We demonstrate here that Aβ potently activates microglia in part through the activation of MAC1 receptors, followed by the production of superoxide free radicals, and leading to neuronal death. These results not only confirm our earlier observations that the presence of microglia potentiated the neurotoxicity of Aβ, but also reveal several novel findings that illustrated in Figure : (1) a role for MAC1 receptor in Aβ-induced microglial activation, (2) involvement of MAC1 receptor in PHOX activation which mediates Aβ-induced neurotoxicity and (3) involvement of PI3K in PHOX activation signals by MAC1 receptor.
Figure 7 β-amyloid signals through microglial MAC1 receptor and PI3K to PHOX. Microglia exposed to Aβ exhibit a respiratory burst leading to the release of superoxide anion (O2-). Release of superoxide is mediated in part through the Aβ (more ...)
In the mature brain, microglia are readily activated in response to certain cues such as brain injury or immunological stimuli. Activated microglia serve diverse beneficial functions essential to neuronal survival [24
]. However, with chronic and over-activation, microglia themselves can be detrimentally neurotoxic by excessive production of a large array of cytotoxic factors such as superoxide [26
]. The signals responsible for microglial dysregulation are diverse, ranging from exposure to environmental toxins to neuronal death due to brain damage or genetic diseases.
There has been considerable interest from several laboratories in identifying microglial surface receptors that interact with Aβ fibrils. Scavenger receptor class A (SR-A), scavenger receptor-BI (SR-BI) and CD36 [28
]El Khoury et al., 1996 J. El Khoury, S.E. Hickman, C.A. Thomas, L. Cao, S.C. Silverstein and J.D. Loike, Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils, Nat andand and other receptors such as macrophage colony-stimulating factor receptor (MCSF) [31
], formyl peptide receptor-1 receptor (FPR-1) [32
], receptor for advanced glycation end products (RAGE) [33
], etc have been reported to be the binding sites for Aβ. A series of papers from Dr. Landreth et al. show that Aβ interacts with a microglial surface receptor complex composed of B-class scavenger receptor CD36, integrin-associated protein/CD47, and α6
MAC1 is a leukocyte integrin expressed on microglia and it is a versatile adhesion and recognition receptor. MAC1 plays a critical role in microglial migration via pathways that include kinase cascades and cytoskeleton rearrangements, culminating in activation critical for antimicrobial leukocyte functions. MAC1 is also reported to cooperatively activate other key adhesion and defense receptors [38
]. In addition to above-mentioned functions, recent work from our laboratory has demonstrated a critical role of MAC1 in mediating the reactivation of microglia in response to neuronal damage/death, so-called reactive microgliosis [17
]. Our studies suggest that MAC1 serves as a pattern recognition receptor mediating the neurodegeneration produced by toxic substances released from damaged or dead neurons. Since Aβ is one of the constituents in Lewy body, damaged dopaminergic neurons may release Aβ to the extracellular space and further activate microglia at least partially through pattern recognition receptor. Results from this study lend additional support to this possibility.
In the current study, we revealed that MAC1 deficiency mitigated the loss of dopaminergic neurons and GABAergic neurons induced by Aβ exposure, which indicates that MAC1 is critical in the Aβ-induced neurotoxic process. MAC1 deficiency did not change the sensitivity of neurons per se because Aβ exerted same degree of toxicity in neuron-enriched cultures from MAC1-/- and MAC1+/+ mice. The presence of microglia with functional MAC1 expression was essential for the differential sensitivity to Aβ neurotoxicity. This conclusion is further supported by the lack of robust microglia activation in neuron-glia cultures from MAC1-/- mice and the in vivo study using MAC1-/- mice, as measured by changes in cell morphology and by the release of pro-inflammatory factors.
In microglia, activated PHOX is a major source of ROS. Most PHOX products are released into the extracellular space, and it is believed that extracellular ROS plays a major role in neurotoxicity associated with microglial activation [20
]. Previous studies in our lab have shown the involvement of PHOX in microglial activation and associated neurotoxicity induced by Aβ [6
]. The neurotoxicity observed in cultures from wild type mice that induced by Aβ was potently attenuated in cultures from PHOX-/-
mice. Our previous findings, together with present results showing that involvement of MAC1 in Aβ-induced neurotoxicity, lend credence to the conclusion that MAC1 receptors are coupled to PHOX for enzymatic activation and subsequent production of superoxide. These conclusions are further supported by the fact that Aβ-induced translocation of p47phox
from cytosol to cell membrane is substantially diminished in MAC1-/-
cultures. This conclusion is consistent with earlier reports of MAC1-mediated PHOX activation in neutrophils [8
] and eosinophils [14
The next question addressed the signaling pathways mediating the coupling the activation between MAC1 and PHOX. As illustrated in Figure , our studies reveal that PI3K is a key mediator for MAC1 to signal the activation of PHOX. PI3K is a class of phosphate kinase that has been implicated in cell signaling pathways that affect cellular death and longevity as well as many other processes that have medically important implications related to disease states [40
]. The preferred substrate of class I PI3K is phosphoinositide(4,5)bisphosphate (PIP2
). Phosphorylation of PIP2
by PI3K generates PIP3
, which is an important second messenger that can promote cell survival, growth, protein synthesis, mitosis, and motility [43
]. Numerous studies showed that PI3K and PIP3
are involved in the regulation of PHOX. As an important component for PHOX activation, Rac-1 is reported to be a point of possible PI3K intervention [45
is believed to bind to p47phox
]. PI3K signaling pathway may also be involved in PKC activation, thus play an important role in the phosphorylation of p47phox
]. The present results not only show the involvement of PI3K in Aβ-induced PHOX activation, but also suggest that PI3K lies downstream of MAC1 receptor (Figure).
Although strong evidence from this study illustrates a critical role for MAC1 receptors in mediating Aβ-induced neurotoxicity, the inhibition of both dopaminergic neuronal loss and production of superoxide in MAC1-deficient mice or microglia was only partial. These findings indicate that other mechanisms are also involved in Aβ-induced effects. In fact, reports from Dr. Landreth's group recently showed that CD14 and Toll-like receptors 2 and 4 are required for fibrillar Aβ-stimulated microglial activation [49
MAC1 has been reported to bind several cell surface and soluble ligands including iC3b, ICAM, fibrinogen, factor X, filamentous hemagglutinin, lipophosphoglycan, and LPS [50
].12. M. Diamond, J. Garcia-Aguilar, J. Bickford, A. Corbi and T. Springer, The I domain is a major recognition site on the leukocyte integrin Mac-1 for four distinct adhesion ligands. J. Cell Biol. It has been demonstrated that MAC1 is colocalized with Aβ plaques in the brain, thus, it is highly likely that Aβ activated microglia partly through the binding to MAC1 receptors [52
]. To confirm this hypothesis, our ongoing project is to determine if there is direct binding of Aβ to MAC1. Preliminary data from binding assays and immunoprecipitation assays both favor our hypothesis that Aβ does bind to MAC1 receptors.
In conclusion, results from this study indicate that MAC1 receptor is involved in Aβ-induced microglial activation and subsequent neurotoxicity. The present findings are important because they identify an early, possibly initiating, event at the microglial plasma membrane. There are substantial interests in identifying microglial surface molecules that serve in effect as pattern recognition receptors transducing the neurodegenerative effects of substances released from damaged or dead neurons. Interactions involving these putative microglial pattern recognition receptors may represent the initial step in reactive microgliosis, leading to progressive neuronal death in neurodegenerative diseases. In this regard, MAC1 may provide a target for therapeutic intervention. Development of specific MAC1 receptor antagonists may reduce the activation of the MAC1-PHOX complex and the subsequent production of proinflammatory factors, extending neuronal survival.