurodegenerative diseases, as described above (i.e. to the Ser 370; Ser 403/404; or Ser 409/401 phosphorylated forms of TDP-43) are also particularly useful in the present invention. These antibodies are powerful tools for biochemical and immunohistochemical analyses of neurodegenerative diseases and for evaluation of cellular or animal models of TDP-43 proteinopathy. Such "phospho-specific" TDP-43 antibodies are particularly useful for the invention since they only detect these pathological forms of TDP-43 and can therefore be used to detect the presence of pathological TDP-43 in a peripheral body fluid sample, preferably plasma.

Examples of such phospho-specific TDP-43 antibodies include those antibodies supplied by Cosmo Bio International

(https://www.cosmobio.co.jp/index_e.asp) under catalogue number TIP-PTD-MOl (Anti phospho TDP-43 (pS409/410-l)).

Hence a preferred embodiment of the invention is wherein a phospho-specific TDP-43 antibody is used to examine the TDP-43 level in the sample from a test subject.

According to a second aspect of the invention, there is provided a kit for identifying a subject predisposed to, or suffering from, a neurodegenerative disease the kit comprising:- (i) means for determining TDP-43 levels in a sample from a test subject; and, optionally

(ii) a reference corresponding to a level of TDP-43 in a sample from an individual who does not suffer from neurodegenerative disease, wherein the kit is used to identify a presence or a raised level of TDP-43 in the sample from the test subject, thereby suggesting that the test subject is predisposed to, or suffers from, a neurodegenerative disease.

By "means for determining TDP-43 levels in a sample from a test subject" we include antibodies that are able to selectively bind to TDP-43 polypeptide. A preferred embodiment of the invention is wherein the antibody can detect the C- terminal breakdown or cleavage fragments of TDP-43 polypeptide that are associated with disease as described further herein. A further preferred embodiment of the invention is wherein the antibody specifically binds to the phosphorylated forms of TDP-43 associated with neurodegenerative diseases, as described above (i.e. to the Ser 370; Ser 403/404; or Ser 409/401 phosphorylated forms of TDP-43). The antibody can be monoclonal or polyclonal; preferably monoclonal.

The method according to the first aspect of the invention is useful for enabling a clinician to make decisions with regards to the best course of treatment for a subject who is suffering from the disease or is suspected of developing a neurodegenerative disease. It is preferred that the diagnostic method is used to enable a clinician to decide how to treat a subject who is suffering from the early stages of a FTLD-U. In addition, the method of the first aspect is useful to a clinician because it allows him or her to monitor the efficacy of a putative treatment for dementia. Hence, the kit according to the second aspect is useful for providing prognostic information with regards a patient's condition, such that the clinician can carry out a treatment. The kit can also be used to monitor the efficacy of a putative treatment. The method and the kit are therefore very useful when planning a treatment regime and for monitoring the efficacy of such a treatment regime. Knowledge of the surprising presence of TDP-43 in peripheral body fluids of subject with neurodegenerative disease has enabled the inventors to develop a screen for identifying whether or not test compounds are able to reduce TDP-43 levels and are therefore putative agents for treating or preventing neurodegenerative diseases.

Thus, according to a third aspect of the present invention there is provided a method of screening a test compound to determine whether the compound has efficacy for treating or preventing neurodegenerative diseases, comprising:

(i) exposing a biological system to the test compound;

(ii) detecting the ability of the compound to reduce the level of TDP-43 in a peripheral body fluid; and,

(iii) comparing the level of TDP-43 in the biological system exposed to the test compound to the level of TDP-43 in a control biological system that was not exposed with the compound;

wherein compounds with efficacy for treating or preventing neurodegenerative diseases reduce the level of TDP-43 relative to controls.

The methods of the third aspect of the invention is a useful screening methods for drugs or lead compounds. The test compound may be a drug-like compound or lead compound for the development of a drug-like compound.

It will be appreciated that the method according to the third aspect of the invention may be adapted such that it is used to test whether or not a compound causes a neurodegenerative disease.

Therefore according to a fourth aspect of the invention there is provided a method of screening a compound, to test whether the compound may cause a neurodegenerative disease, comprising:

(i) exposing a biological system to the test compound; (ii) detecting the ability of the compound to raise the level of TDP-43 in a peripheral body fluid; and, (iii) comparing the level of TDP-43 in the biological system exposed to the test compound to the level of TDP-43 in a control biological system that was not exposed with the compound;

wherein compounds that cause neurodegenerative diseases increase the level of TDP-43 relative to controls.

The screening methods of the invention are based upon the inventors' realisation that the level of TDP-43 in a body fluid may be closely related to the development and progression of neurodegenerative diseases. The screening method of the third aspect of the invention is particularly useful for screening libraries of compounds to identify compounds that may be used as agents for preventing or treating conditions characterised by dementia. The fourth aspect of the invention may be used to identify compounds that cause such diseases. Accordingly the screen according to the fourth aspect of the invention may be used for environmental monitoring (e.g. to test effluents from factories) or in toxicity testing (e.g. to test the safety of putative pharmaceuticals, cosmetics, foodstuffs and the like).

By "biological system" we include any experimental system that would be understood by a skilled person to provide insight as to the effects a compound may have on TDP-43 expression. The system may comprise: (a) an experimental test subject when an in vivo test is to be employed; (b) a biological sample derived from a test subject (for instance: blood or a blood fraction (e.g. serum or plasma), lymph or a cell/biopsy sample); (c) a cell line model (e.g. a cell expressing TDP-43 or a cell engineered to express the protein); or even (d) an in vitro system that comprises the

TDP-43 and simulates the physiological environment such that TDP-43 activity can be measured.

A preferred embodiment of the third and fourth aspects of the invention is wherein the level of TDP-43" in the biological system is measured using antibodies that are able to selectively bind to TDP-43 polypeptide. A preferred embodiment of the invention is wherein the antibody can detect the C-terminal breakdown or cleavage fragments of TDP-43 polypeptide that are associated with disease as described further herein. A further preferred embodiment of the invention is wherein the antibody specifically binds to the phosphorylated forms of TDP-43 associated with neurodegenerative diseases, as described above (i.e. to the Ser 370; Ser 403/404; or Ser 409/401 phosphorylated forms of TDP-43). The antibody can be monoclonal or polyclonal; preferably monoclonal.

As discussed above, recent studies have determined that around 20 to 25% of patients with Alzheimer's Disease have TDP-43 pathological changes, in addition to beta amyloid accumulation and tauopathy, while around half of all cases with FTD have a tau-positive, TDP-43-negative pathology, and half have ubiquitin-positive, TDP-43 -positive pathology (this latter group is pathological similar to motor neuron disease which also displays only a TDP-43 pathology). Hence some neurodegenerative diseases are caused by TDP-43 pathology, rather than tauopathy. However, this is not currently taken in to consideration when selecting subjects for clinical trials of drugs for treating Alzheimer's disease, tauopathy or TDP-43-opathy.

Hence a fifth aspect of the invention provides an in vitro method of selecting a test subject for consideration in a clinical trial for treating neurodegenerative disease, comprising obtaining a peripheral body fluid sample from the subject and examining TDP-43 levels in the sample from a test subject and comparing TDP-43 levels with a reference derived from an individual who does not suffer from a neurodegenerative disease, wherein raised concentration of TDP-43 in the sample from the test subject suggests that the subject is suffering from a neurodegenerative disease or is predisposed to developing a neurodegenerative disease, caused by TDP-43 pathology.

This aspect of the invention may have much utility when screening potential test subjects in clinical trials of drugs for treating neurodegenerative diseases. For example, the method of the invention can be performed as part of an initial screening procedure to determine whether a test subject is suitable for inclusion in the trial. Clearly the inclusion of a test subject with elevated TDP-43 levels in a clinical trial of drugs for treating neurodegenerative disease should be carefully considered.

In one embodiment of this aspect of the invention, any subject with elevated TDP-43 levels would be excluded from a clinical trial of a drug for treating non-TDP-43 related neurodegenerative diseases.

As can be appreciated, the method of the fifth aspect of the invention can use many of the materials and methods described further above in relation to the first aspect of the invention.

A preferred embodiment of the fifth aspect of the invention is wherein the level of TDP-43 in the sample is measured using antibodies that are able to selectively bind to TDP-43 polypeptide. A preferred embodiment of the invention is wherein the antibody can detect the C-terminal breakdown or cleavage fragments of TDP-43 polypeptide that are associated with disease as described further herein. A further preferred embodiment of the invention is wherein the antibody specifically binds to the phosphorylated forms of TDP-43 associated with neurodegenerative diseases, as described above (i.e. to the Ser 370; Ser 403/404; or Ser 409/401 phosphorylated forms of TDP-43). The antibody can be monoclonal or polyclonal; preferably monoclonal.

The invention will be further described, by way of Example, and with reference to the following figures:-

Figure 1 represents a standard curve for the TDP-43 ELISA (data show mean +/- SD).

Figure 2 represents Box-whisker plots for the plasma ELISA results (OD values). The length of the box represents the interquartile range of the sample, the line drawn across the box the median and outliers being denoted by dots. Figure 3 illustrates immunoblotting results. Lane 1, molecular weight markers (numbers to left in kDa); lane 2, protein immunocaptured from a plasma sample with a high ELISA reading.

EXAMPLE 1

The inventors conducted experiments to see whether or not a number of biological molecules associated with neurodegenerative diseases may be found in peripheral body fluid samples. They obtained an antibody against TDP-43 in order that they may assay for this protein although there was no expectation that the protein would be detected outside neurons and neurites in the CNS. However to their surprise, and as described below, they discovered that TDP-43 could be found in plasma and it may be used according to the methods of the invention to discriminate between diseased and control subjects and also to discriminate between subjects with different types of neurodegenerative conditions.

1.1 Materials and Methods

1.1.1 Blood samples

Blood samples were obtained with Ethical permissions from 137 patients, 35 with FTD and 102 with AD, and 85 non-demented control subjects (Table 1). All patients with FTD and AD had been assessed (by DN, JSS) within out-patient clinics at the Cerebral Function Unit, Greater Manchester Neurosciences Centre. All patients had undergone historical interview in the presence of a family caregiver and comprehensive cognitive assessment to obtain information about the nature and progression of cognitive and functional decline. Patients fulfilled the National Institute of Neurological and Communicative Disorders and Stroke - Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) criteria for probable AD,²⁵ and International criteria for FTD.²⁶ Control subjects were either spouses of patients attending CFU clinic (n=13), or a sample of healthy volunteers (n=72) drawn randomly from a cohort of 767 mentally normal people aged >50 years, resident within the same Greater Manchester region from which the AD and FTD patients were recruited.²⁷ The latter comprised a longitudinal ageing study group who had been screened psychologically for absence of cognitive impairment consistent with dementia. All blood samples had been collected with the approval of the local ethics research committee.

Plasma was separated from whole blood samples (5ml blood with EDTA acting as anti-coagulant) by routine methods, and stored in deep freeze (minus 80°C) until assay.

Table 1: Mean (±SD) duration of illness at the time when blood sample was taken, age at onset of illness, and gender ratio for patients with AD and FTD, and control sub ects.

1.1.2 Immunoassay protocol

An antibody-sandwich ELISA was developed to measure the TDP-43 concentration in plasma samples. The ELISA plates (96-well PVC assay plates, Iwaki, Japan) were coated by overnight incubation at 4°C with 0.2 μg/ml anti-TDP monoclonal antibody (H00023435-M01, clone 2E2-D3, Abnova Corporation, Taiwan), 100 μl/well, diluted in 200 mM NaHCO₃ buffer, pH 9.6, containing 0.02% (w/v) sodium azide. The plates were washed 3 times with PBST (PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4) containing 0.05% Tween 20), and incubated with 200 μL/well of blocking buffer (PBS containing 2.5% gelatin and 0.05% Tween 20) for 2 h at 37°C. The plates were again washed 3 times with PBST, and 100 μL of the plasma samples to be tested, diluted 1 :1 with PBS, were added to each of three wells. After washing 3 times with PBST, lOOμl of the detection antibody, TDP rabbit polyclonal antibody (BCOO 1487, ProteinTech Group, Chicago) diluted to 0.2 μg/ml in blocking buffer, was added per well and the plates were incubated at 37⁰C for 2 hrs. After another wash (as before), the plates were incubated with lOOμl/well of goat anti-rabbit secondary antibody coupled to horseradish peroxidase (HRP) (Dako Ltd., Ely, U.K.), diluted 1 : 10,000 in blocking buffer, at 37⁰C for lhr. The plates were then washed again with PBST, before adding 100μl/well Sure Blue TMB Microwell Peroxidase Substrate (KPL Inc, Maryland, USA) and leaving the colour to develop for 30 min at room temperature. Finally, lOOμl/well of stop solution (0.3M H₂SO₄) was added and absorbance values were read at 450 nm in a Victor² multi-function microtitre plate reader. Net absorbance was calculated by deducting the mean value obtained for a triplicate of "blank" wells containing PBS only.

Recombinant TDP-43 protein (MW 54.3 KDa) (AAHO 1487, 1 -261, Abnova Corporation, Taiwan, with 26 KDa GST tag) was used in the standard curve.

1.1.3 Immunoprecipitation of TDP-43 from plasma

Dynabeads covalently coupled with recombinant protein A, were derivatised with rabbit polyclonal anti-TDP-43 antibody (BC001487, ProteinTech Group, Chicago) as recommended by the manufacturer (Dynal Biotech LTD.,Wirral, UK). 300μl plasma was added to the beads and incubated overnight at 4⁰C. The plasma samples were chosen according to the ELISA results, with one sample giving a high signal and the other a low signal. The beads were then washed three times with 0.1 M phosphate buffer, pH 8.2. Any captured TDP-43 was eluted from the beads by boiling for 10 min in NuPAGE LDS sample buffer (Invitrogen LTD., U.K.) and examined by gel electrophoresis and immunoblotting.

1.1.4 Gel electrophoresis and immunoblotting

The protein eluted from the magnetic dynabeads (see above) was separated on NUPAGE Bis-Tris 4-12%, lmm gels (Invitrogen). The separated proteins were transferred to nitrocellulose membranes (0.45 μm, Invitrogen) at 30V, 125mA for 1 hr. Membranes were blocked with 5% Marvel dried skimmed milk, dissolved in PBST for 1 hr. The membranes were probed overnight at 4⁰C with either anti-TDP-43 antibody, rabbit polyclonal (BCOO 1487, ProteinTech Group, Chicago) 1 :1000 in PBST, or anti-TDP-43 mouse monoclonal antibody (H00023435-M01 , clone 2E2-D3, Abnova Corporation, Taiwan) 1:1000 in PBST, as indicated. The membranes were washed three times in PBST, followed by incubation with HRP -conjugated goat anti- rabbit or goat anti-mouse (Dako LTD., Ely, U.K.), 1 :10,000 in PBST, as appropriate, for 1 hr. The protein bands were visualised using ECL reagents (Pierce, Rockford, IL) as described by the manufacturer.

1.1.5 Statistical analyses All data were analysed using SPSS v 14.0. Because OD values for FTD and AD patients, as well as control subjects were not normally distributed according to Kolmogorov-Smirnov test, non-parametric Kruskal-Wallis test was used throughout to compare groups, with post hoc Mann- Whitney test being employed when the results of Kruskal-Wallis test yielded significant group differences.

1.2 Results

A standard curve for the ELISA is shown in Fig. 1. The ELISA data from the plasma samples indicate that in control subjects, in most instances, TDP-43 was barely detectable (mean±SD OD=0.02±0.32), with just 8 individuals (8.8%) having OD values exceeding 0.11 (Fig. 2). This value corresponds to the 99% upper confidence level and was subsequently chosen to represent the 'cut off point of 'normal' TDP-43 values. In AD, TDP-43 was again barely detectable in 79 patients (78%) (Fig. 2), though OD exceeded 0.11 in 23 patients (22%). In FTD, 16/35 patients (46%) patients had OD value exceeding 0.11 (Fig. 2). The proportion of patients with OD greater than 0.11 differed significantly across the groups (χ² = 19.8; p< 0.001) with the proportion of patients with OD values greater than 0.11 being significantly higher in both FTD (χ² = 20.4; p< 0.001) and AD (χ² = 5.8; p=0.016) compared to controls, and in FTD compared to AD (χ² = 6.9; p=0.009). Consequently, the mean OD values for control subjects and patients with FTD (0.26±0.52) and AD (0.13±0.33) were significantly different by Kruskal-Wallis test (χ² = 53.2; p< 0.001), with post hoc Mann- Whitney test showing mean values for both FTD and AD groups to be significantly different from controls (p<0.001), with mean OD in FTD group also being significantly greater (p=0.037) than that in AD.

When protein from a plasma sample with high levels of TDP-43 (based on ELISA results) was immunocaptured with magnetic Dynabeads coupled to the anti-TDP-43 rabbit polyclonal antibody, and then examined by gel electrophoresis and western blotting employing the anti-TDP-43 monoclonal antibody for detection, only a single band migrating at ~43kDa was seen (Fig. 3, lane 2). No specific bands were detected in a similarly treated low-reading plasma sample.

1. 3 Discussion The inventors have shown for the first time that the TAR DNA-binding protein (TDP- 43) can be detected within plasma of normal control people, as well as individuals with neurodegenerative diseases such as AD and FTD.

The ELISA data indicate that TDP-43 is present at extremely low concentrations, or is absent, in the vast majority of normal people, and in AD or FTD there are substantial numbers of patients in whom TDP-43 is likewise barely present.

However, group comparisons by ELISA reveal a highly significant increase (approximately 13-fold) in mean plasma TDP-43 levels in FTD compared to controls, whereas in AD the mean TDP-43 plasma level was about 7-fold increased. The validity of the ELISA method is supported by the immunoblotting results. In accord with the ELISA data, an immunoreactive band migrating at 43 kDa, which corresponds precisely to the molecular weight of full-length TDP-43, ' was detected only in the high-reading plasma sample and not in the low-reading sample. This result not only confirms the specificity of the antibody pair employed for the sandwich ELISA, but also shows that the readout from the ELISA accurately reflects the levels of TDP-43 present in plasma.

From histological studies, it is known that about 50% of patients with FTD have ubiquitin/TDP-43 -based histology,^5"7'^20"24 and consequently it might be expected that a similar proportion of patients with this kind of pathology would be included within the present unselected FTD study group. In the present FTD cohort, there was a highly significant mean increase in plasma TDP-43 levels, with about 46% patients showing 'high' TDP-43 levels (ie above OD of 0.11 corresponding to 99% upper control limit). On the basis of autopsy studies,^5"7 such a proportion of patients would have been anticipated if the presence of TDP-43 in plasma is indeed indexing the presence of TDP-43 pathology in brain. Likewise, in AD it is known that about 20- 25% patients have TDP-43-based pathology,¹⁶ and again it might be anticipated that within the present, group of unselected AD cases, a similar proportion of cases with TDP-43 pathology would exist. In the present AD group 22% patients showed 'high' TDP-43 values, while in most patients no increase was seen. Therefore the proportion of patients showing high plasma TDP-43 levels in both FTD and AD closely match those proportions that would be predicted to occur from histological studies. The few elderly control subjects with high TDP-43 levels may be patients with incipient neurodegenerative disease, most likely AD, since it has long been known that a very high proportion of mentally normal elderly subjects show at autopsy some degree of Alzheimer-type pathological changes in their brains (ie senile plaques and neurofibrillary tangles), and this may, as in AD itself, include some individuals with additional TDP-43 pathology.

The numerical similarities between the proportions of patients with FTD and AD showing 'high' TDP-43 plasma levels and those displaying TDP-43 pathological changes at autopsy strongly imply that measurement of plasma TDP-43 level can index the presence of TDP-43 pathology within the brain of patients with FTD or AD. Therefore, in preferred embodiments of the invention, the test may be employed to distinguish between subjects that have, or are likely to develop a neurodegenerative disorder with TDP-43 pathology. This relationship can be confirmed by comparing plasma TDP-43 levels in FTD patients bearing MAPT or PGRN mutations, since it is known from histological studies that patients with MAPT mutations and tauopathy do not show TDP-43 pathological changes in their brains,⁷ whereas those with PGRN mutation always display TDP-43 pathological changes.⁷'^10"15 Hence, subjects with MAPT mutations should show low (normal) plasma TDP-43 levels and those with PGRN mutations should show high(er) plasma TDP-43 levels.

Although we do not know the biological or functional significance of (the additional) TDP-43 pathology in patients with AD, as with FTD, there are heuristic (future) management or therapeutic values in being able to predict the presence of TDP-43 changes in such patients. Given the possibility of tau- or TDP-43-based therapies becoming available for patients with AD or FTD, knowing who in AD or FTD is potentially able to benefit from tau and/or TDP-43 based therapy, according to predictive knowledge of underlying histology, would be of considerable practical value. This illustrates the usefulness of the screening methods of the third and fourth aspects of the invention.

In summary, these data illustrate, using ELISA-based methodology, that the inventors were able to detect in plasma the presence of the nuclear transcription factor TDP-43, which has, recently been shown to be a major component of the pathological changes in the brains and spinal cords of patients with frontotemporal dementia (FTD), and those with motor neurone disease. They detected elevated levels of TDP-43 protein in plasma of 46% patients with FTD compared to 8% of control subjects. Importantly, plasma TDP-43 levels were also increased in 22% patients with Alzheimer's disease (AD). The proportions of patients with FTD and AD showing raised plasma TDP-43 levels correspond closely to those proportions of patients known from autopsy studies to harbour TDP-43 pathological changes in their brains. This indicates that raised plasma levels of TDP-43 correlates with the presence of TDP-43 pathology within the brain and confirms that TDP-43 is a biomarker for FTD. Plasma TDP-43 levels may be measured accorded to the methods of the first aspect of the invention and are of great practical value for prognostic assessment, diagnostic assessment and for planning future therapeutic strategies aimed at preventing or removing tau or TDP-43 pathological changes from the brain in neurodegenerative disorders.

References

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Claims

1. An in vitro method for identifying a subject predisposed to, or suffering from, a neurodegenerative disease, the method comprising obtaining a peripheral body fluid sample from the subject and examining TDP-43 levels in the sample from a test subject and comparing TDP-43 levels with a reference derived from an individual who does not suffer from a neurodegenerative disease, wherein raised concentration of TDP-43 in the sample from the test subject suggests that the subject is suffering from a neurodegenerative disease or is predisposed to developing a neurodegenerative disease.

2. The method of claim 1 wherein the peripheral body fluid sample is blood or a blood product.

3. The method of claim 2 wherein the peripheral body fluid sample is plasma.

4. The method according to any preceding claim wherein the neurodegenerative disease is characterised by dementia.

5. The method according to any preceding claim wherein the neurodegenerative disease is selected from frontotemporal lobar degeneration, Alzheimer's disease, motor neuron disease, Lewy body diseases or Parkinson Disease

6. The method according to any preceding claim wherein the neurodegenerative disease is characterised by neuronal cytoplasmic inclusions that are immunoreactive for ubiquitin (ub-ir) but not for tau.

7. A use of the method according to any one of claims 1 -6 for distinguishing between subjects with a neurodegenerative disease which is characterised by neuronal cytoplasmic inclusions that are immunoreactive for ubiquitin (ub-ir) and subjects with a neurodegenerative disease characterised by a tauopathy.

8. A kit for identifying a subject predisposed to, or suffering from, a neurodegenerative disease the kit comprising:-

(i) means for determining TDP-43 levels in a sample from a test subject; and, optionally (ii) a reference corresponding to a level of TDP-43 in a sample from an individual who does not suffer from neurodegenerative disease, wherein the kit is used to identify a presence or a raised level of TDP-43 in the sample from the test subject, thereby suggesting that the test subject is predisposed to, or suffers from, a neurodegenerative disease.

9. A method of screening a test compound to determine whether the compound has efficacy for treating or preventing neurodegenerative diseases, comprising:

(i) exposing a biological system to the test compound; (ii) detecting the ability of the compound to reduce the level of TDP-43 in a peripheral body fluid; and,

(iii) comparing the level of TDP-43 in the biological system exposed to the test compound to the level of TDP-43 in a control biological system that was not exposed with the compound; wherein compounds with efficacy for treating or preventing neurodegenerative diseases reduce the level of TDP-43 relative to controls.

10. A method of screening a compound, to test whether the compound may cause a neurodegenerative disease, comprising:

(i) exposing a biological system to the test compound; (ii) detecting the ability of the compound to raise the level of TDP-43 in a peripheral body fluid; and,

(iii) comparing the level of TDP-43 in the biological system exposed to the test compound to the level of TDP-43 in a control biological system that was not exposed with the compound; wherein compounds that cause neurodegenerative diseases increase the level of TDP- 43 relative to controls.

11. An in vitro method of selecting a test subject for consideration in a clinical trial for treating neurodegenerative disease, comprising obtaining a peripheral body fluid sample from the subject and examining TDP-43 levels in the sample from a test subject and comparing TDP-43 levels with a reference derived from an individual who does not suffer from a neurodegenerative disease, wherein raised concentration of TDP-43 in the sample from the test subject suggests that the subject is suffering from a neurodegenerative disease or is predisposed to developing a neurodegenerative disease, caused by TDP-43 pathology.

12. The method of claim 11 wherein a test subject with elevated TDP-43 levels would be excluded from a clinical trial of a drug for treating non-TDP-43 related neurodegenerative diseases.

13. A method of any of the previous claims wherein the level of TDP-43 is detemined using an antibody that is able to selectively bind to TDP-43 polypeptide.

14. The method of claim 13 wherein the antibody can detect C-terminal breakdown or cleavage fragments of TDP-43 polypeptide.

15. The method of claim 13 wherein the antibody specifically binds to the phosphorylated forms of TDP-43.