Diabetes Mellitus Accelerates Aβ Pathology in Brain Accompanied by Enhanced GAβ Generation in Nonhuman Primates

<div><p>Growing evidence suggests that diabetes mellitus (DM) is one of the strongest risk factors for developing Alzheimer’s disease (AD). However, it remains unclear why DM accelerates AD pathology. In cynomolgus monkeys older than 25 years, senile plaques (SPs) are spontaneously and consistently observed in their brains, and neurofibrillary tangles are present at 32 years of age and older. In laboratory-housed monkeys, obesity is occasionally observed and frequently leads to development of type 2 DM. In the present study, we performed histopathological and biochemical analyses of brain tissue in cynomolgus monkeys with type 2 DM to clarify the relationship between DM and AD pathology. Here, we provide the evidence that DM accelerates Aβ pathology <i>in vivo</i> in nonhuman primates who had not undergone any genetic manipulation. In DM-affected monkey brains, SPs were observed in frontal and temporal lobe cortices, even in monkeys younger than 20 years. Biochemical analyses of brain revealed that the amount of GM1-ganglioside-bound Aβ (GAβ)—the endogenous seed for Aβ fibril formation in the brain—was clearly elevated in DM-affected monkeys. Furthermore, the level of Rab GTPases was also significantly increased in the brains of adult monkeys with DM, almost to the same levels as in aged monkeys. Intraneuronal accumulation of enlarged endosomes was also observed in DM-affected monkeys, suggesting that exacerbated endocytic disturbance may underlie the acceleration of Aβ pathology due to DM.</p></div

Cerebral amyloid angiopathy in the brains of aged monkeys with DM.

<p>Images of temporal lobe (TL) sections from normal cynomolgus monkeys (A, B) and cynomolgus monkeys with DM (C, D). Sections were immunostained with anti-Aβ antibody and counterstained with hematoxylin. In the brains of DM-affected aged monkeys, we observed very severe CAA lesions (arrowheads) as compared to normal aged monkeys. CT, normal aged monkeys. DM, DM-affected aged monkeys. Scale bars, 100 μm.</p

Histopathology of adult monkeys with type 2 diabetes mellitus.

<p>(A) Hematoxylin-eosin (HE)-stained section of the pancreas from a 17-year-old cynomolgus monkey with type 2 diabetes mellitus (DM) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117362#pone.0117362.s002" target="_blank">S1 Table</a>, Number 1). Most of the islets were replaced by abundant amyloid deposits. (B) HE-stained section of the pancreas from an 18-year-old cynomolgus monkey with DM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117362#pone.0117362.s002" target="_blank">S1 Table</a>, Number 3). Most of the islets were replaced with severe amyloid deposits. (C) HE-stained section of the pancreas from a 19-year-old cynomolgus monkey with DM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117362#pone.0117362.s002" target="_blank">S1 Table</a>, Number 7) showing hyalinized islets. Very few islet cells remain. (D) Direct fast scarlet-stained section of pancreas from an 18-year-old cynomolgus monkey with DM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117362#pone.0117362.s002" target="_blank">S1 Table</a>, Number 5). Hyalinized islets with severe amyloid deposition were positive for direct fast scarlet staining. (E) HE-stained section of the liver from an 18-year-old cynomolgus monkey with DM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117362#pone.0117362.s002" target="_blank">S1 Table</a>, Number 3). Marked fatty degeneration was observed in the liver. (F) HE-stained section of the aorta from an 18-year-old cynomolgus monkey with DM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117362#pone.0117362.s002" target="_blank">S1 Table</a>, Number 5). Mild atheromatosis with foam cells and sterol clefts was observed in the aorta. Scale bars for a-f, 100 μm.</p

Western blot analyses of axonal motor proteins, cathepsin D heavy chain, autophagosome marker LC3, and neprilysin in the brains of normal and DM-affected adult monkeys.

<p>Western blots showing the amounts of axonal motor proteins, cathepsin D heavy chain (CatD HC), autophagosome marker LC3, and neprilysin (NEP) in the brains of normal and DM-affected adult monkeys. Western blot analyses showed that the level of axonal motor proteins such as dynein heavy chain (DHC), dynein intermediate chain (DIC), kinesin heavy chain (KHC), and kinesin light chain (KLC) unchanged. The level of CatD HC increased in DM-affected monkey brains, and LC3-II showed significant increase in DM-affected adult monkeys. We did not observed DM-related changes in LC3-I and neprilysin (NEP) level. CT, normal adult monkeys; DM, DM-affected adult monkeys.</p

The analyses of Aβ and GAβ in the brains of normal and DM-affected monkeys.

<p>(A) Aβ level in young monkey, normal adult monkey, DM-affected monkey, and normal aged monkey brains were assessed with sandwich ELISA. Aβ level was significantly increased in normal aged monkey brains. In DM-affected monkey brains, Aβ level seemed unchanged. Data obtained from young monkey brains were set as standards. Y-axes show the mean values of the quantified data. Values are means ± SD. *P < 0.02. (B) Image of temporal lobe sections from a 19-year-old normal adult monkey and an 18-year-old cynomolgus monkey with DM. Sections were immunostained with the anti-GAβ-specific antibody 4396C and counterstained with hematoxylin. In the brain of the normal adult monkey, we observed little, if any, immunoreactivity for anti-GAβ antibody. By contrast, in the brain of the DM-affected adult monkey, we observed several neurons immunopositive for anti-GAβ antibody. Scale bars, 100 μm. (C) Quantitative image analysis of GAb-immunopositive area in the sections obtained from female normal adult monkey and DM-affected adult monkey brains. Data obtained from normal adult monkey brains were set as standards. Y-axes show the mean values of the quantified data. Values are means ± SD. *P < 0.02. (D) Dot blots showing the amount of GAβ generated in brains of cynomolgus monkeys of different ages. The blot samples were adjusted to 1 μg, 2.5 μg, or 5 μg of total protein. Dot blot analyses showed that GAβ generation increased in an age-dependent manner. (E) Dot blots showing the amount of GAβ generated in the brains of normal adult monkeys and DM-affected adult monkeys. The blot samples were adjusted to 1 μg, 2.5 μg, or 5 μg of total protein. The amount of GAβ in brains samples from DM-affected monkeys was significantly increased compared to those from normal adult monkeys. CT, normal adult monkeys; DM, DM-affected adult monkeys.</p

Senile plaques in the brains of adult monkeys with DM.

<p>Images of temporal lobe (TL) and frontal lobe (FL) sections from normal cynomolgus monkeys (A-D) and cynomolgus monkeys with DM (E-H). Sections were immunostained with anti-Aβ antibody and counterstained with hematoxylin. In aged monkey brains, we observed SPs immunostained with anti-Aβ antibody, as previously reported (A). In contrast, we did not observe Ab-immunopositive structures in the normal adult monkey brains (B-D). However, we did observe small but obvious Aβ-immunopositive senile plaques (SPs) in the frontal and temporal cortices of DM-affected adult monkeys (E-H). Scale bars, 100 μm. (I) Quantitative image analysis of Ab-immunopositive area in the sections obtained from female normal adult monkey, DM-affected adult monkey, and normal aged monkey brains. Data obtained from normal aged monkey brains were set as standards. Y-axes show the mean values of the quantified data. CT, normal cynomolgus monkeys. DM, DM-affected monkeys.</p

Hypothetical schema of DM-induced Aβ pathology leading to AD onset.

<p>From the results of this study, we propose that DM induces GAβ generation by exacerbating age-dependent endocytic disturbance, resulting in enhanced Aβ pathology in the brain. Although additional studies are needed to clarify the whole mechanisms underlying DM-associated pathology, we hypothesize that, at the very least, enhanced Aβ pathology accompanied by endocytic disturbance might be involved in the development of AD.</p

Additional file 1: of Tau pathology in aged cynomolgus monkeys is progressive supranuclear palsy/corticobasal degeneration- but not Alzheimer disease-like -Ultrastructural mapping of tau by EDX-

Immunoelectron microscopy of tau-positive oligodendroglia-like cells. (JPG 7.94 mb

Analysis of Macular Drusen and Blood Test Results in 945 <i>Macaca fascicularis</i>

<div><p>Age-dependent formation of macular drusen caused by the focal accumulation of extracellular deposits beneath the retinal pigment epithelium precede the development of age-related macular degeneration (AMD), one of the leading causes of blindness worldwide. It is established that inflammation contributes to the pathogenesis of drusen and AMD. However, development of a preemptive therapeutic strategy targeting macular drusen and AMD has been impeded by the lack of relevant animal models because most laboratory animals lack macula, an anatomic feature present only in humans and a subset of monkeys. Reportedly, macular drusen and macular degeneration develop in monkeys in an age-dependent manner. In this study, we analyzed blood test results from 945 <i>Macaca fascicularis</i>, 317 with and 628 without drusen. First, a trend test for drusen frequency (the Cochran–Armitage test) was applied to the quartile data for each parameter. We selected variables with an increasing or decreasing trend with higher quartiles at P < 0.05, to which multivariate logistic regression analysis was applied. This revealed a positive association of age (odds ratio [OR]: 1.10 per year, 95% confidence interval [CI]: 1.07–1.12) and white blood cell count (OR: 1.01 per 1 × 10³/μl, 95% CI: 1.00–1.01) with drusen. When the monkeys were divided by age, the association between drusen and white blood cell count was only evident in younger monkeys (OR: 1.01 per 1 × 10³/μl, 95% CI: 1.00–1.02). In conclusion, age and white blood cell count may be associated with drusen development in <i>M</i>. <i>fascicularis</i>. Systemic inflammation may contribute to drusen formation in monkeys.</p></div

Fundus of a monkey eye with drusen.

<p>Fundus photograph of a 21-year-old male <i>Macaca fascicularis</i> with drusen (open triangle; upper panel). Note retinal hemorrhage is seen inferiorly (filled triangle). Histologic section of an eye with drusen from another 34-year-old monkey. Note an accumulation of extracellular material underneath the retinal pigment epithelium consistent with drusen formation (*).</p