Abstract
BackgroundNeuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer’s disease (AD) pathology. Amyloid beta (Aβ) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aβ activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors.MethodsThe presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca2+]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aβ1–42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain.ResultsAcute applications of human amylin or Aβ1–42 resulted in an increase in [Ca2+]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1β, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1β. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls.ConclusionMicroglial amylin receptors mediate Aβ-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.
Highlights
Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer’s disease (AD) pathology
Bath applications of either monomeric human amylin or soluble oligomeric Aβ1–42 (1 μM) to human fetal microglial (HFM) for 30 s produced a robust Ca2+ increase within 1 min after entry of the peptide within the imaging chamber
The transient elevations in Ca2+ due to human amylin (hAmylin) or Aβ1–42 applications were abolished by prior application of the amylin receptor antagonist, AC253 (Fig. 1h, i), suggesting that these responses
Summary
Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer’s disease (AD) pathology. The main immune cells in the brain, appear central to the initiation and progression of neuroinflammation in AD [2,3,4] These cells are activated and recruited to amyloid plaques, phagocytose Aβ, and secrete cytokines [5]. Initiation of such an inflammatory cascade results from Aβ recruitment of intracellular cytosolic multiprotein complexes termed “inflammasomes” and the subsequent activation of microglial caspase-1 [6,7,8]. Caspase-1 generates inflammatory responses through cleavage and release of the injurious cytokines, IL-1β and IL-18, on to adjacent neurons [6, 10]. We hypothesized that microglial amylin receptors may serve as a portal of expression of Aβ-induced inflammatory responses
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