In transgenic mice carrying AD-linked APP and PS1 mutations, we observed a rapid accumulation of fibrillar Aβ from 6 months of age up to the oldest age studied, 17 months (Fig. 1 A). By immunohistochemical study using Iba1 antibody, we investigated microglial activation in the cerebral cortex of APP/PS1 transgenic mouse at the different time points (3, 6, 10, 14 and 17 months). An aggregate of activated microglia were increased synchronously with Aβ deposition. GFAP-immunopositive reactive astrocytes were also observed in parallel with Aβ deposition. At 14-17 months, microglia and astrocytes were morphologically damaged (Fig. 1 B). Iba1-positive microglia and GFAP-positive astrocyte were abundant and closely associated with fibrillar (thioflavin S-positive) Aβ deposits throughout the cortex (Fig. 1 C).
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Fig. 1. Age-related Aβ deposition and Aβ-associated gliosis in the cerebral cortex of APP/PS1 mice. Tissue sections were obtained from cerebral cortex of APP/PS1 transgenic mouse at the different time points (3, 6, 10, 14 and 17 months). (A) Representative images of thioflavine S staining in the cerebral cortex. Scale bar: 200 μm.
(B) Immunohistochemistry staining with antibodies to Iba1 for microglia and GFAP for astrocyte at the indicated time point. Scale bar: 20 μm. (C) Iba1-positive microglia and GFAP-positive astrocyte immunoreactivity (red) showed in the area surrounding the plaque (thioflavine S, green) at 10 months in the APP/PS1 mice. Scale bar: 20 μm.
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B. Expression of pro-inflammatory mediators and IL-10 in the cerebral cortex of APP/PS1 mice
To elucidate the mechanisms involved in Aβ-mediated inflammation, we investigated the expression of IL-1β, TNF-α, iNOS and IL-10 in the APP/PS1 transgenic mouse. The level of mRNA and protein expression was detected in brain sections from cortex regions at various postnatal ages ranging from 3 to 17 months. The mRNA levels of IL-1β, TNF-α and iNOS were detected from 3 months, and there was increased with the increasing age.
The mRNA of IL-10 was detectable in 6 month-old transgenic mouse, which is 3 months later than the expression of pro-inflammatory mediators (Fig. 2 A, C). The protein levels of IL-1β, TNF-α, iNOS and IL-10 were also similarly reflected at the mRNA expression. The protein levels of IL-1β, TNF-α and iNOS were detected from 3 months, and there was increased with age. The protein levels of IL-10 were detectable in 6 month-old transgenic mouse, which is 3 months later than the expression of pro-inflammatory mediators (Fig. 2 B, D).
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Fig. 2. Expression of pro-inflammatory mediators and IL-10 in the cerebral cortex of APP/PS1 mice. Tissue lysates were obtained from cerebral cortex of APP/PS1 transgenic mouse at the different time points (3, 6, 8, 10, 12, 14 and 17 months). (A) mRNA expression of IL-1β, TNF-α, iNOS and IL-10 in the cerebral cortex. (B) Western blot analysis of IL-1β, TNF-α, iNOS and IL-10 expression. (C) Intensity of PCR bands was quantified using ImageQuant software (*p<0.01). Differences among means ± standard error of means (S.E.M.) were analyzed using ANOVA followed by Tukey-Kramer multiple comparisons test (n=3-5). (D) Intensity of western bands was quantified using ImageQuant software (*p<0.01). Differences among means ± standard error of means (S.E.M.) were analyzed using ANOVA followed by Tukey-Kramer multiple comparisons test (n=3-5).
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C. IL-1β, TNF-α and IL-10 expression in microglia
To identify the specific cell types expressing TNF-α, IL-1β and IL-10 in the cerebral cortex, double immunofluorescence staining with Iba1 for microglia and TNF-α, IL-1β, IL-10 (Fig. 3 A) or GFAP for astrocyte and IL-1β, TNF-α, IL-10 (Fig. 3 B) was performed at 3, 6, 10, 14 and 17 months. Fluorescence images from each channel of the double-labeled sections were merged. The results showed that IL-1β, TNF-α and IL-10 expression was localized mainly in Iba1-immunopositive microglia.
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Fig. 3. IL-1β, TNF-α and IL-10 expression in microglia. Double-immunofluorescence staining with antibodies to (A) Iba1 for microglia and IL-1β, TNF-α and IL-10, (B) GFAP for astrocyte and IL-1β, TNF-α and IL-10 at the different time points (3, 6, 10, 14 and 17 months). Images from the double-labeled tissue were merged. Scale bar: 10 μm.
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D. ROS production and neuronal damage in the cerebral cortex of APP/PS1 mice Aβ1-42 was found to generate ROS through NADPH oxidase, leading to neurodegeneration in rat brain in vivo (Rosales-Corral et al., 2004) and in vitro (Abramov et al., 2007). Thus, we investigated whether fibrillar Aβ mediates the production of ROS in the cerebral cortex of transgenic mouse model of AD (APP/PS1). To examine this possibility, we performed in situ analysis of ROS production by hydroethidine histochemistry (Wu et al., 2003). The fluorescent products of oxidized hydroethidine were detected from 6 months, and there were significantly increased between the ages of 14 months and 17 months (Fig. 4 A). Recent studies have demonstrated that NADPH oxidase is a significant source of ROS in Alzhimer’s disease (Wilkinson et al., 2006).
Double-immunofluorescence staining was performed with a combination of antibodies against Iba1 and gp91phox or GFAP and gp91phox. gp91phox protein was localized to Iba1-immunopositive microglia in the cerebral cortex of 14 month-old APP/PS1 mice (Fig. 4 B). Next, we have investigated the neuronal damage in the APP/PS1 transgenic mouse model of AD. Cerebral cortex of 14, 17-month-old APP/PS1 transgenic mice showed significant decreases in NeuN-positive neurons and MAP2-immunoreactive dendrites (Fig. 4 C).
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Fig. 4. ROS production and neuronal damage in the cerebral cortex of APP/PS1 mice. (A) In situ visualization of O2- and O2--derived oxidant production. Animals were injected with hydroethidine at 3, 6, 8, 10, 12, 14 and 17 months. Confocal micrographs show ethidium fluorescence (red). Nuclei were counterstained with Hocheset33258 (blue).
Scale bar: 100 μm. (B) Double-immunofluorescence staining with antibodies to Iba1 for microglia and gp91phox at 14 months. Images from the double-labled tissue were merged.
Scale bar: 10 μm. (C) Immunohistochemistry staining with antibodies to NeuN (neuronal nuclei) and MAP2 (microtubule associated protein 2) for neuron at the different time points (3, 6, 8, 10, 12, 14 and 17 months). Scale bar: 50 μm., 100 μm.
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. D
Ⅳ ISCUSSION
Microglia and astrocyte are a key component of the inflammatory response inthe brain and are associated with Aβ plaques in Alzheimer'sdisease (AD). Although there is evidence that glial activationis important for the pathogenesis of AD, the role of gliain cerebral amyloidosis remains obscure. The present study wasundertaken to investigate the temporal relationship between Aβ deposition and glial activation in the cerebral cortex of APP/PS1 transgenic mice. In transgenic mice, we showed accumulation of fibrillar Aβ from 6 months of age up to the oldest age studied, 17 months. Activated microglia and astrocytes increased synchronously with Aβ deposits and were abundant and closely associated with senile plaques. At 14-17 months, microglia and astrocytes were morphologically damaged and excessively activated (Fig. 1). Recent studies showed that despite the capability of microglia to degrade amyloid deposits, microglial cells are unable to effectively reduce the Aβ deposition in the brains of APP23 transgenic mice and human AD patients (Bornemann et al., 2001; Fiala et al., 2005).
Previous studies indicate the potentially toxic effects of activated microglia, due to their production of neurotoxic substances, such as glutamate (Piani et al., 1991; Piani et al., 1992), nitric oxide (Chao et al., 1992; Dawson et al., 1994), superoxide anion (Thery et al., 1991), and cytokines with neurotoxic actions (Banati et al., 1993). Pro-inflammatory cytokines such as IL-1β and TNF-α are over-expressed in activated microglia surrounding plaques in AD (McGeer et al., 2001). They can then also stimulate the production of Aβ peptides (Blasko et al., 1999). The balance between pro- and
anti-２２
inflammatory cytokines determines the magnitude of the inflammatory response. IL-10 is known as an inhibitor of the synthesis of pro-inflammatory cytokine, including TNF-α and IL-1β (Bogdan et al., 1992; Wang et al., 1994; Bethea et al., 1999; Sawada et al., 1999). There is evidence that IL-10 has an anti-inflammatory role in the brain (Strle et al., 2001; Lee et al., 2007) and therefore might be important in down-regulating inflammatory processes associated whit AD pathology (Remarque et al., 2001). Of relevance to AD, IL-10 has been shown in vitro to inhibit Aβ-induced cytokine production in differentiated THP-1 monocytes and primary murine microglia (Szczepanik et al., 2001). We examined the endogenous expression of IL-10 in the cerebral cortex of APP/PS1 mice, but did not find of an association between IL-10 expression and down-regulation of brain inflammation. A notable feature is that the expression of IL-10 was detectable in 6 month-old transgenic mouse, which is 3 months later than expression of pro-inflammatory mediators (Fig. 2). Double immunoflorescence staining showed that IL-1β, TNF-α and IL-10 expression was localized mainly in Iba1-immunopositive microglia (Fig. 3). These results suggest that IL-10 production act as a negative feedback regulation in pro-inflammatory mediators expression.
In the present study, reactive oxygen species (ROS) production was detected from 6 months, and there significantly increased between the ages of 14 months and 17 months.
A major subunit of NADPH oxidase, gp91phox protein was localized to Iba1-immunopositive microglia in the APP/PS1 transgenic mice. Additionally, cerebral cortex of 14, 17 months showed significant decrease in NeuN-positive neurons and MAP2-immunoreactice dendrites (Fig 4). These results suggest that activated microglia
２３
surrounding plaques induce excessive expression of pro-inflammatory mediators and ROS production, leading to neuronal damage.
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Ⅴ . CONCLUSION
This study showed that the activated microglia surrounding plaques induced excessive expression of pro-inflammatory mediators such as IL-1β, TNF-α, iNOS and ROS, leading to neuronal damage in the cerebral cortex of APP/PS1 mice. Our previous study demonstrated that LPS-induced endogenous expression of IL-10 in microglia down-regulated brain inflammation and neuronal damage in the rat cerebral cortex. However, in the present study, we did not find of an association between IL-10 expression and down-regulation of brain inflammation. Therefore, further understanding the mechanisms of IL-10 expression in chronic brain inflammation may be an important point to comprehend the pathogenesis of Alzheimer’s disease.
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- 국문요약 -
현이 Iba1-면역양성을 보이는 마이크로글리아에서 대부분 배치됨을 관찰하였 다. 활성산소종 (ROS) 생성은 8개월부터 나타나며 14개월과 17개월 사이에서 두드러지게 증가되었다. NADPH oxidase의 주요 구성물질인 gp91phox 단백질 은 Iba1-면역양성을 보이는 마이크로글리아에서 대부분 배치됨을 관찰하였다.
또한, 14개월과 17개월의 대뇌피질에서 NeuN-양성적 뉴론과 MAP2-면역반 응적인 수지상 돌기가 상당히 감소되어 있음을 관찰하였다. 이 결과들은 알츠 하이머병 뇌조직에서 섬유성 아밀로이드 베타 (fibrillar β-amyloid)의 축적에
또한, 14개월과 17개월의 대뇌피질에서 NeuN-양성적 뉴론과 MAP2-면역반 응적인 수지상 돌기가 상당히 감소되어 있음을 관찰하였다. 이 결과들은 알츠 하이머병 뇌조직에서 섬유성 아밀로이드 베타 (fibrillar β-amyloid)의 축적에