No statistical methods were used to predetermine sample size, the experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Patient recruitment and genotyping

A national referral system for prion diseases was established by the Chief Medical Officer in the UK in 2004. UK neurologists were asked to refer all patients with suspected prion disease jointly to the National CJD Research and Surveillance Unit in Edinburgh and the NHS National Prion Clinic (NPC) in London. All patients with possible CJD who had received cadaver-derived growth hormone were referred to the NHS National Prion Clinic (London, UK) and since 2008 were recruited into the National Prion Monitoring Cohort study.

Next-generation sequencing to exclude mutations known to be causal of Aβ pathology

Deep next-generation sequencing using a custom panel was performed as described previously31. Analysis was done using NextGENe and Geneticist Assistant software (Softgenetics, USA). Variants were assessed for pathogenicity by reference to the published literature, control population allele frequencies (our primary database for allele frequency was the Broad Institute’s ExAC browser (http://exac.broadinstitute.org/)) and in silico predictive tools. The analysis methodology has been validated for the detection of APP duplication31, which was important to exclude. No causal mutations for dementia or Aβ pathology were detected, see Supplementary Table 2. As expected, several rare variants were detected which may modify the risk of various neurodegenerative diseases, see Supplementary Table 2.

Autopsies and tissue preparation

Autopsies were carried out in a post mortem room designated for high risk autopsies. Informed consent to use the tissue for research was obtained in all cases. Ethical approval for these studies was obtained from the Local Research Ethics Committee of the UCL Institute of Neurology/National Hospital for Neurology and Neurosurgery. The anterior frontal, temporal, parietal and occipital cortex and the cerebellum (at the level of dentate nucleus) were dissected during the post mortem procedure and frozen. Samples of the following areas were taken and analysed: frontal, temporal, parietal, occipital, posterior frontal cortex including motor strip, basal ganglia, thalamus, hippocampus, brain stem including midbrain, and cerebellar hemisphere and vermis. Pituitary glands were taken in all cases.

Tissue samples were immersed in 10% buffered formalin and prion infectivity was inactivated by immersion into 98% formic acid for one hour. Tissue samples were processed to paraffin wax and tissue sections were routinely stained with haematoxylin and eosin.

Antibodies and immunohistochemistry

The following antibodies were used: Anti-PrP ICSM35 (D-Gen Ltd, London, UK32,33 1:1,000), Anti-phospho-Tau (AT-8, Innogenetics, 1:100) and anti-βA4 (DAKO 6F3D, 1:50). ICSM35 was stained on a Ventana Benchmark or Discovery automated immunohistochemical staining machine (ROCHE Burgess Hill, UK); βA4 and Tau were stained on a LEICA BondMax (LEICA Microsystems) or a Ventana automated staining instrument following the manufacturer’s guidelines, using biotinylated secondary antibodies and a horseradish-peroxidase-conjugated streptavidin complex and diaminobenzidine as a chromogen.

Immunoblot detection of Aβ in iCJD brain

Biochemical studies were carried out in a microbiological containment level 3 facility with strict adherence to safety protocols. Frozen brain tissue was available from seven of eight patients with growth hormone iCJD (cases 1 and 3–8). 10% (w/v) brain homogenates (grey matter; frontal cortex) were prepared in Dulbecco’s PBS lacking Ca2+ or Mg2+ ions using tissue grinders as described previously34. 20-μl aliquots were treated with 1 µl benzonase nuclease (purity >99%; 25 U ml−1; Novagen) for 15 min at 20 °C. Samples were then mixed with an equal volume of 2× SDS sample buffer (125 mM Tris-HCl, 20% (v/v) glycerol pH 6.8 containing 4% (w/v) SDS, 4% (v/v) 2-mercaptoethanol and 0.02% (w/v) bromophenol blue) and immediately transferred to a 100 °C heating block for 10 min. Electrophoresis was performed on 16% Tris-glycine gels (Invitrogen), run for 70 min at 200 V, before electroblotting to Immobilon P membrane (Millipore) for 16 h at 15 V as described previously34. Membranes were blocked in phosphate buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST) and 5% (w/v) non-fat dried skimmed milk powder. Blots were then probed with anti-human Aβ monoclonal antibodies 6E10 (Covance) and 82E1 (IBL international, Hamburg, Germany) at final concentrations of 0.2 µg ml−1 in PBST for at least 1 h. After washing for 1 h with PBST the membranes were probed with a 1:10,000 dilution of alkaline-phosphatase-conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich no. A2179) in PBST. After washing (90 min with PBST and 5 min with 20 mM Tris pH 9.8 containing 1 mM MgCl 2 ) blots were incubated for 5 min in chemiluminescent substrate (CDP-Star; Tropix Inc.) and visualized on Biomax MR film (Carestream Health Inc.). Anti-human Aβ monoclonal antibody 82E1 recognizes an epitope specific to the amino terminus of Aβ while 6Ε10 recognizes an epitope spanning residues 3–8 of Aβ and cross-reacts with full-length APP or APP fragments that contain the epitope.

Examination of prion pathology

In all iCJD cases there was variably prominent microvacuolar change in the neocortices, deep grey nuclei and cerebellar cortex. Immunostaining for the abnormal prion protein revealed synaptic labelling in all grey matter areas examined. In all but one case, there were also microplaques in all grey matter structures. Variability in the intensity of the immunoreactivity for the abnormal prion protein was evident but detailed comparison between the cases and separately within each case was not feasible as prolonged formalin fixation in some cases significantly attenuated the immunoreactivity. It was apparent that more prominent microvacuolar change and synaptic labelling for abnormal prion protein was more intense in the pre-central gyrus and parietal lobe when compared to the anterior frontal and occipital cortices. Deep cortical layers showed more severe changes. In all cases the microvacuolar degeneration and prion protein deposits in the deep grey nuclei and hippocampal formation was prominent. It was most severe in the caudate nucleus and putamen, and appeared less severe in thalamus and it was least prominent in the globus pallidus. In the cerebellar vermis there was marked granule cell atrophy and often widespread loss of Purkinje cells accompanied by severe Bergmann gliosis, while cerebellar hemispherical cortex showed only patchy loss of Purkinje cells and no significant granule cell loss. Microvacuolar degeneration in the molecular layer was more prominent in the vermis than in the cerebellar hemisphere. No apparent difference in prion protein deposition was seen in vermis and hemisphere. In the dentate nucleus variably intense synaptic prion protein immunoreactivity was present, while the cyto-architecture of the nucleus was well preserved.

Examination, classification and quantification of Aβ pathology

All brains were examined according to the ABC classification35, which assesses the topographic progression of Aβ pathology in the brain (Thal phases36), topographic progression of Tau neurofibrillary tangle pathology (Braak and Braak37) and the density of mature (senile), neuritic plaques in the neocortex (Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) criteria38,39). To allow a more detailed assessment of neocortical Aβ the original Thal phases were modified as follows. Phase 0, no cortical Aβ; phase 0.5, 1–2 neocortical regions affected; phase 1, 3–4 neocortical regions involved; phases 2–5 were scored as published36. In addition we have carried out a semiquantitative assessment of neocortical Aβ load on a standardised region within frontal, temporal, parietal and occipital lobes, and scored as follows. 0, entirely negative; 1, a single small deposit; 2, multiple small deposits, disseminated; 3, multiple small deposits, plus an area with a larger patch; 4, diffuse moderate numbers of deposits; 5, diffuse, frequent numbers of deposits. For each case a cumulative score (0–20) of total semiquantitatively assessed Aβ load in the neocortex was calculated. Cerebral amyloid angiopathy (CAA) was graded (0–3) according to the Vonsattel criteria3. CAA was assessed in leptomeninges and parenchyma of all hemispheric lobes and cerebellum with summary score (0–30) calculated for each case.

Image acquisition and processing

Histological slides were digitised on a LEICA SCN400F scanner (LEICA Milton Keynes, UK) at ×40 magnification and 65% image compression setting during export. Slides were archived and managed on LEICA Slidepath (LEICA Milton Keynes, UK). For the preparation of light microscopy images, 1,024 × 1,024 pixel sized image captures were taken, after matching paired images (Aβ and prion staining) in Slidepath, and overlays in Fig. 1f were prepared using the colour conversion function in conjunction with the image overlay in Slidepath. Laser scanning microscopy of double immunofluorescent tissue preparations was on a ZEISS LSM710 confocal microscope (ZEISS Cambridge, UK). Publication figures were assembled in Adobe Photoshop. Data plots were generated using Prism 5 (GraphPad Software, Inc., La Jolla, USA).

Digital image analysis for cortical Aβ quantification

From all cases Aβ immunostained slides from frontal, temporal, parietal and occipital lobes were digitised as described above. Digital image analysis on 496 whole slides was performed using Definiens Developer 2.3 (Definiens, Munich, Germany). Initial tissue identification was performed at a resolution corresponding to 5× image magnification and stain detection was performed at ×10 resolution. Tissue detection and initial segmentation was done to identify all tissue within the image, separating the sample from background and non-tissue regions for further analysis. This separation was based on identification of the highly homologous relatively bright/white region of background present at the perimeter of each image. A composite raster image produced by selecting the lowest pixel value from the three comprising colour layers (RGB colour model) provided a greyscale representation of brightness. The mean brightness of this background region was used to exclude all background regions from further analysis.

Stain detection (brown) is based on the transformation of the RGB colour model to a HSD representation40. This provides a raster image of the intensity of each colour of interest (brown and blue). A series of dynamic thresholds (T x ) are then used to identify areas of interest (A x ). Initially, following exclusion of intensely stained areas with values greater than 1 arbitrary unit (au) (values range from 0au to 3au in HSD images), the 5th centile ( ) of brown stain intensity was calculated as a baseline. This represents the T brown stain separating the top 5% of A tissue . The standard deviation (C5δ) within the lower 95% of A tissue was used to update the T brown stain as with all pixels above this threshold classed as ‘stain’ (A stain ) and those below as ‘unstained’ (A unstained ). A stain was excluded if the intensity of blue staining was not significantly lower than the level of brown stain (difference less than 0.1au) to remove generically dark areas. The remaining A stain were further categorised using thresholds based on the mean ( ) and standard deviation (Bδ) of brown staining within the A unstained : T brown = (lower threshold); T dark brown = (upper threshold), to give A unstained ≤ T brown > A light brown ≤ T dark brown > A Aβ deposit . Artefacts were then identified as A stain with area greater than 1 mm2, or an area greater than 0.1 mm2 with a standard deviation of brown staining below 0.2au. These A artefacts were then expanded to include surrounding pixels with brown staining greater than . This excludes large areas of homogenous staining and areas of more diffuse, non-specific chromogen deposit.