Summary Amyloidosis is a major problem in over one hundred diseases including Alzheimer's disease (AD). Using the iDISCO visualization method involving targeted molecular labeling, tissue clearing and light-sheet microscopy, we studied plaque formation in the intact AD mouse brain and up to twenty-seven months of age. We visualized amyloid plaques in 3D together with tau, microglia and vasculature. Volume imaging coupled to automated detection and mapping enables precise and fast quantification of plaques within the entire intact mouse brain. The present methodology is also applicable to analysis of frozen human brain samples without specialized preservation. Remarkably, amyloid plaques in human tissues showed greater three-dimensional complexity and surprisingly large three-dimensional amyloid patterns or TAPs. The ability to visualize amyloid in 3D, especially in the context of their micro-environment, while simultaneously analyzing two other markers, and the discovery of large TAPs, may have potentially important scientific and medical implications.

Extracellular a-synuclein (a-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that a-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the a3-subunit of Na + /K + -ATPase (NKA) as a cell surface partner of a-syn assemblies. The interaction strength depended on the state of a-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific a3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing a3-NKA are trapped within a-syn clusters resulting in a3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of a3-NKA, thereby decreasing the efficiency of Na + extrusion following stimulus. Thus, interactions of a3-NKA with extracellular a-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

Background: Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale.

P11 (S100a10), a member of the S100 family of proteins, has widespread distribution in the vertebrate body, including in the brain, where it has a key role in membrane trafficking, vesicle secretion and endocytosis. Recently, our laboratory has shown that a constitutive knockout of p11 (p11 KO) in mice results in a depressive-like phenotype. Furthermore, p11 has been implicated in major depressive disorder (MDD) and in the actions of antidepressants. Since depression affects multiple brain regions, and the role of p11 has only been determined in a few of these areas, a detailed analysis of p11 expression in the brain is warranted. Here we demonstrate that although widespread in the brain, p11 expression is restricted to distinct regions, and specific neuronal and non-neuronal cell types. Furthermore, we provide comprehensive mapping of p11 expression using in situ hybridization, immunocytochemistry and whole-tissue volume imaging. Overall, expression spans multiple brain regions, structures and cell types, suggesting a complex role of p11 in depression.

We demonstrate a concept for how a miniaturized 3-D cell culture in biological extracellular matrix (ECM) or synthetic gels bridges the gap between organ-tissue culture and traditional 2-D cultures. A microfluidic device for 3-D cell culture including microgradient environments has been designed, fabricated, and successfully evaluated. In the presented system stable diffusion gradients can be generated by application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cells. Culture for up to two weeks was performed showing cells still viable and proliferating. The cell tracer dye calcein was used to verify gradient formation as the fluorescence intensity in exposed cells was proportional to the position in the chamber. Cellular response to an applied stimulus was demonstrated by use of an adenosine triphosphate gradient where the onset of a stimulated intracellular calcium release also depended on cell position.