Figure 6: Assessment of γ-secretase activity in WT and in astrocytes lacking βA3/A1-crystallin.

(a) Post-mitochondrial supernatants from WT and Nuc1 astrocytes were layered on 2.5–30% iodixanol gradients and subjected to ultracentrifugation. Western blots identified LAMP1-positive lysosomes in fractions 3, 4 and 5; Rab5-positive endosomes in fractions 6, 7 and 8; and Golgi in fractions 9 and 10. (b) ELISA was performed to quantify β-amyloid (Aβ40) production after incubating above fractions overnight at 37 °C, with or without L-685,458 (γ-secretase inhibitor). In WT astrocytes, γ-secretase activity, as measured by Aβ40 level, was detected in fractions 3, 4 and 5 (lysosomes), and in fractions 6, 7 and 8 (late and early endosomes). γ-Secretase activity was detected at lower level in the Golgi (fractions 9 and 10). (c) In Nuc1 astrocytes, γ-secretase activity was significantly decreased in the endolysosomal compartments (~50% reduction), whereas the activity in Golgi was unchanged. (d) Overexpression of βA3/A1-crystallin in astrocytes cultured from Nuc1 rats rescued γ-secretase activity in endolysosomal compartments, while empty vector had no effect (relative to vehicle). (e) Deletion of Cryba1 from Nuc1 heterozygote astrocytes using a Cryba1 ON-TARGET-plus SMART pool siRNA reduced γ-secretase activity in endolysosomal compartments substantially to levels comparable to that of Nuc1 homozygote astrocytes. There was no effect on activity in Golgi. Overexpression of βA3/A1-crystallin in the same astrocytes, as in e, rescued activity to control levels in the endolysosomal fractions. Scrambled siRNA had no effect (relative to vehicle). (f) Astrocytes from Cryba1-floxed mice were infected with recombinant adenovirus (Ad) constructs (Ad CMV Cre-RSV GFP or Ad CMV-eGFP) followed by βA3/A1-crystallin overexpression. Cryba1-knockout (Ad CMV Cre-RSV GFP) mouse astrocytes show markedly reduced γ-secretase activity in the endolysosomal compartments, with no effect on Golgi. Subsequent overexpression of βA3/A1-crystallin rescued activity to near-normal levels in endolysosomal fractions (relative to Cryba1 flox-Ad CMV-eGFP). In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analysis was performed by a two-tailed Student’s t-test: *P<0.05; **P<0.01.