The invention provides a method of diagnosing, prognosing, staging, and/or monitoring a mammalian amyloidogenic disorder or a predisposition thereto by detecting a protein or polypeptide aggregate in the cortical and/or supranuclear regions of an ocular lens of the mammal.

Description of the Invention

SUMMARY OF THE INVENTION

The invention features a non-invasive antemortem test to aid in the diagnosis, prognosis, staging, and monitoring of a neurodegenerative condition such as AD. Dynamic light scattering (DLS; a.k.a. quasi-elastic light scattering (QLS)), Raman spectroscopy, and other optical instrumentation allow detection of morphological changes in the eye, which are associated with AD.

A method of diagnosing, prognosing, staging, and/or monitoring a mammalian amyloidogenic disorder or a predisposition thereto is carried out by detecting a protein or polypeptide aggregate in the cortical and/or supranuclear region of an ocular lens of the mammal. This determination is compared to or normalized against the same determinations in the nuclear region of the same lens where more general effects of aging are observed. Comparisons are also made to a population norm, e.g., data compiled from a pool of subjects with and without disease. The presence of or an increase in the amount of aggregate in the supranuclear and/or cortical lens regions of the test mammal compared to a normal control value indicates that the test mammal is suffering from, or is at risk of, developing an amyloidogenic disorder. A normal control value corresponds to a value derived from testing an age-matched individual known to not have an amyloidogenic disorder or a value derived from a pool of normal, healthy (non-AD) individuals. An amyloidogenic disorder is one that is characterized by deposition or accumulation of an amyloid protein or fragment thereof in the brain of an individual. Amyloidogenic disorders include AD, Familial AD, Sporadic AD, Creutzfeld-Jakob disease, variant Creutzfeld-Jakob disease, spongiform encephalopathies, Prion diseases (including scrapie, bovine spongiform encephalopathy, and other veterinary prionopathies), Parkinson's disease, Huntington's disease (and trinucleotide repeat diseases), amyotrophic lateral sclerosis, Down's Syndrome (Trisomy 21), Pick's Disease (Frontotemporal Dementia), Lewy Body Disease, neurodegeneration with brain iron accumulation (Hallervorden-Spatz Disease), synucleinopathies (including Parkinson's disease, multiple system atrophy, dementia with Lewy Bodies, and others), neuronal intranuclear inclusion disease, tauopathies (including progressive supranuclear palsy, Pick's disease, corticobasal degeneration, hereditary frontotemporal dementia (with or without Parkinsonism), and Guam amyotrophic lateral sclerosis/parkinsonism dementia complex). These disorders may occur alone or in various combinations. For example, individuals with AD are characterized by extensive accumulation of amyloid in the brain in the form of senile plaques, which contain a core of amyloid fibrils surrounded by dystrophic neurites. Some of these patients exhibit clinical signs and symptoms, as well as neuropathological hallmarks, of Lewy Body disease.

The presence and/or an increase in the amount of an amyloid protein or polypeptide detected in a subject's eye tissue over time indicates a poor prognosis for disease, whereas absence or a decrease over time indicates a more favorable prognosis. For example, a decrease or decrease in the rate of accumulation in amyloid protein or similar changes in the associated ocular morphological features in eye tissue after therapeutic intervention indicates that the therapy has clinical benefit. Therapeutic intervention includes drug therapy such as administration of a secretase inhibitor, vaccine, antioxidant, anti-inflammatory, metal chelator, or hormone replacement or non-drug therapies.

Mammals to be tested include human patients, companion animals such as dogs and cats, and livestock such as cows, sheep, pigs horses and others. For example, the methods are useful to non-invasively detect bovine spongiform encephalopathy (mad cow disease), scrapie (sheep), and other prionopathies of veterinary interest]

For example, the diagnostic test is administered to a human who has a positive family history of familial AD or other risks factors for AD (such as advanced age), or is suspected of suffering from an amyloidogenic disorder, e.g., by exhibiting impaired cognitive function, or is at risk of developing such a disorder. Subjects at risk of developing such a disorder include elderly patients, those who exhibit dementia or other disorders of thought or intellect, or patients with a genetic risk factor.

A disease state is indicated by the presence of amyloid protein aggregates or deposits in the supranuclear or cortical region of a mammalian lens. For example, the amount of amyloid protein aggregates is increased in a disease state compared to a normal control amount, i.e., an amount associated with a non-diseased individual. Amyloid proteins include .beta.-amyloid precursor protein (APP), A.beta., or a fragment thereof (e.g., A.beta..sub.1-42) as well as prion proteins, and synuclein. Protein or polypeptide aggregates may contain other proteins in addition to A.beta. (such as .alpha.-, .beta.-, and/or .gamma.-crystallin). Unlike amyloid protein deposition in brain tissue which is primarily extracellular, ocular deposition in lens cortical fiber cells is cytosolic.

Aggregates are detected non-invasively, i.e., using a device or apparatus that is not required to physically contact ocular tissue. For example, the invention includes a method of diagnosing an amyloidogenic disorder or a predisposition thereto in a mammal, by illuminating mammalian lens tissue with an excitation light beam and detecting scattered or other light signals emitted from the tissue. Aggregates are detected with quasi-elastic light scattering techniques (a.k.a. dynamic light scattering), Raman spectroscopy, fluorimetry, and/or other methods of analyzing light returned from the test tissue. An increase of scattered light emitted from the cortical and/or supranuclear regions of an ocular lens indicates that the mammal is suffering from, or is at risk of developing an amyloidogenic disorder such as AD. Excitation light is in the range of 350-850 nm. Preferably, the excitation light beam is a low wattage laser light such as one with a wavelength of 450-550 nm. Alternatively, the excitation light beam is in the very near-UV (392-400 nm) or visible (400-700 nm) range.

The invention also encompasses a method of monitoring the efficacy of a therapeutic agent or intervention for disease or amyloidogenic disorder by detecting polypeptide aggregates over time, e.g., before therapy begins and at various times after (or during) therapeutic intervention. An increase in the amount or rate of accumulation of aggregates indicates a less favorable prognosis or less favorable response to therapy, whereas a decrease in the amount or rate indicates a favorable response to therapy or a favorable prognosis. For example, a pre-treatment status of the patient is determined, the patient is treated, and then the patient's condition is followed using QLS, Raman techniques, or fluorimetry. An increase in the amount or rate of formation of aggregate or accumulation of amyloidogenic protein or peptides is compared to a normal control value or a prior measurement in the same individual mammal.

Detection of protein aggregation or accumulation or deposition of amyloidogenic proteins or peptides in the supranuclear/cortical region of an ocular lens is ratiometrically, volumetrically, or otherwise mathematically compared to the same or similar measurements in the nuclear or other regions of the lens. The methods are useful to measure protein aggregation or accumulation or deposition of amyloidogenic proteins or peptides in other ocular tissues, including but not limited to the cornea, the aqueous humor, the vitreous humor, and the retina.

A significant advantage of the methods described herein is the ability to reliably and non-invasively diagnose AD antemortem. Prior to the invention, no reliable antemortem diagnostic methods were available. Based on the discovery that an increase in A.beta. is detectable human AD patient lenses compared to normal human lenses, early detection of neurodegeneration is possible. Thus, another advantage of the method is detection of a pathologic state (or pre-pathologic state) prior to any clinical indication of disease, e.g., impaired cognition.

Yet another advantage is the specificity of the diagnostic method. Aggregation in a distinct anatomical region of the lens, i.e., supranuclear and/or cortical region, rather than the nuclear region of the lens indicates a disease state. Neuropathologically confirmed human AD is associated with a relatively uncommon cataract phenotype (the supranuclear/deep cortical cataract). This supranuclear/cortical cataract is distinct from the much more common age-related cataract, which is found in the lens nucleus. A.beta. in the lens of human AD patients was found to be associated with intracellular cytoplasmic aggregated lens particles, which are large enough to scatter light and are evident in the same region of the lens in which the supranuclear/cortical cataract is observed. This same type of cataract occurs in a transgenic mouse model of AD (APP2576) which overexpresses human A.beta. species.

The QLS technique is used to non-invasively detect and quantitate lens protein aggregation in this animal model of AD and in human subjects. An additional advantage to this technique is the ability to monitor disease progression as well as responsiveness to therapeutic intervention. A.beta.-associated lens aggregates are found exclusively in the cytoplasmic intracellular compartment of human lens cells, specifically lens cortical fiber cells in contrast to A.beta. deposits in the brain, which are largely extracellular. A.beta. fosters human lens protein to aggregate through metalloprotein redox reactions and this aggregation by chelation or antioxidant scavengers.

A.beta. and .alpha.B-crystallin crosslink not only in the lens, but also in the brain. Finally, an important advantage of the method is that the amount and rate of progression of A.beta. aggregation and/or crosslinking in the eye closely parallels disease progression in the brain, providing an accurate and reliable determination of pathology in an otherwise inaccessible tissue.

DETAILED DESCRIPTION

This invention provides for a sensitive means to non-invasively, safely, and reliably detect a biomarker of Alzheimer's Disease (AD) in the lens and other ocular tissues using a quasi-elastic light scattering, Raman spectroscopy, fluorometric or other optical technologies. These techniques allow detection and monitoring of amyloid protein deposition in the eye for the diagnosis of neurodegenerative disorders such as AD and prionopathies. Lens protein aggregation is potentiated by human A.beta..sub.1-42 peptide, a pathogenic and neurotoxic peptide species which aggregates and accumulates in AD brain. A.beta. was found to promote protein aggregation in vivo and in vitro. A.beta..sub.1-42 was found specifically in the deep cortex and supranucleus of human lenses and was associated with large molecular weight protein aggregates. The results indicate that the protein aggregation in the lens, e.g., in lens cortical fiber cells, represents an easily accessible peripheral marker of AD pathology in the brain.

Lens Architecture and Protein Aggregation

Beneath an acellular capsule on the anterior side of the lens is a cuboidal monolayer of lens epithelial cells (LEC). The central (axial) LECs do not divide but survive throughout life. The more peripheral LECs divide and migrate peripherally toward the lens equator and there begin a process of terminal differentiation (TD) into cortical fiber cells. During TD the intracellular organelles are lost so that in the nucleus, the cells are devoid of most intracellular organelles. Superficial fiber cells at the equatorial region possess nuclei and organelles in varying stages of disintegration, but deeper cortical fiber cells (and all nuclear fiber cells) are devoid of intracellular organelles. In spite of a general sluggish, largely anaerobic metabolism lens fiber cells maintain protein synthesis throughout life, but they lack means to efficiently or completely clear away post-translationally modified proteins. Consequently lens proteins are the most long-lived proteins in the body and they reflect in their post-translational changes the stresses that have affected the lens throughout life. Protein aggregation is one of the post-translational changes, and A.beta.-associated aggregation in the lens parallels the aggregation that occurs in AD brain.

The unique features of lens fiber cells foster cellular retention and accumulation of protein. A.beta. accumulation and associated protein aggregation within the deep cortical/supranuclear regions of the lens parallels or precedes similar A.beta.-mediated amyloidogenic processes in AD-affected brain, thus providing not only non-invasive but also early (pre-symptomatic) detection of the AD disease process. Thus, non-invasive in vivo quantitative assessment of protein aggregation and opacification within the deep cortical/supranuclear region of the human lens is useful for diagnostic detection and tracking of cerebral A.beta. accumulation in prodromal or established AD.

Lens protein aggregation associated with age-related cataracts (ARC) differ in composition and location from aggregates or cataracts associated with AD. Postmortem human lenses from seven successive donors with severe AD-related neuropathological changes were examined. All of these donors exhibited supranuclear (deep cortical) cataracts. In five of the seven donors, the supranuclear cataracts were evident bilaterally. Supranuclear cataracts are a relatively rare cataract phenotype (0.3% in a series of 1,976 surgically extracted intracapsular cataracts and are anatomically distinct from age-related nuclear cataracts. Based on the presence of supranuclear cataracts in all seven of these cases, the lower limit of the 95% confidence interval for the populational proportion of patients with severe AD-related neuropathological changes who would also exhibit supranuclear cataracts is at least 56% (based on calculation of binomial distribution confidence intervals). Thus, there was a statistically significant correlation of supranuclear/cortical polypeptide aggregation with neurodegenerative disease. This same bilateral cataract phenotype was also observed in amyloid-bearing APP2576 transgenic mice, an art-recognized model for human AD.

In each of these lenses, supranuclear cataracts were either the only form of cataract present or the most prominent form of cataract. Although a simple supranuclear cataract may be age-related, the prevalence of simple (or pure) supranuclear cataract simply as a consequence of aging is very low (0.3% in a series of 1976 extracted age-related cataracts). "Simple" means the only region of opacification present in the lens. Supranuclear cataract as a component of mixed ("mixed" meaning more than one region of the lens opaque) age-related cataracts is higher (approximately 30%). Therefore, in the series of seven pairs of AD lenses the, finding of essentially pure supranuclear cataract in all of them constituted an anomously high, and statistically surprising, rate of supranuclear opacification. The association of supranuclear change with neuropathologically-confirmed AD indicated that the supranuclear opacification or aggregate accumulation is a unique lenticular phenotype or signature of AD evident in the lens. Both human data and animal model data indicate that supranuclear protein accumulation and/or opacification is a manifestation of AD-like degenerative change in the lens.

On a microscopic level, supranuclear opacification is a manifestation of light scattering from areas in which the index of refraction varies greatly over short distances (such as from damaged cellular membranes and low-protein "lakes" that appear in between high-protein fiber cytoplasm). At the interface of the low and high protein areas, light is scattered because the indices of refraction of these two areas are so different. That A.beta. is a pro-oxidant and capable of damaging cellular membranes suggests that increased A.beta. acts like other oxidants (e.g. H.sub.2O.sub.2).

Amyloid Biochemistry in Cataract Formation

As described above, aggregates containing A.beta., the pathogenic protein which accumulates in AD, form supranuclear/deep cortical cataracts within the lenses as well as in the brains of Alzheimer's disease patients. A.beta. deposits in the lens were found to collect as intracellular aggregates within the cytosol of lens cortical fiber cells. Lens A.beta. was quantified and the results showed that it existed as soluble apparent monomeric and dimeric species within the adult human lens at levels comparable to those in normal adult brain. A substantial proportion of lens A.beta. is bound to other lens proteins, including the abundant lens structural protein .alpha.B-crystallin. A.beta. and .alpha.B-crystallin exhibited nanomolar intermolecular binding affinity in vitro and co-immunoprecipitated from formic acid-treated human lens homogenates, indicating strong protein-protein association. Human A.beta..sub.1-42 promotes lens protein aggregation with increased .beta.-sheet content. A.beta.-potentiated lens protein aggregation was blocked by metal chelation or reactive oxygen species scavengers, thus demonstrating that metalloprotein redox reactions are involved in this lens protein aggregation process and supranuclear cataract formation in AD.

These results indicate that a pathologic interaction between A.beta. and lens proteins occurs. Furthermore, these A.beta.-mediated reactions in the lens indicated that amyloidogenic A.beta. species, particularly the human A.beta..sub.1-42 species which is prominently involved in AD pathophysiology, were potent pro-oxidant peptides which fostered lens protein aggregation. and supranuclear/cortical cataract formation.

Methods for Detecting Ocular Protein Aggregates

A method for detecting A.beta.-potentiated protein aggregates using DLS technology was developed and tested in transgenic mice (Tg2576), an art-recognized animal model for Alzheimer's disease. A relationship between hA.beta..sub.1-42 and lenticular protein aggregation was shown to provide a facile means for ocular detection of the early onset stage of AD using DLS (or QLS), in Tg2576 mouse. The data indicated that DLS (or QLS) and/or Raman scattering is useful to detect AD in humans.

The major proteins that can scatter light in a human eye lens are .alpha.-, .beta.-, and .gamma.-crystallins. Since the crystallins are abundant and large molecules (molecular weight .about.10.sup.6 Daltons), they induce the greatest amount of scattering of light, including laser radiation in dynamic light scattering (DLS) measurements. When the lens protein molecules are aggregated, they give rise to lens opacities. The lens gradually becomes cloudy as a result of light scattering and absorbance, thus hindering light transmission and the ability to focus a sharp image on the retina at the back of the eye.

Methods for measuring DLS, are known in the art, e.g., Benedek, G. B., 1997, Invest. Ophthalmol. Vis. Sci. 38:1911-1921; Betelhiem, et al., 1999, J. Biochem. Biophys. Res. Comm. 261(2):292-297; and U.S. Pat. No. 5,540,226. For example, a monochromatic, coherent, low-powered laser is shined into the lens of a subject such as a human patient. Agglomerated particle dispersions within the lens reflect and scatter the light. Light scattering is detected using a variety of known methods such as a photo multiplier tube, a solid-state photo diode or a charge coupling device. Because of random, Brownian motion of the lenticular protein crystallins, the concentration of the crystallins appears to fluctuate and hence, the intensity of the detected light also fluctuates. However, a temporal autocorrelation function of the photo current is mathematically analyzed to reveal the particle diffusivity. The data reveals the composition and extent of cataractogenesis. An increase in light scattering in the supranuclear and/or cortical region of the lens (alone and/or normalized to the scattering in the lens nucleus, where general aging effects on the lens predominate and/or normalized for age) compared to a known normal value or a normal control subject indicates the presence of protein aggregation associated with a neurodegenerative disease such as AD. This finding, in turn, serves as a biomarker for the AD disease process and hence is of clinical utility in the diagnosis, prognosis, staging, and monitoring of AD or other amyloidogenic disorders.

Although A.beta. has been demonstrated in rodent and monkey lens, these earlier studies did not describe its presence in humans, the relationship of deposition relative to a human disease state or severity of the disease. Nor did earlier studies define the presence, localization, or form of a detectable disease-associated phenotype, i.e., aggregates in the supranuclear/cortical lens region (as distinguished from the lens nucleus), a non-invasive diagnostic method for detection of A.beta. aggregates, or methods of distinguishing the AD disease process from ongoing degenerative changes in the lens due to age.

Claim 1 of 21 Claims

1. A method of monitoring the effectiveness of a therapeutic intervention in a person suffering from or at risk for developing an amyloidogenic disorder, the method comprising detecting a polypeptide aggregate in a supranuclear or deep cortical region of an ocular lens, wherein said polypeptide aggregate comprises an amyloid protein selected from the group consisting of .beta.-amyloid precursor protein (APP), A.beta., A.beta..sub.1-42, prion protein, .alpha.-synuclein, and fragments thereof and wherein said polypeptide aggregate is detected using an ophthalmic instrument sensitive to light scattering; and monitoring the amount, the rate, or both the amount and the rate of aggregation over time; wherein a decrease in the amount, the rate, or both the amount and rate of protein aggregation over time following therapeutic intervention indicates that the therapeutic intervention has clinical benefit.

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