Quantifying the effect of human-tau on cortical synaptic

density in a mouse model of Alzheimer’s disease 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, UK2. Massachusetts General Hospital and Harvard Medical School, Cambridge, MassachusettsTau, amyloid-beta and synaptotoxicity in

Alzheimer’s 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

References1. 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 28/04/20147. Marras, S. A. E. et al. http://www.molecular-beacons.org/toto/Marras_energy_transfer.html (n/a) accessed

on 28/04/2014

Hypothesis and experimental aimsTaking the above points into account, we propose that;

Tau exacerbates AβO toxicity through an interaction at the synapsedensity, 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?

Methodology1. 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

Results

Future directions

AcknowledgementsI 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 immunohistochemistrySchematic 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