Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.

Cathepsin D-deficient (CD-/-) mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demonstrated in the neuronal perikarya of CD-/- mouse brains after P20. Autophagosomes and granular osmiophilic deposits were detected in neurons at P0 but were few in number, whereas they increased in the neuronal perikarya within days after birth. Some large-sized neurons having autophagosome/autolysosome-like bodies in the perikarya appeared in the CNS tissues, especially in the thalamic region and the cerebral cortex, at P17. These lysosomal bodies occupied the perikarya of almost all neurons in CD-/- mouse brains obtained from P23 until the terminal stage. Because these neurons exhibited autofluorescence, it was considered that ceroid lipofuscin may accumulate in lysosomal structures of CD-/- neurons. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brain. Moreover, neurons near the terminal stage were often shrunken and possessed irregular nuclei through which small dense chromatin masses were scattered. These results suggest that the CNS neurons in CD-/- mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis.

Developmental changes in the expression of neuronal ceroid lipofuscinoses-linked proteins.

Neuronal ceroid lipofuscinoses (NCL) form a distinct group of storage diseases where the normal development of the central nervous system is interrupted and neurons of the neocortex begin to degenerate. Mutations in genes encoding three lysosomal enzymes are the causes for three early-onset forms of NCLs: palmitoyl-protein thioesterase 1 (PPT1) is deficient in human infantile NCL, tripeptidyl peptidase 1 (TTP1) in late-infantile NCL, and cathepsin D in congenital ovine NCL. We wanted to compare the developmental expression profiles of these enzymes in rat brain. In conclusion, the PPT1 expression pattern differed from the two other lysosomal enzymes implicated in NCL diseases, thus suggesting a distinctive role for PPT1 in brain development.

Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase.

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis.

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.

A lysosomal proteinase, the late infantile neuronal ceroid lipofuscinosis gene (CLN2) product, is essential for degradation of a hydrophobic protein, the subunit c of ATP synthase.

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.

Accumulation of mitochondrial ATP synthase subunit c in muscle in a patient with neuronal ceroid lipofuscinosis (late infantile form).

We report a case late infantile neuronal ceroid lipofuscinosis (NCL). Abnormal granules were found in the skeletal muscle fibers, Schwann cells, perineurial cells, endothelial cells, fibroblasts, and perivascular smooth muscle cells in the sural nerve. Electron microscopy revealed that these granules showed fingerprint profiles, curvilinear profiles or membrane-bound membranous structures. Acid phosphatase reaction was increased in these cells. Immunohistochemical studies for mitochondrial ATP synthase subunit c showed a strong reaction in these cells, suggesting abnormal accumulation of subunit c. Immunohistochemistry for subunit c in muscle may be useful in the diagnosis of late infantile NCL.

Effect of the calcium channel blocker nilvadipine on urinary albumin excretion in hypertensive microalbuminuric patients with non-insulin-dependent diabetes mellitus.

A 24-week study was conducted to evaluate the effects of the dihydropyridine calcium channel blocker nilvadipine on urinary albumin excretion in eight microalbuminuric hypertensive patients with non-insulin-dependent (type II) diabetes mellitus. Blood pressure and urinary albumin excretion measurements before the administration of nilvadipine (8 mg) were compared with those after 4, 8, 12 and 24 weeks of treatment. No significant changes were observed in the mean values of haemoglobin A1C. Systolic blood pressure was significantly reduced from 174 +/- 23 mmHg before treatment to 144 +/- 13 mmHg after 24 weeks of treatment (P < 0.02). Diastolic blood pressure was significantly reduced from 93 +/- 11 mmHg at baseline to 79 +/- 8 mmHg after 24 weeks of treatment (P < 0.05). Urinary albumin excretion was significantly reduced from 65.4 +/- 37.4 mg/g creatinine at baseline to 51.6 +/- 41.1 mg/g creatinine (P < 0.05) after 4 weeks, and to 39.1 +/- 26.9 mg/g creatinine (P < 0.02) after 24 weeks of treatment. These data suggest that in hypertensive microalbuminuric patients with non-insulin-dependent diabetes mellitus, treatment of hypertension with the calcium blocker nilvadipine may slow the progression of diabetic nephropathy.

Decreased lysosomal subunit c-degrading activity in fibroblasts from patients with late infantile neuronal ceroid lipofuscinosis.

We investigated in in-vitro cell-free incubation experiments which factor, lysosomal proteolytic dysfunction or structural alteration of subunit c, is responsible for the specific delay in the degradation of subunit c in patient cells with the late infantile form of neuronal ceroid lipofuscinosis. Experiments using substrates and soluble lysosomal fractions isolated separately from control and patient cells indicated that lysosomes from control cells are able to degrade mitochondrial subunit c either from control or patient cells at much faster rate than lysosomes from patient cells. Subunit c stored in patient cell lysosomes showed much more resistance to proteolytic attack than mitochondrial subunit c, suggesting that conformation of subunit c as well as lysosomal proteolytic dysfunction both participate in the specific lysosomal accumulation of subunit c in the late infantile disease.

Specific delay in the degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis is derived from cellular proteolytic dysfunction rather than structural alteration of subunit c.

Previously we indicated that a specific delay in subunit c degradation causes the accumulation of mitochondrial ATP synthase subunit c in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (NCL). To explore the mechanism of lysosomal storage of subunit c in patient cells, we investigated the mechanism of the lysosomal accumulation of subunit c both in cultured normal fibroblasts and in in vitro cell-free incubation experiments. Addition of pepstatin to normal fibroblasts causes the marked lysosomal accumulation of subunit c and less accumulation of Mn(2+)-superoxide dismutase (SOD). In contrast, E-64-d stimulates greater lysosomal storage of Mn(2+)-SOD than of subunit c. Incubation of mitochondrial-lysosomal fractions from control and diseased cells at acidic pH leads to a much more rapid degradation of subunit c in control cells than in diseased cells, whereas other mitochondrial proteins, including Mn(2+)-SOD, beta subunit of ATP synthase, and subunit i.v. of cytochrome oxidase, are degraded at similar rates in both control and patient cells. The proteolysis of subunit c in normal cell extracts is inhibited markedly by pepstatin and weakly by E-64-c, as in the cultured cell experiments. However, there are no differences in the lysosomal protease levels, including the levels of the pepstatin-sensitive aspartic protease cathepsin D between control and patient cells. The stable subunit c in mitochondrial-lysosomal fractions from patient cells is degraded on incubation with mitochondrial-lysosomal fractions from control cells. Exchange experiments using radiolabeled substrates and nonlabeled proteolytic sources from control and patient cells showed that proteolytic dysfunction, rather than structural alterations such as the posttranslational modification of subunit c, is responsible for the specific delay in the degradation of subunit c in the late infantile form of NCL.

[Batten disease (Neuronal ceroid lipofuscinoses)--accumulation of ATP synthase subunit c caused by the delay of lysosomal degradation].

The neuronal ceroid lipofuscinoses (NCLs) represent a group of recessively inherited neurogenerative diseases of infants, children, and young adults that leads to blindness, seizures, dementia, and premature death. These diseases are pathologically characterized by a massive lysosomal storage of autofluorescent lipopigments in neurons and a wide variety of extraneuronal cells. Linkage studies have shown localization of the infantile disease to chromosome region 1p32 the juvenile onset disease to chromosome 16p12.1-p11.2 and a variant form of late infantile form to chromosome 13q21.1-q32. Recently, protein sequencing and immunochemical studies have identified subunit c of the mitochondrial ATP synthase as a major component of the storage material in the late infantile and juvenile types of NCL, and SAPs in infantile type of NCL. Immunolocalization studies demonstrated a dot-like staining of subunit c in the cells with NCL and the staining pattern of subunit c was similar to that of a lysosomal membrane marker, 1gp120. Pulse-chase experiments revealed that a specific failure occurs in the degradation of subunit c in lysosomes whereas its transport into mitochondria and subsequent sequestration into lysosomes are apparently normal.

New insight into lysosomal protein storage disease: delayed catabolism of ATP synthase subunit c in Batten disease.

Subunit c is normally present as an inner mitochondrial membrane component of the Fo sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. Mechanism for specific accumulation of subunit c in lysosomes is not known. The rate of degradation of subunit c as measured by pulsechase and immunoprecipitation showed a marked delay of degradation in patients fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes, through an autophagic process. There were no large differences of various lysosomal protease activities between control and patient cells. In patient cells sucrose loading caused a marked shift of lysosomal density, but did not a shift of subunit c containing storage body. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.

Abnormal degradative pathway of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid-lipofuscinosis (Batten disease).

Subunit c is normally present as an inner mitochondrial membrane component of the F0 sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid-lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. The rate of degradation of subunit c as measured by pulse-chase and immunoprecipitation showed a marked delay of degradation in patients' fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes through an autophagic process. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.

Specific delay of degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis (Batten disease).

Subunit c is normally present as an inner mitochondrial membrane component of the F0 section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.

Defect of proteolysis of mitochondrial ATP synthase subunit C in neuronal ceroid lipofuscinosis.

Mechanism of lysosomal storage of mitochondrial ATP synthase subunit c in late infantile form of NCL was studied. Morphological and biochemical examinations with patient fibroblasts showed that subunit c, not other mitochondrial proteins was specifically localized in lysosomes. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed a specific delay of degradation of subunit c in patient cells with late infantile form of NCL and lysosomal transfer of radioactive mitochondrial subunit c after chase for 1-2 weeks, suggesting that subunit c is transfered to lysosomes through an autophagic process and accumulated as a consequence of abnormal catabolism in lysosomes.

Purification and characterization of flavine-adenine dinucleotide phosphohydrolase from rat liver lysosomal membranes.

An enzyme hydrolyzing flavine-adenine dinucleotide (FAD) to flavine mononucleotide (FMN) and adenosine monophosphate (AMP) was purified about 460-fold over the isolated lysosomal membranes with 9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and the absence of SDS. Purification procedures included: preparation of crude lysosomal membranes, solubilization with Triton X-100, WGA-Sepharose, Con A-Sepharose, hydroxylapatite chromatography, gel filtration with Superdex 200, DEAE ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme, estimated by gel filtration with Superdex 200, was approximately 560 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular weight of 140,000. The pH optimum for FAD hydrolysis was 8.5 with an apparent Km of 0.1 mM and the isoelectric point was pH 7.3. The activity was inhibited by o-phenanthroline, EDTA, DTT, and NEM and was slightly stimulated by Zn ion, but was not affected by Ca or Mg ions. The purified FADase contained N-linked complex type oligosaccharide chains lacking neuraminic acids. The NH2 terminal 21 amino acid residues of the purified FADase were Ser-Pro-Cys-Val-Cys-Asp-Pro-Val-Val-Val-Cys-Lys-Val-Val-Pro-Cys-Thr-Leu- Ala-Leu .

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.