Mice deficient for the major lysosomal aspartic proteinase cathepsin D, generated by gene targeting, develop normally during the first 2 weeks, stop thriving in the third week and die in a state of anorexia at day 26 +/− 1. An atrophy of the ileal mucosa first observed in the third week progresses towards widespread intestinal necroses accompanied by thromboemboli. Thymus and spleen undergo massive destruction with fulminant loss of T and B cells. Lysosomal bulk proteolysis is maintained. These results suggest, that vital functions of cathepsin D are exerted by limited proteolysis of proteins regulating cell growth and/or tissue homeostasis, while its contribution to bulk proteolysis in lysosomes appears to be non‐critical.
Metachromatic leukodystrophy is a lysosomal sphingolipid storage disorder caused by the deficiency of arylsulfatase A. The disease is characterized by progressive demyelination, causing various neurologic symptoms. Since no naturally occurring animal model of the disease is available, we have generated arylsulfatase A-deficient mice. Deficient animals store the sphingolipid cerebroside-3-sulfate in various neuronal and nonneuronal tissues. The storage pattern is comparable to that of affected humans, but gross defects of white matter were not observed up to the age of 2 years. A reduction of axonal cross-sectional area and an astrogliosis were observed in 1-year-old mice; activation of microglia started at 1 year and was generalized at 2 years. Purkinje cell dendrites show an altered morphology. In the acoustic ganglion numbers of neurons and myelinated fibers are severely decreased, which is accompanied by a loss of brainstem auditory-evoked potentials. Neurologic examination reveals significant impairment of neuromotor coordination.
Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase B (ASB), which is involved in degradation of dermatan sulfate and chondroitin 4-sulfate. A MATERIALS AND METHODSIsolation of a Genomic Clone and Targeting Vector Construction. A 758-bp partial murine ASB cDNA clone (pCABM750) was isolated from a Agtll brain cDNA library (Clontech) by homology screening with a full-length human ASB cDNA probe (4). The nucleotide sequence of pCABM750 was deposited in the GenBank/EMBL data base (accession no. X92096). pCABM750 exhibits 81.8% sequence identity with the human ASB cDNA (4) on nucleotide and 83.1% on deduced amino acid level (data not shown). In EcoRI digested genomic liver DNA of 129/SvJ mice a 9-kbp and a 1.6-kbp DNA fragment are hybridizing with pCABM750. A subgenomic DNA library was constructed by ligation of 8-to 10-kbp EcoRI DNA fragments from genomic liver DNA of 129/SvJ with X-EMBL4 arms and subsequent packaging according to the instructions of the supplier (Stratagene). A 9-kbp genomic clone (pmAB9E, see Fig. la) was isolated from the library by hybridization with pCABM750. For construction of a targeting vector the 6-kbp BamHI DNA fragment from pmAB9E was subcloned into pBlueskript SK-. A linker fragment from the BamHI site to the Sall site of the plasmid vector pUC18 was ligated to the XhoI site at the 5' end of the neomycin (neo) expression cassette of pMClneo poly(A) (ref.13; Stratagene); the neo cassette in pMClneo poly(A) contains a BamHI site at its 3' end. The neo cassette was inserted into the BglII site in exon 5 of the 6-kbp BamHI subclone described above as a BamHI-BamHI restriction fragment resulting in the targeting vector pAB6Bneo (see Fig. la). The insertion of the neo cassette introduces a premature translational stop codon
Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression. Neither the physiological substrates nor the functional significance is known. Mice with a deficiency of LAP generated by targeted disruption of the LAP gene are fertile and develop normally. Microscopic examination of various peripheral organs revealed progredient lysosomal storage in podocytes and tubular epithelial cells of the kidney, with regionally different ultrastructural appearance of the stored material. Within the central nervous system, lysosomal storage was detected to a regionally different extent in microglia, ependymal cells, and astroglia concomitant with the development of a progressive astrogliosis and microglial activation. Whereas behavioral and neuromotor analyses were unable to distinguish between control and deficient mice, ϳ7% of the deficient animals developed generalized seizures. From the age of 6 months onward, conspicuous alterations of bone structure became apparent, resulting in a kyphoscoliotic malformation of the lower thoracic vertebral column. We conclude from these findings that LAP has a unique function in only a subset of cells, where its deficiency causes the storage of a heterogeneously appearing material in lysosomes. The causal relationship of the enzyme defect to the clinical manifestations remains to be determined.
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