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Characterization of Microtubule Polarity in Mouse Alzheimer’s Disease Models
Gunnar Gouras, Associate Professor of Neurology and Neuroscience, Weill Cornell Medical College.
Funding: NIH NIA 5R01AG027140 Intraneuronal Abeta accumulation: mechanism of pathogenesis (8/1/07-4/30/11).

Active intracellular transport is essential for establishing and maintaining the functions of axons and dendrites. Produced in the soma, anterograde transport through neurites delivers cargoes including postsynaptic densities, neurotransmitter receptors, ion channels and specific mRNAs to distal dendritic locations, as well as components of presynaptic terminals, adhesion molecules, and mitochondria to axonal locations. These cargoes are carried by molecular motors that tread along an extensive network of microtubules. Therefore, to understand this directional trafficking, it is essential to know the microtubule polarities involved. Second harmonic generation (SHG) is a nonlinear optical signal due to coherent scattering from non-inversion symmetric structures, such as microtubules which have intrinsic structural polarity (Dombeck et al., 2003). The objective is to understand the trafficking of organelles in neurons and how it may be affected by the neurodegeneration caused by A-b depositions in our mouse models.

An outstanding question in the field has been whether microtubule polarity is different for various neurite types in vivo. To address this question, we prepared acute slices from Thy1-YFP line-H transgenic mice, which labeled a subset of neurons in the hippocampus and neocortex. Using second harmonic generation and multiphoton microscopy, we found that in addition to axons, large stretches of apical dendrites contain uniform polarity microtubules. These arrays can extend for more than 300 um and have a polarity of ~83%. Furthermore, we have also looked at mice at various developmental stages and determined that the microtubule polarity distribution is age-dependent. This work will provide us with a baseline to investigate cases where the microtubule polarity may be compromised, as in Alzheimer’s disease mouse models. Most recently, we have demonstrated that we can simultaneously image microtubules and senile plaques in acute brain slices from Alzheimer’s disease mouse models APPSwe2576 and APPSwe/PS1.

In adult Thy1-YFP line-H transgenic mice we believe we have identified uniform polarity microtubules in the apical dendrites of CA1 and layer V cortical pyramidal neurons. This observation contradicts the conventional assumption that only axons contain uniform polarity microtubules, a hypothesis supported by decade-old electron microscopy experiments. Further work will be carried out in slices and neuronal cultures at well-defined developmental stages to further verify this result. Uniform polarity microtubules in the apical dendrites implicate a different trafficking pattern in these dendrites compared to ones with mixed polarity microtubules. We will combine SHG microscopy with time-lapse imaging in neuronal cultures to trace fluorescently-labeled cargos in identified neurites. Our technique will enable us to determine whether microtubule polarity is compromised near pathological lesions such as senile plaques and neurofibrillary tangles. We have developed a computational framework to characterize the orientation and sizes of collagen fibrils (Williams et al., 2005). One goal of this project is to achieve similar quantitative understanding of the second harmonic signal in microtubules by simulations of the field emitted from various microtubule configurations, in order to better interpret results from neuronal cultures and brain slices.

Uniform polarity microtubule arrays in the neocortex of adult Thy1-YFP mice.  Left panel shows a merged inamge of YFP fluorescence (green) and SHG (red) in layer V cortex of a 11-month old mouse. The same region, shown with SHG only, is shown in the right panel.  Uniform polarity microtubule arrays co-localize with apical dendrites of layer V pyramidal neurons (arrowheads).  Scale bar = 30um.


Dombeck, D. A., K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman and W. W. Webb (2003) "Second Harmonic Generation Microscopy of Uniformly Oriented Microtubules in Native Brain Tissue" PNAS. 100:7081-7086.

Williams, R.M., W.R. Zipfel, and W.W. Webb (2005) "Interpreting Second Harmonic Generation Images of Collagen I Fibrils" Biophysical Journal.88:1377-1386.

Kwan, A.C., Duff, K., Gouras, G.K., and Webb, W.W., “Optical visualization of Alzheimer’s pathology via multiphoton-excited intrinsic fluorescence and second harmonic generation” Optics Express, in press, 2009




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