The ε3 and ε4 Alleles of Human APOE Differentially Affect Tau Phosphorylation in Hyperinsulinemic and Pioglitazone Treated Mice

Impaired insulin signalling is increasingly thought to contribute to Alzheimer's disease (AD). The ε4 isoform of the APOE gene is the greatest genetic risk factor for sporadic, late onset AD, and is also associated with risk for type 2 diabetes mellitus (T2DM). Neuropathological studies reported the highest number of AD lesions in brain tissue of ε4 diabetic patients. However other studies assessing AD pathology amongst the diabetic population have produced conflicting reports and have failed to show an increase in AD-related pathology in diabetic brain. The thiazolidinediones (TZDs), peroxisome proliferator-activated receptor gamma agonists, are peripheral insulin sensitisers used to treat T2DM. The TZD, pioglitazone, improved memory and cognitive functions in mild to moderate AD patients. Since it is not yet clear how apoE isoforms influence the development of T2DM and its progression to AD, we investigated amyloid beta and tau pathology in APOE knockout mice, carrying human APOEε3 or ε4 transgenes after diet-induced insulin resistance with and without pioglitazone treatment.Male APOE knockout, APOEε3-transgenic and APOEε4-transgenic mice, together with background strain C57BL6 mice were kept on a high fat diet (HFD) or low fat diet (LFD) for 32 weeks, or were all fed HFD for 32 weeks and during the final 3 weeks animals were treated with pioglitazone or vehicle.All HFD animals developed hyperglycaemia with elevated plasma insulin. Tau phosphorylation was reduced at 3 epitopes (Ser396, Ser202/Thr205 and Thr231) in all HFD, compared to LFD, animals independent of APOE genotype. The introduction of pioglitazone to HFD animals led to a significant reduction in tau phosphorylation at the Ser202/Thr205 epitope in APOEε3 animals only. We found no changes in APP processing however the levels of soluble amyloid beta 40 was reduced in APOE knockout animals treated with pioglitazone

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

APOE mice develop diet-induced T2DM- like insulin resistance.

<p>Plasma insulin levels were measured in APOE KO, APOEε3, APOEε4 and WT mice over 32 weeks of feeding with A) LFD and B) HFD. Blood samples from the tail vein at each OGTT were collected and plasma was analysed for insulin by ELISA. Values are mean±SEM, n = 10–12. In A) plasma insulin levels in all LFD animals reached *p<0.05 by t-test compared to WT mice. In B) plasma insulin levels in HFD fed animals reached *p<0.05 or ***p<0.001 by t-test compared to WT mice as indicated. Significance is indicated by * APOE KO, ⁺ APOEε3, ∧ APOEε4.</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

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

Body weight increases in HFD fed mice.

<p>Body weight of APOE KO, APOEε3, APOEε4 and WT mice over 32 weeks of A) LFD and B) HFD, starting from 3 months of age. Values are mean±SEM, n = 10-12. Body weights in A) and B) reached ***p<0.001 by t-test at all weeks indicated compared to WT mice. Significance is indicated by * APOE KO, ⁺ APOEε3, ∧ APOEε4.</p

Plasma Transthyretin as a Candidate Marker for Alzheimer's Disease

Abstract. Diagnosis of the progressive neurodegenerative disorder Alzheimer’s disease (AD) can only definitively be made postmortem. The most promising AD biomarkers identified to date are found in cerebrospinal fluid (CSF). Among these, one of the most interesting candidates is transthyretin (TTR), the carrier of thyroxine and retinol, which also binds with amyloid- (A), and it has been suggested that it protects against A deposition. A biomarker detectable in plasma would have great diagnostic value and could be of use for determining disease progression and the monitoring of therapeutic efficacy due to its greater accessibility over CSF-based markers. We aimed to validate TTR as a prognostic marker in AD and to determine its relation with cognitive measures. We examined the plasma protein levels of TTR in 90 people with late-onset AD and 50 age-matched non-demented controls (NDC) by immunoblotting and found lower plasma TTR levels in AD compared to NDC (p = 0.004). We then quantified plasma TTR by enzyme-linked immunosorbent assays in a larger independent cohort (n = 270) including subjects with mild to severe AD. Plasma TTR levels were significantly lower in AD cases with rapid cognitive decline and with severe cognitive impairment. Regression analyses showed plasma TTR levels also predicted cognitive decline over the ensuing 6 months. These data indicate that plasma TTR is a strong candidate AD biomarker that should be included in the development of blood based biomarker panels for disease diagnosis and also suggests that plasma TTR is a marker of diseas