High-resolution 3-dimensional reconstruction reveals early intra-synaptic amyloid fibrils in Alzheimer's transgenic mice

Many lines of evidence support that ß-amyloid (Aß) accumulation is important in Alzheimer's disease (AD) pathogenesis. However, the progression of Aß accumulation leading to synapse damage and plaque formation remains incomplete. Our previous ultrastructural studies showed that aberrant intraneuronal Aß42 accumulation within dystrophic neurites and synaptic compartments was associated with the earliest structural pathology in AD (Takahashi et al., 2002; 2004; 2010). More recently, we reported that damage to synapses as a result of modulating synaptic activity in AD transgenic mice correlated with this intraneuronal pool of Aß rather than with amyloid plaques (Tampellini et al., 2010). High-resolution 3-dimensional (3D) volumetric (HR3DV) imaging allows for higher resolution image analysis of fluorescence confocal microscopy and 3D visualization of early Aß pathology in brain. To better visualize and analyze Aß pathology, thioflavin S staining and immuno-fluorescence using Aß antibodies (against different Aß epitopes and conformations) and structural neuronal proteins (NeuN, Tau, MAP2, SMI32 neurofilament) are performed in brain tissue of Tg2576, Tg19959, wild-type and APP KO mice and human AD. Stacks of 2D images obtained on a Leica SP5 spectral confocal microscope are reconstructed into 3D volumetric datasets using AvizoTM software. HR3DV isosurface and voltex representations are then converted to movies of rotating 3D images. For some studies, confirmatory dual-labeling immuno-electron microscopy is also used. HR3DV imaging clearly demonstrates the intracellular onset of Aß42 accumulation, oligomerization and fibrillization within vulnerable neurites and cell bodies of Tg19959-YFP mouse brains. Remarkably, prior to plaque pathology, early Aß fibrils are evident within individual spines that have a dystrophic appearance. Aß accumulation and aggregation also correlate with declining levels of structural proteins within dendrites. Utilizing a novel 3D reconstruction method to visualize early Aß42 accumulation in brain of AD transgenic mice clearly demonstrates that Aß oligomerization and fibrilization initiate within individual spines, neurites and neuron cell soma. Further, we show that progressive intraneuronal Aß42 aggregation disrupts the normal cytoarchitecture of neurites.