Astrocytes regulate α-secretase-cleaved soluble amyloid precursor protein secretion in neuronal cells: Involvement of group IIA secretory phospholipase A2.

Astrocytes are major supportive cells in brains with important functions including providing nutrients and regulating neuronal activities. In this study, we demonstrated that astrocytes regulate amyloid precursor protein (APP) processing in neuronal cells through secretion of group IIA secretory phospholipase A2 (sPLA2-IIA). When astrocytic cells (DITNC) were mildly stimulated with the pro-inflammatory cytokines, such as TNF α and IL-1ß, sPLA2-IIA was secreted into the medium. When conditioned medium containing sPLA2-IIA was applied to human neuroblastoma (SH-SY5Y) cells, there was an increase in both cell membrane fluidity and secretion of α-secretase-cleaved soluble amyloid precursor protein (sAPPα). These changes were abrogated by KH064, a selective inhibitor of sPLA2-IIA. In addition, exposing SH-SY5Y cells to recombinant human sPLA2-IIA also increased membrane fluidity, accumulation of APP at the cell surface, and secretion of sAPPα, but without altering total expressions of APP, α-secretases and ß-site APP cleaving enzyme (BACE1). Taken together, our results provide novel information regarding a functional role of sPLA2-IIA in astrocytes for regulating APP processing in neuronal cells.

Genetically encoded self-assembly of large amyloid fibers.

"Functional" amyloids are found throughout nature as robust materials. We have discovered that "template" and "adder" proteins cooperatively self-assemble into micrometer-sized amyloid fibers with a controllable, hierarchical structure. Here, Escherichia coli is genetically engineered to express a template protein, Gd20, that can initiate self-assembly of large amyloid fibrils and fibers. Through atomic force microscopy (AFM) we found that expression of Gd20 produces large amyloid fibrils of 490 nm diameter and 2-15 µm length. Addition of an extracellular adder protein, myoglobin, continues self-assembly to form amyloid tapes with widths of ∼7.5 µm, heights of ∼400 nm, and lengths exceeding 100 µm. Without myoglobin the amyloid fibrils are metastable over time. When myoglobin is present, the amyloid fiber continues self-assembling to a width of ∼18 µm and height of ∼1 µm. Experimental results demonstrate that large amyloid fibers with a tailored stiffness and morphology can be engineered at the DNA level, spanning four orders of magnitude.