Raft aggregation with specific receptor recruitment is required for microglial phagocytosis of Aβ₄₂

Abstract

Microglial phagocytosis contributes to the maintenance of brain homeostasis. Mechanisms involved however, remain unclear. Using Aβ 42 solely as a stimulant, we provide novel insight into regulation of microglial phagocytosis by rafts. We demonstrate the existence of an Aβ 42 threshold level of 250pg/ml, above which microglial phagocytic function is impaired. Low levels of Aβ 42 facilitate fluorescent bead uptake, whereas phagocytosis is inhibited when Aβ 42 accumulates. We also show that region-specific raft clustering occurs prior to microglial phagocytosis. Low Aβ 42 levels stimulated this type of raft aggregation, but high Aβ 42 levels inhibited it. Additionally, treatment with high Aβ 42 concentrations caused a redistribution of the raft structural protein flotillin1 from low to higher density fractions along a sucrose gradient. This suggests a loss of raft structural integrity.Certain non-steroidal anti-inflammatory drugs, e.g. the COX-2-specific NSAID, celecoxib, raise Aβ 42 levels. We demonstrated that prolonged celecoxib exposure can disrupt rafts in a manner similar to that seen in an elevated Aβ 42 environment: abnormal raft aggregation and Flot1 distribution. This resulted in aberrant receptor recruitment to rafts and impaired receptor-mediated phagocytosis by microglial cells. Specifically, recruitment of the scavenger receptor CD36 to rafts during active phagocytosis was affected. Thus, we propose that maintaining raft integrity is crucial to determining microglial phagocytic outcomes and disease progression.

Endocytosis of Particle Formulations by Macrophages and Its Application to Clinical Treatment

“…As a result, phagocytic activity toward PSL particles, which are ingested through SRs, decreases. Similarly, recruitment of CD36 to raft domains is necessary for phagocytosis of amyloid  by microglial cells [83]. Lipid rafts are essential for recruitment of phagocytic receptors; and, hence, they are associated with delivery of drug-containing particles by phagocytosis.…”

Isolation of rafts from mouse brain tissue by a detergent-free method

“…Fifteen sequential fractions of 260 ml each were gently removed from the top of the tube and individually aliquoted. High-density fractions 14 and 15 were pooled as they are not normally considered as raft fractions (1,10,28). The fractions were stored at 280°C for a maximum of 6 months.…”

“…High-density fractions (fractions 14 and 15) were pooled. As protein concentration does not provide a true baseline parameter for comparison across fractions, equal sample volume loading was used for the Western blot, as previously described (10,19). This loading method accounts for differential separation of proteins into specific fractions following density ultracentrifugation.…”

“…This arrangement allows for the existence of small, organized membrane microdomains called rafts (1)(2)(3)(4)(5) that orchestrate and regulate a number of signaling processes (6)(7)(8)(9)(10)(11)(12)(13). In order to elucidate mechanisms involved in these processes, it is crucial to understand raft biology.…”

“…These studies, as well as raft interactions for signal transduction, membrane trafficking, and endocytosis, emphasize the value of examining whole tissue in the hope of determining regional brain, genetic, or age-specific effects. The brain represents one target tissue for which the role of membrane rafts in various disease conditions has more recently become of interest (10,31,33,(35)(36)(37), and is therefore, the focus of this protocol. The majority of the work examining the role of rafts in the nervous system has relied on either primary cell cultures or isolated cellular preparations, such as from synaptosomes (38) and microvessicles (39).…”