BackgroundActivation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA2-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes.Methods/ResultsCytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA2-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression.ConclusionThese results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.

The primary molecules for mediating the innate immune response are the Toll‐like family of receptors (TLRs). Recent work has established that amyloid‐beta (Aβ) fibrils, the primary components of senile plaques in Alzheimer’s disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP‐1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Aβ(1–42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam3CSK4) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Aβ(1–42) species. Pre‐treatment of the cells with 10 μg/mL of a TLR2‐specific antibody blocked ∼50% of the cell response to fibrillar Aβ(1–42), completely blocked the Pam3CSK4 response, and had no effect on the LPS‐induced response. A TLR4‐specific antibody (10 μg/mL) blocked ∼35% of the cell response to fibrillar Aβ(1–42), completely blocked the LPS response, and had no effect on the Pam3CSK4 response. Polymyxin B abolished the LPS response with no effect on Aβ(1–42) ruling out bacterial contamination of the Aβ samples. Combination antibody pre‐treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Aβ(1–42) response than the antibodies used alone. These data demonstrate that fibrillar Aβ(1–42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Aβ‐induced tumor necrosis factor alpha production in a human monocytic cell line.

Amyloid β-protein (Aβ) deposits in brains of Alzheimer’s disease (AD) patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aβ. Nucleotides released from apoptotic cells activate P2Y2 receptors (P2Y2Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y2Rs in the phagocytosis and clearance of Aβ. Treatment of mouse primary microglial cells with fibrillar (fAβ1–42) and oligomeric (oAβ1–42)Aβ1–42 aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAβ1–42 and oAβ1–42 treatment for 24 h caused an increase in P2Y2R gene expression. Treatment with fAβ1–42 and oAβ1–42 aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y2R agonists ATP and UTP caused significant uptake of Aβ1–42 by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aβ1–42 uptake by primary microglial cells isolated from P2Y2R−/− mice. Inhibitors of αv integrins, Src and Rac decreased UTP-induced Aβ1–42 uptake, suggesting that these previously identified components of the P2Y2R signaling pathway play a role in Aβ phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aβ1–42 degradation by microglial cells, but not in cells isolated from P2Y2R−/− mice. Taken together, our findings suggest that P2Y2Rs can activate microglial cells to enhance Aβ clearance and highlight the P2Y2R as a therapeutic target in AD.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognitive ability and is a serious cause of mortality. Many of the pathological characteristics associated with AD are revealed post-mortem, including amyloid-β plaque deposition, neurofibrillary tangles containing hyperphosphorylated tau proteins and neuronal loss in the hippocampus and cortex. Although several genetic mutations and risk factors have been associated with the disease, the causes remain poorly understood. Study of disease-initiating mechanisms and AD progression in humans is inherently difficult as most available tissue specimens are from late-stages of disease. Therefore, AD researchers rely on in vitro studies and the use of AD animal models where neuroinflammation has been shown to be a major characteristic of AD. Purinergic receptors are a diverse family of proteins consisting of P1 adenosine receptors and P2 nucleotide receptors for ATP, UTP and their metabolites. This family of receptors has been shown to regulate a wide range of physiological and pathophysiological processes, including neuroinflammation, and may contribute to the pathogenesis of neurodegenerative diseases like Parkinson’s disease, multiple sclerosis and AD. Experimental evidence from human AD tissue has suggested that purinergic receptors may play a role in AD progression and studies using selective purinergic receptor agonists and antagonists in vitro and in AD animal models have demonstrated that purinergic receptors represent novel therapeutic targets for the treatment of AD.

Summary The initiating events that promote tau mislocalization and pathology in Alzheimer’s disease (AD) are not well defined, due partly to the lack of endogenous models that recapitulate tau dysfunction. We exposed wild-type neurons to a neuroinflammatory trigger and examined the impact on endogenous tau. We found that tau re-localized and accumulated within pathological neuritic foci, or beads, comprised of mostly hypo-phosphorylated, acetylated, and oligomeric tau. These structures were detected in aged wild-type mice and were enhanced in response to neuroinflammation in vivo, highlighting a previously undescribed endogenous age-related tau pathology. Strikingly, deletion or inhibition of the cytoplasmic shuttling factor HDAC6 suppressed neuritic tau bead formation in neurons and mice. Using mass spectrometry-based profiling, we identified a single neuroinflammatory factor, the metalloproteinase MMP-9, as a mediator of neuritic tau beading. Thus, our study uncovers a link between neuroinflammation and neuritic tau beading as a potential early-stage pathogenic mechanism in AD.