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Fig. 2 | Molecular Neurodegeneration

Fig. 2

From: Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

Fig. 2

Altered protein expression, maturation and activity of cathepsins in brain of Grn−/− mice. a Schematic presentation of cathepsin maturation [53]. Cathepsins (Cat) are synthesized as an inactive pre-pro-form (pp), translocated into the endoplasmic reticulum (ER) by signal peptide (SP). After SP removal the pro-form (p) becomes co-transitionally modified and is transported to lysosomes predominantly via the manose-6-phosphate pathway. With increasing acidification, the pro-peptide is removed, either autocatalytically or by other enzymes, leading to an active single chain variant (sc, green). For most cathepsins this sc-variant can be further proteolytically processed to a heavy (hc) and a light chain (lc), as long as hc and lc are linked by disulfide bridges or hydrophobic interaction the double chain variant remains active (green) but will be inactivated by separation of hc and lc. Representative blots of brain lysate from 3-month-old and 20-month-old Grn+/+ (wt) and Grn−/− (ko) mice probed for cathepsin D (CatD) (b, d) cathepsin B (CatB) (f, h) and cathepsin L (CatL) (j, l). The molecular weight standards in kilo Daltons (kDa) are indicated on the left side of the blots. Quantification of blots for total cathepsin or maturation variants normalized to Grn+/+ are shown as mean ± SD. N = 5 mice per genotype (b, d, f, h, j, l). Statistical significance was set at *, p < 0.05; **, p < 0.01; and ***, p < 0.001; ns, not significant using an unpaired, two-tailed student’s t-test. In vitro enzyme activity of CatD, CatB and CatL in lysates of mouse brains used for immunoblot analysis. Equal amounts of enzyme optimized brain lysates from Grn+/+ and Grn−/− (n = 3–5) mice were incubated with quenched fluorogenic substrate (c, e, g, i, k, m). The increase of fluorescence signal was continuously measured and for a linear turnover time period normalized to Grn+/+ set as 100% activity, mean ± SD. Statistical significance was set at ***, p < 0.001 and ns, not significant using unpaired, two-tailed student’s t-test

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