Conceived and designed the experiments: JAM RP RDM RMR JIC RET DGH LEG. Performed the experiments: JAM RDM SL MAB JGG ME SJS AM. Analyzed the data: JAM RP RDM MAB ME JK JIC RET DGH LEG. Contributed reagents/materials/analysis tools: JAM RP RDM SL MAB ME RF RMR JK RET DGH LEG. Wrote the paper: JAM RP RDM DGH LEG. Revised the manuscript: JGG AM RF RMR JK JIC RET.
Authors Moncaster, Pineda, Lu, Burton, Ghosh, Ericsson, Soscia, Mocofanescu, Folkerth, Robb, Kuszak, Clark, and Hunter report no conflict of interest. The following authors reported consultant activity during the course of this study: Drs. Tanzi and Goldstein, Neuroptix Corp, Acton, MA; Drs. Moir, Tanzi, and Goldstein, Covance, Dedham, MA. No financial support was provided by any commercial entity for this study.
Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the
Down syndrome (DS,
Lens abnormalities in subjects with clinical features of DS were first reported over a century ago
We previously reported disease-linked supranuclear cataracts that correlate with pathogenic Aβ accumulation, classical amyloid pathology, and co-localizing pathology in lenses obtained from subjects with Alzheimer's disease (AD) but not in those with other neurodegenerative disorders nor in normal aged controls
Lens specimens were obtained from the following sources: (i) subjects with DS requiring cataract surgery (n = 3 males: 36, 46, and 47 years of age) at the Massachusetts Eye and Ear Infirmary; (ii) postmortem specimens from donors with DS (n = 12 total; n = 9 males: 2 to 69 years of age; n = 3 females: 42, 47, and 61 years of age) and normal controls (n = 34 total; n = 20 males: 7 months to 88 years of age; n = 14 females: 2 to 82 years of age) procured through national tissue networks (National Disease Registry Interchange, Philadelphia; PA; Florida Lion's Eye Bank, Miami, FL; Sun Health Research Institute, Sun City, AZ); and (iii) archival lens specimens generously provided by Dr. Richard Robb, Children's Hospital, Harvard Medical School, Boston, MA (DS, n = 4 total; n = 3 males: 1 day, 3 weeks, and 22 years of age; n = 1 female: 7 months of age. Normal controls, n = 3 total; n = 2 males, 1 day and 21 years of age; n = 2 females, 2 days and 4 months of age). An earlier histochemical analysis of lenses from this archival collection was reported in 1978
Ethical review and permission to conduct this investigation were approved by Harvard Medical School, Brigham and Women's Hospital, Massachusetts Eye and Ear Infirmary, and Children's Hospital Boston. This study conforms to applicable regulatory guidelines at Harvard Medical School and principles of human research subject protection in the Declaration of Helsinki. Patients were informed, consent forms signed, and patient samples collected and signed in accordance with an approved IRB protocol (02-08-053X) at the Massachusetts Eye and Ear Infirmary, Boston, MA, USA.
Clinical ophthalmological examinations included slit lamp stereophotomicroscopy and microvideography following routine pupillary dilation. In three DS cases (n = 3 males: 36, 46, and 47 years of age), slit lamp photomicrographs were obtained in conjunction with cataract surgery. Patients were informed and signed consent forms in accordance with an approved IRB protocol at the Massachusetts Eye and Ear Infirmary, Boston, MA, USA. Stereophotomicroscopic analysis of dissected intact human lenses was performed on postmortem specimens obtained from enucleated eyes procured through national tissue bank resources (National Disease Registry Interchange, Philadelphia; PA; Florida Lion's Eye Bank, Miami, FL). Harvested lenses were bathed in buffered artificial aqueous humor (295 mOsm, pH 7.20) at 37°C, visualized with a custom-adapted surgical slit lamp stereophotomicroscope (Carl Zeiss, Thornwood, NY) equipped with a stereo beamsplitter (Urban Engineering, Burbank, CA) and side-arm digital camera assembly (Nikon, Melville, NY).
A portion of the present study included re-analysis of lens specimens from an archival collection generously provided by co-author Richard Robb, M.D., Department of Ophthalmology, Children's Hospital Boston, Harvard Medical School, Boston, MA. Paraffin-embedded specimens in this collection date back to the 1960's and have been previously described
Immunogold electron microscopy (IEM) was conducted as previously described using ultra-thin cryosections without plastic embedding
Lens specimens were homogenized in 70% formic acid, ultra-centrifuged at 100,000×
Analytical quantification of human Aβ40 and human Aβ42 was accomplished using two commercially available human Aβ specific ELISA kits (BetaMark, Covance, Dedham, MA; BioSource, Invitrogen, San Diego, CA). For experiments utilizing SDS extracted tissue, we used a commercially available human Aβ ELISA kit (BetaMark, Covance, Dedham, MA) that is resistant to interference from detergent. This ELISA assay system is compatible with tissue extracts containing SDS with minimal loss in sensitivity. Samples containing SDS were diluted to a final detergent concentration of less than 0.1%. For experiments utilizing formic acid extracted tissue, we used a second commercially available Aβ ELISA kit (BioSource, Invitrogen, San Diego, CA). For immunoblotting analysis, lens subcellular fractions were normalized for protein concentration and analyzed as previously described
Quasi-elastic light scattering (QLS) experiments were conducted using a custom laboratory instrument (Neuroptix Corporation, Acton, MA) that delivers linearly polarized continuous wave near-infrared laser light (λ = 780 nm; focused beam diameter, 30–35 µm; power output = 0.3 mW). The instrument incorporates a rigidly positioned pinhole mirror (aperture diameter = 100 µm) for selective detection of backscattered photons at a fixed scattering angle (θ = 120 degrees). Signal detection and analysis of backscattered light intensity was accomplished with an avalanche photodiode, integrated 256-channel multi-tau digital autocorrelator (τ = 480 ns), and custom software analysis package. Analyses involving aqueous suspensions of water-soluble lens proteins were conducted in cylindrical optical glass specimen vials positioned in a rigid holder mounted to the instrument. Three series of five independent measurements (each 1.3 seconds in duration) were performed for a total of fifteen measurements comprising each averaged test point. To investigate the interaction of Aβ peptides with other cytosolic lens proteins, a membrane-free water-soluble protein extract was prepared from freshly dissected human lenses obtained from postmortem donor eyes (National Disease Registry Interchange, Philadelphia; PA; Florida Lion's Eye Bank, Miami, FL). Dissected lenses were mechanically homogenized, ultracentrifuged at 100,000×g for 1 h at 4°C, and diluted to 1 mg/ml in phosphate buffered saline, pH 7.20. This aqueous lens protein extract is predominantly composed of water-soluble proteins in the cytoplasm of lens fiber cells. Purified synthetic human Aβ42 or Aβ40 (Keck Laboratories, Yale University, New Haven, CT) was added to freshly prepared human lens protein extract and incubated in siliconized Eppendorf tubes in a humidified 37°C 5% CO2-balanced tissue culture chamber prior to light scattering analysis. All samples were incubated in the dark to reduce photodynamic effects. For microscopic visualization, aliquots of the incubated samples were placed on a microscope slide as wet-mount preparations and visualized by conventional photomicroscopy under brightfield and cross-polarized illumination. Immunogold electron microscopic analysis of incubated material was conducted as detailed above.
Slit lamp examination of subjects with karyotype-confirmed DS revealed classical cerulean “blue dot” cataracts (
(
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A representative stereo image of a lens from a 64-year-old male with DS (
The phenotypic correspondence of the distinctive cataracts in DS and AD encouraged us to investigate the possibility of Aβ pathology in DS lenses. Orientation to the anatomy of the adult human lens is shown (
(
Definitive identification of human Aβ in DS lens was accomplished by tryptic digest tandem mass spectrometry peptide sequencing. Biochemical sequencing analysis was conducted on small molecular weight eluates derived from HPLC fractionation of human DS lens protein extract (
Tryptic digest tandem mass spectrometry sequencing of a ∼4 kDa HPLC eluate derived from human Down syndrome lens protein extract. The retention time of the immunopurified HPLC eluate used for sequencing was identical to synthetic human Aβ. The red shade box denotes the detected 12-residue internal tryptic peptide sequence 17LVFFAEDVGSNK28 that uniquely identifies human Aβ. We detected a second unique tryptic peptide 6HDSGYEVHHQK16 in another analysis (purple underline).
See text for details.
(
These data indicate that Aβ accumulates in the cytoplasm of supranuclear lens fiber cells in individuals with DS. We previously showed that Aβ binds (Kapp ∼20 nmol/L) and co-aggregates with αB-crystallin (HspB5)
(
In this report, we identify the origin and pathogenic mechanism of cataractogenesis in DS, establish the distinctive lens phenotype in DS as a genetic cataract, and define the molecular correspondence of DS-associated pathology in the lens and brain. Taken together with our previous findings of supranuclear Aβ lens pathology in late-onset sporadic AD
AD neuropathology is an invariant feature of DS in subjects over the age of 30
In this report, we hypothesize and confirm that this genotype-phenotype concordance extends to Aβ-linked molecular pathology in the supranucleus and deep cortex of the lens. The characteristic cataract phenotype associated with Aβ lens pathology has been identified in only two clinical disorders, Alzheimer's disease
The anatomical localization of the characteristic Down syndrome cataract phenotype (white shading) reflects the temporal origin and natural history of the underlying lens pathology. Fetal lens fiber cells are not involved in this pathogenic process. Parentheses indicate equatorial axial extent of age-dependent disease-linked Aβpathology in the supranuclear subregion of Down syndrome lenses. See text for details.
Distinctive lens pathology is recognized as a characteristic early-onset ocular phenotype in subjects with DS that may be clinically detectable early in life, and in some cases, at birth
The DS-associated cataract phenotype is not observed in other non-AD neurodegenerative diseases, nor in normal aged controls. In this context, it is important to note that DS-associated cataracts are phenotypically distinct from age-related cataracts (ARC) that commonly emerge starting in the fifth decade of life and typically localize in the central nuclear region. In contrast to the lens pathology in ARC, the DS lens phenotype is often clinically detectable early in life, and in some cases, may be present at birth
The results of the present study support a DS-associated pathogenic pathway linking progressive age-dependent Aβ accumulation in the lens and supranuclear cataractogenesis with corresponding cerebral Aβ accumulation and neuropathology in the brain. We propose the following mechanistic pathway leading to expression of the characteristic lens and brain phenotypes associated with DS (
Triplication of human chromosome 21 in Down syndrome results in increased dosage of the
We hypothesize that the Aβ detected in DS lenses is generated endogenously. The human lens expresses the full complement of enzymes and precursor proteins necessary to generate and process Aβ
Our findings suggest an apparent temporal discordance in DS with respect to phenotype expression in the lens and brain, with pathology in the former preceding that in the latter. However, we also observed variability in age-dependent phenotypic expression of Aβ molecular pathology in the lens that comports with analogous variability in the brain of subjects with DS
In this study, we identify Aβ amyloid pathology as the shared molecular etiology of two defining features of DS, namely, supranuclear cataracts in the lens and AD neuropathology in the brain. The proximal pathogenesis of both pathologies likely derives from the primary chromosomal disorder and associated
Stereo image pair demonstrating mature supranuclear pathology in the lens (with intact zonule fibers) from a subject with Down syndrome. Characteristic circumferential supranuclear cataract in the lens of a 64-year-old male subject with Down syndrome. This distinctive cataract is evident as an annular half-toroid band of opacification in the deep cortical and supranuclear subregions of the lens (shown with intact zonules). This same lens specimen is presented as a slit lamp image (
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We thank Mr. Joseph Bertelsen (Signet Laboratories, Covance, Dedham, MA) for generously providing 6E10 and 4G8 monoclonal antibodies. We gratefully acknowledge the National Disease Registry Interchange, Sun Health Research Institute, and the Florida Lion's Eye Bank for their assistance with tissue procurement. We would like to thank the many patients and families who made this work possible.