Fig. 4.

ApoE4-expressing astroglioma cells show more apoE binding and less TFEB binding to CLEAR DNA than apoE3-expressing cells. (A) ApoE is present in the nucleus and the cytosol of apoE-expressing T98G cells. (B) Western-blot analysis of proteins pulled down by binding to CLEAR-motif dsDNA shows elevated apoE4 binding relative to apoE3. (C) ApoE4-expressing cells show less TFEB binding to CLEAR-motif dsDNA relative to cells expressing apoE3. (D) Electrophoretic mobility shift assay (EMSA), in which a ³²P-labeled probe containing the CLEAR motif was incubated with 50 ng of recombinant apoE3 (lane 1, “E3”) or apoE4 (lane 2, “E4”) purified from T98G cells and resolved on a nondenaturing gel. ApoE4 was also tested in the presence of 2 μg of antibody to apoE (lane 3, “E4 Ab”), saturating levels of unlabeled CLEAR dsDNA as cold competition (lane 4, “E4 CC”), or a ³²P-labeled probe of scrambled sequence (lane 5, E4 scram”), demonstrating sequence specificity for the apoE4 isoform-binding to the CLEAR sequence on dsDNA. (E) Schematic of hypothesized modulation of TFEB-CLEAR interactions by apoE. Stresses, such as starvation as we show in these cells, or other stresses that we postulate in AD, evoke nuclear translocation of TFEB. In apoE3-expressing cells, there is reduced binding of apoE to CLEAR sequences, allowing TFEB greater upregulation of transcription of autophagy genes. Conversely, apoE4 competes with TFEB for CLEAR sequences and thus blunts transcription of autophagy mRNAs. This can predispose cells to proteotoxic damage, activation of inflammatory cytokines, and ultimately to cell death; the first is of particular note in neurons as dysfunctions in autophagy result in buildup of aggregates and formation of paired helical filaments of hyperphosphorylated tau. Abbreviations: AD, Alzheimer’s disease; ApoE, apolipoprotein E; CLEAR, coordinated lysosomal expression and regulation; TFEB, transcription factor EB.