Supplementary Materials Ahmed Et Al., (2014)

P301S tau propagation model

Supplementary materials – Ahmed et al., (2014)

Materials and methods

Immunohistochemistry protocol

Following de-paraffinisation and rehydration, antigen retrieval was performed using the Lab Vision PT module system (Thermo Scientific, Cheshire, UK), where sections were heated to 100oC for 20 min in citrate buffer (TA-250-PM1X; Thermo Scientific, Cheshire, UK). After cooling and washing with warm citrate buffer, the slides were transferred to the Lab Vision Autostainer 360 (Thermo Scientific, Cheshire, UK) where the following incubations were performed: 10 min in H2O2 (0.03%); 30 min in normal goat serum (1:20; Vector Laboratories, CA, USA); 60 min in primary antibody (Table 2); 30 min in biotinylated goat anti-mouse or goat anti-rabbit IgG (PK-6101; Vector Laboratories, CA, USA); 30 min avidin-biotin complex solution (PK-6101; Vector Laboratories, CA, USA); 5 min in 3,3′-diaminobenzidine (SK-4100, Vector Laboratories, CA, USA). Apart from the latter, PBS with 0.05% Tween-20 (PBS-T) was used for dilution and washes. Following immunohistochemistry, some sections were counterstained with haematoxylin before cover-slipping.

Homogenisation buffer details

50mM sodium phosphate, pH 7.0, 10mM sodium pyrophosphate, 0.5mM phenylmethylsulphonyl fluoride, 2mM Sodium ortho-vanadate, 2mM EGTA, 2mM EDTA, 20mM sodium fluoride, 1mM DTT, 1:100 dilution Phosphatase Inhibitor Cocktail 1, 1:100 dilution Phosphatase Inhibitor Cocktail 2 and 1 Complete Protease Inhibitor Cocktail Tablet per 50ml.

AlphaScreen assays procedure

Briefly, 10µl / well of optimized antibody/acceptor bead mix were added to 384 well assay plates together with 5µl/well of sample or standard diluted in AlphaScreen assay buffer (0.1% casein in DPBS). After overnight incubation at 4oC 5µl/well of the streptavidin-coated donor beads diluted in AlphaScreen buffer was added to each well and plates incubated in the dark with gentle agitation at room temperature for 4 hours. Plates were read at excitation 680nm and emission 520-620nm using an Envision plate reader.

Concentration of protein and tau in brain extracts

Total protein was quantified by Coomassie plus (Bradford) protein assay (Thermo Scientific, UK) according to the manufactures instructions. Tau protein levels were determined by Alphascreen assays (see above) using total tau (DA9; kind gift from Peter Davies) and AT8 (pSer202 and pThr205; prepared in-house) antibodies. Paired helical filament (PHF) tau purified from Alzheimer’s disease tissue was used to generate a standard curve and tau concentrations were calculated as PHF tau per microgram of protein.

Brain extract / [Protein] ug/ml / [Total tau]* / [AT8 tau]*
P301S / 8416 / 0.0182 / 0.0013
Wild-type / 5394 / 0.0060 / -

*ug PHF tau/ug protein

Results

Time-dependent maturation of pre-tangles into NFTs in P301SBE mice

To investigate the relative proportion of PG-5-positive, silver-negative tau inclusions (pretangles) versus PG-5-positive and silver-positive inclusions (NFTs), the number of Gallyas positive inclusions was counted in CA1 and expressed as the percentage of PG-5 inclusions quantified in an adjacent tissue section (Fig. 2c). The number of PG-5-positive inclusions was consistently greater than that of Gallyas positive inclusions. On the infused side of P301SBE mice at 2 weeks post-infusion, only ~24% of the PG-5-positive inclusions were Gallyas positive, implying that the majority of inclusions were pretangles [1]. The percentage of silver-positive inclusions increased to ~57% at 1 month and ~73% at 2 months post-infusion. A similar time-dependent increase was observed on the contralateral side of P301SBE mice; however, between the 2 week- and 2 month-time-points, the contralateral side had significantly fewer silver-positive inclusions than the infused side. By 2.5 months, the majority of inclusions in P301SBE mice was silver-positive, with no significant difference between the infused (~83%) and contralateral (~78%) sides. In contrast, silver-positive inclusions were a minority in control mice, even at 2.5 months post-infusion; their percentage was substantially lower with no difference between the infused (~33%) and contralateral (32%) sides.

Analysis of tau propagation in the cerebral cortex

The cortex at the level of the infusion was separated into dorsolateral (containing visual and auditory cortices) and ventrolateral (containing perirhinal cortex, entorhinal cortex and the amygdaloid complex) regions. The former does not have direct connections with the hippocampal formation, but is proximally closer to the infusion co-ordinates, whereas the latter is distal to the infusion coordinates, but has strong and complex connections with the hippocampal formation. Examination of both P301SBE and control mice (2.5 months post-infusion) showed mild to moderate tau pathology in the dorsolateral region of the cortex and severe pathology in the ventrolateral region (beginning at the rhinal fissure). Quantification showed evidence of tau propagation in the ventrolateral, but not the dorsolateral region, despite the latter being closer to the infusion co-ordinates (Table 3); however, a subset of P301SBE mice did have PG-5-positive neurons clustered in the general vicinity of the upper infusion coordinate (data not shown). Although tau pathology in the ventrolateral cortex was significantly higher than that seen in age-matched controls, it was no more severe on the infused than the contralateral side (Supplementary Table 1) consistent with a bilateral mode of spread. This data was consistent with the biochemical quantification of tau in similar regions (VI and VC; Supplementary Fig. 3).

Analysis of Iba-1 for evidence of microgliosis

At 1-day post-infusion, the contralateral hippocampus of P301SBE and PBS mice contained microglia with small cell bodies and ramified processes suggestive of a ‘resting’ state [3]. In contrast, the infused hippocampus contained larger, more ameboid microglia with shorter/thicker processes; features indicative of a more ‘reactive’ state [3]. These changes were most prominent in the dentate gyrus and CA1 (especially near the intended infusion co-ordinates), but were also present in CA3 (Supplementary Fig. 6). Microglial burden in these regions, as determined by image analysis, was unchanged between 1 day and 1 month post-infusion. At 2.5 months, there was an increase in microglial burden in the infused hippocampus of both groups, but particularly in P301SBE-infused mice (Supplementary Fig. 6a-c). This P301SBE-asscociated increase was most apparent in the CA3 region where microglia on the infused side had a more ‘reactive’ morphology and showed a mild increase in burden when compared to PBS mice (Supplementary Fig. 6c), although this difference did not reach statistical significance (P =0.08). The subtle increase in microgliosis appeared to be specific to the hippocampus (CA3) of P301SBE mice as qualitative analysis in the LSN and ADthalamus did not identify any changes in microglial burden or morphology, despite these regions showing strong evidence of tau propagation (data not shown).

Figure legends

Supplementary Fig. 1 Regions of interest for neuropathological characterisation and quantification

Tau pathology was analysed at multiple coronal levels both anterior (a-d) and posterior (h-i) to the infusion co-ordinates (e). Regions of interest included the: a) nucleus accumbens (ACB), caudate/putamen (CP; striatum); b) lateral septal nucleus (LSc); c) CA3, dentate gyrus (DG), anteriodorsal thalamus (AD), anterioventral thalamus (AV), globus pallidus (GP), ventrolateral thalamus (VAL), dorsal fornix (df), ventral fornix (f), internal capsule (int); d) Hippocampus region (pale red), dentate gyrus (DG), CA3, CA1, retrosplenial cortex ((RSPv), ventrolateral cortex, dorsolateral cortex, lateral geniculate nucleus (LG); e) infusion co-ordinates (red circles); f) dorsal subiculum, medial-lateral mammillary nucleus (MN), supramammillary nucleus (SUMm); g) dentate gyrus (DG); h) para-hippocampal region (PRE, POST). (Adapted from [2] CD-ROM).

Supplementary Fig. 2 Characterisation of P301SBE-induced hippocampal tau pathology

Quantification of PG-5-positive neurons on the infused (solid shapes) and contralateral (empty shapes) sides of specific hippocampal sub-regions in P301SBE- (red) and WTBE- (blue) or PBS- (green) infused mice 2.5 months post-infusion. Statistics: Group-wise comparison, One-way ANOVA with Bonferroni's post hoc test; ***, P <0.001; ****, P <0.0001. Abbreviations: BE, brain extract; PBS, phosphate buffered saline; WT, wild-type.

Supplementary Fig.3 Tau biochemistry concurs with immunohistochemical characterisation of P301SBE mice

A subset of P301SBE mice (2.5 months post-infusion; n=5) were prepared for biochemical analysis by dissecting (checked lines) the fresh brain as shown in the schematics (a and b). a) Using the adult mouse brain matrix (see methods) the whole brain was dissected in the coronal plane at slots #2, #6 and #11. The olfactory bulb was discarded leaving anterior, middle (b) and posterior segments which were then cut down the midline to give infused and contralateral sides for each segment. b) The middle segment which contains the sites of infusion (yellow dots) was also cut in the horizontal plane to give dorsal and ventral segments, as shown. These individual tissue segments were then fractionated to give S1 (containing soluble tau) and P1 (containing insoluble tau) fractions. c) Biochemical analysis of the P1 fraction using a PG-5 specific AlphaScreen assay showed that this abnormally phosphorylated/insoluble species of tau was significantly increased in the dorsal-infused (DI) segment of P301SBE mice (containing the infused hippocampus) when compared to the dorsal-contralateral (DC) segment (containing the contralateral hippocampus). As similar pattern was observed in the anterior segments of the brain, although the amount of abnormal tau was more variable and the difference between infused (AI) and contralateral (AC) sides was not significant. In contrast, the amount of abnormal tau in ventral (VI and VC) and posterior (PI and PC) segments did not show any differences between the infused and contralateral sides despite containing substantial tau pathology. PI and PC segments contain the cerebellum and brainstem, the latter of which is severely affected in the P301S transgenic line. d) Consistent with Alphascreen results, analysis of the P1 fraction by western blotting using an AT8 specific antibody also showed a significant increase in abnormally phosphorylated tau pathology in the DI segment when compared to DC segment. These biochemical findings were in line with immunohistochemical results showing increased tau pathology in the infused versus the contralateral hippocampus (Figure 1). Statistics: Pair-wise comparison, Student's t test; **, P <0.01. Abbreviations: PC, posterior-contralateral; PI, posterior-infused; AC, anterior-contralateral; AI, anterior-infused; DC, dorsal-contralateral; DI, dorsal-infused; VC, ventral-contralateral; VI, ventral-infused.

Supplementary Fig. 4 Extra-hippocampal regions showing early and severe evidence of tau propagation

The subiculum (sub), retrosplenial cortex (RSC) and medial-lateral mammillary nucleus (mlMN) showed evidence of P301SBE-induced tau pathology as early as 2 weeks post-infusion and were severely affected by 2.5 months. Representative images of PG-5-positive tau pathology in these regions at 2 weeks and 2.5 months post-infusion are shown along with a PBS infused control. Abbreviations: BE, brain extract; PBS, phosphate buffered saline; suMN, supramammillary nucleus; dg, dentate gyrus.

Supplementary Fig. 5 Axonal tau pathology in the ventral hippocampal commissure

The ventral hippocampal commissure (VHC), which can be visualised in a longitudinal section at bregma -0.46, inter-connects the right and left hippocampi. As shown, PG-5 IHC in P301SBE mice at 2.5 months post-infusion identified tau-positive 'dot-like' structures in the VHC similar to those seen in the ventral and dorsal fornix (Fig. 4). The morphology and location of these dot-like structures are compatible with tau aggregates in neuronal axons. Axonal tau pathology was absent or mild in age matched controls. Abbreviations: BE, brain extract; PBS, phosphate buffered saline. Scale bars: 50um.

Supplementary Fig. 6 Increased microgliosis in P301SBE-infused mice

Iba-1-positive microgliosis was analysed as an early marker of neurodegeneration. Subregions of the hippocampus, including the dentate gyrus (a), layer CA1 (b) and layer CA3 (c), were analysed separately, because the dentate gyrus and layer CA1 are disrupted by the needle during the infusion procedure. Microgliosis was slightly greater on the infused side than on the contralateral side in all sub-regions of the hippocampus at 1 day post-infusion, with no difference between P301SBE mice and controls. Microgliosis was unchanged at 1 month post-infusion. At 2.5 months post-infusion, the dentate gyrus and layer CA1 were associated with an increase in microgliosis that was specific to the infused side and was present in both groups. In CA3, microgliosis was increased (P = 0.08) on the infused side of P301SBE mice when compared to controls. Representative images of Iba-1 staining in the CA3 region of P301SBE mice and PBS controls show more reactive (larger, more amoeboid, intensely stained) microglia on the infused side of both groups at 1 day post-infusion. Microglia returned to their ramified state at 1 month post-infusion, but at 2.5 months, microglia on the infused side of P301SBE mice showed signs of activation similar to those seen at 1 day post-infusion. Statistics: Pair-wise comparison, Student's t test; # = P >0.05 and ≤0.08 (trend). Abbreviations: BE, brain extract; PBS, phosphate-buffered saline. Scale bars: 100um.

1

Ahmed et al

P301S tau propagation model

Tables

Supplementary Table 1 Tau propagation to brain regions distal to the infusion site

The density of PG-5-positive neurons or the percentage area occupied by PG-5-positive immunoreactivity (*) was quantified in various brains regions distal to the infusion site in P301SBE- or PBS-infused mice at 2.5 months post-infusion, both on the infused and contralateral sides. For each brain region statistical analysis was performed to identify significant (P <0.05) differences in tau pathology between 1) P301SBE-infused side and PBS controls, 2) P301SBE-contralateral side and PBS controls and 3) infused and contralateral sides of P301SBE mice. Tau propagation was defined as a region showing significantly more tau pathology in P301SBE mice compared to PBS controls (highlighted in red); those that did not are highlighted in green. For comparison, a similar analysis of the hippocampal region representing the infusion site was also included (blue shading). Statistics: Group-wise comparison, One-way ANOVA with Dunnett's post hoc test; Pair-wise comparison, Student's t test; red shading, P <0.05; green shading, P >0.08; yellow shading, P >0.05 and ≤0.08 (trend). Abbreviations: BE, brain extract; PBS, phosphate-buffered saline; Sup., supplementary.

Statistical analysis (P = )
Region of interest / bregma / P301SBE-infused vs PBS-groups / P301SBE-contralateral vs PBS-groups / P301SBE -infused vs -contralateral / Tau propagation? / Fig.
Retrosplenial cortex / -2.50 / 0.000 / 0.000 / 0.000 / Yes / 3a,3m
Para-hippocampal region / -4.50 / 0.000 / 0.031 / 0.003 / Yes / 3c
Hippocampus region / -2.50 / 0.000 / 0.000 / 0.006 / Yes / 1b
Dorsal subiculum* / -2.50 / 0.000 / 0.016 / 0.007 / Yes / 3b
Dentate gyrus / -2.50 / 0.000 / 0.002 / 0.009 / Yes / Sup.2
CA3 / -2.50 / 0.000 / 0.000 / 0.011 / Yes / Sup.2
CA1 / -2.50 / 0.000 / 0.000 / 0.043 / Yes / Sup.2
Medial-lateral mammillary nucleus* / -2.50 / 0.000 / 0.000 / 0.132 / Yes / 3d, 3o
Lateral septal nucleus (dorsal) / 0.00 / 0.000 / 0.000 / 0.589 / Yes / 3f, 3n
Anterioventral thalamus* / -1.00 / 0.001 / 0.002 / 0.026 / Yes / 3h
Dentate gyrus (posterior) / -4.00 / 0.001 / 0.044 / 0.138 / Yes / -
Dentate gyrus (anterior) / -1.00 / 0.001 / 0.013 / 0.088 / Yes / -
CA3 (anterior) / -1.00 / 0.006 / 0.001 / 0.299 / Yes / -
Supramammillary nucleus* / -2.50 / 0.010 / 0.008 / 0.378 / Yes / 3e
Ventrolateral cortex / -2.50 / 0.014 / 0.057 / 0.808 / Yes / -
Anteriodorsal thalamus / -1.00 / 0.032 / 0.928 / 0.079 / Yes / 3g
Nucleus accumbens / 1.00 / 0.035 / 0.166 / 0.432 / Yes / 3i
Dorsolateral cortex / -2.50 / 0.404 / 0.287 / 0.750 / No / -
Caudate/putamen / 1.00 / 0.409 / 0.939 / 0.317 / No / 3l
Globus pallidus / -1.00 / 0.671 / 0.951 / 0.536 / No / 3k
Ventrolateral thalamus* / -1.00 / 0.785 / 0.996 / 0.404 / No / -
Lateral geniculate nucleus* / -2.50 / 0.943 / 0.368 / 0.244 / No / 3j
One-way ANOVA / Unpaired t test

1