Ernest Arnett

Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry, Transparency, and Mechanical Stiffness of the Mouse Lens

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.

Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens

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 α-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, αB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of αB-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.

Automated, computerized, feature-based phenotype analysis of slit lamp images of the mouse lens.

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.

IC-P-127

rescent signals with different lifetimes can be resolved in 3-dimensions. Screening of novel and existing near-infrared fluorophores resulted in several compounds with desirable properties as in vivo contrast agents. Using the new time-domain imaging algorithm, we will image transgenic mouse models of Alzheimer’s disease non-invasively to estimate plaque burden. Post-mortem confirmation of pathology will be used to correlate the in vivo results. Conclusions: These results, from simulations, to phantom measurements and in vivo imaging show that development of contrast agents and imaging approaches will allow sensitive imaging of amyloidbeta deposition in living APP mice with 3-dimensional information. This approach will accelerate pre-clinical drug development in animal models, and would ultimately translate to clinical imaging. Supported by NIH: EB00768, RR14075.

O3-01-02

Background: Passive immunization can consistently deliver the desired titers of therapeutic antibodies to Alzheimer’s Disease (AD) patients while avoiding many of the safety and tolerability issues associated with active vaccination. Intravenous Immunoglobulin (IVIg) is a very promising agent for this purpose. IVIg is a purified human natural immunoglobulin preparation with unique immune-modulating properties that contains elevated titers of polyclonal antibodies against the amyloid beta peptide (A ). IVIg has been used clinically for over 25 years as an approved treatment for various immune-deficiency and auto-immune disorders. Its established safety record can reduce the time and risks associated with its development as a treatment for AD. Methods: In two open-label pilot studies to date involving mild to moderate stage AD, monthly or bimonthly IVIg treatments for six months were well-tolerated, significantly improved dementia symptoms, increased plasma anti-A antibody titers and promoted clearance of A from the cerebrospinal fluid. Conclusions: Ongoing studies are shedding light on IVIg’s mechanisms of action, optimal dosing and the effects of chronic administration in the treatment of mild to moderate AD. The relatively high cost of IVIg, the need for intravenous administration, and current limitations in supplies of IVIg raise concerns about its equitable distribution if ongoing placebo-controlled clinical trials prove IVIg to be a suitable treatment for AD. Nevertheless, these issues are not likely to prove insurmountable, nor should they impede efforts to determine whether immunotherapy with natural human antibodies is an effective means of treating the symptoms and underlying pathology of AD.