1. 1Croft, SP, 1Herrmann, AG, 2Hyman, BT, 2Rudinskiy, N, and 1Spires-Jones, TL
1. Centre for Cognitive and Neural systems, University of Edinburgh, Edinburgh, EH8 9JZ, Scotland, UK
2. Massachusetts General Hospital and Harvard Medical School, Cambridge, Massachusetts
 Alzheimer’s Disease - a progressive dementia characterised by cognitive
impairment1 that affects ̴ 500,000 people in the UK
 Cognitive symptoms correlate strongly with synapse degeneration in the
hippocampal formation and neocortex2
 Misfolded amyloid-beta oligomers (AβOs) and hyperphosphorylated tau
(pTau), both induce synaptotoxicity3, 4
1. Nelson, P. T. et al. J Neuropathol Exp. Neurol. 71(5), 362–381 (2013)
2. Koffie, R. M. et al. Molecular neurodegeneration 6(1), 63 (2011)
3. Koffie, R. M. et al. Proc Natl Acad Sci U S A 106, 4012-4017 (2009)
4. Lasagna-reeves, C. A. et al. Mol. Neurodegen. 6, 39 (2011)
5. Nieva, J. L., Madan, V., and Carrasco, L. Nature Reviews Microbiology 10, 563-574 (2012)
6. http://library.aua.edu.ag/webpath/webpath/tutormul/immuno/immuno.htm (n/a) accessed on 28/04/2014
7. Marras, S. A. E. et al. http://www.molecular-beacons.org/toto/Marras_energy_transfer.html (n/a) accessed
on 28/04/2014
Taking the above points into account, we propose that;
Tau exacerbates AβO toxicity through an interaction at the synapse
density, resulting in synapse loss and cognitive dysfunction
To test this hypothesis, we will address the following questions;
 Is synaptic density in APP/PS1 mice further reduced around plaques with
the addition of a human tau (hTau) transgene? Investigated through synaptic
density calculations using array tomography.
 Are tau and AβO located at the same degenerating synapses?
1. Immunohistochemistry
 Synapses, amyloid plaques
and nuclei labelled with
anti-synaptophysin/AW7
antibodies and DAPI
respectively. DAPI stained
nuclei were used as
reference points
 Serial images of same
cortical region taken on
Nikon confocal microscope
with 60x 1.2 NA objective
and 3x optical zoom
 Images aligned into stacks
(>15 sections) and multiple
crops analysed for synaptic
densities using ImageJ and
Watershed software
I wish to thank Dr Dominic Walsh (Harvard medical school) for his gift
of AW7 antibody, Brad Busse and Stephen Smith for the watershed
program, and the Edinburgh IMPACT facility, for confocal microscope
access and training. Thanks also to ARUK, who have generously
funded this and other work in our lab.
Figure 2: Visual representation of array tomography protocol
(A) 70nm serial sections are stained and imaged, with all images centred on an identical nucleus. (B)
An image of synaptophysin stained synapses and AβOs. (C) The same image without showing the AβO
stain, illustrating loss of synapses in the halo around the plaque. (D) The resultant 3D reconstruction of
all images within the stack.
(A) M55c5 (APP/PS1+, hTau-) 3D reconstructions
(C) M55c5 crops
(B) M61c2 (APP/PS1+, hTau+) 3D reconstructions
(D) M61c2 crops
Figure 3: Immunohistochemically labelled tissue sections and regions of interest
(A) and (B) examples of stained tissue as mentioned in methods. (C) and (D) 5x5μm and 10x10μm
regions of interest (crops) that will be used for synaptic density calculations. White arrows show
reduced synapse staining around plaques.
A B
 Completion of data collection to acquire full dataset, followed by
statistical analysis of synapse density;
1. At different distances from the plaque (synaptotoxicity should
increase nearer the plaque).
2. With/without the effects of hTau addition in an animal model.
 Colocalisation studies will highlight the distribution of hTau/AβOs and
synapses in murine cortex.
 Results will facilitate our understanding of the interplay between the two
major proteins present in Alzheimer’s disease.
i. New mechanisms to exploit in the search for efficacious treatments?
ii. Creation of more faithful models of human disease, negating dependence on
P301L/P301S-FTD tau variants for tau pathology
1. Immunostaining reveals synapse degeneration around amyloid plaques
• Optimized reliable, reproducible protocol for staining synapses and
amyloid pathology
• Evidence of synapse loss around and within plaques and some
colocalisation of synaptophysin/AβOs
2. Density analysis underway
• Synapse density data from 2 pilot animals are in line with previous
datasets, approximately 9E8 synapses/mm3
2. Array tomography
 Allows the visualisation of single synapses – increased Z-plane
resolution vs. other microscopy techniques.
Figure 1: Principles of immunohistochemistry
Schematic of antibody binding complex and fluorescence-
mechanism of fluorophores. Adapted from5-7
Synaptophysin (syn) AW7 DAPI/syn/AW7 merged
Synaptophysin (syn) AW7 DAPI/syn/AW7 merged