Supplemental Material - Introducing the ‘3 Fs model of complexity’ for people with dementia accessing a NHS mental health in-patient dementia assessment ward: An interpretive description study

Supplemental Material for Introducing the ‘3 Fs model of complexity’ for people with dementia accessing a NHS mental health in-patient dementia assessment ward: An interpretive description study by Lesley Jones, Nicky Cullum, Ruth Watson and John Keady in Dementia</p

Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in <i>StHdh</i>^<i>Q7/7</i> and <i>StHdh</i>^{<i>Q111/111</i>} cells at 0 mins and 10 mins of 100ng/ml EGF stimulation, either with or without a prior 2 hour incubation with 500nM AKT inhibitor VIII or 1μM MEK 1/2 inhibitor.

<p>In cases where inhibitors were not used, cells were incubated with the equivalent volume of DMSO for the same amount of time prior to treatment and processing. Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. Grey asterisks indicate genotypic differences. N = 3 replications *p<0.05, ** p<0.01, *** p<0.001.</p

Simplified cartoon of the hypothesised relationship between EGF-stimulated kinase activation, huntingtin localisation and transcriptional regulation.

<p>Upon binding to the EGFR, EGF elicits the phosphorylation of MEK and AKT. In the presence of mutant huntingtin, aberrant interactions with GRB2 and suppressed PHLPP1 expression serve to alter the phosphorylation response. In turn, active MEK and AKT regulate the subcellular localisation of huntingtin, possibly by post-translational modification; however this regulation is impaired due to mutant huntingtin-associated aberrant kinase phosphorylation. Regulation of huntingtin nuclear localisation may then influence transcriptional control via multiple mechanisms. As such, altered nuclear localisation of mutant huntingtin could disrupt the control of these mechanisms and would result in altered transcriptional regulation.</p

mHTT antibody binding in young and old animals from 5 HD mouse models.

<p>R6/1 mice demonstrate some diffuse staining (A) but high levels of frank inclusions from 4 months of age (B). In this mouse line EM48 was overall the most sensitive antibody, with 1C2 and ubiquitin the least. In young YAC128 mice that demonstrate only diffuse staining (C,D), ubiquitin and S830 were the most effective antibodies. As the mice aged MW8 became the most sensitive antibody. In the HdhQ92 mice, EM48 was the most effective antibody in the young animals (E) with S830 the most effective in the old (F) where the other four antibodies demonstrated a consistent level of mHTT detection. For the HdhQ150 strains, both lines were insensitive to EM48 and demonstrated relatively little binding of diffuse staining per se (original line G,H; B6 backcross I,J) with the antibodies detecting inclusions at high levels even at a young age. The original HdhQ150 line demonstrated consistent levels of detection with the four usable antibodies including 1C2, which was ineffective in the B6 line. MW8 was the most sensitive antibody in the B6 line. Significance markers omitted for clarity (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155834#pone.0155834.s001" target="_blank">S1 File</a> for raw data).</p

A. Subcellular localisation of an N-terminal epitope of huntingtin and mutant huntingtin in <i>StHdh</i>^<i>Q7/7</i>, <i>StHdh</i>^{<i>Q7/111</i>} and <i>StHdh</i>^{<i>Q111/111</i>} cell lines. Cells were fixed following 0, 5, 15 and 30 min. of stimulation with 100ng/ml EGF, labelled with Mab2166 against amino acids 181–810, then analysed by confocal microscopy. Scale bar = 20μm. B. Quantitative analysis of immunofluorescence images in <i>A</i>. Nuclear/Cytoplasmic (N/C) and Nuclear/Perinuclear (N/P) mean pixel intensity ratios (MPI) for <i>StHdh</i>^<i>Q7/7</i>, <i>StHdh</i>^{<i>Q7/111</i>} and <i>StHdh</i>^{<i>Q111/111</i>} cells following 0, 5, 15 and 30 min. of stimulation with 100ng/ml EGF.

<p>Mean pixel intensities were calculated from confocal microscopy images using GNU Image Manipulator. All images were randomised and analysed blind to genotype and length of time stimulated with EGF. Each condition consisted of 9 confocal microscopy images taken from 3 separate coverslips. n = 66–91. Error bars = ± SEM. Data representative of 3 experiments; * Denotes a significant difference from 0min.; # Denotes a significant difference from 5mins; */# p<0.05, **/## p<0.01, ***/### p<0.001.</p

Subcellular localisation of an N-terminal epitope of huntingtin and mutant huntingtin in Hdh^Q7/7, Hdh^Q7/111 and Hdh^Q111/111 primary cell lines.

<p>Cells were fixed following 0, 5, 15 and 30 min. of stimulation with 100ng/ml EGF, labelled with Mab2166, then analysed by confocal microscopy. Scale bar = 20μm. <b>B</b>. Quantitative analysis of immunofluorescence images in <b><i>A</i>.</b> Nuclear/Cytoplasmic (N/C) and Nuclear/Perinuclear (N/P) mean pixel intensity ratios (MPI) for Hdh^Q7/7, Hdh^Q7/111 and Hdh^Q111/111 primary cells following 0, 5, 15 and 30 min. of stimulation with 100ng/ml EGF. Mean pixel intensities were calculated from confocal microscopy images using GNU Image Manipulator. All images were randomised and analysed blind to genotype and length of time stimulated with EGF. Each condition consisted of 9 confocal microscopy images taken from 3 separate coverslips. n = 49–70. Error bars = ± SEM. Data representative of 3 experiments; * Denotes a significant difference from 0min.; *p<0.05, ** p<0.01.</p

Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in Hdh^Q7/7 and Hdh^Q111/111 primary cells at 0 mins and 10 mins of 100ng/ml EGF stimulation.

<p>Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. N = 5 replications. *** p<0.001.</p

A. <i>StHdh</i>^<i>Q7/7</i>, B. <i>StHdh</i>^{<i>Q7/111</i>} and C. <i>StHdh</i>^{<i>Q111/111</i>} cells treated with either AKT inhibitor VIII, MEK 1/2 inhibitor, or the equivalent volume of DMSO for 2 hours prior to 0, 5, 15 and 30 mins stimulation with 100ng/ml EGF, then probed with amino-terminal huntingtin antibody Mab2166. Scale bar = 20μm. D-E. Quantification of mean pixel intensity (MPI) from images represented in <i>A-C</i> for the D. Nuclear/Cytoplasmic (N/C) ratio and E. Nuclear/Perinuclear (N/P) ratio.

<p>Error bars = SEM. Light grey bars and asterisks signify statistically significant differences between DMSO conditions. Black asterisks and hashes indicate statistically significant differences between DMSO vs AKT inhibitor conditions and DMSO vs MEK inhibitor conditions, respectively. Data representative of three experiments. n = 85–135. */# p<0.05, **/## p<0.01, ***/### p<0.001.</p

A. The EGFR is present in <i>StHdh</i>^<i>Q7/7</i>, <i>StHdh</i>^{<i>Q7/111</i>} and <i>StHdh</i>^{<i>Q111/111</i>}, cells and has a similar subcellular localisation Scale bar = 20μm. B. Western Blot indicating similar expression of the EGFR protein in all three cell lines, α-tubulin was used as a loading control. C. Quantification of fluorescence intensity of protein bands in <i>B</i> normalised to loading control and expressed as a proportion of <i>StHdh</i>^<i>Q7/7</i> (FI/<i>StHdh</i>^<i>Q7/7</i>); there are no significant differences in the levels of EGFR between <i>StHdh</i>^<i>Q7/7</i>, <i>StHdh</i>^{<i>Q7/111</i>} and <i>StHdh</i>^{<i>Q111/111</i>} cells.

<p>Error bars = ± SEM. Images are representative of multiple experiments. N = 3 replications.</p

High power light microscopy images of mHTT antibody staining of striatal aggregation pathology in early disease HD mice.

<p>The R6/1 mice demonstrated NIIs with all antibodies (A,F,K,P,U), whereas the YAC128 mice demonstrate no NIIs but faint diffuse staining with all antibodies (G,L,Q,V) except S830 (B). The antibodies also failed to detect NIIs in the HdhQ92 line at this age (Photomicrographs C,H,M,R,W) but did demonstrate diffuse staining with all. Both HdhQ150 lines were insensitive to EM48 (N,O) with the original HdhQ150 line also being insensitive to 1C2 (X), but the Photomicrographs for S830 (D), MW8 (I) and ubiquitin (X) in the original line demonstrated good sensitivity for NIIs, as they did for the B6 variant (Photomicrographs E,J, Y respectively). Scale bar = 10 μm.</p