Amylin deposition in the brain: A second amyloid in Alzheimer disease?

Abstract

Objective: Hyperamylinemia, a common pancreatic disorder in obese and insulin-resistant patients, is known to cause amylin oligomerization and cytotoxicity in pancreatic islets, leading to β-cell mass depletion and development of type 2 diabetes. Recent data has revealed that hyperamylinemia also affects the vascular system, heart, and kidneys. We therefore hypothesized that oligomerized amylin might accumulate in the cerebrovascular system and brain parenchyma of diabetic patients.

Methods: Amylin accumulation in the brain of diabetic patients with vascular dementia or Alzheimer disease (AD), nondiabetic patients with AD, and age-matched healthy controls was assessed by quantitative real time polymerase chain reaction, immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay.

Results: Amylin oligomers and plaques were identified in the temporal lobe gray matter from diabetic patients, but not controls. In addition, extensive amylin deposition was found in blood vessels and perivascular spaces. Intriguingly, amylin deposition was also detected in blood vessels and brain parenchyma of patients with late onset AD without clinically apparent diabetes. Mixed amylin and amyloid β (Aβ) deposits were occasionally observed. However, amylin accumulation leads to amyloid formation independent of Aβ deposition. Tissues infiltrated by amylin showed increased interstitial space, vacuolation, spongiform change, and capillaries bent at amylin accumulation sites. Unlike the pancreas, there was no evidence of amylin synthesis in the brain.

Interpretation: Metabolic disorders and aging promote accumulation of amylin amyloid in the cerebrovascular system and gray matter, altering microvasculature and tissue structure. Amylin amyloid formation in the wall of cerebral blood vessels may also induce failure of elimination of Aβ from the brain, thus contributing to the etiology of AD.

Fig 1

Amylin deposition in brain specimens from T2D-D and AD patient groups demonstrated by immunohistochemistry with an anti-amylin antibody on thin tissue sections. A and B are representative images of amylin deposition in the brain of diabetic patients with vascular dementia. C and D show amylin plaques in brains from the AD group (non-diabetics with AD). Amylin is largely present on blood vessel walls (A, C; red arrows) and as small plaques within pericapillary areas (A; blue arrow) and even remote regions from capillaries (B, D; blue arrows). In contrast, brain samples of age-matched healthy humans show only occasional amylin deposition in blood vessels (E; arrow). F is a positive control for amylin deposition in pancreatic islets of T2D patients. G shows amylin deposition in a blood vessel similar to (A) and (C), while H displays the same blood vessel in a brain section incubated only with the secondary antibody demonstrating the specificity of the anti-amylin antibody. I shows the average surface density of amylin deposits in the brain parenchyma of patients in T2D-D, AD, and control groups (N=4 per group). Amylin deposition was assessed on a field of view 0.4 X 1mm. *P < 0.05.

Fig 2

The same blood vessel stained for amylin (A), Congo red (B and C) and Aβ (D). The incorporated amylin in the blood vessel wall exhibits apple-green birefringence under a polarized light microscope (Congo red stain; C) demonstrating that amylin deposition in the blood vessel forms amyloid-like structure. A larger scale view of the area selected in this figure is displayed in Supplemental Fig S8.

Fig 3

Accumulation of oligomeric amylin in supernatant protein homogenates from brains in T2D-D, AD, and control groups demonstrated by ELISA (A) and western blot (B–D). B and C show the relative increase of amylin tetramers (B) and trimers (C) in T2D-D and AD brains versus controls. D displays representative eletrophoretic patterns in these groups. *P < 0.05; **P < 0.01. E shows that the amylin precursor, i.e. proamylin, may also be present in the brain. The test with an anti-proamylin antibody on western blot suggests that the band at ~ 60 kDa (right side of the panel) is specific to proamylin as it is not likely present on the western blot with the amylin antibody (left side of the panel). The specific band may represent a proamylin octamer (MW of proamylin is ~7.5kDa). Additional data are in Supplemental Material.

Fig 4

Amylin and Aβ co-localization demonstrated by co-staining brain sections from AD patients with anti-amylin and anti-Aβ antibodies. Distinct patches of amylin (brown color) and Aβ (red color in A, and dark blue in C) can be observed in blood vessels (A, arrows) and brain parenchyma (B, C). Clusters of small amylin plaques (brown) adjacent to, or surrounded by, much larger Aβ deposits (red in B and dark blue in C) are also present in brain tissues. A and C are brain sections from a diabetic patient with AD (T2D-D group). B is a brain section from an AD patient without clinically demonstrated T2D. In D, we estimate the fraction of mixed amylin-Aβ plaques on a 0.4 X 1mm field of view in T2D-D and AD patient groups. *P < 0.05.

Fig 5

Amylin is observed in the whole (A) or part (B) of the circumference of vessel wall. Co-staining with anti-amylin and anti-Aβ antibodies (C, D) shows a patchy distribution of amylin (brown) which is similar and complementary to that of Aβ (red) suggesting that both peptides are involved in CAA development. A and C are representative images of brain tissue from patients with vascular dementia. B and D are immunohistochemical images of brain tissue from AD patients. The inset in D shows a 40X magnification image of a capillary presenting amylin-Aβ mixed amyloid angiopathy. E is a representative image of tissue in control brain specimens showing lack of amylin deposition in brain parenchyma and blood vessels. F presents the estimated average fraction of the vessel wall circumference covered by amylin in a 0.4 X 1mm field of view of brain samples from T2D-D, AD, and control groups. *P < 0.05; **P < 0.01.

Fig 6

In A, qRT-PCR data show amylin mRNA levels in brain specimens from AD patients and healthy human controls relative to that in the human pancreas (note logarithmic scale and scale break). Rat brain and pancreas samples are negative controls for human amylin transcript. In B, western blot analysis indicates that amylin tetramers exist in both plasma and supernatant brain homogenates from patients in the AD group. In C, amylin was immunoprecipitated from brain specimens corresponding to control, T2D-D, and AD groups. Immunoprecipitates of amylin were then analyzed by western blot using an anti-Aβ antibody. *P < 0.05.