Amyloid-β(1-42) protofibrils formed in modified artificial cerebrospinal fluid bind and activate microglia

Area of Science:

• Neuroscience
• Biochemistry
• Cell Biology

Background:

• Soluble aggregated forms of amyloid-beta (Aβ) protein are implicated in Alzheimer's disease (AD) pathogenesis.
• Protofibrils represent intermediate species in Aβ aggregation and exhibit toxic and inflammatory properties.
• In vitro studies require optimized conditions for preparing and characterizing Aβ protofibrils for cellular assays.

Purpose of the Study:

• To describe the preparation and characterization of Aβ(1-42) protofibrils in a modified artificial cerebrospinal fluid (aCSF).
• To demonstrate the binding and activation of microglial cells by Aβ protofibrils.
• To establish a suitable buffer system for Aβ protofibril biophysical and cellular studies.

Main Methods:

• Aβ(1-42) was reconstituted in a modified aCSF buffer system.
• Protofibrils were isolated using size exclusion chromatography (SEC).
• Protofibril characterization included length, hydrodynamic radii, and concentration determination (BCA assay).
• Microglial activation was assessed by TNFα production and membrane binding assays.

Main Results:

• Modified aCSF provided appropriate ionic strength and cell compatibility without interfering spectroscopic analysis.
• SEC isolation yielded curvilinear β-sheet Aβ(1-42) protofibrils (<100 nm length, 21 nm hydrodynamic radii).
• Protofibrils, but not monomers, significantly stimulated TNFα production in BV-2 and primary microglia.
• Aβ protofibrils demonstrated substantial binding to microglial membranes.

Conclusions:

• A modified aCSF system is suitable for preparing SEC-isolated Aβ(1-42) protofibrils for biological studies.
• Aβ protofibrils exhibit a unique ability to functionally interact with and activate microglia.
• These findings underscore the role of Aβ protofibrils in microglial activation relevant to Alzheimer's disease.