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.

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.

Hermansky-Pudlak syndrome in a Swiss population.

Tyrosinase-positive albinism, previously diagnosed as Hermansky-Pudlak Syndrome (HPS), has been examined in four generations from a village of the canton Valais, Switzerland. Homozygotes, obligate heterozygotes and putative heterozygotes in this geneology yielded lower than normal membrane-associated thioredoxin reductase (TR) activities compared with normal family members and controls. All of the homozygotes and 50% of each the obligate and putative heterozygotes showed an increase in bleeding time associated with storage-pool-deficient platelets lacking dense bodies. The TR activity profile and the platelet-dense body deficiency in the Swiss albinos was the same as that in the HPS population from Puerto Rico. However, in albinos from Puerto Rico, there is an accumulation of ceroid/lipofuscin-like pigment in lysosomal structures causing tissue damage, and, upon kidney involvement, this leads to increased urinary dolichol excretion. Approximately half of the Puerto Rican HPS cases had clinical evidence of storage disease with restrictive lung disease, granulomatous colitis, kidney failure and cardiomyopathy. By comparison, the Swiss HPS geneology had a normal life expectancy with no significant evidence for ceroid accumulation or urinary dolichol excretion. An examination of antioxidant enzymes, catalase, TR and glutathione reductase in epidermal suction blisters from Swiss HPS homozygotes showed a similar result for catalase and TR levels to the depigmented epidermis of patients with vitiligo, except that intracellular TR was found to be calcium free in HPS compared with vitiligo. Intracellular glutathione reductase levels were highest in HPS. Both the Swiss and Puerto Rican HPS homozygotes and heterozygotes have giant melanosomes in skin melanocytes.

Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease).

Immunochemical studies demonstrated the specific accumulation of subunit c of mitochondrial ATP synthase in the brain homogenates of late infantile and juvenile forms of Batten's disease. It is not stored in the infantile form. Storage of subunit alpha of mitochondrial ATP synthase and cytochrome c oxidase subunit IV, an inner membrane protein of mitochondria was not detected in the brains. There was also no difference in the levels of cathepsin B between the two forms of Batten's disease and controls. In cultured skin fibroblasts subunit c accumulates in the late infantile form, whereas it does not in other lysosomal storage diseases. Crude mitochondrial lysosomal preparations of control fibroblasts were separated into high-density fractions rich in a lysosomal marker and low-density fractions rich in a mitochondrial marker on Percoll density gradients. Subunit c was mostly recovered in low-density mitochondrial fractions, but in cells from the late infantile disease a part of subunit c was recovered in the high-density lysosomal fractions. Immunolocalization studies demonstrated a dot-like staining of storage materials for subunit c in the cells from late infantile patients and the staining pattern of subunit c is similar to that of a lysosomal membrane marker, lgp120. Immunostaining failed to detect subunit c in control cells. These results indicate a specific accumulation of subunit c in lysosomes, and suggest that the two forms of Batten's disease are caused by a specific failure in the degradation of subunit c.

Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease).

The ceroid-lipofuscinoses (Batten disease) are neurodegenerative inherited lysosomal storage diseases of children and animals. A common finding is the occurrence of fluorescent storage bodies (lipopigment) in cells. These have been isolated from tissues of affected sheep. Direct protein sequencing established that the major component is identical to the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mitochondrial ATP synthase and that this protein accounts for at least 50% of the storage body mass. No other mitochondrial components are stored. Direct sequencing of storage bodies isolated from tissues of children with juvenile and late infantile ceroid-lipofuscinosis established that they also contain large amounts of complete and normal subunit c. It is also stored in the disease in cattle and dogs but is not present in storage bodies from the human infantile form. Subunit c is normally found as part of the mitochondrial ATP synthase complex and accounts for 2-4% of the inner mitochondrial membrane protein. Mitochondria from affected sheep contain normal amounts of this protein. The P1 and P2 genes that code for it are normal as are mRNA levels. Oxidative phosphorylation is also normal. These findings suggest that ovine ceroid-lipofuscinosis is caused by a specific failure in the degradation of subunit c after its normal inclusion into mitochondria, and its consequent abnormal accumulation in lysosomes. This implies a unique pathway for subunit c degradation. It is probable that the human late infantile and juvenile diseases and the disease in cattle and dogs involve lesions in the same pathway.

Release of arachidonic acid by NMDA-receptor activation in the rat hippocampus.

In hippocampal slices arachidonic acid released after NMDA post-synaptic receptor activation is thought to act as a retrograde trans-synaptic messenger which facilitates the pre-synaptic release of L-glutamate to be involved in the expression of long-term synaptic potentiation (LTP). We measured the mass amount of arachidonic acid released from hippocampal slices incubated under conditions which maintain the electrophysiological responsiveness of the slice. Melittin released arachidonic, oleic and docosahexaenoic acids by phospholipase A2 activation but not palmitic or stearic acids. Of greater interest L-glutamate, N-methyl-D-aspartate and incubation conditions known to induce LTP selectively and rapidly increased the release of arachidonic acid in amounts over basal levels of 200-300 ng/mg protein. This is the first direct determination of the mass amount of arachidonic acid released following NMDA receptor activation in the hippocampus.

Extracellular matrix regulates proliferation and phospholipid turnover in glomerular epithelial cells.

To understand how glomerular epithelial cell (GEC) growth might be regulated in health and disease, we studied the effects of growth factors and extracellular matrix on proliferation and membrane phospholipid turnover in cultured rat GECs. In GECs adherent to type I collagen matrix, epidermal growth factor (EGF), insulin, and serum stimulated DNA synthesis and increased cell number. In addition, GECs proliferated when adherent to type IV collagen, but not to laminin or plastic substrata. Attachment of GECs to the substrata that facilitated proliferation (types I or IV collagen) produced increases in 1,2-diacylglycerol (DAG), an activator of protein kinase C (PKC). Increased DAG was associated with hydrolysis of inositol phospholipids and an increase in inositol trisphosphate and was not dependent on the presence of growth factors. After PKC downregulation (by preincubation with a high dose of phorbol myristate acetate), DNA synthesis was enhanced in GECs adherent to collagen. Thus contact of GECs with collagen matrices is required for serum, EGF, or insulin to induce proliferation. Collagen matrix also activates phospholipase C. As a result, the DAG-PKC signaling pathway desensitizes GECs to the mitogenic effects of growth factors and might promote cell differentiation. Understanding the interaction between GECs, growth factors, and extracellular matrix may elucidate the mechanisms of proliferation during glomerular injury.

Long-term changes of synaptic transmission induced by arachidonic acid in the CA1 subfield of the rat hippocampus.

The effects induced by arachidonic acid upon excitatory synaptic transmission were investigated in the CA1 subfield of rat hippocampal slices. Perfusion with medium containing 50 microM of arachidonic acid induced in 12 of 19 experiments a long-lasting potentiation of the stratum radiatum-induced responses recorded in the cell body and in the apical dendritic layers. In 5 of 19 experiments, arachidonic acid evoked a depression of the same responses. Both effects were antagonized by nordihydroguaiaretic acid which is an inhibitor of lipoxygenase enzymes. These results demonstrate that one or more than one of the arachidonic acid metabolites of the lipoxygenase pathways might be involved in the long-term modulation of synaptic transmission in the hippocampus.

Arachidonic acid cascade and signal transduction.

Since a review on this topic in this Journal appeared (Wolfe, 1982), the CNS has proved to be a major focus in eicosanoid research. Although our knowledge is limited at the moment, the research in this field is rapidly growing. In this short review, we summarize recent progress of research (1982-1989) in this field with special attention directed to eicosanoid metabolism, functions of eicosanoids in the neuroendocrine system and synaptic transmission, current information on eicosanoid receptors, and the link between eicosanoids and cerebral circulation. Knowledge of the eicosanoids has paved the way to a better understanding of intercellular signal transduction systems, including neuronal functions.

Formation of 12-lipoxygenase metabolites in rat cerebral cortical slices: stimulation by calcium ionophore, glutamate and N-methyl-D-aspartate.

The 12-lipoxygenase pathway of arachidonic acid metabolism in rat cerebral cortex slices is stimulated by exogenous arachidonic acid, Ca2+ ionophore A23187, phorbol ester, glutamate, N-methyl-D-aspartate (NMDA) but not by kainate and other neurotransmitters except norepinephrine. The 12-hydroxyeicosatetraenoic acid formed is the (S)-enantiomer. A specific role for 12-lipoxygenase metabolites in NMDA receptor activation and long term potentiation is proposed.

Metabolism of prostaglandin D2 by human cerebral cortex into 9 alpha, 11 beta-prostaglandin F2 by an active NADPH-dependent 11-ketoreductase.

In homogenates of rat cerebral neocortex prostaglandin D2 (PGD2) was found to be quantitatively the main PG biosynthesized by a cytosolic PGD synthetase from endogenously released arachidonic acid. Amounts of 628 ng/g wet weight were found after 30-min incubation periods compared with basal levels of 2.3 ng/g wet weight. In human cerebral cortex, whether obtained at biopsy or postmortem, only small amounts of PGD2 (4.5-11.7 ng/g wet weight/30 min) were formed. Furthermore, PGD2, added to homogenates of human biopsy temporal cortex, was converted efficiently into 9 alpha,11 beta-PGF2 by a NADPH-dependent 11-ketoreductase as has been reported in other human tissues (liver and lung). PGF2 alpha was determined directly as the n-butylboronate derivative. It became clear that 9 alpha,11 beta-PGF2 was formed in considerably greater amounts than PGF2 alpha and that other metabolites are also formed. These results can account for the low amounts of PGD2 found in incubations of human brain tissue. The rat brain does not contain 11-ketoreductase activity. The present results indicate that the 9 alpha,11 beta-PGF2 must be considered along with other eicosanoids in pathophysiological situations in brain.

Lysosomal storage of the DCCD reactive proteolipid subunit of mitochondrial ATP synthase in human and ovine ceroid lipofuscinoses.

The ceroid lipofuscinoses (Batten's disease) are a group of neuro-degenerative lysosomal storage diseases of children and animals that are recessively inherited. In the diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. The material stored in the cells of sheep affected with ceroid lipofuscinosis is two-thirds protein. The stored material does not arise from lipid peroxidation or a defect in lipid metabolism, and the lipid content is consistent with a lysosomal origin for the storage bodies. The major protein stains poorly with Coomassie blue dye and is soluble in organic solvents. It has an apparent molecular weight of 3,500 and its amino acids sequence is identical to that of the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mammalian mitochondrial ATP synthases. Apart from removal of mitochondrial import sequences, it has not been modified post-translationally. At least 50% of the mass of the storage bodies is composed of this protein. A minor protein sequence related to the 17-kDa subunit of vacuolar H(+)-ATPase is also found in storage bodies isolated from pancreas. As in humans and cattle, the ovine protein is the product of two expressed genes named P1 and P2. In normal and diseased animals there are no differences in sequences between P1 cDNAs or P2 cDNAs, nor do levels of mRNAs in liver for P1 or P2 differ substantially between normal and diseased animals. Both normal and diseased sheep also express a spliced pseudogene encoding amino acids 1 to 31 of the mitochondrial import presequence. The peptides they encode differ by one amino acid; arginine-23 is changed to glutamine in the diseased sheep. Storage bodies isolated from brains and pancreas of children affected with the juvenile and late infantile forms of ceroid lipofuscinosis also contain large amounts of material that is identical to subunit c of ATP synthase. However, the protein is not present in storage bodies isolated from brains of patients affected with the infantile form of the disease, and these storage bodies contain other unidentified proteins. It is possible that the cause of ovine, juvenile and late infantile ceroid lipofuscinoses is related to a defect in degradation of the subunit c of mitochondrial ATP synthase.