greatly accelerated [17] and leads to invariant early-onset AD neuropathology and age-dependent neurocognitive sequelae [18,19]. Lens abnormalities in subjects with clinical features of DS were first reported over a century ago [20,21] followed by numerous confirmatory reports since [20,22,23,24]. The distinctive DS lens phenotype clinically manifests as cerulean ‘‘blue dot’’ opacities that often emerge in the first decade of life, and in some cases, may be evident at birth [22,25,26,27]. DS cataracts typically localize to the supranuclear region and are histologically characterized by granular material of uncertain composition [20,24,25]. The molecular origin and pathogenic mechanisms by which the DS aneuploidy is expressed as a distinctive age-dependent lens phenotype are unknown. We previously reported disease-linked supranuclear cataracts that correlate with pathogenic Ab 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 [28]. In AD lenses, Ab accumulates as electron-dense cytosolic aggregates (longest axial dimension, ,5 nm to ,200 nm) that distribute heterogeneously within the cytoplasm of supranuclear and deep cortical lens fiber cells. AD-linked Ab aggregates in the lens qualify as Raleigh scattering centers that clinically manifest as supranu- clear opacities that ultimately progress to frank cataracts. These AD-linked supranuclear cataracts are phenotypically, anatomical- ly, ultrastructurally, and biochemically distinguishable from common age-related nuclear cataracts. Given the association between Ab amyloid lens pathology in AD [28], we hypothesized that subjects with DS would also demonstrate age-related Ab amyloid pathology in the lens. The aim of the present study was to test the hypothesis that DS-linked lens pathology reflects accelerated Ab accumulation and co-localizing amyloid pathology that clinically manifests as the characteristic supranuclear cataract phenotype associated with this common chromosomal disorder. Methods Subjects and Specimens 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 [24]. Postmortem brain specimens (superior temporal cortex) were obtained from the Massachusetts Alzheimer’s Disease Research Center Brain Bank (Massachusetts General Hospital, Boston, MA, USA), and included the following donor groups: AD (CERAD criteria) n = 3 total; n = 1 male: 86 years of age; n = 2 females: 76 and 81 years of age); DS (karyotype confirmed) n = 4 total; n = 4 males: 56, 57, 58, and 60 years of age); normal controls (n = 3 total; n = 1 male: 86 years of age; n = 2 females: 72 and 88 years of age). Clinical information regarding cytogenetic microanalysis and chromosomal Ab in Lens in Down Syndrome subtyping was not available. Lenses obtained from intact postmortem eyes were the only specimens used for biochemical analysis. All lenses obtained from intact, non-traumatic globes were included in the study and assigned to cohorts based on available clinical information. No other selection criteria were applied. 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. Slit Lamp Biomicroscopy 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 37uC, visualized with a custom-adapted surgical slit lamp stereophoto- microscope (Carl Zeiss, Thornwood, NY) equipped with a stereo beamsplitter (Urban Engineering, Burbank, CA) and side-arm digital camera assembly (Nikon, Melville, NY). Histopathology, Immunohistochemistry, Light and Electron Microscopy 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 [24]. Original paraffin blocks in this archival collection were highly friable and required re-embedding in fresh paraffin prior to histological processing. For all histological studies, routine tissue sections (8 mm) were obtained by microtomy, immunostained with Ab monoclonal antibody 6E10 directed against Ab3–8 or 4G8 directed against Ab18–22 (generously provided by Joseph Bertelsen, Signet Laboratories, Covance, Dedham, MA), and processed by conventional immunohistochemistry using a commercial kit (Vector Laboratories, Burlingame, CA) according to the manu- facturer’s instructions. Bennhold’s modified Congo red staining method [29] was used for amyloid histopathology. Brightfield and cross-polarization photomicroscopy were conducted using a Nikon Eclipse E600 upright microscope (Nikon USA, Melville, NY) equipped with a Spot Slider RT digital CCD photomicroscope camera and proprietary image acquisition software (Diagnostic Instruments, Sterling Heights, MI). Immunogold Electron Microscopy Immunogold electron microscopy (IEM) was conducted as previously described using ultra-thin cryosections without plastic PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10659