Liraglutide Reduces Vascular Damage, Neuronal Loss, and Cognitive Impairment in a Mixed Murine Model of Alzheimer's Disease and Type 2 Diabetes

Abstract

Alzheimer's disease is the most common form of dementia, and epidemiological studies support that type 2 diabetes (T2D) is a major contributor. The relationship between both diseases and the fact that Alzheimer's disease (AD) does not have a successful treatment support the study on antidiabetic drugs limiting or slowing down brain complications in AD. Among these, liraglutide (LRGT), a glucagon-like peptide-1 agonist, is currently being tested in patients with AD in the Evaluating Liraglutide in Alzheimer's Disease (ELAD) clinical trial. However, the effects of LRGT on brain pathology when AD and T2D coexist have not been assessed. We have administered LRGT (500 μg/kg/day) to a mixed murine model of AD and T2D (APP/PS1xdb/db mice) for 20 weeks. We have evaluated metabolic parameters as well as the effects of LRGT on learning and memory. Postmortem analysis included assessment of brain amyloid-β and tau pathologies, microglia activation, spontaneous bleeding and neuronal loss, as well as insulin and insulin-like growth factor 1 receptors. LRGT treatment reduced glucose levels in diabetic mice (db/db and APP/PS1xdb/db) after 4 weeks of treatment. LRGT also helped to maintain insulin levels after 8 weeks of treatment. While we did not detect any effects on cortical insulin or insulin-like growth factor 1 receptor m-RNA levels, LRGT significantly reduced brain atrophy in the db/db and APP/PS1xdb/db mice. LRGT treatment also rescued neuron density in the APP/PS1xdb/db mice in the proximity (p = 0.008) far from amyloid plaques (p < 0.001). LRGT reduced amyloid plaque burden in the APP/PS1 animals (p < 0.001), as well as Aβ aggregates levels (p = 0.046), and tau hyperphosphorylation (p = 0.009) in the APP/PS1xdb/db mice. Spontaneous bleeding was also ameliorated in the APP/PS1xdb/db animals (p = 0.012), and microglia burden was reduced in the proximity of amyloid plaques in the APP/PS1 and APP/PS1xdb/db mice (p < 0.001), while microglia was reduced in areas far from amyloid plaques in the db/db and APP/PS1xdb/db mice (p < 0.001). This overall improvement helped to rescue cognitive impairment in AD-T2D mice in the new object discrimination test (p < 0.001) and Morris water maze (p < 0.001). Altogether, our data support the role of LRGT in reduction of associated brain complications when T2D and AD occur simultaneously, as regularly observed in the clinical arena.

Keywords: Alzheimer’s disease; hemorrhage; inflammation; liraglutide; neuronal loss; type 2 diabetes.

FIGURE 1

Long-term liraglutide (LRTG) treatment ameliorates metabolic alterations in T2D and AD-T2D mice. (A) Non-fasting plasma glucose, (B) insulin, and (C) body weight were measured every 4 weeks in controls, APP/PS1, db/db and APP/PS1xdb/db mice treated with vehicle or LGRT 500 μg/kg for 20 weeks (from weeks 6 to 26) (complete statistical analysis included as a Supplementary Material). (A) LRGT significantly reduced postprandial glucose levels in diabetic mice: (^##p < 0.01 Control, Control-LRGT, APP/PS1, APP/PS1-LRGT; p < 0.01 vs. Control; ^††p < 0.01 Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db-LRGT, and APP/PS1xdb/db-LRGT; p < 0.001 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, and db/db-LRGT; p < 0.01 vs. Control-LRGT). (B) Insulin levels were maintained by long-term LRGT treatment. At 6 weeks of age, immediately before the commencement of LRGT treatment, insulin levels were significantly increased in APP/PS1xdb/db mice (^##p < 0.01 Control, Control-LRGT, APP/PS1, APP/PS1-LRGT; p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db, and APP/PS1xdb/db; p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, and APP/PS1xdb/db). (C) LRGT maintained body weight in APP/PS1-LRGT (^##p < 0.01 vs. Control, Control-LRGT, APP/PS1, and APP/PS1-LRGT; ^##p < 0.01 vs. Control, Control-LRGT, APP/PS1, and APP/PS1-LRGT; p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, and APP/PS1xdb/db). Data are representative of 5–12 animals, and differences were detected by one-way ANOVA followed by Tukey’s b or Tamhane test.

FIGURE 2

LRGT treatment reduced cognitive impairment in APP/PS1xdb/db mice. Control, APP/PS1, db/db, and APP/PS1xdb/db animals were analyzed in the new object discrimination test for “what,” “when,” and “where” paradigms (A) as well as in the (B,C) Morris water maze test. Behavioral assessment commenced on week 24, after 18 weeks of LRGT treatment (500 μg/kg/day), and was completed on week 26 (complete statistical analysis included as a Supplementary Material). (A) LRGT improved episodic memory in the new object discrimination test. No differences were observed for the “when” paradigm; however, significant improvement was observed for the “what” (^††p = 0.009 vs. Control, Control-LRGT, and APP/PS1-LRGT) and “where” (**p < 0.001 vs. rest of the groups) paradigms. (B) LRGT also improved the performance along the acquisition phase in the MWM (^††p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db, and db/db-LRGT; ^##p < 0.01 vs. Control, Control-LRGT, APP/PS1, and APP/PS1-LRGT; p < 0.01 vs. Control and Control-LRGT; p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db-LRGT, and APP/PS1xdb/db-LRGT). (C) In the retention of the MWM, we observed that LRGT treatment also improved the performance of APP/PS1xdb/db-LRGT mice (^††p = 0.002 vs. Control, Control-LRGT, and db/db-LRGT) (complete statistical analysis included in Supplementary Material). Data are representative of 5–12 animals, and differences were detected by one-way ANOVA followed by Tukey’s b or Tamhane test.

FIGURE 3

Brain atrophy, neuronal density, and curvature are reduced by LRGT treatment. (A,B) Brain weight, cortex and hippocampal size, (C,D) NeuN/DAPI ratio, and (E,F) axonal curvature ratio were analyzed in all four genotypes (Control, APP/PS1, db/db, and APP/PS1xdb/db) under study and compared with animals by week 26, after 20 weeks on LRGT treatment (500 μg/kg/day) (complete statistical analysis included as a Supplementary Material). (A) Long-term LRGT limited brain weight loss (p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db, and APP/PS1xdb/db). Cortical size was significantly improved by the LRGT treatment (p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db-LRGT, and APP/PS1xdb/db-LRGT; ^††p < 0.01 vs. Control, Control-LRGT, APP/PS1, and APP/PS1-LRGT). No differences were observed in the hippocampus. Data are representative of 4–5 animals. (B) Illustrative example of cresyl violet staining showing reduced cortical size in db/db and APP/PS1xdb/db mice. Scale bar = 200 um. (C) Neuronal density was reduced in the proximity of amyloid plaques in APP/PS1xdb/db mice, and LRGT ameliorated this situation (^††p = 0.008 vs. APP/PS1 and APP/PS1-LRGT). A similar profile is observed in cortical and hippocampal areas with no amyloid plaques (p < 0.001 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db-LRGT, and APP/PS1xdb/db-LRGT; ^##p < 0.01 vs. Control and Control-LRGT; + + p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db, and db/db-LRGT). Data are representative of five animals. (D) Illustrative example of NeuN (red) and DAPI (blue) staining in areas located in the proximity of amyloid plaques (TS staining, green) and in areas without amyloid plaques. Zoom-in images of representative regions are marked by white squares and presented next to the original image, including areas with and without amyloid plaques. Scale bar = 50 μm, insets scale bar = 25 μm. (E) LRGT reduced curvature ratio in the proximity of amyloid plaques (**p = 0.004 vs. rest of the groups) and in areas free of amyloid plaques (**p < 0.01 vs. rest of the groups, ^##p < 0.01 vs. Control and Control-LRGT, p < 0.01. vs. Control) (complete statistical analysis included in Supplementary Material). Data are representative of five animals per group (308–920 neurons/group). (F) Illustrative examples of SMI-312 (red) and TS (green) staining in the proximity of and far from amyloid plaques (yellow lines mark representative neurites). Scale bar = 10 μm.

FIGURE 4

LRGT affects amyloid and tau pathologies in APP/PS1xdb/db mice. (A,B) Amyloid plaque burden and size are quantified by thioflavin S and 4G8 staining. (C) Aβ40, Aβ42, and Aβ aggregates are also determined in APP/PS1 and APP/PS1xdb/db mice after liraglutide treatment (500 μg/kg/day) for 22 weeks. (D,E) Phospho-tau/total tau ratios are also measured in all the genotypes (Control, APP/PS1, db/db, and APP/PS1xdb/db) under study untreated or after LRGT treatment (complete statistical analysis included as a Supplementary Material). (A) LRGT treatment reduced amyloid plaque burden in the cortex from APP/PS1 mice (**p < 0.01 vs. rest of the groups; ^††p < 0.001 vs. APP/PS1xdb/db, and APP/PS1xdb/db-LRGT). No differences were observed in the hippocampus. LRGT treatment also reduced amyloid plaque size in the cortex from APP/PS1xdb/db mice (p < 0.01 vs. APP/PS1-LRGT and APP/PS1xdb/db-LRGT, p < 0.001 vs. APP/PS1-LRGT; **p < 0.01 vs. rest of the groups, ^##p < 0.001 APP/PS1xdb/db-LRGT). No differences were observed in the hippocampus. Data are representative of five animals. (B) Illustrative image of TS (green) and 4G8 (red) staining of amyloid plaques in the cortex from all the groups studied. Scale bar = 50 μm. (C) No differences were observed when soluble Aβ40 or Aβ42 levels were analyzed in the cortex. However, Aβ aggregates were significantly reduced in APP/PS1xdb/db mice on LRGT treatment (†p = 0.046 vs. APP/PS1xdb/db). No statistical differences were observed in the hippocampus for soluble Aβ40 or Aβ42 levels. Insoluble Aβ40 (‡p = 0.026 vs. APP/PS1) and Aβ42 (‡p = 0.011 vs. APP/PS1) levels are reduced in APP/PS1xdb/db when compared with APP/PS1 animals, and LRGT treatment contributes to further reductions in the cortex. No differences were observed in the hippocampus for insoluble Aβ40 or Aβ42. Data are representative of 6–9 animals. (D) Tau phosphorylation was reduced in the cortex after LRGT treatment (^††p = 0.009 vs. Control). Differences did not reach statistical significance in the hippocampus. Data are representative of 3–11 mice. (E) Illustrative example of Western blot for phospho-tau, total tau, and β-actin in the cortex from all the groups studied.

FIGURE 5

Spontaneous bleeding and inflammation are reduced after LRGT treatment while IR-A, IR-B, and IGF-1R mRNA expression is not affected. Prussian blue staining is used to quantify hemorrhage burden and density in the cortex from untreated and treated mice (LRGT, 500 μg/kg/day) (A,B). Microglia burden was quantified by Iba1 immunostaining in the proximity of (<50 μm) and far (>50 μm) from amyloid plaques in untreated and LRGT-treated animals (C,D). IR-A, IR-B, and IGF-1R mRNA expression is also determined in the cortex from untreated and LRGT-treated mice (E) (complete statistical analysis included as a Supplementary Material). (A) LRGT treatment reduces hemorrhage burden in the cortex (^†p = 0.012 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db-LRGT, and APP/PS1xdb/db-LRGT). A similar profile was observed when we analyzed cortical hemorrhage density (^††p < 0.001 vs. Control, Control-LRGT, APP/PS1, db/db-LRGT, and APP/PS1xdb/db-LRGT). No differences were detected in the hippocampus when hemorrhage burden or density was analyzed. Data are representative of 3–5 mice (489–1,012 hemorrhages/group). (B) Illustrative example of cortical hemorrhages stained with Prussian blue. Green arrows point at individual hemorrhages. Scale bar = 100 μm. (C) LRGT treatment reduced cortical microglia burden in APP/PS1 and APP/PS1xdb/db mice, in the proximity of amyloid plaques (**p < 0.01 vs. rest of the groups, p < 0.01 vs. APP/PS1). LRGT also reduced microglia burden in cortical amyloid plaque-free areas in diabetic mice (**p < 0.01 vs. rest of the groups, ^††p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, db/db, and db/db-LRGT, p < 0.01 vs. Control, Control-LRGT, APP/PS1, APP/PS1-LRGT, and db/db, p < 0.01 vs. Control and Control-LRGT). No statistical differences were observed in the hippocampus close or far from amyloid plaques. Data are representative of five mice (cortex 572–748 ROIs/group; hippocampus 108–230, ROIs/group). (D) Illustrative example of cortical immunostaining for Iba1 (microglia, green) and 4G8 (amyloid plaques, red). Scale = 100 μm. Zoom-in images of representative regions are marked by white squares and presented next to the original image. Scale bar = 50 μm, inset scale bar = 10 μm. (E) No differences were observed in the cortex when we analyzed IR-A, IR-B, or IGF-1R mRNA expression. Data are representative of 6–9 mice.