A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration

Abstract

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of neurodegenerative storage diseases characterized by progressive psychomotor retardation, blindness and premature death. Pathologically, there is accumulation of autofluorescent material in lysosome-derived organelles in a variety of cell types, but neurons in the central nervous system appear to be selectively affected and undergo progressive death. In this report we show that a novel form of NCL, congenital ovine NCL, is caused by a deficiency in the lysosomal aspartyl proteinase cathepsin D. A single nucleotide mutation in the cathepsin D gene results in conversion of an active site aspartate to asparagine, leading to production of an enzymatically inactive but stable protein. This results in severe cerebrocortical atrophy and early death, providing strong evidence for an important role of cathepsin D in neuronal development and/or homeostasis.

Fig. 1. Coronal sections of the left cerebral hemisphere of a normal and CONCL lamb. Note the striking reduction in size, lack of distinction between gray and white matter, and the enlarged lateral ventricle in the CONCL brain (scale in millimeters).

Fig. 2. Histochemical and immunocytochemical stainings of paraffin-embedded sections of CONCL and control brain. The storage material had histochemical characteristics typical of NCL (Rapola, 1993), showing positive staining with PAS (A). There is marked loss of neurons and infiltration of macrophages (seen as dark pink in PAS staining) in the cerebral cortex of the affected lambs (A). In the large neurons of basal ganglia of normal sheep, SAP staining shows a punctuate lysosomal pattern (B), whereas intense staining of the storage material filling the whole neuronal cytoplasm is seen in affected sheep (C). The storage material is negative for subunit c of mitochondrial ATP synthase (D). Preimmune sera and PBS were used to verify the specificity of the antisera (not shown). Magnification, ×450.

Fig. 3. Western blot analysis of cathepsin D in four CONCL (–/–), one heterozygous (+/–) and three control (+/+) sheep. Brain homogenates (30 µg protein) were fractionated by SDS–PAGE and transferred to nitrocellulose. The processed single-chain form (48 kDa) and the heavy chain (32 kDa) of the two-chain form of cathepsin D were detected using rabbit antisera and chromogenic substrates. Molecular weight markers are indicated on the right.

Fig. 4. Sequence alignment of the human, ovine and murine cathepsin D. Identical amino acids are shown in black and similar residues in gray. Cathepsin D is synthesized as a preproform with an N-terminal signal sequence and propeptide, which are cleaved to produce the single-chain enzyme. The single-chain enzyme is further processed into the two-chain form, composed of an N-terminal light chain and C-terminal heavy chain. The proteolytic cleavage sites in human cathepsin D are indicated by arrows and the catalytic aspartates by arrowheads. The mutation associated with CONCL results in substitution of an asparagine for the aspartate corresponding to Asp295 of human cathepsin D.