Figure 5.

TFEB Overexpression Reduces Storage in LSD Cells

(A) NSCs isolated from the cerebral cortex of MSD and MPSIIIA mice were nucleofected with either a TFEB plasmid or with an empty plasmid and cultured on coverslips. Alcian blue was then used to stain the GAGs.

(B) Wild-type, MSD, and MPSIIIA NSCs were nucleofected either with a TFEB plasmid or with an empty plasmid. After 16 hr, cells were pulsed with H³-glucosamine in differentiation medium and chased for the indicated time-points. Cell extracts obtained at different time points were quantified to determine the levels of labeled GAGs.

(C) Glia-differentiated MSD and MPS-IIIA NSCs were nucleofected with either TFEB or with an empty vector, fixed on glutaraldehyde, and processed for standard electron microscopy. The number of vacuoles per cell was counted from 20 different cells and displayed as mean ± SEM.

(D) TFEB promotes clearance of lipofuscin in fibroblasts from a patient with Batten disease. Cells were transfected with a vector carrying TFEB-Ruby (continuous red staining). After 24 hr, cells were examined by live imaging confocal analysis. Cells with increased TFEB (i.e., cells with red signal in the picture and outlined by dashed white lines in the middle panel) display highly reduced levels of lipofuscin (punctate green signal) and a normal cellular morphology compared with nontransfected cells (i.e., cells with intense green staining).

(E) Human Pompe disease fibroblasts were transfected with either TFEB or with an empty vector, loaded with the fluorescent sugar 2-NBDG, and analyzed by epifluorescence microscopy.

(F) Secretion efficiency of radioactive GAGs was measured in the culture medium of NSCs from MSD mice. Cells were nucleofected with either TFEB or an empty vector after pulse-chase incorporation of H³-glucosamine. Scale bars represent 100 μm (A), 10 μm (C, MSD cells), 500 nm (C, MPSIIIA cells), and 10 μm (D). Data represent mean ± SEM; ^∗p < 0.05 (B, C, and F).