Increase In Lysosomal Enzymes Research Articles

Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis--an approach for slowing Alzheimer disease?

Previous reports suggest that age-related lysosomal disturbances contribute to Alzheimer-type accumulations of protein species, blockage of axonal/dendritic transport, and synaptic decline. Here, we tested the hypothesis that lysosomal enzymes are upregulated as a compensatory response to pathogenic protein accumulation. In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%. To address whether this selective activation of the lysosomal system represents compensatory signaling, N-Cbz-L-phenylalanyl-L-alanyl-diazomethylketone (PADK) was used to enhance the lysosome response, generating 4- to 8-fold increases in lysosomal enzymes. PADK-mediated lysosomal modulation was stable for weeks while synaptic components remained normal. When PADK and chloroquine were co-infused, chloroquine no longer increased cellular tau levels. To assess pre-existing pathology, chloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species and restored microtubule integrity. Transport processes lost during chloroquine exposure were consequently re-established, resulting in marked recovery of synaptic components. These data indicate that compensatory activation of lysosomes follows protein accumulation events, and that lysosomal modulation represents a novel approach for treating Alzheimer disease and other protein deposition diseases.

Alternative mechanisms for trafficking of lysosomal enzymes in mannose 6-phosphate receptor-deficient mice are cell type-specific.

Viable mice nullizygous in genes encoding the 300 kDa and the 46 kDa mannose 6-phosphate receptors (MPR 300 and MPR 46) and the insulin like growth factor II (IGF II) were generated to study the trafficking of lysosomal enzymes in the absence of MPRs. The mice have an I-cell disease-like phenotype, with increase of lysosomal enzymes in serum and normal activities in tissues. Surprisingly, the ability of MPR-deficient cells to transport newly synthesized lysosomal enzymes to lysosomes and the underlying mechanisms were found to depend on the cell type. MPR-deficient thymocytes target newly synthesized cathepsin D to lysosomes via an intracellular route. In contrast, hepatocytes and fibroblasts secrete newly synthesized cathepsin D. In fibroblasts recapture of secreted lysosomal enzymes, including that of cathepsin D, is limited and results in lysosomal storage, both in vivo and in vitro, whereas recapture by hepatocytes is remarkably effective in vivo and can result in lysosomal enzyme levels even above normal.

The Effect of Hypo- and Hyperthyroidism on Uterine and Cervical Collagens and Glycosidases in Albino Rats

The effect of hypo- and hyperthyroidism on uterine and cervical collagen and glycosidases were studied in mature albino rats. Hypothyroidism significantly increased the salt-soluble, acid-soluble, insoluble and total collagen in the uterus and cervix. However, all of these collagens were significantly decreased in the hyperthyroid condition. The activities of β- and β-galactosidases and glucosidases were significantly decreased in the uterus and cervix of hypothyroid rats, whereas, in hyperthyroid rats, all of these enzymes were significantly increased in the uterus and cervix. The data obtained in the present study suggested that the increase in lysosomal enzymes increased the degradation of uterine and cervical collagen. In contrast, the decrease in lysosomal enzymes in hypothyroid condition increased uterine and cervical collagen by decreasing degradation.

Characterization of the fate of midline epithelial cells during the fusion of mandibular prominences in vivo.

The fusion of the mandibular prominences along the midline is achieved with the absence of medial epithelial cells at the fusion site. Failure of fusion of the mandibular prominences results in median cleft of the lower lip and mandible. Cellular and molecular events controlling mandibular fusion were examined during the fusion process in mouse embryogenesis. Cell lineage analyses at the fusion site revealed that epithelial cells migrated to the surface and oral epithelia. DiI-labeled epithelial cells were not observed within the mandibular mesenchyme at any state of fusion. Examination of the midline region did not reveal cells with ultrastructural changes characteristic of apoptotic cell death. An increase in lysosomal enzymes in the midline epithelial cells, which would be correlated with programmed cell death, was not observed. Mice lacking TGF-beta 3 did not have cleft mandible, but had clefting of the secondary palate as a feature of null mutation phenotype. We interpret our comparisons between wild type and homozygous TGF-beta 3 (-/-) mice to suggest that different developmental processes control palatal vs. mandibular fusion. We hypothesize that medical epithelial cells at the fusion site of mandibular prominences migrate to the surface epithelium during the fusion process and neither transdifferentiate into mesenchyme nor express apoptosis.

Reversible, hepatic, lysosomal phospholipidosis in rat induced by subchronic daily administration of trospectomycin sulfate

Trospectomycin sulfate is an experimental aminocyclitol antibiotic which has been shown previously to induce the formation of cytoplasmic lamellar bodies in rat and dog liver in subchronic experiments. The effect of repeated daily administration of trospectomycin sulfate on hepatic phospholipid levels and activities of marker enzymes for subcellular organelles was examined. Rats were treated for 30 or 90 days with 0, 50, or 250 mg/kg/day of trospectomycin sulfate prior to being killed, and another group was dosed for 90 days and then allowed to recover for 79 days prior to sacrifice. Transmission electron microscopy showed the presence of lamellar bodies in hepatocytes in both 50 and 250 mg/kg groups at 90 days but no other apparent changes in cellular morphology. Total phospholipids were increased significantly (1.6-fold) only at 90 days (P < 0.01) and only in the 250 mg/kg group. Phosphatidylcholine, phosphatidylinositol, and two acidic lysosomal phospholipids, bis(monoacylglycero)phosphate and acylphosphatidylglycerol, accounted for 42, 35, and 21% of the increase in total phospholipids. Changes in the activities of marker enzymes were generally confined to the 250 mg/kg group at 90 days, with the largest and most significant increases being in the lysosomal enzymes acid phosphatase and hexosaminidase (P < 0.01). Levels of all phospholipids and marker enzymes, with the exception of succinate dehydrogenase, were not significantly different from controls 79 days after cessation of dosing, and lamellar bodies had disappeared. We conclude that repeated trospectomycin sulfate treatment in rat induces a reversible, dose- and time-dependent lysosomal phospholipidosis in liver which is characterized by an increase in lysosomal enzymes and selected anionic phospholipids.

Source of Increased Serum Beta-Hexosaminidase in Rat Liver Cirrhosis

Lysosomal enzymes were studied by histo- and biochemical methods in cirrhotic rat livers in order to explain the serum increase of lysosomal enzymes in cirrhosis. Biochemically, the most evident increase was found in isolated hepatocytes. Histochemically, the lysosomal enzymes were increased mainly in rows of macrophages and necrotic or atrophic hepatocytes adjacent to strands of connective tissue at the periphery of the cirrhotic nodules. The results were compared to similar studies in cholestatic rats. It is concluded that most of the increased serum activities of the lysosomal enzymes derive from the hepatocytes.

The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole.

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.

The effect of pregnancy on functions of inflammatory cells in healthy women and in patients with rheumatic disease.

Chemiluminescence (CL) after zymosan stimulated phagocytosis of polymorphonuclear granulocytes (PMN) and mononuclear cells (MNC), random migration of PMN and intra- and extracellular activities of nine lysosomal enzymes were assessed serially in 8 healthy women and 10 women with rheumatic disease during and after pregnancy. A gestational increase of lysosomal enzymes in serum and enhancement of PMN random migration was observed in all women. Significant differences between healthy and rheumatic women were found for CL of phagocytic cells. In healthy women, CL of PMN was slightly enhanced, while it remained unchanged in MNC during pregnancy. In patients, CL of PMN was markedly suppressed, while MNC CL increased during gestation. An inverse relationship between CL and intracellular enzyme activities was noted. Thus, the presence of an inflammatory state seemed to influence the gestational behavior of phagocytic cells.

Tissue content and localization of lysosomal enzymes in cholestatic rats.

Levels of the lysosomal enzymes beta-glucuronidase and beta-hexosaminidase were determined in tissues from rats at 1-7 weeks after permanent bile duct occlusion (28 animals) and sham operation (16 animals). With increasing duration of bile duct occlusion, increasing levels were found in the liver and spleen, whereas levels were unaffected in the pancreas, kidneys, brain, muscle, and skin. Histochemical investigation showed that the lysosomal enzymes accumulated in the hepatocytes and in the increasing numbers of reticuloendothelial cells of the liver and spleen during cholestasis. No significant accumulation of lysosomal enzymes was seen in the pancreas. In preparations of isolated hepatocytes and non-parenchymal cells increased specific activity of lysosomal enzymes in cholestasis appeared in both cellular fractions. It is assumed that the serum increase of lysosomal enzymes in late cholestasis is partly due to a release from activated macrophages in the liver and spleen.

Influence of streptozotocin- and alloxan-induced diabetes in the rat on collagenase and certain lysosomal enzymes in relation to the degradation of connective tissue proteins.

The activity of collagenase and certain lysosomal hydrolases (cathepsin B1, cathepsin D, beta-glucuronidase and beta-N-acetyl glucosaminidase) was studied in serum and tissues of rats with streptozotocin- or alloxan-induced diabetes. The activity of serum lysosomal enzymes was increased in both groups (p less than 0.05). Both streptozotocin- and alloxan-diabetic animals showed significantly higher dermal collagenase activity than those of controls (p less than 0.01), but the liver and spleen showed similar activities; there was a significant decrease in the renal collagenase activity of streptozotocin-diabetic rats (p less than 0.05). Comparison of the alloxan- or streptozotocin-treated groups with control animals showed an increase in lysosomal enzymes (cathepsin B1, cathepsin D, beta-glucuronidase and beta-N-acetyl glucosaminidase in skin, liver and spleen) (p less than 0.05) but beta-N-acetyl glucosaminidase was unchanged in the spleen of streptozotocin-diabetic rats. There was no difference in renal cathepsin B1 and D in control versus alloxan-diabetic rats, but there was an increase in beta-glucuronidase and beta-N-acetyl glucosaminidase (p less than 0.05). The streptozotocin-diabetic animals showed decreased activities of renal lysosomal enzymes (p less than 0.05), but similar activity of cathepsin D to the control animals.

Toxin-induced activation of muscle hydrolases.

Application of batrachotoxin, lysolecithin, tetrodotoxin, or veratridine to mouse skeletal muscle results in an increase in the activity of the following lysosomal enzymes: acid protease, acid phosphatase, and N-acetylglucosarninidase. Batrachotoxin caused the greatest increase in lysosomal enzymes while veratridine resulted in the smallest response. The increase in lysosomal enzymes was most marked within 1–2 days after toxin application and returned to normal values after 8–12 days.

Intracellular distribution of copper in the liver of normal and copper loaded sheep

Thirteen sheep were dosed repeatedly with copper sulphate in order to induce chronic copper poisoning. Twelve similar sheep acted as controls. Three dosed sheep were killed before haemolysis, six at haemolysis and four after cessation of the haemolytic crisis. The subcellular distribution of Cu, Zn, Fe and lysosomal marker enzymes was investigated in the livers of normal sheep and copper loaded sheep. In control sheep the highest concentration of Cu was in the nuclear fraction. Copper loading increased the concentration of Cu in all fractions. The proportion of Cu in the nuclear fraction continued to increase while that in the heavy mitochondrial fraction decreased. Significant increase in lysosomal enzymes along with electron microscopic studies on pellets of fractions indicated: (a) proliferation of lysosomes, (b) possible localisation of Cu in lysosomes, and (c) sedimentation of the denser lysosomes on N and MH fractions in test sheep with the majority being in the N fraction. Thus the increase in Cu observed in the N fraction is at least partly due to the storage of Cu by lysosomes which sediment with the heavier nuclei. The excessive deposition of Cu in lysosomes and nuclei may lead to cell damage. With increase in hepatic Cu concentration the proportion of Zn and Fe in the cytosol decreased.

The cellular origin of lysosomal enzymes in the plaque in multiple sclerosis: a combined histological and biochemical study.

Using a combined biochemical and histological approach certain conclusions can be drawn as to the origin of the increase in lysosomal enzymes in white matter from MS brains. Firstly, there is a gradient of lysosomal enzyme activity, plaque greater than periplaque greater than macroscopically normal white matter, which appears to be independent of the stage of evolution of the plaque. The most consistent change in cellular composition within and around all plaques is a marked astrocytosis. It thus appears that the increases in lysosomal enzymes can be mainly attributed to reactive astrocytes; macrophages and the cells of the perivascular infiltrate making only a limited contribution. Secondly, only one of the enzymes tested was significantly elevated in histologically normal white matter, i.e. n-acetyl-beta-D-glucosaminidase. Since this enzyme appears to be a particularly sensitive marker for astrocytes this may be indicative of proliferation of astrocytes in so-called normal white matter.

Role of the vacuolar apparatus in augmented protein degradation in cultured fibroblasts.

Rat embryo fibroblasts, grown in Eagle's MEM with 10% serum, showed a rapid increase in autophagic vacuoles when placed in MEM with 0-1% serum. Concurrent with this response, degradation of cellular proteins showed a 2-fold increase. We did not find any increases in cathepsin D, beta-glucuronidase, beta-galactosidase, and beta-glucosidase, or proteolytic activity of cell homogenates at pH 3.7 towards endogenous substrates. Homogenates prepared in 250 mM sucrose at pH 7.0 showed a 40% increase in protein breakdown. These data support the hypothesis that the induced increase in proteolysis, characteristic of cells placed in a nutritionally deficient medium, is effected by an activated vacuolar apparatus (lysosomes and autophagic vacuoles). We suggest, however, that this mechanism is distinct from normal protein turnover in the cell, but can be rapidly induced by appropriate alterations in the cellular environment. Finally, this induced proteolytic mechanism is not dependent upon an increase in lysosomal enzymes, but rather a structural alteration within the cell which effects a transfer of cellular proteins into the vacuolar apparatus.

Biochemical changes in rat parotid gland lysosomal enzyme activities after isoprenaline or starvation

The content of several lysosomal enzymes was measured in the rat parotid gland when the normal secretory cycle was altered by either reducing or enhancing secretion. The findings support the concept that lysosomes play a role in regulating the gland content of secretory products. The secretory protein, amylase, began to accumulate when secretion was reduced. This was immediately followed by an increase in lysosomal enzymes (cathepsin D and acid phosphatase). Thereafter, amylase activity decreased rapidly followed by a smaller reduction in lysosomal enzymes. When secretion was enhanced by an injection of isoprenaline, amylase was reduced by 95 per cent within two hours; the lysosomal enzymes were decreased by 15–30 per cent. Within 24 h, amylase was restored and increased by 50 per cent whereas the lysosomal enzymes were increased only 10–30 per cent. The activity of acid phosphatase was measured with two different substrates. Activity measured with β-glycerophosphate paralleled that of cathepsin D whereas that measured with p-nitrophenylphosphate did not when secretion was reduced but did when secretion was enhanced. The results support the hypothesis that acid phosphatase activity measured with β-glycerophosphate is lysosomal whereas that measured with p-nitrophenylphosphate may have another cellular location in addition to lysosomes.

Effect of ethanol administration on the activity of hepatic lysosomal enzymes

The activities of hepatic lysosomal enzymes were determined in rats after the acute administration of ethanol and its feeding for periods of 1 and 6 months. The acute administration of ethanol and its feeding for 1 month did not result in any changes in lysosomal enzymes. By contrast the feeding of ethanol for a period of 6 months resulted in increases in the total activities of β-glucuronidase, β- N-acetylglucosaminidase and arylsulfatase A measured in the homogenates, and in the specific activities of these enzymes plus acid phosphatase measured in the lysosomal fraction. In addition, there were increases in the free activities of β-glucuronidase and acid phosphatase measured in the homogenates. Hyaluronidase activity was not changed by ethanol feeding. The increases in lysosomal enzymes were accompanied by histologic changes consisting only of mild fatty infiltration.

Lysosomes and Related Structures in the Posterior Silk Gland Cells of&lt;I&gt;Bombyx mori.&lt;/I&gt; I. In Late Larval Stadium

Measurements of lysosomal enzyme activities (acid phosphatase, acid RNase, and cathepsin) and observations of electron microscopic cytochemical preparations were correlated with ultrastructural changes in the posterior silk gland cells during the late larval stadium of the silkworm Bombyx mori. In the fourth instar the lysosomal enzymes were synthesized exclusively in the feeding stage when the wet weight of the gland increased exponentially, and the enzymes were probably stored in Golgi bodies. In the molting stage a number of autophagosomes, autolysosomes and autophagic vacuoles appeared in the cells, and the lysosomal enzymes presumably played pivotal roles in the catabolic processes of the silk gland cells. In the early half of the fifth instar (0-96 h), a similar exponential increase in lysosomal enzymes was observed, and the synthesized enzymes were also probably stored in Golgi bodies. In the middle of the fifth instar (96 h) when the metabolism of the silk gland was changed from the growth phase to stationary phase, several characteristic structures appeared, such as lipid droplets, myelin structures and whorl structures of rough ER. In the latter half of the fifth instar, the lysosomal enzyme activities were stabilized temporarily then increased again slightly arriving at a maximum value at the end of the fifth instar.

The Effect of in Vivo Irradiation on Macrophage Function

Abstract In vivo irradiation (660 R) of donor mice before removal of their peritoneal macrophages caused activation of these cells as indicated by increases in lysosomal enzymes but did not significantly affect the manner in which these cells handled keyhole limpet hemocyanin (KLH) in comparison to how macrophages from unirradiated donors handled KLH. The parameters measured included uptake, catabolism, retention and amount of membrane-bound KLH. In syngeneic recipients, however, the immunogenicity of equal amounts of KLH associated with activated macrophages from irradiated donors was significantly increased compared to that of KLH associated with macrophages from unirradiated donors. Also, addition of activated non-KLH-containing macrophages to the inoculum of normal KLH-containing macrophages increased the immune response. Infection of mice with lactic dehydrogenase virus also enhanced the immunogenicity of KLH associated with macrophages from these mice compared to control, uninfected mice.

Hydrolytic enzymes of human blood monocytes and neutrophils with special reference to lysozyme (muramidase).

In order to elucidate the cause of markedly increased serum and urine lysozyme (muramidase) in patients with monocytic leukemia on the cellular basis, monocytes and neutrophils were compared on their hydrolytic enzyme contents and their characteristics. Both types of leukocytes were separated from normal human blood or from patients with monocytic leukemia by means of floatation in dense albumin, utilizing the difference in the specific gravity of both cell types. Equivalent lysozyme and acid phosphatase activities were found to be present in monocytes and neutrophils and very low or negligible activities of alkaline phosphatase in monocytes. The subcellular distributions of these enzymes were comparable in the pattern and specific activity in both cell populations and found to be associated with granules. The phagocytic rate and the readiness of release of lysozyme into the extracellular medium during phagocytosis were similar in both cell populations. Leukemic leukocytes, neutrophils and monocytes, showed lower hydrolase activities than normal counterparts, especially alkaline phosphatase being markedly suppressed in leukemic cells. The possibility was discussed that the mechanism for elevated lysozyme in serum and urine of patients with monocytic leukemia may be accounted for by the developmental potential of monocytes in extravascular sites into macrophages with marked increase in lysosomal enzymes, the relatively ready release of lysozyme from cells, and a stable nature of lysozyme.

Characterization and behaviour of lysosomal enzymes in tail tissue of xenopus larvae during involution

1. The distribution of lysosomal enzymes obtained by density gradient centrifugation of cell-free extracts of Xenopus tail tissue was compared during growth and involution. The particles containing lysosomal enzymes were also characterized by means of electron microscopy. 2. Cytochromeoxidase is recovered to more than 90% in bound form (mitochondria), whereas lysosomal enzymes are characterized by a high proportion of soluble activity (up to 67%). The particle-bound activity ofβ-glucuronidase, cathepsin, and acid RNase show two peaks after centrifugation, in contrast to the activity of acid phosphatase, which is distributed over the whole gradient. During tail involution an increasing proportion of soluble activity is observed, especially for cathepsin and acid RNase. 3. Electronmicroscopical examination reveals a rather heterogeneous composition of the particle populations obtained by density gradient centrifugation. In fractions with highest activity of lysosomal enzymes various types of electron dense granules are found, thus precluding characterization of a specific lysosomal structure. 4. The distribution of enzymes after induction of tail involution with thyroxine was almost similar to the results obtained from spontaneously metamorphosing animals. Since actinomycin D inhibits the increase in lysosomal enzymes and tissue involution it is concluded that the synthesis of soluble acid hydrolases is a prerequisite of hormonal induced tissue involution.