High-resolution 3D reconstruction reveals intra-synaptic amyloid fibrils.

ß-Amyloid (Aß) accumulation and aggregation are hallmarks of Alzheimer's disease (AD). High-resolution three-dimensional (HR-3D) volumetric imaging allows for better analysis of fluorescence confocal microscopy and 3D visualization of Aß pathology in brain. Early intraneuronal Aß pathology was studied in AD transgenic mouse brains by HR-3D volumetric imaging. To better visualize and analyze the development of Aß pathology, thioflavin S staining and immunofluorescence using antibodies against Aß, fibrillar Aß, and structural and synaptic neuronal proteins were performed in the brain tissue of Tg19959, wild-type, and Tg19959-YFP mice at different ages. Images obtained by confocal microscopy were reconstructed into three-dimensional volumetric datasets. Such volumetric imaging of CA1 hippocampus of AD transgenic mice showed intraneuronal onset of Aß42 accumulation and fibrillization within cell bodies, neurites, and synapses before plaque formation. Notably, early fibrillar Aß was evident within individual synaptic compartments, where it was associated with abnormal morphology. In dendrites, increasing intraneuronal thioflavin S correlated with decreases in neurofilament marker SMI32. Fibrillar Aß aggregates could be seen piercing the cell membrane. These data support that Aß fibrillization begins within AD vulnerable neurons, leading to disruption of cytoarchitecture and degeneration of spines and neurites. Thus, HR-3D volumetric image analysis allows for better visualization of intraneuronal Aß pathology and provides new insights into plaque formation in AD.

The clathrin adaptor AP-1A mediates basolateral polarity.

Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.

Volumetric three-dimensional reconstruction and segmentation of spectral-domain OCT.

Despite advances in optical coherence tomography (OCT), three-dimensional (3D) renderings of OCT images remain limited to scanning consecutive two-dimensional (2D) OCT slices. The authors describe a method of reconstructing 2D OCT data for 3D retinal analysis and visualization in a Computer Assisted Virtual Environment (CAVE). Using customized signal processing software, raw data from 2D slice-based spectral-domain OCT images were rendered into high-resolution 3D images for segmentation and quantification analysis. Reconstructed OCT images were projected onto a four-walled space and viewed through stereoscopic glasses, resulting in a virtual reality perception of the retina. These 3D retinal renderings offer a novel method for segmentation and isolation of volumetric images. The ability to manipulate the images in a virtual reality environment allows visualization of complex spatial relationships that may aid our understanding of retinal pathology. More importantly, these 3D retinal renderings can be viewed, manipulated, and analyzed on traditional 2D monitors independent of the CAVE.

Automatic Control of Solvent Density in Grand Canonical Ensemble Monte Carlo Simulations.

We present automated methods for determining the value of Adams' B parameter corresponding to a target solvent density in grand canonical ensemble Monte Carlo simulations. The method found to work best employs a proportional-integral control equation commonly used in industrial process control applications. We show here that simulations employing this method rapidly converge to the desired target density. We further show that this method is robust over a wide range of system sizes. This advance reduces the overall CPU time and user effort in determining the equilibrium excess chemical potential in these systems.