The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.

With increasing human longevity, protein unfolding and aggregation characterize diseases that are among the greatest threats to aging humans and to the health care system. Progress on the development and screening of novel therapeutics is limited by the difficulties of studying the self assembly mechanisms of unfolding proteins in vivo. We report novel methods for overexpression of proteins responsible for aggregation diseases in transparent lens cells where protein unfolding and aggregation can be evaluated and quantified in living animals using inexpensive, noninvasive optical methods including slit lamp biomicroscopy, multiphoton microscopy and dynamic laser scattering spectroscopy (Seeberger et al(2004)J.of Biomed Optics.9:116–120;Greiling & Clark(2008)Seminars in Cell & Devel. Biol 19:94–99; Muchowski et al (2008)J Biol Chem 283:6330).Mouse lenses expressing exon 1 of the huntingtin protein with 72 polyglutamine (polyQ) repeats fused with enhanced green fluorescent protein (EGFP) were examined to determine the spatial distribution of the aggregating protein in vivo. The mobility and size of the huntingtin aggregates was measured using quasielastic light scattering (QLS) in the lens of a living mouse. The huntingtin aggregates labeled with EGFP were measured directly in the whole lens by 2 photon excitation microscopy and in frozen sections using fluorescence microscopy. The novel optical technology identified the earliest stages of protein unfolding and aggregation, when innovative therapies have the highest probability of success against the most prominent causes of neurodegeneration, cardiomyopathy and deteriorating visual function in the aging population. Supported by EY04542 from the NEI.

Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or alpha-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, alpha B-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of alpha B-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.

Longitudinal studies of a variety of transgenic mouse models for lens development can create substantial challenges in database management and analysis. We report a novel, automated, feature-based informatics approach to screening lens phenotypes in a large database of slit lamp images. Digital slit lamp images of normal and abnormal lenses in eyes of wild type (wt), SC1 null and SPARC null transgenic mice were recorded for quantitative evaluation of their structural phenotype. The images were processed to improve the contrast of structural features that corresponded to rings of opacity and fluctuations in scattering intensity in the lenses. Measurable attributes were assigned to the features in the lens images and given as an output vector of 46 dimensions. Characteristic patterns were correlated with the structural phenotype of each mutant and wt lens and a statistical fit for each phenotype was defined. The genotype was identified correctly in nearly 85% of the slit lamp images on the basis of an automated computer analysis of the lens structural phenotype. The automated computer algorithm has the potential to evaluate a large database of slit lamp images and distinguish mouse genotypes on the basis of lens phenotypes objectively using a neural network analysis of the structural features observed in the slit lamp images. The neural network approach is a promising technology for objective evaluation of genotype/phenotype relationships based on structural features and light scattering in lenses. Further improvements in the automated method can be expected to simplify and increase the accuracy and efficiency of the feature based analysis of structural phenotypes linked to genetic variation.

Sensitive, specific, objective diagnostic tests for Alzheimer's disease (AD) are urgently needed for efficient drug development and effective clinical use of emerging therapies. Enabling AD diagnostic technology will accelerate preclinical drug discovery, streamline clinical testing, and facilitate therapeutic intervention. Patient care will be enhanced by an objective means to assess AD risk, establish early diagnosis/prognosis, initiate therapeutic intervention, track disease progression, and monitor treatment response. We previously reported discovery of beta–amyloid peptides, AD–associated amyloid pathology, and unusual co–localizing equatorial supranuclear cataracts in the ocular lenses of patients with AD but not in those without the disorder [Lancet, 2003]. We recently discovered and characterized similar beta–amyloid lens pathology in patients with Down syndrome and in Tg2576 transgenic mice. These findings provide the first evidence of AD–associated beta–amyloid pathology outside the brain and support a direct molecular link between AD–associated pathology in brain and lens. To develop and test novel laser technology for early quantitative detection/monitoring of AD–associated amyloid lens pathology in vivo. Non–invasive infrared quasi–elastic light scattering (QLS), stereophotomicroscopy, quantitative western blot, ELISA. We developed in vivo QLS technology to quantitatively assess amyloid–mediated lens protein aggregation within discrete lens subregions. The instrument is easy to use, reliable, and extremely sensitive. We tested this non–invasive technology in non–anesthetized Tg2576 and age–matched WT control mice. Non–invasive lens QLS measurements (right–angle scattering intensity, It90) completely differentiated Tg2576 vs age–matched WT controls at 10 months of age, when cerebral and lenticular amyloid pathology are minimal. Preclinical testing using systemic or topical administration of a lipophilic amyloid–binding fluorescent ligand demonstrates anatomically localized amyloid–associated lenticular fluorescence in vivo. Non–invasive quantitative laser technologies hold promise for early diagnostic assessment of AD–associated pathology in vivo. NIA (AG024792, LG), NIGMS (GM075986); Alzheimer's Association, Massachusetts Lion's Eye Research Fund, MA Alzheimer's Disease Research Center, Brigham & Women's Hosp, anonymous foundations.

Sensitive, specific, objective diagnostic tests for Alzheimer disease (AD) are urgently needed for efficient drug development and effective clinical use of emerging therapies. Enabling AD diagnostic technology will accelerate preclinical drug discovery, streamline clinical testing, and facilitate therapeutic intervention. Patient care will be enhanced by an objective means to assess AD risk, establish early diagnosis/prognosis, initiate therapeutic intervention, track disease progression, and monitor treatment response. We previously reported discovery of beta–amyloid peptides, AD–associated amyloid pathology, and unusual co–localizing equatorial supranuclear cataracts in the ocular lenses of patients with AD but not in those without the disorder [Lancet, 2003]. We recently discovered and characterized similar beta–amyloid lens pathology in patients with Down syndrome and in Tg2576 transgenic mice. These findings provide the first evidence of AD–associated beta–amyloid pathology outside the brain and support a direct molecular link between AD–associated pathology in brain and lens. To develop and test novel laser technology for early quantitative detection/monitoring of AD–associated amyloid lens pathology in vivo. Non–invasive infrared quasi–elastic light scattering (QLS), stereophotomicroscopy, quantitative western blot, ELISA. We developed in vivo QLS technology to quantitatively assess amyloid–mediated lens protein aggregation within discrete lens subregions. The instrument is easy to use, reliable, and extremely sensitive. We tested this non–invasive technology in non–anesthetized Tg2576 and age–matched WT control mice. Non–invasive lens QLS measurements (right–angle scattering intensity, It90) completely differentiated Tg2576 vs age–matched WT controls at 10 months of age, when cerebral and lenticular amyloid pathology are minimal. Preclinical testing using systemic or topical administration of a lipophilic amyloid–binding fluorescent ligand demonstrates anatomically localized amyloid–associated lenticular fluorescence in vivo. Non–invasive quantitative laser technologies hold promise for early diagnostic assessment of AD–associated pathology in vivo. NIA (AG024792, LG), NIGMS (GM075986); Alzheimer's Association, Massachusetts Lion's Eye Research Fund, MA Alzheimer's Disease Research Center, Brigham & Women's Hosp, anonymous foundations.

Sensitive, specific, objective diagnostic tests for Alzheimer's disease (AD) are urgently needed for efficient drug development and effective clinical use of emerging therapies. Enabling AD diagnostic technology will accelerate preclinical drug discovery, streamline clinical testing, and facilitate therapeutic intervention. Patient care will be enhanced by an objective means to assess AD risk, establish early diagnosis/prognosis, initiate therapeutic intervention, track disease progression, and monitor treatment response. We previously reported discovery of beta–amyloid peptides, AD–associated amyloid pathology, and unusual co–localizing equatorial supranuclear cataracts in the ocular lenses of patients with AD but not in those without the disorder [Lancet, 2003]. We recently discovered and characterized similar beta–amyloid lens pathology in patients with Down syndrome and in Tg2576 transgenic mice. These findings provide the first evidence of AD–associated beta–amyloid pathology outside the brain and support a direct molecular link between AD–associated pathology in brain and lens. To develop and test novel laser technology for early quantitative detection/monitoring of AD–associated amyloid lens pathology in vivo. Non–invasive infrared quasi–elastic light scattering (QLS), stereophotomicroscopy, quantitative western blot, ELISA. We developed in vivo QLS technology to quantitatively assess amyloid–mediated lens protein aggregation within discrete lens subregions. The instrument is easy to use, reliable, and extremely sensitive. We tested this non–invasive technology in non–anesthetized Tg2576 and age–matched WT control mice. Non–invasive lens QLS measurements (right–angle scattering intensity, It90) completely differentiated Tg2576 vs age–matched WT controls at 10 months of age, when cerebral and lenticular amyloid pathology are minimal. Preclinical testing using systemic or topical administration of a lipophilic amyloid–binding fluorescent ligand demonstrates anatomically localized amyloid–associated lenticular fluorescence in vivo. Non–invasive quantitative laser technologies hold promise for early diagnostic assessment of AD–associated pathology in vivo. NIA (AG024792, LG), NIGMS (GM075986); Alzheimer's Association, Massachusetts Lion's Eye Research Fund, MA Alzheimer's Disease Research Center, Brigham & Women's Hosp, anonymous foundations.