WO1991008309A1

WO1991008309A1 - Methods for detection of neurodegenerative disease

Info

Publication number: WO1991008309A1
Application number: PCT/US1990/007116
Authority: WO
Inventors: Barbara R. Talamo; John S. Kauer
Original assignee: New England Medical Center Hospitals, Inc.; Tufts University
Priority date: 1989-12-05
Filing date: 1990-12-05
Publication date: 1991-06-13

Abstract

Methods for the detection of neurodegenerative disease in a living mammal. Methods include obtaining a sample including olfactory receptor epithelium from the nasal passage of the mammal and analyzing the sample for the presence of neurodegenerative disease. Methods of analysis include polymerase chain amplification of a selected nucleic acid sequence and subsequent detection of the amplified sequence as well as the detection of antigens via antigen-antibody interaction wherein presence of antigens and/or nucleic acid sequence is indicative of the neurodegenerative disease.

Description

METHODS FOR DETECTION OF NEURODEGENERATIVE DISEASE Background of the Invention This invention relates to methods for detection of neurodegenerative diseases.

Neurodegenerative diseases include diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's chorea. They also include various degenerative spongiform encephalopathies and diffuse neurodegenerative diseases. Some of these diseases are detected by physical examination of a patient afflicted with the disease. Such physical examination may include observation of generalized dementia, tremors, and difficulty in moving facial or limb muscles. Some of the diseases are diagnosed only by autopsy after death.

Summary of the Invention This invention concerns a method for non- invasively detecting the presence of neurodegenerative disease in a living mammal, for example, a human patient. Generally, a sample is obtained from the olfactory receptor epithelium of the nasal passage near to the cribriform plate, and analyzed by any of a number of procedures to detect the presence or absence of certain molecules associated with the neurodegenerative disease.

In a first aspect, the invention features a method for detecting neurodegenerative disease in a living mammal. The method includes obtaining a sample including olfactory receptor epithelium from the nasal passage of the mammal and analyzing the sample for the presence of a first substance characteristic of neurodegenerative disease.

Examples of neurodegenerative diseases which may be detected by a method of this invention include those listed above. The mammal being examined will generally

SUBSTITUTESHEET be a human patient, but may be any other mammal affected by neurodegenerative disease.

In preferred embodiments, the analyzing step includes contacting the sample with a second substance which has specificity for the first substance, and detecting reaction of the second substance with the sample as an indication of the presence of the disease; the second substance agent is an antibody; the sample is obtained from olfactory receptor epithelium; the sample includes nasal mucus; the step of obtaining a sample includes removing olfactory receptor epithelium from the mammal, or sampling nasal mucus of the mammal.

By "specificity" is meant that the second substance specifically recognizes a molecule, or closely related set of molecules, that is present only in diseased mammals, only in normal mammals, or at a greater or lesser level in a diseased mammal than in a normal mammal.

In a most preferred embodiment, the mammal is a human, the disease detected is Alzheimer's disease

(referred to as AD) , and the step of detecting includes contacting an antibody with the sample.

In other preferred embodiments, the first substance includes target nucleic acid, the second substance is a nucleic acid probe specific for the target nucleic acid, or an enzyme capable of specifically amplifying the target nucleic acid, and the detecting step includes detecting hybridization of the probe with the target nucleic acid, or detecting amplified target nucleic acid. Most preferably the second substance is a DNA polymerase, and the target nucleic acid is responsible for expression of an RNA characteristic of the disease.

In yet other preferred embodiments, the second substance is reactive with an enzyme, which includes the

SUBSTITUTE SHEET first substance, and is useful for specifically detecting the level of the enzyme as a characteristic of the disease. The detecting step includes detecting reaction of the second substance with the enzyme as an indication 5 of the disease. In most preferred embodiments, the enzyme is a protein kinase or a phosphatase, and the second substance is a substrate for the enzyme.

Applicants have provided a simple and non-invasive method for detection of neurodegenerative disease. Prior

10 to this invention, neurodegenerative diseases were either not diagnosed until death of the affected mammal, or were diagnosed only late after onset of the disease. The method of this invention makes early detection of disease possible, and increases the chance of potential treatment

15 of the disease to either prevent further degeneration, or reduce the progress of the disease.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

20 Description of the Preferred Embodiments

There follows an example of the use of the method of this invention for detection of one neurodegenerative disease, Alzheimer's disease ("AD"). This example is not limiting in this invention and is meant as an example

25 only. Those skilled in the art will recognize that other neurodegenerative diseases, as discussed above, can be detected by similar methods using agents which are specific for the disease desired to be detected.

Generally, a sample of tissue from the olfactory

30 receptor epithelium of a mammal, e.g., a human, is obtained. This is the area of the nose which detects i odors, and is in the most dorsal part of the nasal cavity against the cribriform plate extending down laterally and i medially about a centimeter. It extends the full

35 anterior and posterior distance of the cribriform plate.

SUBSTITUTESHEET This epithelium contains nerve cells, nerve processes, proteins and other molecules associated with those processes. It is these molecules that are detected in this invention as an indication of the presence or absence of neurodegenerative disease. To ensure that an appropriate tissue sample is obtained, the sample is generally analyzed to determine whether it contains an olfactory marker protein (OMP) , and an neuronal specific marker of neurons (neuron-specific enolase; NSE) . The sample of neuronal tissue may be obtained by any procedure, for example, that described by Lovell et al.. Arch. Otolarynol., Vol. 108, p. 247, 1982, where a curette having a 1 mm hook at one end is inserted into the nasal cavity (after local anesthesia with xylocaine and cocaine) until the probe reaches the cribriform plate. The hook is rotated against the medial wall and then pulled out to remove a cubic millimeter of tissue. This procedure is repeated three times. The tissue obtained maybe analyzed directly or fixed in a fixative. Sections are then taken and assayed by immunohistochemical techniques, or by any desired biochemical assay.

Molecules which can be usefully detected in the sample include antigenic macromolecules, such as proteins. Preferably the antigenic proteins are associated with growth of abnormal neurons. Such proteins are present in larger amount in AD patients, than in normal patients. Antibodies can be used to detect these proteins. These antibodies may be characteristic of a particular neuronal antigen, or may be an antibody to a growth factor or other molecule which allows neurons to survive in the nasal passage, e.g., a nerve growth factor, fibroblast growth factor, glial or nerve-derived growth factor, or other proteins in the extracellular matrix which surrounds the cells which may

SUBSTITUTE SHEET be important in allowing those cells to grow and survive. If the abnormality results in the number or the presence of these molecules being affected, such abnormality may be detected as an indication of the disease.

We have determined that neurites, which are not observed in normal patients, proliferate in the vicinity of the olfactory epithelium of AD patients, and are characteristic of AD. Antibodies which detect the presence of these neurites are useful for determination of AD. For example, antibodies to phoβphorylβtβd neurofilaments, both to heavy chain and medium chain, recognize the neurofilament protein whether or not it is dephosphorylated or heavily phosphorylated. Antibodies that are particularly useful for detecting AD are those that specifically detect the phosphorylated form of the heavy neurofilament, e.g., those described by Lee, 85 Proc. Natl. Acad. Sci. USA, 7384, 1988. Antibodies to the tau protein, which is a major antigenic component of the neurofibrillary tangle are also useful. This tangle is not present in normal olfactory neurons, but it is present in AD patient neurites. A further useful antibody is that to Alz 50, described by Peter Davies, which detects an antigen termed A68. Antibodies to amyloid protein are also useful in this invention. These proteins are present in plaques found only in AD patients and not in normal patients.

There follows examples of sources of useful antibodies, and other potentially useful antibodies, and methods for preparing antibodies for assay of Alzheimer's disease.

Antibodies that react with a phosphorylated epitope and with the major multiphosphorylation site in neurofilaments (NF) were identified by Lee et al., 85 Proc. Natl. Acad. Sci. USA 1998, 1998. Similar

SUBSTITUTE SHEET antibodies can be isolated by techniques described in this reference, or by using cloned NF genes to express antigenic proteins, to which antibodies can be made. For example, Myers, 6 EMBO J. 1617, 1987 and Robinson et al. 209 FEBS Lett. 203, 1986 describe clones of two NF genes. These clones include sequences known to be useful for making peptides. The multiphosphorylation site is particularly useful for detection of AD.

Examples of antibodies, and the methods of their generation, from preparations of neurofilament H or M chains obtained from cow, human and rat are provided in Lee et al. (1982) Proc. Natl. Acad. Sci., USA 79:6089- 6092; Carden et al. , (1985) J. Biol. Chem. 260:9805- 9817; Lee et al., (1986) J. Neurosci 6:850-858; Lee et al., (1987) J. Neurosci 7:3474-3488; and Lee et al. (1986) J. Neurosci 6:2179-2186.

Other antibodies useful for identifying abnormal neurities in Alzheimer's disease patient nasal epithelium include RM024 (NF-H, P+++; P-form of human NF-H) ; TA51 (NF-H, P++; P-form of human NF-H) ; H014 (NF-M, P+; P- for of human NF-M) described by Lee et al. 7 J. Neurosci. 3474, 1987, and Schmidt et al. 56 Lab Investig. 282, 1987; RM0254 (NF-M, Pind; Pind form of human NF-M); and RMDT2 or RMD09, (NF-H, P- dephosphorylated form of NF-H) . Other useful NF antibodies that distinguish AD neurofibrillary tangles from progressive supranuclear palsy are described by Schmidt et al. 59 Lab Invest. 460, 1988.

Commercially available antibodies that might be useful for identifying neurofilament (phosphorylated) in AD nasal epithelium include SM131 (Sternberger-Meyer) ; SM133 (Sternberger-Meyer) ; NF antibody monoclonal ( absystems) , RT97 (Anderton) , and UCD7H11.

Other useful antibodies are the tau antibodies for identifying AD neurofibrillary tangles and neurites in

SUBSTITUTE SHEET - 7 - nasal epithemium, e.g., Tau46, and Taul4 (referred to by Lee et al. 85 Proc. Natl. Acad. Sci. USA ₇₃₈₄, an_d Trojanowski et al., 37 J. Histochem. Cytochem. ₂₀₉, ₁₉₈₉, as well as tau antibodies used by Grundke-Iqbal et al. ₈₃ Proc. Natl. Acad. Sci. USA 4913, 1986; Kosik et al. ₈₃ Proc. Natl. Acad. Sci. 4044, 1986; Wood et al. ₈₃ Proc. Natl. Acad. Sci. USA 4040, 1986; and Nukina and Ihara, 99 J. Biochem. 1541, 1986, and antibodies to MA_P2, e.g., those described by Kosik et al. 8₁ Proc. Natl. Acad. Sci. USA 7941, 1984.

Other sources of potentially useful antibodies include those raised against neuro ibrillary tanglaβ and plaques, or other identified components of these abnormal structures in AD brain. Useful antibodies can also be generated using synthetic peptide sequences derived from cloned neurofilament subunits, particularly the peptide region involved in phosphorylation which cross reacts with phosphorylation sites on other proteins, tau proteins, or any identified component of the plaques and tangles. There are several forms of tau, yet to be identified fully. Some of these may be particularly important, since some modified form of tau is the major antigenic component of neurofibrillary tangles in AD.

Other antigens (and antibodies to these antigens) that may be useful to selectively identify abnormal neurites in AD nasal epithelium include components of plaques found in AD brain, e.g., α-1 Antichymotrypsin (a component of plaques in AD brain) , β-Amyloid protein, amyloid protein precursors and alternate transcripts, and peptides derived from them, Ubiquitin, GAP43 (B50/F1) growth-associated protein, protease nexin 1 or 2, other cytoskeletal components in addition to neurofilaments and tau, nerve growth factor and its receptor(s), specific neurotransmitters of abnormal neurites such as calcitonin related peptide, or substance P, peptide CGRP, GAP43,

SUBSTITUTE SHEET olfactory marker protein, or other antigens specific to subsets of olfactory neurons, growth factors or extracellular components contributing to abnormal neurite outgrowth, e.g., glial derived growth factors and receptors, fibroblast derived growth factors and receptors, extracellular matrix la in, integrin and families and receptors, and cytokinins such as Interleukin 1 and receptors. Carbohydrates and lipids are also potentially useful markers of AD, and can be directly detected by non-immunological means. Antibodies which identify neurons susceptible to AD also may be useful, e.g., those described by Miller et al. 84 Proc. Natl. Acad. Sci..USA, 8657, 1987.

Other methods, beside immunological detection, for diagnosing abnormalities in neurites and olfactory epithelium include: detection of mRNA for abnormally expressed proteins (e.g., using a polymerase chain reaction for sensitive detection) ; detection of abnormally active protein kinases or phosphatases that are responsible for heavy phosphorylation of cytoskeletal proteins in these neurites (and in some AD brain neurofibrillary tangles), e.g., by use of artificial substrates to assay these enzymes.

It is possible to use nasal mucus, which contains remnants of olfactory epithelium, rather than olfactory epithelium itself, to detect abnormalities. If this is used, the mucus sample is preferably taken after triggering mucus secretion with a drug, such as isoproterenol, which triggers secretery glands to produce mucus. An alternative is simply to feed the patient a mucus stimulating food, such as chili.

Those skilled in the art will recognize that any of these agents may be readily tested by standard procedures to determine whether they are useful in the invention. As shown in the example below, antibodies are

su_BSi^ sHS^r readily tested to determine whether they detect differences characteristic of a neurodegenerative disease, e.g., AD. All the agents listed here, and equivalent agents, are readily tested as in the example, or by analogous techniques.

Biochemical techniques that may be used for detection of molecules indicative of AD include determination of the presence of proteins by Western blot analysis, or by direct visualization with any standard protein staining technique in a polyacrylamide gel or its equivalent. Similarly, nucleic acid indicative of a disease state may be detected by Southern or northern blotting, and may include the use of a polymerase chain reaction to amplify the genomic DNA present within the sample to determine whether a DNA region specific to the neurodegenerative disease is present, or by in situ hybridization using a labeled probe. Example: Detection of AD

Olfactory epithelium of human patients was removed at autopsy by undercutting the cribriform plate and removing it as a unit. Olfactory epithelium and bulbs were placed in 2% paraformaldehyde/10% saturated picric acid in 0.1 M sodium phosphate buffer, pH 7.4 (PB) , for 2-4h (fixation for up to 2 days did not seem to affect the result) . After dissecting away cartilage and bony material, the tissue was rinsed in cold PB, placed in 30% sucrose/PB at 4°C overnight and frozen. Storage as a block or as 8-10 μm frozen sections at -70°C for up to 18 months produced no changes in immunoreactivity. Sections were treated with 3% blocking serum (same species as secondary antibody)/0.1% Triton X-100 in PB for 20-30 min. before incubating with primary antibody, usually for 2 h at 20-25°C or overnight at 4°C. Using standard methods, the appropriate biotinylated secondary antibody was applied in blocking serum followed by

SUBSTITUTESHEET treatment with streptavidin-Texas Red (1:350 in 2% BSA; BRL) . Solutions were buffered in PB except for monoclonal antibodies to NF and ALZ50, which were buffered in Tris. Slides were blocked with 5% non-fatty dry milk for ALZ50. Antibody reactivity was monitored by including known positive control olfactory epithelium or bulb in each run. AD cortex fixed in formalin was the control for ALZ50 staining. In other controls primary antiserum was omitted. Mouse monoclonal antibodies (dilution) used included: RM024 (none); RM0254 (1:10); RMd020 (1:10); glial fibrillary acidic protein (1:10) and rat monoclonal H014 (1:10); tau (5E2) (1:10); ALZ50 (1:5) (P. Davies) ; anti-NF (1:200) (Labysstems) . Polyclonal rabbit antibodies included: NFL (1:500); human NSE

(1:2,500) Polysciences) ; intermediate filament protein 57K (1:300); and goat antibody to OMP (1:200 to 1:1,000). Biotinylated secondary antibodies included: rabbit anti-goat IgG, horse anti-mouse IgG and goat anti- rabbit IgG (Vector) ; goat anti-mouse IgM and anti-rat IgG (Tago) .

Autopsied nasal epithelium from patients with AD contained both sensory and non-sensory areas but, in contrast to control tissue, had increased reactivity to neurofilament antibodies and abnormal neuronal structures. In particular, olfactory nerve axon bundles (identified by location, and staining for OMP) reacted with antibodies to NSE and NFH(P-) and were strongly immunoreactive for phosphorylated forms of NFH, phosphate-independent forms of NFM, and for NFL.

Extensive neurite accummulations were also observed in some sensory regions above and just below the epithelial basal lamina and projecting into the epithelium. The masses of immunoreactive fibres often displaced epithelial cells. These unusual neurites reacted

SUBSTITUTESHEET positively with NSE antibody, with all the NF antibodies that stained axons, with antibodies to NFH(P-), NFM(P+) and, most strongly, with antibodies to NFH(P+++) , to another NF antibody (Labsyste s) that recognized a phosphatase-sensitive epitope, and also to NFM(Pind) . No neurofibrillary tangles were observed in AD olfactory epithelium.

Neuritic masses in AD olfactory epithelium were stained in some cases with antibody ALZ50, which is reported to be completely specific to AD tissue, and with monoclonal antibody to tau protein (5E2, tau 46) . Both these antibodies stain neurofibrillary tangles and neuritic plaques in AD brains. ALZ50, which cross-reacts with tau, had a similar staining pattern to 5E2 near the basal lamina. Deep axon bundles of olfactory neurons did not stain with these antibodies.

It seems that both NF and tau expression are altered in AD olfactory epithelium as the NF antibodies used in our study do not cross-react with tau, nor do they stain tangles (except for RMd020) . NF subunit expression and phosphorylation were increased in both axons and the neuritic network at the basal lamina, whereas tau was present mainly in the neurites and in some unidentified globular structures in the epithelium. These unusual neurite masses in the epithelium may be analogous to the neurites around plaques or dystrophic cortical neuropil structures in AD brain, all of which stain for tau.

This example demonstrates that the nasal epithelium tissue used shows unique immunoreactivity changes characteristic of AD. A simple non-invasive procedure can be used to identify such changes and thus diagnose AD. Other embodiments are within the following claims.

SUBSTITUTE SHEET

Claims

Claims 1. A method for detecting neurodegenerative disease in a living mammal, comprising the steps of: obtaining a sample comprising olfactory receptor epithelium from the nasal passage of the mammal; and analyzing said sample for the presence of a first substance characteristic of a neurodegenerative disease.

2. The method of claim 1, wherein said analyzing step comprises contacting said sample with a second substance which has specificity for said first substance; and detecting reaction of said second substance with said sample as an indication of the presence of the disease.

3. The method of claim 2 wherein said second substance is an antibody.

4. The method of claim 1, wherein said sample is obtained from an olfactory receptor epithelium.

5. The method of claim 1, wherein said sample comprises nasal mucus.

6. The method of claim l, wherein said sample obtaining step comprises removing olfactory receptor epithelium from the mammal.

7. The method of claim 4, wherein said sample obtaining step comprises sampling nasal mucus of the mammal.

8. The method of claim 2, wherein the mammal is a human, the disease is Alzheimer's disease, and said

SUBSTITUTE SHEET 3 detecting comprises contacting said antibody with said

4 sample.

1 9. The method of claim 2, wherein said first

2 substance comprises a target nucleic acid, said second

3 substance is a nucleic acid probe specific for said

4 target nucleic acid, or an enzyme capable of specifically

5 amplifying said target nucleic acid, and said detecting

6 step comprising detecting hybridization of said probe

7 with said target nucleic acid, or detecting amplified

8 target nucleic acid.

1 10. The method of claim 9, wherein said second

2 substance is a DNA polymerase.

1 11. The method of claim 10, wherein said target

2 nucleic acid is DNA responsible for expression of an RNA

3 characteristic of the disease.

1 12. The method of claim 1, wherein said second

2 substance is reactive with an enzyme comprising said

3 first substance and which is characteristic of the

4 disease;

5 and said detecting step comprises detecting

6 reaction of said second substance with said enzyme.

1 13. The method of claim 12, wherein said enzyme

2 is a protein kinase or a phosphatase, and said second

3 substance is a substrate for said enzyme.

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SUBSTT C- E-^HEET