Early induction of interleukin-6 mRNA in the hippocampus and cortex of APPsw transgenic mice Tg2576.

The neuropathological changes in Alzheimer's disease (AD) include the occurrence of activated microglia and astrocytes. Activated microglia expressing interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) immunoreactivity have been observed in close vicinity of the amyloid plaques in post-mortem tissue from AD patients. In order to further analyze the inflammatory process in relation to amyloidosis, we have studied the levels of markers for inflammation in the brain of Tg(HuAPP695K670N/M671L)2576 transgenic mice (Tg2576) that express high levels of human beta-amyloid precursor protein with the Swedish double mutation and develop prominent AD type neuropathology. The mRNA levels for IL-1beta, IL-1beta-converting enzyme (ICE/caspase-1) and IL-6 were analyzed by semi-quantitative reverse transcription-polymerase chain reaction in the cerebral cortex, hippocampus and cerebellum from Tg2576 and wild type (wt) mice. The levels of mRNA for IL-1beta and caspase-1 were not significantly increased in either young (4 months) or aged (18 months) Tg2576 mice as compared to the age-matched wt mice. However, we observed an increase in IL-6 mRNA levels in the hippocampus and cortex of both 4- and 18-month-old transgenic mice as compared to wt mice. The increase in IL-6 mRNA levels in Tg2576 animals thus occurs before amyloid plaques can be detected (9-10 months). This would indicate that IL-6 mRNA induction is an early event in a beta-amyloid-induced immune response cascade or that it may be involved in the amyloidosis.

Increased gene expression of interleukin-1alpha and interleukin-6 in rat primary glial cells induced by beta-amyloid fragment.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques. The main constituent of the amyloid plaques is the amyloid beta-peptide (A beta) shown to activate glial cells in vitro. A growing body of evidence suggests that these cells contribute to neurotoxicity through production of inflammatory cytokines, chemokines, and neurotoxic substances, such as reactive oxygen species (ROS). In this study, mRNA levels of the inflammatory cytokines interleukin (IL)-1alpha and beta, and IL-6 were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) in rat primary mixed glial cells after treatment with A beta(25-35), a biologically active fragment of A beta peptide with neurotoxic properties. Clear morphological changes of the astrocytes, as well as proliferation and clustering of microglial cells were observed by light and immunofluorescence microscopy after 24 h treatment. Significant increases in IL-1alpha and IL-6 mRNA levels were detected after 24 and 72 h, whereas significantly increased levels of IL-1beta mRNA could only be detected after 4 h treatment. The most pronounced effect was seen on IL-6 mRNA expression, which increased approx two- to threefold after treatment. In addition, increased secretion of IL-6 was detected after 96 h exposure. Recently, association of IL-1alpha and IL-6 gene polymorphism with AD was reported, suggesting that these cytokines may play an important role in the development of the disease. The increased mRNA levels of IL-1alpha and IL-6 in parallel with the morphological changes in the mixed glial-cell cultures support that these cytokines may be involved in A beta-induced gliosis and in the pathogenesis of AD.

Increased expression of mRNA encoding interleukin-1beta and caspase-1, and the secreted isoform of interleukin-1 receptor antagonist in the rat brain following systemic kainic acid administration.

Kainic acid, an analogue of glutamate, injected systemically to rats evokes seizures that are accompanied by nerve cell damage primarily in the limbic system. In the present study, we have analyzed the temporal profile of the expression of the cytokines interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra), and the related IL-1beta-converting enzyme (ICE/caspase-1), in different regions of the rat brain in response to peripheral kainic acid administration (10 mg/kg, i.p.). In situ hybridization histochemistry experiments revealed that IL-1beta mRNA-expressing cells, morphologically identified as microglial cells, were mainly localized to regions showing pronounced neuronal degeneration; hippocampus, thalamus, amygdala, and certain cortical regions. The strongest expression of IL-1beta mRNA was observed after 12 hr in these regions. A weak induction of the IL-1beta mRNA expression was observed already at 2 hr. Similar results were obtained by RT-PCR analysis, showing a significantly increased expression of IL-1beta mRNA in the hippocampus and amygdala after 12 hr. In addition, RT-PCR analysis revealed that IL-1ra mRNA, and specifically mRNA encoding the secreted isoform of IL-1ra (sIL-1ra), was strongly induced in the hippocampus and amygdala at 12 and 24 hr post-injection. RT-PCR analysis of mRNA encoding caspase-1 showed a significantly increased expression in the amygdala after 12 hr. In conclusion, in response to systemic kainic acid injection IL-1beta mRNA is rapidly induced and followed by induction of IL-1ra mRNA and caspase-1 mRNA, supporting a role of the IL-1 system in the inflammatory response during excitotoxic damage.

Regulation of GTPase and adenylate cyclase activity by amyloid beta-peptide and its fragments in rat brain tissue.

Modulation of GTPase and adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) activity by Alzheimer's disease related amyloid beta-peptide, A beta (1-42), and its shorter fragments, A beta (12-28), A beta (25-35), were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by A beta (25-35), whereas A beta (12-28) did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. A beta (1-42), similar to A beta (25-35), stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, A beta (1-42) did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of A beta (25-35) and A beta (1-42), decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of A beta (25-35) was pertussis toxin sensitive, suggesting that this effect is mediated by Gi/G(o) proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased A beta (25-35) stimulated adenylate cyclase activity in both tissues. Lys-A beta (16-20), a hexapeptide shown previously to bind to the same sequence in A beta-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by A beta (25-35), however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that A beta (25-35) was active in a monomeric form. Our data strongly suggest that A beta and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood.