US 6,130,048 AGrant
Method of Diagnosing Alzheimer's Disease
Issue Date:2000-10-10
•5 Claims
•4 Drawing Sheets
Abstract
Disclosed is a method of diagnosing Alzheimer's disease in a patient by measuring the level of a lysosomal hydrolase or lysosomal protease inhibitor in the patient's cerebrospinal fluid. The invention also features methods of measuring the progression of the disease and screening therapeutic compositions for treating Alzheimer's disease.
Metadata
Assignee
- McLean Hospital Corporation
Inventor
- Ralph A. Nixon
Application Information
Application Number:US 9667563
Filing Date:1997-11-10
Priority Date:1994-07-28
Art Unit:165
Classifications
IPC:
G01N 3353
Field of Search:
4354364;7.1;7.92;7.93;7.94;7.95518;527;529;530;531;63;811
Patent Drawings (4 sheets)
Description
Background of the Invention
This invention relates to diagnosing Alzheimer's disease.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder which is characterized by memory loss, impairment in abstract thinking, impaired judgment, personality change, and insidious onset of the disease in the absence of other specific causes of dementia. Eventually, the AD patient becomes completely incapacitated, and death results from debilitation or infection.
Under the guidelines established by the National Institute of Neurological and Communicative Disorders and Stroke, and the Alzheimer's Disease and Related Disorders Association (NINCDS/ADRDA), a clinical diagnosis of probable AD involves the administration of a series of neuropsychologic tests (see Table 1). Many of the signs of AD occur as part of the normal process of aging or may result from other forms of dementia, and thus the present NINCDS/ADRDA standards require the absence of an alternative explanation for the symptoms.
Table 1: NINCDS/ADRDA Criteria for Clinical Diagnosis of Alzheimer's Disease
Criteria for the clinical diagnosis of probable Alzheimer's disease include
Dementia established by clinical examination and documented by the Mini-Mental State Examination, Blessed Dementia Scale, or some similar examination and confirmed by neuropsychologic tests
Deficits in two or more area of cognition
Progressive worsening of memory and other cognitive functions
No disturbance of consciousness
Onset between ages 40 and 90, most often after age 65
Absence of systemic disorders or other brain diseases that could account for the progressive deficits in memory and cognition
The diagnosis of probable Alzheimer's disease is supported by
Progressive deterioration of specific cognitive functions such as language (aphasia), motor skills (apraxia), and perception (agnosia)
Impaired activities of daily living and altered patterns of behavior
Family history of similar disorders, particularly if confirmed neuropathologically
Laboratory results as follows: normal puncture as evaluated by standard techniques; normal pattern or nonspecific changes in EEG, such as increased slow-wave activity; and evidence of cerebral atrophy of CT with progression documented by serial observation
Other clinical features consistent with the diagnosis of probable Alzheimer's disease, after exclusion of causes of dementia other than Alzheimer's disease, include
Plateaus in the course of progression of the illness
Associated system of depression, insomnia, incontinence, delusions, illusions, hallucinations, sexual disorders, weight loss, and catastrophic verbal, emotional, or physical outbursts
Other neurologic abnormalities in some patients, especially with more advance disease and including motor signs such as increased muscle tone, myocionus, or gait disorder
Seizures in advanced disease
CT normal for age
Features that make the diagnosis of probable Alzheimer's disease uncertain or unlikely include
Sudden, apoplectic onset
Focal neurologic finds such as hemiparesis, sensory loss, visual field deficits, and incoordination early in the course of the illness
Seizures or gait disturbances at the onset or very early in the course of the illness
Clinical diagnosis of possible Alzheimer's disease
May be made on the basis of the dementia syndrome, in the absence of other neurologic, psychiatric, or systemic disorders sufficient to cause dementia and in the presence of variations in the onset, presentation, or clinical course
May be made in the presence of a second systemic or brain disorder sufficient to produce dementia, which is not considered to be the cause of the dementia
Should be used in research studies when a single, gradually progressive, severe cognitive deficit is identified in the absence of another identifiable cause
Criteria for diagnosis of definite Alzheimer's disease are
The clinical criteria for probable Alzheimer's disease
Histopathologic evidence obtained from a biopsy or autopsy
Classification of Alzheimer's disease for research purposes should specify features that may differentiate subtype of the disorder, such as
Familial occurrence
Onset before age 65
Presence of trisomy 21
Coexistence of other relevant conditions, such as Parkinson's disease
Note: NINCDS=National Institute of Neurological and Communicative Disorders and Stroke; ADRDA=Alzheimer's Disease and Related Disorders Association
Under the current standards established by the Consortium to Establish a Registry for Alzheimer's Disease (CERAD), a post-mortem diagnosis of definite AD involves a series of histopathologic test on biological samples obtained from the patient. Tables 2 and 3 indicate that the CERAD neuropathologic diagnosis is derived from an evaluation of senile plague frequency and a clinical history of dementia.
Summary of the Invention
I have, for the first time, detected the lysosomal hydrolase cathepsin D (CD) in human cerebrospinal fluid (CSF), and I have discovered that the CSF of AD patients contains elevated levels of CD relative to non-AD patients.
Accordingly, the invention features a method of diagnosing AD in a patient, involving determining the level of a lysosomal hydrolase in the patient's CSF and comparing the level of the lysosomal hydrolase to normal levels. Thus, lysosomal hydrolases provide biochemical markers for the diagnosis of AD; an increase in the level of a lysosomal hydrolase, relative to the normal level, is indicative of AD. The invention provides a rapid and convenient method of distinguishing AD from other dementias.
In a related aspect, the invention features a method of diagnosing AD in a patient by measuring the level of a lysosomal protease inhibitor in the CSF of a patient. An increase in the level of the inhibitor, relative to the normal level of the inhibitor, is indicative of AD.
In another aspect, the invention features a method of following the rate of AD progression, involving assaying the CSF for changes in the level of a lysosomal hydrolase over the course of time. An increase in the level of the lysosomal hydrolase signifies that the state of disease has advanced; a decrease in the lysosomal hydrolase level indicates amerlioration of the disease.
In a related aspect, the rate of AD progression is monitored by assaying the CSF of the patient for changes in the levels of an inhibitor of a lysosomal protease inhibitor. An increase in the level of the lysosomal protease inhibitor indicates that the disease state has advanced, while a decrease in the inhibitor level signifies amelioration of the disease.
The invention further features a method of determining whether a candidate therapeutic composition is useful for treating AD. The level a lysosomal hydrolase in the CSF of a patient diagnosed with AD is measured prior to, and following, administration of the candidate therapeutic composition. A decrease in the level of lysosomal hydrolase indicates that the composition is useful for treating AD.
In a related aspect, the invention features a method of determining whether a candidate therapeutic composition is useful for treating AD, involving measuring the level of a lysosomal protease inhibitor in the CSF of the patient prior to, and following, administration of the candidate therapeutic composition.
Preferred lysosomal hydrolases include cathepsin D, cathepsin G, cathepsin L, cathepsin H, hexosaminidase A, and hexosaminidase B. Preferred lysosomal protease inhibitors include cystatin C and stefin A.
CSF useful in the invention includes ventricular and lumbar CSF. Thus, the invention can be used for clinical and postmortem diagnoses of AD.
In preferred embodiments, the level of the lysosomal hydrolase or lysosomal protease inhibitor is measured in an immunoassay (e.g., Western blot, ELISA, inhibition ELISA, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, and fluorescent immunoassays). Antibodies which are immunoreactive with a chosen lysosomal hydrolase or lysosomal protease inhibitor are appropriate for use in such assays. For example, antibodies which are raised by immunization of a mammal with antigenic determinants of the chosen lysosomal hydrolase or lysosomal protease inhibitor are useful in immunoassays. Antibodies for detecting each of the lysosomal hydrolases and lysosomal protease inhibitors useful in the invention are currently available and known in the art (e.g., for anti-CD antibodies, see Nixon and Marotta, 1984, J. Neurochem. 43:507-516). Also useful in the invention are combinations of antibodies to detect a CSF lysosomal hydrolase or lysosomal protease inhibitor. Such combinations of antibodies can be used, for example, to enhance the detection of a lysosomal hydrolase or lysosomal protease inhibitor, or to detect more than one lysosomal hydrolase or lysosomal protease inhibitor in the CSF sample. Thus, a diagnosis of AD can be made on the basis of the detection of more than one lysosomal hydrolase and/or lysosomal protease inhibitor in the CSF of the patient.
In other embodiments, non-immunologic techniques, such as SDS-PAGE and activity assays in polyacrylamide gels can be used to measure the level of the lysosomal hydrolase or lysosomal protease inhibitor. Generally, the level of lysosomal hydrolase or lysosomal protease inhibitor is measured in the CSF of a patient suspected of having AD, and compared with the level found in non-AD patients. An elevated level of the lysosomal hydrolase or lysosomal protease inhibitor is indicative of AD. The absolute levels of the lysosomal hydrolases and lysosomal protease inhibitors are expected to differ from protein to protein, and the extent to which each protein is elevated in the CSF of AD patients (relative to normal patients) also will vary from protein to protein. Generally, a CSF lysosomal hydrolase or lysosomal protease concentration which is at least 1.5 times, or more preferably 2 times, or most preferably 3 times the concentration in normal patients is indicative of AD. For examples, a CD concentration which is two times the normal level of CD indicates AD in the tested patient. Generally, a ventricular CSF CD level of at least 1.7 .mu.g/ml, more preferably 2.0 .mu.g/ml, indicates AD.
The level of lysosomal hydrolase can also be determined by measuring its enzymatic activity. For example, CD cleaves proteins preferentially between two large hydrophobic amino acid residues; thus, the level of CD in a sample can be determined by assaying for protein fragments produced by the proteolytic cleavage of substrates such as hemoglobin (Hb) (Kirschke and Barrett, Lysosomes: Their Role in Protein Breakdown, 193-238, Academic Press, 1987). In one preferred embodiment, the ability of the lysosomal hydrolase to cleave a protein substrate is measured in the presence and absence of an inhibitor of its activity. For example, the level of CD can be measured as pepstatin-inhibitable hydrolysis of hemoglobin at the optimal pH of 3.2. The combination of a specific inhibitor of aspartyl proteinases (pepstatin) and the acid pH optimum pH 3.2 distinguishes the hydrolysis of hemoglobin by CD from hydrolysis by other enzymes. Generally, a CD activity level of at least 1.5 times, or more preferably 2 times, the activity level in non-AD patients is indicative of AD.
Prior to measuring the level of the lysosomal hydrolase or lysosomal protease inhibitor by any method, the protein concentration of the CSF can be increased by any of the well known protein concentration techniques (e.g., ammonium sulfate precipitation; see, e.g., Ausbel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 1989). The chosen lysosomal hydrolase or lysosomal protease inhibitor in the CSF sample can be partially purified by standard protein purification methods (e.g., ion exchange chromatography, affinity chromatography, high performance liquid chromatography, and immunoprecipitation; see, e.g., Ausubel et al., supra).
By "cerebrospinal fluid" is meant the fluid which surrounds the bulk of the nervous system, as described in Physiological Basis of Medical Practice (ed. J. B. West, Williams and Wilkins, Baltimore, Md., 1985). Cerebrospinal fluid, as used herein, includes ventricular and lumbar CSF.
By "lysosomal hydrolase" is meant any of the acid hydrolases which are normally present intracellulary in the endosomes, lysosomes, related endosomal-lysosomal compartments, and residual bodies of cells, including neurons and other neural cells. Lysosomal hydrolases include, without limitation, lysosomal proteases such as cathepsins B, G, H, and L; non-protease lysosomal hydrolases such as hexosaminidases A and B; and other proteins of various enzyme classes.
By "lysosomal protease inhibitor" is meant a compound which prevents or inhibits the activity of a lysosomal protease. Examples of lysosomal protease inhibitors are cystatin C and stefin A.
By "normal levels" of a lysosomal hydrolase or lysosomal protease inhibitor is meant the normal range of amounts of the lysosomal hydrolase or lysosomal protease inhibitor present in healthy individuals who do not suffer from AD or any other disease in which the level in CSF is affected.
By "immunoassay" is meant any method of detecting proteins which involves the use of antibodies (e.g., Western blot, ELISA, inhibition ELISA, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, and fluorescent immunoassay).
By "activity" of a lysosomal protease is meant the ability of the protease to hydrolyze peptide bonds (e.g., CD cleaves the internal peptide bonds of proteins, usually between two hydrophobic residues).
By "activity" of a non-protease lysosomal hydrolase is meant the ability of the enzyme to cleave a substrate (e.g., an oligosaccharide) with the addition of H.sub.2 O at the point of cleavage.
By "activity" of a lysosomal protease inhibitor is meant the ability of the compound to prevent or inhibit cleavage by a lysosomal protease as defined above.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Detailed Description
The drawings will first be briefly described.
FIG. 1a is a graph showing that CD immunoreactivity on immunoblots of CSF is linearly related to the weight of purified human brain CD.
FIG. 1b is a graph showing that CD activity in CSF is linearly related to the weight of purified human brain CD.
FIG. 2a is a schematic representation of CD content in AD and non-AD patients.
FIG. 2b is a schematic representation of CD activity in AD and non-AD patients.
FIG. 2c is a schematic representation of CD specific activity in AD and non-AD patients.
FIG. 3 is a histogram of the elution profile of ventricular CSF CD from a pepstatin column.
The following examples are meant to illustrate, but no limit, the methods of the present invention. Other suitable modifications and adaptations of the conditions normally encountered in immunodiagnostics which are obvious to those skilled in the art are within the scope of the invention.
Collection of CSF
Techniques for collecting CSF are well known in the art (see, e.g., Appleyard et al., 1987, Brain 110: 1309; and Wester et al., 1990, J. Neurochem. 54: 1148). In the present example, CSF was collected from the lateral ventricle of the brain using at 16-gauge spinal needle within 24 hours of death. Cellular debris in the sample was removed by centrifugation at 15,0000.times.g. In several cases, the pelleted material was analyzed for CD, and the level of CD in the pellet was less than 1% of the CD in the CSF sample. Lumbar CSF can be isolated using a similar technique (see, e.g., The Merck Manual, 1746-1748, 12th edition D. N. Holvey, ed., Merck, Sharp, and Dohne Research Publishing, N.J., 1972).
Elevated Cd Concentrations in Ad Patients
A 20 ml sample of ventricular CSF was collected after death from 30 AD patients, 14 patients with Huntington disease (HD), and 7 patients with other neurodegenerative diseases (including 2 with diffuse Lewy body disease, 3 with Pick's disease, 1 with non-Alzheimer neurofibrillary degeneration, and 1 with multiple sclerosis). All of the Alzheimer's patients met the neuropathologic criteria for definite AD developed by the Consortium to Establish a Registry for Alzheimer's Disease (see Mirra et al., 1991, Neurology, 41: 479-486). HD is accompanied by a gradual loss of the mental faculties ending in dementia, and thus HD patients and patients suffering from other neurological disorders are a useful control group for identifying biochemical markers which can distinguish AD from other dementias. These late-onset dementias are sometimes difficult to distinguish clinically from AD.
The concentration of CD in 100 .mu.l samples of CSF was determined by Western blot immunoassay using a 1:5,000 dilution of polyclonal sheep antiserum directed against human brain CD (Nixon and Marotta, 1984, J. Neurochem. 43: 507-516). In addition, anti-CD monoclonal antibodies (Oncogene Science, Cambridge, Mass.) and polyclonal antisera (DAKO, Carpinteria, Calif.; Cortex Biochem, San Leandro, Calif.; and Athens Research, Athens, Ga.) were used in the Western blot immunoassays to confirm the identity of CD. Purified human brain CD was used as a protein standard (see Nixon and Marotta, supra and Takekuchi et al., 1992, J. Neurochem. 58: 1526-1532). In these Western blot immunoassays, immunoreactivity of the mature 32-kD polypeptide chain of CD was linearly related to the weight of purified human brain CD within the range of 50 and 400 ng (see FIG. 1a).
As shown in Table 4, the level of immunoreactive CD (1.98 .mu.g/ml CSF) was more than four times higher in the AD patients than in patients suffering from other late-onset neurological diseases (0.44 .mu.g/ml CSF). In 90% of the AD patients, the level of CD exceeded the mean value for CD in the neurologic disease controls, and all of the control subjects had CD levels which were lower than the mean level for the AD subjects. The results of these studies are independent of post-mortem variables and patient age. The data presented in Table 4 also indicate that the CD level expressed as ng/mg total protein was elevated almost 4-fold in AD patients. Generally, a CD level of at least 1.7 ng/mg total CSF protein is indicative of AD. Thus, these data indicate that the level of CD immunoreactive protein can also serve as a clinical marker of AD.
Elevated Cd Activity Levels in Ad Patients
CD activity levels in the CSF of 30 AD, 14 HD patients, and 7 patients afflicted with other neurodegenerative diseases were measured after death as pepstatin-inhibitable hydrolysis of [.sup.14 C]-hemoglobin (Hb) at ph3.2. CD activity was linearly related to the weight of purified human brain CD within the range of 18 and 150 ng (FIG. 1b). In AD patients, the activity of CD was elevated approximately two-fold relative to the CD activity in HD patients. As shown in Table 4, the CD activity level in Alzheimer's CSF was 1.92 mg Hb/hr/ml CSF, and in non-Alzheimer's CSF, the level was 1.01 mg Hb/hr/ml. The observation that CD activity levels are higher in AD patients than in patients afflicted with other neurologic disorders supports the use of CD activity level as an indicator of AD. Regression analysis indicated that CD activity in CSF was unrelated to post-mortem interval (r=0.026 and 0.044, respectively) and age (r=0.239 and 0.018, respectively). The data presented in Table 4 also indicate that the CD specific activity is lower in AD patients than in non-AD patients (1.81 mg Hb/hr/ml in Alzheimer's CSF and 4.98 mg Hb/hr/ml in non-Alzheimer's CSF), suggesting that the abnormally accumulated CD of AD patients includes both enzymatically active and inactive forms.
The CSF of AD patients contains elevated levels of CD relative to patients who do not suffer from AD. Thus, the invention provides a method for using biochemical markers to diagnose AD, monitor the rate of AD progression, and screen for AD therapeutic compositions.
In diagnosing AD, the CSF of the patient is collected, and the level of CD is compared with normal CD levels, Generally, a CSF CD level of at least 1.7 .mu.g/ml is indicative of AD. A variety of protein detection methods (e.g., immunoassays, SDS-PAGE, and activity assays) can be used to assay for CD, and an elevated CD level indicates probable AD. A diagnosis of AD may involve an assay of CD levels in conjunction with the detection of any of the AD symptoms outlined in Tables 1, 2, and 3. By measuring the change in CD levels as a function of time, the progression of AD can be monitored; lumbar CSF is particularly useful for this purpose. An increase in CD levels over time signifies that the state of the disease has advanced; a decrease in CD levels is indicative of amelioration of the disease. In addition, the invention facilitates the screening of therapeutic compositions, with a decrease in the CD level signifying that a composition is useful for treating AD.
Other Embodiments
Other embodiments are within the following claims. For example, if desired, CD or the other lysosomal hydrolases and lysosomal protease inhibitors can be partially purified from the CSF sample. Partial purification of the protein facilitates detection of the protein in CSF samples having low levels of immunoreactive or enzymatically active forms of the protein. Any of the methods commonly available to those skilled in the art (e.g., ion exchange chromatography, affinity chromatography, and immunoprecipitation) may be used for partial purification.
For example, CD has been partially purified from a 1-5 ml CSF sample as follows. The CSF sample was adjusted to 0.2 M NaCl and pH 4.5 with 0.1 volume of 1 M acetate buffer (pH 4.5). This fraction was mixed for 2 hours with 0.5 g of diaminodipropylamine-pepstatinyl-Sepharose 6 B (Pierce Chemicals; Rockport, Ill.) equilibrated with 0.1 M acetate buffer (pH 4.5) containing 0.2 M NaCl. The mixture then was poured into a 5.times.0.25 cm column, and, after washing the column with the same buffer (10 ml/1hour), the enzyme was eluted with 0.1 M Tris-HCl (pH 8.6) containing 0.2 M NaCl. FIG. 3 is a histogram which shows that all of the CD activity in CSF binds to the column and is eluted in the first two 1 ml fractions by the pH 8.6 buffer. In this case, the recovery of the enzyme from the column is at least 90% of the activity measured in the sample prior to chromatography. In addition, recovery of the immunoreactive CD also is greater than 90%.
Also within the invention is the use of proteins other than CD to diagnose and monitor the progression of AD. The brains of AD patients accumulate a variety of lysosomal hydrolases and lysosomal protease inhibitors extracellularly within senile plaques (Cataldo et al., 1991, PNAS 88:10998-11002). Such lysosomal hydrolases and lysosomal protease inhibitors include cathepsins D, B, G, L, and H; hexosaminidases A and B; cystatin C; and stefin A. I have determined, for the first time, that the lysosomal hydrolase CD is present in CSF. This observation suggests that the CD which accumulates extracellularly in AD brains in cleared via the CSF. Accordingly, the other lysosomal proteins which accumulate extracellularly in AD brains should be present in CSF as well. Indeed, I have detected several of these enzymes, including cathepsins B and H, cystatin C, and hexosaminidase A in CSF obtained from AD patients. Partial protein purification using standard methods will facilitate detection of other lysosomal proteins as well. Using the guidance provided herein, the protein levels which are indicative of AD can be readily determined by one skilled in the art.
Also within the invention is the use of lumbar CSF in the diagnosis and monitoring of AD. As is the case for detection of the diagnostic proteins in ventricular CSF, protein purification methods may be used to facilitate detection of the lysosomal hydrolases and lysosomal protease inhibitors in lumbar CSF. Indeed, I have detected CD in 100 .mu.L samples of lumbar CSF; as expected, CD levels are lower in lumbar CSF than in ventricular CSF. Western blot immunoassays indicate that the concentration of CD in AD patients is approximately 0.1 .mu.g/ml lumbar CSF, and this level of CD is elevated relative to the CD level found in lumbar CSF of non-AD patients. In addition, I have detected CD activity in lumbar CSF of non-AD patients; in these patients, the CD activity level is approximately 0.05 mg Hb/hr/mg total protein. Using the guidance provided herein, one skilled in the art can readily determine the lumbar CSF protein levels which are indicative of AD.
Claims
Certificate of Correction present
The claims shown below may not include correction changes. Use the corrected PDF for authoritative text.
What is claimed is:
1. A method for diagnosing probable Alzheimer's disease in a patient, said method comprising determining the level of cystatin C in cerebrospinal fluid of said patient, and comparing said level of cystatin C to normal levels, wherein an increase in cystatin C levels relative to normal indicates said diagnosis of probable Alzheimer's disease in said patient.
2. The method of claim 1, wherein said cerebrospinal fluid is ventricular cerebrospinal fluid.
3. The method of claim 1, wherein said cerebrospinal fluid is lumbar cerebrospinal fluid.
4. A method of measuring Alzheimer's disease progression in a patient, comprising determining the level of cystatin C in cerebrospinal fluid of said patient at a first time point, and determining the level of said cystatin C in cerebrospinal fluid at a second time point, wherein an increase in said cystatin C at said second time point signifies that the state of Alzheimer's disease has progressed.
5. The method of claim 4, wherein said cerebrospinal fluid is lumbar cerebrospinal fluid.
Patent Citations (1)
| Patent | Date | Inventor | Cited By |
|---|---|---|---|
| US5387742 | 1995-02-01 | Cordell |
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