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.

DETAILED DESCRIPTION OF THE INVENTION

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.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 Aggreation

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 differentation (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.1 42 promots 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 aggegation 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 35 Claims

1. A method of diagnosing an amyloidogenic disorder or a predisposition thereto in a mammal, 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 an increase in the amount of said aggregate compared to a normal control value indicates that said mammal is suffering from or is at risk of developing an amyloidogenic disorder.

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