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

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

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

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

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

Effects of Mycobacteria Major Secretion Protein, Ag85B, on Allergic Inflammation in the Lung

<div><p>Many epidemiological studies have suggested that the recent increase in prevalence and severity of allergic diseases such as asthma is inversely correlated with <i>Mycobacterium bovis</i> bacillus Calmette Guerin (BCG) vaccination. However, the underlying mechanisms by which mycobacterial components suppress allergic diseases are not yet fully understood. Here we showed the inhibitory mechanisms for development of allergic airway inflammation by using highly purified recombinant Ag85B (rAg85B), which is one of the major protein antigens secreted from <i>M. tuberculosis</i>. Ag85B is thought to be a single immunogenic protein that can elicit a strong Th1-type immune response in hosts infected with mycobacteria, including individuals vaccinated with BCG. Administration of rAg85B showed a strong inhibitory effect on the development of allergic airway inflammation with induction of Th1-response and IL-17and IL-22 production. Both cytokines induced by rAg85B were involved in the induction of Th17-related cytokine-production innate immune cells in the lung. Administration of neutralizing antibodies to IL-17 or IL-22 in rAg85B-treated mice revealed that IL-17 induced the infiltration of neutrophils in BAL fluid and that allergen-induced bronchial eosinophilia was inhibited by IL-22. Furthermore, enhancement of the expression of genes associated with tissue homeostasis and wound healing was observed in bronchial tissues after rAg85B administration in a Th17-related cytokine dependent manner. The results of this study provide evidence for the potential usefulness of rAg85B as a novel approach for anti-allergic effect and tissue repair other than the role as a conventional TB vaccine.</p></div

Innate immune cells that secrete Th17-related cytokines are induced by rAg85B administration in BAL fluid.

<p>OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. BAL cells were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of BAL cells from PBS-treated (upper) and rAg85B protein-treated (lower) OVA-sensitized mice stained with anti-CD3, anti-CD4, anti-CD8, anti-Gr-1, anti-γδ TCR, anti-NKp46, anti-CD11c, anti-CD127 (IL-7R) and Lineage specific marker (CD3, CD19, Gr-1, CD11b, CD11c). Numbers in quadrants indicate percent of cells in each (<i>A</i>). Intracellular IL-17 and IL-22 staining in indicated cells by flow cytometry (dot plots) and absolute numbers of those cell populations (side graphs) in the BAL fluid (<i>B, C, D, E, F, G</i>). Data are representative of at least two independent experiments (**P<0.01 compared with OVA control. error bars, s.d.; n = 6 mice).</p

Schematic illustration of the proposed effects of rAg85B in a mouse model of allergic inflammation.

<p>IFN-γ and IL-17-producing Th cells are induced in regional lymph nodes by rAg85B challenge, however, Th17 cells do not enter the lung unlike Th1 cells. Th17-related cytokine-secreting cells in lungs from rAg85B-administered mice are innate immune cells including γδT cells, IL-7R⁺ Lin⁻ cells, CD3⁻ NKp46⁺ cells and CD11c⁺ cells. IL-17 and IL-22 induced by rAg85B in an allergic environment have crucial roles in not only anti-allergic effects but also regulation of tissue homeostatic reactions.</p