Figure 1
From: Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity
α-syn accumulates in neuronal cell bodies in p25 Ctsd -/- cortex. a. Immunohistochemical detection of α-syn and ubiquitin in p25 Ctsd+/+ (+/+) and Ctsd-/- (-/-) cortex. Scale bar = 20 micron. Arrows point to intense α-syn and ubiquitin immunoreactive cells. b. α-syn accumulation occurs in NeuN+ neuronal cell bodies. α-syn = Cy3 (red), NeuN = FITC (green). Wildtype (+/+) brains exhibit diffuse α-syn staining consistent with a synaptic distribution. Ctsd-/- brains showed neurons with cytoplasmic accumulation of α-syn immunoreactivity. Scale bar = 10 micron. c. α-syn does not exhibit pronounced accumulation in GFAP+ cells. α-syn = Cy3 (red). GFAP = FITC (green). Scale bar = 10 micron. d. Accumulation of high molecular weight α-syn and ubiquitinated proteins in both the TritonX-100 soluble and the insoluble fractions of the Ctsd-/- mice. Intensity of α-syn monomer, α-syn oligomers, and Ub-positive smears were quantified and compared between Ctsd+/+ and Ctsd-/- extracts. Truncated 12 kDa and 10 kDa α-syn fragments are reduced in Ctsd-/- extracts. e. Quantification of the western results. n = 3 mice each genotype. *p < 0.05 compared to Ctsd+/+ by Student t-test. S = TritonX-100 soluble. IS = TritonX-100 insoluble. f. A small fraction of accumulated α-syn in Ctsd-/- brains being mono-ubiquitinated. Western blot analysis of TritonX-100 soluble Ctsd-/- brain extract (Input), pulldown product by a polyclonal α-syn antibody C-20 (Santa Cruz) (middle lane), and pulldown product by a same-isotype control antibody (IgG). Immunoblot was probed with mAb1510 against ubiquitin. Shown are the 25 kDa bands of ubiquitin immunoreactivity that were pulled down by the C-20 anti-α-syn antibody. g. Western blot analysis of ubiquitin with p21 Ctsd+/+ and Ctsd-/- cortical extracts. The intensity of each lane was quantified and shown in the bar graph. *p < 0.05 Student t-test. h. Western blot analyses of Ctsd+/+ and Ctsd-/- cortical extracts, together with human DLB brain extracts using anti-phospho-α-syn antibody provided by Dr. Iwatsubo [33, 36, 38]. Arrows indicate positions of immunoreactive bands that are increased in Ctsd-/- mice and the position of α-syn monomer.