Additional file 2: of Red blood cell indices and anaemia as causative factors for cognitive function deficits and for Alzheimer’s disease

Figure S1. Anaemia has a significant effect on four cognitive test measures. Figure S2. Two sample MR results replicating the direction of effect for MCH on the verbal–numeric reasoning outcome. Figure S3. Rate of change as a measure of red blood cell count. (PDF 371 kb

Additional file 1: of Red blood cell indices and anaemia as causative factors for cognitive function deficits and for Alzheimer’s disease

Table S1. Sample set description. Table S2. MR replication in release 2 of the UK Biobank data. Table S3. MR results for RET trait. Table S4. General linear model results show an association between AD patients and red blood indices in UK Biobank. Table S5. Significantly enriched KEGG pathways for MCH decline correlated genes. (XLSX 18 kb

Older flies are more vulnerable to chronically induced Arctic Aβ42, despite a lower lifetime exposure to the peptide.

<p>(A) UAS-Arctic Aβ42/+; elavGS/+ females were chronically treated with 200 µM RU486 from 2-days (RU 200 [2d chronic]) and 20-days (RU 200 [20d chronic]) post eclosion. Total (soluble and insoluble) Arctic Aβ42 protein levels were assayed every 5 days until flies started dying. Data are presented as means ± SEM and were analysed by two-way ANOVA and Tukey's HSD (n = 3). Aβ42 levels were higher in RU 200 [2d chronic] versus RU 200 [20d chronic] flies at all time-points up to 25 days post-induction (<i>P</i><0.05, Tukey's HSD) except day 5. (B) A crude measure of the lifetime Arctic Aβ42 load (from induction until the start of decline in survival) was determined in 2d and 20d-induced flies by calculating the area under the curves in (A). Data are presented as the means ± SEM (n = 3). The lifetime Arctic Aβ42 load was significantly lower in RU 200 [20d chronic] than in RU 200 [2d chronic]-treated flies (**<i>P</i><0.01, student's t-test). (C) Survival, expressed as a percentage of non-RU486-treated controls, plotted from the age of RU486 induction. RU 200 [20d chronic] flies exhibited a significant reduction in relative survival following RU486 treatment compared to RU 200 [2d chronic] flies (<i>P</i><0.05, Wilcoxon matched pairs sign rank test). Original survival curves are depicted in Fig S4B.</p

Ageing increases Arctic Aβ42 peptide accumulation and toxicity when transcript levels are equalised at different ages.

<p>UAS-Arctic Aβ42/+; elavGS/+ females were pulsed, for 1 week, either at 2 or 5 days old with 50 µM RU (RU 50 [2–9d] or [5–12d]) or 20 days old with 200 µM RU (RU 200 [20–27d]). Aβ42 mRNA, lifespan and protein levels were then measured. Data are presented as means ± SEM. (A) Arctic Aβ42 transcripts were measured at the end of the induction period (see methods). Aβ42 transcript levels were not significantly different between RU 50 [5–12d] and RU 200 [20–27d] flies under these induction conditions (<i>P</i> = 0.5, student's t-test, n = 4). A more detailed analysis of Aβ42 expression levels at varying RU486 concentrations can be found in Fig S1. (B) Arctic Aβ42 peptide accumulates in older flies at equivalent levels of Aβ42 mRNA. Total (soluble and insoluble) Aβ42 peptide was extracted using GnHCl (see methods), and levels were measured by ELISA at the end of the RU486 pulse (0d) then 1, 2 and 3 weeks following the switch to RU486-free medium. Data were analysed by two-way ANOVA followed by Tukey's HSD post-hoc (n = 4). RU 200 [20–27d] flies contained significantly more Aβ42 peptide than RU 50 [5–12d] flies at all time-points measured (<i>P</i><0.0001). No significant difference in protein levels was observed up to 3 weeks following switch-off at either age of induction (<i>P</i> = 0.382). (C) Equivalent levels of Aβ42 transcripts reduced survival only when induced in old flies (RU 200 [20–27d]). Arrows indicate the period of RU486 treatment. Median lifespans were: 60 days for −RU, 58 days for RU 50 [5–12d], and 53 days for RU 200 [20–27d] flies. <i>P</i> = 0.18 comparing −RU to RU 50 [5–12d] flies, <i>P</i><0.0001 comparing −RU to RU 200 [20–27d] flies (log-rank test). (D) Protein translation, as measured by ³⁵S-methionine incorporation, decreases with age in heads of flies conditionally over-expressing Arctic Aβ42. Data were analysed by Student's t-test (*<i>P</i><0.05, comparing RU 50 [2–9d] to RU 200 [20–27d] flies, n = 5). (E) Proteasome activity, as measured using the fluorogenic peptide substrate LLVY-AMC, is reduced with age in the heads of flies conditionally over-expressing Arctic Aβ42 as well as non-RU-treated controls. Data are presented as mean activities (pmoles/min/mg protein) ± SEM (n = 5). *** <i>P</i><0.0001 comparing 9 versus 27 day-old flies, <i>P</i> = 0.429 comparing + versus – RU486 (two-way ANOVA); <i>P</i><0.05 comparing RU 50 [2–9d] to RU 200 [20–27d] flies and –RU day 9 to –RU day 27 flies (Tukey's HSD).</p

Effects of varying RU486 exposure time on Aβ42 levels and toxicity.

<p>2-day-old UAS-Arc Aβ42/+; elavGS/+ females were treated with 200 µM RU486 for 2, 4, 7 or 14 days before being transferred to RU486-free medium (see table S1 for pulse conditions). Flies maintained chronically on – RU486 (−RU) and 200 µM RU486 (+RU) were used as negative and positive controls, respectively. Data are presented as means ± SEM and were analysed by ANOVA followed by Tukey's honestly significant difference (HSD) post-hoc test. (A & B) Removal of RU486 resulted in rapid clearance of Arctic Aβ42 mRNA as well as soluble, but not insoluble, protein levels. (A) Aβ42 mRNA levels were measured by quantitative RT PCR prior to RU486 treatment (−RU), at the end of treatment (+RU 7 d), and at the indicated time-points following the switch to RU486-free food (see methods). ***<i>P</i><0.001 comparing the level of Arctic Aβ42 mRNA at the end of RU treatment to all other conditions (n = 3). (B) Soluble and insoluble Arctic Aβ42 was fractionated (see methods) and measured by ELISA at the indicated time-points (n = 3). <i>P</i><0.0001 comparing soluble and insoluble Aβ42 fractions (two-way ANOVA). Soluble Aβ42 was reduced to baseline levels following switch-off (see inset; *<i>P</i><0.05 comparing +RU 7d to 2 or 7d following transfer to −RU food and no significant difference comparing 2 or 7d following switch to −RU to non-RU-treated controls; Tukey's HSD), whereas insoluble Aβ42 was highly stable for 1 week following cessation of transgene expression (no significant difference between +RU 7d and 2 or 7d following switch to −RU, Tukey's HSD). (C–F) Arctic Aβ42 protein levels correlate with increasing duration of RU486 exposure and associate with impairments in function. (C) Arctic Aβ42 mRNA levels were quantified at the end of each RU486 pulse, as indicated. Aβ42 transcript was significantly increased under all RU treatment conditions compared to untreated controls (***<i>P</i><0.001, n = 5, Tukey's HSD), with no significant difference (n.s.) between the varying RU486 pulse conditions. (D) Total (soluble and insoluble) Aβ42 peptide was extracted using Guanidine HCl and levels were measured by ELISA at the end of each RU486 pulse (see methods). Total Arctic Aβ42 protein levels increased with the length of RU486 exposure (<i>P</i><0.0001, one-way ANOVA, n = 3). *<i>P</i><0.05 comparing 2, 4 and 7 day pulses, but no significant difference between 7 and 14 day pulses. <i>P</i><0.05 comparing −RU to all + RU treatments conditions (Tukey's HSD). (E) Climbing ability was assessed at the indicated time-points. Arrows indicate the period of RU486 treatment for each pulse. Data were analysed by two-way ANOVA and Tukey's HSD (n = 3, number of flies per group = 39–45). Negative geotaxis decreased with increasing RU486 exposure length. Pulse lengths ≥4 days were significantly different from −RU controls, whereas pulse lengths ≤4 days were significantly different from chronically treated (+RU) controls (<i>P</i><0.05, Tukey's HSD). A 2-day pulse of RU486 did not significantly induce climbing defects compared to untreated flies, whereas a 7-day pulse did not significantly improve the reduced climbing ability compared to chronically treated +RU controls. (F) Survival decreased with increasing RU486 exposure time. Median lifespans were: 62 days for –RU (chronic), 55 days for +2d RU, 52 days for +4d RU, 45 days for +7d RU and 27 days for + RU (chronic). All survival curves were significantly different from each other (<i>P</i><0.001, log rank test).</p

Older flies are more vulnerable to a standardised dose of Arctic Aβ42 peptide.

<p>(A & B) UAS-Arctic Aβ42/+; elavGS/+ females were treated, for 2 weeks, with 100 µM RU486 from 5 days (RU 100 [5–19d]) and 200 µM RU486 from 20 (RU 200 [20–34d]) and 30 (RU 200 [30–44d]) days post-eclosion, and were maintained on RU486-free medium prior to and following the pulse period. (A) Total Aβ42 protein levels (soluble and insoluble as measured by GnHCl extraction) were equivalent at the end of the RU pulse when comparing each age of induction. Data are expressed as means ± SEM (n = 5) and were compared using one-way ANOVA and Tukey's HSD post-hoc. No significant differences were observed. (B) Survival, expressed as a percentage of non-RU486-treated controls, plotted from the age of RU486 induction. Arrow indicates the induction period. Older flies exhibit a significantly greater reduction in relative survival following RU treatment (<i>P</i><0.05 comparing RU 200 [20–34d] and RU 200 [30–44d] with RU 100 [5–19d] and RU 200 [20–34d] with RU 200 [30–44d], Wilcoxon matched pairs sign rank test). Raw survival curves are depicted in Fig S4A. (C) UAS-Arctic Aβ42/+; elavGS/+ females were pulsed for 1 week with 200 µM RU486 from 5 (RU 200 [5–12d]) and 20 (RU 200 [20–27d]) days post-eclosion, to equalise the total Aβ42 level in young versus old-induced flies (see Fig S3). Soluble and insoluble Aβ42 was fractionated (see methods) and measured by ELISA at 1 and 7 days following induction, then 7, 21 and, in the case of RU 200 [5–12d] flies, 35 days following the switch to RU486-free food. Data are expressed as means ± SEM and were analysed by two-way ANOVA (n = 4). No significant differences were observed comparing RU 200 [5–12d] versus RU 200 [20–27d]-induced flies for either soluble (<i>P</i> = 0.428) or insoluble (<i>P</i> = 0.258) Aβ42 fractions. Equivalent levels of soluble and insoluble Aβ42 were observed 1 day following induction (n.s. difference between insoluble Aβ42 at day 1 versus soluble Aβ42 at days 1 and 7 following RU treatment). Soluble Aβ42 levels did not vary significantly during the induction period (<i>P</i> = 0.546 comparing flies on RU486 for 1 day versus 7 days). Soluble Aβ42 was significantly reduced following switch-off (<i>P</i><0.001 comparing flies at the end of the 7-day induction period with flies switched to −RU food), reaching baseline levels within 7 days (<i>P</i> = 0.99 comparing RU 200 [5–12d] flies switched to −RU food for 7, 21 and 35 days and <i>P</i> = 0.628 comparing RU 200 [20–27d] flies switched to −RU for 7 and 21 days). Insoluble Aβ42 levels increased significantly during the RU486 induction period (<i>P</i> = 0.016 comparing flies on RU486 for 1 day versus 7 days, <i>P</i><0.05 comparing insoluble Aβ42 levels in flies on RU for 7 days versus soluble Aβ42 levels at days 1 and 7 following induction). Insoluble Aβ42 was not significantly reduced following switch-off (<i>P</i> = 0.886 comparing flies at the end of the 7-day induction period with flies switched to −RU food) and remained stable for several weeks following transfer to RU486-free food (<i>P</i> = 0.744 comparing RU 200 [5–12d] flies switched to −RU food for 7, 21 and 35 days and <i>P</i> = 0.748 comparing RU 200 [20–27d] flies switched to −RU for 7 and 21 days).</p

Pioglitazone treatment selectively lowers tau phosphorylation at the Ser202/Thr205 epitope.

<p>APOE mice fed HFD for 32 weeks were treated with pioglitazone or vehicle for the final 3 weeks. A) ptau Ser396, B) Ser202/Thr205 (AT8), C) Thr231 (AT180). *p<0.05 by t-test.</p

Levels of soluble Aβ40 and Aβ42 in HFD fed mice treated with or without pioglitazone.

<p>Aβ A) x-40 and B) x-42 was analysed by ELISA in the frontal cortex of APOE mice fed HFD and with or without pioglitazone treatment. *p<0.05 by t-test.</p

Tau phosphorylation is reduced in HFD fed APOE mice, independent of genotype.

<p>The frontal cortex from APOE mice were homogenised in sucrose homogenisation buffer, lysates were then immunoblotted with the indicated antibodies. A) Western blot of tau antibodies in frontal cortex of APOE KO, APOEε3, APOEε4 and WT mice fed LFD or HFD. B) Density of phosphorylated tau normalised against total tau. C) Tau phosphorylation is dependent on diet. Phosphorylated tau is normalised to total tau, data is grouped by diet. *p<0.05; **p<0.01; ***p<0.001 by t-test.</p

Animals develop glucose intolerance over 32 weeks of HFD.

<p>Oral glucose tolerance tests (OGTT) were conducted at baseline and then at 6 weekly intervals. Animals were fasted overnight. The next morning, animals were placed in a warming chamber prior to blood sampling and blood was taken by direct venopuncture. Mice were then given a single oral dose of glucose (3 g/kg p.o dose volume 10 ml/kg) and serial blood samples collected from the tail tip post-dose at 30, 60, 90, 120 and 180 mins. Glucose levels were accessed using a blood glucose meter. Graphs show plasma glucose concentrations at the beginning, at week 6, week 24 and week 32 of APOE KO, APOEε3, APOEε4 and WT mice fed A, C, E, G) LFD and B, D, F, H) HFD. Values are mean±SEM, n = 10–12. *p<0.05, **p<0.01; ***p<0.001 versus WT mice by t-test. Significance is indicated by * APOE KO, ⁺ APOEε3, ∧ APOEε4.</p