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Abstract Background Compelling evidence has implicated neuroinflammation in the pathogenesis of a number of neurodegenerative conditions. Chronic activation of both astrocytes and microglia leads to excessive secretion of proinflammatory molecules such as TNFα, IL-6 and IL-1β with potentially deleterious consequences for neuronal viability. Many signaling pathways involving the mitogen-activated protein kinases (MAPKs), nuclear factor κB (NFκB) complex and the Janus kinases (JAKs)/signal transducers and activators of transcription (STAT)-1 have been implicated in the secretion of proinflammatory cytokines from glia. We sought to identify signaling kinases responsible for cytokine production and to delineate the complex interactions which govern time-related responses to lipopolysaccharide (LPS). Methods We examined the time-related changes in certain signaling events and the release of proinflammatory cytokines from LPS-stimulated co-cultures of astrocytes and microglia isolated from neonatal rats. Results TNFα was detected in the supernatant approximately 1 to 2 hours after LPS treatment while IL-1β and IL-6 were detected after 2 to 3 and 4 to 6 hours, respectively. Interestingly, activation of NFκB signaling preceded release of all cytokines while phosphorylation of STAT1 was evident only after 2 hours, indicating that activation of JAK/STAT may be important in the up-regulation of IL-6 production. Additionally, incubation of glia with TNFα induced both phosphorylation of JAK2 and STAT1 and the interaction of JAK2 with the TNFα receptor (TNFR1). Co-treatment of glia with LPS and recombinant IL-6 protein attenuated the LPS-induced release of both TNFα and IL-1β while potentiating the effect of LPS on suppressor of cytokine signaling (SOCS)3 expression and IL-10 release. Conclusions These data indicate that TNFα may regulate IL-6 production through activation of JAK/STAT signaling and that the subsequent production of IL-6 may impact on the release of TNFα, IL-1β and IL-10.

Macrophages are key cells in tissue defense in the periphery and, under certain circumstances, infiltrate the central nervous system, where they may play a similar role in the brain, perhaps supporting the function of microglia. Macrophages have been shown to adopt different activation states in response to various stimuli. Specifically, when exposed to inflammatory stimuli such as interferon (IFN)γ, the cells adopt the M1 phenotype, whereas when exposed to anti-inflammatory cytokines such as interleukin (IL)-4 or IL-13, the M2 phenotype is adopted. While M1 macrophages are associated with tissue defense and destruction of invading pathogens, M2 macrophages are involved in tissue repair and in terminating inflammation. It is well known that an inflammatory microenvironment exists in the brain of aged animals and also in the brain of mice that overexpress amyloid-β protein precursor (AβPP) and presenilin 1 (PS1; AβPP/PS1 mice), a commonly-used model of Alzheimer's disease (AD). Recent studies have revealed that immune cells, including macrophages, infiltrate the brain in both circumstances raising the possibility that these cells adopt the M1 activation state and contribute to the already-existing neuroinflammation. We set out to examine the responses of bone marrow-derived macrophages prepared from wildtype and AβPP/PS1 mice and demonstrate that cells from AβPP/PS1 mice, even after several days in culture, respond more profoundly to IFNγ than those from wildtype mice. We suggest that this propensity to respond to M1-polarizing stimuli, together with the described changes in the brain of AβPP/PS1 mice, contribute to the development of chronic neuroinflammation. [ABSTRACT FROM AUTHOR]

Abstract: The amyloid precursor-like protein-1 (APLP1) is a member of a protein family that includes the Alzheimer''s disease-associated amyloid precursor protein (APP). While much is known about the proteolytic processing of APP, fewer details are available about APLP1. Using Chinese hamster ovarian cells stably transfected with human APLP1 and a novel juxtamembrane anti-APLP1 antibody, we demonstrate the detection of a secreted ∼3.5 kDa APLP1-derived peptide (ALP-1). The production of this peptide is abolished by inhibition of γ-secretase, but not β-secretase, suggesting that ALP-1 is analogous to the p3 fragment produced from APP. However, unlike p3 or Aβ, ALP-1 shows no obvious propensity for aggregation and is not toxic to neuronal cells. Moreover, using two distinct experimental paradigms, we demonstrate that neither cell-derived nor chemically synthesized ALP-1 influences the oligomerization or aggregation of Aβ. [Copyright &y& Elsevier]

Amyloid-β (Aβ) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of Aβ. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of Aβ on cultured cells; in particular, the evidence suggests that Aβ-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of Aβ[sub (1-40)] on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CAI pyramidal cell synapses in vivo. We report that Aβ[sub (1-40)] induced activation of JNK in CA1 and that this was coupled with expression of the proapoptotic protein, Bax, cytosolic cytochrome c, poly-(ADPribose) polymerase cleavage, and Fas ligand expression in the hippocampus. These data indicate that Aβ[sub (1-40)] inhibited expression of long term potentiation, and this effect was abrogated by administration of the JNK inhibitor peptide, D-JNKI1. In parallel with these findings, we observed that Aβ-induced changes in caspase-3 activation and TdT-mediated dUTP nick-end labeling staining in neuronal cultured cells were inhibited by D-JNKI1. We present evidence suggesting that interleukin (IL)-1β plays a significant role in mediating the effects of Aβ[sub (1-40)] because Aβ([sub 1-40)] increased hippocampal IL-1β and because several effects of Aβ[sub (1-40)] were inhibited by the caspase-1 inhibitor Ac-YVAD-CMK. On the basis of our findings, we propose that Aβ-induced changes in hippocampal plasticity are likely to be dependent upon IL-1β-triggered activation of JNK. [ABSTRACT FROM AUTHOR]

Whereas the classical histological hallmarks of Alzheimer's disease (AD) are deposition of amyloid-containing plaques and development of neurofibrillary tangles, there is also clear evidence of inflammatory changes accompanied by the presence of activated microglia and astrocytosis. However, at this time, it remains uncertain whether inflammatory changes contribute to pathogenesis of the disease or if they are secondary to deposition of amyloid-β or other pathological changes. A greater understanding of the sequence of events would clearly improve development of strategies to delay progression of the disease. There is a realistic expectation that advances in imaging technology may provide the key to uncovering this sequence. In this study, we employed non-invasive imaging techniques to examine changes in tissue state in hippocampus and cortex of transgenic mice which overexpress amyloid-β protein precursor and presenilin 1 and show that the observed increase in T1 relaxation time was associated with astrogliosis while the decrease in T2 relaxation time was associated with microglial activation. We explored the possibility that interferon-γ might trigger glial activation and demonstrate a genotype-related infiltration of macrophages and natural killer cells, which release interferon-γ. The evidence suggests that IFNγ triggers glial activation and expression of proinflammatory cytokines, and these changes, in turn, contribute to the decrease in long-term potentiation.

The amyloid precursor family of proteins are of considerable interest, both because of their role in Alzheimer's disease pathogenesis and because of their normal physiological functions. In mammals, the amyloid precursor protein (APP) has two homologs, amyloid precursor-like protein (APLP) 1 and APLP2. All three proteins undergo ectodomain shedding and regulated intramembrane proteolysis, and important functions have been attributed to the full-length proteins, shed ectodomains, C-terminal fragments and intracellular domains (ICDs). One of the proteases that is known to cleave APP and that is essential for generation of the amyloid beta-protein is the beta-site APP-cleaving enzyme 1 (BACE1). Here, we investigated the effects of genetic manipulation of BACE1 on the processing of the APP family of proteins. BACE1 expression regulated the levels and species of full-length APLP1, APP and APLP2, of their shed ectodomains, and of their membrane-bound C-terminal fragments. In particular, APP processing appears to be tightly regulated, with changes in beta-cleaved APPs (APPsbeta) being compensated for by changes in alpha-cleaved APPs (APPsalpha). In contrast, the total levels of soluble cleaved APLP1 and APLP2 species were less tightly regulated, and fluctuated with BACE1 expression. Importantly, the production of ICDs for all three proteins was not decreased by loss of BACE1 activity. These results indicate that BACE1 is involved in regulating ectodomain shedding, maturation and trafficking of the APP family of proteins. Consequently, whereas inhibition of BACE1 is unlikely to adversely affect potential ICD-mediated signaling, it may alter other important facets of amyloid precursor-like protein/APP biology.

The amyloid precursor-like protein-1 (APLP1) is a member of a protein family that includes the Alzheimer's disease-associated amyloid precursor protein (APP). While much is known about the proteolytic processing of APP, fewer details are available about APLP1. Using Chinese hamster ovarian cells stably transfected with human APLP1 and a novel juxtamembrane anti-APLP1 antibody, we demonstrate the detection of a secreted approximately 3.5 kDa APLP1-derived peptide (ALP-1). The production of this peptide is abolished by inhibition of gamma-secretase, but not beta-secretase, suggesting that ALP-1 is analogous to the p3 fragment produced from APP. However, unlike p3 or Abeta, ALP-1 shows no obvious propensity for aggregation and is not toxic to neuronal cells. Moreover, using two distinct experimental paradigms, we demonstrate that neither cell-derived nor chemically synthesized ALP-1 influences the oligomerization or aggregation of Abeta.

We report the development of a high-level bacterial expression system for the Alzheimer's disease-associated amyloid beta-peptide (Abeta), together with a scaleable and inexpensive purification procedure. Abeta(1-40) and Abeta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Abeta(1-40) and Met-Abeta(1-42), referred to as Abeta(M1-40) and Abeta(M1-42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Abeta peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Abeta1-40 and Abeta1-42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.

The age-related deficit in long-term potentiation (LTP) in the dentate gyrus is positively correlated with hippocampal concentration of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta). Previous evidence also indicates that the inhibition of LTP induced by intracerebroventricular injection of amyloid-beta(1-40) (Abeta) is accompanied by increased hippocampal IL-1beta concentration and IL-1beta-stimulated signalling, specifically activation of the stress-activated protein kinase, c-jun N-terminal kinase (JNK). We considered that the underlying age-related neuroinflammation may render older rats more susceptible to Abeta administration and, to investigate this, young, middle-aged and aged rats were injected intracerebroventricularly with Abeta or vehicle. Hippocampal IL-1beta concentration, JNK phosphorylation, expression of the putative Abeta receptor, Receptor for advanced glycation end products (RAGE) and the microglial cell surface marker, CD40 were assessed. We report that Abeta inhibited LTP in a concentration-dependent manner in young rats and that this was accompanied by concentration-dependent increases in hippocampal IL-1beta and expression of phosphorylated JNK, RAGE and CD40. While 20 micromol/L Abeta exerted no significant effect on LTP in young rats, it inhibited LTP in middle-aged and aged rats and the increased vulnerability of aged rats was associated with increased IL-1beta concentration. Treatment of rats with eicosapentaenoic acid attenuated the inhibitory effect of 60 micromol/L Abeta on LTP in young rats and the effect of 20 micromol/L Abeta in middle-aged and aged rats. We present evidence which indicates that the effect of eicosapentaenoic acid may be linked with its ability to stimulate activation of peroxisome proliferator-activated receptor gamma.