Figure 8: Intracellular processing and degradation of Notch receptor.

(a) WT and Nuc1 astrocytes transfected with myc-tagged full-length Notch1 receptor were monitored for receptor clearance when cocultured with astrocytes overexpressing Jagged1. Top panels show myc labelling (red) after transfection. Labelling decreased quickly in WT astrocytes, with little remaining after 3 h. Degradation was minimal in the Nuc1 astrocytes. Scale bar, 20 μm. (b) Subcellular fractionation showed Notch1 present in LAMP1-positive late endosomes/lysosomes (interface 1) and Rab5-positive early endosomes (interface 2) in both WT and Nuc1 astrocytes, but more strongly so in Nuc1. Overexpression of βA3/A1-crystallin in Nuc1 astrocytes (right panel) stimulated Notch1 degradation, decreasing Notch1 to the range present in WT astrocytes. Notch1 was not present in endoplasmic reticulum (calnexin-positive lane). (c) Immunostaining of WT and Nuc1 astrocytes for βA3/A1-crystallin (red), revealed abundant staining in the cytosol in both cell types. Co-localization (yellow) with LAMP1-positive lysosomes (green) was perinuclear in WT; little co-localization was seen in Nuc1 and lysosome distribution was abnormal. Scale bar, 20 μm. (d) Subcellular fractionation revealed both βA3- and βA1-crystallins in the cytosol of WT and Nuc1 astrocytes. They were more strongly associated with lysosomes (Interface 1) in WT astrocytes. (e) Western analysis demonstrated substantially reduced levels of Cathepsin D heavy (~50%) and light (~60%) chains in Nuc1 homozygote astrocytes relative to WT, consistent with decreased active enzyme in the mutant astrocytes. Quantification as described in Methods. (f) Overexpression of βA3/A1-crystallin in Nuc1 astrocytes increased activity from 30 to 75% of WT level (*P=0.037). (g) Cathepsin D activity in Nuc1 heterozygote astrocytes was decreased 80% by Cryba1 siRNA knockdown of βA3/A1-crystallin (**P=0.011); subsequent overexpression of βA3/A1-crystallin rescued activity to control values (**P=0.009). (h) In astrocytes from Cryba1-floxed mice, deletion by Cre-recombinase reduced Cathepsin D activity (~60%) (*P=0.031); subsequent overexpression of βA3/A1-crystallin rescued the activity (*P=0.018). (i) Schematic model of Notch signalling and βA3/A1-crystallin. Steps affected by loss of βA3/A1-crystallin shown by red dotted arrows. Impaired endolysosomal acidification inhibits Notch receptor processing and degradation, decreases NICD, ultimately reducing transcription of Notch target genes. 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<0.01; *P<0.05.