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Effects of carboxyl-terminal fragment of Alzheimer’s amyloid precursor protein and amyloid ß-peptide on the production of cytokines and nitric oxide in glial cells¹

Department of Pharmacology, College of Medicine, National Creative Research Initiative Centre for Alzheimer’s Dementia and Neuroscience Research Institute, Medical Research Centre, Seoul National University, Seoul 110–799, Republic of Korea; and
^* Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 156–756, Republic of Korea

²Correspondence: Department of pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer’s Dementia, Seoul National University, Seoul, 110–799, Republic of Korea. E-mail: yhsuh{at}plaza.snu.ac.kr

SPECIFIC AIMS

To investigate the role of carboxyl-terminal fragments of amyloid precursor protein (CTs) and amyloid ß-peptide (Aß) in inflammatory processes possibly linked to neurodegeneration associated with Alzheimer’s disease (AD) at the submicromolar level, we examined the effects of the carboxyl-terminal fragment of APP with 105 amino acid (CT105) and Aß at 100 nM on the alterations of inflammatory mediators in rat cortical astrocytes and microglia.

PRINCIPAL FINDINGS

1. CT105 induced the proinflammatory cytokines interleukin 1ß (IL-1ß) and tumor necrosis factor (TNF-) at 100 nM whereas Aß_1-42 did not
To check whether CT105 or Aß_1–42 induces the production of proinflammatory cytokines, we measured the levels of IL-1ß and TNF- from rat cortical astrocytes and microglial cells after treatment with CT105 or Aß_1–42 at 100 nM for 0, 3, 6, 9, 12, and 24 h. The mRNA levels of IL-1ß and TNF- were significantly increased by treatment with CT105 (100 nM) in astrocytes or microglia whereas the same dose of Aß_1–42 did not.

2. Astroglial expression of iNOS and NO induced by CT105 might be mediated by IL-1 and NF-B
To examine the effects of CT105 on NO production and iNOS expression, we checked the levels of NO and iNOS after treatment with CT105 or Aß_1–42 at 100 nM for 12 h in rat cortical astrocytes. We also checked the effects of IL-1 receptor antagonist (IL-1ra) or pyrrolidine dithiocarbamate (PDTC) on iNOS expression and NO production. iNOS induced by 100 nM CT105 in astrocytes was blocked by pretreatment with IL-1ra (100 ng/ml) 30 min before CT105 treatment (Fig. 1a ). We have checked whether NF-B was activated by treatment with CT105 at 100 nM in rat primary astrocytes using an immunocytochemistry method. As shown in Fig. 1b , NF-B was gradually translocated into the nucleus by treatment with 100 nM CT105 for 4 to 8 h. Accumulated nitrite by CT105 was also dramatically reduced when 100 ng/ml of IL-1ra, 20 µM of PDTC, or 1 µM of N^G-nitro-L-arginine methyl ester (NAME) was pretreated, as shown in Fig. 1c .

View larger version (25K): [in this window] [in a new window]   Figure 1. a) Effect of IL-1ra on CT105-induced iNOS expression. Pretreatment of the IL-1 receptor antagonist IL-1ra 30 min before CT105 was added almost blocked CT105-induced iNOS expression and NO. *P<0.05, **P<0.01: significantly different from control and Aß1–42-treated group by Student’s t test, n = 8. b) Effect of CT105 on translocation of NF-B into the nucleus. Astrocytes were incubated with 100 nM CT105 for 4 h (center) or 8 h (right), cytospun on slide glasses, and incubated with NF-B antibodies (1:100) for 1 h in 10% normal serum. c) Effect of IL-1ra on CT105-induced NO accumulation. NO was released significantly in astrocytes incubated with CT105 (100 nM) for 24 h whereas Aß1–42 did not induce NO production. The release of NO induced by CT105 recovered by 30 min preincubation of IL-1ra (100 ng/ml) or NAME (1 µM) to control level. Pretreatment of 20 µM PDTC for 2 h decreased NO production to the control level. *P < 0.05, significantly different from control group by Student’s t test, n = 16.

3. Conditioned media from CT105-treated astrocytes were toxic to rat cortical neurons
To examine the effects of molecules released from astrocytes treated with CT105 or Aß_1–42 at 100 nM on neuronal survival, media from CT105 or Aß_1–42 -treated astrocytes (conditioned media) or CT105 were added into cultured cortical neurons. MTT reduction of 100 nM CT105 or conditioned medium from Aß_1–42 (100 nM) -treated astrocytes was not reduced in cultured neurons, but conditioned media from CT105-treated astrocytes significantly reduced neuronal MTT reduction (Fig. 2a ). IL-1ra, NAME were pretreated at 30 min on cultured astrocytes and PDTC was treated 2 h before CT105 was added; these conditioned media were then transferred to the cortical neurons. The level of MTT reduction of the neuron by the conditioned media from CT105-treated astrocytes recovered almost to the control level as shown in Fig. 2b .

View larger version (31K): [in this window] [in a new window]   Figure 2. Effects of conditioned media of CT105 or Aß1–42-treated astrocytes on primary rat neuron viability. Conditioned media of CT105 (100 nM) -treated astrocytes exert toxicity on the rat cortical neuron in a IL-1ß- and NO-dependent manner. a) Conditioned media of CT105 (100 nM) -treated astrocytes significantly reduced MTT reduction whereas conditioned media from Aß1–42-treated astrocytes did not. *P > 0.05, significantly different from control group by Student’s t test, n = 8. b) Rat cortical neuronal death was prevented by addition of conditioned media from astrocytes pretreated with 1 µM of NAME, 100 unit/ml of IL-1ra, and 20 µM of PDTC 30 min, 30 min, and 2 h, respectively, before treatment of CT105. *P < 0.05, P < 0.01: significantly different from conditioned media-treated group, by Student’s t test, n = 8.

4. 100 nM CT105 induced chemokines such as MIP-1, MCP-1, and RANTES, which are potent in vitro microglial chemoattractants from astrocytes, whereas 100 nM Aß_1–42 did not
To examine the effects of CT105 or Aß_1–42 on the changes of chemokines, another family of small proteins that play important roles in inflammatory reaction—the relative mRNA levels of MIP-1, MCP-1, and RANTES—were checked by RT-PCR in astrocytes and microglia incubated with 100 nM CT105 or Aß_1–42 for 0, 3, 6, 9, 12, and 24 h. 100 nM CT105 significantly induced mRNA levels for MIP-1, MCP-1, and RANTES, whereas 100 nM Aß_1–42 did not.

CONCLUSIONS

Several studies suggest that the CTs were released from several different cells and/or more easily released from the damaged neurons into the media or extracellular fluids.

In this study, we demonstrated that CT105 at a relatively low concentration (100 nM) induced the proinflammatory cytokines IL-1ß, TNF-, and NO whereas the same concentration of Aß_1–42 did not. Our data also showed that astroglial expression of iNOS and NO production by CT105 might be mediated by IL-1ß and NF-B. 30 min pretreatment with 100 ng/ml IL-1ra completely blocked the expression of iNOS. The amount of nitrite derivatives accumulated by CT105 in the medium was also significantly decreased by IL-1ra, NAME, and PDTC pretreatment. A signal transduction pathway activated by IL-1ß ultimately leads to NF-B activation, stimulates iNOS expression, and involves the TNF--receptor-associated factor-6 (TRAF6). TRAF6 activates NF-B-inducing kinase (NIK); NIK can complex with and activate the IB kinase signalsome complex (,–ß,–). Activated IB kinase signalsome complex phosphorylates IB, the inhibitor of NF-B, directing the inhibitor to proteasome-mediated degradation and allowing NF-B to translocate to the nucleus, where it binds to specific promoter response element sequence and stimulates gene transcription. Activated NF-B has been found in the AD brain. Other potential signal transduction pathways activated during inflammation, such as p38 MAPK and c-jun amino-terminal kinase/stress-activated protein kinase, may also participate in iNOS production in astrocytes. Stress kinase pathways that target the AP-1 transcription factor could contribute to iNOS promoter activity.

To evaluate the effects of the molecules released from astrocytes on the rat cortical neuron, MTT reduction of neuron incubated with conditioned media from astrocytes was examined. Our data clearly show that this conditioned media from CT105-treated astrocytes were toxic to cortical neurons and suggest that some soluble factors released from CT105-treated astrocytes contribute to neuronal death. Pretreatment with IL-1ra, NAME, and PDTC prevented neuronal death induced by the conditioned media from CT105-treated astrocytes, suggesting that NO plays a role in exerting toxicity on rat cortical neuron through IL-1ß-NF-B-dependent pathway.

There are also several lines of evidence that inflammatory markers, including cytokines and acute-phase proteins, are associated with amyloid deposits by activated microglia and astrocytes in AD.

High concentrations (50 µM) of Aß_1–42 have been reported to activate astrocytes and oligodendrocytes to produce cytokines and chemokines; in this study, however, a much lower concentration (100 nM of Aß_1–42) did not induce cytokines or chemokines, whereas the same concentration of CT105 did significantly induce them. Therefore, our data suggest that CTs might contribute to the activation of glial cells more strongly than Aß_1–42 in cultured cells as well as in AD brain.

Chemokines are representative small (8–10 kDa) proteins functionally associated with inflammatory cell recruitment in host defense and have been proved to alter the permeability of blood-brain barrier (BBB) so that monocytes migrate across BBB more easily. In this study, CT105 induced chemokines such as MIP-1, MCP-1, and RANTES, which function as potent in vitro microglial chemoattractants from astrocytes or microglia, indicating that microglial or macrophage migration could be induced by CT105. However, 100 nM Aß_1–42 did not induce chemokines.

In conclusion, nanomolar CT105 induced IL-1ß and TNF- expression whereas Aß_1–42 did not, and IL-1ß induced by CT105 up-regulated iNOS gene expression through NF-B activation in astrocytes and microglial cells. CT105 also induced astroglial and microglial chemokines such as MIP-1, MCP-1, and RANTES, which play roles in persuading microglial accumulation around amyloid plaques. Accumulated microglia may induce NO and other toxic genes in response to the component of the plaques. Released cytokines, NO, and chemokines from astrocytes and microglia might contribute to cell death in neighboring neurons, which could be an another cell death mechanism involved in the propagation of neuronal death in AD (Fig. 3 ).

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of the glia-mediated neuronal death caused by CT105. CT105-induced gliosis includes the production of cytokines, which induce both iNOS expression and direct neuronal death. These pathways can be amplified by CT105-induced chemokine production.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0724fje ; to cite this article, use FASEB J. (April 18, 2001) 10.1096/fj.00-0724fje

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