Differential contribution of isoaspartate post-translational modifications to the fibrillization and toxic properties of amyloid ß and the Asn23 Iowa mutation.

Mutations within the Aß (amyloid ß) peptide, especially those clustered at residues 21-23, are linked to early-onset AD (Alzheimer's disease) and primarily associated with cerebral amyloid angiopathy. The Iowa variant, a substitution of an aspartic acid residue for asparagine at position 23 (D23N), associates with widespread vascular amyloid and abundant diffuse pre-amyloid lesions significantly exceeding the incidence of mature plaques. Brain Iowa deposits consist primarily of a mixture of mutated and non-mutated Aß species exhibiting partial aspartate isomerization at positions 1, 7 and 23. The present study analysed the contribution of the post-translational modification and the D23N mutation to the aggregation/fibrillization and cell toxicity properties of Aß providing insight into the elicited cell death mechanisms. The induction of apoptosis by the different Aß species correlated with their oligomerization/fibrillization propensity and ß-sheet content. Although cell toxicity was primarily driven by the D23N mutation, all Aß isoforms tested were capable, albeit at different time frames, of eliciting comparable apoptotic pathways with mitochondrial engagement and cytochrome c release to the cytoplasm in both neuronal and microvascular endothelial cells. Methazolamide, a cytochrome c release inhibitor, exerted a protective effect in both cell types, suggesting that pharmacological targeting of mitochondria may constitute a viable therapeutic avenue.

Correction to: Nrf2 activation through the PI3K/GSK-3 axisprotects neuronal cells from Aß-mediatedoxidative and metabolic damage.

After the publication of this article [1], we became aware that there were errors in Figs. 4 and 31.

Nrf2 activation through the PI3K/GSK-3 axis protects neuronal cells from Aß-mediated oxidative and metabolic damage.

BACKGROUND: Mounting evidence points to a crucial role of amyloid-ß (Aß) in the pathophysiology of Alzheimer's disease (AD), a disorder in which brain glucose hypometabolism, downregulation of central elements of phosphorylation pathways, reduced ATP levels, and enhanced oxidative damage coexist, and sometimes precede, synaptic alterations and clinical manifestations. Since the brain has limited energy storage capacity, mitochondria play essential roles in maintaining the high levels of energy demand, but, as major consumers of oxygen, these organelles are also the most important generators of reactive oxygen species (ROS). Thus, it is not surprising that mitochondrial dysfunction is tightly linked to synaptic loss and AD pathophysiology. In spite of their relevance, the mechanistic links among ROS homeostasis, metabolic alterations, and cell bioenergetics, particularly in relation to Aß, still remain elusive. METHODS: We have used classic biochemical and immunocytochemical approaches together with the evaluation of real-time changes in global energy metabolism in a Seahorse Metabolic Analyzer to provide insights into the detrimental role of oligAß in SH-SY5Y and primary neurons testing their pharmacologic protection by small molecules. RESULTS: Our findings indicate that oligomeric Aß induces a dramatic increase in ROS production and severely affects neuronal metabolism and bioenergetics. Assessment of global energy metabolism in real time demonstrated Aß-mediated reduction in oxygen consumption affecting basal and maximal respiration and causing decreased ATP production. Pharmacologic targeting of Aß-challenged neurons with a set of small molecules of known antioxidant and cytoprotective activity prevented the metabolic/bioenergetic changes induced by the peptide, fully restoring mitochondrial function while inducing an antioxidant response that counterbalanced the ROS production. Search for a mechanistic link among the protective small molecules tested identified the transcription factor Nrf2-compromised by age and downregulated in AD and transgenic models-as their main target and the PI3K/GSK-3 axis as the central pathway through which the compounds elicit their Aß protective action. CONCLUSIONS: Our study provides insights into the complex molecular mechanisms triggered by oligAß which profoundly affect mitochondrial performance and argues for the inclusion of small molecules targeting the PI3K/GSK-3 axis and Nrf2-mediated pathways as part of the current or future combinatorial therapies.

Variations in intraocular lens injector dimensions and corneal incision architecture after cataract surgery.

PURPOSE: To evaluate the differences in intraocular lens (IOL) injectors and to assess the effect of IOL insertion on injector tips and eyes after cataract surgery in a rabbit model. SETTING: Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida, USA. DESIGN: Experimental study. METHODS: A modified optical comparator was used to measure the tips of 13 IOL injector models to determine the perimeter, tip angle, and cone angle of each. Injectors were analyzed before and after IOL insertion. Surgery was performed on rabbits with 71 IOL injectors of 13 models, and custom gauges were used to determine the incision size before and after surgery. RESULTS: The injector dimensions varied by model; tip diameter, tip angle, and cone angle ranged from 1.44 to 2.12 mm, 29.7 to 66.5 degrees and 0.6 to 10.8 degrees, respectively. The incision size through which surgery was successfully performed also varied by injector model; the initial incision sizes ranged from 2.0 to 2.63 mm. For all injectors, there was wound enlargement after IOL insertion that ranged from a 0.1 to 0.65 mm increase in incision length. CONCLUSIONS: The dimensions and injection systems varied with each IOL injector. All injectors led to postoperative wound stretch after IOL insertion, with no final incision measuring less than 2.0 mm. These findings suggest that the clear cornea incision should have a width corresponding to the injector diameter.

A new, specular reflection-based, precorneal tear film stability measurement technique in a rabbit model: viscoelastic increases tear film stability.

PURPOSE: To develop a safe, noninvasive, noncontact, continuous in vivo method to measure the dehydration rate of the precorneal tear film and to compare the effectiveness of a viscoelastic agent in maintaining the precorneal tear film to that of a balanced salt solution. METHODS: Software was designed to analyze the corneal reflection produced by the operating microscope's coaxial illumination. The software characterized the shape of the reflection, which became distorted as the precorneal tear film evaporated; characterization was accomplished by fitting an ellipse to the reflection and measuring its projected surface area. Balanced salt solution Plus (BSS+) and a 2% hydroxypropylmethylcellulose viscoelastic were used as the test agents. The tear film evaporation rate was characterized and compared over a period of 20 minutes in 20 eyes from 10 New Zealand white rabbits. RESULTS: The ellipse axes ratio and surface area were found to decrease initially after each application of either viscoelastic or BSS+ and then to increase linearly as the tear film began to evaporate (P < 0.001) for eyes treated with BSS+ only. Eyes treated with BSS+ required 7.5 ± 2.7 applications to maintain sufficient corneal hydration during the 20-minute test period, whereas eyes treated with viscoelastic required 1.4 ± 0.5 applications. The rates of evaporation differed significantly (P < 0.043) between viscoelastic and BSS+. CONCLUSIONS: The shape and surface area of the corneal reflection are strongly correlated with the state of the tear film. Rabbits' corneas treated with viscoelastic remained hydrated significantly longer than corneas treated with BSS+.