Intraperitoneal injection of the pancreatic peptide amylin potently reduces behavioral impairment and brain amyloid pathology in murine models of Alzheimer's disease

Abstract

Amylin, a pancreatic peptide, and amyloid-beta peptides (Aβ), a major component of Alzheimer's disease (AD) brain, share similar β-sheet secondary structures, but it is not known whether pancreatic amylin affects amyloid pathogenesis in the AD brain. Using AD mouse models, we investigated the effects of amylin and its clinical analog, pramlintide, on AD pathogenesis. Surprisingly, chronic intraperitoneal (i.p.) injection of AD animals with either amylin or pramlintide reduces the amyloid burden as well as lowers the concentrations of Aβ in the brain. These treatments significantly improve their learning and memory assessed by two behavioral tests, Y maze and Morris water maze. Both amylin and pramlintide treatments increase the concentrations of Aβ1-42 in cerebral spinal fluid (CSF). A single i.p. injection of either peptide also induces a surge of Aβ in the serum, the magnitude of which is proportionate to the amount of Aβ in brain tissue. One intracerebroventricular injection of amylin induces a more significant surge in serum Aβ than one i.p. injection of the peptide. In 330 human plasma samples, a positive association between amylin and Aβ1-42 as well as Aβ1-40 is found only in patients with AD or amnestic mild cognitive impairment. As amylin readily crosses the blood-brain barrier, our study demonstrates that peripheral amylin's action on the central nervous system results in translocation of Aβ from the brain into the CSF and blood that could be an explanation for a positive relationship between amylin and Aβ in blood. As naturally occurring amylin may play a role in regulating Aβ in brain, amylin class peptides may provide a new avenue for both treatment and diagnosis of AD.

Figure 1

Amylin treatment of 5XFAD mice reduces the amyloid burden and improves their learning and memory. At 3.5 months of age, 5XFAD mice were treated by intraperitoneal injection of phosphate-buffered saline (PBS) or amylin (200 pg kg⁻¹) daily for 10 weeks (n=10 per group) (Supplementary Table S1). (a) Dense-cored Aβ plaque burden is reduced in the whole brain, including the cerebral cortex and the hippocampus. (b) Amylin-treated mice had a significant reduction in Aβ amyloid burden in the cortex, hippocampus and thalamus quantitated by average amyloid intensity × plaque size. (c) Compared with PBS treatment, the amylin-treated mice also had fewer plaques in the cortex and thalamus, while the hippocampus region only showed a tendency. Compared with the PBS treatment, the amylin-treated mice had lower concentration of Aβ1-42 (pg per 10 μg brain protein) in the brain (d) (P=0.005) but had higher concentrations of Aβ1-42 in CSF (pg ml⁻¹) (e) (P=0.04). The amylin-treated 5XFAD mice illustrated improved cognition by showing increased percentage of alternation in the Y maze test (f) (P=0.001) and by showing shortened times in Morris water maze test (g) in finding the hidden platform at day 10 (D10) (P=0.005), in memory at day 12 (D12) after the completion of training and skipping day 11 (P=0.002) and in the probe trial (P=0.03). Mean±s.e. was used with *P<0.0001; **P<0.01; ***P<0.05.

Figure 2

Treatment of Tg2576 mice with amylin or its analog pramlintide reduces the amyloid burden and improves their learning and memory. At 9 months of age, Tg2576 mice were treated with intraperitoneal injection of phosphate-buffered saline (PBS), amylin (200 pg kg⁻¹) or pramlintide (200 pg kg⁻¹) daily for 10 weeks (n=8 per group) (Supplementary Table S2). Both amylin- and pramlintide-treated mice had reduced concentrations of (a) Aβ1-42 (measured as pg per 10 μg brain protein) and (b) Aβ1–40 in the brain compared with PBS-treated controls. (c) Both amylin- and pramlintide-treated mice had increased concentrations of Aβ1–42 (pg ml⁻¹) in CSF, but (d) neither treatment significantly affected the concentration of Aβ1–40. (e) Compared with PBS treatment, both amylin- and pramlintide-treated mice exhibited improved cognition by showing increased percentage of alternation in the Y maze test. Mean±s.e. was used with *P<0.05.

Figure 3

A single injection of amylin or pramlintide induces a change of serum Aβ in the amyloid precursor protein (APP) transgenic mice. After one intraperitoneal (i.p.) injection of phosphate-buffered saline vs amylin (a) or pramlintide (b) (200 μg kg⁻¹) in Tg2576 mice, changes in levels of serum Aβ1–42 and Aβ1–40 were observed. A single i.p. injection of amylin induced an increase in serum Aβ1–42 but not Aβ1–40 in 5XFAD mice (c); in contrast, i.p. injection of amylin induced an increase in serum Aβ1–40 but not Aβ1–42 in Dutch APP mice (d). In 5XFAD mice, the changes in serum Aβ1–42 in response to a challenge with amylin were dose dependent (e). Significance of changes in Aβ levels before and after the injection was determined by Student's t-test. *P<0.05.

Figure 4

Change in serum Aβ levels induced by amylin challenge and Aβ pathology in the brain. Serum samples were isolated before and after 5XFAD mice, which were at different ages, were challenged by a single intraperitoneal (i.p.) injection of amylin. The mice were killed immediately after the amylin challenge procedure, and the brain protein was extracted. The percentage of changes in serum Aβ1–42 induced by a single i.p. injection of amylin significantly correlated with the amounts of Aβ1–42 in their brains (r=0.83, P=6.7 × 10⁻⁶) (a). Amylin-induced serum Aβ1-42 changes induced by i.p. injection or intracerebroventricular (i.c.v.) injection in the 5XFAD mice were compared (b). Higher levels of increased serum Aβ1–42 induced by i.c.v. injection of amylin than by i.p. injection of amylin were observed (P=0.03) in these mice.

Figure 5

Characterization of the effect of amylin on amyloid precursor protein (APP) processing and BACE1. (a) Detection of full-length APP (fAPP) and APP processed products by 6E10 antibody in western blotting in brain homogenates from Tg2576 mice treated with phosphate-buffered saline (PBS) or amylin (n=6 per group), and (b) quantitation of the results (mean±s.e.) are shown. Although there were no differences in the levels fAPP and secreted APP cleaved by α secretase (sAPPα) between PBS control and the amylin treatment, there was an increase in C99 fragment from APP cleaved by BACE1 in the brains treated with amylin compared with PBS treatment (*P=0.004). (c) In vitro assay using purified BACE1 showed that amylin significantly inhibited BACE1 activity, but pramlintide had little inhibition for BACE1. The concentration of amylin necessary to inhibit 50% BACE1 activity (IC50) in this assay is 6.9 μM (d).