In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11
Fig 1
Homozygous trapped mice represent Spatacsin knockout mice.
(A) Partial genomic structure of the targeted Spg11 locus and the predicted mutant fusion protein as compared to wild-type Spatacsin; rectangles: exons, SA: splice acceptor, βgeo: β-galactosidase and neomycin fusion cassette, pA: polyadenylation site. The black arrowhead indicates the position of the epitope for antibody generation. CHC: clathrin heavy chain. (B-H) LacZ stainings of sections of the cortex (B-D), hippocampus (E), cerebellum (F), brain stem (G), and spinal cord (H) from 2-month-old heterozygous trapped mice shows that Spg11 expression follows a neuronal pattern. DG: dentate gyrus; GL: granular layer, PCL: Purkinje cell layer, ML: molecular layer; SOC: superior olivary complex, IOC: inferior olivary complex. Scale bars: 100 μm. (I) Northern blot analysis of total brain RNA from wild-type (WT) mice shows a 7.6 kb WT-transcript which is absent in RNA isolated from homozygous trapped mice (KO). Gapdh served as a loading control. (J) Western Blot analysis with an affinity-purified monoclonal antibody directed against the deleted part of the Spatacsin protein (the position of the epitope is indicated in (A) by an arrowhead) detects a band of the predicted size in WT brain lysates, which is absent in brain lysates of homozygous trapped mice. Tubulin served as a loading control. (K) While Zfyve26 levels are diminished in brain lysates of Spatacsin KO mice, levels of the beta subunit of the AP-5 complex (Ap5b1) are not changed (n = 3; Student’s t-test: * indicates p<0.05; n.s.: not significant).