WO2007068105A1 - Method of diagnosing amyotrophic lateral sclerosis - Google Patents

Abstract

A method of diagnosing ALS in a mammal is provided comprising the step of analyzing a tau-containing sample obtained from the mammal to determine whether the sample includes pT175 tau, wherein phosphorylation at position 175 of tau is indicative of ALS. Antigenic pT175 tau polypeptides may be used in the method, as well as antibodies prepared using the antigenic polypeptides.

Description

METHOD OF DIAGNOSING AMYOTROPHIC LATERAL SCLEROSIS

Field of the Invention

[0001]The present invention relates to a method of diagnosing amyotrophic lateral sclerosis (ALS). In particular, the invention relates to the identification of a novel biomarker for diagnosing ALS, as well as antibodies selective for the biomarker.

Background of the Invention

[0002]ALS has been considered an age-dependant neurodegenerative disease state in which the neuropathological process is restricted to the motor system (1 ;2). The contemporary view of ALS is, however, that of a multisystems disorder in which motor neuron degeneration remains the core neuropathological feature, but in which a frontotemporal lobar degeneration (FTLD) can either accompany the motor neuron degeneration, or in some cases, precede its onset (3). This process may be reflected in mild cognitive deficits typical of a frontal dysexecutive syndrome, as behavioural impairment with a disinhibition syndrome, or as a frank frontotemporal dementia (FTD) (4). The prevalence of FTD in ALS has been estimated to range from 30% to 50% of cases, with most recent studies suggesting that this may approach 70% (5-7).

[0003] While the neuropathological substrate of the FTLD in ALS remains to be fully defined, ubiquitin immunoreactive intraneuronal inclusions are frequently observed in the dentate granule cells, the superficial frontal and temporal cortical layers, and in the entorhinal cortex hippocampus of ALS patients. Ubiquitin immunoreactive dystrophic neurites are evident in the extramotor cortices, but with a predominance of involvement in the frontal, temporal and hippocampal cortex. These features are not specific to ALSci cases and can be observed in other forms of neurodegeneration (8-11). However, these ubiquitin immunoreactive inclusions are unique in lacking immunoreactivity to either microtubule associated protein tau or α-synuclein, suggesting that such inclusions may be pathogenomic for ALSci (8- 10; 12- 14). The neuropathological correlates of the FTLD in ALS include superficial linear spongiosus, astrogliosis and microglial proliferation, and tau protein aggregation within the frontal cortex (5; 15). [0004]The absence of tau pathology in ALSci has recently been re-evaluated. Tau immunoreactive aggregates are observed in neurons, astrocytes, as neuritic threads, and in rare instances, as oligodendroglial coiled bodies within cortical layers II and III, deeper cortical layers and subcortical white matter in both cognitively impaired (ALSci) and cognitively intact ALS patients (15). Tau immunoreactive thread-like structures have been described in the neurophil and in glial cells (as coiled bodies) in the hippocampus, parahippocampal gyrus and amygdala of ALS patients (16). Tau protein aggregation was present in the absence of an alteration in the expression of either the 3 R or 4R isoforms of tau, suggesting a potential post-translational alteration in tau expression (15). Moreover, these pathological changes of tau aggregation are outside that which would be expected as a function of normal aging (17).

[0005]Based on these findings, the categorizaton of ALSci has been examined within the current nosology of the FTLDs in which both the neuropathological characteristics and the molecular characterization of the tau protein are critical components (18). The FTLDs, thus, can be considered as either reflective of a tauopathy, or not. In those individuals in which perturbations of tau protein metabolism are evident, a "signature" tau neuropathology can be developed based on the relative presence of either the 3R or 4R isoforms of tau (both by Western blotting and by immunohistochemical analysis) and on the presence or absence of specific neuronal or glial inclusions. For example, corticobasal degeneration (CBD) can be characterized by the deposition of hyperphosphorylated tau as filamentous inclusions in neurons and glia, with 4R-tau as the predominant isoform (19). The sarkosyl -insoluble tau fraction in both the grey and white matter contains predominantly the hyperphosphorylated 4R-tau isoforms, with the isoforms recognized by the monoclonal antibody AT8 (recognizing phosphorylation at Ser-202/Thr-205) specifically increased in the white matter. This is in distinction to Pick's disease which is marked by prominent frontotemporal degeneration clinically, and neuropathologically by discrete frontal and anterior temporal cortical atrophy with achromatic neurons (Pick cells) and intraneuronal argentophilic inclusions (Pick bodies). The biochemical "signature" of this tauopathy is the accumulation of both 3R and 4R-tau isoforms within both grey and white matter sarkosyl-insoluble fraction (20). At the level of the immunohistochemical analysis of the intraneuronal aggregates, both CBD and progressive supranuclear palsy (PSP) can be differentiated from Pick's disease by the observation of 4R-tau immunostaining of intraneuronal aggregates, a feature not observed in Pick's neuronal aggregates (although observed within glial aggregates) (21). A similar process can be adopted for a number of the remaining tauopathies, including argyrophilic grain disease, dementia lacking distinctive histopathology (DLDH), and Alzheimer's disease (22-25).

[0006]It would, thus, be desirable to identify a unique "signature" for ALS, including both cognitively impaired and cognitively intact forms, that would be useful in the diagnosis of this disease.

Summary of the Invention

[0007]It has now been found that a phosphorylated isoform of Tau protein specifically phosphorylated at Threonine 175 selectively exists in the brain tissue of patients having ALS. Thus, in a first aspect of the present invention, a method of diagnosing ALS in a mammal is provided comprising the step of analyzing a tau-containing sample obtained from the mammal to determine whether the threonine at position 175 of tau is phosphorylated, wherein phosphorylation at position 175 of tau is indicative of ALS.

[0008]In another aspect of the invention, a polypeptide useful to generate an antibody immunospecific to a Tau protein phosphorylated at threonine 175 is provided. The polypeptide comprises the sequence RIPAK[pT]PPAPK, wherein [pT] represents a phosphorylated threonine.

[0009]In another aspect of the invention, a phosphospecific T175-Tau antibody is provided. Methods of making such antibodies, and hybridomas used to make them, are also provided herein. [001O]In a further aspect of the invention, kits for use in the diagnosis of ALS are provided comprising a pT175 antigenic polypeptide comprising the sequence, RIPAK[pT]PPAPK, or comprising a phosphospecific T175-Tau antibody.

[001 l]These and other aspects of the invention are described by reference to the detailed description that follows, and by reference to the drawings in which:

Brief Description of the Drawings

Figure 1 illustrates the results of Western blot analysis exhibiting a unique Tau protein expression profile for ALS in comparison with Alzheimer's;

Figure 2 illustrates the results of Western blot analysis which shows that ALS and ALSci patients have prominent insoluble tau in both frontal grey and white matter tau protein isolates in contrast to corresponding samples in neurologically intact controls;

Figure 3 illustrates the determination of extent and stability of tau phosphorylation at amino acids S202 and T205 in AD, ALS and ALSci isolates of frontal grey matter when exposed to dephosphorylation by lambda alkaline phosphatase (A) andbovine alkaline phosphatase (B);

Figure 4 is a schematic illustration of soluble tau phosphoepitopes for control, AD, ALS and ALSci;

Figure 5 illustrates the amino acid sequence alignment between different isoforms of human Tau protein;

Figure 6 shows the expression of tau protein phosphorylated solely at Tl 75 or S217, or tau protein simultaneously phosphorylated at S208/S210 in isolates from the white matter of patients suffering from AD or ALS; and Figure 7 illustrates the immunohistochemical analysis of samples from AD or ALS white matter showing the distribution of Tau protein phosphorylated at T175, S217, or S208/S210.

Detailed Description of the Invention

[0012] An ALS-specific T175-phosphorylated isoform of Tau protein is provided that is useful in a method of diagnosing ALS in a mammal. The method comprises analyzing a tau-containing sample obtained from the mammal to determine whether or not the threonine at position 175 of tau is phosphorylated. Phosphorylation at position 175 of tau is indicative of ALS.

[0013]Tau protein is a highly soluble microtubule-associated protein (MAP). The major tau protein in the human brain is encoded by 11 exons. Exon 2, 3 and 10 are alternative spliced, allowing six combinations (2-3-10-; 2+3-10-; 2+3+10-; 2-3-10+; 2+3-10+; 2+3+10+). Thus, in the human brain, the tau proteins constitute a family of six iso forms having from 352-441 amino acids. They differ in the number of inserts of 29 amino acids at the N-terminal part (exon 2 and 3) including either none, 1 or 2 inserts, and/or the number of repeat-regions at the C-terminal end (exon 10). The longest isoform in the CNS has four repeats (Rl, R2, R3 and R4) and two inserts (441 amino acids total), while the shortest isoform has three repeats (Rl, R3 and R4) and no insert (352 amino acids total). The amino acid sequences of four human Tau isoforms are set out in Figure 5.

[0014]The term "Tl 75" is used herein to denote the threonine at position 175 of a tau protein, including any tau isoform as described above. Phosphorylated threonine is denoted by pT or [pT], and phosphorylated threonine at position 175 of a tau protein is denoted by pT175 or [pT]175.

[0015]The term "tau-containing sample" as used herein is meant to encompass any mammalian biological sample containing at least one tau isoform that exists in the brain. Samples may be obtained from the cerebrum cortex, hypothalamus, amygdala, hippocampus, thalamus and basal ganglia Such a tau-containing sample is obtained from a living mammal via cerebral spinal fluid, or alternatively, serum. Less desirably, the sample may be obtained from tissue obtained by biopsy. [0016]The term "mammal" is used herein to refer to human and non-human mammals, including both domestic and wild animals.

[0017]Once an appropriate tau-containing sample is obtained, it may be necessary to purify the sample prior to analyzing it for Tl 75 phosphorylation of tau. Purification techniques well- established in the art may be used for this purpose as one of skill in the art will appreciate including, for example, filtration and centrirugation.

[0018]Determination of phosphorylation at position 175 of tau may be conducted using any one of a number of techniques. For example, an immunological method of determining site-specific phosphorylation may be employed using an antibody based on a phosphorylated Tl 75 immunogenic fragment or antigen.

[0019]The raising of antibodies to a desired peptide or immunogenic fragment can be achieved, for polyclonal antibody production, using immunization protocols of conventional design, and any of a variety of mammalian hosts, such as sheep, goats and rabbits. For monoclonal antibody production, immunocytes such as splenocytes can be recovered from the immunized animal and fused, using hybridoma technology, to myeloma cells. The fusion cell products, i.e. hybridoma cells, are then screened by culturing in a selection medium, and cells producing the desired antibody are recovered for continuous growth, and antibody recovery. Selected hybridoma cells may be implanted into the peritoneum of a mouse, for example, and monoclonal antibodies subsequently produced can be collected from the ascites produced by the mouse in response to implantation of the hybridoma.

[0020]Recovered antibody can then be coupled covalently to a reporter molecule, i.e. a detectable label, such as a radiolabel, enzyme label, luminescent label or the like, optionally using linker technology established for this purpose. [0021]The antibody may be farther modified, for example, to enable its use in humans for therapy. Techniques well-established in the art may be utilized to humanize the antibody, utilizing CDR (complementarity determining region (CDR)) grafting, in which non-human CDR loops are grafted onto human framework regions may be used as well more recent methodology as described by Studnicka et al. (Protein Engineering vol. 7 no. 6 pp. 805-814, 1994 Oxford Journals).

[0022]Once obtained, antibodies may be used to diagnose ALS, to monitor the progression of ALS and to determine the effectiveness of therapies designed to treat ALS. For example, detectably labelled pT175 antibody may be used to identify the existence of phosphorylation of Tl 75 of a tau protein in a tau-containing sample obtained from a mammal. The labelled antibody is added to the sample and incubated under conditions suitable for an immunological interaction between antibody and pT175 tau in the sample. The sample is then treated or washed to remove free antibody (e.g. antibody that did not interact). The presence of antibody in the washed sample is then determined using techniques suitable to identify the label on the antibody. Detection of label in the sample is indicative of pT175, and thus, of ALS. In a similar fashion, the pT175 antibody can be used to monitor the progression of ALS and to determine the effectiveness of therapies designed to treat ALS by determining the level of pT175 over time in subsequent samples obtained from the mammal being diagnosed/treated.

[0023]In accordance with an aspect of the invention, an isolated tau polypeptide useful to generate an antibody immunospecific to a pT175 Tau protein is provided comprising the sequence, RIP AK[pT] PPAPK (SEQ ID NO:1) in accordance with the 3-letter amino acid code. Polypeptides which are functionally equivalent to the polypeptide of SEQ ID No:l are also encompassed. The term "isolated" is used herein to refer to peptides which are essentially pure and free from extraneous cellular material including other proteins or peptide fragments.

[0024]Thus, polypeptides comprising SEQ ID NO: 1 may include additional peptide sequence at either or both ends thereof as long as pT175 antigenic property is retained. Such additional peptide sequence is not particularly restricted with respect to composition or length, but will generally yield an antigenic polypeptide of at least about 12 amino acids in length. Such additional sequence may be added to bestow on the polypeptide a desirable property such as increased stability, or to enhance cellular uptake. Preferred pT175 antigenic polypeptides may range in size from 12 to about 100 amino acids, but will preferably be 12 to about 50 amino acids in length or smaller.

[0025]Polypeptides which are "functionally equivalent" to polypeptides comprising the amino acid sequence set out in SEQ ID NO: 1 include peptides comprising one or more amino acid deletions, additions or substitutions, but which retain the antigenic property thereof. Thus, functionally equivalent variants of a tau polypeptide according to SEQ ID NO: 1 include analogues, fragments and derivatives thereof. Variants in accordance with the present invention are not necessarily restricted by size, as long as they retain pT175 antigenic activity. Generally, variant peptides range in size from about 10 to about 100 amino acids, preferably from about 10 to about 50 amino acids, and more preferably from about 10 to about 20 amino acids.

[0026] A functionally equivalent variant of a tau polypeptide may include one or more amino acid substitutions, particularly a conservative amino acid substitution. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glutamine and glutamic acid, between asparagine and aspartic acid, and between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

[0027] A functionally equivalent variant may also include a fragment of a tau polypeptide comprising a sequence that corresponds with SEQ ID NO:1 but which is truncated by one or more amino acid residues. Fragments in accordance with the invention include fragments of SEQ ID No:l which retain pT175 antigenic activity. [0028] A functionally equivalent variant may additionally include a derivative of a tau polypeptide in accordance with the present invention comprising a sequence that corresponds with SEQ ID NO:1 in which one or more of the amino acid residues therein is chemically derivatized. Functionally equivalent derivatives also encompass analogues or fragments comprising one or more derivatized amino acid residues. The amino acids may be derivatized at the amino or carboxy groups, or alternatively, at the side "R" groups thereof. Derealization of amino acids within the peptide may render a peptide having more desirable qualitities such as increased stability or activity. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form, for example, amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form, for example, O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids, for example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Terminal modification of a peptide to protect against chemical or enzymatic degradation may also include acetylation at the N-terminus and amidation at the C-terminus of the peptide.

[0029] Antigenic tau polypeptides in accordance with the present invention may be made using well-established techniques of protein synthesis which may include automated methods utilizing a peptide synthesizer, or manual techniques. The present inhibitors may also be made using any one of a number of suitable techniques based on recombinant technology. It will be appreciated that such techniques are well-established by those skilled in the art, and involve the expression of the nucleic acid encoding the selected tau polypeptide in a genetically engineered host cell.

[0030]Isolated nucleic acid encoding tau polypeptides in accordance with the present invention is also encompassed by the invention. Thus, nucleic acid, including DNA and RNA, encoding a polypeptide comprising the sequence, RIPAK[ρT]PPAPK (SEQ ID NO.l), is provided. It will be appreciated that more than one nucleic acid sequence will encode each tau polypeptide according to the invention, given the degeneracy that exists in the genetic code. Nucleic acid encoding functionally equivalent variants of a tau polypeptide is also encompassed by the present invention.

[0031 ] DNA encoding a tau polypeptide may be synthesized de novo by techniques well-known in the art. Generally, gene synthesis may be conducted by the successive 3' to 5' coupling of appropriately protected nucleotide reagents in an automated synthesizer, followed by recovery of the deprotected polynucleotide. Sequences obtained by de novo synthesis may be amplified using the polymerase chain reaction as described in United States Patent No. 4,683,195.

[0032]Recombinant techniques for producing a tau polypeptide generally involve insertion of a tau peptide-encoding DNA sequence into a suitable expression vector which is subsequently introduced into an appropriate host cell (such as Chinese hamster ovary cells (CHO cells) or human embryonic kidney cells of the 293 lineage (ATCC CRL 1573)) for expression. Suitable expression vectors are those vectors which will drive expression of the inserted tau-encoding DNA in the selected host. Typically, expression vectors are prepared by site-directed insertion of the DNA construct therein. The DNA construct is prepared by replacing a coding region, or a portion thereof, within a gene native to the selected host, or in a gene originating from a virus infectious to the host, with the tau DNA. In this way, regions required to control expression of the tau DNA, which are recognized by the host, including both a 5' region to drive expression and a 3' region to terminate expression, are inherent in the DNA construct. To allow selection of host cells stably transformed with the expression vector, a selection marker is generally included in the vector which takes the form of a gene conferring some survival advantage on the transformants such as antibiotic resistance. Thus, following culture of the transformed cells under suitable conditions, the desired tau polypeptide is isolated and purified for use, e.g. to make pT175 antibodies.

[0033] Antibodies prepared using a pT175 antigenic polypeptide are provided in another aspect of the present invention. [0034]In another aspect, a kit for use in the diagnosis of ALS is provided. The kit may include one or more pT175 tau protein antigens comprising the sequence, RIPAK[pT]PPAPK (SEQ ID NO: 1), or a functionally equivalent variant thereof as described above. In another embodiment, the kit may include an antibody directed to pT175 tau protein prepared using conventional methodology as set out above.

[0035] All references referred to herein are incorporated by reference.

[0036]Embodiments of the invention are described by reference to the following specific examples which are not to be construed as limiting.

Example 1

Materials and Methods

[0037]Protein isolation - Tau protein was extracted from archival frozen frontal cortex in 2 cases of neurologically normal controls (mean age 73.4 ± 3.1 years, mean post mortem interval 17.1 ± 3.7 hours), 4 ALS patients with no antemortem evidence for cognitive dysfunction (mean age 67.0 ± 18.3 years, mean post mortem interval 11.3 ± 9.3 hours), and 4 ALS patients with clinical evidence of cognitive dysfunction (mean age 58.9 ± 5.5 years, mean post mortem interval 4.8 ± 1.1 hours). At the time of autopsy, the right cerebral hemisphere was isolated, sectioned in 1.0 cm coronal slices, and frozen at -8O⁰C. The clinical and neuropathological characteristics of these patients have been previously reported (15). In addition to the neurologically normal control, a similar isolation was performed from the cortex of a patient with neuropathologically documented Alzheimer's disease. The latter served as a control for the technical aspects of the tau isolation and for the immunostaining studies. For the isolation of grey and white matter, blocks of tissue were cut from the anterior cingulate gyrus and superior frontal gyrus at the level of the anterior corpus callosum. Leptomeninges and blood vessels were dissected away, and the grey matter separated from the white matter using blunt dissection.

[0038]Tau protein was isolated from 1.0 gm. of either grey or white matter, using the technique of Hanger et al with minor modifications (26). In brief, tissue was homogenized in 1.0 ml of MES buffer, pH 6.5, centrifuged at 27,000 X g for 60 minutes at 4⁰C and the pellet discarded. The supernatant was further centrifuged at 95,000 X g for 60 minutes at 4⁰C and the supernatant saved (containing the soluble tau isoforms). The pellet (containing insoluble tau) was solubilized in 150 μl 4 M guanidine HCl with a brief sonication (1 hours, room temperature) and dialyzed against 50 raM Tris-HCl, pH 7.5, 1 mg/ml PMSF overnight at 4⁰C. The dialysate was centrifuged at 15,000 X g for 60 minutes at 4⁰C and the supernatant retained (containing insoluble tau). The pellet was discarded. The supernatant containing soluble tau isoforms was boiled at 100⁰C for 10 minutes and then centrifuged at 15,000 X g for 30 minutes at 4⁰C. The supernatant was brought to approximately 3.0 ml in 50 mM Tris-HCl, 1.35 gm ammonium sulphate added, and then cooled on ice for 15 minutes. Precipitated proteins were collected following centrifugation at 15,000 X g for 30 minutes at 4⁰C and resuspended in 150 μl 50 mM Tris-HCl, pH 7.5. The suspension was dialyzed against 50 MM Tris-HCl, pH 7.5, 1 mg/ml PMSF overnight at 4°C and then dialysate clarified by centrifugation at 15,000 X g for 30 minutes at 4⁰C.

[0039]Immunoblot analysis - In the initial set of studies, the solubility characteristics of tau were examined, both pre and post dephosphorylation. For these studies, immunoblots were performed using mouse monoclonal antibodies directed against tau, including T46 (1 : 1,000) and Tl 4 (1 :3,000) (Zymed Laboratories Inc., San Francisco, CA) with overnight incubation at 4⁰C. For all immunoblots, antigen antibody localization was undertaken using enhanced chemiluminescence.

[0040]Dephosphorylation studies - The extent of phosphorylation was examined by two techniques. In the first, aliquots of either soluble or insoluble tau in 50 mM Tris-HCl, pH 7.5 were incubated with lambda alkaline phosphatase (20 U/μl, 6 hours, 3O⁰C) (Sigma). Reactions were stopped by the addition of 2x electrophoresis buffer. Equal aliquots of supernatant were electrophoresed on 7.5% SDS.PAGE and electrophoretically transferred to nitrocellulose membrane. A complete loss of immunoreactivity to tau phosphoepitopes was achieved within 3 hours using isolates from control tissue (data not shown) and thus subsequent studies were performed at 3 hours incubation. In the second technique, following electrophoretic transfer of protein isolates to nitrocellulose membrane, whole membranes were incubated with bovine alkaline phosphatase (196 U/ml) at room temperature for 15 hours. The monoclonal antibody AT8 (requiring phosphorylation of both S²⁰² and T²⁰⁵) recognizes isolates of control, AD, ALS and ALSci, and thus immunoblots were performed using the AT8 antibody. [004l]Identification ofTau Phosphopeptides - Clarified dialysates of soluble tau prepared as described above from frontal cortex of control, ALS, ALSci and AD brains were buffer exchanged to 0.2M NH₄HCO₃ with 1OK Nanosep ultrafiltration units (Pall). Protein solutions were dried in the Speed Vac (Savant) and protein residue (200 μg protein for soluble tau and 100 μg for insoluble tau from control, ALS and ALSci) were taken up in 100 μl 8M urea/0.4M NH₄HCO₃ for reduction, alkylation, tryptic digestion, SPE clean up, immobilized metal affinity chromatography (IMAC) and LC-MS/MS analysis as previously described (27). In addition to digestion with trypsin, AD soluble tau protein (200 μg) was also digested with subtilisin (10 ug, Boehringer Mannheim) and after acidification with 12ul TFA, pepsin (10 μg, Boehringer Mannheim) as described above for trypsin digestion. AD samples were analyzed by LC/MS/MS utilizing an extended HPLC gradient of 2-50% acetonitrile (0.1% HCOOH) over 240 min. MS/MS spectra were searched against a database consisting of the human tau proteins utilizing the SEQUEST program (Thermo) for identification of phosphopeptides and specific phosphorylated residues as previously described.

[0042]In addition, a set of soluble tau from frontal cortex of control, ALS and ALSci brains (200 μg total protein each) was processed as above except that the digests were methylated prior to IMAC. Methylated digests were divided into two equal aliquots (100 μg total protein equivalent) which were subjected to (1) gallium IMAC utilizing a Phosphopeptide Isolation Kit (Pierce) as previously described (28) or (2) Iron (III) IMAC utilizing PHOS-Select iron affinity gel (Sigma) as previously described (27).

RESULTS

[0043]Both 3 R and 4R tau isoforms are expressed in ALS - Abnormal tau protein deposition has previously been shown with neurons and astrocytes, as well as extraneuronal deposits in ALSci within the frontal cortex (15). The present analysis was focused on frontal tissue and it was first confirmed that the isolation technique used provided consistent results with regards to the purification of tau proteins. Using the Hangar et al. technique, it was observed that tau protein isolated from Alzheimer's brain demonstrated 3 prominent isoforms in the insoluble fraction, with dephosphorylation inducing the expected increase in migration rate (Figure 1). When this technique was applied to the ALS tissue, all six alternatively spliced tau isoforms containing either 3 (3R) or 4 (4R) microtubule binding domains were expressed in both the soluble and insoluble fractions. Expression of both 3R and 4R isoforms was confirmed using a monoclonal antibody specific to these tau variants (31) (data not shown). Thus, while insoluble tau isoforms existed in ALS tissue, they were not homologous to that isolated in Alzheimer's disease.

[0044]Solubility characteristics of tau protein isolated from ALS frontal cortex - Tau solubility characteristics were examined across both frontal grey and white matter (Figure 2). Within the grey matter tau isolates, both control cases demonstrated prominent tau protein expression in the soluble grey fractions, with only faint tau irnmunostaining in the insoluble fractions. The subcortical white matter, however, showed tau protein only in the soluble fractions, and not within the insoluble fractions. In contrast, ALS cases demonstrated prominent irnmunostaining in both soluble and insoluble fractions, although the intensity of irnmunostaining varied amongst samples. This was observed in all of the ALSci cases and in 3 of the 4 ALS and ALSci samples. The most striking difference between ALS, ALSci and control cases was the observation of prominent white matter insoluble tau immunostaining.

[0045]Both soluble and insoluble tau isoforms are phosphatase resistant in ALS - To determine whether the extent of tau phosphorylation differed between ALS, ALSci, control and AD tissues, dephosphorylation studies were conducted. Regardless of the technique utilized to assess the extent of phosphorylation, soluble tau isolated from ALS and ALSci showed a significant resistance to dephosphorylation. While control tau isolates lost all immunoreactivity to the AT8 monoclonal antibody, recognizing phosphorylation at S202 and T205, ALS samples were resistant and could not be fully dephosphorylated. This suggests an excessive amount of phosphorylation, or alternatively, a post-translational modification to the tau protein that renders it relatively phosphatase resistant.

[0046]Soluble ALS-deήved tau protein is hyperphosphorylated with unique Tl 75, S208 and S210 phosphoepitopes - Because a difference in the extent of dephosphorylation between soluble tau from ALS, ALSci, control and AD was observed, the phosphoepitopes of soluble tau protein in ALS were characterized. Tau protein from frontal cortex of ALS, ALSci, AD and normal control brains was digested with trypsin and the resulting digests subjected to immobilized metal affinity chromatography (IMAC) for phosphopeptide enrichment and LC-MS/MS analysis for the global identification of phosphopeptides and their phosphorylated residues (constituting a phosphoproteomics analysis). AD samples were analyzed utilizing an extended HPLC gradient of 2-50% acetonitrile (0.1% HCOOH) over 4 hr which is designed to maximize phosphopeptide detection (32). Phosphopeptides thus identified are listed in Table 1 and are seen to exhibit excellent agreement between the observed and calculated molecular weights of the identified phosphopeptides. Also listed are methylated phosphopeptides that were identified from a set of sample digests that were methylated prior to IMAC in order to improve the efficiency of the phosphopeptide enrichment step (33). In addition, Table 1 lists non-tryptic AD phosphopeptides that were identified from soluble AD tau protein digested with two essentially non-specific enzymes (subtilisin and pepsin) in order to increase phosphorylation coverage (34).

Table 1: LC-MS/MS identification of phosphopep tides from tau fractions isolated from frontal cortex of ALS, ALSci and AD brains.

Tryptic Tau Phosphopeptides

Methylated Tryptic Tau Phosphopeptides'

O

Pepsin Cleaved Tau Phosphopeptides

Tau protein preparations isolated from cerebral cortex of Normal control, ALS, ALSci and AD brains were analyzed as described in Methods. Columns 5-8 indicate the sample from which the phosphopeptide was identified. All phosphopeptides were identified by the SEQUEST program as described in methods and the references contained therein. ^a Within groups, phosphopeptides are listed in order of their sequence in tau. ^b Monoisotopic mass. * Indicates phosphorylated residue. ^c Whenever there is uncertainty in the assignment of a phosphorylated residue, the sequence that encompasses the potentially phosphorylated residues is indicated by brackets (e.g. [TPPSS]* indicates that one of the one of the serines or the threonine is phosphorylated). ^d Phosphorylated residues or pairs of phosphorylated residues are listed in order of decreasing probability as listed in the SEQUEST output report. (First residue or pair is most probable.) ^e Partially methylated phosphopeptide. ^f From both IMAC materials (see Methods section).

[0047]Examination of the table reveals a total of 31 unique tau phosphopeptides, of which 18 are tryptic phosphopeptides and 13 are products of the non-specific proteolytic enzymes, pepsin and subtilisin. Also listed are 13 unique methylated tryptic tau phosphopeptides, of which 12 are seen to be methylated analogues of the previously listed tryptic tau phosphopeptides. When the four matched soluble tau samples (200 μg protein each) from control, ALS, ALSci and AD brains were compared, the ALS and ALSci samples displayed an increase in the number of phosphopeptides identified, 11 and 13 respectively, as compared to 9 each for the control and AD samples. The same trend was observed for the insoluble tau samples. Although fewer phosphopeptides were identified from those samples, 5 unique phosphopeptides were identified from each of the ALS and ALSci samples as compared to 1 from the normal control.

[0048]Finally, a similar trend was observed among the methylated phosphopeptides listed, where the three matched soluble tau samples (100 Dg protein each) from normal, ALS and ALSci were compared. The ALS and ALSci samples each displayed a modest increase of 12 phosphopeptides identified as compared to 7 for the control sample. In addition, a unique methylated phosphopeptide, (R)SGYSSPGSPGTPGS*RS*R (SEQ ID NO: 2), was identified only in the ALS sample.

[0049]Upon focusing at specific phosphorylation sites, soluble tau isolates from ALS, ALSci, AD and control brains exhibit unequivocal phosphorylation at Tl 81, Tl 99, S202, S205 (not observed unequivocally in AD isolate), T217, T231, S396 and S404. However, the Tl 75 site is observed to be unequivocally phosphorylated in both ALS and ALSci as compared to normal control and AD. In addition, based on the identification of one methylated phosphopeptide, (R)SGYSSPGSPGTPGS*RS*R, the S208 and S210 sites are observed to be unequivocally phosphorylated in the ALS as compared to the control and ALSci. Finally, unequivocal phosphorylation was observed in the soluble tau isolate at T52, S237 and S238. However, these sites are not unique to ALS since T52 is based on a phosphopeptide resulting from pepsin cleavage which was not done on the other tau isolates and the S237 and S238 were observed as equivocal sites in tryptic phosphopeptides from the other tau isolates. [0050]With respect to the dual AT8 (S202 & T205) and PHFl (S396 & S404) sites, the results shown above indicate that all samples had phosphorylation at these sites. Upon searching for a diphosphopeptide or triphosphopeptide that exhibits simultaneous phosphorylation at both phosphorylation sites of the PHFl motif, as seen in Table 1, several non-methylated and methylated tryptic diphosphopeptides having the AKTDHGAEIVYKSP VVSGDTSPR (386-406) sequence were identified from ALS tau isolates (and also from normal control). Although all of these peptides exhibit unequivocal S396 phosphorylation and equivocal S404 phosphorylation, some are ranked by SEQUEST as having S404 as their most likely second phosphorylated residue. However, when searching for a diphosphopeptide or triphosphopeptide that exhibits simultaneous phosphorylation at both phosphorylation sites in the AT8 motif, as seen in Table 1 , one methylated tryptic diphosphopeptide having the SGYSSPGSPGTPGSR (195-209) sequence was identified only from the ALS tau isolate. This peptide exhibits unequivocal S205 phosphorylation and possible S 198, S202 or S 199 phosphorylation.

[005I]To confirm the expression of the unique ALS phosphoepitopes highlighted in the phosphopeptide analysis, phosphospecific polyclonal antibodies were generated as described below in Example 2. These antibodies were designed to specifically recognize tau protein phosphorylated solely at Tl 75 or S217 or tau protein simultaneously phosphorylated at S208 and S210. These antibodies were used to probe soluble and insoluble tau isolates from the white matter of patients suffering from AD or ALS. This analysis demonstrated that S208/S210 was predominantly found in AD, while S217 appeared to be equally present in both AD and ALS. This analysis also demonstrated that phosphorylation of T 175 was predominantly found in ALS.

[0052]To further analyze the distribution of Tau protein phosphorylated at T175, S217, or S208/S210, antibodies specific to these phosphoepitopes were used for immunohistochemical analysis of samples from AD or ALS white matter. Similar to the immunoblot analysis the Tl 75 phosphepitope was found to be unique to the ALS sample. DISCUSSION

[0053]In this study, the nature of altered tau phosphorylation in ALS was examined in samples from both cognitively intact and cognitively impaired patients. All tau isoforms were expressed, including 3 R and 4R tau isoforms, in both soluble and insoluble tau isolates from ALS and ALSci. There was an apparent increased amount of insoluble tau within the ALS and ALSci samples as compared to control. This is most evident in the subcortical white matter samples, where no insoluble tau was observed in control samples. A relative resistance to dephosphorylation for tau protein isolated from both ALS and ALSci was also observed most evident in the soluble tau isolate.

[0054]Although three unique phosphorylation sites, T175, S208 and S210, were putatively identified in tau isolates from ALS brain, the most striking observation from phosphoproteomic analysis is the increased number of unique phosphopeptides identified in ALS tau isolates as compared to the AD and normal control. Since almost all the phosphorylation sites in the increased number of phosphopeptides identified in ALS tau are the same sites observed to be phosphorylated in normal control and AD tau, it was concluded that to a large degree the nature of the observed ALS hyperphosphorylation is indicative of greater stoichiometric phosphorylation at those same sites. This higher degree of phosphorylation leads to increased isolation and detection of phosphopeptides from ALS tau.

[0055] A number of phosphoepitopes in soluble tau were found to be common to controls, ALS, ALSci and AD, including Tl 81, T202, T231, S396 and S400 (Figure 4). Control and ALS tissue, but not soluble AD tau, were also observed to be phosphorylated at S 199 and T217 (Table 1). This is of considerable interest in that PHF tau derived from AD (within the Sarkosyl insoluble fraction) has been reported to be phosphorylated at these sites, suggesting that these sites may be critical in altering solubility characteristics (29). Unique to soluble tau derived from ALS and ALSci are phosphopeptides at T175, S208 and S210, with only T175 and S210 being reported in insoluble PHF tau (29).

Example 2 - Preparation of a pT175 antibody

[0056]Using standard methodology (as per 21^st Century Biochemicals protocol: Standard Antibodies, a robust protocol, 72 days in length using 2 rabbits, 5 innoculations, 5 production bleeds/animal and provide ~160-200ml of serum.), antibodies to the following peptides (0306, 0307 and 0308) were made and compared to appropriate controls as indicated.

0306

Bleed 5

Ac-G [pS] R [pS] RTPSLP-Ahx-C-amide tau-MJS- 1

C9346 ~ 1 : 22 , 000

C9347 ~ 1 : 50 , 000

Control for 0306

Ac-GSRSRTPSLP-Ahx-C-amide C9346 -1:4,000 C9347- 1:20,000

0307

Bleed 5

Ac-SLP [pT] PPTREPC-amide tau-MJS-2

C9348 -1:65,000

C9349 ~ 1:200,000

Control for 0307

Ac-SLPTPPTREPC-amide tau-MJS-2 NP C9348- 1:4,000 C9349 - 1:25,000

0308

Bleed 5

Ac-RIPAK [pT] PPAPKC-amide tau-MJS-3

C9350 - 1:40,000

C9351 - 1:40,000 Control for 0308

Ac-RIPAKTPPAPKC-amide tau-MJS-3B NP C9350- 1:20,000 C9351- 1:10,000

[0057]The antibodies were used to identify phosphorylation at specific sites in tau. Antibody 0308 was successfully used to identify the unique pT175 in ALS samples.

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Claims

CLAIMS What is claimed is:

1. A method of diagnosing ALS in a mammal, comprising the step of analyzing a tau- containing sample obtained from the mammal to determine whether the sample includes pT175 tau, wherein phosphorylation at position 175 of tau is indicative of ALS.

2. A method as defined in claim 1, wherein an antibody is used to determine pT175 in the sample.

3. A method as defined in claim 2, wherein the antibody is based on an immunogenic fragment comprising the sequence, RIP AK[pT] PPAPK, or a functionally equivalent variant thereof, wherein [pT] represents a phosphorylated threonine.

4. A polypeptide useful to generate an antibody immunospecific to a Tau protein phosphorylated at threonine 175, said polypeptide comprising the amino acid sequence, RIP AK[pT]PP APK, or a functionally equivalent variant thereof, wherein [pT] represents a phosphorylated threonine.

5. A polypeptide as defined in claim 4, having the amino acid sequence RIPAK[pT]PPAPK, wherein [pT] represents a phosphorylated threonine.

6. A phosphospecific Tl 75-Tau antibody.

7. A hybridoma that secretes an antibody as defined in claim 6.

8. A kit for use in the diagnosis of ALS comprising a pT175 antigenic polypeptide comprising the sequence, RIP AK[pT] PPAPK, or a functionally equivalent variant thereof, wherein [pT] represents a phosphorylated threonine.

9. A kit for use in the diagnosis of ALS comprising a phosphospecific Tl 75-Tau antibody.

10. A method of making a phosphospecific T175-Tau antibody comprising the steps of immunizing a non-human mammal with an antigenic polypeptide as defined in claim 4, and collecting the antibodies generated by the mammal.