Scavenger Receptor Class A and Macrophage Receptor with Collagenous Structure Mediate Oligomeric Amyloid-beta Clearance in Microglia

• Main Authors: Cheng-Ning Yang, 楊振寧
• Other Authors: Huey-Jen Tsay
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/20430700741105623980

博士 === 國立陽明大學 === 神經科學研究所 === 100 === The accumulation of soluble oligomeric amyloid- peptide (oA) proceeds the formation of senile plaques and contributes to synaptic and memory deficits in Alzheimer’s disease (AD). The mechanism of microglial oA clearance remains unclear and thought to be mediated by scavenger receptors (SRs). SRs bind to their ligands in a subtype-specific manner. The study aims at identifying the specific subtypes of SRs for oAinternalization and oA degradation mechanism in microglia. Here, sequence-specific small interfering RNAs (siRNAs) targeting at SR-A, CD36, and macrophage receptor with collagenous structure (MARCO) were used. The involvement of lysosomes in oAdegradation was investigated. Our results suggest that SR-A, a member of class A scavenger receptors is the prominent receptor for oA internalization by siRNA knockdown approach and SR-A knockout mice. Internalized oA vesicles were degraded effectively to acidified lysosomes, instead of recycling. The involvement of CD36 in oAinternalization was ruled out. In addition to SR-A, human MARCO (hMARCO), another member of class A scavenger receptors was also colocalized with internalized oAvesicles. MACRO mediated approximately 20% of oA internalization in microglia. Overexpressed hMARCO in HEK293T cells enhanced the surface binding of oA. To verify the oA binding sites of hMARCO, hMARCO variants with the deletion and point mutations in scavenger receptor cysteine-rich (SRCR) domain were constructed and were overexpressed in HEK293T cells. SRCR domain, but not collagenous domain of MARCO was required for the surface targeting and oA binding. Furthermore, the critical residues in SRCR domain determining translation/degradation and surface targeting were identified. Leucine residue at position 463 of SRCR domain was critical for oA binding. MARCO contains two putative N-glycosylation sites at position N83 and N136. Our results indicate that both N83 and N136 were critical for protein expression. Mutated N83 impaired the surface targeting even more severely compared with mutated N136. Mutant at both N83 and N136 completely abolished the surface targeting. However, the relative intensity of bound oA on MARCO-positive cells was not different among wild-type, N83, and N136 mutants. In conclusion, our study suggest that SR-A and MARCO are responsible for the majority of microglial oAinternalization, and oAis sequentially degraded by lysosomes. SRCR domain of MARCO on oAinternalization and N-glycosylation play critical roles for the protein expression and surface targeting. During aging, impaired SR-A and MARCO or lysosomes in microglia could contribute to oA accumulation in the initiation stage of AD.