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Methods for Studying Aggregation of Beta-Amyloid and Alpha-synuclein in vitro and in vivo.

The DRBIO center has a long history of successful collaborative projects with the laboratory of Dr. Brad Hyman.  In our earlier collaborations we assisted the Hyman group’s move into multiphoton in vivo imaging studies of mouse model of Alzheimer’s disease (Christie, et al, 2001; Bacskai, et al, 2001).  Dr. Hyman was also an important collaborator on our early research into the use of multiphoton-excited intrinsic tissue emissions for studying disease (Zipfel, et al 2003).  

The objective of our current collaborative work is to develop new techniques to quantify the aggregation state of proteins such as a-beta and alpha-synuclein. The specialized burst and fluctuation analysis capabilities developed in Core R&D project 4, Aim 1 are essential to this collaboration.  We are also fabricating and applying micro- and nanoscale fluidic channels coupled to highly optimized fluorescence detection to create a system with the sensitivity to accurately detect the integer number of fluorophores passing through the small focal volume within the channel.  Using this system we hope to be able to acquire accurate histograms of the oligomer size distribution formed during the in vitro aggregation of labeled a-beta, for example, under different environmental conditions or in the presence of pharmaceuticals thought to inhibit aggregation.   

We have also used the hardware/software infrastructure of Core R&D project 4 Aim1 to carry out studies alpha-Synuclein-GPF aggregation in cells and on the modulation of alpha-Synuclein-GPF aggregation by co-expressed heat shock factor (HSF) (Spiegel et al, 2009). This is accomplished by fluorescent burst measurements and sFCS measurements on cellular extracts taken at increasing time points from cell expressing these proteins (Fig. 1). HSPs respond to a variety of cellular stresses and are known to stabilize proteins during folding, assembly, degradation, and movement within the cell. Coexpression of HSPs with a-synuclein in flies and with a-synuclein in mice leads to an amelioration of the disease phenotype.

Figure 1. Total photon bursts (from aggregated proteins) above small particle background as a function of time after transfection.


Christie, R.H., B.J. Bacskai, W.R. Zipfel, R.M. Williams, S.T. Kajdasz, W.W. Webb and B.T. Hyman, (2001) “Growth arrest of individual senile plaques in a model of Alzheimer’s disease observed by in vivo multiphoton microscopy,” J. Neurosci. 21(3), 858–864.

Bacskai, B.J., S. T. Kajdasz R. H. Christie, W. R. Zipfel, R. M. Williams, K.A. Kasischke, W. W. Webb and B. T. Hyman,  (2001) Chronic imaging of amyloid plaques in the live mouse brain using multiphoton microscopy.  Proceedings of SPIE 4262:125-133.

Zipfel, W.R., R. M. Williams, R. Christie, A. Y. Nikitin, B.T. Hyman, and W. W. Webb. (2003) Live tissue intrinsic emission microscopy using multiphoton excited native fluorescence and second harmonic generation. PNAS 100(12), 7075-7080.

Spiegel, E.T., Jones, P, McLean, P, Hyman, B, and W.R.  Zipfel  (2003) Probing the effect of Heat Shock Protein 70 on the aggregation of α-Synuclein  Biophys. J. 96(3):92a 





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