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. 2002 Jun 11;99(12):7883-8.
doi: 10.1073/pnas.112632299. Epub 2002 Jun 4.

Neuronal loss and brain atrophy in mice lacking cathepsins B and L

Ute Felbor  1 , Benedikt Kessler, Walther Mothes, Hans H Goebel, Hidde L Ploegh, Roderick T Bronson, Bjorn R Olsen
Affiliations

Neuronal loss and brain atrophy in mice lacking cathepsins B and L

Ute Felbor et al. Proc Natl Acad Sci U S A. .

Abstract

Cathepsins B and L are widely expressed cysteine proteases implicated in both intracellular proteolysis and extracellular matrix remodeling. However, specific roles remain to be validated in vivo. Here we show that combined deficiency of cathepsins B and L in mice is lethal during the second to fourth week of life. Cathepsin B(-/-)/L(-/-) mice reveal a degree of brain atrophy not previously seen in mice. This is because of massive apoptosis of select neurons in the cerebral cortex and the cerebellar Purkinje and granule cell layers. Neurodegeneration is accompanied by pronounced reactive astrocytosis and is preceded by an accumulation of ultrastructurally and biochemically unique lysosomal bodies in large cortical neurons and by axonal enlargements. Our data demonstrate a pivotal role for cathepsins B and L in maintenance of the central nervous system.

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Figures

Figure 1
Figure 1
Early death in cathepsin B−/−/L−/− mice. (a) Genotyping of wild-type (lanes 1), cathepsin B−/−/L−/− (lanes 2), and cathepsin B+/−/L+/− (lanes 3) mice by PCR. (b) 125I-LHVS-PhOH and 125I-JPM565 active-site labeling of brain homogenates from cathepsin B+/−/L+/+ (lane 1), cathepsin B+/−/L+/− (lane 2), cathepsin B+/−/L−/− (lane 3), cathepsin B+/+/L+/− (lane 4), cathepsin B+/−/L+/− (lane 5), and cathepsin B−/−/L+/− (lane 6) mice. Cathepsins B and L (CTSB and CTSL) and their corresponding proforms were identified according to previous studies (19, 24). (c) Mortality diagram of wild-type (n = 20, dashed line) and cathepsin B−/−/L−/− mice (n = 14, solid line). Generally, the lifetime could be extended by only a few days through reduction of litter size to 3 to 5 pups at day 7 in further breedings (II) (n = 34). Importantly however, these breedings yielded 8 mice aged 21 to 50 days in which brain shrinkage became evident.
Figure 2
Figure 2
Cerebral and cerebellar atrophy in cathepsin B−/−/L−/− mice. (a–c) Luxol fast blue (LFB)-stained coronal sections of P50.5 littermate control (a), P17.5 (b), and P50.5 (c) cathepsin B−/−/L−/− cerebrum demonstrating cortical shrinkage accompanied by white-matter (wm) reduction between days 17 and 50 in double-mutants (cx = cortex; scale bar in a = 1 mm). The deep cerebral white matter is not yet myelinated in 17-day mutants (b). (d–f) LFB-stain of P50.5 wild-type (d), P17.5 (e), and P50.5 (f) cathepsin B−/−/L−/− cerebellum (m = molecular layer, g = granular layer; scale bar = 1 mm). (h–j) H&E stain of the above samples at ×100. Note the dying bright red Purkinje cell in i, the complete absence of the Purkinje cell layer (p), and the striking reduction in cellular density in the granule cell layer (g) in j. (g) Nuclear pyknosis, karyorrhexis, and brightly red-stained angular cytoplasm in dying cortical neurons at P24.5. Healthy neurons exhibit a lightly stained nucleus and a darkly stained nucleolus as seen in the bottom part of g. (k) Lipid-laden macrophages in the corpus callosum at P50.5 (×1250 in g–k).
Figure 3
Figure 3
Positive TUNEL-labeling indicative of apoptotic cell death and positive GFAP immunostaining demonstrating reactive gliosis in response to neuron death in cathepsin B−/−/L−/− mice. (a–c) Control (a) and cathepsin B−/−/L−/− P24.5 (b and c) cryosections of the cerebral cortex showing small TUNEL-positive globules in the center of a shrunken cathepsin B−/−/L−/− neuron in b and division into apoptotic cell fragments in c. (d and e) Control (d) and cathepsin B−/−/L−/− (e) P24.5 granule layer of the cerebellar central lobe shows TUNEL-positive material around the periphery of double-mutant nuclei, suggesting chromatin margination. (f–i) GFAP-immunostaining of 6-μm coronal paraffin sections reveals abundant star-shaped hypertrophic astrocytes in the 50-day-old double-mutant cerebral cortex (g) not present in the control cortex (f). Control (h) and double-mutant (i) 50-day-old cerebellum shows the picture of an isomorphic gliosis in the latter. The processes of the Bergmann glia are in parallel orientation. Note the tiny healthy area in i with a residual Purkinje cell (arrow). [Scale bars = 7 μm (a–e), and 0.1 mm (f–i).]
Figure 4
Figure 4
Accumulation of amorphous and membranous material in neuronal perikarya, dendrites, and axons in cathepsin B−/−/L−/− mice. (a and b) LFB stain of P11.5 and P15.5 double-mutant cortical neurons. At P11.5 (a), only a few inclusions are observed in some neuronal perikarya (arrow). The arrowhead points to a normal neuron. Only a few days later, at P15.5 (b), the perikarya are expanded and filled with LFB-positive inclusions. (c–e) Electron micrographs of inclusions in P12.5 (c and d) and P24.5 (e) cathepsin B−/−/L−/− cortical neurons. (d) Higher magnification of the inclusion indicated by an arrow in c. (f) Sagittal section of the P11.5 double-knockout brain shown in a revealing a dense accumulation of eosinophilic globules in cerebral white matter (H&E stain). (g and h) Magnification of the area surrounding the asterisk shown in f stained with H&E and LFB, respectively. Normal white matter is seen in the upper right corner. All LFB stainings were performed simultaneously. (i) Electron micrograph of membranous inclusions in a swollen axon in the corpus callosum of a P12.5 cathepsin B−/−/L−/− mouse. [Scale bars = 15 μm (a, b, g, h), 2 μm (c), 100 nm (d), 1.6 μm (e), 0.15 mm (f), and 500 nm (i).]
Figure 5
Figure 5
Endosomal and lysosomal compartments accumulate proportionally in null mice because processing of the short-lived 53-kDa precursor of cathepsin D into the 47-kDa intermediate form and the mature enzyme composed of a 31-kDa and a 14-kDa fragment is not altered in cathepsin B−/−/L−/− brain lysates. Ten micrograms of brain homogenates from wild-type (WT), 10-, and 18-day-old cathepsin B−/−/L−/− mice (MUT) were separated on 7.5–17.5% SDS/PAGE gels, electroblotted onto nitrocellulose, and probed with an anti-cathepsin D antibody.
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